Metabolic Diseases PDF

Physician’s Guide to the
Diagnosis, Treatment, and

Follow-Up of Inherited
Metabolic Diseases
Nenad Blau
Marinus Duran
K. Michael Gibson
Carlo Dionisi-Vici
Editors
123
Physician’s Guide to the Diagnosis, Treatment,
and Follow-Up of Inherited Metabolic Diseases
Nenad Blau • Marinus Duran
K. Michael Gibson • Carlo Dionisi-Vici
Editors
Physician’s Guide to the

Diagnosis, Treatment,
and Follow-Up of Inherited
Metabolic Diseases
Editors
Nenad Blau K. Michael Gibson
Division of Inborn Metabolic Diseases Section of Clinical Pharmacology
Department of General Pediatrics Washington State University
University Children’s Hospital Spokane, WA
Heidelberg USA
Germany
Carlo Dionisi-Vici
Marinus Duran Division of Metabolism
Laboratory Genetic Metabolic Diseases Department of Pediatric Medicine
University of Amsterdam Bambino Gesù Children’s
Academic Medical Center Research Hospital
Amsterdam Rome
The Netherlands Italy
ISBN 978-3-642-40336-1 ISBN 978-3-642-40337-8 (eBook)

DOI 10.1007/978-3-642-40337-8
Springer Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014933397
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Springer is part of Springer Science+Business Media (www.springer.com)

Milan Blaskovics (1928–2013)
The Editors take this opportunity to acknowledge Dr. Milan Blaskovics,

one of the founding members of the Editorial team who passed away on
13 December 2013, for his longstanding efforts and contributions to our
series Physician’s Guide and Laboratory Guide in Metabolic Disease, as well
as his longstanding involvement in improving the life of children and adults
with inherited metabolic diseases, especially phenylketonuria. In recognition
of his constant guidance and valuable contributions, we wish to dedicate this
edition of the Physician’s Guide to the Diagnosis, Treatment, and Follow-Up
of Inherited Metabolic Diseases to his memory.
Nenad Blau
Marinus Duran
K. Michael Gibson
Carlo Dionisi-Vici
Foreword
In a recent study of urea cycle disorders, two of the main findings were that many patients
presented as adults, and at all ages there were frequently long delays before the correct diag-
nosis was made. It is to be hoped that this book, aimed at physicians responsible for all ages,
children, adolescents, and adults, will reduce delays in the diagnosis of inborn errors of metab-
olism. This is vital as early and rapid diagnosis is often the key to a good outcome.
This new book brings up-to-date and combines two earlier volumes, Physician’s Guide to
the Treatment and Follow-Up of Metabolic Diseases and Physician’s Guide to the Laboratory
Diagnosis of Metabolic Diseases. It is not a conventional textbook but rather a book for refer-
ence with information in a form that is readily accessible. The format is broadly the same as
before with some text and much of the information presented in tables. This single volume is
an important step forward as the diagnosis and management of inborn errors are highly depen-
dent on laboratory investigations, so it makes sense to combine the clinical with the laboratory
aspects. The clinical chapters cover the full range of inborn errors covering clinical symptoms
and signs with management. The chapters on investigations range from the simple to the com-
plex and include imaging as well as laboratory tests.
Most inborn errors are rare so experience is generally spread quite thinly. This volume will
undoubtedly be useful for busy physicians faced with difficult clinical problems that need
quick decisions.
Oxford, UK James Leonard
vii
Preface
The editors feel that the present edition of the Physician’s Guide to the Diagnosis, Treatment,
and Follow-Up of Metabolic Diseases takes clinical practice in rare metabolic disorders to the
next level. Exactly 100 experts in the field authored 55 chapters dealing with a total of 530
inherited metabolic disorders. The structure of the book and chapters was redesigned, and
many new chapters and new disorders were added to the current edition. Additionally, the
entire content of this edition is stored in a single database, which will become the foundation
for the future knowledgbase of inborn errors of metabolism, IEMBASE.
The major goals of this edition, however, remain comparable to those of earlier editions.
The book presents the signs and symptoms of most of the recognized inborn errors of metabo-
lism in relation to age. There is a chronological sequence of signs and symptoms from infancy
through childhood, adolescence, and adulthood, and in addition normal and pathological val-
ues are provided for each of the disorders so that one does not have to question the significance
of laboratory tests and reported values.
Recognized authorities have described each disorder. Based upon their experience, they
have created flow charts and diagnostic algorithms and have recommended a variety of confir-
matory tests and initial treatment schemes to help those practitioners who do not have exten-
sive experience in inborn errors of metabolism. The second part of each chapter describes the
treatment of groups of disorders in more detail. With regard to the latter, the current edition
utilizes expanding progress with computer technology to make accessing all of the data in the
book seamless. In addition to the hardcover version, an ebook will be available that will allow
the user to rapidly locate a disorder utilizing standard searches with keywords. It is the hope of
the editors that the readers will find this edition helpful, both now and in the future, for the
treatment and care of patients with inborn errors of metabolism.
Heidelberg, Germany Nenad Blau

Amsterdam, The Netherlands Marinus Duran
Spokane, WA, USA K. Michael Gibson
Rome, Italy Carlo Dionisi-Vici
ix
How to Use This Book
This book is meant to supply clinicians and clinical biochemists with data that should facilitate
the diagnosis of an inherited metabolic disease. No information about detailed laboratory
methods is given; rather, the relationship between laboratory data and clinical signs and symp-
toms is highlighted. Subsequently, the current knowledge on the immediate emergency inter-
vention, standard treatment, and experimental options are given. Entry to the book is achieved
by scanning either of the indices, i.e., the signs and symptoms index, the tests index, or the
disorders index. Due to the great clinical variability of inherited metabolic diseases, one should
not restrict oneself to one disorder when observing a given symptom or sign.
Most chapters have a uniform layout as given below. In a few chapters, however, this was
not possible and information is given for the entire related group of disorders in the chapter.
Introduction
The introduction gives a brief overview of the clinical conditions described in the chapter and
relates them to the biochemical abnormalities. Key references for further reading are
provided.
Nomenclature
Disorders in each chapter are numbered in accordance with the corresponding OMIM number
[1], gene symbols and gene products and chromosomal localization if known.
Metabolic Pathway
Disorders are identified by corresponding reference numbers at the step where the defect is
localized. Pathological metabolites (“markers”) are given in most chapters.
Signs and Symptoms

The tables describe most, if not all, of the signs and symptoms for each disorder, including its
reference number, and the most important laboratory tests, in relation to age. In all instances,
the signs and symptoms are in the untreated (natural) state. The signs written in bold are char-
acteristic feature of the particular disease.
± indicates that a sign or symptom may occur but is not inevitably present.
+ indicates that a sign or symptom is always or nearly always present. If there are signifi-
cant clinical signs and symptoms which exceed the usual, or if changes occur, this is indicated
with + to + + +, etc.
n (normal) is used only when it is significant and may be useful in distinguishing one condi-
tion from another.
Relative increases or decreases of substances, compounds, metabolites, etc., are indicated
with the use of arrows; for example, metabolite X ↑ to ↓↓↓. Where metabolite X may change,
it would be indicated by n-↑ for a possible increase or ↓-n for a possible decrease, whichever
the case.
In all tables, the test substance, material, compound, metabolite, etc., are listed and the
source—(U), (B), (CSF), (P), (RBC), etc.—is given in parentheses, with an arrow or arrows
indicating increase/decrease or relative increase/decrease.
xi
xii How to Use This Book
Body fluids, cells, tissues, etc., are defined as:

P Plasma CV Chorionic villi
S Serum AF Amniotic fluid
B Blood AFC Amniocytes
U Urine CCV Cultured chorionic villi
CSF Cerebrospinal fluid PLT Platelets
RBC Red blood cells WBC White blood cells
LYM Lymphocytes Hb Hemoglobin
FB Fibroblasts creat Creatinine
BM Bone marrow
Age groups are defined as:

Neonatal Birth to 1 month
Infancy 1–18 months
Childhood 1.5–11 years
Adolescence 11–16 years
Adulthood >16 years
Normal Values/Pathological Values/Differential Diagnosis

Reference and pathological values are listed for all parameters relevant to the diagnosis accord-
ing to the specimen (e.g., P, U, CSF) and age. For some parameters, normal values depend on
methodology and may differ from chapter to chapter. Methods are specified where necessary.
Pathological values are listed either as absolute values or with symbols (e.g., ↑, ↓) according to
the disorder. Values are limited to the analyses which can be performed in a laboratory experi-
enced in selective screening. Data on enzyme studies are not given in most cases, which can be
found in the pertinent literature.
Loading Tests
There is a brief description of the tests, with a table or figure to illustrate the interpretation.
Diagnostic Flow Chart

The flow charts use simple yes/no algorithms to demonstrate the sequence for differential
diagnosis, starting with clinical symptoms or general tests and proceeding to specific tests and
a final diagnosis.
Specimen Collection
This table lists preconditions, material, handling, and pitfalls for each parameter used in the
diagnosis.
Prenatal Diagnosis
This table lists the tissue or specimen, timing, and pitfalls for each disorder.
DNA Analysis
This table lists the tissue or specimen and methodology for each disorder.
Treatment and Follow-Up

This section outlines urgent treatment to consider before a definitive diagnosis is established
for each (or each group of) disorder(s). Long-term treatment and alternative therapeutic options
are highlighted in this book.
How to Use This Book xiii
Indices
Three indices are included: (1) disorders, (2) signs and symptoms, and (3) tests and medica-
tions. Each entry is linked to the corresponding disorder or page.
Reference
1. OMIM (2013) Online Mendelian Inheritance in Man®, http://www.omim.org
Contents
Part I Amino Acids
1 Disorders of Phenylalanine and Tetrahydrobiopterin Metabolism. . . . . . . . . . . 3

Nenad Blau and Francjan J. van Spronsen
2 Tyrosine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Elisabeth Holme and Grant A. Mitchell
3 Sulphur Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Ivo Barić and Brian Fowler
4 Hyperammonemias and Related Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Johannes Häberle and Vicente Rubio
5 Disorders of Glycine, Serine, GABA, and Proline Metabolism . . . . . . . . . . . . . . 63
Johan L.K. Van Hove and Janet A. Thomas
6 Amino Acid Transport Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Manuel Palacín and Stefan Broer
Part II Organic Acids
7 Disorders of Leucine, Isoleucine, and Valine Metabolism . . . . . . . . . . . . . . . . . . 103

Ina Knerr, Jerry Vockley, and K. Michael Gibson
8 Cerebral Organic Acidurias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Stefan Kölker, Eduard A. Struys, Marjo S. van der Knaap,
and Cornelis Jakobs
9 Ethylmalonic Encephalopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Alberto Burlina and Massimo Zeviani
Part III Vitamins, Cofactors, and Metals
10 Disorders of Folate Metabolism and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Fernando Scaglia and Nenad Blau
11 Vitamin B6-Dependent and Responsive Disorders . . . . . . . . . . . . . . . . . . . . . . . . 179
Barbara Plecko, Eduard A. Struys, and Cornelis Jakobs
12 Molybdenum Cofactor Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Günter Schwarz and Alex Veldman
13 Vitamin B12 Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Matthias R. Baumgartner and Brian Fowler
xv
xvi Contents
14 Biotin Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Bruce A. Barshop
15 Thiamine Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Frédéric Sedel
16 Riboflavin and CoQ Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Rita Horvath and Anne Lombès
Part IV Energy Metabolism
17 Mitochondrial Fatty Acid Oxidation Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Ute Spiekerkoetter and Marinus Duran
18 Disorders of Carbohydrate Metabolism and Glucose Transport . . . . . . . . . . . . 265
René Santer, Joerg Klepper, and G. Peter A. Smit
19 Pyruvate Carboxylase and Pyruvate Dehydrogenase Deficiency . . . . . . . . . . . . 303
Linda De Meirleir
20 Disorders of the Krebs Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Eva Morava and Rosalba Carrozzo
21 Hyperinsulinism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Khalid Hussain and Pascale De Lonlay
22 Mitochondrial Oxidative Phosphorylation Disorders . . . . . . . . . . . . . . . . . . . . . 337
Paul de Laat, Richard Rodenburg, and Jan Smeitink
23 Disorders of Ketone Body Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Jörn Oliver Sass and Sarah C. Grünert
Part V Organelles
24 Peroxisomal Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Bwee Tien Poll-The and Ronald J.A. Wanders
25 Lysosomal Storage Disorders Including Neuronal
Ceroid Lipofuscinoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Carla Hollak, Matthias Kettwig, Lars Schlotawa, and Robert Steinfeld
26 Oligosaccharidoses and Sialic Acid Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Zoltan Lukacs and Michael Beck
27 The Mucopolysaccharidoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Giancarlo Parenti and Edward J. Wraith
28 Hyperoxalurias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Bernd Hoppe and Nenad Blau
29 Cystinosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
Elena Levtchenko and Francesco Emma
30 Congenital Disorders of Glycosylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
Jaak Jaeken and Lambert van den Heuvel
Part VI Selected Disorder
31 Neurotransmitter Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

Thomas Opladen and Georg F. Hoffmann
Contents xvii
32 Creatine Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529

Sylvia Stöckler, Olivier Braissant, and Andreas Schulze
33 Heme Synthesis Defects and Porphyrias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Ulrich Stölzel, Thomas Stauch, and Manfred O. Doss
34 Disorders of Bile Acid Synthesis and Biliary Transport . . . . . . . . . . . . . . . . . . . 555
Hugh A. Lemonde, Paul Gissen, and Peter T. Clayton
35 Disorders of Polyol Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Mirjam M.C. Wamelink, Vassili Valayannopoulos, and Cornelis Jakobs
36 Cholesterol Synthesis Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
Richard I. Kelley and Lisa Kratz
37 Disorders of Adrenals and Gonads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
Anna Lauber-Biason
38 Leukotrienes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
Ertan Mayatepek
39 Disorders of Copper and Zinc Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
Peter M. van Hasselt and Roderick H.J. Houwen
40 Iron Metabolism Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
Vineta Fellman
41 Purine and Pyrimidine Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
Jörgen Bierau and Ivan Šebesta
42 Disorders of Glutathione and γ-Glutamyl Cycle . . . . . . . . . . . . . . . . . . . . . . . . . 661
Nenad Blau and Carlo Dionisi-Vici
43 Disorders of Lipoprotein Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671
Robert A. Hegele and Serena Tonstad
44 Biochemical Phenotypes of Questionable Clinical Significance. . . . . . . . . . . . . . 691
Stephen I. Goodman and Marinus Duran
Part VII General Subjects and Profiles
45 Emergency Diagnostic Procedures and Emergency Treatment . . . . . . . . . . . . . 709

Stephanie Grünewald, James Davison, Diego Martinelli, Marinus Duran,
and Carlo Dionisi-Vici
46 Newborn Screening for Inborn Errors of Metabolism . . . . . . . . . . . . . . . . . . . . . 719
Carol L. Greene and Dietrich Matern
47 Genetic Counseling for Inborn Errors of Metabolism . . . . . . . . . . . . . . . . . . . . . 737
Johannes Zschocke and Sigrid Tinschert
48 Simple Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743
K. Michael Gibson and Marinus Duran
49 Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749
Marzia Pasquali and Nicola Longo
50 Organic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
Isabel Tavares de Almeida and Marinus Duran
51 Acylcarnitines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775
Dietrich Matern
xviii Contents
52 Lysosomals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
Giancarlo la Marca
53 Proton NMR Spectroscopy of Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Udo Engelke, Angelina Goudswaard, and Ron Wevers
54 MRI and In Vivo Spectroscopy of the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803
Alessandro Burlina and Renzo Manara
55 SSIEM Classification of Inborn Errors of Metabolism . . . . . . . . . . . . . . . . . . . . 817
Johannes Zschocke
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
Contributors
Ivo Barić Division for Metabolic Diseases, Department of Pediatrics,

University Hospital Center Zagreb and University of Zagreb, School of Medicine,
Zagreb, Croatia
Bruce Barshop Department of Pediatrics, UCSD School of Medicine, La Jolla, CA, USA
Matthias Baumgartner Division for Metabolic Diseases, University Children’s Hospital,
Zürich, Switzerland
Michael Beck Villa Metabolica, Universitäts-Kinderklinik, Mainz, Germany
Jörgen Bierau Laboratory Biochemical Genetics, Department of Clinical Genetics,
Maastricht University Medical Centre, Maastricht, The Netherlands
Nenad Blau Division of Inborn Metabolic Diseases, Department of General Pediatrics,
University Children’s Hospital, Heidelberg, Germany
Division of Metabolism, University Children’s Hospital, Zürich, Switzerland
Olivier Braissant Service of Biomedicine, Department of Laboratories,
University Hospital of Lausanne, Lausanne, Switzerland
Stefan Broer Research School of Biology, Australian National University,
Canberra, Australia
Alberto Burlina Division of Metabolic Disorders, Department of Pediatrics,
University Hospital, Padova, Italy
Alessandro Burlina Neurology Unit, St. Bassiano Hospital, Bassano del Grappa, Italy
Rosalba Carrozzo Molecular Genetic, Bambino Gesù Children’s Hospital, Rome, Italy
Peter Clayton Institute of Child Health, University College London and Great Ormond
Street Hospital, London, UK
James Davison Metabolic Unit, Great Ormond Street Hospital for Children,
NHS Foundation Trust and Institute for Child Health, London, UK
Paul de Laat Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders,
Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands
Pascale De Lonlay Departments of Pediatrics, Hôpital Necker Enfants Malades,
Université Paris-Descartes, Faculté de Médecine, Paris, France
Linda de Meirleir Department of Pediatric Neurology, VZ-Brussel, Brussels, Belgium
Carlo Dionisi-Vici Division of Metabolism, Department of Pediatric Medicine,
Bambino Gesù Children’s Research Hospital, Rome, Italy
xix
xx Contributors
Manfred O. Doss German Competence Center for Porphyria Diagnosis and Consultation,
Marburg an der Lahn, Germany
Marinus Duran Laboratory Genetic Metabolic Diseases, Academic Medical Center,
Amsterdam, The Netherlands
Francesco Emma Division of Nephrology and Dialysis, Ospedale Pediatrico
Bambino Gesù, IRCCS, Rome, Italy
Udo Engelke Department of Laboratory Medicine, Laboratory of Genetic, Endocrine and
Metabolic Diseases (HP 830), Radboud University Medical Center, Nijmegen,
The Netherlands
Vineta Fellman Department of Pediatrics, Lund University, Lund, Sweden
Brian Fowler Division for Metabolic Diseases, University Children’s Hospital, Zürich,
Switzerland
K. Michael Gibson Section of Clinical Pharmacology, Washington State University,
Spokane, WA, USA
Paul Gissen Institute of Child Health, University College London and Great Ormond Street
Hospital, London, UK
Stephen I. Goodman Department of Pediatrics, University of Colorado,
Denver School of Medicine, Aurora, CO, USA
Angelina Goudswaard Department of Laboratory Medicine, Laboratory of Genetic,
Endocrine and Metabolic Diseases (HP 830), Radboud University Medical Center,
Nijmegen, The Netherlands
Carol Greene Department of Pediatrics, University of Maryland, Baltimore, MD, USA
Stephanie Grünewald Metabolic Unit, Great Ormond Street Hospital for Children,
NHS Foundation Trust and Institute for Child Health, London, UK
Sarah C. Grünert Zentrum für Kinder- und Jugendmedizin, Universitätsklinikum Freiburg,
Freiburg, Germany
Johannes Häberle Division of Metabolism and Children’s Research Center,
University Children’s Hospital, Zürich, Switzerland
Robert A. Hegele Blackburn Cardiovascular Genetics Laboratory, Robarts Research
Institute, University of Western Ontario, London, ON, Canada
Peter M. van Hasselt Department of Metabolic Diseases and Pediatric Gastroenterology,
University Medical Center Utrecht, Utrecht, The Netherlands
Susan J Hayflick Department of Molecular and Medical Genetics,
Oregon Health and Science University, Portland, OR, USA
Georg F. Hoffmann Department of Pediatrics, University Children’s Hospital,
Heidelberg, Germany
Carla Hollak Division of Endocrinology and Metabolism, Department of Internal Medicine,
F5-170, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
Elisabeth Holme Department of Clinical Chemistry, Sahlgrenska University Hospital,
Göthenburg, Sweden
Roderick Houwen Department of Metabolic Diseases and Pediatric Gastroenterology,
University Medical Center Utrecht, Utrecht, The Netherlands
Contributors xxi
Bernd Hoppe Division of Pediatric Nephrology, Department of Pediatrics,

University Hospital, Bonn, Germany
Rita Horvath Mitochondrial Research Group, Institute for Aging and Health,
Newcastle University, Newcastle, UK
Khalid Hussain Department of Endocrinology, Great Ormond Street, Children’s Hospital,
London, UK
Jaak Jaeken Division of Metabolic Diseases, Department of Pediatrics,
University Hospital Gasthuisberg, Leuven, Belgium
Cornelis Jakobs Metabolic Unit, Department of Clinical Chemistry,
VU University Medical Center Amsterdam, Amsterdam, The Netherlands
Richard I. Kelley Department of Metabolism and Pediatrics, Kennedy Krieger Institute,
Johns Hopkins University, Baltimore, MD, USA
Matthias Kettwig Department of Pediatrics, University Medical Center Göttingen,
Göttingen, Germany
Joerg Klepper Kinderklinik, Klinikum Aschaffenburg, Am Hasenkopf, Aschaffenburg,
Germany
Ina Knerr National Centre for Inherited Metabolic Disorders, Children’s University
Hospital, Dublin, Ireland
Stefan Kölker Division of Inborn Metabolic Diseases, Department of General Pediatrics,
Lisa Kratz Department of Neurogenetics, Kennedy Krieger Institute, Baltimore, MD, USA
Giancarlo la Marca Department of Neurosciences, Psychology, Pharmacology
and Child Health, University of Florence, Florence, Italy
Newborn Screening, Clinical Chemistry and Pharmacology Lab, Meyer Children’s Hospital,
Florence, Italy
Anna Lauber-Biason Faculty of Science, Department of Medicine, University of Fribourg,
Fribourg, Switzerland
Hugh A. Lemonde Institute of Child Health, University College London
and Great Ormond Street Hospital, London, UK
James Leonard Department of Clinical and Molecular Genetics,
UCL Institute of Child Health, Oxford, UK
Elena Levtchenko Department of Pediatric Nephrology, University Hospitals Leuven,
Catholic University Leuven, Leuven, Belgium
Anne Lombès APHP, Biochimie Métabolique, GH Pitié-Salpêtrière,
Inserm UMRS 1016 Institut Cochin, CNRS UMR 8104, Université Paris Descartes,
Paris, France
Nicola Longo Division of Medical Genetics, Department of Pediatrics,
University of Utah, Salt Lake City, USA
Zoltan Lukacs Metabolic Laboratory, Department of Pediatrics,
Institute of Clinical Chemistry, University Medical Center, Hamburg, Germany
Renzo Manara Neuroradiology, University of Salerno, Salerno, Italy
Diego Martinelli Division of Metabolism, Department of Pediatric Medicine,
Bambino Gesù Children’s Research Hospital, Rome, Italy
xxii Contributors
Dietrich Matern Division of Laboratory Genetics, Departments of Laboratory Medicine

and Pathology, Medical Genetics, and Pediatric and Adolescent Medicine,
Mayo Clinic College of Medicine, Rochester, MN, USA
Ertan Mayatepek University Children’s Hospital, Düsseldorf, Germany
Grant Mitchell Service de Génétique Médicale, CHU Sainte-Justine,
Université de Montréal, Montréal, QC, Canada
Eva Morava Hayward Genetics Center SL#31, Tulane University Medical School,
New Orleans, LA, USA
Thomas Opladen Division of Inborn Metabolic Diseases, Department of Pediatrics,
Manuel Palacín Department of Biochemistry and Molecular Biology,
Institute for Research in Biomedicine (IRB), Centre for Biomedical Network Research
on Rare Diseases (CIBERER), U731, University of Barcelona, Barcelona, Spain
Giancarlo Parenti Department of Pediatrics, Federico II University, Naples, Italy
Marzia Pasquali Department of Pathology, University of Utah and Arup Laboratories,
Salt Lake City, UT, USA
Barbara Plecko Department of Pediatric Neurology, Children’s Hospital,
University of Zürich, Zürich, Switzerland
Bwee Tien Poll-The Department of Pediatrics/Pediatric Neurology,
Academic Medical Centre, Amsterdam, Netherlands
Richard Rodenburg Department of Pediatrics, Nijmegen Centre for Mitochondrial
Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Vicente Rubio Instituto de Biomedicina de Valencia of the Spanish National Research
Council (CSIC) and Centre for Biomedical Network Research on Rare Diseases (CIBERER),
Valencia, Spain
René Santer Department of Pediatrics, Univ Medical Center Hamburg Eppendorf,
Hamburg, Germany
Jörn Oliver Sass Division of Clinical Chemistry and Biochemistry,
University Children’s Hospital Zürich, Zürich, Switzerland
Ivan Šebesta Institute of Medical , Biochemistry and Laboratory Medicine,
Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University,
Prague 2, Czech Republic
Fernando Scaglia Department of Molecular and Human Genetics,
Baylor College of Medicine, Houston, TX, USA
Andreas Schulze Division of Clinical and Metabolic Genetics, Department of Pediatrics,
The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
Lars Schlotawa Department of Pediatrics, University Medical Center Göttingen,
Göttingen, Germany
Günter Schwarz Institute of Biochemistry, University of Cologne, Köln, Germany
Frederic Sedel Département de Neurologie, Unité neuro-métabolique et centre de référence
des maladies lysosomales, GRC13UPMC, Université Pierre et Marie Curie-Paris 6,
Paris, France
Contributors xxiii
Jan Smeitink Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders,

Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
G. Peter A. Smit Department of Metabolic Diseases, Beatrix Children’s Hospital,
Groningen, The Netherlands
Ute Spiekerkoetter Department of General Pediatrics and Neonatology,
University Children’s Hospital, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Robert Steinfeld Department of Pediatrics, University of Göttingen, Göttingen, Germany
Thomas Stauch Department of Clinical Chemistry and Toxicology, German Competence
Center for Porphyria Diagnosis and Consultation, MVZ Labor PD Dr. Volkmann und
Kollegen GbR, Karlsruhe, Germany
Sylvia Stöckler Division of Biochemical Diseases, BC Children’s Hospital,
University of British Columbia, Vancouver, BC, Canada
Ulrich Stölzel Department of Internal Medicine II, Gastroenterology, Hepatology,
Endocrinology, Metabolic Disorders, Oncology, Saxony Porphyria Center,
Klinikum Chemnitz gGmbH, Chemnitz, Germany
Eduard A. Struys Metabolic Unit, Clinical Chemistry, VUmc Medical Center, Amsterdam,
The Netherlands
Isabel Tavares de Almeida Metabolism and Genetics, iMed.UL,
Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, Lisboa, Portugal
Janet A. Thomas Clinical Genetics and Metabolism, The Children’s Hospital, Aurora,
CO, USA
Sigrid Tinschert Division of Human Genetics, Medical University Innsbruck,
Innsbruck, Austria
Serena Tonstadt Section of Preventive Cardiology, Department of Preventive Medicine,
Oslo University Hospital, Oslo, Norway
Department of Health Promotion and Education, Loma Linda University,
Loma Linda, CA, USA
Lambert van den Heuvel Department of Pediatrics, Center for Metabolic Disease,
University Hospital Gasthuisberg, Leuven, Belgium
Department of Laboratory Medicine, Institute for Genetic and Metabolic Disease,
Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands
Marjo S. van der Knaap VU University Medical Center, Amsterdam, The Netherlands
Johan Van Hove Clinical Genetics and Metabolism, The Children’s Hospital, Aurora,
CO, USA
Francjan J. van Spronsen Section of Metabolic Diseases, Beatrix Children’s Hospital,
University Medical Center of Groningen, University of Groningen, Groningen,
The Netherlands
Vassili Valayannopoulos Reference Center for Inherited Metabolic Disorders,
Necker-Enfants Malades Hospital and Paris Descartes University, Paris, France
Alex Veldman Colbourne Pharmaceuticals GmbH, Niederkassel, Germany
Jerry Vockley Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center,
University of Pittsburgh, Pittsburgh, PA, USA
xxiv Contributors
Mirjam M. C. Wamelink Metabolic Unit, Department of Clinical Chemistry,

VU University Medical Center, Amsterdam, The Netherlands
Ronald Wanders Lab Genetic Metabolic Diseases, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Ron Wevers Department of Laboratory Medicine, Laboratory of Genetic, Endocrine and
Metabolic Diseases (HP 830), Radboud University Medical Center,
Nijmegen, The Netherlands
Edward J. Wraith Manchester Academic Health Sciences Centre (Genetic Medicine),
St. Mary’s Hospital, Manchester, UK
Massimo Zeviani Unit of Molecular Neurogenetics, Fondazione Istituto Neurologico
“Carlo Besta”, Milan, Italy
Johannes Zschocke Division of Human Genetics, Medical University Innsbruck,
Innsbruck, Austria
Abbreviations
17βHSD 17-beta-hydroxysteroid dehydrogenase

17OHP 17-hydroxyprogesterone
1H-MRS Proton magnetic resonance spectroscopy
2KG 2-ketoglutaric acid
2OGA 2-oxoglutaric acid
3β-HSDH 3-beta-hydroxysteroid dehydrogenase
3HBD D-3-hydroxy-n-butyrate dehydrogenase
3MCC 3-methylcrotonyl-CoA carboxylase; 3-methylcrotonylglycinuria
3MT 3-methoxytyramine
3-OH-GA 3-hydroxyglutaric acid
3OMD 3-O-methyldopa
5-ALA 5-aminolevulinate
5HIAA 5-hydroxyindoleacetic acid
5HTP 5-hydroxytryptophan
5-LO 5-lipoxygenase
5-LOAP 5-lipoxygenase-activating protein
5MTHF 5-methyltetrahydrofolate
7DHC 7-dehydrocholesterol
AADC Aromatic L-amino acid decarboxylase
AAs Aminoacidopathies
AASA Alpha-aminoadipic semialdehyde
AASS 2-aminoadipic semialdehyde synthase
ABAT GABA transaminase gene
ABCA1 ATP-binding cassette, subfamily member A1
ABCC8 ATP-binding cassette, subfamily C; hyperinsulinism of infancy
ABCD1 ALD protein
ABCD4 Lysosomal export of cobalamin
ABL Abetalipoproteinemia
ABS Antley-Bixler syndrome
ACAD2 Isovaleryl-CoA dehydrogenase gene
ACAD8 Isobutyryl-CoA dehydrogenase gene
ACAD9 Acyl-CoA dehydrogenase 9 gene
ACADM Medium-chain acyl-CoA dehydrogenase gene
ACADS Short-chain acyl-CoA dehydrogenase gene
ACADSB 2-methylbutyryl-CoA dehydrogenase gene
ACADVL Very-long-chain acyl-CoA dehydrogenase gene
ACAT1 Methylacetoacetyl-CoA thiolase gene
ACAT2 Cytosolic acetoacetyl-CoA thiolase gene
ACC Acetyl-CoA carboxylase
ACOX1 Peroxisomal acyl-CoA oxidase 1
ACSF3 Acetyl-CoA-synthase family 3
ACT Aspartate carbamoyltransferase
xxv
xxvi Abbreviations
ACTH Adrenocorticotropic hormone

ADA Adenosine deaminase
ADCK3 AARF domain-containing kinase 3
ADK Adenosine kinase
ADKD Adenosine kinase deficiency
AdoCbl Adenosylcobalamin
AdoHcy Adenosylhomocysteine
AdoMet Adenosylmethionine
ADSL Adenylosuccinate lyase
AEZ Acrodermatitis enteropathica
AFC Amniotic fluid cells
AGA Glycosylasparaginase
αGalA α-Galactosidase A
AGAT Arginine:glycine amidinotransferase
AGAT L-arginine:glycine amidinotransferase
AGC1 Global cerebral hypomyelination due to AGC1 defect
AGL Amylo-1,6-glucosidase
AGPS Alkyl-DHAP synthase
AGT Alanine-glyoxylate aminotransferase
AGU Aspartylglucosaminuria
AGXT Alanine-glyoxylate aminotransferase
AHCY S-adenosylhomocysteine hydrolase
AICAR 5′-phosphoribosyl-5-aminoimidazole-4-carboxamide
AICART 5′-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase
AIP Acute intermittent porphyria
AIS Androgen insensitivity syndrome
AKR1C 3α-hydroxysteroid dehydrogenase deficiency
AKR1D1 Delta(4)-3-oxosteroid-5 beta-reductase
AKU Alkaptonuria
ALA 5-aminolevulinic acid
ALAD 5-aminolevulinate dehydratase
ALAS 5-aminolevulinate synthase
ALAS2 5-aminolevulinate synthase 2
ALD Adrenoleukodystrophy
ALDH18A1 Pyrroline-5-carboxylate synthetase gene
ALDH4A1 Pyrroline-5-carboxylate dehydrogenase gene
ALDH5A1 Succinic semialdehyde dehydrogenase gene
ALDH6A1 Methylmalonate semialdehyde dehydrogenase gene
ALDH7A1 Alpha-aminoadipic semialdehyde dehydrogenase gene
Aldo Aldosterone
ALDOA Aldolase A
ALDOA-D Glycogen storage disease type XII
ALDOB Fructose-1-phosphate aldolase
ALG 12 Mannosyltransferase 8
ALG 2 Mannosyltransferase 2
ALG 8 Glucosyltransferase 2
ALG1 Mannosyltransferase 1
ALG1 Mannosyltransferase 7-9
ALG11 Mannosyltransferase 4-5
ALG3 Mannosyltransferase 6
ALG6 Glucosyltransferase 1
ALP Alkaline phosphatase
ALSDP Doss porphyria
Abbreviations xxvii
AMACR 2-methylacyl-CoA racemase

AME Apparent mineralocorticoid excess
AMN Adrenomyeloneuropathy; amnionless
AMP Adenosine monophosphate
AMPD Adenosine monophosphate deaminase deficiency
AMPD1 Adenosine monophosphate deaminase
AMPDA Adenosine-5′-monophosphate deaminase
AMPK-A Constitutional AMP-activated protein kinase activation
AMRF Action myoclonus-renal failure syndrome
α-NAGA α-N-acetylgalactosaminidase
ANCL Adult neuronal ceroid lipofuscinoses
ANGPTL3 Angiopoietin-like protein 3
AOA1 Ataxia oculomotor apraxia 1 (AOA1)
AP1S1 Adaptor-related complex protein 1; Mednik syndrome
APOA1 Apolipoprotein A-I
APOB Apolipoprotein B
APOC2 Apolipoprotein C-II
APOE Apolipoprotein E
APRT Adenine phosphoribosyltransferase
APTX Aprataxin
AR Aldose reductase; androgen receptor
ARC Arthrogryposis, renal dysfunction, and cholestasis
ARG1 Arginase 1
ARH1 Autosomal recessive hypercholesterolemia (ARH)
ARO Aromatase deficiency
ARSA Arylsulfatase
ARSB N-acetylgalactosamine-4-sulfatase
ASA Arylsulfatase A; argininosuccinic acid
ASAH1 Acid ceramidase
ASL Adenylosuccinate lyase; argininosuccinic aciduria
ASPA Aspartoacylase (aminoacylase 2)
ASS1 Argininosuccinate synthetase
AThDP Adenosine thiamine diphosphate
AThTP Adenosine thiamine triphosphate
ATIC AICAR transformylase/IMP cyclohydrolase
ATP Adenosine triphosphate
ATP13A2 Lysosomal type 5 P‐type ATPase gene
ATP6V0A2 Vesicular H(+)-ATPase subunit a2 gene
ATP6V0A2 ATPase H + transporting V0 subunit 2 gene
ATP7A Copper-transporting P-type ATPase gene
ATP8B1 ATP8B1 (type-4 P-type ATPase) gene
AUH 3-methylglutaconyl-CoA hydratase
B Corticosterone
B0AT Sodium-dependent neutral amino acid transporter
B3GALTL O-fucose-specific beta-1,3-N-glucosyltransferase gene
B4GALT1 Beta-1,4-galactosyltransferase 1 gene
B4GALT7 Beta-1,4-galactosyltransferase 7 gene
BA CoA LD Bile acid-CoA ligase deficiency
BAAT Bile acid-CoA amino acid N-acyltransferase
BAT BCAA aminotransferase
BCAA Branched-chain amino acids
BCAT BCAA aminotransferase deficiency
BCAT1 Branched-chain amino acids transporter
xxviii Abbreviations
BCAT2 Branched-chain amino acids transporter

BCKDC Branched-chain a-keto acid dehydrogenase complex
BCKDHA Branched-chain alpha-keto acid dehydrogenase complex
BH2 7,8-dihydrobiopterin
BH4 Tetrahydrobiopterin
BHMT Betaine-homocysteine methyltransferase
Bio Biopterin
BKT Alpha-methylacetoacetic aciduria
BMT Betaine-homocysteine methyltransferase
BSEP Bile salt export pump
BTD Biotinidase
BVVLS Brown-Vialetto-Van Laere syndrome
BWS Beckwith-Wiedemann syndrome
C27-3β-HSD 3β-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase
C5-DC Glutarylcarnitine
C7orf10 Glutaryl-CoA oxidase
CACT Carnitine acylcarnitine translocase
CAH 21-hydroxylase deficiency
CAH 11-Beta-hydroxylase type I deficiency
CAH Congenital adrenal hyperplasia
CaOx Calcium oxalate
CARN Carnosinemia and homocarnosinosis
Cbl Vitamin B12
cblA Adenosylcobalamin synthesis defect-cbl A
cblB Adenosylcobalamin synthesis defect-cbl B
cblC Adenosylcobalamin and methylcobalamin synthesis defect-cblC
cblD-HC Methylcobalamin synthesis defect-cblD-HC
cblD-MMA Adenosylcobalamin synthesis defect-cblD-MMA
cblD-MMA/HC Combined MMAuria and homocystinuria
cblE Methionine synthase reductase deficiency-cblE
cblF Adenosylcobalamin and methylcobalamin synthesis defect-cblF
cblG Methionine synthase deficiency-cblG
cblJ Adenosylcobalamin and methylcobalamin synthesis defect-cblJ
Cbl-TC Holotranscobalamin
CBS Cystathionine beta-synthase
CCHLND SLC33A1 deficiency with low serum copper and ceruloplasmin
CD Canavan disease
CD Collecting duct
CD320 CD32 receptor
CDG Congenital defects of glycosylation
CDG Ie GDP-Man:Dol-P mannosyltransferase subunit 1 deficiency-CDG Ie
CDG-Id Mannosyltransferase 6 deficiency-CDG-Id
CDG-If Dol-P-Man utilization 1 deficiency
CDG-Ih Glucosyltransferase 2 deficiency-CDG-Ih
CDG-Ii Mannosyltransferase 2 deficiency-CDG-Ii
CDG-Ij UDP-GlcNAc:Dol-P-GlcNac-P transferase deficiency-CDG-Ij
CDG-Il Mannosyltransferase 7-9 deficiency-CDG-IL
CDO Cysteine dioxygenase
CDPX2 3β-hydroxysteroid-delta8, delta7-isomerase deficiency; chondrodysplasia
punctata 2
CEP Congenital erythropoietic porphyria
CETP Cholesteryl ester transfer protein
CFD Cerebral folate deficiency
Abbreviations xxix
CHI Congenital hyperinsulinism

CHILD Congenital hemidysplasia, ichthyosis, and limb defects
CHP Chronic hepatic porphyria
CHSY1 Chondroitin sulfate synthase 1
CHSY1-CDG Chondroitin sulfate synthase 1 deficiency
CI Complex I deficiency
CII Complex II deficiency
CIII Complex III deficiency
CIV Complex IV deficiency
CKS CK syndrome
CLN1 Ceroid lipofuscinosis, neuronal, 1
CLN12/ PARK9 Ceroid lipofuscinosis, neuronal, 12
CLN14/ EPM3 Ceroid lipofuscinosis, neuronal, 14
CLN3 Lysosomal transmembrane CLN3 protein
CLN4A Ceroid lipofuscinosis, neuronal, 4A
CLN4B Ceroid lipofuscinosis, neuronal, 4B
CLN8-EPMR Ceroid lipofuscinosis, neuronal, 8, Northern epilepsy variant
CM Chylomicron
CMAMMA Combined malonic and methylmalonic aciduria
CMO Corticosterone methyl oxidase deficiency
CNCL Congenital neuronal ceroid lipofuscinoses
CNDP1 Carnosinase
COG1 COG complex 1
COG5 COG complex 5
COG6 COG complex 6
COG7 COG complex 7
COG8 COG complex 8
COMT Catecholamine methyltransferase
CoQ Coenzyme Q
CoQ10 Coenzyme Q10; ubiquinone
COQ2 4-hydroxybenzoate-polyprenyltransferase
COQ6 CoQ6 monooxygenase
COQ9 Coenzyme Q10
COX Cytochrome C oxidase
COXPD1 Combined oxidative phosphorylation defect 1
CP Ceruloplasmin
CPOX Coproporphyrinogen oxidase
CPS1 Carbamoyl phosphate synthetase I
CPS2 Carbamoyl phosphate synthetase 2
xxx Abbreviations
CPT I Carnitine palmitoyltransferase I

CPT II Carnitine palmitoyltransferase II
CPT1 Carnitine palmitoyltransferase
CPT2 Carnitine palmitoyltransferase 2
CR Carbonyl reductase
CRD Cortisone reductase deficiency
CRD/ARD Refsum disease (classic, adult)
CrT Creatine transporter
CSAT Cysteine sulfinate α-oxoglutarate aminotransferase
CSD Cysteine sulfinate decarboxylase
CSE Cystathionine γ-lyase
CT Cytosolic acetoacetyl-CoA thiolase
CTH Cystathionine gamma-lyase; cystathionase
CTLN1 Citrullinemia type I
CTLN2 Citrullinemia type II
CTNS Cystinosin
CTNS Cystinosin
CTSA Protective protein/cathepsin A
CTSD Cathepsin D
CTSF Cathepsin F
CTX Sterol 27-hydroxylase deficiency
CUBN Cubilin
CV Chorionic villi
CVD Cardiovascular disease
CyD Cysteine dioxygenase
CYP11A1 P450 side-chain cleavage
CYP11B1 P450 11 beta-hydroxylase type 1
CYP11B1/B2 11-beta-hydroxylase I/II
CYP11B2 Corticosterone methyl oxidase
CYP17A1 Cytochrome P450 17 alpha-hydroxylase
CYP19A1 P450 aromatase
CYP21A2 P450 21-hydroxylase
CYP27A1 Sterol 27-hydroxylase
CYP51 Lanosterol demethylase deficiency
CYP7A1 Cholesterol 7α-hydroxylase
CYP7B1 Oxysterol 7α-hydroxylase
CySD Cysteine sulfinate decarboxylase
CYSTA Cystathioninuria
D2HG D-2-hydroxyglutaric acid
D2HGA D-2-hydroxyglutaric aciduria
D2HGA I D-2-hydroxyglutaric aciduria type I
D2HGA II D-2-hydroxyglutaric aciduria type II
D2HGDH D-2-hydroxyglutarate dehydrogenase
D4A Androstenedione
DA Dicarboxylic aminoaciduria
DAT Dopamine transporter
DBH Dopamine beta-hydroxylase
DBL Dysbetalipoproteinemia
DBP D-bifunctional protein deficiency
DBS Dried blood spot
DCT Distal convoluted tubule
DDC Aromatic L-amino acid decarboxylase gene
dGUOK Deoxyguanosine kinase
Abbreviations xxxi
DHCA Dihydroxycholestanoic acid

DHCR14 3β-hydroxysteroid-delta14-reductase
DHCR24 3β-hydroxysteroid-delta24-reductase; desmosterolosis
DHCR7 7-dehydrocholesterol reductase
DHEA Dehydroepiandrosterone
DHF Dihydrofolate
DHFR Dihydrofolate reductase
DHO Dihydroorotase
DHODH Dihydroorotate dehydrogenase
DHP Dihydropyrimidinase
DHPR Dihydropteridine reductase deficiency
DHT Dihydrotestosterone
DK1 Dolichol kinase
DK1-CDG Dolichol kinase deficiency
DLD Dihydrolipoyl dehydrogenase
DLP1 Dynamin-like protein 1
DMGDH Dimethylglycine dehydrogenase
DMGLY Dimethylglycinuria
DNPH 2,4-dinitrophenylhydrazine
DOC Deoxycorticosterone
DOPS Dihydroxyphenylserine
DPAGT1 UDP-GlcNAc:Dol-P-GlcNac-P transferase
DPD Dihydropyrimidine dehydrogenase
DPM1 GDP-Man:Dol-P mannosyltransferase subunit 1
DPM3 GDP-Man:Dol-P mannosyltransferase 3
DPYD Dihydropyrimidine dehydrogenase
DPYS Dihydropyrimidinase
DRD Dopa-responsive dystonia
DS Dermatan sulfate
DSD Defects of salt-water homeostasis and sexual development
dTMP Deoxythymidine monophosphate
dUMP Deoxyuridine monophosphate
DWI Diffusion-weighted images
E Epinephrine
E2 Estradiol
E3BP Pyruvate dehydrogenase complex deficiency E3 X
EA6 Episodic ataxia due to EAAT1 glutamate transporter defect
EAAT3 Excitatory amino acid transporter 3
EBP 3β-hydroxysteroid-delta8, delta7-isomerase
EE Ethylmalonic encephalopathy
EFEMP2 Fibulin 4
EIEE3 Early infantile epileptic encephalopathy 3; neonatal myoclonic epilepsy due
to mitochondrial glutamate carrier GC1 defect
EIHI Exercise-induced hyperinsulinism
ELN Elastin
EMA Ethylmalonic acids
ENO3 Beta-enolase
EPP Erythropoietic protoporphyria
ER Endoplasmic reticulum
ERT Enzyme replacement therapy
ESR1 Estrogen resistance
ESRF End stage renal failure
ETF Multiple acyl-CoA dehydrogenase deficiency
xxxii Abbreviations
ETF Electron transfer flavoprotein

ETFA Electron transfer flavoprotein A
ETFB Electron transfer flavoprotein B
ETFDH ETF-ubiquinone oxidoreductase, ETF dehydrogenase; electron
transfer flavoprotein dehydrogenase; myopathic form of CoQ10
deficiency
ETF-DH Electron transfer flavoprotein dehydrogenase
ETF-DH Multiple acyl-CoA dehydrogenase deficiency DH
ETHE1 Ethylmalonic encephalopathy
EXT1 Exostosin 1
EXT2 Exostosin 2
F Cortisol
FA Fumaric acid
FAD Flavin adenine dinucleotide
FAH Fumarylacetoacetase
FAICAR Formyl-5′-phosphoribosyl-5-aminoimidazole-4-carboxamide
FAO Fatty acid oxidation
FAODs Fatty acid oxidation disorders
FAOs Fatty acids oxidation disorders
FB Fibroblasts
FBP1 Fructose-1,6-bisphosphatase
FBS Fanconi-Bickel syndrome
FCH Familial combined hypolipidemia (ANGPTL3)
FECH Ferrochelatase
FED Familial LCAT deficiency (partial)
FEV Forced expiratory volume
FFA Free fatty acids
FGAR Formyl-5′-phosphoribosylglycinamide
FGE Cα-formylglycine-generating enzyme
FH1 Fumarase deficiency
FID Flame-ionization detector
FIGLU Glutamate formiminotransferase
FITHFCH Formimino-THF cyclodeaminase
FK-D Essential fructosuria; fructokinase deficiency
FMN Flavin mononucleotide
FMO3 Flavin-containing monooxygenase
FOLR1 Folate receptor alpha
FP False-positive
FRα Folate receptor alpha
FSH Follicle-stimulating hormone
FTCD Formiminotransferase
FTHFDH Formyl-THF dehydrogenase
FTHFI Formyl-THF isomerase
FTHFS 10-formyl-THF synthase
FUCA1 Alpha-L-fucosidase
FUCO Fucosidosis
FUM Fumarase
G6PC Glucose-6-phosphatase
G6PT1 (SLC37A4) Glucose-6-phosphate translocase
GA Glutaric acid
GA3 Glutaric aciduria type 3
GAA Guanidinoacetate
GABAT GABA transaminase deficiency
Abbreviations xxxiii
GAGs Glycosaminoglycans
GA-I Glutaric aciduria type I
GALC Galactocerebrosidase
GALE Uridine diphosphate galactose-4-epimerase
GALE Galactose-1-phosphate uridyltransferase
GALK Galactokinase
GALK-D Galactokinase deficiency
GALNS N-acetylgalactosamine-6-sulfatase
GALNT3 Polypeptide N-acetylgalactosaminyltransferase 3
GALT Galactose-1-phosphate uridyltransferase
GALT-D Galactosemia
GAMT Guanidinoacetate methyltransferase; arginine:glycine
amidinotransferase
GAR 5′-phosphoribosylglycinamide
GARTF 5′-phosphoribosylglycinamide transformylase
GBA Glucocerebrosidase
GBE1 Glycogen branching enzyme
GCCR Glucocorticoid resistance
GCDH Glutaryl-CoA dehydrogenase
GCH Global cerebral hypomyelination
GCH1 GTP cyclohydrolase I gene
GCK Glucokinase (hexokinase-4)
GCLC Gamma-glutamylcysteine synthetase
GCMS Gas chromatography/mass spectrometry
GCS γ-Glutamylcysteine synthetase
GCS1 Glucosidase 1
GDH Glutamate dehydrogenase
GEPH Gephyrin
GGCS Gamma-glutamylcysteine synthetase deficiency
GGCT γ-Glutamyl cyclotransferase
GGM Intestinal glucose-galactose malabsorption
GGT1 Gamma-glutamyl transpeptidase
GGUOK Mitochondrial deoxyguanosine kinase
GIF Transcobalamin III
GK Glycerol kinase
GKD Glycerol kinase deficiency, isolated
GLA Alpha-galactosidase
GLB1 Beta-galactosidase
GlcNAc N-acetylglucosamine conjugate
GLD Krabbe disease
GLDC; AMT; GCSH P protein; T protein; H protein
Gluc Glucuronide
GLUD1 Glutamate dehydrogenase-1
GLUL Glutamine synthetase
GLUT1 Glucose transporter-1
GLUT1-D Glucose transporter-1 deficiency
GLYCTK Glycerate kinase
GLYCTK-D Glycerate kinase deficiency
GLYT2 Neuronal glycine transporter
GMAP210 Golgi-microtubule-associated protein
GNE UDP-GlcNAc epimerase/kinase
xxxiv Abbreviations
GNMT Glycine N-methyltransferase

GNPAT Dihydroxyacetone phosphate acyltransferase
GNPTAB Alpha-/beta-subunit of N-acetylglucosamine-1-phosphotransferase
GNPTG Gamma-subunit of N-acetylglucosamine-1-phosphotransferase
GNS N-acetylglucosamine-6-sulfatase
GORAB SCYL1 binding protein
GRA Glucocorticoid suppressible hyperaldosteronism
GRACILE GRACILE syndrome
GRHPR D-glycerate dehydrogenase
GRHPR D-glycerate dehydrogenase and hydroxypyruvate reductase
GRN Progranulin
GS Glutathione synthetase
GSD Glycogen storage disorder
GSD-0a Glycogen storage disease type 0 a
GSD-0b Glycogen storage disease type 0 b
GSD-I non-a Glycogen storage disease type I non-a
GSD-Ia Glycogen storage disease type I a
GSD-IIa Glycogen storage disease type II a
GSD-IIb Glycogen storage disease type II b
GSD-III Glycogen storage disease type III
GSD-IV Glycogen storage disease type IV
GSD-IXa-c Glycogen storage disease type IX a-c
GSD-IXd Glycogen storage disease type IX d
GSD-V Glycogen storage disease type V
GSD-VI Glycogen storage disease type VI
GSD-VII Glycogen storage disease type VII
GSD-X Glycogen storage disease type X
GSD-XIII Glycogen storage disease type XIII
GSD-XIV Glycogen storage disease type XIV
GSD-XV Glycogen storage disease type XV
GSH Glutathione
GSL Galactosialidosis
GSS Glutathione synthetase
GUSB Beta-glucuronidase
GYG1 Glycogenin-1
GYS1 Muscle glycogen synthase
GYS2 Liver glycogen synthase
HADH Short-chain L-3-hydroxyacyl-CoA dehydrogenase
HADH Hydroxyacyl-Coenzyme A dehydrogenase
HADHA Long-chain 3 hydroxyacyl-CoA dehydrogenase
HADHB 3-oxothiolase; long-chain 3-ketoacyl-CoA thiolase
HAL Histidase
HAMP Hepcidin
HAT Heteromeric amino acid transporters
HAWK Hawkinsinuria
HBL Hypobetalipoproteinemia (APOB)
HC Hereditary coproporphyria
HC Haptocorrin; transcobalamin 1
HCD Haptocorrin deficiency
HCHOLA2 Familial defective apolipoprotein B (APOB)
HCHOLA3 Autosomal dominant hypercholesterolemia
HCP Hereditary coproporphyria
HCSD Holocarboxylase synthetase deficiency
Abbreviations xxxv
hCys Homocysteine
HD Hartnup disorder
HD Hemodialysis
HDL High-density lipoprotein
HE Hyperekplexia due to Gly transporter GLYT2 defect
HeFH Familial hypercholesterolemia heterozygous (LDLR)
HEMD Greenberg skeletal dysplasia
HEXA Alpha-subunit of hexosaminidase
HEXA β-Hexosaminidase A subunit
HEXB Beta-subunit of hexosaminidase
HF Hemofiltration
HFE Hereditary hemochromatosis
HFE2A Hereditary hemochromatosis (type 2a)
HFE2B Hereditary hemochromatosis (type 2b)
HFE3 Hereditary hemochromatosis (type 3)
HFI Hereditary fructose intolerance
HFM Hereditary folate malabsorption
HGD Homogentisate 1,2-dioxygenase
HGPRT Hypoxanthine guanine phosphoribosyltransferase
HHF2 Hyperinsulinism
HHH HHH syndrome
HI Hyperinsulinism
HI/HA Hyperinsulinism and hyperammonemia
HIBCH 3-hydroxyisobutyryl-CoA deacylase
HIDS Hyper Ig D syndrome
HJV Hemojuvelin
HJV Hemojuvelin
HLCS Holocarboxylase synthetase
HLP Hyperlipoproteinemia
HLP1 Lipoprotein lipase deficiency (LPL)
HMBS Hydroxymethylbilane synthase
HMGA 3-hydroxy-3-methylglutaryl
HMGCL 3-hydroxy-3-methylglutaryl-CoA lyase
HMG-CoA 3-hydroxy-3-methylglutaryl-CoA
HMG-CoAL 3-hydroxy-3-methylglutaryl-CoA lyase
HMG-CoAS 3-hydroxy-3-methylglutaryl-CoA synthase
HMGCS2 3-hydroxy-3-methylglutaryl-CoA synthase 2
HMGL 3-hydroxy-3-methylglutaryl-coenzyme A lyase
HML Hereditary myopathy with lactic acidosis
HNF1A Hepatocyte nuclear factor 1 alpha
HNF4A Hepatocyte nuclear factor 4 alpha
HoFH Familial hypercholesterolemia homozygous
HOGA1 4-hydroxy-2-oxoglutarate aldolase
HOPS Congenital hypophosphatasia
HOT Hydroxyacid:oxoacid transhydrogenase
HP II Hyperprolinemia type II
HP1 Hyperprolinemia type I
HPA Hyperphenylalaninemia
HPD 4-hydroxyphenylpyruvate hydroxylase
HPRT Hypoxanthine guanine phosphoribosyltransferase
HRSA Health Resources and Service Administration
HS Heparan sulfate
HSCT Hematopoietic stem cell transplantation
xxxvi Abbreviations
HSD10 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency

HSD10 17beta-hydroxysteroid dehydrogenase type 10
HSD11B1/H6PDH 11beta-hydrosteroid dehydrogenase type 1
HSD11B2 11beta-hydroxysteroid dehydrogenase type 2
HSD17B4 D-bifunctional protein
HSD3B2 3β-hydroxysteroid dehydrogenase type II
HSD3B7 3β-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase
HSP Hereditary spastic paraplegia
HTG Hypertriglyceridemia
hTHTR1 Human thiamine transporter 1
hTHTR2 Human thiamine transporter 2
HTOx Hypotaurine:NAD; oxidoreductase
HVA Homovanillic acid
HYAL1 Hyaluronidase
HYPO Hydroxyprolinemia
IBD Isobutyryl-CoA dehydrogenase deficiency
IBDH Isobutyryl-CoA dehydrogenase
ICD Implanted cardiac defibrillator
IDH2 Isocitrate dehydrogenase 2
IDL Intermediate-density lipoprotein
IDS Iduronate 2-sulfatase
IDUA Alpha-iduroanidase
IFD Intrinsic factor deficiency
IG Iminoglycinuria
IGFBP-1 Insulin growth factor binding protein 1
IGS Cubilin deficiency
IGS Amnionless deficiency
IGS Imerslund-Gräsbeck syndrome
IMM Inner mitochondrial membrane
IMPDH Inosine-5′-monophosphate dehydrogenase
IMPDH1 Inosine monophosphate dehydrogenase
INCL Infantile neuronal ceroid lipofuscinoses
IP Intestinal alkaline phosphatases
IRD Infantile Refsum disease
ITPA Inosine-5′-triphosphate pyrophosphohydrolase
IVA Isovaleric acidemia
JNCL Juvenile neuronal ceroid lipofuscinoses
KAA 2-ketoadipic and 2-aminoadipic academia
KCNJ11 Kir6.2 subunit of the inwardly rectifying potassium channel
KCTD7 Potassium channel tetramerization domain‐containing protein 7
KHK Fructokinase
KS Keratan sulfate
KSS Kearns-Sayre syndrome
KYN Hydroxykynureninuria
KYNU Kynureninase
L2HG L-2-hydroxyglutaric acid
L2HGA L-2-hydroxyglutaric aciduria
L2HGDH L-2-hydroxyglutarate dehydrogenase
L2HGDH L-2-hydroxyglutaric acid dehydrogenase
LA Lactic acid
LAD2 GDP-fucose transporter deficiency
Abbreviations xxxvii
LAMAN Mannosidosis
LAMM Labyrinthine aplasia, microtia and microdontia
LAMP2 Lysosome-associated membrane protein-2
LBMAN Beta-mannosidosis
LBSL Leukoencephalopathy with brainstem and spinal cord involvement and
lactate elevation
LCAT Lecithin cholesterol acyl transferase
LCHAD Isolated deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase
LDH Lactate dehydrogenase
LDHA-D Glycogen storage disease type XI
LDL Low-density lipoprotein
LDLR Low-density lipoprotein receptor
LDLRAP1 Low-density lipoprotein receptor associated protein 1
LFNG O-fucose-specific beta-1,3-N-acetylglucosaminyltransferase
LH Luteinizing hormone
LHON Leber Hereditary Optic Neuropathy, LHON
LIMM Lethal Infantile Mitochondrial Myopathy
LINCL Late-infantile neuronal ceroid lipofuscinoses
LIPA Acid lipase
LIPC Hepatic triglyceride lipase
LKAT Long-chain 3 ketoacyl-CoA thiolase
LLO Lipid-linked oligosaccharides
LMBRD1 Lysosomal export of cobalamin?
LN11 Ceroid lipofuscinosis, neuronal, 11
LPA Apolipoprotein(a) moiety of Lp(a)
LPI Lysinuric protein intolerance
LPL Lipoprotein lipase
LS Leigh syndrome
LSFC Leigh syndrome with French-Canadian Ethnicity
LT Leukotriene
LTA4H Leukotriene A4 hydrolase
LTC4D Cysteinyl leukotriene synthase 4 deficiency
LTC4S Cysteinyl leukotriene synthase 4
MA Malonic aciduria
MADD Multiple acyl-CoA dehydrogenation defect
MAGT1 Magnesium transporter 1
MAN2B1 Alpha-mannosidase B
MANBA Beta-mannosidase
MAO Monoamine oxidase
MAOA Monoamine oxidase A
MAT Methionine S-adenosyltransferase
MAT Methylacetoacetyl-CoA thiolase; β-ketothiolase
MAT I/III Methionine adenosyltransferase I/III deficiency
MBCD 2-methylbutyrylglycinuria
MBD 2-methylbutyryl-CoA dehydrogenase
MBDD Membrane-bound dipeptidase deficiency
MCAD Medium-chain acyl-CoA dehydrogenase
MCCC1 or 2 Methylcrotonyl-CoA carboxylase
MCCD 3-methylcrotonyl-CoA carboxylase deficiency
MCD Multiple carboxylase defect
MCEE Methylmalonyl-CoA epimerase
MCM Methylmalonyl-CoA mutase
MCSU Molybdenum cofactor sulfurylase
xxxviii Abbreviations
MCT-1 Monocarboxylate transporter

MDH Malate dehydrogenase
MDR3 Multidrug resistance protein 3
MeCbl Methylcobalamin
MEGDEL MEGDEL syndrome
MELAS Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like
episodes
MEN1 Multiple endocrine neoplasia syndrome type 1
MERRF Myoclonic epilepsy associated with ragged red fibers
MET Metanephrine
MFSD8 Major facilitator superfamily domain-containing protein-8 (MFSD8)
MGA 3-methylglutaconic aciduria
MGA1 Methylglutaconic aciduria type I
MGA2 Barth syndrome gene
MGA3 Costeff syndrome gene
MGA4 Methylglutaconic aciduria type IV
MGAT2 N-acetylglucosaminyltransferase 2
MGAT2-CDG N-acetylglucosaminyltransferase 2 deficiency-CDG-IIa
MHBD 2-methyl-3-hydroxybutyryl-CoA dehydrogenase
mHMGS Mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase
MHPG 3-methoxy-4-hydroxyphenylglycol
MIDD Maternally inherited deafness and diabetes
MKD Mevalonate kinase deficiency
MLASA Myopathy Lactic Acidosis and Sideroblastic Anemia
MLD Metachromatic leukodystrophy
MLYCD Malonyl-CoA decarboxylase
MMA Methylmalonic acidemia
MMA/MA Combined MMA and MA
MMAB Adenosyltransferase
MMAE Methylmalonyl-CoA epimerase deficiency
MMSDH Methylmalonate semialdehyde dehydrogenase deficiency
MNGIE Thymidine phosphorylase deficiency
MNK (MK) Menkes disease
MoCD Molybdenum cofactor deficiency
MOCD-A Molybdenum cofactor deficiency A
MOCD-B Molybdenum cofactor deficiency B
MOCD-C Molybdenum cofactor deficiency C
MPDU1 Dol-P-Man utilization 1
MPI Phosphomannose isomerase
MPI-CDG Phosphomannose isomerase deficiency-CDG-Ib
MPS I Hurler, Scheie disease
MPS II Hunter disease
MPS IIIA Sanfilippo A disease
MPS IIIB Sanfilippo B disease
MPS IIIC Sanfilippo C disease
MPS IIID Sanfilippo D disease
MPS IVA Morquio A disease
MPS IVB Morquio B disease
MPS IX Hyaluronidase deficiency
MPS VI Maroteaux-Lamy disease
MPS VII Sly disease
MPS3C Acetyl-CoA alpha-glucosaminide acetyltransferase
MPST 3-mercaptopyruvate sulfurtransferase
Abbreviations xxxix
MRI Magnetic resonance imaging

MSD Multiple sulfatase deficiency
MSD Multiple sulfatase deficiency
MSPT 3-mercaptopyruvate sulfurtransferase
MSUD Maple syrup urine disease
MT Methyltransferase
mtDNA Mitochondrial DNA
MTDP8A Mitochondrial ribonucleotide reductase subunit 2 deficiency
MTDP8B Mitochondrial ribonucleotide reductase subunit 2 deficiency
MTDPS1 Mitochondrial depletion syndrome 1
MTDPS2 Thymidine kinase 2 deficiency
MTDPS3 Deoxyguanosine kinase deficiency
MTDPS4A Mitochondrial depletion syndrome 4A
MTDPS8A Mitochondrial depletion syndrome 8A
mTFP Mitochondrial trifunctional protein complex
mTFP, MTP Mitochondrial trifunctional protein deficiency
MTHF 5-methyltetrahydrofolate
MTHFCH Methenyl-THF cyclohydrolase
MTHFD1 5,10-Methylenetetrahydrofolate dehydrogenase/5,10-ethenyltetrahydrofolate
cyclohydrolase/formyltetrahydrofolate synthetase
MTHFR 5,10-methylenetetrahydrofolate reductase
MTHFS 5,10-methenyltetrahydrofolate synthetase
MTP Microsomal triglyceride transfer protein
MTP Mitochondrial trifunctional protein
MTR Methionine synthase, 5-methyltetrahydrofolate-homocysteine
methyltransferase
MTRR Methionine synthase reductase
MTS Mohr-Tranebjaerg syndrome
MUT Methylmalonyl-CoA mutase
MVK Mevalonate kinase
N/A Apolipoprotein A-I deficiency (APOA1)
NAA N-acetylaspartic acid
NAGA Schindler disease type I
NAGA Kanzaki disease
NAGA Schindler disease type III
NAGA α-N-acetylgalactosaminidase
NAGS N-acetylglutamate synthase
NAGU N-acetyl-α-D-glucosaminidase
NALD Neonatal adrenoleukodystrophy
NARP Neuropathy ataxia and retinitis pigmentosa
NBIA1 Neurodegeneration with brain iron accumulation 1
NBS Newborn screening
NCL Neuronal ceroid lipofuscinoses
nDNA Nuclear DNA
NE Norepinephrine
NEFA Nonesterified fatty acids
Neo Neopterin
NEU Sialidosis
NEU1 Alpha-neuraminidase
xl Abbreviations
NH Neonatal hemochromatosis
NH2TP Dihydroneopterin triphosphate
NKH Nonketotic hyperglycinemia
NME Neonatal mitochondrial encephalocardiomyopathy
NMN Normetanephrine
NMR Nuclear magnetic resonance
NPC1 Niemann-Pick disease type C1
NPC1 Niemann-Pick type C
NPC2 Niemann-Pick disease type C2
NR3C1 Glucocorticoid receptor
NR3C2 Mineralocorticoid receptor
NSDHL 3-Beta-hydroxysteroid dehydrogenase
OAs Organic acidurias
OAT Ornithine aminotransferase
OAT Ornithine aminotransferase deficiency
OCTN2 Organic cation carnitine transporter 2
OGDH 2-ketoglutarate dehydrogenase
OGDH 2-oxoglutarate dehydrogenase
OHS Occipital horn syndrome
OMIM Online Mendelian Inheritance in Man
OMPDC OMP decarboxylase
OPA1 Childhood-onset autosomal dominant optic atrophy
OPLAH Oxoprolinase
OPRT Orotate phosphoribosyltransferase
ORNT1 HHH syndrome gene
ORNT1 Mitochondrial ornithine transporter
OTC Ornithine transcarbamylase
OXCT1 Succinyl-CoA:3-oxoacid-CoA transferase
OXPHOS Oxidative phosphorylation system
P450c17 17-α-hydroxylase gene
P450scc Cholesterol side-chain cleavage
P5CDH Hyperprolinemia type II
P5CS Pyrroline-5-carboxylate synthetase
P6C L-Δ1-piperideine-6-carboxylate
PA Propionic acidemia
PA Propionic acidemia
PAH Phenylalanine hydroxylase
PANK2 Pantothenate kinase
PAP Pulmonary alveolar proteinosis
PBD Peroxisome biogenesis disorders
PBG Porphobilinogen
PC Pyruvate carboxylase
PC Phosphatidylcholine
PCBD Pterin carbinolamine-4a-dehydratase gene
PCC Propionyl-CoA carboxylase
PCCA or B Propionyl-CoA-carboxylase deficiency
PCD Pterin carbinolamine-4a-dehydratase deficiency
PCD Pyruvate carboxylase deficiency
PCSK9 Proprotein convertase subtilisin/kexin type 9
PCSK9D PCSK9 deficiency with low LDL
PCT Proximal convoluted tubule
Abbreviations xli
PCT Porphyria cutanea tarda

PDE Pyridoxine-dependent epilepsy
PDH Pyruvate dehydrogenase complex
PDHA1 Pyruvate dehydrogenase E1α subunit
PDHB Pyruvate dehydrogenase E1β subunit
PDHC E2 Pyruvate dehydrogenase complex deficiency E2
PDHC E3 Pyruvate dehydrogenase complex deficiency E3
PDHDD Dihydrolipoyl transacetylase
PDHPD Pyruvate dehydrogenase complex deficiency PDHP
PDHX Pyruvate dehydrogenase E3 binding protein
PDP1 Pyruvate dehydrogenase phosphatase
PDSS1 Prenyl diphosphate synthase
PDSS2 Prenyl diphosphate synthase
PEO Progressive external ophthalmoplegia
PEPD Peptidase D
PEX Different proteins
PFIC Progressive familial intrahepatic cholestasis
PFIC 2 ABCB11 deficiency
PFIC1 ATP8B1 deficiency
PFKM Phosphofructokinase
PGAM2 Phosphoglycerate mutase-2
PGK1 Phosphoglycerate kinase
PGR Progesterone resistance
PH Primary hyperoxaluria
PH 1 Primary hyperoxaluria type I
PH 2 Primary hyperoxaluria type II
PH 3 Primary hyperoxaluria type III
PHA1 Pseudohypoaldosteronism
PHGDH Phosphoglycerate dehydrogenase
PHHI Persistent hyperinsulinemic hypoglycemia of infancy
PHKA1 Muscle phosphorylase kinase
PHKA2 Liver phosphorylase kinase
PHYH Phytanoyl-CoA hydroxylase
PIGM Phosphatidylinositol glycan, class M
PIGV Phosphatidylinositol glycan, class V
PK Phosphate kinase
PKU Phenylketonuria
PL Phospholipid
PLP Pyridoxal 5′-phosphate
PMM2 Phosphomannomutase 2
PNP Purine nucleoside phosphorylase deficiency
PNP Purine nucleoside phosphorylase
PNP Purine nucleoside phosphorylase
PNPO Pyridox(am)ine 5′-phosphate oxidase
POADS Dihydroorotate dehydrogenase deficiency
POLG Polymerase gamma
POMGNT1 O-mannose beta-1,2-N-acetyglucosaminyltransferase
POMT1 O-mannosyltransferase 1
POMT2 O-mannosyltransferase 2
POR P450 oxidoreductase
POX Hydroxyproline oxidase
PPOX Protoporphyrinogen oxidase
PPP Pentose phosphate pathway
xlii Abbreviations
PPT1 Lysosomal palmitoyl protein thioesterase-1

Pri Primapterin
PRKAG2 AMP-activated protein kinase
PRODH Proline dehydrogenase
PRPPS Phosphoribosyl pyrophosphate synthetase
PRPS1 Phosphoribosyl pyrophosphate synthetase 1
PSAP Saposin A-D
PSAPD Combined saposin deficiency
PSAT Phosphoserine aminotransferase deficiency
PSAT1 Phosphoserine aminotransferase
PSPH Phosphoserine phosphatase
PSPHD Phosphoserine phosphatase deficiency
PST Proximal straight tubule
PTP 6-pyruvoy-tetrahydroptrin
PTPS 6-pyruvoyl-tetrahydropterin synthase
PTS 6-pyruvoyl-tetrahydropterin synthase gene
PTS Peroxisome targeting signals
PV Porphyria variegata
PYCS Pyrroline-5-carboxylate synthase
PYGL Liver glycogen phosphorylase
PYGM Muscle glycogen phosphorylase
q-BH2 Quinoid-dihydrobiopterin
QDPR Dihydropteridine reductase gene
RBC Red blood cells
RCDP Rhizomelic chondrodysplasia punctata
RCDP1 Rhizomelic chondrodysplasia punctata type 1
RFC1 Reduced folate carrier 1
RFT1 Flippase of Man5GlcNAc2-PP-Dol
RFT1-CDG Flippase of Man5GlcNAc2-PP-Dol deficiency-CDG-In
RPI Ribose-5-phosphate isomerase
RPIA Ribose-5-phosphate isomerase deficiency
RR Ribonucleotide reductase
RRM2B Mitochondrial ribonucleotide reductase
SA Succinic acid
SAHH S-adenosylhomocysteine hydrolase
SAM S-adenosylmethionine
SANDO Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis
SARDH Sarcosine dehydrogenase
SBCAD 2-methylbutyrylglycinuria, benign
SBCAD Short-/branched-chain acyl-CoA dehydrogenase
SC4MOL Sterol C4-methyloxidase
SC5D Lathosterolosis; sterol C5-desaturase
SCAD Short-chain acyl-CoA dehydrogenase deficiency
SCARB1 Scavenger receptor B1
SCARB2/LIMP-2 Scavenger receptor class B, member 2/Limp-2
SCHAD Short-chain 3-hydroxyacyl-CoA dehydrogenase
SCOT Succinyl-CoA:3-oxoacid-CoA transferase
SCOX Straight-chain acyl-CoA oxidase
SCP Sterol carrier protein
SCS Succinyl-CoA synthetase
SDD Sarcosinemia
Abbreviations xliii
SDH Hyperlysinemia and saccharopinuria

SDO Sulfur dioxygenase
SEC23B COPII component SEC23B
SEC23B-CDG, CDA II COPII component SEC23B deficiency
SERC1 MEGDEL
SGLT Sodium-dependent glucose transporter
SGLT2-D Renal glucosuria
SGSH Heparan-N-sulfatase
SLC16A1 Monocarboxylate transporter gain-of-function
SLC16A1 Monocarboxylate transporter 1
SLC17A5 Salla disease
SLC17A5 Sialin
SLC18A2 Vesicular monoamine transporter 2
SLC19A2 THTR1 transporter
SLC19A3 Wernicke-like encephalopathy and BRBG
SLC19A3 Reduced folate family of micronutrient transporter
SLC1A1 Neuronal/epithelial high affinity glutamate transporter, excitatory
amino acid transporter 3
SLC1A3 Glutamate/aspartate transporter (GLAST), excitatory amino acid
transporter 3
SLC22A5 Organic cation carnitine transporter 2
SLC25A12 Neuronal- and muscle-specific mitochondrial aspartate/glutamate
transporter 1
SLC25A13 Aspartate glutamate carrier
SLC25A19 Bilateral striatal necrosis
SLC25A19 Amish microcephaly
SLC25A20 Carnitine acylcarnitine translocase
SLC25A22 Mitochondrial glutamate/H + symporter 1
SLC27A5 Bile acid-CoA ligase
SLC2A1 Glucose transporter-1
SLC35A1 CMP-sialic acid transporter
SLC35A1-CDG CMP-sialic acid transporter deficiency
SLC35C1 GDP-fucose transporter
SLC35D1 UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter
SLC35D1-CDG UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter
deficiency
SLC36A2 Amino acid transporter
SLC3A1 Amino acid transport system
SLC46A1 SLC46A1 transporter
SLC52A3 Riboflavin transporter 2
SLC6A19 Sodium-dependent neutral amino acid transporter
SLC6A5 Neuronal glycine transporter GLYT2
SLC6A8 CrT, creatine transporter
SLC7A7 Amino acid transport system (LAT1)
SLOS Smith-Lemli-Opitz syndrome
SMAX3 X-linked distal spinal muscular atrophy
SMPD1 Acid sphingomyelinase (ASM)
SO Sulfite oxidase
SOX Sulfite oxidase
SPR Sepiapterin reductase gene
SQR Sulfide CoQ reductase
xliv Abbreviations
SR Sepiapterin reductase
SRB1D Scavenger receptor B1 deficiency (SCARB1)
SRD5A2 5-alpha-reductase type II
SRD5A3 Steroid 5 alpha-reductase 3
SRD5B1 Δ4-3-Oxosteroid-5β-reductase
SRT Substrate reduction therapy
SSADH Succinic semialdehyde dehydrogenase
SSC S-sulfocysteine
ST Sulfurtransferase
ST3GAL5 Lactosylceramide alpha-2,3-sialyltransferase
ST3GAL5-CDG Lactosylceramide alpha-2,3-sialyltransferase
StAR Steroidogenic acute response protein
StAR Lipoid adrenal hyperplasia gene
SUCLA2 Succinate-CoA ligase beta-subunit
SUCLG1 Succinate-CoA ligase alpha-subunit
SUMF1 Formylglycine-Generating Enzyme
SUOX Sulfite oxidase
SUR1 Sulfonylurea receptor 1
TALDO Transaldolase
TAT Tyrosine aminotransferase
TC II Transcobalamin II
TCD Transcobalamin deficiency
TCN1 B12-binding alpha-globulin
TCN2 Vitamin B12-binding protein 2
TCR Transcobalamin receptor defect
TD, FHA Tangier disease (ABCA1)
TF Transferrin, atransferrinemia
TFR2 Transferrin receptor 2
TG Triglyceride
TH Tyrosine-3-hydroxylase
THAN Transient hyperammonemia of the newborn
THCA Trihydroxycholestanoic acid
tHcy Total homocysteine
THF Tetrahydrofolate
THF Tetrahydrofolate
ThTP Thiamine triphosphate
THTR1 Thiamine-responsive megaloblastic anemia syndrome (SLC19A2)
TKS Thymidine kinase 2
TLC Thin-layer chromatography
TMA Trimethylaminuria
TMA Trimethylamine
TMAO Trimethylamine N-oxide
TMEM70 Complex V assembly protein
TMS Tandem mass spectrometry
TNSALP Tissue nonspecific alkaline phosphatase
TP Thymidine phosphorylase
TPK Thiamine pyrophosphokinase
TPMT Thiopurine S-methyltransferase
TPP Thiamine pyrophosphate
TPP tr Thiamine triphosphate transporter
TPP1 Lysosomal tripeptidyl-peptidase-1
TRMA Thiamine-responsive megaloblastic anemia
TRPV1 Transient receptor potential channel vanilloid subfamily member 1
Abbreviations xlv
TS Thymidylate synthase
TSD Tay-Sachs disease
TUSC3 Oligosaccharyltransferase subunit tusc 3
TUSC3-CDG Oligosaccharyltransferase subunit tusc 3 deficiency
TYMP Thymidine phosphorylase
TYR1 Tyrosinemia type I
TYR2 Tyrosinemia type II
TYR3 Tyrosinemia type III
UCD Urea cycle disorders
UCP Uncoupling protein deficiency
UCP2 Uncoupling protein 2 deficiency
UCP2 Uncoupling protein 2
UCP2 Mitochondrial uncoupling protein 2
UMPH1 Pyrimidine-5′-nucleotidase I
UMPH1 Uridine-5′-monophosphate hydrolase
UMPS Uridine monophosphate synthase
UP ß-Ureidopropionase
UPB1 Beta-ureidopropionase
UPD Uniparental disomy
UPLC Ultra performance liquid chromatography
UROC1 Urocanase
UROD Uroporphyrinogen decarboxylase
UROS Uroporphyrinogen III synthase
USF1 Upstream stimulatory factor
VLA Vanillactic acid
VLCAD Very-long-chain acyl-CoA dehydrogenase
VLCFA Very-long-chain fatty acids
VLCS Very-long-chain acyl-CoA synthase
VLDL Very low-density lipoprotein
VMA Vanillylmandelic acid
VMAT2 Dopamine-serotonin vesicular transport
VP Variegate porphyria
WHO World Health Organization
WND (WD) Wilson disease
X-ALD and AMN X-linked adrenoleukodystrophy and adrenomyeloneuropathy
XDH Xanthine dehydrogenase (oxidase)
XDH/AO Combined xanthine oxidase and aldehyde oxidase
XLDPP X-linked dominant protoporphyria
XLPP X-linked protoporphyria
XLSA X-linked sideroblastic anemia
X-MT S-adenosylmethionine-dependent transmethylation reactions
ZSD Zellweger spectrum disorders
Part I
Amino Acids
Disorders of Phenylalanine
and Tetrahydrobiopterin Metabolism 1
Nenad Blau and Francjan J. van Spronsen
Contents 1.1 Introduction

1.1 Introduction ...................................................................... 3
Hyperphenylalaninemia (HPA), a disorder of phenylalanine
1.2 Nomenclature.................................................................... 4
catabolism, is caused primarily by a deficiency of the hepatic
1.3 Metabolic Pathway ........................................................... 5 phenylalanine-4-hydroxylase (PAH) or by one of the enzymes
1.4 Signs and Symptoms ........................................................ 5 involved in its cofactor tetrahydrobiopterin (BH4)
1.5 Reference Values ............................................................... 8
biosynthesis (GTP cyclohydrolase I (GTPCH) and
6-pyruvoyl-tetrahydropterin synthase (PTPS)) or regeneration
1.6 Pathological Values/Differential Diagnosis .................... 9
(dihydropteridine reductase (DHPR) and pterin-4a-
1.7 Loading Tests .................................................................... 10 carbinolamine dehydratase (PCD)) (Blau et al. 2001). BH4 is
1.8 Diagnostic Flowcharts...................................................... 11 known to be the natural cofactor for PAH, tyrosine-3-
hydroxylase, and tryptophan-5-hydroxylase as well as all
1.9 Diagnostic Flowchart in the Differential
Diagnosis of Non-HPA Variants ...................................... 15 three isoforms of nitric oxide synthase (NOS) (Werner et al.
2011), the latter two being the key enzymes in the biosynthesis
1.10 Specimen Collection ......................................................... 16
of the neurotransmitters dopamine and serotonin. Thus, with
1.11 Prenatal Diagnosis ............................................................ 16 two exceptions (see below) any cofactor defect will result in a
1.12 DNA Analysis .................................................................... 16 deficiency of biogenic amines accompanied by HPA. Because
phenylalanine is a competitive inhibitor of the uptake of tyro-
1.13 Treatment .......................................................................... 16
sine and tryptophan across the blood-brain barrier and of the
1.14 Follow-Up/Monitoring ..................................................... 20 hydroxylases of tyrosine and tryptophan, depletion of cate-
References .................................................................................... 21 cholamines and serotonin occurs in untreated patients with
PAH deficiency. Both groups of HPA (PAH and BH4 defi-
ciency) are heterogeneous disorders varying from severe, e.g.,
classical phenylketonuria (PKU), to mild and benign forms
(see Sect. 1.4). Because of the different clinical and biochemi-
cal severities in this group of diseases, the terms “severe” or
“mild” will be used based upon the type of treatment and
involvement of the CNS. For the BH4 defects, symptoms may
manifest during the first weeks of life but usually are noted
N. Blau (*)
Division of Inborn Metabolic Diseases, within the first half year of life. Birth is generally uneventful,
Department of General Pediatrics, University Children’s Hospital, except for an increased incidence of prematurity and lower
Im Neuenheimer Feld 430, Heidelberg 69120, Germany birth weights in severe PTPS deficiency (Opladen et al. 2012).
Division of Metabolism, University Children’s Hospital, Two disorders of BH4 metabolism may present without
Zürich, Switzerland HPA. These are dopa-responsive dystonia (DRD; Segawa dis-
e-mail: nenad.blau@med.uni-heidelberg.de
ease) (Segawa 2011) and sepiapterin reductase (SR) deficiency
F.J. van Spronsen (Friedman et al. 2012). While DRD is caused by mutations in
Section of Metabolic Diseases, Beatrix Children’s Hospital,
the GTPCH gene and is inherited in an autosomal dominant
University Medical Center of Groningen, University of Groningen,
30.001, 9700 RB Groningen The Netherlands manner, SR deficiency is an autosomal recessive trait. Both
e-mail: f.j.van.spronsen@umcg.nl diseases evidence severe biogenic amine deficiencies. DRD
N. Blau et al. (eds.), Physician’s Guide to the Diagnosis, Treatment, and Follow-Up of Inherited Metabolic Diseases, 3
DOI 10.1007/978-3-642-40337-8_1, © Springer-Verlag Berlin Heidelberg 2014
4 N. Blau and F.J. van Spronsen
usually presents with a dystonic gait and diurnal variation, while BH4 deficiencies presenting without HPA are detectable
many patients with SR deficiency have initial diagnosis of cere- only by investigations for neurotransmitter metabolites and
bral palsy. At least two reports describe heteroallelic patients pterins in CSF or by clinical signs and symptoms. In DRD, a
with DRD suggesting a wide spectrum of GTPCH variants. phenylalanine loading test, a trial with l-dopa, and enzyme
A diagnosis of HPA is usually based upon the confirma- activity measurement in cytokine-stimulated fibroblasts and
tion of an elevated blood phenylalanine level obtained on a molecular testing are confirmatory for the diagnosis. SR
normal diet, following a positive newborn screening test. deficiency can be definitely diagnosed by an enzyme assay
Normal breast milk or formula feeding for only 24 h is suffi- of cultured fibroblasts or DNA testing, but phenylalanine
cient to raise the baby’s blood phenylalanine sufficiently to loading test is also positive.
trigger a positive test level (>120 μmol/l). In general, an infant The goals of treatment are to control HPA in PAH and
will be found to have a positive screening test 12 h postnatal. BH4 deficiencies and to restore CNS neurotransmitter
The tandem mass spectrometry (TMS) is today the method of homeostasis in BH4 deficiencies (Blau et al. 2010). To that
choice for newborns screening. A detection as early as aim, dietary restriction in phenylalanine intake, supplementa-
possible is essential in order to introduce appropriate treat- tion with BH4, and oral administration of dopamine and sero-
ment to prevent effects on mental development. tonin precursors (l-dopa/carbidopa and 5-hydroxytryptophan,
In PAH and BH4 deficiencies, factors like a relatively high respectively), as well as some other drugs are available
phenylalanine intake or catabolic situations may be responsi- (Opladen et al. 2012). In this respect it should be taken into
ble for high phenylalanine concentrations in blood. Once HPA account that some patients with PAH deficiency, historically
has been detected, a sequence of quantitative tests (see only treated by diet, can be treated with BH4 (sapropterin
Sect. 1.8) enables the differentiation between variants, i.e., dihydrochloride). At the same time, in patients with DPHR
BH4-non-responsive PKU (usually the patients with the most deficiency, in whom historically the HPA was not treated with
severe PAH deficiency), BH4-responsive PKU (Heintz et al. BH4, the diet restricting phenylalanine intake is the treatment
2013), and BH4 deficiencies. Because the BH4 deficiencies of choice. Only about 20 % of DHPR-deficient patients are on
are actually a group of diseases which may be detected because BH4 treatment (Opladen et al. 2012).
of HPA, but not simply and routinely identified by neonatal Late detection of PAH or BH4 deficiencies and late intro-
mass screening, selective screening for a BH4 deficiency is duction of treatment lead to irreversible brain damage. In
essential in every newborn with even slightly elevated phenyl- contrast to early and continuously treated patients with PAH
alanine levels. Differential testing for BH4 deficiencies should deficiency, some patients with BH4 deficiencies show
be done in all newborns with plasma phenylalanine levels progressive neurological deterioration despite treatment.
greater than 120 μmol/l (2 mg/dl), as well as in older infants Patients with PCD deficiency are at risk for developing early-
and children with neurological signs and symptoms. onset diabetes in puberty.
1.2 Nomenclature
Alternative Gene Chromosomal
No. Disorder Name Abbreviation Symbol Localization Affected Protein OMIM No. Sub Type
1.1 Phenylalanine Classic Classic PKU PAH 12q22-24.1 Phenylalanine 261600 Mild to
hydroxylase phenylketonuria hydroxylase severe
deficiency
1.2 GTP cyclohydrolase arGTPCH GCH1 14q22.1-22.2 GTP 233910 Autosomal
deficiency cyclohydrolase I recessive
1.3 6-Pyruvoyl- PTPS PTS 11q22.3-23.3 6-Pyruvoyl- 261640
tetrahydropterin tetrahydropterin
synthase deficiency synthase
1.4 Dihydropteridine DHPR QDPR 4p15.3 Dihydropteridine 261630 Moderate
reductase deficiency reductase and severe
1.5 Pterin- Primapterinuria PCD PCBD1 10q22 Pterin- 264070 Benign
4a-carbinolamine 4a-carbinolamine HPA
dehydratase dehydratase Early-onset
deficiency diabetes
1.6 Dopa-responsive Segawa disease adGTPCH, GCH1 14q22.1-22.2 GTP 600225
dystonia DRD cyclohydrolase I
1.7 Sepiapterin SR SPR 2p14-p12 Sepiapterin 182125
reductase deficiency reductase
1 Disorders of Phenylalanine and Tetrahydrobiopterin Metabolism 5
1.3 Metabolic Pathway
Fig. 1.1 Biosynthesis and GTP

regeneration of tetrahydrobiopterin GTPCH (1.2 & 1.6)
(BH4) including possible metabolic Neopterin
NH2TP
defects in hyperphenylalaninemia
(HPA) and catabolism of PTPS (1.3)
phenylalanine. 1.1 phenylalanine-
4-hydroxylase (PAH), 1.2/1.6 GTP PTP
cyclohydrolase I, 1.3 6-pyruvoyl- non- SR (1.7)
tetrahydropterin synthase (PTPS), enzymatic
1.4 dihydropteridine reductase Sepiapterin Oxo-PH4
(DHPR), 1.5 pterin-4a-
SR CR SR (1.7) Phenylalanine
carbinolamine dehydratase (PCD),
1.7 sepiapterin reductase, carbonyl DHFR (10.4)
7,8-BH2 BH4
reductase (CR), aldose reductase
(AR), dihydrofolate reductase Phe Tyr Trp
(DHFR), aromatic amino acid DHPR (1.4)
decarboxylase (AADC), tyrosine
hydroxylase (TH), and tryptophan PAH (1.1) O2 TH (31.1) O2 TPH O2
hydroxylase (TPH). 7,8-BH2: q-BH 2
7,8-dihydrobiopterin; PTP:
6-pyruvoyl-tetrahydropterin; HVA: Tyr L-Dopa 5-OH-Trp
PCD (1.5)
homovanillic acid; 5HIAA: HO-BH 4 AADC (31.2)
5-hydroxyindoleacetic acid.
Biopterin Dopamine Serotonin
Pathological metabolites used as
specific markers in the differential Primapterin HVA 5HIAA
diagnosis are marked in squares
1.4 Signs and Symptoms
Table 1.1 Phenylalanine hydroxylase deficiency, classic PKU

System Symptom Neonatal Infancy Childhood Adolescence Adulthood
CNS Autism ± ± ±
Hypertonia ± ± ±
Irritability ± ± ± ±
Mental retardation ± + + + +
Seizures ± ± ±
Dermatological Hypopigmentation + + + + +
Skin rash ± ± ± ± ±
Digestive Vomiting ± ±
Musculoskeletal Head circumference ↓-n ↓-n
Height ↓-n ↓-n ↓-n
Microcephaly + + + +
Other Odor (urine and body) ± + + + +
Birth weight ↓-n
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓-n ↓-n ↓ ↓
BH4 test n n n n n
Homovanillic acid, HVA (CSF) ↓-n ↓-n ↓ ↓
MRI: brain ± ± ± ±
Phenylalanine (P, U, CSF) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Phenylpyruvic acid (U) n-↑ ↑ ↑ ↑ ↑
Table 1.2 GTP cyclohydrolase deficiency

Autonomic system Temperature instability + + +
CNS Hypertonia, extremities + + + +
Hypotonia, axial + + + +
Mental retardation + + +
Seizures, myoclonic + +
Digestive Drooling + + +
Feeding difficulties + + +
Musculoskeletal Microcephaly + + + +
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓ ↓ ↓ ↓
Biopterin (U, CSF, DBS) ↓↓ ↓↓ ↓↓ ↓↓
GTPCH activity, cytokine-stimulated (FB) ↓ ↓ ↓ ↓ ↓
Homovanillic acid, HVA (CSF) ↓↓ ↓↓ ↓↓ ↓↓
Neopterin (U, CSF, DBS) ↓↓ ↓↓ ↓↓ ↓↓
Phenylalanine (P, U, CSF) n-↑ ↑ ↑ ↑
Tetrahydrobiopterin (BH4) loading test +++ +++ +++ +++
Table 1.3 6-Pyruvoyl-tetrahydropterin synthase deficiency

CNS Choreoathetosis + + +
Retardation, psychomotor + +
Dermatological Hypopigmented hair + +
Rash, eczematous + + +
Digestive Drooling + + + +
Musculoskeletal Microcephaly + + +
Respiratory Pneumonia + +
Other Birth weight ↓-n
Sudden death ± ±
Routine laboratory EEG: abnormal + + + + +
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓↓ ↓↓ ↓↓ ↓ ↓
Biopterin (U, CSF, DBS) ↓↓↓ ↓↓↓ ↓↓ ↓↓ ↓↓
Homovanillic acid, HVA (CSF) ↓↓ ↓↓ ↓↓ ↓↓ ↓
MRI: cortical and subcortical atrophy + + + + +
Neopterin (U, CSF, DBS) ↑↑↑ ↑↑↑ ↑↑ ↑↑ ↑↑
Phenylalanine (P, U, CSF) ↑ ↑ ↑ ↑ ↑
Prolactin (P) ↑ ↑ ↑ ↑ ↑
PTPS activity (RBC, FB) ↓ ↓ ↓ ↓ ↓
Tetrahydrobiopterin (BH4) loading test ++ ++ ++ ++ ++
Table 1.4 Dihydropteridine reductase deficiency

CNS Choreoathetosis + + +
Hypotonia, axial + + +
Table 1.4 (continued)

Dermatological Hypopigmented hair + +
Rash, eczematous + + +
Digestive Drooling + + + +
Musculoskeletal Microcephaly + + +
Respiratory Pneumonia + +
Other Sudden death + +
Routine laboratory CT scan: basal ganglia calcifications + + +
EEG: spike wave discharges and generalized + + + + +
slowing, abnormal
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓↓ ↓↓ ↓↓ ↓ ↓
Biopterin (U, CSF, DBS) n-↑ n-↑ n-↑ ↑ ↑
Dihydrobiopterin (CSF) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Dihydropteridine reductase (DBS) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Homovanillic acid, HVA (CSF) ↓↓ ↓↓ ↓↓ ↓↓ ↓
MRI: cortical and subcortical atrophy (+) + + + +
Neopterin (U, CSF, DBS) n n n n n
Phenylalanine (P, U, CSF) ↑ ↑ ↑ ↑ ↑
Tetrahydrobiopterin (BH4) loading test + + + + +
Table 1.5 Pterin-4a-carbinolamine dehydratase deficiency

CNS Hypotonia, mild +
Transient alteration in tone +
Endocrine Diabetes MODY3-like ± ±
Routine laboratory Glucose (P) n-↑ n-↑
Magnesium (P) ↓-n ↓-n
Special laboratory Neopterin (U) ↑↑ ↑
Phenylalanine (P) ↑ (↑) (↑) n n
Primapterin (U) ↑↑ ↑↑ ↑
BH4 loading test + + + + +
Table 1.6 Dopa-responsive dystonia

CNS Bradykinesia ± ± ± ±
Diurnal fluctuation of symptoms ± + + ±
Dyskinesia ± ±
Hypertonia ± ± ± ± ±
Hypokinesia + ++ ++ ++
Hypotonia ± ± ± ±
Parkinsonism ± ± ±
Spasticity ± ± ± ± ±
Tendon reflexes, increased ± ± ± ± ±
Tremor + +
Digestive Dysphagia ± ± ± ±
Musculoskeletal Pes equinovarus ± ±
Rigidity ± + + + +
Scoliosis ± ±
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓-n ↓-n ↓-n ↓-n ↓-n
Biopterin (CSF) ↓ ↓ ↓ ↓ ↓
Homovanillic acid, HVA (CSF) ↓ ↓ ↓ ↓ ↓
Neopterin (CSF) ↓ ↓ ↓ ↓ ↓
Phe loading test + + + + +
Phenylalanine (P) n n n n n
Table 1.7 Sepiapterin reductase deficiency

CNS Behavior, psychotic ± ± ± ±
Cerebral palsy ± ± ±
Diurnal fluctuation of symptoms + + ± ±
Dysarthria ± ± ± ±
Hypo or hypertonia ± ± ± ±
Hypokinesia + ++ ± ± ±
Hypotonia, axial ++ ++ ++ +
Language difficulties ++ ++ +
Parkinsonism ± ± ±
Retardation, psychomotor ++ ++
Tendon reflexes, increased ± ± ±
Digestive Gastrointestinal dysmotility ± ± ±
Eye Eye movements, abnormal, oculogyric crisis ± ± ±
Musculoskeletal Muscle weakness + ± ± ±
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓
Biopterin (CSF) ↑ ↑ ↑ ↑ ↑
Biopterin (U) n n n n n
Dihydrobiopterin (CSF) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Homovanillic acid, HVA (CSF) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Neopterin (U, CSF, DBS) n n n n n
Phe loading test + + + +
Phenylalanine (P) n n n n n
Sepiapterin (CSF) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
1.5 Reference Values
Serum, urine, and dried blood spots

Phe (S) Neo (S) Bio (S) Neo (U) Bio (U) Neo (DBS) Bio (DBS)
Age μmol/l nmol/l nmol/l mmol/mol Creat mmol/mol Creat nmol/g Hb nmol/g Hb
Newborns <120 3–11 4–18 1.1–4.0 0.5–3.0 0.19–2.93 0.08–1.20
0–1 years <80 3–11 4–18 1.1–4.0 0.5–3.0 0.19–2.93 0.08–1.20
2–4 years <80 3–11 4–18 1.1–4.0 0.5–3.0 0.19–2.93 0.08–1.20
5–10 years <80 3–11 4–18 1.1–4.0 0.5–3.0 0.19–2.93 0.08–1.20
11–16 years <70 3–11 4–18 0.2–1.7 0.5–2.7 0.19–2.93 0.08–1.20
>16 years <70 3–11 4–18 0.2–1.7 0.5–2.7 0.19–2.93 0.08–1.20
CSF
5HIAA (CSF) HVA (CSF) 5MTHF (CSF)
Age BH4 (CSF) nmol/l BH2 (CSF) nmol/l Neo (CSF)a nmol/l Bio (CSF)a nmol/l nmol/l nmol/l nmol/l
Newborns 25–121 4–18 15–35 20–70 144–800 300–1,000 64–182
0–1 years 24–59 4–18 12–30 15–40 114–336 295–932 64–182
2–4 years 20–61 4–18 9–20 10–30 105–299 211–871 63–111
5–10 years 20–49 4–18 9–20 10–30 88–178 144–801 41–117
11–16 years 20–49 4–18 9–20 10–30 74–163 133–551 41–117
>16 years 18–53 4–18 9–20 10–30 66–141 115–488 41–117
a
Total neopterin or biopterin
Amniotic fluid, amniocytes, and fibroblasts

Phe Neo Bio 5-HIAA HVA
Age μmol/l nmol/l nmol/l nmol/l nmol/l
Amniotic fluid Fetus <120 16–40 6–21 32–135 50–144
Amniocytes Fetus <14b <115b
Fibroblastsa Infants (0–12 m) 11–73b 183–303b
Children and adults 18–98b 154–303b
a
In cytokine-stimulated cells
b
pmol/mg
Enzymes
DHPR (RBC) PTPS (RBC) GTPCH (FB)a PTPS (FB) DHPR (FB) SR (FB)
Age mU/mg Hb µU/g Hb µU/mg protein µU/mg protein mU/mg protein µU/mg protein
Fetus 2.3–3.8 35–77 1.5–1.9 3.0–3.3 5.8–8.8
Newborns (0–1 months) 1.8–4.8 34–64
Infants (0–12 months) 1.8–4.8 1.7–4.9 0.5–1.7 6.3–8.7 97–185
Children and adults 1.8–4.8 11–29 1.4–6.5 0.4–1.6 4.5–8.3 99–185
a
In cytokine-stimulated cells
1.6 Pathological Values/Differential

Diagnosis
Plasma Actual Phea Neo (S) Bio (S)

μmol/l nmol/l nmol/l
<200 3–11 4–18
200–600 2–32 12–46
600–1,200 9–27 24–39
a
Plasma neopterin and biopterin values depend strongly upon the actual blood Phe concentrations
Plasma, urine, DBS, and CSF

Phe (S) Neo (U) Bio %Bioc Neo Bio Neo Bio 5HIAA HVA 5-MTHF
Variant (U) (DBS) (DBS) (CSF) (CSF) (CSF) (CSF) (CSF)
μmol/l mmol/mol Creat nmol/g Hb nmol/l
1.1 PAH def. >1,200 1.1–16.9 1.2–8.1 ~50 0.15–4.62 0.08–1.68 9–118 15–143 14–471 47–1,174 n
(classical)
1.1 PAH def. 600–1,200 1.1–16.9 1.2–8.1 ~50 0.15–4.62 0.08–1.68 9–118 15–143 n n n
(variant)
1.1 PHA def. 120–600 1.1–16.9 1.2–8.1 ~50 0.15–4.62 0.08–1.68 n n n n n
(benign)
1.2 GTPCH 120–1,200a <0.2 <0.2 ~50 <0.15 <0.08 0.05–3.0 1.5–7.5 61–183 15–48 n
def.
1.3 PTPS def. 250–2,500 5.0–51.2 <0.5 <5 2.2–6.3 0 47–402 1.0–16.0 5–113 5–223 n
(severe)
1.3 PTPS def. 240–2,200 5.0–51.2 <0.5 <5 2.2–6.3 0 25–230 13–56 93–420 249–998 n
(mild)
1.4 DHPR def. 180–2,500 0.5–23.2 3.8– >80 0.47–2.1 0.67–1.5 11–70 43–117d 4–75 19–204 ↓
(severe) 25.6
1.4 DHPR def. 280–600 0.5–23.2 3.8– >80 0.47–2.1 0.67–1.5 11–70 43–117d 21–66 n ↓-n
(mild) 25.6
1.5 PCD def. 180–1,200 4.1–22.5 0.7– <50 – – 43–117 16–96 n n n
(benign) 1.5b
1.6 DRD <120 n n ~50 n n 1.1–6.2 3.1–7.6 48–97 120–239 n
1.7 SR def. <120 n n ~50 n n 14–51 72–102d 3–15 49–111 n
a
Several patients were missed in the newborn screening due to the negative Guthrie test
b
Primapterin (7-Bio) ↑
c
%Bio = 100 * Bio/(Neo + Bio)
d
7,8-Dihydrobiopterin ↑
1.7 Loading Tests
Loading test with BH4
Fig. 1.2 Proposed algorithms for a

the BH4 (sapropterin
dihydrochloride; Kuvan) Day 1
challenge, screening, and initiating Check blood Phe
treatment in BH4-responsive PKU 24, 16, and 8 h before test
patients. (a) Initial screening test
with blood Phe monitoring on the
first day and BH4 (sapropterin Maintain constant protein intake Day 2
dihydrochloride) administration BH4 (Kuvan®)
Initial Screening Test

(20 mg/kg) on two following days. 20 mg/kg
(b) Efficiency testing in BH4-
reponsive patients over several
weeks with BH4 doses adjusted
individually according to Phe Check blood Phe
tolerance and therapeutic blood 8, 16, and 24h after BH4
Phe levels. Combined Phe Phe ↓ ≥ 30%
(100 mg/kg) and BH4 (20 mg/kg)
loading test is sometimes difficult
to interpret and is therefore not
recommended (Blau et al. 2011)
Day 3
BH4 (Kuvan®)
20 mg/kg
b Check blood Phe No

Non-
8, 16, and 24h after BH4
responder
Phe ↓ ≥ 30%
Reduce or stop diet
Efficiency Testing
Yes
Adjust dose to 5–20mg/kg/day to keep blood
Phe in therapeutic range
Monitor blood Phe frequently

Monitor Phe tolerance
Loading tests with Phe
a
Fig. 1.3 Oral phenylalanine loading with 100 mg Phe/kg 14,0
body weight is performed, as previously described by **
Hyland et al. (1997). Samples were collected at time T-0, 12,0
*
T-1h, T-2h, T-4h, and T-6h. Plasma was frozen immediately
10,0
and kept in the dark; DBS were kept in dry conditions at
phe/tyr ratio
4 °C. The clearest differentiation measured by the

8,0 *
standardized mean difference between the patients and
controls was seen 2 h after Phe administration (Opladen
6,0
et al. 2010). See also the flowchart with cut-off values.
*
(a) Means and standard deviations (SDs) of phenylalanine/
4,0
tyrosine (Phe/Tyr) ratios in pediatric patients after Phe
loading [open circles dopa-responsive dystonia (DRD)
2,0
patients; black squares controls; * p < 0.05; **p < 0.01]
(b) Means and standard deviations (SDs) of biopterin 0,0
concentration in pediatric patients after phenylalanine 0 1 2 4 6
loading [open circles dopa-responsive dystonia (DRD) time [h]
patients; black squares controls; **p < 0.01] (Opladen et al.
2010) controls (n=17) DRD patients (n=17)
Fig. 1.3 (continued) b

40
**
35 **
30
**
25
nmol/i
20
15
10
0
0 1 2 4 6
time [h]
controls (n=17) DRD patients (n=17)
1.8 Diagnostic Flowcharts 4. DNA testing

The first two tests are essential and will allow the differ-
Differential diagnosis of HPAs entiation between all variants with BH4 deficiencies. With
Screening for a BH4 deficiency should be done in all new- some limitations (DHPR def.), the BH4 loading test is an
borns and children with even slight HPA (plasma Phe additional useful diagnostic tool for the rapid discrimination
>120 μmol/l) as well as in older children without HPA but between classical PKU and biopterin variants. This test is
with neurological symptoms suggestive of a neurotransmit- also useful for identifying the recently described BH4-
ter deficiency. The following protocol is suggested: responsive PAH deficiency. For the interpretation and deter-
1. Analysis of pterins in urine mination of the various disorders based upon loading tests,
2. Measurement of DHPR activity in blood from a Guthrie see Sect. 1.6.
card Biochemical and loading test data are summarized in
3. Analysis of phenylalanine and tyrosine in serum or Fig. 1.5 (Blau et al. 2010):
plasma before and after a BH4 challenge
Phe↑ In newborns screening

(DBS)
Phe and Tyr

(Plasma)
BH4 loading test Phe ↑ Tyr n ↑

Phe ↑ Tyr n -↓ Phe normal
(Phe In Plasma) Repeat amino acids
Before Introduction of diet!

Pterins (DBS or U)
DHPR (DBS)
Pterins normal Pterins ± normal

DHPR normal DHPR ↓
DHPR def.
PAH def. Neo n; Bio n-↑
Initiate treatment with low-Phe diet.
(PKU) folinic acid and neurotransmitter
precursors
Pterins abnormal
NBS false positive
DHPR normal
GTPCH def. PTPS def. PCD def.

Neo ↓; Bio ↓ Neo ↑; Bio ↓ Neo ↑; Bio n- ↓; Pri ↑
Initiate treatment with BH4 and Initiate treatment with BH4 and Initiate treatment with BH4
neurotransmitter precusors neurotransmitter precusors
Fig. 1.4 Diagnostic flowchart for the laboratory diagnosis of PKU neopterin and biopterin), 6-pyruvoyl-tetrahydropterin synthase
and BH4 deficiencies (Blau et al. 2011). Dried blood spots (DBS) or (PTPS) deficiency (high neopterin and low or no detectable biop-
random urine (U) can be used for the differential diagnosis and terin), dihydropteridine reductase (DHPR) deficiency (normal neop-
depending on the profile of neopterin (Neo), biopterin (Bio), and terin and normal or elevated biopterin and no DHPR activity), and
primapterin (Pri) and dihydropteridine reductase (DHPR) activity in pterins-4acarbinolamine dehydratase (PCD) deficiency (elevated
DBS, diagnosis of following BH4 deficiencies can be established: neopterin, low-normal biopterin, and elevated primapterin). N nor-
GTP cyclohydrolase I (GTPCH) deficiency (low or no detectable mal (Blau et al. 2011)
Newborn screening (TMS) BH4-loading test BH4-loading test

Phenylanine > 120 μmol/L (DBS) (Europe) (USA)
Phenylalanine >120 μmol/L Day 1 ,7, and 14

(plasma) Phenylalanine >400 μmol/L Day 1
BH4 (20 mg/kg per day)
Blood phenylalanine
Blood phenylalanine
–8, –16, –24 h
(1xday)
Pterins (DBS)+DHPR (DBS) Day 2 Week 3

BH4 (20 mg/kg) BH4 (20 mg/kg per day)
Blood phenylalanine Blood phenylalanine
Neo ↓ 0, 8,16, 24 h (1xweek)
GTPCH deficiency
Bio ↓
Day 3 Week 4
Neo ↑ BH4 (20 mg/kg) BH4 (20 mg/kg per day)
PTPS deficiency
Bio ↓ Blood phenylalanine Blood phenylalanine
0, 8,16, 24 h 1xweek
Neo ↑
Bio n or ↓ PCD deficiency
Pri ↑
Blood phenylalanine ↓ ≥30% BH4 responsive PKU
Neo n Blood phenylalanine ↓ >85%
Bio n or ↑ DHPR deficiency Blood phenylalanine ↓ 20–30%
DHPR ↓ 1–3 week BH4 trial
Blood phenylalanine ↓ <20%
Stop test
BH4 deficiency
Fig. 1.5 Flow-chart for the differential diagnosis of BH4 responsive PKU GTPCH GTP-cyclohydrase I, PTPS 6-pyruvoyl-tetrahydropterin
and BH4 deficiencies. Phe phenylalanine, DBS dried blood spots, DHPR synthase, PCD pterin-4a-carbinolamine dehydratase, BH4 tetrahydrobi-
dihydropteridine reductase, Neo neopterin, Bio biopterin, Pri primapterin, opterin. (Blau et al. 2010)
Interpretation of the Phe loading test
Fig. 1.6 Phenylalanine (Phe) loading

in children: revised shortened test Phe/Tyr & Bio (DBS or P)
to
procedure and test interpretation (Bio
100 mg Phe/kg BW
biopterin, Phe/Tyr Phe/tyrosine ratio,
DBS dried blood spot, P plasma, BW
body weight, dd diagnostic decision)
(Opladen et al. 2010)
t1 Phe/Tyr & Bio (DBS or P)
Bio (P) Bio (P)

≤14.0 nmo/I > 14.0 nmo/I
or or
Bio (DBS) Bio (DBS)
≤ 16.2 nmo/I > 16.2 nmo/I
t2 Phe/Tyr & Bio (DBS or P) Phe/Tyr & Bio (DBS or P)
Phe/Tyr Phe/Tyr
≥ 4.6 DBS < 4.6 DBS
≥ 5.4 P <5.4 P
Test invalid
Insufficient
dd DRD Normal Phe loading
Phe intake.
Test repetition!
Fig. 1.7 Diagnostic algorithm for patients with a possible disorder of and false negatives may still occur. Therefore, this alternative evaluation
neurotransmitter metabolism. Sepiapterin reductase (SR) deficiency and route should be reserved for cases in which CSF analysis is not available
other disorders of neurotransmitter metabolism should be considered in or is declined, or in which other clinical features lead to suspicion of a
patients with (1) developmental delay with hypotonia, (2) suspect but specific diagnosis. (2) In a patient with unexplained CP or CP with atypi-
unexplained cerebral palsy (CP) or CP with atypical features, and (3) cal features, a disorder of neurotransmitter metabolism should be consid-
uncharacterized l-dopa-responsive motor disorders. (1) In a patient with ered and diagnostic algorithm, as outlined above, should be followed.
developmental delay and hypotonia, if oculogyric crises, diurnal fluctua- Atypical or unexplained features suggesting need for further metabolic
tion, sleep disturbance, or extrapyramidal or autonomic signs exist, a dis- investigation in a child with possible CP include lack of adequate anteced-
order of neurotransmitter biosynthesis is likely and cerebrospinal fluid ent, nondiagnostic magnetic resonance imaging, progressive symptoms,
(CSF) analysis should be done. If no other signs are present, CSF analysis familial occurrence, episodic encephalopathy, and features not expected in
should be considered if standard workup for hypotonia is unrevealing. If the CPs such as diurnal variation, sleep disturbance, autonomic symp-
CSF analysis is abnormal, then mutational screening and/or measurement toms, or oculogyric crises. (3) All patients with an l-dopa-responsive
of enzymatic activity can be targeted to confirm the specific disorder sug- motor disorder should be evaluated for a disorder of neurotransmitter
gested by the pattern of CSF abnormalities. If CSF evaluation is impracti- metabolism. CSF analysis (after discontinuation of l-dopa therapy for at
cal, alternative evaluation may include l-dopa trial and/or phenylalanine least 10 days) is the recommended first step. If l-dopa withdrawal is
load. If negative, CSF analysis must still be done to exclude a disorder of impractical, the results of CSF analyses may still be informative if either
neurotransmitter metabolism. If l-dopa trial and/or phenylalanine loading pterins or 5-hydroxyindoleacetic acid levels are abnormal. Alternatively,
are positive, CSF analysis will allow targeted mutational screening; how- molecular investigations can be done, guided either by results of phenyl-
ever, one should keep in mind that phenylalanine (Phe) load can be posi- alanine loading test or clinical symptoms with caveats as noted above.1
tive in heterozygote carriers for phenylketonuria. Alternatively, CSF CSF analysis should consist of homovanillic acid, 5-hydroxyindoleacetic
analysis may be skipped and broad mutational screening undertaken. acid (5HIAA), pterins (neopterin, biopterin, and sepiapterin), and
Mutational and gene dosage screening may be time-consuming and costly, 5-methyltetrahydrofolate (Blau et al. 2010)
1.9 Diagnostic Flowchart in the Differential

Diagnosis of Non-HPA Variants
1) Development Delay
and
Hypotonia
Oculogyric Crises
or
Dystonia/Other Extrapyramidal Signs
or
Diurnal Fluctuation
or
Sleep Disturbance
or
Autonomic Symptoms
NO
YES
Standard Workup for (+)
Hypotonia/Developmental Delay Alternate Diagnosis
(depending on exam)
(–)
2) Unexplained
3) L-Dopa-Responsive
or Atypical Possible Disorder of Neurotransmitter Metabolism
Motor Disorder
Cerebral Palsy
CSF
L-Dopa Trial (–)
Neurotransmitter
and/or Alternate Diagnosis
and Pterins
Phe Load (+ or –) (prefered)
(+) (+)
CONFIRMATORY TESTS
Targeted Mutational Analysis
and/or
Enzymatic Activity in Fibroblasts
SR Deficiency or Other Neurotransmitter Disorder

1.10 Specimen Collection
Test Preconditions Material Handling Pitfalls

Phe Free diet Guthrie card, serum/plasma, Keep cool (−20 °C) for serum or
CSF plasma
Guthrie card at RT
Neo Free diet, Phe in plasma Random urine, spotted urine Keep cool, dark (−20 °C) Infections (Neo ↑)
Bio high enough Oxidized sample, dark, RT
Serum/plasma Keep cool, dark (−20 °C)
CSF EDTA tube (−20 °C)
BH4, BH2 CSF DTE/DETAPAC tube (−80 °C)
HVA 1 h before medication, CSF (−80 °C)
5HIAA withdraw first 0.5 ml
5-CH3-THF
DHPR Erythrocytes from Frozen (−20 °C)
heparinized blood
Guthrie card RT
Fibroblasts RT
Min. 50 mg Chorionic villi Frozen (−80 °C)
PTPS Before medication, no Erythrocytes from frozen (−20 °C)
BH4 heparinized blood
Min. 50 mg Chorionic villi Frozen (−80 °C)
GTPCH Fibroblasts RT
SR Fibroblasts RT
RT room temperature
1.11 Prenatal Diagnosis 1.13 Treatment
PAH or BH4 deficiency present either with or without

Disorder Material Timing, Trimester
HPA. In those presenting with HPA (1.1–1.5; see below),
1.1 Fetal DNA I
the main goal of treatment is to reduce or normalize blood
1.2 Amniotic fluid, liver, DNA II
phenylalanine levels without causing deficiencies of other
1.3 Amniotic fluid, liver, II
erythrocytes, amniocytes, DNA amino acids and other nutrients that are usually received
CV I by intake of natural intake (Belanger-Quintana et al.
1.4 Amniotic fluid, liver, II 2011). This can be done by introduction of the low-phe-
erythrocytes, amniocytes, DNA nylalanine (or low-natural protein) diet; in some patients
CV I administration of the synthetic cofactor BH4 can relax or
even replace the diet (Keil et al. 2013). In such cases it is
important to be sure that patients use normal amounts of
1.12 DNA Analysis natural protein and not causing deficiencies of amino
acids.
DNA analysis is possible for PAH and all BH4 deficiencies In PAH deficiency, one of the areas that need consensus is
whether to start treatment at blood phenylalanine levels
above 360–600 μmol/l (van Spronsen et al. 2011). Treatment
Disorder Material Method targets have not reached consensus yet, but for the first
1.1 Genomic DNA PCR/RFLP/SSCP/ sequencing decade, more or less every center will try to keep phenylala-
1.2 Genomic DNA/FB PCR/DGGE/sequencing nine below 360 μmol/l or even lower (Blau et al. 2010). Very
1.3 Genomic DNA/FB PCR/DGGE/sequencing recent data even show that outcome is even better when phe-
1.4 Genomic DNA/FB PCR/RFLP/DGGE/sequencing nylalanine is kept below 240 μmol/l (Jahja R et al., J Pediatr,
1.5 Genomic DNA PCR/sequencing in press). The next issue to decide on is the search for BH4-
1.6 Genomic DNA/FB PCR/DGGE/sequencing responsive patients. To this end we refer to paragraph 1.7.
1.7 Genomic DNA/FB PCR/sequencing All three existing methods (i.e., DNA, 7–24 days BH4
loading test (Levy 2007), and the 48-h BH4 loading test policies in various countries and centers (Opladen et al. 2012).
(Blau et al. 2011; Anjema et al. 2013)) have their own con- In addition, patients with HPA due to a cofactor defect need
siderations. For all tests, long-term data are necessary to more strict blood phenylalanine control and additional supple-
proof the predictive correctness of the method. Other issues mentations with neurotransmitter precursors l-dopa and
that need attention are the amount of total protein (natural 5-hydroxytryptophan in a combination with the peripheral
protein plus amino acids), giving extra tyrosine in case of decarboxylase inhibitor carbidopa. Patients with dihydropteri-
low tyrosine concentrations (especially in case of preg- dine reductase deficiency (DHPR, 1.4) need additional folinic
nancy), and the question how strict treatment should be dur- acid substitution. In patients revealing levodopa-induced peak-
ing adulthood (especially in males and in females after dose dyskinesia, slow-release forms of drugs can be used, and
reproductive age). reaching the upper therapeutic limits of l-dopa may be an indi-
One should be aware that there are individuals with cation for the use of MAO and/or COMT inhibitors.
“severe” PKU mutations that have escaped severe mental Patients with dopa-responsive dystonia (DRD, dominant
retardation despite high blood phenylalanine levels and very GTPCH cyclohydrolase I deficiency, 1.6) and sepiapterin
poor dietary control. One explanation for this phenomenon is reductase deficiency (SR, 1.7) respond to low dosage l-dopa/
that they have near normal brain phenylalanine levels, despite carbidopa therapy and patients with SR deficiency need
high blood phenylalanine levels. A number of studies have additional supplementation with 5-hydroxytryptophan and
now demonstrated considerably variability in blood versus probably also BH4 (Friedman et al. 2012).
brain phenylalanine levels in PKU patients. Outcome in Prognosis and outcome strongly depend on the age when
PKU appears to be related to both blood and brain phenylala- the diagnosis is made and treatment introduced but also on
nine levels. This, in all probability, will assume greater the type of mutation (Jäggi et al. 2008).
importance in making decisions about the strictness and Recommendations for treatment and monitoring are not
duration of dietary control in the future. completely uniform worldwide. Therefore, where possible
In BH4 deficiencies, the mode of treatment depends on the and necessary, recommendations have been combined and
type of disease, may differ with the patient’s age, and the ranges of values indicating lower and upper limits are reported.
1.1 Phenylalanine hydroxylase deficiency (PKU)
Protein Target blood Phe (μmol/l) Phe-free AAM

requirement Phe tolerance
Age (g/kg BW/day)a (mg/day) Germany Netherlands UK USA Type g/dayb
0–3 months 2.1–2.7 ~130–400 40–240 120–360 120–360 120–360 1 3–10
4–12 months 2.1–2.0 ~130–400 40–240 120–360 120–360 120–360 1 3–10
1–2 years 1.7 ~130–400 40–240 120–360 120–360 120–360 2 20–50
2–3 years 1.7 ~200–400 40–240 120–360 120–360 120–360 2 20–50
4–6 years 1.6 ~200–400 40–240 120–360 120–360 120–360 2 20–50
7–9 years 1.4 ~200–400 40–240 120–360 120–480 120–360 2 20–50
10–12 years 1.1 ~350–800 40–900 120–360 120–480 120–360 2 50–90
13–15 years 1.0 ~350–800 40–900 120–600 120–700 120–600 2 50–90
>16 years 0.9 ~450–1,000 40–1,200 120–600 120–700 120–900 3 60–150
Protein requirement for PKU diet is assigned higher than recommendations for healthy people since bioavailability of amino acid mixtures is
equivalent to natural protein
a
DGE 1985; RDA; WHO
b
Spread as evenly as possible through the 24 h
Non-PKU Hyperphenylalaninemia
Protein Target
Treatment only necessary for pregnant women with blood
requirement Phe tolerance blood Phe mg BH4/
Phe levels >250–360 μmol/l (see below). Clinical monitor- Age g/kg BW/day mg/day μmol/l kg BWa
ing is advised for all patients with Phe >360 μmol/l All ages See 1.1 Near normal See 1.1 5–20
Tetrahydrobiopterin (BH4)-Responsive PKU/HPA a
This applies to BH4 responsive PKU, but there it is not necessary to
There are no specific recommendations for the treatment distribute in >1 time daily (usually); no long-term clinical experience;
of this group of HPA. The following table summarizes the BH4 tablets contain (per 100 mg BH4) 100 mg ascorbic acid
current knowledge based on several experimental trials.
Maternal PKU/HPA 1.3 6-Pyruvoyl-tetrahydropterin synthase deficiency

(mild form)
Protein Target Phe-free AAM
requirement Phe blood
g/kg BW/ tolerance Phe Dosage
Trimester day mg/day μmol/l Type g/daya (mg/kg/ Dosages
No. Symbol Age Medication/diet day) per day
1 1.1 ~180–1,600 120–360 3 60–150
1.3 PTPS All ages Tetrahydrobiopterin 5–10 2
2–3 1.3–1.5 ~180–1,600 120–360 3 60–150 (mild) (BH4)a
a
Spread as evenly as possible through the 24 h a
BH4 tablets contain (per 100 mg BH4) 100 mg ascorbic acid
1.2 GTP cyclohydrolase I deficiency and 1.3 6-Pyruvoyl-

tetrahydropterin synthase deficiency (severe form)
No. Symbol Age Medication/diet Dosage (mg/kg/day) Dosages per day

1.2 GTPCH Newborn l-Dopa 1–3 3–6
1.3 PTPS (severe) Carbidopa 10–25 %a 3–6
5-Hydroxytryptophan 1–2 3–6
Tetrahydrobiopterin (BH4) 5–10 3
<1–2 years l-Dopa 4–7 3–6
Carbidopa 10–25 %a 3–6
>1–2 years l-Dopa 8–15 3–6
a
Percentage compared to l-dopa
Beware/Pitfalls
due to gastrointestinal side effect. In these cases mono-
1. Patients are on an unrestricted (i.e., protein-rich) therapy with l-dopa/carbidopa may be sufficient.
diet. 4. l-Dopa/carbidopa/5-hydroxytryptophan therapy
2. BH4 may significantly reduce plasma and CSF may reduce CSF folates (CH3-group trapping by
tyrosine levels. Consider nutrition and tyrosine l-dopa to 3-O-methyl-dopa). Determine 5-methyl-
supplementation. tetrahydrofolate in CSF. Consider folinic acid
3. l-Dopa/carbidopa/5-hydroxytryptophan therapy (5-formyltetrahydrofolate, leucovorin) substitution
should be introduced slowly but continuously increased (10–20 mg/day).
according to the clinical picture in steps of 1 mg/kg/day 5. Drugs like trimethoprim-sulfamethoxazoles or
per week. 5-Hydroxytryptophan may not be tolerated methotrexate may induce hyperphenylalaninemia
by inhibiting DHPR.
1.4 Dihydropteridine reductase deficiency

1.4 DHPR Newborn l-Dopa 1–3 3–6
Folinic acid 15–20 mg/day 1–2
Diet (see 1.1 PKU)
<1–2 years l-Dopa 4–7 3–6
Diet (see 1.1 PKU)
>1–2 years l-Dopa 8–15 3–6
Diet (see 1.1 PKU)
a
Beware/Pitfalls
1. Patients are on low-Phe diet (see Table 1.1); how-
ever, blood Phe levels should be close to normal.
These patients are more sensitive to high Phe levels
than other PKU.
2. l-Dopa/carbidopa/5-hydroxytryptophan therapy
should be introduced slowly but continuously
increased according to the clinical picture in steps
of 1 mg/kg/day per week. 5-Hydroxytryptophan
may not be tolerated due to gastrointestinal side
effect. In these cases monotherapy with l-dopa/car-
bidopa may be sufficient.
3. Drugs like trimethoprim-sulfamethoxazoles or
methotrexate may induce hyperphenylalaninemia
by inhibiting DHPR.
1.5 Pterin-4a-carbinolamine dehydratase deficiency

1.5 PCD Newborn Tetrahydrobiopterin (BH4)a 5–10 2
>1 years No treatmentb
a
BH4 tablets contain (per 100 mg BH4) 100 mg ascorbic acid
b
Due to the possible development of diabetes type MODY3-like, periodic control necessary
Beware/Pitfalls
1. Patients are on an unrestricted (i.e., protein-rich)
diet.
2. BH4 may significantly reduce plasma and CSF
tyrosine levels. Consider tyrosine supplementation.
3. Drugs like trimethoprim-sulfamethoxazoles or
methotrexate may induce hyperphenylalaninemia
by inhibiting DHPR.
1.6 Dopa-responsive dystonia/autosomal dominant

GTPCH deficiency Beware/Pitfalls
1. Administration of MAO-B or COMT inhibitors
Medication/ Dosage (mg/ Dosages allows a 30 % reduction of the daily dosage of neu-
No. Symbol Age diet kg/day) per day rotransmitter precursors.
1.6 DRD Newborn l-Dopa 1–3 3–4
>1 years l-Dopa 4–12 3–4
1.14 Follow-Up/Monitoring
a
1.1. PAH deficiency
Beware/Pitfalls
Biochemical Intellectual and
1. l-Dopa/carbidopa therapy should be introduced monitoring Phe Clinical personality
slowly but continuously increased according to the Age and Tyr monitoringa development
clinical picture in steps of 1 mg/kg/day per week. 0–3 months Weekly 1–3 monthly
4–12 months Weekly 1–3 monthly X
1–2 years Weekly 2–6 monthly
2–3 years Weekly 2–6 monthly X
1.7 Sepiapterin reductase deficiency 4–6 years Fortnightly 3–6 monthly X
7–9 years Fortnightly 6 monthly
10–12 years Monthly 6 monthly X
Dosage 13–15 years Monthly 6 monthly X
(mg/kg/ Dosages
No. Symbol Age Medication/diet day) per day Adolescents/ Monthly 6–12 monthly X
adults
1.7 SR Newborn l-Dopa 1–3 3–4
Maternal PKU Weeklyb Bimonthlyc
Carbidopa 10– 3–4
a
25 %a Nutrient intake, body growth, general health, as well as laboratory
tests: blood count, calcium, phosphate, magnesium, iron, liver and kid-
ney function tests, alkaline phosphatase, total protein, albumin, pre-
>1 years l-Dopa 4–10 3–4 albumin, cholesterol, triglycerides, vitamins
Carbidopa 10– 3–4 b
Plasma AA, albumin, cholesterol, ferritin, folate, vitamin B12
25 %a c
Nutrient intake including micronutrients, body growth, general health
a
Standard protocol for intercurrent illness
Aim the best possible intake of fluid, energy, and Phe-free
AAM, with special attention for higher need of energy, while
Beware/Pitfalls
taking AAM in these periods may be a real.
1. l-Dopa/carbidopa/5-hydroxytryptophan therapy
should be introduced slowly and increased in steps 1.2–1.7. BH4 deficiencies
of not more than 1 mg/kg over days/weeks.
Plasma Phe and Tyr are monitored in all forms of HPA, CSF
investigations only in disorders affecting BH4 metabolism
with and without HPA (1.2–1.7).
Alternative therapies/experimental trials
No. Deficiency symbol Age Medication Dosage (mg/kg/day) Dosages per day
1.1 BH4-responsive PKU >4 years BH4a 10–20 1–2
1.3–1.5 GTPCH, PTPS, DHPR All ages Deprenylb 0.1–0.3 3–4
Entacaponec ~30 1–2
1.7 SR All ages Deprenylb 0.07–0.14 3–4
Sertralined 0.71–2.14 2–3
Melatonine 0.01–0.03 1–2
Bromocriptine Not reported Not reported
a
Tetrahydrobiopterin (BH4; sapropterin dihydrochloride, Kuvan®) treatment has been recently introduced as a standard therapy for children with
phenylalanine hydroxylase deficiency that showed a decrease of Phe levels after BH4 loading (see above)
b
MAO-B inhibitor (Selegiline)
c
COMT inhibitor
d
Serotonin reuptake inhibitor
e
Product of serotonin metabolism
Test Age Frequency Comments

Phe and Tyr (blood) 1–3 years Weekly to fortnightly Phe levels: 40–240 (360) μmol/la
4–10 years Fortnightly to monthly
11–16 years Monthly Phe levels: 40–900 μmol/la
>16 years Every 2–3 months Phe levels: 40–1,200 μmol/la
Neopterin <1 month Fortnightly Close to normal range
Biopterin
5HIAA
HVA
Folates (CSF)b
1 month–1 years Every 4–8 weeks Close to normal range
<1 years Monthly to yearly Close to normal range
Glucose (P) >5 years Yearly Only in PCD-deficient patients
a
In DHPR-deficient patients, Phe levels should be close to 240–360 μmol/l at all ages
b
Lumbar puncture in the morning before medication. Discard first 0.5 ml and collect the next 1–2 ml (−80 °C)
References Jäggi L, Zurflüh MR, Schuler A et al (2008) Outcome and long-term

follow-up of 36 patients with tetrahydrobiopterin deficiency. Mol
Genet Metab 93:295–305
Anjema K, van Rijn M, Hofstede FC, et al (2013) Tetrahydrobiopterin
Keil S, Anjema K, van Spronsen FJ et al (2013) Long-term follow-up
responsiveness in phenylketonuria: prediction with the 48-hour
and outcome of phenylketonuria patients on sapropterin: a retro-
loading test and genotype. Orphanet J Rare Dis 10:103
spective study. Pediatrics 131:e1881–e1888
Belanger-Quintana A, Burlina A, Harding CO, Muntau AC (2011) Up
Levy HB, Burton S, Cederbaum C, Scriver (2007) “Recommendations
to date knowledge on different treatment strategies for phenylketon-
for evaluation of responsiveness to tetrahydrobiopterin (BH(4)) in
uria. Mol Genet Metab 104(Suppl):S19–25
phenylketonuria and its use in treatment.” Mol Genet Metab 92(4):
Blau N, Thöny B, Cotton RGH, Hyland K (2001) Disorders of tetrahy-
287–291
drobiopterin and related biogenic amines. In: Scriver CR, Beaudet al,
Opladen T, Okun JG, Burgard P, Blau N, Hoffmann GF (2010)
Sly WS, Valle D, Childs B, Vogelstein B (eds) The metabolic and
Phenylalanine loading in pediatric patients with dopa-responsive
molecular bases of inherited disease. McGraw-Hill, New York,
dystonia: revised test protocol and pediatric cut-off values. J Inerit
pp 1725–1776
Metab Dis 33:697–703
Blau N, Van Spronsen FJ, Levy HL (2010) Phenylketonuria. Lancet
Opladen T, Hoffmann FG, Blau N (2012) An international survey of
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patients with tetrahydrobiopterin deficiencies presenting with
Blau N, Hennermann JB, Langenbeck U, Lichter-Konecki U (2011)
hyperphenylalaninaemia. J Inerit Metab Dis 35:963–73
Diagnosis, classification and genetics of phenylketonuria and tetra-
Segawa M (2011) Hereditary progressive dystonia with marked diurnal
hydrobiopterin (BH4) deficiencies. Mol Genet Metab 104:S2–9
fluctuation. Brain Dev 33:195–201
Friedman J, Roze E, Abdenur JE et al (2012) Sepiapterin reductase
van Spronsen FJ, Huijbregts SC, Bosch AM, Leuzzi V (2011) Cognitive,
deficiency: a treatable mimic of cerebral palsy. Ann Neurol
neurophysiological, neurological and psychosocial outcomes in
71:520–530
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action on phenylalanine hydroxylase and importance of genotypes
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Werner ER, Blau N, Thöny B (2011) Tetrahydrobiopterin: biochemistry
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Neurology 48:1290–1297
Tyrosine Metabolism
2
Elisabeth Holme and Grant A. Mitchell
Contents Summary
2.1 Introduction ...................................................................... 24 The tyrosine degradation pathway includes five enzymatic
steps. Inherited disorders have been identified at four of
2.2 Nomenclature.................................................................... 25
these steps. Under normal conditions the level of tyrosine is
2.3 Metabolic Pathway ........................................................... 26 regulated by the first enzyme (tyrosine aminotransferase) of
2.4 Signs and Symptoms ........................................................ 26 the pathway, but acquired or inherited deficiency of the sec-
2.5 Reference Values............................................................... 27
ond enzyme (4-hydroxyphenylpyruvate dioxygenase) also
results in hypertyrosinaemia. Tyrosine is mainly degraded
2.6 Pathological Values/Differential in the liver but also to a minor extent in the kidney. In tyros-
Diagnosis ........................................................................... 28
inaemia type I, the primary defect is in the last enzyme of
2.7 Diagnostic Flow Chart in the pathway. This enzyme deficiency results in accumula-
Hypertyrosinaemias ......................................................... 28
tion of toxic metabolites, and the hypertyrosinaemia in
2.8 Specimen Collection ......................................................... 29 this disorder is caused by secondary deficiency of
2.9 Prenatal Diagnosis ............................................................ 29 4-hydroxyphenylpyruvate dioxygenase, which also is found
2.10 DNA Testing ...................................................................... 29
in severe liver disease of other causes and in the immature
liver. There is no common phenotype to the different disor-
2.11 Treatment and Monitoring Summary ............................ 29 ders of tyrosine degradation. The occurrence of corneal and
2.12 Summary/Comments ....................................................... 31 skin lesions, as seen in tyrosinaemia type II, is a direct effect
References ...................................................................................... 31 of high tissue tyrosine. Mental retardation and cognitive
impairment is common in tyrosinaemia type II and III. Most
patients with tyrosinaemia type I have normal development
but some reports of developmental delay have appeared.
The liver and kidney diseases of tyrosinaemia type I are
caused by accumulation of toxic metabolites (fumarylaceto-
acetate and its derivatives) and can be prevented by an
inhibitor (nitisinone) of tyrosine degradation at the level of
4-hydroxyphenylpyruvate dioxygenase. In alkaptonuria
there is no increase in tyrosine level and the degradation of
tyrosine proceeds at a normal rate to produce homogentis-
E. Holme (*) ate. Upon oxidation, homogentisate forms reactive interme-
Department of Clinical Chemistry, Sahlgrenska University diates and pigment, which is deposited in various tissues
Hospital, Göthenburg 41345, Sweden particularly in joints and connective tissue. In hawkinsin-
e-mail: elisabeth.holme@clinchem.gu.se
uria, a very rare condition, data based on small numbers of
G.A. Mitchell observations all suggest that an aberrant metabolism of
Service de Génétique Médicale,
4-hydroxyphenylpyruvate leads in some cases to failure to
CHU Sainte-Justine, Université de Montréal,
3175 Côte Sainte-Catherine, Montréal, QC H3T 1C5, Canada thrive, acidosis and excretion of a characteristic metabolite
e-mail: grant.mitchell@recherche-ste-justine.qc.ca pattern.
24 E. Holme and G.A. Mitchell
2.1 Introduction is grossly elevated in an otherwise normal amino acid profile.

The presence of characteristic eye or skin lesions in a hyper-
Hypertyrosinaemia in the newborn is usually not due to tyrosinaemic patient strongly suggests the diagnosis of
inborn errors of tyrosine metabolism, but rather to immaturity TYR2. (Of note, in this situation it is necessary to confirm
of 4HPD (transient tyrosinaemia of the premature newborn). specifically that such a patient is not receiving nitisinone,
At any age, liver dysfunction of any cause or postprandial because subepithelial corneal lesions have been described in
sampling can give mild increases in tyrosine levels. association with hypertyrosinaemia in such patients.)
Sustained isolated hypertyrosinaemia is strongly sugges- In asymptomatic patients referred for hypertyrosinaemia,
tive of an inborn error of tyrosine degradation. discovered by neonatal screening or in the course of an inves-
The phenotype of tyrosinaemia type I (TYR1) is highly tigation for another problem, it is urgent to eliminate type I
variable, and the severity of the disease varies with age at the disease by showing normal liver function and that succinyl-
onset of symptoms (van Spronsen et al. 1994). Newborns acetone is not elevated. After this, the distinction between
detected by neonatal screening are asymptomatic, although tyrosinaemia types II and III may be problematic. The tyro-
elevations of succinylacetone in blood and urine and of sine level in TYR2 tends to be higher, but there is consid-
plasma alpha-foetoprotein are present and most children erable overlap in the plasma tyrosine values seen in these
have mild prolongation of coagulation tests. In nonscreened conditions and plasma tyrosine is not a reliable diagnostic
patients, the most common presentation is severe liver dis- criterion. In practice, in both type II and type III patients, it
ease or failure at 2–4 months of age, often preceded by a seems reasonable to restrict dietary phenylalanine and tyro-
period of nonspecific failure to thrive. Typically there is a sine intake so as to reduce plasma tyrosine concentration.
pronounced coagulopathy with highly increased prothrom- To obtain a specific diagnosis in chronically hypertyros-
bin time and sometimes thrombocytopenia, with a dispropor- inaemic patients without skin or eye signs, molecular testing
tionately moderate increase in transaminases and normal or is the approach of choice, complemented by expression stud-
mildly elevated bilirubin. Plasma α-foetoprotein is markedly ies when required (Huhn et al. 1998). Enzyme diagnosis is
increased. Sepsis is not uncommon. There may be early not a standard practice to perform because the assay of tyro-
signs of hypophosphataemic rickets secondary to renal tubu- sine aminotransferase (TYR2) or of 4-hydroxyphenylpyruvate
lopathy. Plasma tyrosine levels show variable elevations. In dioxygenase (TYR3) requires a liver biopsy. Today, the ther-
most cases there is also a moderate to marked increase in apeutic impact of differentiating between types II and III is
methionine. In the more chronic forms of the disease, symp- minimal. (They respond to similar dietary measures. It can
toms may be subtle. However, there are most often clinical be argued that in theory some pyridoxine-responsive TYR2
and laboratory signs of liver disease, although some patients patients may exist, which could lead to a difference in treat-
present with rickets and few signs of liver disease. Some ment approach.) However, it will likely be useful in the
adolescents and young adults have a predominantly renal future to know the precise diagnosis in order to assess the
presentation with progressive glomerulopathy. There is a effects of treatment and to delineate the phenotypes of these
high risk for hepatocellular carcinoma, which increases with two disorders.
age. The older the patient is at diagnosis, the higher the risk Tyrosinaemia type III (TYR3) has hitherto only been
of malignancy or premalignant changes at diagnosis. A com- reported in nine cases (Ruetschi et al. 2000; Tomoeda et al.
mon complication of TYR1 is occurrence of porphyria-like 2000) and five additional cases have been confirmed in one
neurologic crises characterised by pain in the limbs and author’s laboratory (EH). Patients have been detected
sometimes by paralysis which can require mechanical venti- because of high plasma tyrosine levels in an otherwise nor-
lation but that is reversible. Neurologic crises are caused by mal amino acid profile, performed in the investigation of
elevation of succinylacetone, which is a powerful inhibitor of neurological symptoms, mental retardation or in neonatal
porphobilinogen synthase. screening programmes. Eye or skin lesions have not been
Tyrosinaemia type II (TYR2) is characterised by eye reported. Some degree of mental retardation seems to be
lesions (painful recurrent corneal lesions, frequently with associated with the disorder, although the strong recruitment
dendritic morphology, that may be diagnosed as herpetic bias inherent in the way that the samples were obtained
keratitis if the diagnosis is not suspected), skin disease makes it difficult to conclude with certainty. Enzymatic diag-
(hyperkeratosis of the pressure points of palms, finger pads nosis is not generally performed because it requires a liver
and soles of the feet) and/or developmental delay or intellec- sample. Mutation analysis is possible, and in the near future,
tual deficiency. The disorder usually presents during infancy it may be feasible to complement this with expression stud-
but may become manifest at any age. Tyrosine concentration ies. As discussed above for tyrosinaemia type II, it seems
2 Tyrosine Metabolism 25
reasonable to reduce tyrosine levels by dietary phenylalanine with tyrosinaemia type III and the other p.Asn241Ser
and tyrosine restriction, especially during early childhood. (Item et al. 2007). The latter mutation changes the reaction
Hawkinsinuria (HAWK): This rare and incompletely mechanism of 4HPD so that quinol acetate is formed in place
understood disorder is characterised by failure to thrive and of homogentisate (Brownlee et al. 2010). Quinol acetate
acidosis in some affected infants. No symptoms have been reacts readily with thiols to form hawkinsin.
reported after infancy. Autosomal dominant transmission is Alkaptonuria (AKU): The earliest physical sign of alkap-
described. A slight increase in plasma tyrosine has been tonuria is abnormal darkening of urine on standing. This sign
noted in a minority of patients but this is not a good diagnosis frequently overlooked by parents and often not explored
tic marker. The diagnosis is based on identification of by physicians. It should lead to investigation of the excretion
hawkinsin (2-cystenyl-1,4-dihydroxycyclohexenylacetate), of homogentisate. The finding is most likely to be noticed in
formed from a reactive tyrosine metabolite that has been diapers and is typically not detectable in older patients who
detoxified by reaction with glutathione. Depletion of gluta- live in areas with modern plumbing. Symptoms like patchy
thione and the resulting high excretion of 5-oxoproline is greyish pigmentation of the sclera, a blue-grey aspect to the
probably responsible for the acidosis in hawkinsinuria. cartilage of the ear and arthritis, most often in the hip and
Hawkinsin is ninhydrin positive and can be identified by knee, do not appear until adulthood. Periods of acute inflam-
analysis of urinary amino acids (Lehnert et al. 1999). After mation may resemble rheumatoid arthritis. The arthritis may
infancy 4-hydroxycyclohexylacetate appears in urine in be severe and disabling in middle-aged adults. Ankylosis of
addition to hawkinsin. 4-Hydroxycyclohexylacetate is the lumbosacral region is common. The specific diagnosis
detected by analysis of urinary organic acids and analysis of may be suggested by radiologists who note reduced interver-
parents to a sick infant may help in the diagnostic work-up tebral spacing in the lumbar spine, with calcification of the
(Niederwieser et al. 1978). The condition has responded to intervertebral disc and marked degenerative changes in the
restriction of dietary protein. A fascinating patient was shoulder and hip joints. Diagnosis is confirmed by identifica-
recently described, a genetic compound of two 4HPD mutation of homogentisate in urine, often present in millimolar
tions, one an inactivating mutation known to be associated amounts.
2.2 Nomenclature
Gene Chromosomal OMIM

No. Disorder Alternative name Abbreviation symbol localisation Affected protein No. Subtype
2.1 Tyrosinaemia Fumarylacetoacetase TYR1 FAH 15q25.1 Fumarylacetoacetase 276700 All forms
type I deficiency
2.2 Tyrosinaemia Tyrosine aminotransferase TYR2 TAT 16q22.2 Tyrosine aminotransferase 276600 All forms
type II deficiency
2.3 Tyrosinaemia 4-hydroxyphenylpyruvate TYR3 HPD 12q24.31 4-hydroxyphenylpyruvate 276710 All forms
type III dioxygenase deficiency dioxygenase
2.4 Hawkinsinuria 4-hydroxyphenylpyruvate HAWK HPD 12q24.31 4-Hydroxyphenylpyruvate 140350 1 case
dioxygenase change of hydroxylase
function
2.5 Alkaptonuria Homogentisate 1,2 – AKU HGD 3q13.33 Homogentisate 203500 All forms
dioxygenase deficiency 1,2 – dioxygenase
Fig. 2.1 Tyrosine degradation TYROSINE

pathway. Diagnostically
important metabolites are framed. 2.2
The sites of known metabolic
disorders are indicated as filled
4-OH-phenyllactate 4-OH-phenylpyruvate 4-OH-phenylacetate
boxes. 2.1 fumarylacetoacetase,
2.2 tyrosine aminotransferase, 2.3
2.3; 2.4
4-hydroxyphenylpyruvate
NTBC
dioxygenase, 2.5 homogentisate
dioxygenase. Inhibition by Homogentisate
succinylacetone and nitisinone
(NTBC) are indicated by crosses. 2.5
5-ALA 5-aminolevulinate
Maleylacetoacetate
Succinylacetoacetate
CO2 Fumarylacetoacetate
Succinylacetone
2.1
Fumarate + Acetoacetate
5-ALA Porphobilinogen
Table 2.1 Tyrosinaemia type I

CNS Neuropathy − ± ± ± ±
Porphyria-like neurological crisis − ± ± ± ±
Digestive Liver carcinoma, hepatocellular − ± + + +
Liver failure, acute ± ± − − −
Musculoskeletal Rickets − − ± ± ±
Renal Nephrocalcinosis − − − ± ±
Renal enlargement ± + + − −
Renal failure, chronic − − − ± ±
Renal tubulopathy ± + + + +
Special laboratory 4-Hydroxyphenylacetate (U) ↑ ↑ ↑ ↑ ↑
4-Hydroxyphenyllactate (U) ↑ ↑ ↑ ↑ ↑
4-Hydroxyphenylpyruvate (U) ↑ ↑ ↑ ↑ ↑
5-Aminolevulinic acid (U) ↑ ↑ ↑ ↑ ↑
Alpha-foetoprotein (S) ↑↑ ↑↑ ↑ (↑) (↑)
Methionine (P) (↑) (↑) (↑) – –
Porphobilinogen synthase (RBC) ↓ ↓ ↓ ↓ ↓
Succinylacetone (U, P)a ↑ ↑ ↑ ↑ ↑
Tyrosine (P) ↑ ↑ ↑ ↑ ↑
Succinylacetone is measured rapidly in order to exclude tyrosinaemia type I. Decreased activity of porphobilinogen synthase activity in RBC is a
sensitive and easily performed test that reflects increased plasma succinylacetone concentrations and may serve as a convenient first line diagnostic
test before the levels of succinylacetone and related metabolites are available. Of note, increased excretion of phenolic tyrosine metabolites is
present in sustained hypertyrosinaemia of any cause and is of no differential diagnostic value
a
Succinylacetone and/or succinyl acetoacetate and/or 4-oxo-6-hydroxyheptanoate (U)
Table 2.2 Tyrosinaemia type II

CNS Mental retardation − ± ± ± ±
Dermatological Blisters, erosion, hyperkeratosis on palms and soles − − ± ± ±
Eye Corneal erosion − ± + + +
Lacrimation − + + + +
Photophobia − ± + + +
Special laboratory 4-Hydroxyphenylacetate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
4-Hydroxyphenyllactate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
4-Hydroxyphenylpyruvate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Tyrosine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 2.3 Tyrosinaemia type III

CNS Mental retardation − ± ± ± ±
Special laboratory 4-Hydroxyphenylacetic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
4-Hydroxyphenyllactate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
4-Hydroxyphenylpyruvate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Tyrosine (P) ↑↑ ↑↑ ↑↑ ↑↑ ↓↓
Table 2.4 Hawkinsinuria

Digestive Unspecified hepatopathy − + − − −
Other Failure to thrive, acidosis − + − − −
Special 4-Hydroxycyclohexylacetate (U) − − ↑ ↑ ↑
laboratory 4-Hydroxyphenylacetate (U) − ↑ − − −
4-Hydroxyphenyllactate (U) − ↑ − − −
4-Hydroxyphenylpyruvate (U) − ↑ − − −
5-Oxoproline (U) − ↑ − − −
Hawkinsin (U) − ↑ ↑ ↑ ↑
Tyrosine (P) n (↑)a n n n
a
Reported in a single patient
Table 2.5 Alkaptonuria

Cardiovascular Mitral and aortic valvulitis − − − − ±
Dermatological Pigmentation − − − − ±
Eye Scleral pigmentation − − − − +
Musculoskeletal Arthritis − − − − +
Lumbosacral disc degeneration − − − − +
Ochronosis − − − − +
Other Urine darkening on standing + + + + +
Special laboratory Homogentisate (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Succinylacetone Porphobilinogen Hawkinsin Homogentisate

Age Tyr (P) Met (P) activitya (P) synthase (RBC)b Succinylacetone (U) 5-Aminolevulinate (U) (U) (U)
Years μmol/L nkat/g Hgb mmol/mol creatinine in random samples
Newborn 50–150 10–60 <0.1 0.58–1.25 <0.1 <20 n.d. <1
1–12 30–130 10–50 <0.1 0.58–1.25 <0.1 <12 n.d. <1
>12 30–100 10–40 <0.1 0.58–1.25 <0.1 <3 n.d. <1
a
Enzymatic method. Inhibition of porphobilinogen synthase by boiled plasma given in the equivalent succinylacetone concentration
b
Enzymatic method. Reference values vary with the methodology
2.6 Pathological Values/Differential

Diagnosis
Porphobilinogen
Succinylacetone synthase activity Succinylacetone 5-Aminolevulinate Hawkinsin Homogentisate
No Disorder Tyr (P) Met (P) activity (P) (RBC) (U) (U) (U) (U)
% of Normal
μmol/L mean mmol/mol Creatinine in random samples
2.1 Tyrosinaemia I 150–1,300 20–1,300 0.5 to >100 1–50 0.5 to >1,000 20 to >100 nd <1-trace
2.2 Tyrosinaemia II 800 to >2,000 Normal <0.1 Normal <1 Normal nd <1
2.3 Tyrosinaemia III 500–1,200 Normal <0.1 Normal <1 Normal nd <1
2.4 Hawkinsinuria Normal– Normal – – <1 – 200–2,000 –
moderate
increase
2.5 Alkaptonuria Normal Normal Normal Normal Normal Normal nd >1,000
Increased tyrosine concentration is caused by inborn or of TYRI is caused by secondary deficiency of

acquired deficiency of the first two enzymes of the tyrosine 4-hydroxyphenylpyruvate dioxygenase)
degradation pathway (the increased tyrosine concentration
2.7 Diagnostic Flow Chart

in Hypertyrosinaemias
Diagnostic flow chart in hypertyrosinemias
Clinical situation Symptoms and signs Diagnosis
Neonatal screening asymptomatic Tyrosinemia type I if succinylacetone
(Succinylacetone) mild coagulopathy confirmed
Neonatal screening Transaminases (±) Tyrosinemia type I
(Hypertyrosinemia) α-Fetoprotein (+) (tyr(P) 120-1300 μmol/L)
Prothrombine time (±)
Porphobilinogen synthase
deficiency (+)
Succinylacetone (+)
Persistent No liver disease Tyrosinemia type II

Succinylacetone (-) (tyr(P) 800->2000 μmol/L
Tyrosinemia type III
(tyr(P) 500-1300 μmol/L)
↓
Mutation analysis
(Rarely, liver enzyme assay)
Failure to thrive Signs of liver disease (+) Tyrosinemia type I

Succinylacetone (+)
Acidosis (+) Hawkinsinuria

Hawkinsin (+)
5-oxoproline (+)
Liver disease Prothrombin time (+) Tyrosinemia type I

α-Fetoprotein (+)
Bilirubin (normal-100 μmol/L)
Succinylacetone (U)(+)
Rickets Hypophosphatemia (+) Tyrosinemia type I
General aminoaciduria (+)
Complete Fanconi syndrome (+)
Signs of liver disease (+)
Succinylacetone (U) (+)
Fig. 2.2 Clinical and biochemical signs Mental retardation Eye and/or skin symptoms (+) Tyrosinemia type II
suggestive of hereditary tyrosinaemias No other symptoms Tyrosinemia type II or III
Test Precondition Material Handlinga Pitfalls

Tyr (P) – Plasma Ambient temp Liver disease, any cause; false negatives in diet
treated patients
Met (P) – Plasma Ambient temp Liver disease, any cause
4-Hydroxyphenylpyruvate, – Urine Ambient temp Nonspecific elevations in liver disease of any cause.
4-Hydroxyphenyllactate and Urinary levels highly variable with diet and not
4-Hydroxyphenylacetate generally useful for patient follow-up
Succinylacetone (P) – Plasma (heparin) Ambient temp Slight increase in severe liver disease; possible
false positive on dichloroacetate, treatment
Porphobilinogen synthase (RBC) – Heparinised blood Ambient temp May be close to normal in TYR1
Primary deficiency of porphobilinogen synthase
Succinylacetone (U) – Urine Ambient temp False negatives possible in dilute urine specimens
(Frozen −20 °C) with some techniques
Hawkinsin – Urine Frozen −20 °C Failure to recognise hawkinsin and related
metabolites if the specific diagnosis is not
suggested on requisition
Homogentisate – Urine Frozen −20 °C –
FAH activity – Fibroblasts Ambient temp False positive: Pseudodeficiency
Lymphocytes Frozen (−70 °C) False negative: Back mutation with significant FAH
Liver Frozen (−70 °C) activity in revertant areas (common in liver).
Insensitivity: very low normal activity in most
available extrahepatic tissues renders enzymatic
diagnosis difficult
TAT activity – Liver Frozen (−70 °C) False positive: Highly regulated enzyme with wide
normal range of activity depending on
physiological state
HPD activity – Liver Frozen (−70 °C) False positive: Secondary deficiency in cirrhotic
liver.
Late maturation prenatally; may be low in
premature infants
In general plasma and urine are stored frozen until assay. 2.10 DNA Testing
One author (EH) has found that untreated blood and urine
samples retain acceptable quality if transported at ambient DNA analysis is available for all diseases of tyrosine degra-
temperature by same day or overnight courier. This offers the dation. Standard molecular diagnostic procedures can be
additional advantage of providing cells for measurement of applied, using genomic DNA of any source. If the patient
porphobilinogen synthase activity comes from a background with a known ethnic founder
effect or if the mutation for which he or she is at risk is
known, this can be tested directly. Otherwise, gene sequenc-
2.9 Prenatal Diagnosis ing and, if necessary, deletion/duplication analysis are the
methods of choice.
DNA analysis is the preferred method for prenatal diagnosis
of all diseases of tyrosine degradation. The mutation of each
biological parent must be known before proposing this tech- 2.11 Treatment and Monitoring Summary
nique. Molecular diagnosis can be performed on cells
obtained directly or by culture after amniocentesis, from Diet Therapy
chorionic villus samples or at the preimplantation stage. In all forms of hypertyrosinaemia, a mainstay of treatment is
Measurement of succinylacetone in amniotic fluid is an dietary restriction of phenylalanine and tyrosine intake, plus
acceptable approach to diagnosis of tyrosinaemia type I for provision of adequate amounts of other nutrients in a palat-
couples in whom the causal mutations are not known. Rare able form. Close supervision is necessary in order to avoid
affected pregnancies with normal amniotic fluid succinylac- dietary deficiencies. Compliance is an important long-term
etone levels have been reported. issue, especially because hypertyrosinaemia itself does not
directly confer a sense of discomfort. In TYR1, expert guide- Patients who develop findings suggestive of hepatic
lines have recently suggested that plasma tyrosine be main- malignancy such as hepatic nodules, particularly if these
tained below 400 or 500 μmol/L for plasma tyrosine (De abnormalities are accompanied by an increase in alpha-
Laet et al. 2013). This is viewed as reasonable by expert con- foetoprotein, should be considered for possible hepatic trans-
sensus but formal data to support these suggestions are lack- plantation. In rare patients with liver and kidney failure,
ing. In types II and III, the ideal levels of plasma tyrosine and combined transplantation of liver and kidney has been per-
phenylalanine are even less established. Eye and skin lesions formed with success.
have rarely been seen in TYR2 patients with plasma tyrosine
level <800 μmol/L, suggesting that levels should be main- TYR2 and TYR3
tained below this level. However, the repeated observation of These patients are offered dietary treatment as above.
developmental delay in many TYR2 patients and some Cutaneous and ocular complications are usually rapidly
TYR3 patients suggests that a lower level, perhaps 400– reversed when dietary control of plasma tyrosine is achieved.
500 μmol/L, may be appropriate. Symptomatic treatment can provide some immediate benefit.
It is reasonable to consider that the developmental delay
TYR1-Nitisinone Treatment seen in some TYR2 and TYR3 patients may be related to
Nitisinone combined with dietary therapy is the medical increased levels of plasma tyrosine. Controlled trials in large
treatment of choice for patients with TYR1 (Holme and numbers of patients are not available at present and will not be
Lindstedt 1998). To date, after over 15 years of neonatal available in the near future because of the rarity of these condi-
screening and early treatment with nitisinone, no instance of tions and the lack of consistent documentation of outcome.
liver cancer has occurred in patients detected by neonatal This issue should be discussed with patients and families, and
screening and treated with nitisinone rapidly thereafter treatment should be offered accordingly. The authors treat
(Larochelle et al. 2012). Late-treated patients are at greater patients with these conditions in a similar fashion to the rec-
risk of liver cancer, but no acute episodes of liver failure or ommendations for nitisinone-treated TYR1 patients (De Laet
neurological crises occurred during nitisinone treatment. et al. 2013). For all such patients, developmental assessment
Nitisinone has a long half-life in adults (54 h), permitting should be performed and plasma tyrosine and phenylalanine
twice or even once daily administration. A recent consensus levels should be monitored regularly in order that evidence-
statement (de Laet et al. 2013) recommended an initial dose of based conclusions regarding development and metabolic con-
1 mg/kg/day. The dose thereafter is titrated individually, by trol can be attained in the future. If developmental delay is
measuring the suppression of the level of succinylacetone and/ present, appropriate educational help is offered.
or the plasma level of nitisinone. As for the level of plasma
tyrosine and phenylalanine, there is no universal agreement Alkaptonuria (AKU)
about the optimal level of nitisinone. Some authors have Currently most treatment of AKU is symptomatic, concen-
reported that levels as low as 30 μmol/L may suffice in some trating upon the relief of the symptoms of arthritis and joint
patients, whereas others have noted increased succinylacetone pain. Preliminary studies have shown a marked reduction in
excretion at levels below 50–60 μmol/L, which probably is the homogentisate excretion in patients who receive even small
level required for complete block of tyrosine degradation. doses of nitisinone. It must be recalled that nitisinone treat-
Patients feel well on doses of nitisinone that provide subopti- ment for alkaptonuria would imply the development of
mal control and modestly elevated levels of succinylacetone. hypertyrosinaemia. However, in adults on a small dose of
This situation presumably reflects a state of increased liver nitisinone, a normal or a slightly protein-restricted diet is
cancer risk. It is important to monitor dose and compliance sufficient to maintain plasma tyrosine below 800 μmol/L
clinically and by repeated laboratory measurements. (personal observation (EH)). It is not known when or to
For TYR1 patients, monitoring of biochemical control what extent homogentisate has to be reduced in order to pre-
(plasma amino acids, succinylacetone, nitisinone), liver vent the major complications of alkaptonuria, but if nitisi-
function (coagulation, alpha-foetoprotein) and imaging none treatment is started in infancy or childhood, long-term
(magnetic resonance and ultrasound) are performed regu- dietary therapy with phenylalanine and tyrosine restriction
larly. Organs that are less frequently affected in TYR1 are as described above would have to be considered.
tested at greater intervals (cardiac ultrasound, glomerular fil-
tration rate). Although monitoring must be individualised, in Hawkinsinuria (HAWK)
one author’s centre (GM), for patients who are otherwise Reported patients have become asymptomatic after infancy.
well, biochemical monitoring is performed a minimum of Patients should be given symptomatic treatment for acidosis.
every 3 months, ultrasound imaging is obtained every Temporary phenylalanine and tyrosine restriction could be
6 months and magnetic resonance testing every 12 months. considered in symptomatic patients.
Emergency Treatment (TYR1) hypertyrosinaemia. Expanded newborn screening does not

Nitisinone should be given as an emergency treatment to currently detect most of the inborn errors of tyrosine degra-
infants with unexplained liver failure in whom TYR1 has not dation and a high index of suspicion by clinicians and diag-
been eliminated. Nitisinone is available only for oral admin- nostic laboratory personnel is necessary to ensure early
istration. In acutely ill patients with suspected or proven diagnosis and therapy.
TYR1, all efforts should be made to administer nitisinone
immediately, as soon as adequate samples for diagnostic
confirmation have been obtained.
Acutely ill infants with acute liver failure and TYR1 are References
treated in the intensive care unit and receive general support-
Brownlee JM, Heinz B, Bates J, Moran GR (2010) Product analysis and
ive therapy. Protein intake should be restricted/stopped ini-
inhibition studies of a causative Asn to Ser variant of
tially and efforts should be made to reverse the catabolic 4-hydroxyphenylpyruvate dioxygenase suggest a simple route to the
state. Sepsis should be considered and the risk of bleeding treatment of Hawkinsinuria. Biochemistry 49:7218–7226
should be diminished as necessary by replacement of De Laet C, Dionisi-Vici C, Leonard JV, McKiernan P, Mitchell G,
Monti L, de Baulny HO, Pintos-Morell G, Spikerkötter U (2013)
coagulation proteins.
Recommendations for the management of tyrosinemia type 1.
Some severely ill TYR1 patients do not have a rapid Orphanet J Rare Dis. doi:10.1186/1750-1172-8-8
clinical response to nitisinone treatment. In these infants Holme E, Lindstedt S (1998) Tyrosinaemia type I and NTBC (2-(2-nitro-
there may be no reversal of the coagulopathy after several 4-trifluoromethylbenzoyl)-1,3-cyclohexanedione). J Inherit Metab
Dis 21:507–517
days and increasing jaundice; hepatic encephalopathy may
Huhn R, Stoermer H, Klingele B, Bausch E, Fois A, Farnetani M, Di
develop. In such patients, urgent liver transplantation is Rocco M, Boue J, Kirk JM, Coleman R, Scherer G (1998) Novel
required for survival. and recurrent tyrosine aminotransferase gene mutations in tyrosin-
Acute porphyria-like neurologic crises in the non- emia type II. Hum Genet 102(3):305–313
Item CB, Mihalek I, Lichtarge O, Jalan A, Vodopiutz J, Muhl A,
nitisinone-treated TYR1 patient are a medical emergency.
Bodamer OA (2007) Manifestation of hawkinsinuria in a patient
Nitisinone is administered immediately. Anabolism is compound heterozygous for hawkinsinuria and tyrosinemia III. Mol
encouraged by nutrition and treatment of concurrent compli- Genet Metab 91:379–383
cations like infections. As for other hepatic porphyrias, treat- Larochelle J, Alvarez F, Bussières JF, Chevalier I, Dubois J, Faucher F,
Fenyves D, Gooyer P, Grenier A, Holme E, Laframboise R, Lambert
ment with barbiturates and other compounds that stimulate
M, Lindstedt S, Maranda B, Melançon S, Merouani A, Mitchell J,
cytochrome P450 is carefully avoided for several days after Parizeault G, Pelletier L, Phan V, Rinaldo P, Scott CR, Scriver C,
receiving the first dose of nitisinone. Mitchell GA (2012) Effect of nitisinone (NTBC) treatment on the
clinical course of hepatorenal tyrosinemia in Québec. Mol Genet
Metab 107:49–54
Lehnert W, Stögmann W, Engelke U, Wevers RA, van den Berg GB
2.12 Summary/Comments (1999) Long-term follow up of a new case of hawkinsinuria. Eur J
Pediatr 158:578–582
There are five defined inborn errors of tyrosine metabolism: Lindstedt S, Holme E, Lock EA, Hjalmarson O, Strandvik B (1992)
Treatment of hereditary tyrosinaemia type I by inhibition of
TYR1, TYR2, TYR3, HAWK and AKU. The most common
4-hydroxyphenylpyruvate dioxygenase. Lancet 340:813–817
and serious of these disorders is TYR1. For this disorder effi- Niederwieser A, Wadman SK, Danks DM (1978) Excretion of cis- and
cient drug treatment is available with nitisinone, based on trans-4-hydroxycyclohexylacetic acid in addition to hawkinsin in a
inhibition of tyrosine degradation at a level prior to the for- family with a postulated defect of 4-hydroxyphenylpyruvate dioxy-
genase. Clin Chim Acta 90:195–200
mation of hepatotoxic metabolites (Lindstedt et al. 1992).
Ruetschi U, Cerone R, Perez-Cerda C, Schiaffino MC, Standing S,
Early institution of nitisinone therapy is desirable, before Ugarte M, Holme E (2000) Mutations in the 4-hydroxyphenylpyruvate
complications occur. Succinylacetone is the most sensitive dioxygenase gene (HPD) in patients with tyrosinemia type III. Hum
and specific marker for neonatal screening of TYR1 and this Genet 106:654–662
Tomoeda K, Awata H, Matsuura T, Matsuda I, Ploechl E, Milovac T,
is now practised in many countries. The outcome in tyros-
Boneh A, Scott CR, Danks DM, Endo F (2000) Mutations in the
inaemia type I patients detected by neonatal screening and 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for
treated early with nitisinone is very promising to date tyrosinemia type III and hawkinsinuria. Mol Genet Metab
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van Spronsen FJ, Thomasse Y, Smit GP, Leonard JV, Clayton PT, Fidler
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V, Berger R, Heymans HS (1994) Hereditary tyrosinemia type I: a
Controlled dietary restriction of tyrosine and phenylalanine new clinical classification with difference in prognosis on dietary
is a reasonable approach to all forms of hereditary treatment. Hepatology 20:1187–1191
Sulphur Amino Acids
3
Ivo Barić and Brian Fowler
Contents Summary
3.1 Introduction...................................................................... 33 The conversion of methionine to inorganic sulphate involves
3.2 Nomenclature ................................................................... 35 the formation of homocysteine involving transmethylation fol-
lowed by transsulphuration. Several inherited enzyme deficien-
3.3 Metabolic Pathway .......................................................... 35
cies within this pathway have been described. Those causing
3.4 Signs and Symptoms ........................................................ 37 hypermethioninaemia may be confused with the many known
3.5 Diagnosis ........................................................................... 37 secondary causes of increased methionine demanding diagnos-
3.6 Reference Values .............................................................. 40
tic expediency. Most of the disorders have been described in
only a few patients so that the full clinical spectrum of these is
3.7 Pathological Values .......................................................... 40 not known. Exceptions are methionine adenosyltransferase
3.8 Specimen Collection ........................................................ 41 deficiency which is mainly but not always a benign disorder and
3.9 Treatment Summary........................................................ 41 cystathionine ß-synthase deficiency which causes classical
homocystinuria and is the second most common amino acid
3.10 Emergency Treatment ..................................................... 41
disorder in some populations. While methionine adenosyltrans-
3.11 Standard Treatment ........................................................ 42 ferase deficiency is only in some patients symptomatic causing
3.12 Experimental Treatment ................................................. 44 different neurological problems and glycine N-methyltransferase
3.13 Follow-Up and Monitoring ............................................. 45
deficiency affects liver function, other diseases causing hyper-
methioninaemias are often associated with a multisystem dis-
References .................................................................................... 45
ease of varying severity and progression. The enzymes and
genes have been characterised for each of the known disorders.
Besides sulphite oxidase deficiency, for which sulphocysteine
and sulphite analysis is crucial, diagnosis centres on measure-
ment of plasma and urine amino acids and total homocysteine
backed up by determination of adenosylmethionine and adeno-
sylhomocysteine with confirmation by enzyme assay and muta-
tion analysis. Treatment depends mostly on dietary restriction
of precursors and substitution of essential products. In particu-
lar about half of patients with cystathionine synthase deficiency
respond to varying degree to pharmacologic doses of vitamin
B6. Existing clinical abnormalities may not fully resolve, and
I. Barić (*)
long-term outcome can be poor. Prenatal diagnosis can be reli-
Division for Metabolic Diseases,
Department of Pediatrics, University Hospital Center Zagreb and ably performed in those disorders where this is indicated.
University of Zagreb, School of Medicine,
Kispaticeva 12, Rebro, Zagreb 10000, Croatia
e-mail: ivo.baric@zg.t-com.hr
3.1 Introduction
B. Fowler
Division for Metabolic Diseases,
Sulphur-containing amino acids include methionine,
University Children’s Hospital, Steinwiesstrasse 75,
Zürich 8032, Switzerland homocysteine, cystathionine, cysteine and taurine. Related
e-mail: brian.fowler@kispi.uzh.ch inherited disorders include deficiencies of enzymes in the
34 I. Barić and B. Fowler
transsulphuration pathway that converts sulphur from methi- MAT I/III deficiency is asymptomatic in the vast majority
onine via homocysteine and cysteine to sulphate and in the of patients. However, hypermethioninaemia, the biochemical
remethylation of homocysteine to methionine (Fig. 3.1). hallmark of this disease, if very high, is itself associated with
Remethylation defects, disorders of renal tubular transport (cys- increased risk of various neurological problems regardless of
tinuria), lysosomal transport of cystine (cystinosis) and molyb- the primary cause (Braverman et al. 2005; Mudd et al. 2003)
denum cofactor deficiency are described in other chapters of (see Sect. 3.4). The most characteristic brain imaging
this book. Although not primarily a disorder of sulphur-con- changes are demyelination with oedema of subcortical and
taining amino acids, adenosine kinase deficiency disrupts the deep white matter, more pronounced in dorsal brain stem and
methionine cycle, and it is, therefore, included in this chapter. resulting in separation of myelin layers—the so-called vacu-
All disorders described in this chapter are autosomal olating myopathy (Braverman et al. 2005; Chamberlin et al.
recessively inherited. The only exception is that autosomal 1996; Devlin et al. 2004). Patients with no residual activity,
dominant inheritance has also been described for methionine whose plasma methionine is higher, frequently above
adenosyltransferase (MAT) deficiency, mostly when caused 1,000 μmol/L, have increased risk for the aforementioned
by the R264H mutation, which has a dominant negative clinical problems. This concentration of methionine may
effect on the wild type allele (Chamberlin et al. 1997). The serve as a rough indicator of possible clinical problems.
true incidence of sulphur-containing amino acid disorders is Glycine N-methyltransferase (GNMT) deficiency has
not known. Reported incidences vary greatly due to different been so far described in only three children (Mudd et al.
ethnic backgrounds, consanguinity rate and are probably too 2001b; Augoustides-Savvopoulou et al. 2003). Two of them
low because of suboptimal cut-off values for methionine in were sibs and had mild to moderate hepatomegaly, while the
neonatal screening programmes. Lowering of the cut-off to third one was asymptomatic. Their aminotransferase activi-
the 99th centile resulted in much higher incidence than ties ranged from borderline to fivefold increased. Liver biop-
expected and suggested that the proportion of milder cases is sies showed almost normal finding and mild fibrosis,
higher than expected. Current efforts to combine methionine respectively. So far the patients have remained clinically well
and total homocysteine in neonatal screening programmes during follow-up (Mudd 2011).
may generate more reliable data on incidence. S-adenosylhomocysteine hydrolase deficiency has been
Pathophysiology of disorders of sulphur-containing proven so far in nine patients from five families (Barić et al.
amino acids is complex, only partly understood and depends 2004, 2005; Buist et al. 2006; Grubbs et al. 2010). Two sibs
on widely varying metabolic consequences of the individual had foetal hydrops, liver synthetic failure and muscular
enzyme deficiencies (Mudd et al. 2001a). In disorders asso- hypotonia leading to respiratory failure and death in early
ciated with hypermethioninaemia, very high values can be infancy. They also showed brain abnormalities including cer-
harmful themselves, primarily for the brain. In MAT defi- ebellar and pontine hypoplasia, hypoplastic corpus callosum
ciency, low adenosylmethionine (AdoMet) and subsequent and hypomyelination. Muscle disease with high creatine
deficient methylation could be a contributing factor. It is pos- kinase was present also in another seven patients with milder
sible that mild to moderate hyperhomocysteinaemia, found phenotype. They also had in various combinations develop-
in many patients with MAT deficiency, may be harmful in mental delay, behavioural abnormalities, myelination delay,
the long term. In adenosylhomocysteine (AdoHcy) hydro- strabismus, coagulopathy and liver disease.
lase deficiency, high values of AdoHcy inhibit numerous Cystathionine beta-synthase (CBS) deficiency is charac-
methyltransferases with very variable clinical consequences. terised primarily by increased risk of thrombosis and embo-
This mechanism could also be important in classical homo- lism, osteoporosis and other skeletal abnormalities,
cystinuria, where a major pathogenetic mechanism seems to developmental delay, mental insufficiency, myopia and lens
be elevation of homocysteine with its adverse effect on con- dislocation (Mudd et al. 1985). Clinical variability is signifi-
nective tissue, lens, coagulation, vessels and secondary to cant and seems to depend on the homocysteine concentra-
vascular changes on many other tissues. Clinical conse- tion. Some patients may present with a thromboembolic
quences of sulphite oxidase deficiency are likely due to toxic event after a long asymptomatic period (de Franchis et al.
effects of sulphite, S-sulphocysteine and thiosulphate on 1998; Mudd 2011).
brain. Several putative pathogenetic mechanisms of adenos- Isolated sulphite oxidase deficiency is characterised by
ine kinase deficiency are related to increased adenosine and refractory convulsions starting in the neonatal or early infan-
its various toxic effects on mammalian cells. Another mech- tile period, severe psychomotor retardation and early death.
anism could be inhibition of numerous methyltransferases Lens dislocation occurs after the neonatal period. Milder and
caused by secondary elevation of S-adenosylhomocysteine. late-onset cases have been reported. Severity of the clinical
Clinical presentation can occur at any age and varies course depends primarily on the nature of mutations and
widely in its severity. Cystathionase deficiency is probably residual activity, but other factors may be important, as well
benign. (Tan et al. 2005).
3 Sulphur Amino Acids 35
Adenosine kinase deficiency has been described so far neurological symptoms or muscle disease or liver disease or
in only six patients from three families. All had severe any other symptom attributable to diseases from this group
developmental delay, early onset epilepsy and liver dis- (see Sect. 3.4). Measurement of plasma total homocysteine
ease characterised mostly by cholestasis. All had fron- and amino acids (methionine, S-sulphocysteine) should be
tal bossing and all but one macrocephalus. Three patients sufficient as the first step to detect all diseases from this
had cardiac anomalies and two had hearing impairment group. The exception could be only AdoHcy hydrolase defi-
(Bjursell et al. 2011). ciency, where hypermethioninaemia is not always/necessary
Since most of the mentioned diseases are at least partly present. For the same reasons, all patients with unexplained
treatable if diagnosed early and may have rapid course, the hypermethioninaemia and/or hyperhomocysteinaemia need
diagnostic work-up in suspected cases should also be rapid. to be rapidly and carefully evaluated (see Diagnostic flow
Suspicion should be raised in all patients having unexplained chart, Fig. 3.2 and Fig. 3.3).
3.2 Nomenclature

No. Disorder Alternative name Abbreviation symbol localisation Affected protein No. Subtype
3.1 Methionine MAT deficiency MAT I/III MAT1A 10q22 Methionine 250850 All forms
adenosyltransferase adenosyltransferase
I/III deficiency
3.2 Glycine GNMT deficiency GNMT GNMT 6p12 Glycine 606664 All forms
N-methyltransferase N-methyltransferase
deficiency
3.3 S-adenosylhomocysteine SAHH deficiency AHCY AHCY 20q11.22 Adenosylhomocysteinase, 613752 All forms
hydrolase deficiency S-adenosylhomocysteine
hydrolase
3.4 Cystathionine beta- Classical CBS CBS 21q22.3 Cystathionine 263200 All forms
synthase deficiency homocystinuria beta-synthase
3.5 Cystathionase Cystathionine CTH CTH 1p31.1 Cystathionine 219500 All forms
deficiency gamma-lyase gamma-lyase
deficiency
3.6 Sulphite oxidase Isolated sulphite SUOX SUOX 12q13.13 Sulphite oxidase 272300 Isolated
deficiency oxidase deficiency
3.7 Adenosine kinase Hypermethioninaemia ADKD ADK 10q22.2 Adenosine kinase 614300 All forms
deficiency due to adenosine
kinase deficiency
3.3 Metabolic Pathway transsulphuration pathway takes over whereby homocys-

teine is condensed with serine to form cystathionine via a
Methionine metabolism is summarised in Fig. 3.1. reaction catalysed by the pyridoxal-phosphate-requiring
Methionine, homocysteine and cysteine are linked by the cystathionine ß-synthase. Cystathionine is cleaved to cys-
methylation cycle (see Chap. 13 for details) and the trans- teine and α-ketobutyrate by another pyridoxal-phosphate-
sulphuration pathway. The essential amino acid methio- dependent enzyme, γ-cystathionase. The first step in the
nine is derived from the diet or catabolism of proteins. It subsequent oxidation of the sulphur atom of cysteine is its
is first converted to S-adenosylmethionine by methionine- conversion to cysteine sulphinic acid by cysteine dioxy-
S-adenosyltransferase. S-adenosylmethionine is the methyl- genase followed by transamination with α-oxoglutarate
group donor in a wide range of transmethylation reactions, yielding pyruvate and sulphite. The oxidation of sulphite
for example, glycine N-methyltransferase. These reactions to sulphate is catalysed by the molybdenum-containing
yield S-adenosylhomocysteine, which is a strong inhibi- enzyme sulphite oxidase. Cysteine sulphinic acid can also
tor of transmethylation reactions and must be cleaved to be decarboxylated by a decarboxylase forming hypotau-
adenosine and homocysteine by S-adenosylhomocysteine rine which is then oxidised by the NAD-requiring enzyme,
hydrolase. Depending on a number of factors, about 50 % hypotaurine:NAD+oxidoreductase resulting in the forma-
of available homocysteine is recycled into methionine by tion of taurine, an important amino acid with neurotrans-
the remethylation cycle. Alternatively, the degradative mitter function.
Fig. 3.1 Pathway of methionine Proteins

metabolism. MAT I/III methionine Methionine
adenosyltransferase, X-MT MAT I/III (3.1)
S-adenosylmethionine-dependent
transmethylation reactions, GNMT S-adenosyl-methionine
glycine methyltransferase, AHCY Methylation
MS Glycine (DNA,RNA
S-adenosylhomocysteine X-MT
hydrolase, BMT betaine- GNMT (3.2) Hormones
BMT MeCbl
homocysteine methyltransferase, Sarcosine Lipids
MS 5-methyltetrahydrofolate- Proteins
S-adenosyl-homocysteine
Creatine-P
homocysteine methyltransferase;
AHCY (3.3) etc.)
ADKD adenosine kinase, CBS,
cystathionine ß-synthase, CTH Betaine
Homocysteine Adenosine
cystathionine γ-lyase, CyD
cysteine dioxygenase; CySD Serine
CBS (3.4) PLP ADKD (3.7) AMP
cysteine sulphinate decarboxylase,
HTOx hypotaurine:
Cystathionine
NAD+oxidoreductase, CSAT
cysteine sulphinate α-oxoglutarate CTH (3.5) PLP
aminotransferase, SUOX sulphite CySD HTOx
oxidase; MeCbl methylcobalamin, Cysteine-
Cysteine Hypotaurine Taurine
PLP pyridoxal phosphate, AMP Sulphinate
CyD
adenosine monophosphate
CSAT
β-Sulphinyl pyruvate
Sulphite Sulphocysteine
SUOX (3.6)
Sulphate
high methionine
isolated amino acid abnormality or

with elevated tHcy only
high tyrosine with signs of liver failure
tHomocysteine >60 µmol/L* tHomocysteine <60 µmol/L**
tyrosinemia type 1 or another non

primary sulfur containing amino acid
disorder
low-normal AdoMet high AdoMet
CBS
deficiency
normal or near high AdoHcy
*in non-treated CBS tHcy is usually >60 µmol/L normal AdoHcy
but may be lower in mild cases, in particular when
on vitamin supplementation (non-pharmacological)
doses SAHH or ADK
**in non-CBS hypermethioninemias tHcy is usually MAT GNMT deficiency-
Fig. 3.2 Diagnostic flow-chart in normal or only mildly elevated, and values of about deficiency deficiency or distinguish by
patients with elevated plasma 50 µmol/L are exceptionally seen mtDNA enzyme or gene
*** tyrosine can be elevated depletion*** analysis
methionine
Elevated tHcy
Methionine elevated Methionine low-normal
tHcy mildly to
tHcy moderately to tHcy mildly to tHcy moderately tHcy severely
moderately elevated
severely elevated >60 moderately elevated to highly elevated >100
15−60
15−60 elevated 60−100
MAT, SAHH,
ADK or CBS*
deficiency** Most probably: remethylation
severe folate or
CBS Variants of MTHFR, B12 deficiency, defects
See Elevated deficiency renal failure, See Chapter 13
remethylation
methionine iatrogenic causes, (MTHFR and
flowchart milder folate or B12 Cbl) defects
deficiency
tHcy- plasma total homocysteine (µmol/L)

MAT- methionine adenosyltransferase
ADK-adenosine kinase *in non-treated CBS tHcy is usually significantly >60 μmol/
L, but may be lower in mild cases, in particular when on
CBS- cystathionine beta synthase vitamin supplementation (non-pharmacological) doses
SAHH- S-Adenosylhomocysteine hydrolase **in non-CBS hypermethioninemias tHcy is usually normal
or only mildly elevated, and values of about 50 μmol/L are
MTHFR- methylenetetrahydrofolate
exceptionally seen
reductase
Cbl- cobalamine
Fig. 3.3 Diagnostic flow-chart in patients with elevated plasma total homocysteine
Table 3.1 Methionine System Symptom Neonatal Infancy Childhood Adolescence Adulthood
adenosyltransferase I/III CNS Cognitive dysfunction n n ± ± ±
deficiency Demyelination n n ± ± ±
Developmental delay n n ± ± ±
Dysdiadochokinesis n n ± ± ±
Dysmetria n n ± ± ±
Dystonia n n ± ± ±
Headache n n ± ± ±
Language difficulties n n ± ± ±
Tendon reflexes, increased n n ± ± ±
Tremor n n ± ± ±
Vacuolating myelopathy n n ± ± ±
Eye Nystagmus n n ± ± ±
Other Odorous breath due to n ± ± ± ±
dimethylsulphide
Special Methionine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
laboratory S-Adenosylhomocysteine (P) n n n n n
S-Adenosylmethionine (P) n n-↓ n-↓ n-↓ n-↓
Total homocysteine (P) n-↑ n-↑ n-↑ n-↑ n-↑
3.4 Signs and Symptoms 3.5 Diagnosis
The clinical presentation and characteristic laboratory find- Measurements of the key amino acids, homocysteine, methi-
ings of each disease are summarised in Tables 3.1, 3.2, 3.3, onine, cystine and cystathionine in plasma or urine using
3.4, 3.5, 3.6 and 3.7. classical ion exchange chromatography or tandem MS, as
Table 3.2 Glycine N-methyltransferase deficiency

Digestive Failure to thrive n ± n n n
Hepatomegaly ± ±
Routine ASAT/ALAT (P) ↑ ↑ ↑
laboratory
Special Methionine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
laboratory S-Adenosylhomocysteine (P) n n n n n
S-Adenosylmethionine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Total homocysteine (P) n n n n n
Table 3.3 S-adenosylhomocysteine hydrolase deficiency

CNS Attention deficit disorder n ± ± − ++ ± − ++ ± − +++
Behaviour, aggressive n n ± ± ±
Cerebellar hypoplasia ± ± n n n
Developmental delay ± − +++ + − +++ + ± ±
Hyperactivity n n ± ± ±
Hypoplasia of pons ± ± n n n
Myelination delay ± ± ± n n
Digestive Liver dysfunction ± − +++ ± − +++ ± ± ±
Eye Strabismus ± ± ± ±
Haematological Coagulopathy ± − +++ ± − +++ ± − ++ ±
Musculoskeletal Absent/weak tendon reflexes ++ ++ ++ ++ ++
Hypotonia + − +++ + − +++ + − ++ + − ++ ++
Muscle weakness + − +++ + − +++ + − ++ + − ++ ++
Myopathy + − +++ + − +++ ++ ++ ++
Respiratory Respiratory insufficiency ± − +++ ± − +++ n n n
Other Foetal hydrops ± − ++ n n n n
Routine laboratory Albumin (P) ↓↓-n ↓↓-n n n n
ASAT/ALAT (P) n-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
Creatine kinase (P) ↑-↑↑ ↑-↑↑ ↑↑ ↑↑ ↑↑
Protein synthesis reduced ± − +++ ± − +++ ± n n
Prothrombin time n-↑↑ n-↑↑ n-↑ n-↑
Special laboratory Methionine (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
S-Adenosylhomocysteine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
S-Adenosylmethionine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 3.4 Cystathionine beta-synthase deficiency

Cardiovascular Thromboses, infarcts n ± ± ± ±
CNS Developmental delay n n-± n-± n-± n-±
Mental retardation n n-+++ n-+++ n-+++
Psychiatric symptoms n n ± ± ±
Seizures n ± ± ± ±
Stroke n ± ± ± ±
Dermatological Malar flush n n ± ± ±
Eye Ectopia lentis n ± ± ± ±
Iridodonesis n ± ± ± ±
Myopia n ± ± ± ±
Haematological Thromboembolism n ± ± ± ±
Musculoskeletal Arachnodactyly n n ± ± ±
Genu valgum n n ± ± ±
Kyphosis n n ± ± ±
Marfanoid features n n ± ± ±
Osteoporosis n ± ± ± ±
Pes cavus n n ± ± ±
Scoliosis n n ± ± ±
Sternal deformities n n ± ± ±
Special laboratory Cystine (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Methionine (P) n-↑↑ ↑↑↑ ↑↑↑ n-↑↑ n-↑↑
S-Adenosylhomocysteine (P) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
S-Adenosylmethionine (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Total homocysteine (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Table 3.5 Cystathionase deficiency

Other None n n n n n
Special laboratory Cystathionine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Cystathionine (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Total homocysteine (P) n n n n n
Table 3.6 Sulphite oxidase deficiency

CNS Axial hypotonia/peripheral ± + +
hypertonia
Movement, abnormal ± + +
Retardation, psychomotor + − +++ + − +++ + − +++
Seizures, pharmacoresistant + − +++ + − +++ + − +++
Digestive Feeding difficulties + + +
Eye Ectopia lentis n ± ± ± ±
Musculoskeletal Microcephaly ± + +
Routine laboratory Uric acid (U,P) n n n
Special laboratory Methionine (P) n n n n n
MRI/CT: cerebral and cerebellar ± + +
atrophy, cystic white matter
changes, ventriculomegalia
Sulphocysteine (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Sulphocysteine (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Total homocysteine (P) ↓ ↓ ↓ ↓ ↓
Table 3.7 Adenosine kinase deficiency

Cardiovascular Cardiac, anomalies/malformations ± ± ± ± ±
CNS Developmental delay ++ ++ ++ ++
Epilepsy n ± ± − ++ ++ ++
Digestive Liver dysfunction + + + + +
Ear Hearing loss, sensorineural n ± − ++ ± − ++ ± − ++ ± − ++
Musculoskeletal Frontal bossing n + + + +
Macrocephaly n ± − ++ ± − ++ ± − ++ ± − ++
Muscle weakness, progressive ±−+ ± − ++ ± − ++ ± − ++
Routine laboratory ALAT (P) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
Creatine kinase (P) n-↑ n-↑ n-↑ n-↑ n-↑
Prolonged conjugated + + – – –
hyperbilirubinaemia
Prothrombin time n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Adenosine (U) ↑ ↑ ↑ ↑ ↑
Methionine (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
S-Adenosylhomocysteine (P) n-↑ n-↑ n-↑ n-↑ n-↑
S-Adenosylmethionine (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
well as total homocysteine in plasma or less reliably in urine, plasma. Sulphite as a marker for sulphite oxidase deficiency
by various methods including HPLC and immunoassays, are must be tested for in fresh urine although S-sulphocysteine is
central to the diagnosis of this group of disorders. much more stable and can be measured by usual amino acid
Homocysteine in urine is increased in classical forms of CBS analysis. Urinary levels of adenosine have been reported
deficiency but its lack of detection is not sufficient to rule out elevated in adenosine kinase deficiency.
this disorder. Differentiation of the hypermethioninaemias Confirmation of diagnosis depends on enzyme assays
can be achieved by measurement of S-adenosylmethionine, and/or mutation analysis.
S-adenosylhomocysteine and sarcosine (rarely available) in

Infant Child Adolescent Adult
Analyte <1 year 1–12 years 12–18 years >18 years
Plasma amino acids (μmol/L)
Total homocysteine 3.3–8.3 4.7–10.3 <15 <15
Methionine 11–31 11–30 16–23 14–34
S-adenosylmethionine (nmol/L) 29.6–118
S-adenosylhomocysteine (nmol/L) 11.8–39.4
Urinary amino acids (mmol/mol creatinine)
Total homocyst(e)ine 0.2–3.7
3.7 Pathological Values

Methionine Homocysteine Homocyst(e) Cystathionine Sulphocysteine
Disorder (P) (P) ine (U) AdoMet (P) AdoHcy (P) Cystine (P) (U) (U)
3.1 MAT I/III ↑↑↑ n-↑ n n n n n n
3.2 GNMT ↑↑↑ n-↑ n ↑↑↑ n n n n
3.3 SAHH n-↑↑↑ n-↑ n ↑↑↑ ↑↑↑ n n n
3.4 CBS n-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑↑ ↑↑ ↓↓ n n
3.5 CTH n n n n n ↑↑↑ n
3.6 SUOX n ↓ – n n ↓ n ↑↑↑
3.7 ADKD ↑↑ n-↑ n ↑-↑↑ n-↑ n
Prenatal diagnosis is only relevant for SAHH, ADK, AdoMet supplementation, especially if methionine intake
CBS and SO deficiencies. Generally the first choice of is limited, may be necessary. It seems that GNMT defi-
method for each of these is mutation analysis in chorionic ciency and cystathionase deficiency do not require treat-
villous material provided that disease causing mutations and ment. In AdoHcy hydrolase deficiency, low-methionine diet
their parental origin has been confirmed. Alternatively can decrease plasma AdoMet and AdoHcy, which can have
enzyme assay in cultured amniocytes can be performed. a positive effect on methylation and clinical and biochemi-
cal abnormalities (Barić et al. 2005). Phosphatidylcholine
supplementation seems justified, while creatine and cyste-
3.8 Specimen Collection ine supplementation may be useful. CBS deficiency is ini-
tially treated with high doses of pyridoxine to test the
Overview on methods and required samples for enzyme and responsiveness. Responsive patients continue with pharma-
mutation analysis cological doses of pyridoxine. Regardless of responsive-
ness, folate and cobalamin should be added to avoid vitamin
Disorder Metabolite: sample depletion and stimulate homocysteine remethylation.
Total homocysteine: plasmaa Betaine is frequently needed, as is a low-methionine diet
Amino acids: plasma or serumb
(protein restriction plus methionine-free amino acid mix-
S-adenosylmethionine: plasmac, whole blood
ture enriched with cysteine). The diet is necessary in pyri-
S-adenosylhomocysteine: plasmac, whole blood
doxine nonresponsive patients and is combined with
Enzyme assaysd: method
MAT I/III Livere
vitamins and betaine. Anti-aggregation drugs may be indi-
GNMT Livere cated. In isolated sulphite oxidase deficiency, partly suc-
SAHH Cultured fibroblasts, erythrocytes, liver cessful treatment with low-protein diet combined with
ADKD ? methionine- and cysteine-free amino acid mixture has been
CBS Cultured fibroblasts reported but only in late-onset patients (Touati et al. 2000).
CTH Livere For adenosine kinase deficiency, no successful causal treat-
SUOX Cultured fibroblasts ment has been reported.
Mutation analysis
All disorders DNA
a
Immediate separation of plasma essential 3.10 Emergency Treatment
b
Rapid deproteinisation essential
c
EDTA blood on ice, immediate separation of plasma and
3.1 Methionine Adenosyltransferase I/III Deficiency
deproteinisation
d
In general, enzymatic work-up as a first step is advised. In some cases If unexplained neurological signs are present with very high
(common mutation or small gene), mutation analysis as first step methionine level, discontinuance of methionine intake for
e
Liver biopsy is not routinely justified 1–3 days followed by low-methionine diet until symptoms
disappear, in combination with AdoMet supplementation
(for instance, at a dose of 400 mg twice daily, Surtees et al.
3.9 Treatment Summary 1991) seems to be indicated. See also the commentb below
the standard treatment table.
The general treatment goal for sulphur-containing amino
acid disorders with relevant clinical consequences is correct- 3.3 S-adenosylhomocysteine Hydrolase Deficiency
ing biochemical abnormalities in order to suppress their Severe cases, as those presented with foetal hydrops, insuf-
adverse effects. These measures consist primarily of high ficiency of liver synthetic function and severe muscular
doses of cofactors, low-protein or low-methionine diet and hypotonia leading to respiratory insufficiency, may poten-
supplementation of metabolites behind the enzymatic block. tially benefit from strict methionine restriction and choline
Betaine is a useful additional means to decrease homocyste- and cysteine supplementation in combination with vigorous
ine concentration (Lawson-Yuen and Levy 2006). symptomatic treatment.
MAT deficiency requires regular clinical and biochemi- For other disorders from this group emergency situations,
cal monitoring. In symptomatic patients and those with which are both amenable to specific emergency treatment
brain imaging changes, methionine restriction is indicated. and related to these diseases are not likely. Perhaps, only in
Reduced methionine intake may be justified also in those mild sulphite oxidase deficiency, a diet low in protein and an
patients with severe deficiency and very high methionine amino acid mixture without cystine and methionine may be
and patients with persistent hyperhomocysteinaemia. helpful.
3.11 Standard Treatment

Disease Comment Medication/diet Dosagea Goals
3.1. MAT Only in symptomatic patients and Low-methionine diet In infancy ~15–20 mg/kg/ Disappearance/prevention of
deficiency those with brain imaging changes is day clinical symptoms
methionine restriction indicated. Normalisation of brain imaging
Limited methionine intake may be findings
justified also in those with severe Plasma methionine well below
deficiency and very high methionine 1,000 μmol/L
and patients with persistent
hyperhomocysteinaemia
AdoMet supplementation, especially S-adenosylmethionine 2–3 × 400 mg daily per osb Clinical improvement,
if methionine intake is limited, may normalisation of plasma and/or
be necessary CSF AdoMet; normalisation of
hyperhomocysteinaemia
3.2. GNMT It is questionable if the therapy is Low-methionine diet In infancy ~15–20 mg/kg/ Correction of biochemical
deficiency necessary; low-methionine diet can day abnormalities
correct biochemical abnormalities
3.3. AHCY Due to small number of patients, Low-methionine diet In infancy natural protein Clinical improvement and
deficiency these recommendations are based intake of ~10–20 mg/kg/day decrease of AdoMet and
only on pathogenetic hypotheses and of methionine, depending on AdoHcy, while avoiding
limited clinical experience. In the severity of the disease protein malnutrition
patients on diet close monitoring is and biochemical findings, in
necessary to avoid methionine combination with
deficiency and related consequences methionine-free amino acid
of protein malnutrition mixture to meet needs for
proteins
Phosphatidylcholine 3 × 600–1,200 mg/day
N-acetylcysteine 3 × 100–200 mg/day
Creatine (may be 3–5 g/day
useful theoretically)
Disease Comment Medication/diet Dosagea Goals

3.4. CBS Test of pyridoxine responsiveness A. Pyridoxine Decrease of the homocysteine
deficiency should be done at the beginning of responders as close to normal as possible,
the treatment Pyridoxine 50–200 (−500?) mg/day in i.e. below 20 μmol/L; in
1–2 oral doses pyridoxine fully responsive
Folate 1–5 mg daily in single dose persons, this is possible; in
others, this is not realistic and
Before test possible folate deficiency Hydroxocobalamin 1 mg daily per os, or 1 mg
values of 50–70 μmol/L may
should be corrected to assure proper i.m. monthly
be an acceptable target (Schiff
assessment of the test results. Test Low-methionine diet Less restrictive than in and Blom 2011). If reached
should start with oral dose of 100 mg (for partly responsive responders (see below) early, they significantly reduce
daily. If no responses, a higher dose patients) the risk of thromboembolism
of 200 and then 500 mg daily should Betaine 150–250 mg/kg/day in and other major clinical
be given. Rarely, only higher doses infants and 6–9 g daily in complications of CBS
are efficient. Most guidelines older children and adults in deficiency (Yap et al. 2001a, b).
recommend waiting period between 1 two doses Methionine values should be
and 2 weeks. If significant response
kept in acceptable range, which
has been achieved, the dose should be
is probably below
gradually tapered to the lowest
1,000 μmol/L
efficient dose. About half of the
patients respond to the treatment but
the great majority only partly
Guidelines for protein intake in B. Pyridoxine
methionine restricted diet are very nonresponders
approximate. Diet must be adjusted, Pyridoxine (although range is same as in
in combination with other measures, contradictory it can be responders, usual doses are
to achieve therapeutic goal, if given since it may 100–200 mg daily
possible, but should not jeopardise improve betaine effect)
the patient; therefore strict Folate 2–5 mg/day in single dose
monitoring is necessary
In nearly all CBS-deficient patients, Cobalamin 1 mg daily per os, or 1 mg
remethylation is overused to the i.m. monthly
extent that folate and/or cobalamin Low-methionine diet In infancy 2–2.5 g/kg of
depletion is present or very likely; natural protein daily; later
therefore, folate and cobalamin 1.5–1 g/kg daily;
should be added to the therapy methionine-free amino acid
mixture may be necessary
Betaine 150–250 mg/kg/day in
infants and 6–9 g daily in
older children and adults in
two doses
3.5. CTH The disorder seems benign and the – – –
deficiency therapy not necessary
3.6. Isolated The treatment has been useful only in Low-methionine and – Clinical improvement, decrease
SUOX milder forms of the disease. The diet low-cysteine diet of toxic metabolites
deficiency must be carefully monitored to avoid Dextrometorphan 12.5 mg/kg daily (dosage (S-sulphocysteine,
protein malnutrition (NMDA receptor reported in patient with thiosulphate)
antagonist) molybdenum cofactor
deficiency; largely variable
dosage has been reported in
nonketotic
hyperglycinaemia)
3.7. ADK No etiological treatment has been
deficiency reported so far. It seems that even the
effect of antiepileptic drugs is poor
a b
Given doses are arbitrary and frequently not tested in a sufficient num- The dosage and route are only for rough orientation. Information about
ber of patients for each given indication; therefore, they must be AdoMet treatment in children is very limited. In adults daily doses from
adjusted individually according to the diagnosis, patients needs and 50 mg to 3 g have been used. Intramuscular and intravenous forms of
results of clinical and biochemical monitoring. A common problem is the drug do exist and should be considered in every patient
finding a proper balance between the wish to achieve desired therapeu- individually
tic goals and avoidance of potentially serious side effects of higher
doses than recommended/tested
Besides specific measures listed in table for CBS

deficiency, anti-aggregation drugs may be indicated and
very high methionine values (Stabler et al. 2002;
other risk factors for thromboembolism checked and, if
Linnebank et al. 2005).
needed, treated. Vitamin C may protect nitric oxide-depen-
2. In patients treated with low-methionine diet, care-
dent vasodilatation in CBS patients. Theoretically, phospha-
ful clinical and biochemical monitoring is neces-
tidylcholine and creatine may inhibit transfer of methyl
sary to avoid consequences of protein malnutrition.
groups and thereby diminish the production of
S-adenosylhomocysteine and homocysteine. 3. Long-term folate therapy in high doses may be
Folinate is probably generally a better option than folate, associated with increased cancer risk.
but folate should be satisfactory in most cases, except in 4. Sensory neuropathy has been reported with doses
those where parenteral use is necessary (folinate is available of pyridoxine greater than 1,000 mg/day so that this
for parenteral use and folate is not). should be kept at the lowest dose able to achieve
adequate metabolic control. In particular in infancy,
doses should not exceed 300 mg/day. In consider-
3.12 Experimental Treatment ing effective or dangerous doses of pyridoxine
intake should be related to body weight.
No experimental treatment specific for this group of disor- 5. A major potential problem of betaine therapy in
ders is currently available, although molecular chaperones CBS deficiency and other disorders with both ele-
for CBS deficiency are being investigated. vated tHcy and methionine is potential increase of
methionine to the concentrations that may be toxic
for brain, leading to cerebral oedema and other con-
Warning Boxes/Pitfalls sequences of excessive hypermethioninaemia
1. In MAT-deficiency homocysteine can be sufficiently described above (Lawson-Yuen and Levy 2006).
elevated to mimic CBS deficiency, probably due to 6. Accidental inhalation of betaine in powder form
less than normal stimulation of CBS by low AdoMet can cause pulmonary problems.
and inhibition of betaine-homocysteine methyltrans- 7. Due to irreversible deactivation of methionine syn-
ferase, N5-methyltetrahydrofolate-homocysteine thase, nitrous oxide should be avoided in anaesthesia.
methyltransferase and cystathionine gamma-
lyase by methionine, in particular in patients with
3.13 Follow-Up and Monitoring
Recommendations given in the table below are only rough guidelines and should be adjusted individually according to age,
severity of the disease, compliance and other factors.
Disease Clinical follow-up and monitoring Biochemical follow-up and monitoring

3.1. MAT I/III Both for untreated patients and those on In untreated patients without symptoms checking of methionine, AdoMet and
deficiency therapy: Any neurological sign or total homocysteine (tHcy) are justified. The frequency depends on the severity
symptom should be considered as of enzyme deficiency. In patients with severe deficiency and previous plasma
possible sign of the disease and reason methionine close to 1,000 μmol/L or more or abnormal tHcy values checking
for further clinical (including brain should be more frequent (i.e. in infancy every 3 months, later every
imaging) and metabolic evaluation. For 6–12 months and after significant changes in dietary methionine intake). In mild
patients on low-methionine diet, deficiency, if highest methionine values, which should be checked after normal
additionally, signs of protein and high protein intake, are not close to 1,000 μmol/L only sporadic checking,
malnutrition should be regularly looked when symptoms attributable to MAT deficiency appear. In patients on low-
for methionine diet, regular monitoring of protein status and plasma amino acids is
indicated (in infancy at least every 3 months, later every 6–12 months and after
significant changes in dietary methionine intake). In hyperhomocysteinaemic
patients in similar intervals tHcy should be measured and thrombophillia screen
should be performed at least once
3.2. GNMT Sporadic evaluation if mild to moderate Liver function testsa
deficiency hepatomegaly is the only symptom
3.3. AHCY Careful evaluation of all body systems, Protein status, amino acids, AdoMet, AdoHcy, liver function tests, creatine
deficiency particularly of the nervous system and kinase, coagulation tests. In infancy every 1–3 months, if indicated even more
development, muscles, liver and frequently, later every 3–6 months
coagulation. In infancy every
1–3 months, if indicated even more
frequently, later every 3–6 months
3.4. CBS Neurological and, depending on age, Plasma amino acids and total homocysteine in infancy every 1–3 months, if
deficiency developmental or mental evaluation in indicated even more frequently, later every 3–6 months. In patients on
infancy every 3 months, later every low-methionine diet protein status in the same intervals. Unless on
6–12 months. Ophthalmology supplementation, serum cobalamin and folate every 3–6 months. Thrombophilia
examination yearly. Bone mineral screening once. Lipids first time at age 2–3 years, afterwards, if normal, every
density once in 1–3 years. Vascular 2–3 years, if not every 3 months alongside therapy
status every 6–12 months, depending on
the severity of the disease and clinical
course
3.5. CTH Not necessary
deficiency
3.6. Isolated SUOX General, and particularly, neurological If on diet, protein status, amino acids, S-sulphocysteine, thiosulphate, sulphite
deficiency and developmental evaluation in infancy in infancy every 1–3 months, if indicated more frequently, later every
every 1–3 months, later every 3–6 3–6 months
months, if indicated more frequently.
Ophthalmology every 6 months, if
indicated, more frequently
3.7. ADK Taking into account lack of specific therapy, neither clinical nor biochemical monitoring directed specifically to the
deficiency metabolic defect seems indicated. According to the clinical presentation standard paediatric palliative care, with emphasis
on neurological and liver aspects of the disease seems justified
a
In mice, in the long run significant, liver disease may take place, including hepatocellular carcinoma; therefore, liver ultrasound and liver tumour
markers may be justified
Bjursell MK, Blom HJ, Cayuela JA et al (2011) Adenosine kinase defi-

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N-methyltransferase deficiency: A new patient with a novel Braverman NE, Mudd SH, Barker PB, Pomper MG (2005) Characteristic
mutation. J Inherit Metab Dis 26:745–759 MR imaging changes in severe hypermethioninemic states. AJNR
Barić I, Fumić K, Glenn B et al (2004) S-adenosylhomocysteine Am J Neuroradiol 26:2705–2706
hydrolase deficiency in a human: a genetic disorder of methionine Buist NR, Glenn B, Vugrek O et al (2006) S-Adenosylhomocysteine hydro-
metabolism. Proc Nat Acad Sci U S A 101:4234–4239 lase deficiency in a 26-year-old man. J Inherit Metab Dis 29:538–545
Barić I, Ćuk M, Fumić K et al (2005) S-Adenosylhomocysteine hydrolase Chamberlin ME, Ubagai T, Mudd SH, Wilson WG, Leonard J, Chou JY
deficiency: a second patient, the younger brother of the index patient, (1996) Demyelination of the brain is associated with methionine
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Chamberlin ME, Ubagai T, Mudd SH, Levy H, Chou JY (1997) Mudd SH, Cerone R, Schiaffino MC et al (2001b) Glycine
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with a mutation in the human methionine adenosyltransferase 1A tent isolated hypermethioninemia. J Inherit Metab Dis 24:448–464
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de Franchis R, Sperandeo MP, Sebastio G et al (1998) The clinical oninemia and hyperhomocysteinemia due to high methionine
aspects of cystathionine β-synthase deficiency: how wide is the intake: a diagnostic trap. Mol Genet Metab 79:6–16
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Grubbs R, Vugrek O, Deisch J, Wagner C, Stabler S, Allen R, Barić I, homocysteine in severe methionine adenosyltransferase I/III defi-
Rados M, Mudd SH (2010) S-adenosylhomocysteine hydrolase ciency. Metabolism 51:981–988
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Hyperammonemias and Related
Disorders 4
Johannes Häberle and Vicente Rubio
Contents Summary
4.1 Introduction ...................................................................... 48 The term hyperammonemia describes a clinical situation
marked by increased plasma ammonia concentration. It is
4.2 Nomenclature ................................................................... 49
generally due to decreased ammonia detoxification as urea.
4.3 Metabolic Pathways ......................................................... 50 Patients can be of any age. Since ammonia is neurotoxic,
4.4 Signs and Symptoms ........................................................ 51 life and mental status can be threatened within a few hours
of breaking the balance between ammonia production and
4.5 Reference Values Table .................................................... 55
detoxification, as in neonatal urea cycle defects patients when
4.6 Pathological Values Table ................................................ 56 they stop relying on maternal ammonia detoxification upon
4.7 Diagnostic Flow Chart ..................................................... 57 birth. The urea cycle, fully expressed exclusively in periportal
4.8 Specimen Collection ......................................................... 58 hepatocytes, is the main pathway to detoxify ammonia; it can
be primarily affected by an inherited enzyme or transporter
4.9 Prenatal Diagnosis Table and Sample Requirements ... 59
defect (primary hyperammonemia) or secondarily by toxic
4.10 DNA Testing...................................................................... 59 metabolites or substrate depletion (secondary hyperammo-
4.11 Treatment.......................................................................... 59 nemia). Hyperammonemia in infants and children is mainly
References .................................................................................... 62
due to inborn metabolic errors causing primary or secondary
hyperammonemia. In adults, hyperammonemia is more often
due to acquired hepatic disease. The clinical manifestations
of hyperammonemia are generally unspecific and are mainly
neurological, gastrointestinal, or psychiatric. The hallmark of
hyperammonemia is an unexplained change in consciousness;
thus, in any unexplained encephalopathy hyperammonemia
must be excluded without delay to avoid severe neurological
sequelae. Treatment of acute hyperammonemia most urgently
requires the establishment of anabolism and the avoidance of
protein catabolism, with rapid initiation of parenteral glucose
infusion and protein restriction. Additional urgent measures
include the use of dialysis to remove ammonia and of medi-
cations that can improve residual urea cycle function (carba-
J. Häberle (*) mylglutamate and either l-arginine or l-citrulline) and that
Division of Metabolism and Children’s Research Center,
allow circumventing the urea cycle (the nitrogen scavengers
University Children’s Hospital,
Steinwiesstrasse 75, Zürich 8032, Switzerland sodium benzoate and/or sodium phenylbutyrate). Liver trans-
e-mail: johannes.haeberle@kispi.uzh.ch plantation is curative. Prognosis of acute hyperammonemia
V. Rubio remains poor but can be improved by increasing awareness
Structural Enzymopathology Unit, Department of Genomics and of healthcare professionals. This chapter focuses on hyper-
Proteomics, Instituto de Biomedicina de Valencia of the Spanish ammonemia due to urea cycle defects but also deals with the
National Research Council (CSIC) and Centre for Biomedical
very rare deficiencies of pyrroline-5-carboxylate synthetase
Network Research on Rare Diseases (CIBERER),
C/ Jaime Roig 11, Valencia 46010, Spain and glutamine synthetase and with the transient hyperammo-
e-mail: rubio@ibv.csic.es nemia of the newborn.
48 J. Häberle and V. Rubio
4.1 Introduction supply of ornithine is compromised. We also mention here

two hyperammonemias of more obscure nature, the tran-
In clinical hyperammonemia, plasma ammonia exceeds sient hyperammonemia of the newborn (Table 4.10) and the
upper normal limits (100 μmol/L in newborns; 50 μmol/L in hyperinsulinism-hyperammonemia syndrome. The latter
older individuals (Colombo et al. 1984)) because of increased syndrome, which will be treated in detail in the chapter on
ammonia production or decreased ammonia detoxifica- hyperinsulinism (Chap. 21), results from gain-of-function
tion to urea (Bachmann 2002). Ammonia, the nitrogenous mutations affecting glutamate dehydrogenase and has auto-
waste product of protein and amino acid catabolism, crosses somal dominant inheritance. The hyperammonemia of this
biological membranes and reaches the brain, where it is a syndrome has been attributed to exacerbated glutamate oxi-
potent neurotoxin. It causes brain edema possibly because dation or to depressed acetylglutamate synthesis from gluta-
of increased ammonia-dependent glutamine synthesis/accu- mate, although a renal origin for this hyperammonemia has
mulation in glial cells, with concomitant osmotically medi- also been postulated (Palladino and Stanley 2010).
ated water influx. Oxidative damage may also contribute to Although the urea cycle has a high capacity and is
ammonia neurotoxicity (Häussinger and Görg 2010). The ultimately responsible for essentially all ammonia detoxi-
effects of hyperammonemia are mainly dependent on its fication, its low affinity for ammonia requires the coopera-
duration and severity. tion of glutamine synthetase, the high-affinity, low-capacity
Ammonia disposal proceeds mainly by its conversion to ammonia-detoxifying system, which is present exclusively
urea in the periportal hepatocytes of the liver, followed by in perivenous hepatocytes. These hepatocytes, by converting
urinary urea excretion. Periportal hepatocytes are the only ammonia to glutamine, further reduce the ammonia level in
cells having all the enzymes of the urea cycle (Fig. 4.1) the blood leaving the liver. Indeed, a deficiency of glutamine
(Grisolia et al. 1976). They extract ammonia from the por- synthetase, which is also discussed here (Table 4.9), will pro-
tal blood (which is much richer in ammonia than peripheral duce hyperammonemia. In summary, the combined action of
blood) or produce it from glutamine by glutaminase glutamine-consuming, urea-forming periportal hepatocytes
(Häussinger 1990b) or from glutamate by oxidative deami- and of glutamine-forming perivenous hepatocytes makes
nation catalyzed by glutamate dehydrogenase (Fig. 4.1). possible the final detoxification of ammonia as urea while
One molecule of ammonia and another molecule of bicar- ensuring that the ammonia levels reaching the brain are low
bonate enter the urea cycle inside the mitochondria of (Häussinger 1990a).
periportal hepatocytes, where the first two steps of the urea When ammonia production exceeds urea synthesis, as in
cycle, catalyzed by carbamoyl phosphate synthetase I the inborn errors of the urea cycle, blood ammonia levels
(CPS1) and ornithine transcarbamylase (OTC), take place. escalate and can reach in few hours even millimolar concen-
The remaining three urea cycle steps, catalyzed by arginino- trations. The clinical consequences of hyperammonemia
succinate synthetase, argininosuccinate lyase, and arginase, develop in parallel, with rapid escalation towards coma, ren-
occur in the cytosol, incorporating another ammonia mol- dering time crucial in the management of these patients. In
ecule donated by aspartate (Fig. 4.1). In this way, urea pro- roughly half of the patients, urea cycle defects present within
duction from ammonia requires the concerted action of five 2–3 days from birth with somnolence and lethargy accompa-
enzymes and of one mitochondrial transporter (the orni- nied by vomiting, temperature instability, and seizures, with
thine/citrulline mitochondrial antiporter) (Fig. 4.1). Defects rapid progression to coma (Brusilow and Horwich 2001;
in these enzymes and transporter as well as in Leonard and Morris 2002). The other half of the patients has
N-acetylglutamate synthase (NAGS), the enzyme that pro- later presentation (Summar et al. 2008) that can be as severe
duces the essential activator of CPS1, N-acetylglutamate as in newborns. Indeed, urea cycle disorders can manifest at
(Table 4.1–4.7), cause primary hyperammonemia. Except any age.
OTC deficiency, which exhibits X-linked inheritance, all Diagnosis of the underlying disorder relies on studies of
these defects present autosomal recessive inheritance. metabolites (Fig. 4.2) followed by enzymatic and/or molecu-
In addition, urea cycle function can be impaired by lar genetic confirmation (Häberle et al. 2012). Only in the
enzyme inhibition or substrate deficiency leading to second- transient hyperammonemia of the newborn, no confirmatory
ary hyperammonemia (Häberle 2011). Two diseases consid- diagnostic test is available.
ered in this chapter, i.e., citrullinemia type II (Table 4.8) and Treatment of acute hyperammonemia aims at immedi-
pyrroline-5-carboxylate synthetase (P5CS) deficiency (Table ate lowering of the ammonia levels by (1) preventing pro-
4.11), cause secondary hyperammonemia. Citrullinemia tein catabolism and establishing anabolism by starting soon
type II is characterized by inappropriate aspartate supply parenteral glucose infusion and stopping protein intake, (2)
because of a defect in citrin, the mitochondrial aspartate/ administering the nitrogen scavengers sodium benzoate and/
glutamate antiporter. In P5CS deficiency, the endogenous or sodium phenylbutyrate to eliminate nitrogen by alternative
4 Hyperammonemias and Related Disorders 49
pathways that circumvent the urea cycle (benzoate conju- recommended for patients with neonatal onset (Leonard and
gates with glycine, yielding hippurate that is excreted in the McKiernan 2004).
urine, the same happens for glutamine with phenylacetate, of Prognosis is still bleak, particularly in cases with pro-
which phenylbutyrate is a precursor) (Batshaw et al. 2001), longed, severe, or recurrent episodes of metabolic decom-
and (3) by stimulating residual urea cycle function by giving, pensation (Gropman and Batshaw 2004; Gropman et al.
depending on the disorder, l-arginine or l-citrulline (these 2007), since the duration and frequency of comatose epi-
are urea cycle intermediates, Fig. 4.1) and, in the rare NAGS sodes determine the severity of neurological sequelae (Picca
deficiency, by giving N-carbamylglutamate (a commercial et al. 2001). However, prognosis can be improved by early
N-acetylglutamate analog that is effective orally (Rubio and recognition of new patients and prompt specific therapy
Grisolia 1981)) (Häberle et al. 2012). Severe hyperammo- (Nassogne et al. 2005). Hopefully, enhanced awareness will
nemia may require extracorporeal dialysis to rapidly lower lead in the near future to more widely available diagnosis,
ammonia. Chronic treatment includes protein restriction improved care, and better prognosis, while increased access
(Leonard 2001; Singh 2007); supplementation of essential to liver transplantation should expand the reach of curative
amino acids, trace elements, and vitamins; and use of nitro- therapy. Hepatocyte infusions, gene repair, chemical chaper-
gen scavengers and of l-arginine or l-citrulline or, in NAGS oning, nonsense mutation therapeutics, and gene or enzyme
deficiency, of N-carbamylglutamate. The only definite cure substitution might move soon from research into clinical
of most of these conditions is liver transplantation, strongly practice.
4.2 Nomenclature
Gene Chromosomal
No. Disordera Alternative name Abbreviation symbol localization Affected protein OMIM no. Subtype
4.1 N-Acetylglutamate NAGS deficiency NAGS NAGS 17q21.31 N-Acetylglutamate 237310 All
synthase deficiency deficiency synthase forms
4.2 Carbamoyl CPS1 deficiency CPS I CPS1 2q35 Carbamoyl phosphate 237300 All
phosphate deficiency synthetase I forms
synthetase I
deficiency
4.3 Ornithine Ornithine OTC OTC Xp11.4 Ornithine 311250 All
transcarbamylase carbamoyltransferase deficiency transcarbamylase forms
deficiency deficiency
4.4 Citrullinemia type I Argininosuccinate CTLN1 ASS1 9q34.11 Argininosuccinate 215700 All
synthetase deficiency synthetase forms
4.5 Argininosuccinic Argininosuccinate ASL ASL 7q11.21 Argininosuccinate lyase 207900 All
aciduria lyase deficiency deficiency forms
4.6 Argininemia Arginase 1 deficiency ARG1 ARG1 6q23.2 Arginase 1 207800 All
deficiency forms
4.7 HHH syndrome Hyperammonemia- HHH ORNT1 13q14.11 Mitochondrial ornithine 238970 All
hyperornithinemia- syndrome; transporter (ORNT1) forms
homocitrullinuria ORNT1 SLC25A15
syndrome deficiency
4.8 Citrullinemia type II Citrin deficiency CTLN2 SLC25A13 7q21.3 Aspartate glutamate 605814, All
carrier (SLC25A13) 603471 forms
4.9 Glutamine GS deficiency GS GLUL 1q25.3 Glutamine synthetase 610015 All
synthetase deficiency forms
deficiency
4.10 Transient Transient THAN – – – – Early
hyperammonemia hyperammonemia of infantile
of the newborn prematurity form
4.11 Pyrroline-5- P5CS deficiency P5CS ALDH18A1 10q24.1 Pyrroline-5-carboxylate 219150 All
carboxylate deficiency synthetase forms
synthetase
deficiency
a
The hyperinsulinism-hyperammonemia syndrome is not described in detail in this chapter but the reader is referred to Chap. 21.
4.3 Metabolic Pathways
Portal blood
Glutamine NH3 + HCO3– Alanine ALT Pyruvate LDH Lactate

α-Ketogultarate Glutamate NADH NAD
GLNase Citrin Aspartate AST Oxalacetate MDH Malate
α-Keto- + 1 ATP
glutarate Aspartate
Citrulline ASS
GDH NAD(P)H Aspartate cycle
AST ORNT1
NAD(P)
Glutamate Oxalacetate Citruline Arginino succinate
FUM
+ 2 ATP Nitric oxide
AcetyCoA
NAGS iNOS + H2O
N-Acetyl- ASL
CPS1 OTC
L-glutamate Urea cycle
ATP Carbamoyl- Fumarate
NADPH + Ornithine
phosphate
Arginine
P5CS OAT
+ H2O
L-Glutamate-
γ-semialdehyde ORNT1 ARG1
UMP Uridine, Uracil
Chemical Ornithine
Δ1-Pyrroline- Urea OMP Orotidine

P5CR Proline
Mitochondrion 5-carboxylate
Cytosol Carbamoyl phospate Orotic acid
Suprahepatic blood
Urine
Fig. 4.1 The urea cycle and associated pathways. For simplicity not antiporter (ORNT1) has been illustrated transporting separately orni-
all the substrates and products of each reaction are shown. The main thine and citrulline, although it exchanges ornithine for citrulline. A
nitrogen-containing compounds taken by urea-synthesizing periportal double-lined arrow next to an encircled plus sign indicates allosteric
hepatocytes include ammonia, glutamine, and other amino acids of activation of CPS1 by N-acetyl-l-glutamate. “De novo” ornithine syn-
which alanine is shown as the paradigm. Glutaminase (GLNase) and thesis from glutamate by Δ1-pyrroline-5-carboxylate synthetase (P5CS)
glutamate dehydrogenase (GDH) can supply ammonia from glutamine and δ-ornithine aminotransferase (OAT) is shown also, although it
and glutamate, respectively, for entry into the urea cycle at the CPS1 occurs mostly in the intestine and it is only important for ureagenesis
step. The various transaminases (of which alanine aminotransferase, during fasting. Proline synthesis is illustrated also, since the P5CS reac-
ALT, is shown as the paradigm), coupled to aspartic transaminase tion is also the first step of proline synthesis (pyrroline-5-carboxylate
(AST), provide the other N atom of urea as the aspartate used in the ASS reductase, P5CR, is responsible for the other catalyzed step, as illus-
reaction. Aspartate can also be provided by exporting it from mitochon- trated). The figure also shows nitric oxide synthase (iNOS in the case of
dria by citrin (in exchange for glutamate and a proton, not shown for hepatocytes) in its position across the cytosolic part of the urea cycle. In
simplicity). The fumarate produced in the ASL reaction is recycled and macrophages and vascular cells, a reduced cycle involving ASS, ASL,
reconverted to aspartate in the “aspartate cycle,” which combines the and NOS appears to be operative. NAD(P) and NAD(P)H denote that
actions of fumarase (FUM), malate dehydrogenase (MDH), and AST. GDH can use both NAD and NADP (and its reduced forms); “chemi-
This cycle can be carried out in the cytosol if there is an adequate cyto- cal” denotes a non-catalyzed step. OMP orotidine monophosphate,
solic supply of NAD as when utilizing alanine (thanks to the conversion UMP uridine monophosphate (This figure summarizes information
of alanine-derived pyruvate to lactate by lactate dehydrogenase, LDH). from many sources all of which cannot be listed because of space con-
However, if there is little cytosolic NAD supply, the malate produced straints. A summary of much knowledge is provided in Grisolia et al.
by fumarase enters the mitochondria (in exchange for α-ketoglutarate), (1976), with more recent data from Häussinger (1990b) and Mori et al.
where it is converted to aspartate, which is then exported to the cyto- (1998). Data for the citrin transporter is reviewed in Imamura et al.
sol via citrin (for simplicity, this variant of the aspartate cycle is not (2003) and Mutoh et al. (2008))
shown). For convenience, the mitochondrial ornithine/citrulline
Table 4.1 N-Acetylglutamate synthase deficiency

Autonomic system Temperature instability ± n n n n
CNS Coma +++ +++ ++ ++ ++
Developmental delay n ±-++a ±-++a ±-++a ±-++a
Encephalopathic crisis, acute +++ +++ ++ ++ ++
Encephalopathy +++ +++ ++ ++ ++
Digestive Failure to thrive ± ± ± ± ±
Feeding, protein aversion ++ ++ ++ ++ ++
Vomiting +++ ++ ++ ++ ++
Routine laboratory Ammonia (B) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Special laboratory Arginine (P) ↓ ↓ ↓ ↓ ↓
Citrulline (P) ↓↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Glutamine (P, CSF) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
Orotic acid (U) ↓-n ↓-n ↓-n ↓-n ↓-n
a
If untreated
Table 4.2 Carbamoyl phosphate synthetase I deficiency

CNS Coma +++ +++ ++ ++ ++
Developmental delay n ±-++ ±-++ ±-++ ±-++
Feeding, protein aversion ++ ++ ++ ++ ++
Vomiting +++ ++ ++ ++ ++
Argininosuccinate (U) n n n n n
Citrulline (P) ↓↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Glutamine (P, CSF) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
Orotic acid (U) ↓-n ↓-n ↓-n ↓-n ↓-n
Table 4.3 Ornithine transcarbamylase deficiency

CNS Asterixis n n n n ±
Ataxia n n ± ± ±
Coma +++ +++ ++ ++ ++
Confusion, episodic n n ± ± ±
Stroke-like episodes ± ± ± ± ±
Digestive Feeding, protein aversion ++ ++ ++ ++ ++
Liver failure, acute n ± ± ± ±
Vomiting +++ ++ ++ ++ ++
Eye Vision, impaired n n n ± ±
(continued)

ASAT/ALAT (P) (↑) (↑) (↑) n n
Urea (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Citrulline (P) ↓↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Glutamine (P, CSF) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
Homocitrulline (U) (↑) (↑) (↑) (↑) (↑)
Orotic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Table 4.4 Citrullinemia type I

CNS Ataxia n n ± ± ±
Coma +++ +++ ++ ++ ++
Digestive Feeding, protein aversion ++ + + + +
Liver failure, acute n ± ± n n
Vomiting ++ ++ + + +
ASAT/ALAT (P) (↑) (↑) (↑) n n
Urea (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Citrulline (P,U) ↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Glutamine (P, CSF) ↑↑↑ ↑↑↑ ↑↑ ↑↑ ↑↑
Orotic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Table 4.5 Argininosuccinic aciduria

Coma +++ ++ + + +
Developmental delay n ± + + +
Encephalopathic crisis, acute +++ ++ + + +
Encephalopathy +++ ++ + + +
Digestive Feeding, protein aversion ++ + + + +
Vomiting ++ + + + +
ASAT/ALAT (P) n-↑ n-↑ n-↑ n-↑ n-↑
Urea (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Argininosuccinate (P)a ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Argininosuccinate (U)a ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Citrulline (P) ↑ ↑ ↑ ↑ ↑
Glutamine (CSF) ↑↑↑ ↑↑↑ ↑↑ ↑↑ ↑↑
Glutamine (P) ↑↑ ↑↑ ↑ ↑ ↑
a
Argininosuccinate is converted spontaneously to its two anhydrides particularly in the urine, depending on the pH and storage conditions of the
sample. Modern methods of amino acid analysis identify ASA and its two anhydrides. Quantification is usually based on the ASA peak;
argininosuccinate levels are generally higher in CSF than in plasma
Table 4.6 Argininemia

Developmental delay n + + + +
Spastic diplegia n ± ± + +
Digestivea Feeding, protein aversion n ± ± ± ±
Vomiting ± ± ± ± ±
Routine laboratory Ammonia (B) n-↑ ↑ ↑ ↑ ↑
Urea (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory Arginine (P) n-↑ ↑↑ ↑↑ ↑↑ ↑↑
Citrulline (P) n-↑ n-↑ n-↑ n-↑ n-↑
Glutamine (P) ↑ ↑ ↑ ↑ ↑
a
In addition, a few neonatal cases presenting liver pathology (intrahepatic cholestasis) have been reported
Table 4.7 HHH syndrome

CNS Asterixis n n n n ±
Ataxia n n ± ± ±
Coma ± ± ± ± ±
Encephalopathic crisis, acute ± ± ± ± ±
Encephalopathy ± ± ± ± ±
Seizures ± ± ± ± ±
Spastic paresis/pyramidal signs n n ± ± ±
Digestive Failure to thrive n ± ± ± ±
Feeding, protein aversion ± ± ± ± ±
Liver dysfunction ± ± ± ± ±
Liver failure, acute n ± ± n n
Eye Vision, impaired n n n ± ±
Routine laboratory Ammonia (B) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
ASAT/ALAT (P) (↑) (↑) (↑) (↑) (↑)
Urea (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory 14
C-Ornithine incorporation (FB) ↓ ↓ ↓ ↓ ↓
Arginine (P) n n n n n
Citrulline (P) n n n n n
Creatine (P) n ↓-n ↓-n ↓-n ↓-n
Factor VII and X ↓-n ↓-n ↓-n ↓-n ↓-n
Glutamine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Homocitrulline (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Ornithine (P) n-↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 4.8 Citrullinemia type II

CNS Ataxia n n n ± ±
Coma n n n + +
Confusion, episodic n n n ± ±
Consciousness disturbance n n n ± ±
Developmental delay n n n ± ±
Encephalopathy n n n + +
Digestive Cholestasis ++ + n n n
Jaundice ± n n n n
Liver dysfunction + + n n n
Liver steatosis ± ± n n n
Low-carbohydrate, high-protein, n n n + +
and high-fat intake
Routine laboratory Ammonia (B) n-(↑) n-(↑) n-(↑) ↑ ↑
ASAT/ALAT (P) n-↑ n-↑ n-↑ n n
Special laboratory Arginine (P) ↑ ↑ ↑ (↑) (↑)
Citrulline (P) n-↑↑ n-↑↑ n-↑↑ ↑↑ ↑↑
Galactose (P) ↑ (↑) n n n
Galactose (U) ↑↑ ↑ n n n
Glutamine (CSF) n-↑ n-↑ n-↑ ↑ ↑
Glutamine (P) (↑) (↑) (↑) (↑) (↑)
Methionine (P) ↑ ↑ (↑) (↑) (↑)
Threonine (P) ↑ ↑ (↑) (↑) (↑)
Tyrosine (P) ↑ ↑ (↑) (↑) (↑)
Two forms, neonatal and later form, are distinguished. The neonatal form is dominated by intrahepatic cholestasis and jaundice, generally waning
out with carbohydrate-devoid formula. The adult form is dominated by the symptoms and signs of hyperammonemia. Both forms are caused by
mutations in the same gene
Table 4.9 Glutamine synthetase deficiency

CNS Absent head control + ++ ++
Cerebellar hypoplasia ±
Developmental delay + +++ +++
Encephalopathy, epileptic ++ ++ ++
Epilepsy, intractable ++ ++ ++
Dermatological Erythema, necrotizing ± ± ±
Routine laboratory Ammonia (B) ↑ ↑ ↑
EEG: abnormal + ++ ++
Special laboratory Glutamic acid (P, CSF) n n n
Glutamine (CSF) ↓↓ ↓↓ ↓↓
Glutamine (P) ↓↓ ↓↓↓ ↓↓↓
Glutamine (U) ↓ ↓ ↓
Recent description. No data for adolescence and adulthood
Table 4.10 Transient hyperammonemia of the newborn

CNS Coma ++ n n n n
Encephalopathic crisis, acute ++ n n n n
Digestive Vomiting ± n n n n
Respiratory Respiratory failure ++ n n n n
Other Prematurity + Not applicable Not applicable Not applicable Not applicable
Routine Laboratory Ammonia (B) ↑↑↑ n n n n
Special Laboratory Glutamine (P) n-↑ n n n n
Glutamine (P)/ammonia (B) ratio <1.6 n n n n
Table 4.11 Pyrroline-5-carboxylate synthetase deficiency

Cardiovascular Valvulitis, mitral + + +
CNS Developmental delay n ± ±
Dermatological Wrinkly skin ++ ++ +
Digestive Failure to thrive n ± +
Eye Cataract + +
Musculoskeletal Hernias n ± ±
Joint laxity ± ± ±
Othera Intrauterine growth retardation + N/A N/A N/A N/A
Progeroid appearance ++ + +
Routine laboratory Ammonia (B)b n-↑ n-↑ n-↑
Special laboratory Arginine (P)c ↓-n ↓-n ↓-n
Citrulline (P)c ↓-n ↓-n ↓-n
Ornithine (P)c ↓-n ↓-n ↓-n
Proline (P) ↓-n ↓-n ↓-n
The extreme rarity of this disease prevents giving data for adolescence and adulthood
a
Cerebral vascular tortuosity and low creatine phosphate levels (MRS) have been reported in one patient
b
Abnormal levels only after fasting
c
Laboratory changes in metabolite levels observed only in two of the five families reported; make sure that sampling is done in fasting state
4.5 Reference Values Table
Plasma (μmol/L)
Age <1 month 1 month–12 years >12 years
Ammonia, fasting (enzymatic)a <100 <50 <50
Arginineb 37–71 32–142 28–96
Argininosuccinate <1 <1 <1
Citrulline 8–47 8–47 19–47
Ornithineb 66–116 27–96 55–135
Lysine 154–246 85–218 135–243
Glutaminec 475–746 475–746 466–798
Alanine 274–384 148–475 146–494
Urine (mmol/mol creatinine)

Age <1 month 1–6 months >6 months
Arginine 0.2–14 0.2–14 0.2–14
Argininosuccinate <0.5 <0.5 <0.5
Citrulline 0.2–11 0.2–11 0.2–11
Ornithine 0.2–26 0.2–26 0.2–26
Lysine 15–199 15–199 13–80
Glutaminec 52–205 52–205 45–133
Alanine 75–244 72–206 41–130
Orotate <3.4 <2.35 <1.44
Orotidine <5.0 <1.3 <0.6
Uracil 10–50 10–50 10–50

Age <7 days 8 days–4 months 5 months–2 years 3–10 years >10 years
Homocitrulline <135 <15 <4 <11 <8
Cerebrospinal fluid (μmol/L)

Age <1 year >1 year
Glutaminec 390–824 352–680
Except the values for ammonia, cerebrospinal fluid, and urinary homocitrulline, these reference values are from the Metabolic Diseases Unit of the
Vall d’Hebrón Hospital of Barcelona (Drs. J.A. Arranz and E. Riudor)
a
Preanalytical problems: tourniquet, convulsions, or very heavy resistance. Cooling of the blood and prompt cell removal and ammonia analysis
are essential
b
Cells should be separated soon, since erythrocyte arginase can hydrolyze the arginine and increase the ornithine. Hemolysis must be avoided
c
Glutamine is unstable on standing unless plasma, CSF or urine is frozen
4.6 Pathological Values Table
Disorder Ammonia (P) Citrullinea (P) Arginineb (P) Glutamine (P) Ornithine (P) ASA (U) Orotic acid (U) Homocitrullinec (U)
NAGS def. ↑↑↑ ↓ ↓-n ↑-↑↑ n nd n-↓ n
CPS1 def. ↑↑↑ ↓ ↓-n ↑-↑↑ n nd n-↓ n
OTC def. ↑↑↑ ↓ ↓-n ↑-↑↑ n nd ↑↑-nd n-↑
ASS def. ↑↑ ↑↑↑ ↓ ↑ n nd ↑-n n
ASL def. ↑↑ ↑↑ ↓ ↑-n n ↑↑↑ ↑-n n
ARG1 def. ↑-↑↑ ↑ n-↑↑ in ↑-n n nd ↑-n n
(not always) neonates;
↑↑↑ in infants,
children, and
adults
HHH ↑↑ n n ↑-↑↑ ↑↑ but not nd ↑-n ↑↑
syndrome in neonates
Citrin def. ↑↑ ↑-↑↑ (↑)-↑ n n nd n n
(not in neonates)
GS def. ↑ n n ↓↓ n nd n n
THAN ↑↑↑ n n-↓ n n nd Unknown n
P5CS def.e ↑ (fasting) -n ↓-n ↓-n ↑-n ↓-n nd n n
Ammonia is determined most often enzymatically using glutamate dehydrogenase. Bedside ammonia determination uses a reflectrometric method.
Amino acids can be determined in different ways, although the classical method is based on ion exchange chromatography with post-column
derivatization with ninhydrin and colorimetric detection
Def. deficiency, nd not detectable, ASA argininosuccinate
a
Watermelon contains citrulline, and its consumption in high amount was reported to increase citrulline levels. Take also into account the possibil-
ity that citrulline may have been administered therapeutically to the patient
b
Exclude arginine load or take into account the possibility that arginine may have been administered therapeutically to the patient
c
Exclude canned food or milk as a source of the homocitrulline
d
Normal urinary level of orotic acid has been found in neonatal onset OTC patients
e
Plasma proline levels may also be decreased in P5CS deficiency (together with arginine, citrulline, and ornithine) but this might be missed if
sampling is not done in a fasting state
Hyperammonemia
Glutamine Glutamine
Glutamine
low elevated
normal
↓ Gln Citrulline Citrulline Citrulline Citrulline

Preterm Full Term
GS deficiency ↓ ↑ ↑↑ ↑↑↑
normal AA ↓ Arg, Lys, Orn ↑↑↑ ASA (U)

profile Organic acids ↑↑ Citrulline
↑ ASA, ↓ Arg
Acylcarnitines ↑ ↑↑ orotic acid (U) orotic acid (U) ↑ Arg, Lys, Orn (U)
THAN normal elevated ↑ orotic acid (U) ↑ orotic acid (U)
LPI ASL deficiency
hypoglycemia ↑ Ala, ↓ Arg

acidosis
HIHA syndrome NAGS deficiency Ornithine Ornithine ↓ Arg
Organic acidurias
HMG-CoA-lyase defect CPS1 deficiency ↑ normal ↑ Cit (U)
↑ orotic acid (U)
↓ Arg, ↑ Ala ASL deficiency
hypoglycemia OTC deficiency
FAO defects ↑ Met, Tyr, Arg ↓ Orn, Pro, Arg
↑ α-FP, galactose fasting hyper-NH3
Citrin deficiency P5CS deficiency
↑↑↑ Arg
↑ homocitrulline ARG1 deficiency
lactic acidosis HHH syndrome
PC deficiency
Orn in newborns
↑ Orn
transiently normal
OAT deficiency
OAT deficiency
Fig. 4.2 This diagnostic flow chart describes the typical situation for lyase 3-hydroxy-3-methylglutaryl-CoA lyase, α-FP α-fetoprotein, PC
each disorder; however, some patients will present with different bio- pyruvate carboxylase, OAT ornithine aminotransferase, LPI lysinuric
chemical findings. For this reason, the flow chart should guide diagnosis protein intolerance, Orn ornithine, ASA argininosuccinic acid; other
but must not be taken for absolute exclusion of a disorder. Unless indi- amino acids are abbreviated according to the international three-letter
cated with a (U) (urine), the figure refers to plasma levels. Abbreviations code (The figure was adapted from Häberle et al. (2012))
not defined in the disease table: FAO fatty acid oxidation, HMGA-CoA
Overview on methods and required samples for enzyme and mutation analysis
Metabolites
For all disorders Ammonia in plasma (arterial blood preferable; use tourniquets minimally; do not request
muscular contractions; refrigerate blood and process at 4 °C; assay within 30 min of blood
drawing)
Amino acids in plasma (for routine analysis, draw blood 3–4 h after a meal; use Na heparin;
harvest plasma promptly; avoid hemolysis; freeze plasma if analysis is to be delayed, particularly
since glutamine is unstable; ASA may not be seen in certain amino acid analyzers; in these cases
it can be converted first to its anhydrides by heating; watermelon contains citrulline and can
increase its level)
Orotic acid in urine (if urine is refrigerated or frozen, it can precipitate out; heat urine at 37ºC
until precipitate dissolved)
Drug levels (benzoate, phenylacetate) in serum if monitoring is required
ASL deficiency Argininosuccinate in the urine (make certain that method detects ASA)
HHH syndrome Homocitrulline in the urine
Mutation analysis
For all disorders (apart from THAN) DNA
RNA (preferred for CPS1 genetics)
Enzyme analysis
Disorder Method Sample
NAGS deficiency Stable isotope dilution assay with GCMS Liverb
CPS1 deficiency Colorimetrica or radiometric OTC-coupled assay Liverb, small intestine
OTC deficiency Colorimetric assaya Liverb,c, small intestinec
ASS deficiency Radiometric assay (cultured fibroblasts) Skin fibroblasts, kidney, liverb
14
C-Citrulline incorporation (cultured fibroblasts)
Colorimetric ASL-arginase-coupled assay (liver)
ASL deficiency Colorimetric, arginase-coupled assay Skin fibroblasts, red blood cellsd,
(erythrocytes, liver, kidney) liverb, kidney
14
C-Citrulline incorporation (cultured fibroblasts)
ARG1 deficiency Colorimetric assay Red blood cells, liverb
14
HHH syndrome C-Ornithine incorporation Skin fibroblasts, liverb
Citrullinemia type II Transport activity assayed radiometrically in liposomes Artificial liposomes
incorporating the recombinant transporter expressed in
Escherichia coli
GS deficiency Colorimetric assay Liver
Transient hyperammonemia of the newborn Not an enzymatic disorder –
P5CS deficiency Incorporation of glutamate into protein proline was not Skin fibroblasts
diagnostic
Table adapted from Häberle et al. (2012)
Bold: first choice if analysis in more than one tissue is possible
a
Treatment with carbamylglutamate interferes with some colorimetric assays of citrulline
b
Needle biopsy (>10 mg) sufficient either for NAGS assay or for assay of the other five UC enzymes; Liver tissue should be snap frozen and kept
at −80 °C until analysis
c
Reliable in males but less so in females due to X-mosaicism in all tissues
d
Caution: conflicting results
4.9 Prenatal Diagnosis Table and Sample Requirements
Recommended analyses and sample requirements for prenatal diagnosis

Disorder Tests recommended
NAGS deficiency Mutation analysis on DNA from CVS or AFCa
CPS1 deficiency Mutation analysis on DNA from CVS or AFC
Enzyme analysis, late fetal liver biopsyb
OTC deficiency Mutation analysis on DNA from CVS or AFCc
Enzyme analysis, late fetal liver biopsyb, d
ASS deficiency Mutation analysis on DNA from CVS or AFC
Citrulline in amniotic fluid
Enzyme analysis, intact or cultured CVS, or cultured AFC
ASL deficiency Mutation analysis on DNA from CVS or AFC
Argininosuccinate and its anhydrides in amniotic fluid
Enzyme analysis, intact or cultured CVS, or cultured AFC
ARG1 deficiency Mutation analysis on DNA from CVS
Enzyme assay in fetal blood erythrocytes (mid-gestation sampling)
HHH syndrome Mutation analysis on DNA from CVS or AFC
Enzyme assay in CVS or cultured AFC
Citrullinemia type 2 Mutation analysis on DNA from CVS or AFC
GS deficiency Mutation analysis on DNA from CVS or AFC
Transient hyperammonemia of the newborn Not applicable
P5CS deficiency Mutation analysis on DNA from CVS or AFC
CVS chorionic villous sampling, AFC amniotic fluid cells
Bold: first choice
a
In case of a request for prenatal testing one should keep in mind that NAGSD is a treatable disorder
b
Described in single patients but not widely available and very limited experience
c
In the female fetus the genotype is only able to exclude OTC mutations. Because of the lyonization, it has no predictive value for the resulting
phenotype if affected.
d
Feasible in male, but interpretation not clear in females due to X-mosaicism
4.10 DNA Testing type II, in which carbohydrates are contraindicated, high-
dose intravenous glucose (with or without insulin) should
Mutation analysis is feasible in all disorders described in this be given to supply high energy and to prevent endogenous
chapter apart from THAN in which no gene locus is known. protein catabolism. The nitrogen scavengers sodium ben-
In all disorders, DNA from peripheral blood leukocytes can zoate and sodium phenylacetate/phenylbutyrate are given
be used for PCR and direct sequencing; due to the size of the to increase urinary nitrogen excretion bypassing the urea
CPS1 gene, some labs prefer using RNA from cultured fibro- cycle, being excreted as conjugates of glycine and glu-
blasts or from stimulated lymphocytes for mutation analysis. tamine, respectively (Batshaw et al. 2001; Enns 2010).
See also the table in Sect. 4.8. Administration of l-arginine (oral or intravenous) or
l-citrulline (oral) enhances residual urea cycle function
and urinary excretion of urea cycle intermediates (Brusilow
4.11 Treatment 1984). Carbamylglutamate administration (oral) restores
CPS1 function in NAGS deficiency. Nitrogen scavenger
Summary drugs and l-arginine are given at the beginning of treatment
Acute symptomatic hyperammonemia is always an emer- of acute hyperammonemia as boluses followed by continu-
gency that must be promptly treated (Häberle 2011). ous infusions (Häberle et al. 2012).
Treatment aims at minimizing ammonia production by pre- A central venous line is desirable for administration of
venting protein breakdown and at the same time increas- high glucose concentrations especially if parenteral nutrition
ing ammonia removal. Dietary protein should be withheld is needed for more than 1 or 2 days, also facilitating fluid
immediately but not for longer than 48 h (give thereafter management and continuous supply of nitrogen scavengers
at least essential amino acids). Except in citrullinemia and l-arginine (Häberle et al. 2012).
If plasma ammonia exceeds 400 μmol/L, extracorporeal et al. 2008), and in NAGS deficiency, which only requires
detoxification should be performed to remove ammonia, by oral carbamylglutamate, chronic treatment of all other
either venovenous hemodiafiltration (infants and children) or defects requires lifelong protein restriction, use of scaven-
hemodialysis (adults) (Picca et al. 2001). ger drugs and of l-arginine/l-citrulline, and preparation to
Except in citrullinemia type II, which is treated with cope with catabolic situations. Liver transplantation pro-
high-protein, low-carbohydrate diet and l-arginine and vides a definitive cure for all urea cycle disorders and for
pyruvate supplementation (Imamura et al. 2003; Mutoh citrullinemia type 2.
Emergency Treatment Table and Medication Requirements
Dosages of drugs to be used in acute hyperammonemia and acute decompensations

Sodium PBA/sodium N-Carbamylglutamate
Sodium benzoate (to be given phenylacetate (to be given l-Arginine hydrochloride (to be (only available as oral/
Disordera IV in glucose 10 %) IV in glucose 10 %) given IV in glucose 10 %) enteral drug)
NAGS deficiency 250 mg/kg as bolus in – 250 mg/kg (1.2 mmol/kg) as 100 mg/kg bolus per
90–120 min, then bolus in 90–120 min, then nasogastric tube then
maintenance 250–500 mg/kg/ maintenance 250 mg/kg/day 25–62.5 mg/kg every 6 h
dayb; >20 kg bw 5.5 g/m2/day (1.2 mmol/kg/day)
CPS1 deficiency Same 250 mg/kg as bolus Same –
and OTC deficiency in 90–120 min, then
maintenance 250–500 mg/
kg/dayb
ASS deficiency Same Same Same –
ASL deficiencyc Same Same 200–400 mg/kg (1–2 mmol/kg) –
as bolus in 90–120 min, then
maintenance 200–400 mg/kg/
day (1–2 mmol/kg/day)
ARG1 deficiencyd Same – Avoid –
HHH syndrome Same Same – –
Citrullinemia type 2 – – 10–15 g/daye –
GS deficiency Not applicable
Transient 250 mg/kg as bolus in – 250(–400) mg/kg (1–2 mmol/ ?
hyperammonemia 90–120 min, then kg) as bolus in 90–120 min,
of the newborn maintenance 250–500 mg/kg/ then maintenance 250 mg/kg/
dayb day (1.2 mmol/kg/day)
P5CS deficiency Not applicable
Maximal daily drug dosages: sodium benzoate 12 g/day, sodium PBA 12 g/day, l-arginine 12 g/day (15 g/day in citrullinemia type 2)
Note: The doses indicated can be used at the start of treatment but must be adapted depending on plasma ammonia and amino acids
Sodium benzoate and sodium PBA/phenylacetate should be given in parallel in severe acute decompensation. In less severe cases, a stepwise
approach with initial sodium benzoate and if hyperammonemia persists or worsens, the addition of sodium PBA/phenylacetate can be chosen
a
In undiagnosed patients, consider additional use of carnitine 100 mg/kg IV, hydroxycobalamin 1 mg IM/IV, and biotin 10 mg IV/PO
b
If on hemodialysis/hemodiafiltration, maintenance doses should be increased by 50% without exceeding the top limit given
c
In ASL deficiency, l-arginine therapy for acute decompensations might be sufficient for some patients
d
The risk for acute hyperammonemic decompensation is low in ARG1 deficiency
e
Limited data in literature; dose according to Imamura et al. (2003)
Standard Treatment Table and Medication Requirements
Dosages of drugs to be used per orally for long-term treatment

Sodium Carbamyl
benzoatea Sodium PBAa, b, c l-Argininea (hydrochloride L-Citrullinea glutamatea
Disorder Diet (mg/kg/day) (mg/kg/day) and/or free base) (mg/kg/day) (mg/kg/day) (mg/kg/day)
NAGS deficiency Normal – – – – 10–100
CPS1 deficiency Protein-restricted Up to 250d, e <20 kg, up to 250d, e <20 kg, 100–200d mg/kg/day 100–200f –
max. 12 g/day >20 kg, 5 g/m2/day or, 0.5–1 mmol/kg/day Max. 6 g/day
max. 12 g/day >20 kg, 2.5–6 g/m2/day
max. 6 g/day
OTC deficiency Protein-restricted Same Same Same as for CPS1 deficiency 100–200f –
Max. 6 g/day
ASS deficiency Protein-restricted Same Same <20 kg, 100–300d,e mg/kg/day – –
or, 0.5–1.5 mmol/kg/day
>20 kg, 2.5–6 g/m2/daye
max. 8 g/day
ASL deficiency Protein-restricted Same – Same as for ASS deficiency – –
ARG1 deficiency Protein-restricted Same Same – – –
HHH syndrome Protein-restricted Same Same <20 kg, 100–200d mg/kg/day 100–250f –
>20 kg, 2.5–6 g/m2/day Max. 6 g/day
max. 6 g/day
Citrullinemia type 2 Low-carbohydrate For children: fat-soluble vitamins and use of lactose-free formula
and protein-rich For adults: l-arginine, pyruvate, liver transplantation
GS deficiency Normal No treatment available (consider oral/enteral use of glutamine)
Transient Not applicable
hyperammonemia
of the newborn
P5CS deficiency Normal Consider ornithine and/or arginine supplementation
All medications should be divided into three to four doses daily taken with meals and distributed as far as possible throughout the day. Food intake
and protein intake is also recommended to be divided in frequent small meals, considering a light evening snack to minimize night fasting
catabolism
a
100 mg equal 0.694 mmol sodium benzoate; 0.537 mmol sodium PBA; 0.475 mmol arginine hydrochloride; 0.574 mmol arginine base; 0.571 mmol
citrulline; 0.532 mmol carbamylglutamate, respectively
b
PBA is the second choice drug for long-term treatment and should be given in patients not responsive to benzoate alone
c
Glycerol phenylbutyrate is being introduced as an alternative to Na phenylbutyrate. One ml delivers 6.22 mmol of phenylbutyrate
d
Serum/plasma levels of benzoate/PBA and plasma levels of arginine should be monitored
e
In some patients higher doses are needed, according to expert advice
f
If citrulline is given, there is usually no need for concomitant use of l-arginine
Experimental Treatment can be performed. Currently, clinical studies are ongoing

In severe urea cycle disorders, liver cell transplantation that evaluate the effect of these experimental approaches
and more recently stem cell transplantation have been and patients should be included in studies if this treatment
suggested as bridging therapy until liver transplantation is considered.
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O, Saheki T (2003) Effectiveness of carbohydrate-restricted diet
Batshaw ML, MacArthur RB, Tuchman M (2001) Alternative path-
and arginine granules therapy for adult-onset type II citrullinemia: a
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with and without the disease. Hepatol Res 26:68–72
Brusilow SW (1984) Arginine, an indispensable amino acid for
Leonard JV (2001) The nutritional management of urea cycle disorders.
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Disorders of Glycine, Serine, GABA,
and Proline Metabolism 5
Johan L.K. Van Hove and Janet A. Thomas
Contents Summary
5.1 Glycine Disorder .............................................................. 63 In addition to the role as components of protein synthesis,
5.2 Serine Disorders ............................................................... 64 several amino acids have other functions in the brain such as
building blocks of other brain molecules or a role in neuro-
5.3 Gamma-Aminobutyric Acid (GABA) Disorders .......... 65
transmission. Disorders in catabolism of glycine and of proline
5.4 Proline Disorders ............................................................. 66 are known. The disorders of the synthesis of serine and proline
5.5 Disorders of the Renal Handling of Proline, cause severe abnormalities. Serine is required for the synthe-
Hydroxyproline, and Glycine.......................................... 68 sis of white matter compounds such as specialized lipids, and
5.6 Nomenclature ................................................................... 68 its deficiency results in severe hypomyelination. Proline is
required for the synthesis of connective tissue proteins, and
5.7 Metabolic Pathways ......................................................... 69
its deficiency results in laxity of skin and joints. Early treat-
5.8 Signs and Symptoms ........................................................ 72 ment of synthetic defects such as serine has shown promise to
5.9 Diagnostic Flow Charts ................................................... 77 avoid severe symptoms. Disturbance of the neurotransmitter
5.10 Specimen Collection and Interpretation Pitfalls ........... 78 roles of GABA, glycine, and 4-hydroxybutyric acid results in
severe neurological symptoms. The pathophysiology of these
5.11 Normal and Pathological Values .................................... 79
disorders is complex, as has been shown in the mouse model
5.12 Prenatal Diagnosis ........................................................... 80 of 4-hydroxybutyric aciduria. In most disorders, diagnostic
5.13 Treatment ......................................................................... 81 studies rely on careful measurement of metabolites using age-
appropriate reference ranges, followed by molecular analysis.
5.14 Treatment Summary........................................................ 82
References .................................................................................... 82
5.1 Glycine Disorder
Nonketotic hyperglycinemia (glycine encephalopathy). The

primary disorder of glycine is a deficiency of the main cata-
bolic enzyme, the glycine cleavage enzyme. Glycine is part
of many biochemical pathways, but deficiency of the glycine
cleavage enzyme removes the main catabolic breakdown of
glycine resulting in increased levels of glycine. The disorder
is known as nonketotic hyperglycinemia (NKH) or glycine
encephalopathy. All forms of the disorder are characterized
by cerebral dysfunction.
The glycine cleavage enzyme breaks glycine down into car-
J.L.K. Van Hove (*) • J.A. Thomas bon dioxide and ammonia, and a methyl group is transferred to
Clinical Genetics and Metabolism, The Children’s Hospital, tetrahydrofolate creating methylene-tetrahydrofolate (Fig. 5.1).
Education 2 South, L28-4122, 13121 E. 17th Avenue, The enzyme consists of four subunits: the P-protein (pyridoxal-
Aurora, CO 80045, USA
e-mail: johan.vanhove@childrenscolorado.org; containing subunit), the H-protein (hydrogen carrier protein),
janet.thomas@childrenscolorado.org the T-protein (tetrahydrofolate-requiring protein), and the
64 J.L.K. Van Hove and J.A. Thomas
l-protein (lipoamide dehydrogenase protein). The P-protein tion of blood or serum as demonstrated by absence of red
requires pyridoxal-phosphate, and disorders that affect the blood cells and normal protein content (Korman and Gutman
availability of pyridoxal-phosphate (such as PNPO) result in 2002). Once CSF glycine is elevated, an increased ratio of
secondary deficiency of the enzyme activity. The H-protein CSF/plasma glycine aids in discrimination of nonketotic
is lipoylated and disorders in the biogenesis of the lipoylation from ketotic hyperglycinemia (Perry et al. 1975). Organic
result in variant forms of nonketotic hyperglycinemia. acidurias must be excluded. Valproate use can iatrogenically
Typical nonketotic hyperglycinemia (NKH) can be divided raise glycine levels including the ratio.
into three main clinical presentations based on the severity of Diagnosis is confirmed by mutation analysis of the GLDC
the condition. The most severe form is classic, severe NKH. gene encoding the P-protein and the AMT gene encoding the
Patients with residual activity, but presenting in the first year T-protein (Kure et al. 2006) (Fig. 5.5). No mutations have
of life, have attenuated NKH. Patients presenting later than been identified in the GCSH gene encoding the H-protein or
1 year of age usually have mild NKH. The primary differ- in the L-protein. Up to 20 % of mutations in the GLDC gene
ence is related to outcome and is based on the amount of are exonic deletions which must be identified by either
residual activity of the particular mutation (Dinopoulos et al. MLPA or array CGH. Patients with mutations that confer
2005; Hennermann et al. 2012). Signs and symptoms of substantial residual activity such as A802V and A389V have
NKH are listed in Table 5.1. attenuated or mild NKH, whereas patients with two muta-
Patients with NKH most often present neonatally in the tions without any activity (deletions, nonsense mutations, or
first week of life (Hennermann et al. 2012). They develop frameshift mutations) have severe NKH. About 4 % of
lethargy, fail to feed, and progress to coma with frequent hic- patients do not have mutations in these two genes. Enzyme
cupping and seizures. They have severe hypotonia. They assay is possible in liver biopsy and prenatally in uncultured
often have a burst suppression pattern on EEG. These chorionic villi although a small rate of false-negative diag-
patients spontaneously recover from respiratory failure and nosis is possible.
regain spontaneous breathing within the first 3 weeks of life. Some patients presenting with neonatal seizures have
Patients presenting during infancy have hypotonia, lethargy, transient elevation of glycine in the neonatal period. Other
and seizures (spasms or myoclonic seizures) with either mul- patients have been identified by newborn screening without
tifocal epilepsy or hypsarrhythmia on EEG. any symptoms with very elevated glycine levels. No muta-
Patients with severe NKH develop spasticity in the first tions have been found in the GLDC or AMT genes in any of
6 months of life. They develop progressively therapy- these patients. This likely represents a phenocopy.
resistant seizures, requiring multiple anticonvulsants. They Variant forms of NKH have atypical presentations with
lose any developmental skills with only spontaneous smiling either less or more severe neurological involvement and
maintained. Most require tube feeding. sometimes with leukodystrophy or pulmonary hypertension.
Patients with attenuated NKH have developmental delay These patients can have additional symptoms such as lactic
(IQ 20–70) but still make progress (Dinopoulos et al. 2005; acidosis, cardiomyopathy, or leukodystrophy. The pyruvate
Hennermann et al. 2012). They may learn to sit, walk, and com- dehydrogenase and the 2-ketoglutarate dehydrogenase
municate. They have better receptive than expressive language enzyme are also deficient. The genes responsible are involved
skills and often use signing. They remain hypotonic, with cho- in the lipoate synthesis, either in the lipoate synthase gene
rea, hyperactivity, and intermittent ataxia. Half the patients LIAS or in the synthesis of its iron-sulfur cluster such as
have seizures, which are often treated with a single anticonvul- BOLA3, GLRX5, and NFU1 (Cameron et al. 2011; Navarro-
sant. They can have intermittent episodes of severe lethargy Sastre et al. 2011; Mayr et al. 2011; Baker et al. 2012).
and depression, which are related to increased glycine levels.
Patients with mild NKH have a range of learning disabilities
spanning from mild mental retardation to normal intelligence. 5.2 Serine Disorders
They have attention deficit hyperactivity disorder and inter-
mittent episodes of ataxia, chorea, lethargy, and poor school Serine is obtained from diet and is synthesized endogenously
performance. They usually do not have a seizure disorder. starting from the glycolytic intermediate 3-phosphoglycerate
On brain MRI almost all patients with severe NKH have in three steps using the enzymes phosphoglycerate dehydro-
diffusion restriction of the posterior limb of the internal cap- genase (gene PHGDH), phosphoserine aminotransferase
sule and/or the long tracts in the brain stem, which disappears (PSAT1), and phosphoserine phosphatase (PSPH) (Fig. 5.2).
in the first months. Half the patients with severe NKH have Three disorders of serine biosynthesis are known affecting
brain malformations: agenesis or severe hypoplasia of the cor- each of the steps in this serine biosynthetic pathway
pus callosum, simplification of gyration, and hydrocephalus (Tabatabaie et al. 2010). Characteristic for all three disorders
with posterior fossa malformations (Hennermann et al. 2012). is the decreased biosynthesis of serine resulting in serine defi-
The diagnosis is made by finding elevated glycine in ciency. Serine is an essential component of phosphatidylser-
serum and in CSF. The CSF should be without contamina- ine, sphingolipids, and ceramides and is necessary for myelin
5 Disorders of Glycine, Serine, GABA, and Proline Metabolism 65
development (de Koning et al. 2003). l-Serine is converted 5.3 Gamma-Aminobutyric Acid (GABA)
through the racemase to d-serine, which is an NMDA recep- Disorders
tor activator. Through the serine hydroxymethyltransferase
enzyme, serine is converted into glycine and provides methy- Two disorders affect the catabolism of GABA: GABA trans-
lene-tetrahydrofolate which is important for thymidine aminase deficiency and succinic semialdehyde dehydroge-
synthesis. nase deficiency (Fig. 5.3).
3-Phosphoglycerate dehydrogenase deficiency (PHGDH) GABA transaminase deficiency. Two families have been
is an autosomal recessive condition and the most frequently reported with GABA transaminase deficiency (Jaeken et al.
reported cause of serine deficiency syndrome (Table 5.2 ). 1984; Tsuji et al. 2010) (Table 5.5). The patients had axial
In the severe infantile form, it presents in the neonatal hypotonia, spasticity, severe convulsions, and feeding
period with congenital microcephaly, intractable seizures problems necessitating tube feeding. Patients in the first
starting shortly after birth, and severe psychomotor retarda- family had accelerated growth and increased growth hor-
tion. A wide variation of seizure types are reported including mone secretion. In the second family, MRI showed diffu-
West syndrome. The EEG patterns include hypsarrhythmia, sion restriction in the internal and external capsule and
multifocal epilepsy, and Lennox-Gastaut syndrome. They subcortical white matter areas (Tsuji et al. 2010).
develop spastic tetraparesis, with adducted thumbs, and Biochemically, patients have elevation of free GABA in
hyperexcitability. Variable symptoms observed in some cerebrospinal fluid but also elevation of homocarnosine
patients include cataracts, hypogonadism, megaloblastic and β-alanine. Elevated GABA can be recognized on mag-
anemia, and nystagmus. The MRI of the brain shows a strik- netic resonance spectroscopy (Tsuji et al. 2010). The
ing reduction in the volume of the white matter and very enzyme activity was deficient in liver and lymphocytes,
delayed to absent myelination. The cerebral white matter on and mutations were identified in the ABAT gene.
T2 has a higher signal intensity than the cortex, indicative of Succinic semialdehyde dehydrogenase deficiency
a lack of myelin. There is also cortical and subcortical atro- (SSADH) or 4-hydroxybutyric aciduria (Table 5.6). Patients
phy. MRS shows a decreased level of N-acetylaspartate/cre- with succinic semialdehyde dehydrogenase deficiency
atine and increased choline/creatine in the white matter. accumulate succinic semialdehyde derived from GABA
Rare late-onset patients with milder mutations have been transamination, which is converted by succinic semialde-
reported. Two teenagers had absence seizures, moderate devel- hyde reductase into 4-hydrobybutyric acid and excreted in
opmental delay, and normal head circumference. An adult urine. Most patients present in the first 2 years of life, and
patient presented with a chronic axonal sensorimotor polyneu- whereas 26 % of patients have problems in the neonatal
ropathy compatible with Charcot-Marie-Tooth type 2, which period, an equal number have normal early development
had started at age 8 years. He also had congenital cataracts, (Gibson et al. 1997).
mild psychomotor retardation, and slight cerebellar ataxia. These patients present with a static neurological picture
Biochemically, low values of serine and of glycine are of developmental delay and intellectual disability with
recorded in plasma after an overnight fast and in CSF, with prominent deficits in expressive language, motor delay,
the CSF values more reliable (Fig. 5.6). Also, hypotonia, and nonprogressive ataxia, each present in more
5-methyltetrahydrofolate can be low in CSF. Mutations than 70 % of patients (Gibson et al 1997; Pearl et al. 2003).
throughout the protein have been reported, but a common Neuropsychiatric symptoms are frequent and include
mutation, p.V490M, has been reported in several families hyperactivity, inattention, and anxiety. Sleep disorders are
from variable ethnicities. very common and include excessive daytime sleepiness,
Phosphoserine aminotransferase deficiency (PSAT1) was prolonged REM latency, and reduced REM sleep (Pearl
reported in a single family (Hart et al. 2007) (Table 5.3 ). et al. 2009). Seizures are present in 48 % of patients con-
They had acquired microcephaly, intractable seizures since sisting mostly of generalized tonic-clonic and atypical
early infancy, and hypertonia. Brain MRI showed general- absence seizures and myoclonic seizures in a minority.
ized atrophy, a hypoplastic cerebellar vermis, and poor white EEG abnormalities were noted in 26 % of patients. About
matter development. Serine and glycine were deficient in 10 % of patients have a degenerative course with myoclo-
plasma and CSF. Diagnosis was confirmed by sequencing, as nus and extrapyramidal movements of chorea and dystonia
the enzyme assay was not deficient. (Pearl et al. 2009). Neuroimaging shows increased T2 sig-
Phosphoserine phosphatase deficiency (PSPH) has been nal intensity in the globus pallidus, cerebellar dentate
reported in a single patient who also had Williams syndrome nucleus and brain stem, and subcortical white matter
(Jaeken et al. 1997) (Table 5.4). The child had growth and (Gibson et al. 1997; Pearl et al. 2003). There may also be
psychomotor retardation, but no seizures. His fasting plasma cerebellar and cerebral atrophy. NMR spectroscopy is usu-
serine levels were low–normal, and his CSF serine levels ally normal, unless special edited sequences for GABA and
were low. He was homozygous for a missense mutation that GABA metabolites are done, which show increases in these
decreased the enzyme activity. compounds (Pearl et al. 2003).
The diagnosis is suspected on finding 4-hydoxybutyric contrast to hyperprolinemia type II. There is a large varia-
acid on urine organic acids analysis and quantified by stable tion of the serum levels of proline in the same patient. There
isotope dilution assay. Other accumulating metabolites is little transport of proline across the blood-brain barrier
include the β-oxidation product 3,4-dihydroxybutyric acid and brain proline levels reflect primarily endogenous syn-
and the condensation product 4,5-dihydroxyhexanoic acid. thesis and catabolism. Spinal fluid proline levels were ele-
Secondary elevations in glycine, dicarboxylic acids, and vated 5-fold in a hyperprolinemic patient with a known
3-hydroxypropionic acid are sometimes seen. The CSF total severe genotype.
and free GABA is mildly elevated, and CSF glutamine can A recurrent 350 kb deletion, encompassing the PRODH
be decreased. Some patients also excrete an increased and DGCR6 genes, mediated by flanking low copy repeats is
amount of D-2-hydroxyglutaric acid formed through the present at a frequency of 1/250 in the general population.
hydroxyacid-oxoacid dehydrogenase enzyme. False-positive Many polymorphic mutations exist in the population that
results are seen with 4-hydroxybutyric acid use as a sedative usually do not affect the proline oxidase activity for more
drug. Confirmation can be done by enzyme assay or molecu- than 30 % reduction in activity. Most patients with velocar-
lar analysis of the ALDH5A1 gene (Akaboshi et al. 2003). diofacial syndrome are hemizygous deleted for the PRODH
gene (Guilmate et al. 2010). A subset of these patients has
increased proline levels, and in half of these patients, a sec-
5.4 Proline Disorders ond mutation in the PRODH gene was identified, mostly in
those patients with the highest proline levels. Mutations in
There are three types of disorders that affect proline metabo- the PRODH gene have been classified into three categories
lism (Fig. 5.4). The first group of disorders affects the catab- based upon the effect on proline oxidase activity in vitro:
olism of proline and is characterized by excessive proline. type I <30 % residual activity, type II >30 and <70 % resid-
This includes the disorders hyperprolinemia type I caused by ual activity, and type III >70 % residual activity (Bender
deficiency of proline oxidase and hyperprolinemia type II et al. 2005). There is a weak, but clear correlation between
caused by Δ1-pyrroline-5-carboxylate dehydrogenase defi- residual activity and levels of proline and phenotype.
ciency. The second group of disorders is characterized by a Some patients with hyperprolinemia have been asymp-
deficiency of proline due to defects in proline synthesis. tomatic giving the impression that this may be a non-disease
These disorders include deficiency of the first step Δ-1- and that the observed phenotype may be due to selection
pyrroline-5-carboxylate synthase (P5CS) and of the second bias. This is likely the case for the originally reported renal
step Δ-1-pyrroline-5-carboxylate reductase. The last group symptoms. Multiple series of patients, however, with a neu-
of disorders includes accumulation of proline-containing rological phenotype consisting of mild to moderate mental
peptides. This group is exemplified by prolidase deficiency. retardation, psychosis, and therapy-resistant epilepsy have
Finally, renal transporters of proline and glycine are involved been reported (Di Rosa et al. 2008). The phenotype seems to
in the benign condition of iminoglycinuria. be most commonly seen in patients with very high serum
levels of proline and the presence of severe mutations, such
Disorders of Proline Catabolism as a type I mutation L441P or deletions. In addition, in
Hyperprolinemia type I (Proline oxidase deficiency). Proline patients with velocardiofacial syndrome, increased proline is
is catabolized by proline oxidase into Δ-1-pyrroline-5- an independent risk factor for lower IQ, psychotic pheno-
carboxylate (P5C). This enzyme is primarily present in the type, and seizures (Raux et al. 2007) (Table 5.7).
liver, kidney, and brain. It has a conserved α8β8 barrel struc- A role of proline in the brain and particularly in the gluta-
ture in which FAD and proline bind. The PRODH gene is minergic synapse has been documented, and a mouse model
located in the 22q11 region and is often involved in larger exists.
deletions of the velocardiofacial syndrome (22q11 microde- Hyperprolinemia type II is described in the chapter on
letion syndrome). A pseudogene ψPRODH is located 1.4 Mb pyridoxine metabolism.
telomeric on chromosome 22 and has a 13.1 kb internal dele-
tion (exon 2–7) and contains several missense mutations, Disorders of Proline Synthesis
which have been transferred to the PRODH gene by apparent Δ-1-Pyrroline-5-carboxylate synthase (P5CS) deficiency. The
gene conversion (Bender et al. 2005). first step in proline synthesis is the synthesis of Δ1-pyrroline-
Hyperprolinemia type I is associated with an increase in 5-carboxylate. Δ-1-Pyrroline-5-carboxylate synthase (P5CS)
plasma proline. A continuum of plasma levels of proline is an enzyme with a dual action. The N-terminal γ-glutamyl
ranging from the upper limit of normal to >1,500 μM have kinase domain catalyzes the phosphorylation of glutamate
been reported. In hyperprolinemia type I, proline levels are with ATP to γ-glutamylphosphate, an unstable intermediate,
less elevated than in hyperprolinemia type II, and in addi- and the C-terminal γ-glutamylphosphate reductase domain
tion, there is no increase in P5C in hyperprolinemia type I in catalyzes the subsequent reduction with NAD(P)H to
γ-glutamic semialdehyde, which is in tautomeric nonenzy- other biochemical abnormalities (Bicknell et al. 2008). Skin
matic equilibrium with Δ-1-pyrroline-5-carboxylate (P5C). biopsy showed thin dermis with collagen bundles of vari-
P5C is then acted upon by the subsequent enzymes, ornithine able diameters and rarefaction and size decrease of elastic
aminotransferase, to produce ornithine or by Δ-1-pyrroline-5- fibers, but normal biochemical analysis of collagen fibers
carboxylate reductase to produce proline. P5CS has two iso- (Martinelli et al. 2012; Skidmore et al. 2011). Brain imaging
forms caused by alternative splicing and differing in two showed atrophy and thin corpus callosum and a decreased
amino acids on the N-terminal side. Both isoforms have P5CS creatine on MRS (Martinelli et al. 2012).
enzymatic activity. A short isoform is highly active in the gut A similar phenotype was seen in patients with missense
and participates in synthesis of ornithine and arginine, with mutations in the γ-glutamyl kinase domain affecting cataly-
feedback noncompetitive inhibition by ornithine and stimula- sis (e.g., R84Q or G39R) (Baumgartner et al. 2005; Martinelli
tion by N-acetylglutamate (Hu at al. 1999; Pérez-Arellano et al. 2012), those affecting the multimerization such as
et al. 2010). A long isoform is present in most tissues for the H784Y (Bicknell et al. 2008), as in those where the mutation
synthesis of proline and is not inhibited by ornithine. affected splicing and protein abundance (e.g., c.1923+1G>A)
There have been four reports of patients with a deficiency (Skidmore et al. 2011). Incorporation of 3H-glutamate into
in the enzyme Δ-1-pyrroline-5-carboxylate synthase. Two protein proline by fibroblasts was decreased in some, but not
siblings presented with chronic vomiting and failure to all (Baumgartner 2005; Bicknell et al. 2008).
thrive (Baumgartner et al. 2005). They had developmental Δ-1-Pyrroline-5-carboxylate reductase (P5CR) defi-
delay with a DQ of 40. They had hypotonia, severe joint lax- ciency. The mitochondrial Δ-1-pyrroline-5-carboxylate
ity, including pes planus and dislocated hips, skin hyper- reductase (P5CR) enzyme catalyzes the reduction with
elasticity, and osteoporosis. They developed bilateral NADPH of pyrroline-5-carboxylate to proline. Two paralogs
zonular cataracts. In childhood, their mental and motor exist, PYCR2 and PYCRL.
skills deteriorated, and they had abnormal behavior, severe Reported patients presented with intrauterine growth
hypotonia, dystonia, a pyramidal syndrome, and an axonal retardation, lax and wrinkly skin, connective tissue weak-
motor neuropathy. Another patient at age 6 months had joint ness, and mild to moderate mental retardation. Several
laxity with pes planus and coxa valga, bilateral inguinal her- patients had osteopenia without bone fractures, hip disloca-
nia, lax but not hyperelastic skin showing superficial veins, tion, inguinal hernias, camptodactyly, and corpus callosum
sparse hair, subcapsular cataracts, and clonic seizures at day agenesis (Reversade et al. 2009; Guernsey et al. 2009;
10 of age (Martinelli et al. 2012). Imaging studies showed Yildirim et al. 2011; Kouwenberg et al. 2011; Lin et al.
wormian bones, osteopenia, and limb shortness. Four 2011). Rare symptoms are wormian bones, cataracts, and
affected siblings presented with very wrinkled and lax, but dystonia. Visible subcutaneous veins and wrinkly skin can
not hyperelastic, skin, which disappeared with age by ado- resemble a progeroid appearance. Skin wrinkling is most
lescence (Bicknell et al. 2008). There was short stature and pronounced on the dorsum of hands and feet. Mild dysmor-
joint laxity, and two had hip dislocation. They had congeni- phic features include triangular face, broad forehead, micro-
tal microcephaly and static neurodevelopmental delay since cephaly, sagging cheeks, large fontanel, and large ears.
early infancy, with seizures in two individuals. Brain MRI Electron microscopy of skin biopsy shows rarefaction and
showed paucity of the white matter. One child had cataracts. fragmentation of elastic fibers (Reversade et al. 2009). Serum
Finally, a last patient was reported with prenatal contrac- proline levels and urine proline excretion were within the
tures, short stature, and microcephaly. He had progeroid normal range (Reversade et al. 2009; Guernsey et al. 2009).
features, a thin and wrinkled skin with visible veins and Fibroblasts show abnormal mitochondrial morphology,
sparse hair (Skidmore et al. 2011). He had cloudy cornea mitochondrial fragmentation, and cell death with oxidative
with absent Bowman’s membrane. The brain MRI showed stress (Reversade et al. 2009). The diagnosis is made by gene
tortuous blood vessels. He developed seizures at 2 weeks of sequencing with multiple mutations reported (Reversade
age, poor feeding, bilateral inguinal hernias, and severe fail- et al. 2009; Guernsey et al. 2009; Yildirim et al. 2011;
ure to thrive. He made little developmental progress and Kouwenberg et al. 2011).
died at age 6 months. Differential diagnostic syndromes for the P5CR synthase
Patients in two families had metabolic abnormalities: they and P5CR reductase genes include the clinical cutis laxa syn-
developed low fasting plasma levels of proline, citrulline, dromes of geroderma dysplasticum, de Barsy syndrome, and
arginine, and ornithine (Baumgartner et al. 2005; Martinelli autosomal recessive cutis laxa. Mutations in the following
et al. 2012). Postprandial plasma levels and urine levels genes have been described in these closely related syn-
were normal, emphasizing the subtlety of the abnormali- dromes: ATP6V0A2 (ATPase H+ transporting V0 subunit 2),
ties. There was fasting hyperammonemia, which paradoxi- COG7CDG, GORAB (SCYL1 binding protein), EFEMP2
cally decreased postprandially. In contrast, the third family (fibulin 4), FBLN5 (fibulin 5), ELN (elastin), and ATP7A for
showed only mild postprandial low ornithine levels, but no Menkes and occipital horn syndrome. Testing for copper,
ceruloplasmin, and glycosylation of glycoprotein analysis the urine. The most common are glycylproline and alanylp-
(e.g., transferrin and apo-CIII), and a fasting proline level are roline. Iminodipeptides can also be found in hypophospha-
good tests to consider prior to molecular testing in cutis laxa temic rickets or in hyperparathyroidism but at much lower
syndromes particularly when combined with developmental levels than in prolidase deficiency (Kelly et al. 2010). Proline
delay (Reversade et al. 2009) (Fig. 5.7). levels in serum are normal. In red blood cells, prolidase
activity measured after activation with manganese, is
Disorders of Proline Peptide Metabolism reduced. It is also reduced in serum, leucocytes, and fibro-
Prolidase deficiency. Prolidase, also called peptidase D, is a blasts. Mutations in the gene PEPD encoding peptidase D
peptidase that cleaves carboxy-terminal proline residues, are found and have been summarized (Lupi et al. 2006).
whereas the enzyme prolinase cleaves peptides with amino-
terminal proline residues. The proline-containing peptides
are particularly frequent in connective tissue proteins such as 5.5 Disorders of the Renal Handling of
collagen. The gene for prolidase, PEPD, has 15 exons. Proline, Hydroxyproline, and Glycine
There is substantial phenotypic variability between and
within families, even with the same genotype (Lupi et al. Iminoglycinuria. Iminoglycinuria is characterized by the
2006; Falik-Zaccai et al. 2010) (Table 5.8). Typical features excretion of large quantities of the imino acids, proline, and
are dermatologic and immune. Skin lesions are pruritic hydroxyproline and of glycine in the urine. The intestinal
eczematous lesions, erythematous papular eruptions, telangi- absorbance of these amino acids is unaffected. The condition
ectasis, lymphedema, impetigo-like eruptions, and necrotic is generally asymptomatic. Patients identified through new-
papules. Characteristic chronic, slowly healing ulcerations, born screening have been asymptomatic. It is generally
particularly on the legs and feet, develop. They are tender, assumed that the symptoms initially reported with the condi-
covered with granulomatous tissue, and can be a source of tion of iminoglycinuria or glycinuria likely reflect ascertain-
infection. Splenomegaly, recurrent pulmonary infections, and ment bias (Coşkun et al. 1993; Bröer et al. 2008). The
chronic ear and sinus infections, sometimes mimicking cystic condition is inherited as a codominant trait. The primary gene
fibrosis, are often reported. A malar rash and the presence of involved is the high-affinity transporter for proline, hydroxy-
anti-DNA antibodies can lead to a diagnosis of systemic lupus proline, and glycine in the proximal renal tubulus SLC36A2
erythematosus (Klar et al. 2010). Mild to severe mental retar- (Bröer et al. 2008). Patients who are homozygous for a null
dation is common. Facial dysmorphisms include low hairline allele in SLC36A2, such as IVS1+1G>A, have iminoglycin-
and hirsutism, ocular hypertelorism, micrognathia, mandibu- uria, whereas the heterozygous carriers have isolated glycin-
lar protrusion, and high palate. Rare symptoms include ane- uria with variable expression. Patients who have a mild
mia and thrombocytopenia, likely due to hypersplenism. Most mutation in SLC36A2 such as G87V have iminoglycinuria if
patients present early, but late-onset cases are known. Clusters they also are heterozygous for a polymorphism in SLC6A19
of patients have been reported in Ohio Amish families and in which impairs the distal renal tubular transport of the imino
Druze and Palestinian families (Falik-Zaccai et al. 2010). acids or in SLC6A18 which affects glycine transport (Bröer
Patients excrete multiple dipeptides and tripeptides et al. 2008). There is no need for treatment and no specific
containing proline and to a lesser extent hydroxyproline in treatment for this condition exists. See also Chap. 44.
5.6 Nomenclature
Chromosomal
No. Disorder Alternative name Abbreviation Gene symbol localization Affected protein OMIM no. Subtype
5.1 Nonketotic Glycine cleavage NKH GLDC; 9p24.1, P-protein; 238300, All forms
hyperglycinemia deficiency AMT; GCSH 3p21.31, T-protein; 238310,
16q23.2 H-protein 238330,
605899
5.2 Phosphoglycerate PHGDH PHGDH PHGDH 1p12 Phosphoglycerate 606879, All forms
dehydrogenase deficiency dehydrogenase 601851
deficiency
5.3 Phosphoserine PSPHD PSPH 7p11.2 Phosphoserine 172480, All forms
phosphatase deficiency phosphatase 614023
5.4 Phosphoserine PSAT deficiency PSAT PSAT1 9q21.2 Phosphoserine 610992, All forms
aminotransferase aminotransferase 610936
deficiency
Chromosomal
No. Disorder Alternative name
Abbreviation Gene symbol localization Affected protein OMIM no. Subtype
5.5 GABA transaminase GT ABAT 6p13.2 GABA 137150, All forms
deficiency transaminase 613163
5.6 Succinic semialdehyde
4-Hydroxybutyric SSADH ALDH5A1 6p22.3 Succinic 271980, All forms
dehydrogenase aciduria semialdehyde 610045
deficiency dehydrogenase
5.7 Hyperprolinemia I Proline oxidase PRODH PRODH 22q11.2 Proline oxidase 239500, All forms
deficiency 606810
5.8 Pyrroline-5-carboxylate Hypoprolinemia P5CS PYCS 10q24.1 Pyrroline-5- 138250, All forms
synthase deficiency type I carboxylate 219150
synthase
5.9 Δ1-pyrroline-5- Hypoprolinemia P5CR PYCR1 17q25.3 Δ1-pyrroline-5- 179035, All forms
carboxylate reductase type II carboxylate 612940,
deficiency reductase 614438
5.10 Prolidase deficiency Iminopeptiduria PEPD 19q13.11 Peptidase D 170100, All forms
613230
serine
NFU1
SHMT IBA57
ISC1,2
HSPAS9 BOLA3
NH3 glycine HSCB
H4-folate CH2=floate
GSH
GLRX5
NH2 Fe-S
T cluster
AMT SAM
CH2
S SH LIAS
lipoyl-H octanoyl-H opo-H

H
GCSH HS HS
LIPT2
P LIPT1
CO2 GLDC Apo-H
L NAD+
GMP-lipoate octanoyl-mtACP
S S
pyridoxal-P
NH2 ACSM
NADH + H+
CH2
mt lipoate mt FAS II
COOH
glycine
Fig. 5.1 Biochemistry of glycine metabolism. CH2 = folate is methy- GLRX5” (Baker et al. 2013) Brain 2013:10.1093/brain/awt328 with
lene-tetrahydrofolate. Reproduced from “Variant non-ketotic hypergly- permission from Oxford University Press
cinaemia is caused by mutations in LIAS, BOLA3, and the novel gene
Fig. 5.2 Biochemistry of serine glycolysis glycolysis

metabolism. PHGDH is glucose D-glyceraldehyde-3-phosphate pyruvate
3-phosphoglycerate NAD+
dehydrogenase, PSAT1 is PHGDH
phosphoserine aminotransferase,
PSPH is phosphoserine NADH + H+
phosphatase, SHMT is serine 3-phosphohydroxypyruvate
hydroxymethyltransferase, THF is
tetrahydrofolate, DHF is glutamate
dihydrofolate, DHFR is PSAT1
dihydrofolate reductase, TYMS is PLP
thymidylate synthase 2-ketoglutarate
3-phosphoserine
H2O
PSPH
Mg2+
Pi
SHMT
glycine L-serine
SR
Methylene-THF THF D-serine

DHFR
DHF
dUMP dTMP phosphatidyl-serine
TYMS sphingolipids
ceramides
uracil beta-alanine malonic semialdehyde

4,5-dihydroxyhexanoic
GABAT
SSADH
glutamine glutamic acid GABA Succinic semialdehyde Succinate Krebs cycle
D-2-hydorxyglutarate
3-oxo-4-hydroxybutanoic
2-ketoglutarate
2-oxo-4-hydroxybutanoic 4-hydroxybutyric acid
3-hydroxypropionic
Fig. 5.3 Biochemistry of GABA metabolism. GABA is gamma-aminobutyric acid, GABA-T is GABA transaminase, SSADH is succinic acid
semialdehyde dehydrogenase
glutamic
P5CS glutamate
γ-glutamyl-P NADP + H+
NH3
P5CDH
NADPH + H+
ALDH4A1
P5CS
NAD+
NADP OAT
glutamic-semialdehyde Δ1-pyrroline-5-carboxylate ornithine citrulline
P5C
FADH2 NADP + H+
PRODH P5CR
arginine ASA
NADP
FAD
collagen glycyl-proline proline

alanyl-proline
X-proline PEPD
X-OH-proline
OH-proline Δ1-pyrroline-3-hydroxy-
5-carboxylate
PEPD PRODH
Fig. 5.4 Biochemistry of proline and hydroxyproline. P5CS is Δ-1-pyrroline-5-carboxylate synthetase, P5CR is Δ-1-pyrroline-5-carboxylate
reductase, P5C is Δ1-pyrroline-5-carboxylate, PRODH is proline dehydrogenase, ASA is argininosuccinic acid
Table 5.1 Nonketotic hyperglycinemia

CNS Apnea ++ n n n n
Ataxia ± ++ ++ ++
Chorea + ++ ++ ++
Hiccups ++ ± n n n
Hydrocephalus + + + +
Hyperactivity n + ++ ++
Hypotonia +++ +++ ++ + +
Lethargy +++ +++ ++ + +
Neurologic deterioration ± ++ + +
Retardation, psychomotor +++ +++ +++ +++ +++
Seizures +++ +++ +++ ++ ±
Spasticity ± + ++ +++ +++
Digestive Feeding difficulties +++ +++ +++ ++ +
Eye Optic atrophy ± ± ± ±
Routine laboratory EEG: burst suppression, +++ ++
abnormal
EEG: hypsarrhythmia, ++
abnormal
EEG: multifocal epilepsy, + ++ ++ ++ +
abnormal
Special laboratory Glycine (CSF) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Glycine ratio (CSF:P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
MRI: agenesis or ++ ++ ++ ++ ++
hypoplasia of the corpus
callosum
MRI: posterior fossa + + + + +
abnormalities
MRI: simplification of the + + + + +
gyral pattern
Table 5.2 Phosphoglycerate dehydrogenase deficiency

CNS Ataxia ± ± ± + +
Axonal sensory motor ± ± ± + ++
polyneuropathy, chronic
Hyperexcitability + ++ ++ ++
Retardation, psychomotor ++ +++ +++ ++ +
Seizures +++ +++ ++ ++ +
Spastic tetraplegia + +++ +++ +++
Endocrine Hypogonadism + + + +
Eye Cataract + + + +
Nystagmus ++ ++ ++ +
Hematological Anemia, megaloblastic + + + +
Musculoskeletal Adducted thumbs + ++ ++ ++
Microcephaly +++ +++ +++ +++
Routine laboratory EEG: hypsarrhythmia, +++ ++
abnormal
EEG: Lennox-Gastaut ++ ++ +
syndrome, abnormal
EEG: multifocal epilepsy, +++ +++ +++ +++
abnormal
Special laboratory Serine (CSF) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Serine (P) – fasting ↓ ↓ ↓ ↓ ↓
5-Methyl-THF (CSF) ↓ ↓ ↓ ↓ ↓
Glycine (CSF) ↓ ↓ ↓ ↓ ↓
Glycine (P) – fasting ↓ ↓ ↓ ↓ ↓
MRI: cortical and subcortical ++ +++ +++ +++
atrophy
MRI: delayed to absent +++ +++ +++ +++
myelination
MRS: decreased N-acetylaspartate/ + + + +
creatine ratio
MRS: increased choline/creatine + + + +
ratio
Table 5.3 Phosphoserine aminotransferase deficiency

CNS Retardation, ++ ++ ++
psychomotor
Musculoskeletal Growth retardation ++ ++ ++
Special laboratory Serine (CSF) ↓↓ ↓↓ ↓↓
Serine (P) ↓-n ↓-n ↓-n
Table 5.4 Phosphoserine phosphatase deficiency

CNS Hypertonia + ++ ++
Seizures, intractable ++ ++ ++
Musculoskeletal Microcephaly n ++ ++
Special laboratory Glycine (CSF) ↓ ↓ ↓
Glycine (P) ↓ ↓ ↓
MRI: cerebellar vermis + ++ ++
hypoplasia
MRI: generalized atrophy + ++ ++
Serine (CSF) ↓↓ ↓↓ ↓↓
Serine (P) ↓ ↓ ↓
Table 5.5 GABA transaminase deficiency

CNS Hypotonia ++ ++ ++
Lethargy ++ + +
Pitched cry, high ++ ++ +
Retardation, psychomotor + +++ +++
Seizures +++ +++ +++
Spasticity ± + ++
Tendon reflexes, increased ++ ++ ++
Digestive Feeding difficulties ++ ++ ++
Other Accelerated growth + ++ ++
Routine laboratory Growth hormone ↑ ↑
Special laboratory Beta-alanine (CSF) ↑ ↑ ↑
GABA-free (CSF) ↑↑ ↑↑ ↑↑
Homocarnosine (CSF) ↑ ↑ ↑
MRI: diffusion restriction in the + + +
internal and external capsule
and subcortical white matter
MRS: GABA ↑ ↑ ↑
Table 5.6 Succinic semialdehyde dehydrogenase deficiency

CNS Anxiety + ++ ++
Ataxia + ++ ++
Attention disorder + ++ ++
Behavior, aggressive + ++ ++
Hyperactivity + ++ ++
Hypotonia ++ ++ ++ +
Language difficulties ++ ++ ++
Oculomotor dyspraxia + ++
Retardation, psychomotor + ++ ++ ++
Seizures ++ ++ ++
Sleep disturbances + ++ ++ ++
Tendon reflexes, decreased + ++ ++ +
Digestive Feeding difficulties + + +
Eye Strabismus + + +
Routine laboratory EEG: spike wave discharges and + ++ ++ ++
generalized slowing, abnormal
Special laboratory 4-Hydroxybutyric acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑
MRI: cerebellar and cerebral atrophy + + + +
MRI: increased T2 signal in the globus + ++ ++ ++
pallidus, cerebellar dentate nucleus, brain
stem, and subcortical white matter
Table 5.7 Hypoprolinemia

CNS Behavior difficulties + ++ ++
Dystonia + + +
Hypotonia + ++ ++
Neuropathy, axonal motor ++ +
Pyramidal signs + ++
Retardation, psychomotor + ++ +
Seizures + + + +
Dermatological Cutis laxa ++ +++ +++ +
Sparse hair + ++ +
Digestive Feeding difficulties ++ ++ ++
Eye Cataract + ++ ++ ++
Corneal clouding ++ ++
Musculoskeletal Hernias + + +
Hip dislocation + ++ +
Joint contractures +
Joint laxity ++ ++ ++ +
Laxity + ++ ++ +
Microcephaly ++ ++ ++ ++
Osteopenia + + +
Osteoporosis + ++ ++
Pes planus + ++ +
Rhizomelia + +
Stature, short + ++ ++
Wormian bones + + +
(continued)

Special laboratory Pro, Cit, Arg, Orn, fasting (P) ↓-n ↓-n ↓-n
Ammonia, fasting (P) ± ± ±
MRI: paucity of white matter + +
MRI: thin corpus callosum + +
MRI: tortuous blood vessels +
MRS: creatine ↓-n ↓-n ↓-n
Table 5.8 Prolidase deficiency

CNS Retardation, psychomotor + + + +
Dermatological Erythematous, papular eruptions ++ + + +
Itching, eczematous lesions ++ + + +
Lymphedema + + + +
Malar flush + + + +
Skin ulceration + ++ +++ +++
Telangiectasia + ++ ++ ++
Ear Recurrent otitis media ++ ++ +
Hematological Anemia + + + +
Splenomegaly + ++ ++ ++
Thrombocytopenia + + + +
Musculoskeletal Dysmorphic features + + + + +
Respiratory Respiratory infections ++ ++ ++ +
Special laboratory Alanylproline (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Glycylproline (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Proline, after acid hydrolysis (U) ↑ ↑ ↑ ↑ ↑
Anti-DNA antibodies ± ± ±
5.9 Diagnostic Flow Charts
Elevated glycine in serum and CSF
present
CSF blood or protein , valproate↑ use? False diagnosis, reconsider
decreased
Pyridoxal-P in CSF Vitamin B6 disorder: PNPO, PDE
normal
Atypical symptoms: lactic acidosis

cardiomyopathy, optic atrophy, leukodystrophy
present
absent
Typical brain MRI:
DWI of internal capsule, hypoplasia corpus callosum
Likely typical NKH Likely variant NKH
mutation absent
AMT, GLDC, GCSH GCE enzyme assay in liver,
PDH enzyme assay in fibroblasts
Sequence and del/dup analysis
l
ma both enzymes deficient
H nor
mutation
t, PD
present ien
defic BOLA3, NFU1, LIAS, GLRX5
GCE Molecular analysis
Diagnosis GCE NKH mutation present

Diagnosis: Variant NKH
Fig. 5.5 Diagnosis of glycine disorders. GCE is glycine cleavage enzyme, DWI is diffusion-weighted images, PDE is pyridoxine-dependent
epilepsy, PNPO is pyridox(am)ine phosphate oxidase, NKH is nonketotic hyperglycinemia, PDH is pyruvate dehydrogenase
Fig. 5.6 Diagnosis of serine Low fasting serum serine

deficiency disorders Intractable seizures
Hypomyelination
CSF serine
use correct control values
Serine deficient
mutations present Diagnosis: deficiency of

Sequence PHGDH gene 3-phosphoglycerate dehydrogenase
No mutations
Sequence PSAT1, PSPH

Treatment: serine and glycine
Fig. 5.7 Differential diagnosis of deficient ATP7A

proline deficiency disorders. CDG is Copper & ceruloplasmin Menkes disease
congenital disorder of glycosylation
abnormal ATP6VOA2
Wrinkly skin COG7
Hyperlaxity joints Transferrin glycosylation
CDG syndromes
Wormian bones
Developmental delay
decreased
Seizures Proline levels fasting P5C-synthetase
Skin biopsy:
abnormal elastin fibers P5C-reductase
GORAB
EFEMP2
FBLN5
FBLN4
ELN
5.10 Specimen Collection and Interpretation

Pitfalls
Metabolite Material Handling Pitfalls
Glycine Serum Serum or plasma. Store frozen Do not use tubes containing
thrombin ↑
Valproate ↑
Kwashiorkor ↑
Irrigation fluids with glycine ↑
IV immunoglobulins with glycine
buffer ↑
Glycine CSF Store frozen; tap without blood Spinal fluid containing blood or
or protein; use age-appropriate serum ↑
reference range Valproate ↑↑
Other anticonvulsants vigabatrin ↗
Hypoxic-ischemic brain injury ↑
Pyridoxine responsive seizures,
pyridoxal-P-responsive
encephalopathy ↑

Glycine CSF/plasma ratio Obtain blood and CSF glycine Valproate ↑
simultaneously CSF containing blood or serum ↑
Hypoxic-ischemic encephalopathy ↑
Glycine Urine Infant
Hyperprolinemia
Immaturity of transporter or
polymorphism in transporter
Serine Serum/plasma Freeze at −20 °C Obtain sample after overnight fast to
identify deficiency
Serine CSF Freeze at −20 °C; tap without Use age-appropriate reference
blood or protein intervals (Moat et al. 2010)
GABA Serum Freeze immediately; store at Vigabatrin ↑
−70 °C
GABA CSF Freeze immediately; store at Vigabatrin↑
−70 °C Hyperprolinemia
Immaturity of transporter or
polymorphism in transporter
4-Hydroxybutyric acid Urine Freeze at −20 °C Treatment with 4-hydroxybutyric
acid ↑
Vigabatrin ↓
4-Hydroxybutyric acid CSF Freeze at −20 °C Treatment with 4-hydroxybutyric
acid ↑
Vigabatrin ↓
Proline Serum Obtain fasting Hypoprolinemia in P5CS only
present in fasting state
Proline-containing dipeptides Urine Confirmation by large increase in
proline and hydroxyproline after
acid hydrolysis
Prolidase enzyme activity Red blood cells, leucocytes, Red blood cell requires
fibroblasts preincubation with Mn
5.11 Normal and Pathological Values
Nonketotic hyperglycinemia
Normal Classic NKH Attenuated NKH Variant NKH
Glycine
Plasma μmol/L <1 month, 230–450 261–2,127 324–1,000 445–1,035
>1 month, 100–350
CSF μmol/L 5–20 neonate 96–440 43–230 15–180
3–12 older
CSF/plasma ratio <0.02 0.09–0.49 0.05–0.22 0.02–0.09
Serine deficiency syndromes

Normal PHGDH severe PHGDH mild PSAT1/ PSPH
Serine
Plasma μmol/L <1 month, 230–450 28–64 63, 58, 33 30–80
>1 month, 100–350
CSF μmol/L <1 month, 31–82 <2 years, 4–18 9, 13 5–18
1 month–1 year, 24–70
1–5 years, 20–59 >2 years,3–12
>5 years, 17–44
CSF glycine <3 months, 5–20 1–4 Normal <1 to normal
>3 months, 3–12
GABA disorders Normal SSADH GABA-T

4-Hydroxybutyric acid
Urine mmol/mol creatinine 0–7 100–1,200
Plasma μmol/L 0–3 35–600
CSF μmol/L 0–2 100–850
GABA-free
Serum μmol/L 0.12–0.50 2.9; 2.1
CSF μmol/L <2 years, 0.017–0.067 0.12–2.33 1.26
>2 years, 0.032–0.167
GABA total
CSF μmol/L <2 years, 4.2–13.4 38
>2 years, 3.3–12.2
Beta-alanine
Plasma μmol/L <12 23
CSF μmol/L <0.1 0.48
Proline disorders
Normal Hyperprolinemia 1 Hyperprolinemia 2 P5CS Prolidase
Proline
Plasma <1 month, 110–420 500–2,000 >2,000 91 ± 18;
107 ± 49
>1 month, 50–350 120–197
8–20a
Peptide bound Urine μmol/L
Proline 430–1,250 5,700–27,000
Hydroxyproline 250–725 1,400–2,100
Prolidase enzyme activity: prolidase <10 %
a
Results from individual patients
5.12 Prenatal Diagnosis

Condition Prenatal diagnosis Pitfalls
Nonketotic Mutation analysis Gene and mutations must be known
hyperglycinemia Intragenic linkage if only one allele is known
Enzyme assay uncultured CVS 1 % false-negative diagnosis

Variant NKH Mutation analysis Gene and mutation must be known
Enzyme assay of GCS and PDH Limited experience
Serine deficiency Mutation analysis Gene known
GABA-T Mutation analysis
SSADH Metabolite: 4-hydroxybutyric in Stable isotope dilution assay
amniotic fluid
Enzyme assay in CVS or cultured
amniocytes
Mutation analysis
Hyperprolinemia 1 Mutation analysis
P5CS Mutation analysis
P5CR Mutation analysis
Prolidase Mutation analysis
Enzyme assay in amniocytes Prediction of clinical severity not possible
5.13 Treatment Phosphoserine phosphatase deficiency (PSPH) has been

reported in a single patient who also had Williams syndrome
Nonketotic hyperglycinemia (glycine encephalopathy). In the (Jaeken et al. 1997). Treatment with l-serine resulted in slow
neonatal period during the apneic phase, withdrawal of inten- improvement of head growth, but no improvement in height
sive care should be discussed with the parents (Boneh et al. or weight.
2008). Treatment consists of reduction in glycine by using GABA transaminase deficiency. There is no effective
sodium benzoate and inhibition of the action of glycine on treatment for this condition.
the NMDA receptor. The dose of benzoate must be individu- Succinic semialdehyde dehydrogenase deficiency
ally adjusted to bring the glycine level down to the therapeu- (SSADH). Treatment is symptomatic. Antiepileptic drugs
tic range of 120–300 μM. Most patients with severe NKH that are most helpful include carbamazepine and lamotrig-
require 500–750 mg/kg/day, whereas most patients with ine. Valproate is contraindicated since it inhibits the enzyme.
attenuated NKH require much lower doses (250–450 mg/kg/ Methylphenidate, thioridazine, risperidone, and fluoxetine
day) (Wolff et al. 1986; Van Hove et al. 2005; Hennermann have been helpful in some patients for psychiatric symptoms
et al. 2012). Dosing must be divided in at least three doses, (Pearl et al. 2009). Clonidine has been used to improve REM
more in infants. Secondary carnitine deficiency is possible sleep. Vigabatrin has variable clinical responses and is no
during treatment with high doses of benzoate in the first year longer indicated. GABA(B) receptor antagonists are under
of life. Blocking of the NMDA receptor can be done with experimental investigation (Pearl et al. 2009). Although tau-
dextromethorphan (5–10 mg/kg/day) or with ketamine. rine was promising in animal experiments, it did not show
Treatment improves lethargy and seizure control. It does not efficacy in an open-label clinical trial.
avoid severe mental retardation in patients with severe NKH. Hyperprolinemia type I. At this moment, a specific treat-
However, in patients with attenuated or mild NKH, early ment is not available. Given the endogenous brain metabo-
treatment improves neurodevelopmental outcome, often suf- lism of proline, a dietary restriction does not seem indicated
fices for seizure control, and prevents lethargic attacks during as a treatment option.
intercurrent illness. Valproate must be avoided in such Δ-1-Pyrroline-5-carboxylate synthase (P5CS) deficiency.
patients. Difficult to control seizures can sometimes benefit Treatment with arginine (150 mg/kg/day) corrected the brain
from ketogenic diet or from vagal nerve stimulator. MRS creatine levels; improved levels of ammonia, proline,
3-Phosphoglycerate dehydrogenase deficiency (PHGDH). and ornithine; and was associated with improved psychomo-
Treatment with supplementation with serine and glycine shows tor development (Martinelli et al. 2012). Treatment with
improvement in seizures and increased white matter volume citrulline and proline has been suggested, but is without data
and myelination, but little effect on psychomotor development (Baumgartner et al. 2005).
unless treatment is started prenatally (De Koning et al. 2002). Δ-1-Pyrroline-5-carboxylate reductase (P5CR) defi-
Some patients also need folinic acid treatment. Mild patients ciency. A specific treatment is not available. Treatment is
responded well to treatment with low doses of serine. High- symptomatic.
dose serine improved the adult patient subjectively. Prolidase deficiency. No effective treatment exists. Red
Phosphoserine aminotransferase deficiency (PSAT1). blood cell transfusions with manganese incubation to acti-
Treatment with serine and glycine supplementation resulted vate the enzyme did not work. High-dose pulsed systemic
in marginal improvement in seizure control and responsive- steroids and growth hormone systemic or topical treatment
ness in the older patient, but the patient treated from the first had a transient improvement on skin ulcers. Ointments with
day of life has had normal development and has remained proline, or proline and glycine, have limited benefit. Enzyme
symptom-free. replacement therapy is currently under development.
5.14 Treatment Summary

Condition Treatment Comments
Nonketotic hyperglycinemia: classic severe Benzoate 500–750 mg/kg/day (for severe) Dosing of benzoate individualized
Glycine restricted diet Effect on outcome for seizure control but
limited in severe
Dextromethorphan 5–10 mg/kg/day Diet limited impact
Multiple anticonvulsants
Nonketotic hyperglycinemia: attenuated Benzoate 250–500 mg/kg/day (for attenuated) Avoids neurodepressive episodes
Dextromethorphan 5–10 mg/kg/day Prevents seizures
Improves neurocognitive outcome
Effect of dextromethorphan persists
Benzoate dose individualized to maintain
serum glycine <300 μM
Effect on outcome in developmental progress
in attenuated if started early
Variant nonketotic hyperglycinemia None known Theoretical treatment with lipoic acid of
limited benefit
Serine deficiency syndromes Serine 200–600 mg/kg/day Improves head growth and myelination
Glycine 200–300 mg/kg/day Improves seizures
Neurodevelopment improved if started prenatal
or first day of life
Lower doses in mild serine deficiency
GABA transaminase deficiency None known
SSADH deficiency Carbamazepine or lamotrigine; psychotropic Most effective anticonvulsants
drugs, physical and occupational therapy Treatment with vigabatrin or taurine not
consistently helpful
Hyperprolinemia type 1 None known
P5C synthetase deficiency Arginine 150 mg/kg/day Improved serum ammonia, proline, ornithine,
and brain creatine and development
P5C reductase deficiency None known
Prolidase deficiency None known Transient and inconsistent improvement with
topical proline-containing ointment, steroids,
and growth hormone
Boneh A, Allan S, Mendelson D et al (2008) Clinical, ethical and legal

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Genet 43:e58 134–140
Amino Acid Transport Defects
6
Manuel Palacín and Stefan Broer
Contents Summary
6.1 Introduction ...................................................................... 85 Disorders associated with the malfunction of amino acid
transporters mainly affect the function of the intestine, kid-
6.2 Nomenclature.................................................................... 87
ney, brain, and liver. Mutations of brain amino acid trans-
6.3 Metabolic Pathways ......................................................... 88 porters, for example, alter neuronal excitability. Examples
6.4 Signs and Symptoms ........................................................ 89 presented in this chapter are episodic ataxia due to EAAT3
defect, hyperekplexia due to GLYT2 deficiency, global cere-
bral hypomyelination due to AGC1 deficiency, and neonatal
6.6 Pathological Values .......................................................... 93 myoclonic epilepsy due to GC1 deficiency. Mutations of
6.7 Diagnostic Flow Charts.................................................... 94 renal and intestinal amino acid transporters cause renal prob-
6.8 Specimen Collection ......................................................... 96 lems (cystinuria and dicarboxylic aminoaciduria) and
malabsorption that can affect whole-body homoeostasis
6.9 Prenatal Diagnosis ............................................................ 96
(Hartnup disorder, lysinuric protein intolerance, and hyper-
6.10 DNA Testing ...................................................................... 96 dibasic aminoaciduria type 1). Inborn errors associated with
6.11 Treatment Summary ........................................................ 96 the mitochondrial SLC25 family with a liver phenotype such
References .................................................................................... 98
as the ones affecting SLC25A13 (aspartate/glutamate trans-
porter 2), citrin deficiency and SLC25A15 (ornithine–citrul-
line carrier 2), homocitrullinuria, hyperornithinemia, and
hyperammonemia will be dealt with in Chap. 4.
6.1 Introduction
Amino acid transporters are essential for the absorption of

amino acids from nutrition, mediating the interorgan and
intercellular transfer of amino acids and the transport of
amino acids between cellular compartments (Broer and
Palacín 2011). To date, 11 SLC families are known to com-
prise amino acid transporters. Defects due to mutations in
M. Palacín (*)
Institute for Research in Biomedicine (IRB Barcelona), amino acid transporters in 4 of these families affecting renal
Centre for Biomedical Network Research on Rare Diseases tubular reabsorption (Fig. 6.1) and intestinal absorption, neu-
(CIBERER, U731) and University of Barcelona, rotransmitter reuptake in the synapse, and mitochondrial oxi-
Baldiri i Reixac 12, Barcelona 08028, Spain
dation are considered in this chapter: (1) Mutations in the
e-mail: manuel.palacin@irbbarcelona.org
members of the glutamate transporter family SLC1A1
S. Broer
(excitatory amino acid transporter 3, EAAT3) and SLC1A3
Research School of Biology, Australian National University,
Canberra ACT 0200, Australia (excitatory amino acid transporter 1, EAAT1) cause the
e-mail: stefan.broeer@anu.edu.au primary inherited aminoaciduria named dicarboxylic
86 M. Palacín and S. Broer
aminoaciduria and episodic ataxia type 6, respectively (4F2hc/y+LAT1) is mainly expressed in the basolateral
(Bailey et al. 2011; Jen et al. 2005). (2) Mutations in the het- plasma membrane of the epithelial cells of the kidney prox-
eromeric amino acid transporters (HAT) SLC3A1/SLC7A9 imal convoluted tubule (Fig. 6.1) and the small intestine
(rBAT/b0,+AT) and SLC3A2/SLC7A7 (4F2hc/y+LAT1) cause and in white blood cells. The SLC6 family comprises 20
the primary inherited aminoacidurias cystinuria and lysin- members in humans that can be grouped into four subfami-
uric protein intolerance (LPI), respectively (Calonge et al. lies, namely, the monoamine transporter branch, the GABA
1994; Feliubadaló et al. 1999; Torrents et al. 1999; Borsani (γ-aminobutyric acid) transporter branch, and the amino
et al. 1999). In cystinuria, mutations are found in either of acid transporter branches I and II. SLC6A5 (GLYT2) and
the two subunits (SLC3A1 or SLC7A9), whereas in LPI SLC6A19 (B0AT) belong to the two latter subfamilies and
mutations are only present in the light subunit SLC7A7. (3) mediate reuptake of glycine in synapses and the uptake of
Mutations in the members of the neurotransmitter transporter neutral amino acids in epithelial cells, respectively. GLYT2
family SLC6A5 (neuronal glycine transporter GLYT2) and is mainly found in the spinal cord where it terminates inhib-
SLC6A19 (sodium-dependent neutral amino acid transporter itory neurotransmission. B0AT1 is found in the apical mem-
B0AT) cause hyperekplexia and the primary inherited amino- brane of kidney proximal tubular epithelial cells (Fig. 6.1)
aciduria named Hartnup disorder, respectively (Seow et al. and enterocytes of the small intestine. Finally, the SLC25
2004; Kleta et al. 2004; Rees et al. 2006). (4) Mutations in family comprises a total of ~30 members, including three
the mitochondrial transporters SLC25A12 (neuronal- and ADP/ATP carrier isoforms, five uncoupling protein iso-
muscle-specific mitochondrial aspartate/glutamate transforms, and six amino acid transporters. In the inner
porter 1, AGC1) and SLC25A22 (mitochondrial glutamate/ mitochondrial membrane, SLC25A12 (AGC1) exchanges
H+ symporter 1, GC1) cause global cerebral hypomyelin- glutamate and aspartate, and SLC25A22 (GC1) mediates
ation and neonatal myoclonic epilepsy, respectively (Wibom proton-dependent transport of glutamate. AGC1 is highly
et al. 2009; Molinari et al. 2005). Finally, for dibasic amino- expressed in the inner mitochondrial membrane in the
aciduria type 1, only two families have been described and brain, heart, and skeletal muscle. GC1 is more highly
the causing gene is unknown (Kihara et al. 1973). expressed in brain astrocytes than in neurons.
The principal biochemical and structural characteristics 6.1 Cystinuria. Renal reabsorption and intestinal absorp-
of these amino acid transporters have been reviewed tion of cystine, lysine, arginine, and ornithine. Metabolites
recently (Broer and Palacin 2011). SLC1 family members important for diagnosis: cystine, lysine, arginine, and orni-
(glutamate/aspartate transporters named EAAT for excit- thine in urine.
atory amino acid transporters 1–5) mediate high-affinity 6.2 Dicarboxylic aminoaciduria. Reuptake of neurotrans-
sodium- and potassium-dependent uptake of glutamate and mitter glutamate, absorption of glutamate and aspartate in
aspartate in mammalian cells. SLC1A1 (EAAT3) is the intestine, and reabsorption of glutamate in the kidney.
expressed in the apical membrane of epithelial cells of the Metabolites important for diagnosis: glutamate and aspartate
kidney proximal convoluted tubule (Fig. 6.1) and the small in the urine.
intestine and in the brain cortex, particularly in the hippo- 6.3 Hartnup disorder. Renal reabsorption and intestinal
campus, the basal ganglia, and the olfactory bulb. SLC1A3 absorption of neutral amino acids. Metabolites important for
(EAAT1) is found throughout the brain. It is the main glu- diagnosis: neutral amino acids in the urine. Glycine is usu-
tamate transporter in the cerebellum, inner ear, circumven- ally normal.
tricular organs, and retina. Expression in peripheral organs 6.4 Lysinuric protein intolerance. Renal reabsorption and
is limited (Danbolt 2001). HAT are composed of a heavy intestinal absorption of dibasic amino acids. Metabolites
subunit (SLC3 family) and a light subunit (SLC7 family), important for diagnosis: dibasic amino acids in the plasma
which are linked by a conserved disulfide bridge (Fig. 6.1). (decreased) and urine (increased) and orotic acid in urine
Both subunits are required to form functional transporters (increased).
at the cell surface. Transporter SLC3A1/SLC7A9 (rBAT/ 6.5 Dibasic aminoaciduria type 1. Renal reabsorption and
b0,+AT) is expressed in the apical membrane of epithelial intestinal absorption of lysine, arginine, and ornithine.
cells of the kidney proximal convoluted tubule (Fig. 6.1) Metabolites important for diagnosis: lysine, arginine, and
and the small intestine. Transporter SLC3A2/SLC7A7 ornithine in urine.
6 Amino Acid Transport Defects 87
6.6 Episodic ataxia due to EAAT1 defect. Reuptake of mitochondria for myelin formation. Metabolite important
neurotransmitter glutamate. for diagnosis: N-acetylaspartate in the brain (magnetic
6.7 Hyperekplexia. Reuptake of inhibitory neurotransmit- resonance 1H spectrum in cerebral areas).
ter glycine. 6.9 Neonatal myoclonic epilepsy due to mitochondrial
6.8 Global cerebral hypomyelination due to AGC1 defect. glutamate carrier GC1 defect. Mitochondrial glutamate
Malate–aspartate shuttle for mitochondrial oxidation of import/metabolism and neuronal excitability.
cytosolic NADH and efflux of aspartate from neuronal
6.2 Nomenclature
Gene Chromosomal
No. Disorder Alternative name Abbreviation symbol localization Affected protein OMIM no. Subtype
6.1 Cystinuria Cystinuria SLC3A1, 2p16.3, 19q12 Amino acid transport 220100 All forms
SLC7A9 system b(0,+), composed of
proteins rBAT (SLC3A1)
and b(0,+)AT (SLC7A9)
6.2 Dicarboxylic DA SLC1A1 9p24 Neuronal/epithelial 222730 All forms
aminoaciduria high-affinity glutamate
transporter, excitatory
amino acid transporter 3
(EAAT3)
6.3 Hartnup disorder HD SLC6A19 5p15.33 Sodium-dependent neutral 234500 All forms
amino acid transporter
(B0AT)
6.4 Lysinuric protein Hyperdibasic LPI SLC7A7 14q11.2 Amino acid transport 222700 All forms
intolerance aminoaciduria system y + L (4F2hc/
type 2 y+LAT1)
6.5 Dibasic – – – – 222690 All forms
aminoaciduria
type 1
6.6 Episodic ataxia Episodic ataxia EA6 SLC1A3 5p13 Glutamate/aspartate 612656 All forms
due to EAAT1 type 6 transporter (GLAST),
glutamate excitatory amino acid
transporter defect transporter 3 (EAAT3)
6.7 Hyperekplexia Startle disease, HE SLC6A5 11p15.1 Neuronal glycine 149400 All forms
due to Gly familial transporter GLYT2
transporter
GLYT2 defect
6.8 Global cerebral Aspartate/ AGC1 SLC25A12 2q31.1 Neuronal- and muscle- 612949 All forms
hypomyelination glutamate deficiency specific mitochondrial
due to AGC1 transporter 1 aspartate/glutamate
defect (AGC1) transporter 1 (AGC1;
deficiency Aralar)
6.9 Neonatal Early infantile EIEE3 SLC25A22 11p15.5 Mitochondrial glutamate/ 609304 All forms
myoclonic epileptic H+ symporter 1 (glutamate
epilepsy due to encephalopathy-3 carrier 1, GC1)
mitochondrial (EIEE3)
glutamate carrier
GC1 defect
Fig. 6.1 Principal epithelial transporters involved in amino acid reab- from additional modifying mutations in the high-affinity proline trans-
sorption, which are mutated in human aminoacidurias. A nephron is porter SLC6A20 when SLC36A2 is not completely inactivated (Broer
depicted (inset), showing the glomerulus, proximal convoluted tubule et al. 2008). Iminoglycinuria is not further discussed in this chapter
(PCT), proximal straight tubule (PST), distal convoluted tubule (DCT), because it is a benign condition with no associated pathology. Mutations
and collecting duct (CD). A cross section of the proximal convoluted in the neutral amino acid transporter, SLC6A19, are responsible for
tubule (white square indicated with an arrow) is represented in the main Hartnup disorder (Kleta et al. 2004; Seow et al. 2004). The neutral
diagram. Four of the aminoacidurias, including DA, iminoglycinuria, amino acid transport defect can also be caused by a kidney-specific loss
Hartnup disorder, and cystinuria, manifest at the apical surface of the of heterodimerization of mutant SLC6A19 with TMEM27 (Kowalczuk
renal tubule, while lysinuric protein intolerance manifests at the baso- et al. 2008). Finally, no mutations have been identified in SLC3A2, cod-
lateral surface (see text for details). Iminoglycinuria results from com- ing for the ancillary protein of y+LAT1 (SLC7A7) in patients with LPI
plete inactivation of SLC36A2, a proline and glycine transporter, or (Broer and Palacín 2011) (Figure extracted from Bailey et al. (2011))
Table 6.1 Cystinuria

Renal Obstructive uropathy − ± ± ± ±
Renal failure, chronic − − ± ± ±
Urinary infections − ± ± ± ±
Urolithiasisa − ± ± ± ++
Urolithiasis, cystine stones − ± ± ± ++
Routine laboratory Hematuria − ± ± ± ±
Leukocytes (U) − ± ± ± ±
Special laboratory Cys, Lys, Arg, Orn (P) ↓-n ↓-n ↓-n ↓-n
Cystine (U) ↑↑ ↑↑ ↑↑ ↑↑
Cystine crystals (U) + + + + +
Lysine, arginine, ornithine (U)b ↑↑ ↑↑ ↑↑ ↑↑
a
Cystine stones usually appear in the third decade of life. A small proportion of patients, with the 2 mutated alleles in any of the two cystinuria
genes, do not produce them
b
In isolated cystinuria there is no hyperexcretion of dibasic amino acids in urine (Eggermann et al. 2007)
Table 6.2 Dicarboxylic aminoaciduria

CNS Behavior, psychotic − − ± ± ±
Mental retardationb − ± ± ± ±
Renala Urolithiasis, glutamate stones − ± ± ± ±
Special laboratory Aspartic acid (U) ↑ ↑ ↑ ↑ ↑
Glutamic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
No characteristic clinical findings in DA
a
Two out of the three patients presented in Bailey et al. (2011) presented stones of unknown nature
b
Reported in several cases, but most likely ascertainment bias
Table 6.3 Hartnup disorder

CNS Ataxia − ± ± ± −
Psychosis − ± ± ± −
Dermatological Photosensitivity − ± ± ± −
Special laboratory Glutamic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Neutral amino acids (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 6.4 Lysinuric protein intolerance

Cardiovascular Valvulitis, mitral − − ± ± ±
CNS Coma, hyperammonemic − ± ± ± ±
Mental retardation − − ± ± ±
Dermatological Sparse hair − − ± ± ±
Digestive Diarrhea − ± ± ± ±
Hepatosplenomegaly ± ± ± ± ±
Protein intolerance − − ± ± ±
Vomiting − ± ± ± ±
Hematological Hemophagocytic − − ± ± ±
lymphohistiocytosis, macrophage
activation syndrome
Musculoskeletal Bone growth n n ↓-n ↓-n ↓-n
Osteoporosis − − ± ± ±
(continued)

Renal Glomerulonephritis − − ± ± ±
Hypertension − − − ± ±
Renal failure, end stage − − − ± ±
Respiratory Interstitial changes (chest − − ± ± ±
radiographs)
Pulmonary alveolar proteinosis − – ± ± ±
Respiratory insufficiency − − ± ± ±
Other Combined hyperlipidemia ± ± ±
Special laboratory Arginine, ornithine (U) − – ↑ ↑ ↑
Gln, Ala, Gly, Pro, Cit (P) − − ↑ ↑ ↑
Lys, Arg, Ornithine (P) n n ↓-n ↓-n ↓-n
Lysine (U) − − ↑↑ ↑↑ ↑↑
Orotic acid (U) − ↑ ↑ ↑ ↑
Table 6.5 Dibasic aminoaciduria type 1

CNS Athetosis − ± +
Dysarthria − ± +
Hyperactivity − + ++
Mental retardation − + ++
Other Adverse reaction to phenothiazines − ++ ++
Special laboratory Cys, Lys, Arg, Orn (P) n n n
Lysine, arginine, ornithine (U)a ↑ ↑
Lysine, intestinal absorption ↓
Signs and symptoms correspond to the only suspected homozygous patient described (Kihara et al. 1973). The parents of the patient, as well
members of another family, showed moderated hyperdibasic aminoaciduria (lysine, arginine, and ornithine) and presented no pathological signs
and therefore had been considered heterozygotes. The patients described by Whelan and Scriver (1968) were free of characteristic symptoms
a
The patient showed a moderated urine hyperexcretion of cystine
Table 6.6 Episodic ataxia due to EAAT1 glutamate transporter defect

CNS Ataxiaa − − + + +
Epilepsy − − + + +
Hemiplegic migraine − − + + +
Interictal nystagmus − − + + +
Digestive Nausea/vomiting − − + + +
Eye Photophobia − − + + +
Signs and symptoms according to a very small number of cases described by Jen et al. (2005) and de Vries et al. (2009)
a
Ataxia episodes lasting hours to days. Normal routine laboratory
Table 6.7 Hyperekplexia due to Gly transporter GLYT2 defect

CNS Head-retraction reflexa ↑ ↑ ↑ ↑ ↑
Mental retardation, mild − ± ± ± ±
Startle reflex + + + + +
Stiffness + + ± ± ±
Sudden infant death ± ± − − −
Musculoskeletal Hernias − ± ± ± ±
Hip dislocation ± ± ± ± ±
Periodic limb movement during slip ± ± ± ± ±
Signs and symptoms according to de-Koning-Tijssen and Rees (2007) and Bakker et al. (2006)
a
Short period of stiffness following startle response. Normal routine laboratory
Table 6.8 Global cerebral hypomyelination due to AGC1 defect

CNS Apneaa − − +
Muscular hypotonia − − ++
Retardation, psychomotorb − + +
Spasticityc − − ++
Sporadic tonic seizures − + +
Routine laboratory Lactate (P) ↑
Special laboratory ATP production (muscle biopsy)d ↓↓
Cerebrum volumee ↓
Hypomyelination, globalf ++
Mitochondrial respiratory complexesg n
N-Acetylaspartate (CNS) ↓↓
Signs and symptoms correspond to the only patient identified so far, a 3-year-old girl (homozygous for the missense mutation Q590R)
(Wibom et al. 2009)
a
Episodic
b
Affecting mainly motor skills
c
With generalized hyperreflexia
d
Using glutamate + succinate or glutamate + malate as substrates
e
Detected by magnetic resonance imaging in the cerebral hemispheres with reduced cerebral volume (cerebellum, brainstem, and thalami
normal)
f
Detected by magnetic resonance single-volume spectroscopy (1H spectrum) in the left basal ganglia, occipital midline, and frontal lobe
g
Activities of complexes I, I + III, II, II + III, and IV in muscle biopsy
Table 6.9 Neonatal myoclonic epilepsy due to mitochondrial glutamate carrier GC1 defect
CNS Encephalopathy, progressivea − ± ++
Hypotonia ++ ++ ++
Myoclonic epilepsyb ++ ++ ++
Spasticity − − ±
Vegetative statec − − ±
Musculoskeletal Microcephaly − − +
Routine laboratory EEG: abnormald ++ ++ ++
ERG: abnormale ±
MRI (CNS): abnormalf ++
Special laboratory Glutamate oxidation (FB)g ↓↓
Signs and symptoms correspond to the few patients identified, four affected children in one family (homozygous for the missense mutation P206L)
and one affected child in another family (homozygous for the missense mutation G236W) (Molinari et al. 2005, 2009)
a
One of the patients showed no psychomotor development at the age of 10 years
b
Refractory to vigabatrin, carbamazepine, stiripentol, and phenobarbital
c
One of the identified patients died at the age of 8 years
d
Abnormal EEG with burst suppression pattern and hypsarrhythmia suggesting West syndrome
e
Electroretinogram with progressive alteration and abolition of macular and peripheral responses
f
Abnormal magnetic resonance imaging with cerebellar hypoplasia and other alterations
g
Strongly defective mitochondrial oxidation of glutamate, but not succinate, in cultured skin fibroblasts
Fasting plasma amino acid levels (mM) (range limits) measured by

amino acid analyzer
Neonatal Infancy Childhood to adulthood
Amino acid Fasting Fasting Fasting
Alanine 0.19–.34 0.17–0.44 0.23–0.42
Arginine 0.05–0.08 0.05–0.12 0.05–0.10
Asparagine 0.05–0.12 0.05–0.12 0.03–0.11
Aspartate <0.02 <0.02 <0.02
Citrulline <0.04 0.01–0.05 0.01–0.05
Cystine 0.02–0.06 0.02–0.06 0.02–0.06
Glutamate <0.08 <0.08 <0.08
Glutamine 0.42–0.75 0.33–0.67 0.33–0.63
Glycine 0.11–0.29 0.11–0.29 0.11–0.29
Histidine 0.05–0.10 0.05–0.10 0.05–0.10
Isoleucine 0.04–0.09 0.04–0.09 0.04–0.09
Leucine 0.08–0.15 0.08–0.15 0.06–0.16
Lysine 0.07–0.20 0.12–0.24 0.11–0.23
Methionine 0.01–0.04 0.01–0.04 0.01–0.04
Ornithine 0.04–0.11 0.04–0.11 0.04–0.11
Phenylalanine 0.04–0.07 0.04–0.07 0.04–0.07
Proline 0.09–0.27 0.09–0.27 0.12–0.23
Serine 0.10–0.20 0.10–0.20 0.10–0.20
Threonine 0.06–0.29 0.06–0.29 0.10–0.20
Tyrosine 0.04–0.11 0.04–0.08 0.04–0.09
Valine 0.13–0.29 0.13–0.29 0.13–0.29
Reference values from the Hospital San Joan de Deu, Barcelona, Spain
(Rafael Artuch)
Urine amino acid levels Neonatal Infancy Childhood Adolescence Adulthood

(mmol/mol creatinine) (5–95
Amino acid Fasting Fasting Fasting Fasting Fasting
percentile limits) measured by
amino acid analyzer Alanine 75–244 36–206 33–130 17–92 16–68
Arginine <14 <11 <9 <6 <5
Asparagine <84 <58 <32 <24 <23
Aspartate 2–12 2–16 2–10 1–10 2–7
Citrulline <10 <10 <10 <10 <10
Cystine 24–78 12–48 8–30 7–23 6–34
Glutamate <30 <29 <11 <9 <12
Glutamine 52–205 63–229 45–236 20–112 20–76
Glycine 283–1,097 210–743 110–356 64–236 43–173
Histidine 80–295 72–342 61–287 43–184 26–153
Isoleucine <6 <6 <6 <6 <4
Leucine 3–25 4–16 3–18 3–16 2–11
Lysine 22–171 13–199 10–69 10–56 7–58
Methionine 7–27 6–29 5–29 3–17 2–16
Ornithine <31 <31 <14 <14 <8
Phenylalanine 4–32 7–28 6–31 5–20 2–19
Proline 21–213 <130 <13 <9 <9
Serine 80–262 42–194 32–124 23–69 21–50
Threonine 20–138 14–92 9–62 8–28 7–29
Tyrosine 6–55 11–54 9–48 6–26 2–23
Valine 3–26 4–19 3–21 3–17 3–13
6.6 Pathological Values 6.4 Lysinuric protein intolerance. Altered amino acid
plasma concentration (mM) in 20 patients (the range of
6.1 Cystinuria. Altered urine amino acid levels (5–95 lower–upper values is shown) (Simell 2001). Altered urine
percentile limits in mmol/mol creatinine) in cystinuria excretion values (mmol/mol creatinine) of amino acids and
(Font-Llitjos et al. 2005). orotic acid after overnight fasting in one LPI patient (pro-
Amino acid Controlsa Cystinuria patientsb vided by R. Artuch from ref. Gómez et al. 2006). Reference
Cystine 3–12 73–385 values for urine orotic acid excretion (1.2–6.9 mmol/mol
Lysine 4–56 300–1,315 creatinine).
Arginine <5 26–946 Amino acid Plasma Urine
Ornithine 1–8 66–389 Alanine 0.42–1.02 116
Urine samples randomly collected (morning or 24 h) from 83 controls Arginine 0.01–0.06 661
and 34 patients of any age Citrulline 0.14–0.53 53
a
Controls were relatives of cystinuria patients without mutations in Glutamine 3.64–7.16 149
SLC3A1 and SLC7A9
b
Patients with two mutated SLC3A1 alleles (cystinuria type A); similar Glycine 0.39–0.53 240
range of values were obtained from the urine of patients with cystinuria Lysine 0.03–0.18 1,040
type B (two mutated SLC7A9 alleles) Ornithine <0.08 112
Carbamoyl phosphate metabolite
6.2 Dicarboxylic aminoaciduria. Altered urine amino acid Orotic acid – 30
levels (lower and upper values in mmol/mol creatinine) in
DA (Bailey et al. 2011). 6.5 Hyperdibasic aminoaciduria type 1. Altered urine
Reference values DA patients amino acid levels (range of values in 6 determinations
Amino acid Infancy Adulthood Infancy Adulthood within 2 years) of the only identified patient with
Aspartate <26 <15 50 46–51 hyperdibasic aminoaciduria type 1 (mmol/mol creatinine)
Glutamate <63 <36 1,225 1,052–1,377 (Kihara et al. 1973).
Urine samples obtained by random collection Amino acid Controlsa Patient
Cystine 3 22–45
6.3 Hartnup disorder. Altered urine amino acid levels (lower Lysine 12 400–803
and upper values in mmol/mol creatinine) in adults with HD Arginine 2 11–56
(Potter et al. 2002). Ornithine 9 21–83
a
Amino acid HD patients Control reference values in the Pacific State Hospital (California,
Alanine 384–1,436 USA) at the time of the study
Glutamate 15–29
Glutamine 515–2,010 6.6 Episodic ataxia due to EAAT1 defect and 6.7 hyperek-
Glycine 159–708 plexia. Not applicable.
Histidine 325–653 6.8 Global cerebral hypomyelination due to AGC1
Isoleucine 14–194 defect. In the only identified patient, plasma lactate was
Leucine 16–200 elevated (6 mM), and magnetic resonance single-volume
Lysine 2–88 spectroscopy (1H spectrum) in the left basal ganglia, occipi-
Methionine 5–51 tal midline, and frontal lobe in the only patient described
Phenylalanine 12–122 with AGC1 deficiency showed severely reduced peak of
Serine 546–842
N-acetylaspartate (ratio N-acetylaspartate/creatine = 0.7)
Threonine 233–665
(Wibom et al. 2009).
Tyrosine 2–281
6.9 Neonatal myoclonic epilepsy due to mitochondrial
Valine 43–566
glutamate carrier GC1 defect. Not applicable.
Urine samples obtained by random collection. Glutamate and lysine are
usually slightly elevated, but not in all HD patients
6.7 Diagnostic Flow Charts 6.4 Lysinuric protein intolerance

Drowsiness
6.1 Cystinuria Hyperammonemic coma
(after high-protein food)
Urinary tract infection
Urolithiasis
Lysine, Arginine,
Ornithine, Aortic acid (U) ↑
Hexagonal cystine crystals (U)
Y N
Y N
Cystinuria + LPI Other causes

dibasic amino acids (U)↑ Other causes
Fig. 6.4 Diagnostic flow chart for lysinuric protein intolerance
Y N 6.5 Hyperdibasic aminoaciduria type 1. The principal

differences between the described patient with hyperdibasic
Cystinuria Other causes aminoaciduria type 1 and LPI patients are (i) absence of
hyperammonemia or protein intolerance and (ii) moderate
Fig. 6.2 Diagnostic flow chart for cystinuria
hyperdibasic aminoaciduria in the obligated heterozygotes
(e.g., parents) in the former.
If patients are presented with neonatal seizures, hypoto- 6.6 Episodic ataxia due to EAAT1 defect. Typically, epi-
nia, developmental delay, and/or facial dysmorphism, the sodes of ataxia can be triggered by startle, stress, or exertion
very rare hypotonia–cystinuria syndrome (OMIM 606407) (Fig. 6.5).
due to homozygous deletion on chromosome 2p21 should be
discarded. Ataxia
6.2 Dicarboxylic aminoaciduria. Usually detected retro-
Episodic Progressive
spectively, consider in cases of obsessive–compulsive disor-
der. Mental retardation most likely diagnosed due to
ascertainment bias. Short episodes
6.3 Hartnup disorder. Usually detected in children by (sec–min) Spinocerebellar
Myokymia between attacks ataxias
urine amino acid analysis when presenting with photoderma-
titis of unknown origin (Fig. 6.3). Y N
Photodermatitis
Ataxia (some cases) EA1 Other EA’s
(EA3)
Genetic Genetic
testing testing
Amino aciduria
(neutral AA) Fig. 6.5 Diagnostic flow chart for ataxia
Y N
Hartnup Other
disorder causes
Fig. 6.3 Diagnostic flow chart for Hartnup disorder

6.7 Hyperekplexia. Diagnosis of HE requires three key

symptoms: generalized stiffness after birth, excessive startle
reflex particularly after auditory stimuli and short periods of
stiffness after the startle response (Fig. 6.6).
Fig. 6.6 Diagnostic flow chart for hyperekplexia Excessive startle reflex
stiffness after startle response
Stiffness at birth
Family history
Y N
Hereditary
Hyperekplexia Sporadic hyperekplexia
Symptomatic hyperekplexia
Neuropsychiatric startle syndromes
Mutations in GLR1A
Y (80%) N (20%)
Hyperekplexia Genetic testing

due to GLR1A SLC6A5
mutation GLRB
GPHN
ARHGEF9
6.8 Global cerebral hypomyelination due to AGC1 6.9 Neonatal myoclonic epilepsy due to mitochondrial
defect. Diagnostic flow chart followed by the authors in glutamate carrier GC1 defect
the only published reference of AGC1 deficiency
(Wibom et al. 2009). CNS symptoms
and global hypomyelination
(Table 6.8)
Early infantile
epileptic encephalopathy (EIEE)
with progressive microcephalia N-acetyl aspartate (CNS)↓↓
Lactate (P)↑
Y N
Mutations in AGC1 ATP production Other causes

from gluctamate
Y N (muscle biopsy) ↓
Y N
AGC1 deficiency Genetic testing Normal activity of

Other causes
ARX mitochondrial
Munc 18-1 complexes
Y N
Y N
GC1 mutations Other causes
EIEE Other causes Y N

(OMIM No 612164)
GC1
Other causes
Fig. 6.7 Diagnostic flow chart for AG1 deficiency deficiency
Fig. 6.8 Flow chart for GC1 deficiency

6.8 Specimen Collection adults relate to the prevention of stone formation by reducing
the absolute amount and increasing the solubility of the
6.1 Cystinuria, 6.2 dicarboxylic aminoaciduria, 6.3 Hartnup poorly soluble cystine that is excreted in the urine by dietary
disorder, 6.4 lysinuric protein intolerance, and 6.5 hyperdi- measures and alkalization of urine. If these conservative
basic aminoaciduria type 1. Standard urine sample collected approaches fail, thiol-chelating drugs that reduce cystine to
for 24 h or as single sample where amino acid concentration more soluble cysteine adducts may be administered. The
is referred to creatinine amount. 6.6 Episodic ataxia. Not goal is to maintain cystine urine concentration below 1
applicable. 6.7 Startle disease. Not applicable. 6.8 AGC1 mmol/l (~250 mg/l) and excretion below <100 μmol/mmol
deficiency. Muscle biopsy could be use to demonstrate of creatinine (Knoll et al. 2005; Chillarón et al. 2010).
reduced ATP production from glutamate. 6.9 GC1 defi- Treatment is not required for dicarboxylic aminoaciduria.
ciency. Skin specimen to prepare cultured fibroblast could be Photodermatitis in Hartnup disorder is treated with oral nico-
used to demonstrate deficient glutamate oxidation. tinamide. For LPI they are two main directions of therapy
(Sebastio et al. 2011). The first is aimed to reduce the risk of
hyperammonemia [low-protein diet and l-citrulline supple-
6.9 Prenatal Diagnosis mentation (to refill urea cycle intermediates) or administra-
tion of ammonium scavengers (e.g., sodium benzoate)]. The
Prenatal diagnosis is not recommended in cystinuria, dicar- second is aimed at the specific treatment of the severe com-
boxylic aminoaciduria, Hartnup disorder, and lysinuric pro- plications. Acetazolamide should be tried in any patient with
tein intolerance. For at risk pregnancies of EA and AGC1 EA, but not all are responsive. Clonazepam is used to treat
and GC1 deficiencies, if mutations have been identified in hyperekplexia. Epilepsy in the unique case reported on
the family, DNA sequence analysis can be performed by a AGC1 deficiency is treated with carbamazepine and leveti-
research laboratory. racetam. There is no treatment for GC1 deficiency.
Emergency Treatment
6.10 DNA Testing 6.1 Cystinuria. Urological interventions are often indicated
for the management of cystine stones >5 mm in diameter
DNA testing can be performed but is not necessary for diag- (Chillarón et al. 2010). The almost noninvasive extracorpo-
nosis of cystinuria, dicarboxylic aminoaciduria, and Hartnup real shock wave lithotripsy should be the treatment of choice
disorder. In cystinuria, if one mutated allele is already identi- at least in children (Knoll et al. 2005).
fied in one of the two cystinuria genes (SLC3A1 or SLC7A9),
the second mutated allele most probably will be identified in
Pitfalls
the same gene because digenic inheritance in cystinuria has
Some cystine stones have crystalline structures (e.g.,
not been demonstrated. A small proportion (~4 %) of carriers
smooth appearance or low degree of radiopacity),
of one mutated allele (mainly SLC7A9 heterozygotes) pres-
which make them resistant to extracorporeal shock
ent with cystine lithiasis. LPI: Neonatal DNA screenings for
wave lithotripsy. Ureteroscopy and percutaneous neph-
the unique mutation present in Finland (Finnish mutation
rostolithotomy may be preferable in these patients to
1181-2A→T) and a northern part of Iwate (Japan) (mutation
remove the stones.
R410X) with an incidence in the population of 1:60,000 have
been established due to the benefits of an early therapy. For
early infantile epileptic encephalopathy, DNA sequencing of 6.6 Episodic ataxia due to EAAT1 defect. Antiepileptic
GC1, ARX, and Munc18 will explain part of the cases, but drugs: carbamazepine (up to 1,600 mg/day), sulthiame
this information has no impact on therapy. DNA testing for (50–200 mg/day), and diphenylhydantoin (150–300 mg/day).
common forms of hyperekplexia and episodic ataxia is avail-
able. For the cases associated with SLC6A5 and SLC1A3,
Dangers/Pitfalls
respectively, sequencing is only available through research
Drugs should be used with caution due to significant
laboratories.
side effects.
6.11 Treatment Summary 6.7 Hyperekplexia due to GLYT2 defect. Sudden infant
death due to apnea (stiffness of the respiratory muscles) can
Methods to reverse the defect in transport that causes the dis- occur in cases of hyperekplexia. This can be prevented by the
orders discussed in this chapter have not been developed. Vigevano maneuver (flexing of the head and limbs toward
Treatment and management of cystinuria in children and the trunk; Vigevano et al. 1989).
Standard Treatment
6.1 Cystinuria
Objective Strategy Neonatal Infancy Childhood Adolescence Adulthood
Decrease in cystine excretion Dietary sodium intake (upper limit; g/24 h) – – 2 2 2
Animal protein intake (g/kg BW • 24 h) – – – <1 <1
Decrease in urine cystine Fluid intake (L/24 h) 0.5–1 2 3–4 4 4–5
concentration
Increase in cystine solubility in Urine alkalization (pH 7.5) Potassium citrate – 10–20 10–60 10–60 40–90
urine (mmol/24 h) (divided in 2–3 doses/day)
Tiopronina (mg/kg BW • 24 h)2 – – 20–40 20–40 45–60
a
Alternative treatment with tiopronin (α-mercaptopropionylglycine) produces adducts with cysteine that increase the solubility of cysteine 50-fold
Dangers/Pitfalls The incidence of adverse effects is similar for both

A high nocturnal fluid intake will delay the achievement agents (tiopronin (fever, proteinuria, and hyperlipidemia),
of urinary control in childhood. At least two nocturnal d-penicillamine (rash, fever, immune-complex-mediated
fluid intakes are recommended, but good compliance is glomerulonephritis, leucopenia, thrombocytopenia, and
difficult to achieve in adolescents and adults. taste loss)) but is slightly lower with tiopronin. Monitoring
Potassium citrate administration is recommended in 3 of liver enzymes, complete blood cell count, and urinary
doses (one-quarter of the daily dose in the morning, one- protein excretion should be performed regularly while
quarter at lunchtime, and half in the evening), which is patients are on tiopronin or d-penicillamine therapy.
recommended to monitor urine pH (>7.5) with indicator
paper three times per day to adjust treatment.
6.3 Hartnup disorder. Oral nicotinamide treatment

(50–100 mg/day) may prevent or resolve photodermatitis in
Hartnup disorder.
6.4 Lysinuric protein intolerance
Objective Strategy Neonatal Infancy Childhood Adolescence Adulthood

Prevention of Low-protein diet (upper limit; g/kg BW •24 h) – – 0.8–1.5 0.8–1.5 0.5–0.8
hyperammonemia Citrulline supplementation (mg/kg BW • 24 h) – – 100 100 100
(in 4 divided doses)
Sodium benzoate (mg/kg BW • 24 h) – – 100–250 100–250 100–250
(in 4 divided doses)
l-Carnitine (mg/kg BW • 24 h) (in 2 or more – – 25–50 25–50 25–50
divided doses)a
Additional hGH (for patients with GH deficiency) – – Careful evaluation for patients –
treatments with severe growth retardation
and delayed bone age
l-Lysine (mg/kg BW • 24 h) (in presence of – – 10–40 10–40 10–40
marked hypolysinemia)
Statins (to treat combined hyperlipidemia) – – Standard protocols
Severe Pulmonary alveolar proteinosis (PAP) – Bronchoalveolar lavage in specialized center.
complications Immunosuppressors
Chronic renal disease – – – Standard guidelines under the
direction of specialist
Hemophagocytic lymphohistiocytosis – – Standard guidelines under the direction of
specialist
a
Hypocarnitinemia, which strongly correlates with renal insufficiency, low-protein diet, and the use of ammonia scavenger drugs, might present in
LPI. Therefore, l-carnitine is supplemented after measurement of plasma carnitine levels (Sebastio et al. 2011)
Dangers/Pitfalls Dangers/Pitfalls
Urinary orotic acid can be used to monitor the protein Hyperekplexia can be misdiagnosed as seizures, but
tolerance and the urea cycle functioning but is not commonly used anticonvulsants are ineffective. The
totally reliable. effectiveness of valproic acid, clobazam, and fluox-
Nutritional deficiency due to the low-protein diet etine has been reported in a few sporadic cases of
might require supplementation of calcium, vitamin D, unknown genetic etiology (Zhou et al. 2002), but has
iron, and zinc. not been tested in controlled studies.
Recurrent fatal pulmonary alveolar proteinosis after
heart–lung transplantation in a child highlighted that
this complication of LPI is caused by factors external 6.8 AGC1 deficiency. Epilepsy in the unique case reported
to the lung, most likely macrophages (Santamaria et al. on AGC1 deficiency is treated with carbamazepine and
2004). levetiracetam.
Pulmonary alveolar proteinosis (PAP) of differ- 6.9 GC1 deficiency. There is no treatment, even for the
ent origins is usually treated by whole lung lavage or epilepsy associated.
granulocyte–macrophage colony-stimulating factor
(GM-CSF) administration. GM-CSF is a hematopoi- Experimental Treatment
etic growth factor known to stimulate stem cells to 6.1 Cystinuria. Real-time in situ atomic force microscopy
proliferate into granulocytes or monocytes, promote bas been used to identify cystine derivatives (l-cystine
differentiation of monocytes into alveolar macro- dimethylester and l-cystine methylester) that binds to the
phages, increase the catabolism within alveolar mac- surface and reduce the growth of cystine microcrystals in
rophages, and increase the innate immune potential of vitro (Rimer et al. 2010). Proof of principle of their antilithi-
neutrophils. Although GM-CSF may have therapeutic asic activity in vivo is yet lacking.
advantage in certain types of PAP, it may not be suit- 6.4 Lysinuric protein intolerance. Therapy with bisphos-
able for treating LPI-associated PAP because of the phonates (alendronate 10 mg/kg body weight • 24 h) has
tendency of LPI alveolar macrophages to form granu- been proposed for severe osteoporosis in LPI (Gómez et al.
lomas (Douda et al. 2009). 2006), but a standardized protocol is lacking.
6.6 Episodic ataxia due to EAAT1 defect. See emergency
treatment.
6.6 Episodic ataxia due to EAAT3 defect. Typical treat-
ment is acetazolamide (125–1,000 mg/day) (Pessia and Acknowledgements The authors thank Dr. Christian Lueck
Hanna 2010). Acetazolamide is a carboanhydrase inhibitor (Canberra Hospital) for clarification of differential diagnosis in cases
of episodic ataxia. The authors thank Dr. Rafael Artuch (Hospital
and particularly effective in episodic ataxia type 2 caused by
San Joan de Deu, Barcelona) for reference values of plasma amino acid
mutations in the calcium channel gene CACNA1A (Jen et al. concentration.
2007). Whether bicarbonate homeostasis is deranged in epi-
sodic ataxias is unclear.
4-Aminopyridine has been shown to be effective and
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Part II
Organic Acids
Disorders of Leucine, Isoleucine,
and Valine Metabolism 7
Ina Knerr, Jerry Vockley, and K. Michael Gibson
Contents Summary
7.1 Introduction...................................................................... 103 Disorders in the catabolic pathways of the branched-chain
amino acids (BCAA) leucine, isoleucine, and valine encom-
7.2 Nomenclature ................................................................... 106
pass diverse organic and aminoacidurias. Clinical severity
7.3 Metabolic Pathway .......................................................... 107 may range from asymptomatic findings in some to life-
7.4 Signs and Symptoms ........................................................ 107 threatening episodes and multiorgan involvement in others.
7.5 Reference Values .............................................................. 122
Several of these defects reflect a complex pathogenesis
related to mitochondrial dysfunction, particularly the
7.6 Pathological Values .......................................................... 124 3-methylglutaconic acidurias. As a general rule, treatment
7.7 Diagnostic Flow Chart..................................................... 127 includes the following: (1) dietary restriction of the precur-
7.8 Specimen Collection, Prenatal Diagnosis, sor BCAA along with optimal nutritional supply, (2) adjunct
and DNA Analysis ............................................................ 128 therapy (e.g., with L-carnitine, appropriate cofactors, other
7.8.1 Specimen Collection .......................................................... 128 conjugating compounds), (3) rapid intervention for meta-
7.8.2 Prenatal Diagnosis ............................................................. 128
bolic decompensation. Late complications of these diseases
7.8.3 DNA Analysis .................................................................... 129
must be anticipated, such as liver and renal failure. In asymp-
7.9 Treatment Summary........................................................ 130 tomatic individuals, instructions regarding risks for meta-
References .................................................................................... 140 bolic stress and fasting avoidance, along with clinical
monitoring, represent appropriate prophylactic interventions
at this time.
7.1 Introduction
The catabolic pathways of branched-chain amino acids

(BCAA), namely, leucine, isoleucine, and valine, consist of
multiple steps including transamination, oxidative decarbox-
ylation, and dehydrogenation. Due to irreversible steps early
I. Knerr (*) in metabolism, elevated levels of these amino acids do not
National Centre for Inherited Metabolic Disorders,
occur in those disorders that result from blocks in the path-
Children’s University Hospital, Temple Street, Dublin 1, Ireland
e-mail: ina.knerr@cuh.ie ways distal to the site of MSUD. Rather, these disorders are
associated with organic academia and aciduria. Severe forms
J. Vockley
Department of Pediatrics, Children’s Hospital of Pittsburgh, of these disorders usually present as acute, overwhelming ill-
University of Pittsburgh Medical Center, University of Pittsburgh, ness in the neonatal period if not detected through newborn
4401 Penn Avenue, Pittsburgh, PA 15224, USA screening. Milder variants may be episodic and not become
e-mail: vockleyg@upmc.edu
symptomatic until late childhood or even adult life. Moreover,
K.M. Gibson some patients are asymptomatic and identified only through
Section of Clinical Pharmacology,
family studies or newborn screening. Some disorders of
Washington State University, SAC 525M,
412 E. Spokane Falls Blvd, Spokane, WA 99202-2131, USA BCAA metabolism are exceedingly rare, and the clinical
e-mail: mike.gibson@wsu.edu experience in managing these cases is still being defined.
104 I. Knerr et al.
The existence of branched-chain amino acid (BCAA) leucine degradation pathway. Barth (MGA2) syndrome is an
transferase (BCAT1 and BCAT2) deficiency in humans X-linked multisystem disorder with cardiomyopathy, myop-
remains in question. There is some evidence that transamina- athy, neutropenia, and 3-methylglutaconic aciduria. Costeff
tion of valine and transamination of leucine and isoleucine syndrome (MGA3) is characterized by optic atrophy and neu-
may be affected differentially. Early reports have described rological symptoms. MEGDEL syndrome is a recessive dis-
patients with hyperleucine-isoleucinemia and hypervalin- order featuring sensorineural deafness and encephalopathy
emia, attributed to a defect of branched-chain amino acid with neuroradiological evidence of Leigh disease. Neonatal
transamination, who presented with failure to thrive and mitochondrial encephalocardiomyopathy is caused by iso-
mental retardation (Reddi et al. 1977). lated mitochondrial ATP synthase deficiency, associated
Maple syrup urine disease (MSUD) results from deficient with mutation in the TMEM 70 gene. 3-Methylglutaconic
activity of the branched-chain α-keto acid dehydrogenase aciduria type IV refers to conditions in which the primary
complex (BCKDC). During episodes of metabolic decom- etiologies remain to be elucidated.
pensation, the BCAA and their corresponding branched- 3-Hydroxy-3-methylglutaric acidemia (HMG-CoA lyase
chain α-keto acids (BCKA) accumulate excessively. The deficiency) is a dual defect in leucine degradation and keto-
pathophysiology of MSUD is thought to be related primarily genesis that often presents with neonatal hypoketotic hypo-
to leucine (Knerr et al. 2012). The enzyme complex BCKDC glycemia, metabolic acidosis, and hyperammonemia (Gibson
consists of three catalytic components (E1, E2, and E3) et al. 1988). Milder forms of the disorder, including presen-
encoded by four different genetic loci. Mutations in all four tation in adulthood, have also been reported. Overwhelming
of the catalytic loci have been associated with clinical dis- metabolic decompensation and organic aciduria is associated
ease (MSUD 1A, 1B, 2, 3, respectively). with a characteristic absence of ketone bodies, a hallmark of
Five clinical forms of MSUD exist, differentiated by the this disorder.
amount of residual enzymatic activity, age and severity of 2-Methylbutyrylglycinuria or 2-methylbutyryl-CoA
onset, and responsiveness to thiamine. These include classic, dehydrogenase (MBD) deficiency is a defect in isoleucine
intermediate, intermittent, and thiamine-responsive MSUD, degradation caused by short/branched-chain acyl-CoA dehy-
in addition to E3 (lipoamide dehydrogenase) deficiency. The drogenase (SBCAD) deficiency (Andresen et al. 2000).
latter associates with combined deficiencies of the pyruvate Diverse clinical symptoms such as hypotonia, mental retar-
dehydrogenase complex and 2-oxoglutarate dehydrogenase dation, autistic features, hypoglycemia, or metabolic acido-
complexes, which share the E3 component with BCKDC. sis have been reported, but most individuals (especially those
Isovaleric acidemia (IVA) results from deficient activity identified by newborn screening) have remained asymptom-
of isovaleryl-CoA dehydrogenase, ranging from acute neo- atic (Gibson et al. 2000; Sass et al. 2008). A founder muta-
natal to an intermittent or chronic presentation. Asymptomatic tion in the Hmong Chinese population has been described
individuals with only subtle biochemical abnormalities are (Alfardan et al. 2010; Matern et al. 2003).
detected through newborn screening (Vockley and Ensenauer 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD)
2006; Ensenauer et al. 2004). In patients with a severe phe- deficiency (or 17beta-hydroxysteroid dehydrogenase type 10
notype, the odor of “sweaty feet” may be detected during (HSD10) deficiency) was first reported in a 2-year-old boy
metabolic crisis, associated with marked ketoacidosis, bone with neurodegenerative symptoms (Zschocke et al. 2000).
marrow suppression, and hyperammonemia. This defect was also documented in a female patient who
3-Methylcrotonyl-CoA carboxylase (3MCCC) deficiency was less severely affected, suggesting skewed X-inactivation
(MCCD) also features a highly variable phenotype, from (Perez-Cerda et al. 2005). The disease-causing gene,
severe to asymptomatic presentation in patients detected via HSD17B10, encodes 17beta-hydroxysteroid dehydrogenase
newborn screening or family association studies (Gibson type 10 (HSD10), the latter essential for structural and func-
et al. 1998). Thus far, there are no reliable markers to iden- tional integrity of mitochondria (Rauschenberger et al. 2010).
tify the few individuals at risk for clinical symptoms. Beta-ketothiolase deficiency (alpha-methylacetoacetic
Appropriate testing (urinary organic acids, acylcarnitine pro- aciduria, 3-oxothiolase deficiency) is associated with recur-
file, and eventually mutation analysis/enzymatic assay) is rent episodes of vomiting, ketosis, and metabolic acidosis.
necessary to differentiate 3MCCC deficiency from the mul- Hypoglycemia, neutropenia, and thrombocytopenia have
tiple carboxylase deficiencies due to defects in biotin metab- been observed in infants.
olism (Chap. 14). Isobutyryl-CoA dehydrogenase deficiency (IBD defi-
3-Methylglutaconic aciduria may occur in multiple ciency) is a disorder of valine degradation. The original
forms. Only type I (MGA1), associated with reduced activ- patient presented with anemia and dilated cardiomyopathy
ity of 3-methylglutaconyl-CoA hydratase, is a disorder of the (Roe et al. 1998). Thus far, at least 22 individuals have been
7 Disorders of Leucine, Isoleucine, and Valine Metabolism 105
described who are mostly asymptomatic, largely identified Isoleucine and valine share the propionate pathway for
via newborn screening (Koeberl et al. 2003; Sass et al. 2004; their terminal steps of catabolism, and propionic acidemia
Oglesbee et al. 2007). (PA, propionyl-CoA carboxylase deficiency) and methylma-
3-Hydroxyisobutyryl-CoA deacylase deficiency (or meth- lonic acidemia (MMA, methylmalonyl-CoA mutase defi-
acrylic aciduria) has been confirmed in only two patients. ciency) are disorders of propionate degradation derived in
They presented with failure to thrive, developmental delay, part from the catabolism of isoleucine and valine, as well
and neurological symptoms in infancy. One patient had as other propionate precursors (threonine, methionine,
congenital malformations including vertebral abnormali- odd-chain fatty acids, and cholesterol). PA usually presents
ties, tetralogy of Fallot, and agenesis of the corpus callo- with life-threatening episodes of ketoacidosis, hyperam-
sum and died at 3 months. A second patient had episodes of monemia, encephalopathy, and hematological manifesta-
ketoacidosis and an abnormal brain MRI with signal abnor- tions. Late complications of these disorders include renal
malities in the globi pallidi and in the midbrain. Unusual failure (particularly in MMA), cardiomyopathy, long QT
cysteine and cysteamine conjugates of methacrylic acid syndrome, basal ganglion infarction, optic neuropathy, or
were detected in urine. In fibroblasts from both patients, impaired hearing ability (particularly in PA) (Grünert et al.
3-hydroxyisobutyryl-CoA hydrolase activity was deficient 2013). Respiratory chain deficiencies have been demon-
and molecular analysis revealed mutations in the HIBCH strated in both disorders (De Keyzer et al. 2009). In addition
gene (Loupatty et al. 2007). to the primary deficiencies of propionyl-CoA carboxylase
3-Hydroxyisobutyric aciduria may be caused by a pri- (PCC) and methylmalonyl-CoA mutase (MUT), secondary
mary defect of 3-hydroxyisobutyrate dehydrogenase, active defects of these enzymes can be associated with genetic dis-
in valine metabolism, or via secondary inhibition of orders in the metabolism of their cofactors, including biotin
3-hydroxyisobutyrate dehydrogenase in selected respiratory (Chap. 14) and cobalamin (Chap. 13).
chain defects (Loupatty et al. 2006). The clinical phenotype Malonic aciduria (MA) is caused by malonyl-CoA decar-
is variable, but the majority of patients reported in the litera- boxylase deficiency leading to a block in fatty acid metabo-
ture presented with dysmorphic features, neurodevelopmen- lism. Patients may present with cardiomyopathy,
tal symptoms, encephalopathy, ketoacidotic episodes, and developmental delay, short stature, seizures, hypoglycemia,
lactic acidemia. and metabolic acidosis (Salomons et al. 2007).
Methylmalonate semialdehyde dehydrogenase (MMSDH) Combined malonic and methylmalonic aciduria
deficiency is a rare disorder of the valine catabolic pathway (CMAMMA) is a rare recessive disorder caused by a defect
associated with vomiting and dehydration. So far, only one in the ACSF3 gene which encodes an acyl-CoA synthetase.
patient has had a mutation identified in the MMSDH gene Patients present with developmental delay, seizures, cogni-
(Chambliss et al. 2000). tive decline, cardiomyopathy, and metabolic acidosis.
106 I. Knerr et al.
7.2 Nomenclature
No. Disorder Alternative name Abbreviation symbol localization Affected protein no. Subtype
7.1 BCAA aminotransferase BCAA transaminase BCAT BAT BCAA 238340 All forms
deficiency aminotransferase
7.2 Maple syrup urine disease Branched-chain MSUD BCKDHA; 19q13.1–13.2; Branched-chain 248600 All forms
alpha-keto acid BCKDHB; 6q14.1; alpha-keto acid
dehydrogenase DBT; DLD 7q31–q32; dehydrogenase
complex deficiency 1p31 complex
7.3 Isovaleric acidemia Isovaleryl-CoA IVA ACAD2 15q14–q15 Isovaleryl-CoA 243500 All forms
dehydrogenase dehydrogenase
deficiency
7.4 Methylcrotonylglycinuria Methylcrotonyl-CoA MCC A; MCCC1; 3q27; Methylcrotonyl-CoA 210200 All forms
carboxylase MCC B MCCC2 5q12–13.2 carboxylase
deficiency
7.5 Methylglutaconic aciduria 3-Methylglutaconyl- MGA1 AUH 9q22.31 3-Methylglutaconyl- 250950 All forms
type I CoA hydratase CoA hydratase
deficiency
7.6 Barth syndrome Methylglutaconic MGA2 Xq28 Taffazin 302060 All forms
aciduria type II
7.7 MEGDEL syndrome MEGDEL SERAC1 6q25.3 MEGDEL 614739 All forms
7.8 Neonatal mitochondrial NME TMEM70 8q21.11 Complex V assembly 614052 All forms
encephalocardiomyopathy protein
7.9 Costeff syndrome Methylglutaconic MGA3 OPA3 19q13.2–13.3 OPA3 protein 258501 All forms
aciduria type III
7.10 Methylglutaconic aciduria 3-Methylglutaconic MGA4 ? 250951 Idiopathic
type IV aciduria
7.11 3-Hydroxy-3- 3-Hydroxy-3- HMGCL HMGCL 1p35–36 3-Hydroxy-3- 246450 All forms
methylglutaric aciduria methylglutaryl-CoA methylglutaryl-CoA
lyase deficiency lyase
7.12 2-Methylbutyrylglycinuria 2-Methylbutyryl-CoA MBCD ACADSB 10q26.13 2-Methylbutyryl-CoA 610006 All forms
deficiency
7.13 2-Methyl-3- 17-beta- HSD10 HSD17B10 Xp11.2 17-Beta- 300438 All forms
hydroxybutyryl-CoA hydroxysteroid hydroxysteroid
dehydrogenase deficiency dehydrogenase type dehydrogenase type 10
10 deficiency
7.14 Alpha-methylacetoacetic Beta-Ketothiolase BKT HADHB 2p23 3-Oxothiolas 203750 All forms
aciduria deficiency
7.15 Isobutyryl-CoA Isobutyrylglycinuria IBD ACAD8 11q25 Isobutyryl-CoA 611283 All forms
dehydrogenase deficiency dehydrogenase
7.16 3-Hydroxyisobutyryl-CoA Methacrylic aciduria HIBCH HIBCH 2q32.2 3-Hydroxyisobutyryl- 250620 All forms
deacylase deficiency deficiency CoA deacylase
7.17 3-Hydroxyisobutyrate 3-Hydroxyisobutyric HIBADH HIBADH 7p15.2 3-Hydroxyisobutyrate 236795 All forms
dehydrogenase deficiency aciduria deficiency dehydrogenase
7.18 Methylmalonate Combined MMSDH ALDH6A1 14q24.3 Methylmalonate 603178 All forms
semialdehyde semialdehyde semialdehyde
dehydrogenase deficiency dehydrogenase dehydrogenase
deficiency
7.19 Propionic acidemia Propionyl-CoA PA PCCA; 13q32; Propionyl-CoA 232000 All forms
carboxylase PCCB 3q21–q22 carboxylase deficiency
deficiency
7.20 Methylmalonic acidemia Methylmalonyl-CoA MMA MUT 6p12.3 Methylmalonyl-CoA 251000 Functional
mutase deficiency mutase
7.21 Malonic aciduria Malonyl-CoA MA MLYCD 16q24 Malonyl-CoA 248360 All forms
decarboxylase decarboxylase
deficiency
7.22 Combined MMA and MA MMA/MA ACSF3 16q24.3 Acetyl-CoA synthase 614245 All forms
family 3
L-Leucine Leucine L-Isoleucine Isoleucine L-Valine Valine
7.1 Aminotransferase 7.1 Aminotransferase Alloisoleucine 7.1 Aminotransferase

2-Oxoisocaproate 2-Oxoisovalerate
2-Oxoisocaproate 2-Hydroxyisocaproate 2-Oxo-3-methylvalerate
2-Oxo- 2-Oxo-
2-Hydroxyisovalerate
3-methylvalerate isovalerate
3-Hydroxyisovalerate 2-Hydroxy-
7.2 BCKDH thiamine Isovalerylglycine 7.2 BCKDH thiamine
3-methylvalerate 7.2 BCKDH thiamine
Isovalerylglucuronide
Isovaleryl-CoA Isovaleric acid 2-Methylbutyryl-CoA Isobutyryl-CoA Isobutyrylglycine
2-Methylbutyrylglycine
4-Hydroxyisovaleric acid Isobutyrylcarnitine
2-Ethylhydracrylic acid
7.3 IVD Isovalerylcarnitine 7.12 MBD 2-Methylbutyrylcarnitine 7.15 IBD Methacrylic acid
3-Hydroxyisovalerate conjugates
3-Methylcrotonyl-CoA Tiglyl-CoA Tiglylglycine Methacrylyl-CoA (S-2-carboxypropyl-
3-Methylcrotonylglycine
C5-Hydroxyacylcarnitine cysteamine, S-2-
7.4 MCC Hydratase Hydratase carboxypropylcysteine)
Tiglylglycine
3-Hydroxyisovalerate 2-Methyl-3- 2-Hydroxyisovalerate
3-Methylglutaconate hydroxybutyrate 3-Hydroxyiso- 3-Hydroxyiso-
3-Methyl- 3-Methylglutarate 2-Methyl-3-hydroxybutyrylcarnitine
Tiglylcarnitine butyryl-CoA
glutaconyl-CoA C5-Hydroxyacylcarnitine butyryl-CoA
2-Methyl-3-
3-Methylglutarylcarnitine hydroxybutyrylcarnitine 7.16 Deacylase 3-Hydroxyisobutyrate
7.5 Hydratase 3-Hydroxyisovalerate 7.13 MHBD 2-Hydroxyisovalerate
3-Methylglutaconate 3-Hydroxyisobutyrate 3-Hydroxyisobutyryl-
3-Methylglutarate 2-Methylacetoacetic carnitine
3-Hydroxy-3-methyl- 3-Hydroxy-3-methyl- acid 7.17 DH
2-Methylaceto- Tiglylglycine 3-Hydroxyisobutyrate
glutaryl-CoA glutarate
acetyl-CoA 2-Methyl-3- 3-Aminoisobutyric acid
C5-Hydroxyacylcarnitine hydroxybutyrate Methylmalonate 3-Hydroxypropionic acid
3-Methylglutarylcarnitine Beta- Tiglylcarnitine semialdehyde Beta-alanine
7.11 HMG-CoA lyase 7.14 Ethylmalonic acid
3-Hydroxyhexanoyl- ketothiolase 2-Methyl-3-hydroxy-
carnitine butyrylcarnitine 3-Hydroxypropionate
7.18 DH R,S-Methylcitrate
Propionylglycine
Acetoacetate Tiglylglycine
3-Hydroxybutyrate Acetyl-CoA Propionyl-CoA
Malonate/Methylmalonate Propionylcarnitine
Ketone Bodies
Malonyl-CoA 7.19 PCC biotin Methylmalonate
7.21
decarboxylase 7.22 ACSF3 R,S-Methylcitrate
Malonic acid
Ethylmalonic acid Malonyl-CoA Methylmalonyl-CoA 3-Hydroxypropionate
Methylmalonic acid Propionylcarnitine
Malonylcarnitine 7.20 Mutase cobalamin Methylmalonylcarnitine
Fatty acid Methylmalonic acid
metabolism Malonic acid Succinyl-CoA Krebs cycle
Malonylcarnitine
Fig. 7.1 Metabolic pathway
Table 7.1 BCAA aminotransferase deficiency

CNS Hyperkinesia ± ± ±
Hypotonia ± ± ±
Movement, decreased spontaneously ± ± ±
Retardation, psychomotor ± + ±
Dermatological Alopecia ± ± ±
Digestive Failure to thrive ± + ±
Feeding difficulties ± + ±
Vomiting, episodic ± ± ±
Eye Nystagmus ± ± ±
Hair Loss of hair ± ± ±
Routine laboratory Ketoacidosis ± ± ±
Metabolic acidosis ± ± ±
(continued)
108 I. Knerr et al.

Special laboratory Acylcarnitines, all (P) n n n
Allo-isoleucine (P) n n n
Arginine (P) n-↑↑ n-↑↑ n-↑↑
Glycine (P) n-↑ n-↑ n-↑
Isoleucine (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Leucine (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Valine (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Table 7.2 Maple syrup urine disease

CNS Apnea ± ± ± ± ±
Ataxia ± ± ± ± ±
Axonal sensory motor polyneuropathy, ± ± ± ± ±
chronic
Brain edema ± ± ± ± ±
Brain edema, cytotoxic ± ± ± ± ±
Dystonia ± ± ± ± ±
Hypo- or hypertonia ± ± ± ± ±
Irritability, episodic ± ± ± ± ±
Lethargy, coma (during ketoacidotic + + + + +
episodes)
Opisthotonus ± ± ± ± ±
Retardation, psychomotor ± ± ± ± ±
Feeding difficulties + + + ± ±
Pancreatitis ± ± ± ± ±
Vomiting, episodic + + + ± ±
Musculoskeletal Hypotonia, muscular ± ± ± ± ±
Other Hypothermia during crisis ± ± ±
Routine laboratory Ammonia (B) n-↑ n-↑ n-↑ n-↑ n-↑
Anion gap + + + + +
Glucose (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Ketoacidosis + + + + +
Metabolic acidosis + + + + +
Odor of maple syrup ± ± ± ± ±
Osmolality (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Sodium (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory 2,4-Dinitrophenylhydrazine test (U) ± ± ± ± ±
Allo-isoleucine (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
BCKDC activity (FB) ↓ ↓ ↓ ↓ ↓
Branched-chain oxoacids (P, U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Ferric chloride test (U) ± ± ± ± ±
Leu, Ile, Val (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
MRI: brain edema ± ± ± ± ±
MRI: brainstem and cerebellar edema ± ± ± ± ±
MRI: cerebral atrophy ± ± ± ± ±
MRI: deep gray matter structural lesions ± ± ± ± ±
MRI: delayed myelination ± ± ± ± ±
MRI: vacuolating myelinopathy ± ± ± ± ±
MRI: white matter changes ± ± ± ± ±
MRS: low NAA peak during crisis + + + + +
Table 7.3 Isovaleric acidemia

Cardiovascular Cardiac arrhythmia ± ± ± ± ±
CNS Altered consciousness ± ± ± ± ±
Ataxia ± ± ± ±
Developmental delay ± ± ± ± ±
episodes)
Dermatological Alopecia ± ±
Hepatomegaly ± ± ± ± ±
Vomiting, episodic + + + ± ±
Hematological Leukopenia ± ± ± ± ±
Neutropenia ± ± ± ± ±
Pancytopenia ± ± ± ± ±
Thrombocytopenia ± ± ± ± ±
Odor of sweaty feet ± ± ± ± ±
Anion gap + + + + +
Calcium (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Lactate (P) n-↑ n-↑ n-↑ n-↑ n-↑
Uric acid (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory 3-Hydroxyisovaleric acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
C5-Acylcarnitine (P, B) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Carnitine, esterfied (P) ↑ ↑ ↑ ↑ ↑
Carnitine, free (DBS, P) ↓ ↓ ↓ ↓ ↓
Glycine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Isovaleric acid (P) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
Isovaleryl-CoA dehydrogenase (FB) ↓ ↓ ↓ ↓ ↓
Isovalerylglycine (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
MRI: abnormalities of the globus pallidus ± ± ± ± ±
MRS: increased ratio of lactate to NAA ± ± ± ± ±
Table 7.4 Methylcrotonylglycinuria

Cardiovascular Cardiomyopathy ± ± ± ± ±
CNS Encephalopathy acute, precipitated ± ± ± ± ±
by infection
Hypo or hypertonia ± ± ± ± ±
Metabolic stroke ± ± ± ± ±
(continued)
110 I. Knerr et al.

Hematological Neutropenia ± ± ± ± ±
Musculoskeletal Muscle pain. ± ± ± ± ±
Muscle weakness ± ± ± ± ±
Other Highly variable phenotype incl ± ± ± ± ±
asymptomatic individuals
Odor, acrid (Urine) ± ± ± ± ±
Anion gap ± ± ± ± ±
Base excess ± ± ± ± ±
Ketoacidosis ± ± ± ± ±
Metabolic acidosis ± ± ± ± ±
3-Methylcrotonylcarnitine n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
3-Methylcrotonylglycine (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
C5-OH-acylcarnitine (P, B) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Carnitine, esterfied (P) n-↑ n-↑ n-↑ n-↑ n-↑
Carnitine, free (DBS, P) ↓-n ↓-n ↓-n ↓-n ↓-n
Methylcrotonyl-CoA carboxylase (FB) ↓ ↓ ↓ ↓ ↓
Table 7.5 Methylglutaconic aciduria type I

CNS Ataxia ± ± ± + +
Athetosis ± ± ± ± ±
Cerebellar abnormalities ± ± ± ± ±
Dementia ± ± ± ± ±
Fits ± ± ±
Leukoencephalopathy ± ± + + +
Retardation and regression + + + + +
Retardation, psychomotor ± + + ± ±
Seizures, febrile ± ± ±
Spasticity, limbs ± ± ± ± +
Digestive Hepatomegaly ± ± ± ± ±
Eye Nystagmus ± ± ± ± ±
Optic atrophy ± ± ± ± ±
Hematological Thrombocytopenia ± ± ±
Creatine kinase (P) n-↑ n-↑ n-↑
Glucose (P) ↓-n ↓-n ↓-n
Metabolic acidosis ± ± ±

3-Methylglutaconic acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Methylglutaconyl-CoA hydratase (FB) ↓ ↓ ↓ ↓ ↓
3-Methylglutaric acid (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
C6-unsaturated acylcarnitine (P, B) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
MRI: basal ganglia lesions ± ± ± ± ±
MRI: cerebellar abnormalities ± ± ± ± ±
MRS: accumulation of ± ± ± ± ±
3-hydroxyisovaleric acid
MRS: decrease in N-acetylaspartate ± ± ± ± ±
Table 7.6 Barth syndrome

Cardiovascular Cardiac arrhythmia ± ± ± ± ±
Cardiomyopathy + + + + +
Cardiomyopathy, dilated ± + + + +
Clots, stroke ± ± ± ± ±
Heart Failure ± ± ± ± ±
Left ventricular non-compaction ± + + + +
Dermatological Chronic aphthous ulceration ± ± ± ± ±
Digestive Feeding difficulties + + ± ± ±
Hematological Neutropenia + + + + +
Sepsis ± ± ± ± ±
Musculoskeletal Exercise Intolerance ± ± ± ± ±
Growth retardation ± + + + +
Hypotonia, muscular + + + ± ±
Myopathy + + + + +
Respiratory Respiratory distress ± ± ± ± ±
Other “Cherubic” face ± ±
Mild dysmorphic features ± ± ± ± ±
Cholesterol (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Creatine kinase (P) ± ± ± ± ±
Hypoglycemia, episodic ± ± ± ± ±
Special laboratory 2-Ethylhydracrylic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
3-Methylglutaconic acid (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
3-Methylglutaric acid (U) ↑ ↑ ↑ ↑ ↑
Abnormal cardiolipin profile + + + + +
Cardiolipin (tissue, FB) ↓ ↓ ↓ ↓ ↓
MRI: occasionally cerebral atrophy ± ± ± ± ±
112 I. Knerr et al.
Table 7.7 MEGDEL syndrome

CNS Basal ganglia abnormalities + + + + +
Behavior difficulties ± + + + +
Bilateral sensory hearing loss + + + + +
Dystonia + + + + +
Encephalopathy + + + + +
Epilepsy ± ± ± ± ±
Extrapyramidal signs + + + + +
Leigh syndrome + + + ± ±
Metabolic stroke ± + + + +
Regression, motor ± + + + +
Retardation, psychomotor + + + + +
Spasticity ± + + + +
Digestive Failure to thrive + + + ± ±
Ear Deafness, sensorineural ± + + + +
Hematological Sepsis + + + ± ±
Routine laboratory Anion gap + ± + + +
Lactate (CSF) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Lactate (P) n-↑↑↑ n-↑↑↑ n-↑↑↑ n-↑↑↑ n-↑↑↑
Special laboratory 3-Hydroxyisovaleric acid (U) n n n n n
3-Methylglutaconic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Filipin test + + + + +
MRI: bilateral hyperintensities + + + + +
of basal ganglia
MRI: cerebral and cerebellar atrophy + + + + +
MRI: Leigh-like lesions + + + + +
Table 7.8 Neonatal mitochondrial encephalocardiomyopathy

Cardiovascular Cardiomyopathy, hypertrophic + + + + +
Wolf-Parkinson-White syndrome ± ±
CNS Apnea ± ± ±
Cortical-subcortical atrophy ± ± ± ± ±
Encephalopathy + + + + +
Digestive Failure to thrive + + + +
Gastrointestinal dysmotility ± ± ± ± ±
Hepatomegaly + + ± ± ±
Eye Cataract ± ± ± ± ±
Genitourinary Cryptorchidism ± ± ± ± ±
Hypospadias ± ± ± ± ±
Musculoskeletal Contractures ± ± ± ± ±
Hypotonia, muscular + + + + +
Renal Renal tubulopathy ± ± ± ± ±
Respiratory Respiratory insufficiency ± ± ± ± ±
Other Facial dysmorphism ± ± ± ± ±

Routine laboratory Ammonia (B) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Anion gap + + + + +
Creatine kinase (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Lactate (CSF) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Lactate (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Lactic acidosis + + ± ± ±
Metabolic acidosis + + + + ±
Special laboratory 3-Methylglutaconic acid (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
MRI: mild cerebellar hypoplasia ± ± ± ±
Pronounced ketonuria during crisis + + + + +
Table 7.9 Costeff syndrome

CNS Ataxia ± ± ± ± ±
Chorea ± ± ± ±
Cognitive dysfunction ± ± ± ± ±
Extrapyramidal movement disorder ± ± ± + +
Mental retardation, mild ± ± ± ±
Movement disorder, complex ± ± ± ± ±
(Paroxysmal)
Spastic paraplegia ± ± ± ±
Eye Nystagmus ± + + + +
Optic atrophy ± + + + +
Special laboratory 3-Methylglutaconic acid (U) ↑ ↑ ↑ ↑ ↑
3-Methylglutaric acid (U) ↑ ↑ ↑ ↑ ↑
Table 7.10 Methylglutaconic aciduria type IV

CNS Basal ganglia abnormalities ± ± ± ± ±
Encephalopathy ± ± ± ± ±
Intellectual disability ± ± ± ± ±
Hematological Anemia, macrocytic ± ± ± ± ±
Creatine kinase (P) n-↑ n-↑ n-↑ n-↑ n-↑
Lactate (U) n-↑ n-↑ n-↑ n-↑ n-↑
(continued)
114 I. Knerr et al.

Special laboratory 3-Methylglutaconic acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Methylglutaric acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Citric acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Glycogen (heart, muscle) n-↑ n-↑ n-↑ n-↑ n-↑
Lipids (heart) n-↑ n-↑ n-↑ n-↑ n-↑
Lipids (liver) n-↑ n-↑ n-↑ n-↑ n-↑
Lipids (muscle) n-↑ n-↑ n-↑ n-↑ n-↑
Methionine (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Table 7.11 3-Hydroxy-3-methyl glutaric aciduria

Cardiovascular Cardiomyopathy, dilated ± ± ± ±
CNS Cerebral infarction/stroke-like ± ± ± ± ±
encephalopathy
Convulsions + +
Hypotonia, muscular ± ± ± ± ±
Lethargy, coma (during crisis) + + + + +
Digestive Hepatomegaly + + ± ± ±
Liver dysfunction + + + + +
Vomiting, episodic + + ± ± ±
Free fatty acids (S) n-↑ n-↑ n-↑ n-↑ n-↑
Hypoketotic hypoglycemia + + + + +
Ketones (U, P) ↓ ↓ ↓ ↓ ↓
Lactic acidosis + + + + +
Special laboratory 3-Hydroxy-3-methylglutaric acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Hydroxy-3-methylglutaryl-CoA ↓ ↓ ↓ ↓ ↓
lyase (FB)
3-Hydroxyisovaleric acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Methylcrotonylglycine (U) n-↑ n-↑ n-↑ n-↑ n-↑
Adipic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
C6:1 Acylcarnitine (B, P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
C6DC Acylcarnitine (B, P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Glutaric acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
MRI/CT: white matter abnormalities ± ± ± ± ±
MRI: occipital lesions ± ± ± ± ±
MRS: decreased N-acetylaspartate/ ± ± ± ± ±
creatine ratio
Sebacic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Suberic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Table 7.12 2-Methylbutyrylglycinuria

CNS Autistic spectrum disorder ± ± ± ± ±
Decreased cortical sulci ± ±
Hypotonia, muscular ± ± ± ± ±
Other Patients may remain asymptomatic ± ± ± ± ±
Routine laboratory Anion gap ± ± ± ± ±
Special laboratory 2-Ethylhydracrylic acid (P, B) n-↑ n-↑ n-↑ n-↑ n-↑
2-Methylbutyric acid (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
2-Methylbutyrylcarnitine (P, B) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
2-Methylbutyryl-CoA dehydrogenase ↓ ↓ ↓ ↓ ↓
(FB)
2-Methylbutyrylglycine (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
Table 7.13 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency

Cardiovascular Cardiomyopathy ± + + ± ±
CNS Basal ganglia abnormalities ± ± ± ± ±
Brain atrophy ± ± ± ± ±
Choreoathetosis ± ± ± ± ±
Dysarthria ± ± + + +
Dystonia ± + + + +
Frontotemporal atrophy ± ± ± ± ±
Movement disorder ± ± ± ± ±
Retardation, psychomotor ± + + + +
Rigidity ± ± ±
Seizures ± + + ± ±
Ear Hearing loss, sensorineural ± ± ± ± ±
Eye Vision, decreased ± ± ± ± ±
Other Most patients are male + + + + +
Routine laboratory Glucose (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Lactate (CSF) n-↑ n-↑ n-↑ n-↑ n-↑
Lactic acidosis ± + + ± ±
Special laboratory 17-Beta-hydroxysteroid dehydrogenase ↓ ↓ ↓ ↓ ↓
type 10 (FB)
2-Methyl-3-hydroxybutyric acid (U) ↑ ↑ ↑ ↑ ↑
2-Methyl-3-hydroxy-butyrylcarnitine n-↑ n-↑ n-↑ n-↑ n-↑
(P, B)
MRI: frontotemporal atrophy ± ± + + +
MRI: periventricular white matter changes ± ± ± ± ±
Tiglylcarnitine (P, B) n-↑ n-↑ n-↑ n-↑ n-↑
Tiglylglycine (U) ↑ ↑ ↑ ↑ ↑
116 I. Knerr et al.
Table 7.14 Alpha-methylacetoacetic aciduria

Basal ganglia abnormalities ± ± ± ± ±
Lethargy, coma (during ketoacidotic ± ± ± ± ±
episodes)
Movement disorder ± ± ± ± ±
Pyramidal signs ± ± ± ± ±
Hepatomegaly + + + ± ±
Liver dysfunction ± + + ± ±
Vomiting, episodic + + + + +
Hematological Neutropenia ± ± ± ± ±
Other Acetone-like odor to the breath ± ±
Anion gap + + + + +
Free fatty acids (S) n n n
Glucose (P) ↓-↑ ↓-↑ ↓-↑ ↓-↑ ↓-↑
Ketones (U, P) ↑ ↑ ↑ ↑ ↑
Special laboratory 2-Ethylhydracrylic acid (U) ↑ ↑ ↑ ↑ ↑
2-Methyl-3-hydroxybutyric acid (U) ↑ ↑ ↑ ↑ ↑
2-Methyl-3-hydroxy-butyrylcarnitine ↑ ↑ ↑ ↑ ↑
(P, B)
2-Methylacetoacetic acid (U) ↑ ↑ ↑ ↑ ↑
3-Hydroxy-n-butyric acid (U, B) ↑ ↑ ↑
3-Oxothiolase activity (FB) ↓ ↓ ↓ ↓ ↓
Acetoacetate (U, B) ↑ ↑ ↑
Dicarboxylic acids (U) n n n n n
Glycine (P) n-↑ n-↑ n-↑ n-↑ n-↑
MRI: Dentate nucleus lesions ± ± ± ± ±
MRI: Hyperintensities (T2) ± ± ± ± ±
of the globi pallidi
MRI: signal abnormalities within the ± ± ± ± ±
putamina
MRS: Occasional increase of lactate ± ± ± ± ±
and choline
Tiglylcarnitine (P, B) ↑ ↑ ↑ ↑ ↑
Table 7.15 Isobutyryl-CoA dehydrogenase deficiency

Cardiovascular Cardiomyopathy, dilated ± ± ± ± ±
Digestive Vomiting, episodic ± ± ± ± ±
Hematological Anemia ± ± ± ± ±
Other Most patients appear to be asymptomatic ± ± ± ± ±
Special laboratory C4-acylcarnitine ↑ ↑ ↑ ↑ ↑
Isobutyryl-CoA dehydrogenase (FB) ↓ ↓ ↓ ↓ ↓
Isobutyrylglycine (U) ↑ ↑ ↑ ↑ ↑
Table 7.16 3-Hydroxyisobutyryl-CoA deacylase deficiency

CNS Developmental delay + ± + + +
Encephalopathy acute, precipitated ± ± ± ± ±
by infection
Retardation and regression ± ± ± ± ±
Truncal ataxia ± ± ± ± ±
Digestive Feeding difficulties + + + + +
Other Dysmorphism ± ± ± ± ±
Routine laboratory Anion gap ± ± ± ± ±
Special laboratory 2-Hydroxyisovaleric acid (U) ↑ ↑ ↑ ↑ ↑
3-Hydroxyisobutyrylcarnitine (P, B) ↑ ↑ ↑ ↑ ↑
Methacrylic acid conjugates (U) ↑ ↑ ↑ ↑ ↑
MRI: abnormalities in the globus pallidus ± ± ± ± ±
MRI: abnormalities in the midbrain ± ± ± ± ±
MRI: agenesis of the cingulate gyrus ± ± ± ± ±
MRI: agenesis of the corpus callosum ± ± ± ± ±
S-2-carboxypropyl-cysteamine (U) ↑ ↑ ↑ ↑ ↑
S-2-carboxypropyl-cysteine (U) ↑ ↑ ↑ ↑ ↑
Test 3-Hydroxyisobutyryl-CoA ↓ ↓ ↓ ↓ ↓
deacylase (FB)
Table 7.17 3-Hydroxyisobutyrate dehydrogenase deficiency

CNS Developmental delay ± ± ± ± ±
Microcephaly ± ± ± ± ±
Digestive Failure to thrive ± + + ± ±
Other Dysmorphic features ± ± ± ± ±
Routine laboratory Ketoacidosis + + + + ±
Metabolic acidosis + + + + ±
Special laboratory 2-Hydroxyisovaleric acid (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
3-Hydroxyisobutyric acid (U) ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑ ↑-↑↑
3-Hydroxyisobutyryl-carnitine (P, B) n-↑ n-↑ n-↑ n-↑ n-↑
CT: intracerebral calcification ± ± ± ± ±
MRI: Focal white matter lesions ± ± ± ± ±
Test 3-hydroxyisobutyrate dehydrogenase (FB) ↓ ↓ ↓ ↓ ↓
118 I. Knerr et al.
Table 7.18 Methylmalonate semialdehyde dehydrogenase deficiency

CNS Developmental delay ± ± ± ± ±
Vomiting, episodic ± ± ± ± ±
Routine laboratory Lactate (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory 2-Aminoisobutyrate n-↑ n-↑ n-↑ n-↑ n-↑
3-Aminoisobutyric acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
3-Hydroxyisobutyric acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
3-Hydroxypropionic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Beta-alanine (U) n-↑ n-↑ n-↑ n-↑ n-↑
Ethylmalonic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Methionine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Methylmalonic semialdehyde ↓ ↓ ↓ ↓ ↓
dehydrogenase (FB)
Table 7.19 Propionic acidemia

Cardiovascular Arrhythmia ± ± ± ± ±
Cardiomyopathy ± ± ± ± ±
Cardiomyopathy, dilated ± ± ±
QT interval prolongation ± ± ± ± ±
Encephalopathy acute, precipitated + + + + +
by infection
Extrapyramidal signs ± ± ± ± ±
Hypo or hypertonia + + + + +
episodes)
Feeding difficulties + + + + +
Ear Hearing loss, sensorineural ± ± ± ± ±
Endocrine Decreased body height ± ± ± ± ±
Hypogonadism (in females), ± ±
hypergonadotropic
Eye Optic atrophy ± ± ± ± ±
Hematological Anemia + + + + +
Myelodysplasia ± ± ± ± ±
Neutropenia + + + + +
Thrombocytopenia + + + + +
Musculoskeletal Hypotonia, muscular + + + ± ±
Osteopenia ± ± ± ±
Renal Renal failure, chronic ±
Temporary impairment of renal function ± ± ± ± ±

Other Low body temperature during crisis ± ± ±
Anion gap + + + + +
Hyperglycemia ± ±
Ketones (U, P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Lactate (U) ↑ ↑ ↑ ↑ ↑
Special laboratory 3-Hydroxypropionic acid (U) ↑ ↑ ↑ ↑ ↑
Glutamine (CSF) n-↑ n-↑ n-↑ n-↑ n-↑
Glutamine (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Glycine (P, U, CSF) ↑ ↑ ↑ ↑ ↑
Methylcitric acid (U) ↑ ↑ ↑ ↑ ↑
MRS: elevated lactate ± ± ± ± ±
MRS: elevation of glutamine/glutamate ± ± ± ± ±
Propionylcarnitine (P, B) ↑ ↑ ↑ ↑ ↑
Propionyl-CoA carboxylase activity ↓ ↓ ↓ ↓ ↓
(WBC, FB)
Propionylglycine (U) ↑ ↑ ↑ ↑ ↑
Table 7.20 Methylmalonic acidemia

Encephalopathy acute, precipitated + + + + +
by infection
Extrapyramidal signs ± ± ± ± ±
episodes)
(continued)
120 I. Knerr et al.

Endocrine Decreased body height ± ± ± ± ±
Eye Optic neuropathy ± ± ± ± ±
Hematological Anemia + + + + +
Thrombocytopenia + + + + +
Musculoskeletal Hypotonia, muscular + + + ± ±
Osteopenia ± ± ± ±
Renal Progressive renal impairment ± + + + +
Reduced glomerular filtration rate ± ± + + +
Renal tubulopathy ± ± ± ± ±
Tubulointerstitial nephritis ± ± ± ± ±
Anion gap + + + + +
Hyperglycemia ± ±
Ketones (U, P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Lactate (U) ↑ ↑ ↑ ↑ ↑
Special laboratory 14C-Propionate incorporation assay (FB) ↓ ↓ ↓ ↓ ↓
3-Hydroxypropionic acid (U) ↑ ↑ ↑ ↑ ↑
Glutamine (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Glycine (P, U) ↑ ↑ ↑ ↑ ↑
Methylmalonic acid (P, U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Methylmalonylcarnitine (P, B) ↑ ↑ ↑ ↑ ↑
Methylmalonyl-CoA mutase activity ↓ ↓ ↓ ↓ ↓
(FB)
MRS: elevated lactate ± ± ± ± ±
Propionylcarnitine (P, B) ↑ ↑ ↑ ↑ ↑
Table 7.21 Malonic aciduria

Cardiovascular Cardiomyopathy ± + + + +
CNS Developmental delay ± + + ± ±
Encephalopathy acute, precipitated ± ± ± ± ±
by infection
Vomiting + + + ± ±

Base excess ± ± ± ± ±
Ketones (U, P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Lactate (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Special laboratory 3-Hydroxybutyric acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Adipic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Ethylmalonic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Fumaric acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Malic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Malonic acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Malonylcarnitine ↑ ↑ ↑ ↑ ↑
Methylmalonic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
MRI/CT: Frontotemporal atrophy ± ± ± ± ±
MRI/CT: white matter abnormalities ± ± ± ± ±
Sebacic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Suberic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Succinic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Urine MMA/MA ratio ↓ ↓ ↓ ↓ ↓
Test Malonyl-CoA decarboxylase activity ↓ ↓ ↓ ↓ ↓
Table 7.22 Combined MMA and MA

CNS Autistic spectrum disorder ± ± ± ± ±
Coma, lethargy ± ± ± ± ±
Developmental delay ± + + ± ±
Loss of speech ± ± ± ±
Memory problems ± ± ± ±
Microcephaly ± ± ±
Migraine, ocular ± ± ±
Feeding difficulties ± ± ± ± ±
Other Mild dysmorphic features ± ± ± ± ±
Routine laboratory Base excess ± ± ± ± ±
Lactate (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
(continued)
122 I. Knerr et al.

Special laboratory Carnitine, free (DBS, P) ↓-n ↓-n ↓-n ↓-n ↓-n
Malonic acid (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Malonylcarnitine ↑ ↑ ↑ ↑ ↑
Methylmalonic acid (P, U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
MRI: hyperintensities (T2) ± ± ± ± ±
Propionylcarnitine (P, B) n n n n n
Urine MMA/MA ratio ↑ ↑ ↑ ↑ ↑

Urine/spot tests
2,4-Dinitrophenylhydrazine
(DNPH) test Ferric chloride test
Normal No precipitate No color change
Plasma quantitative amino acids (μmol/l) (Ion exchange column

chromatography or high-performance liquid chromatography, HPLC)
Age Valine Isoleucine Leucine Alloisoleucine
Newborna 86–220 23–102 48–180 0
Child 64–320 30–105 59–180 0
Adult 99–286 30–108 61–162 0
Age Glycine Methionine Threonine

Newborna 220–500 6–46 60–200
Child 130–400 9–45 60–200
Adult 120–550 9–59 43–130
a
The national standards and local reference ranges for newborn
screening or selective screening tests should be used
7
Organic acids in urine (mmol/mol creatinine), blood plasma or serum (μmol/l), or dried blood spots (DBS, μmol/l) (gas chromatography/mass spectrometry, GC/MS)
Urine 2-oxoiso- Urine 2-oxo-3-methyl- Urine 2-oxoiso- Urine 2-hydroxyisovaleric Urine 2-hydroxy- Urine 2-hydroxy-3- Venous blood lactate
Age caproic acid valeric acid valeric acid acid isocaproic acid methyl-valeric acid Urine lactate (mmol/l)
All <2 <2 <2 <2 <2 <2 <197 <2.2
Plasma/serum Urine Urine Urine Urine Urine Urine Urine 3-hydroxy-

isovaleric DBS isovalery- 3-hydroxyisovaleric 4-hydroxyisovaleric Urine 3-methyl- 3-methylglutaconic 3-methyl- 3-methylglutaric
Age acid isovalerylglycine glycine acid acid isovalerylglucuronide crotonyl-glycine acid glutaric acid acid
All <10 <0.5 <10 <16 <2 ND <2 <6 <7 <36
Urine Urine Urine

Urine DBS Urine propionyl- Urine 2-methyl Urine DBS 2-methyl-3- Urine 2-methyl-
Age 3-hydroxypropionate 3-hydroxypropionate methyl-citrate glycine butyrylglycine methylmalonate methylmalonate hydroxybutyrate 3-hydroxyisobutyrate acetoacetate
Disorders of Leucine, Isoleucine, and Valine Metabolism
All <2 ND <2 <2 <2 <2 <0.4 <12 <24 <2
Urine Urine S-2-carboxypropyl-

Urine 2-methyl-3- Urine Urine cysteamine and Urine
Age Urine 2-methylbutyrylglycine 2-ethylhydracrylic acid hydroxybutyrate tiglylglycine isobutyrylglycine S-2-carboxypropyl-cysteine 3-hydroxyisobutyrate
All <2 <2 <11 <2 <3.8 ND <33
Urine Urine
Urine DBS methyl- Urine Urine Urine DBS Plasma/serum malonic
Age 3-hydroxypropionate 3-hydroxypropionate citrate tiglylglycine propionylglycine methylmalonate methylmalonate methylmalonate acid
All <10 ND <8 <2 <2 <2 <0.4 <0.2 <5
Acylcarnitines in dried blood spots (DBS, μmol/l) (tandem mass spectrometry)

Free C3-/ C4-DC-/ C5-/isovaleryl- C5:1-/ C5-OH-/3-hydroxyisovaleryl-
carnitine propionyl- C3-DC/ C4-/ methylmalonyl C4-OH-/3-OH- /2-methylbutyryl pentenoyl-/ /2-methyl-3-OH-
Age (C0) carnitine malonylcarnitine butyrylcarnitine + isobutyrylcarnitine carnitine isobutyrylcarnitine carnitine tiglylcarnitine butyrylcarnitine
Alla 10–60 <3.60 <0.20 <0.50 <0.54 <0.39 <0.20 <0.03 <0.36
The national standards and local reference ranges for newborn screening or selective screening tests should be used
a
Of note, acylcarnitine reference values are age dependent (see special literature)
123
124 I. Knerr et al.
Urine/spot tests
Disorder 2,4-Dinitrophenylhydrazine (DNPH) testa Ferric chloride testa
7.2 Maple syrup disease (MSUD) Yellow precipitate Greenish-gray color
a
This test has historical importance but has since been replaced (e.g., through newborn screening or selective metabolic screening using tandem
mass spectrometry)
Plasma quantitative amino acids (μmol/l) (ion exchange column chromatography or high-performance liquid chromatography, HPLC)
Disorder Valine Isoleucine Leucine Alloisoleucine % normal activity of BCKDa complex
7.1. BCAT deficiency 220–1,500 Increased Increased – Normal
7.2 MSUD
Clinical forms:
Classic To 7,550 To 4,800 To 10,800 ≥5–970 0–2
Intermediateb To 1,000 To 1,000 To 2,000 2–220 3–30
Intermittentb To 1,000 To 1,000 To 4,000 2–220 3–30
Thiamin responsive To 1,000 To 1,000 To 5,000 Present 2–40
BCKD Branched-chain α-keto acid dehydrogenase
a
b
Levels may only be abnormal during acute episodes of ketoacidosis in the intermittent/intermediate form
Comments/Additions
1. Leucine, isoleucine, and valine are usually not elevated in 2. Secondary findings, such as elevated plasma glycine and
metabolic defects distal from the BCKD complex as this alanine concentrations, are not listed separately.
complex catalyzes an irreversible enzymic reaction.
Organic acids in urine (mmol/mol creatinine), blood plasma or serum (μmol/l), or dried blood spots (DBS, μmol/l) (gas chromatography/mass
spectrometry, GC/MS)
Urine Urine
Urine 2-oxo-3- Urine Urine Urine 2-hydroxy-3- Venous blood
2-oxoiso- methyl-valeric 2-oxoiso- 2-hydroxy- 2-hydroxymethyl-valeric Urine lactate
Disorder abbreviation caproic acid acid valeric acid isovaleric acid isocaproic acid acid lactatea (mmol/l)a
7.1 BCAA – – – – – –
transferase deficiency
7.2 MSUD To 4,400 To 2,500 To 800 To 3,600 To 80 To 400 n-↑ n-↑
Urine Urine Urine

Disorder Plasma/serum DBS Urine 3-hydroxy- 4-hydroxy- Urine 3-methyl-
abbreviation isovaleric acid isovalerylglycine isovalerylglycine isovaleric acid isovaleric acid isovalerylglucuronide crotonyl-glycine
7.3 IVA 600–5,000 (with 1.3–80.0 To 4,980 To 2,000 20–300 Detectableb –
episodes);
10–50 (between
episodes)
7.4 MCCD – – – 96–8,850 – – 40–4,042
Urine Urine
3-hydroxy- 3-methyl- Urine 3-methylglutaconic Urine Urine 3-hydroxy-3-
Disorder abbreviation isovaleric acid crotonyl-glycine acid 3-methylglutaric acid methylglutaric acid
7.5 MGA1 47–3,840 – 168–2,900 4.5–9.0 –
7.6 Barth syndrome – – 18–600 10–85 –
7.7 MEGDEL syndrome – 16–196 n-↑
7.8 Neonatal mitochondrial – 12–361 n-↑
encephalocardiomyopathy
7.9 Costeff syndrome – – 9–187 (combined excretion –
with 3-methylglutaric acid)
7.10 MGA4 – – 23–1,793 5–215 –
7.11 HMG-CoA lyase deficiency 60–9,600 0–400 140–24,200 14–3,000 200–11,000
7
Urine Urine Urine Urine

Disorder Urine 2-ethylhydracrylic 2-methyl-3- Urine 2-methyl- Urine S-2-carboxypropyl-cysteamine + S-2- Urine 3-hydroxy-
abbreviation 2-methylbutyrylglycine acid hydroxybutyrate tiglylglycine acetoacetate isobutyrylglycine carboxypropyl-cysteine isobutyrate
7.12 MBD 3–37 ↑ – – –
deficiency
7.13 MHBD – n-↑ 11–30 ↑ – –
deficiency
7.14 BKT – 11–4,400 2–1,000 2–650 – –
deficiency
7.15 IBD – – – – ↑ –
deficiency
Disorders of Leucine, Isoleucine, and Valine Metabolism
7.16 HIBCH – – ↑
deficiency
7. 17 – – 60–600 (between
HIBADH episodes); up to 10,000
deficiency (with episodes)
Disorder Urine DBS Urine Urine Plasma/(DBS) Urine malonic

abbreviation 3-hydroxypropionate 3-hydroxypropionate methyl-citrate Urine tiglylglycine propionyl-glycine Urine methylmalonate methylmalonate acid
7.18 MMSDH n-↑ n-↑ n (-↑) n (-↑)
deficiencyc
7.19 PA 20–2,000 69–107 150–2,800 13–497 2–450 – –
7.20 MMA 4–1,000 11–32 To 2,800 (↑) (↑) 20–16,543 24–6,129 –
(2–2,920)
7.21 MA 17–210 n-↑ 100–5,440
7.22 CMAMMA 21–1,830 0.4–48 3–600
Levels may only be abnormal in variant forms during metabolic decompensation. Range of (but not all) metabolites are given
a
As an elevated concentration of lactate is a frequent finding, lactate is therefore not listed repeatedly
b
Isovalerylglucuronide is more likely to be excreted when the concentration of 3-hydroxyisovaleric acid is high
c
3-Hydroxy-/3-aminoisobutyric acids, 2-aminoisobutyrate, 3-hydroxypropionic acids, and ethylmalonate may also be increased in methylmalonate semialdehyde dehydrogenase deficiency
125
126
Acylcarnitines in dried blood spots (DBS, μmol/l) (tandem mass spectrometry)

C5-/ C5-OH-/3-
Free C3-/ C4-/ C4-DC-/ isovaleryl-/2- C5:1-/ hydroxyisovaleryl-
carnitine propionyl- butyrylcarnitine + C3-DC/ methylmalonyl C4-OH-/3-OH- methylbutyryl pentenoyl-/ /2-methyl-3-OH-
Disorder abbreviation (C0) carnitine isobutyrylcarnitine malonylcarnitine carnitine isobutyrylcarnitine carnitine tiglylcarnitine butyrylcarnitine
7.1 BCAA transferase deficiency n
7.2 MSUD n
7.3 IVA ↓-n ↑
7.4 MCCD ↓-n ↑
7.5 MGA1 ↓-n ↑
7.6 Barth syndrome ↓-n n-(↑)
7.7 MEGDEL syndrome ?
7.8 Neonatal mitochondrial ?
encephalocardiomyopathy
7.9 Costeff syndrome (n) n-(↑)
7.10 MGA4 ↓-n n-(↑)
7.11 HMG CoA lyase deficiency ↓-n ↑
7.12 MBD deficiency ↓-n ↑
7.13 MHBD deficiency ↓-n ↑ ↑
7.14 BKT deficiency ↓-n ↑ ↑
7.15 IBD deficiency ↓-n ↑
7.16 HIBCH deficiency ↓-n ↑
7.17 HIBADH deficiency ↓-n ↑
7.18 MMSDH deficiency (n)
7.19 PAa ↓-n ↑
7.20 MMAa ↓-n ↑ ↑
7.21 MA ↓-n ↑
7.22 CMAMMA ↓-n n ↑
Levels may only be abnormal in patients with variant forms during metabolic decompensation
Acylcarnitine concentration may increase following L-carnitine supplementation
a
Molar ratios should be applied for diagnostic purposes, e.g., C3/acetylcarnitine (C2) and C3/C0
I. Knerr et al.
7.7 Diagnostic Flow Chart spectrometry. In patients not screened as newborns, nonspe-
cific symptoms such as failure to thrive or developmental
A positive newborn screening test for leucine should be con- delay should trigger a metabolic evaluation that may iden-
firmed by quantitative plasma amino acid analysis and is tify a diagnostic metabolite. Blood acylcarnitines and uri-
usually only seen in MSUD. High-risk newborns require an nary organic acid profiling are again essential for the correct
early newborn screening, e.g., at day 1 along with preventa- differential diagnosis followed by confirmatory analysis.
tive dietary intervention. Abnormal blood acylcarnitine spe- The disorders of isoleucine and valine metabolism are also
cies may be seen in the other disorders in this pathway, and detected in a sequential process that begins with the evalua-
making the correct diagnosis depends on the analysis of uri- tion of the symptoms and signs displayed by the patient
nary organic acids by combined GC-MS. Further diagnostic (selective metabolic screening) or in some countries on
information is obtained through analysis of urinary acylgly- newborn screening. Clinical chemistry is helpful in the
cines and (in selected instances) intact fibroblast oxidation assessment of ketogenesis by urinary tests for ketone bodies
studies or direct enzyme analyses. Essentially, the detection or quantification of 3-hydroxybutyrate and acetoacetate in
of unusual body odor (maple syrup, sweaty feet), acidosis, the blood. The electrolytes, blood gases, and pH provide
ketosis, elevated ammonia, hypoglycemia, or carnitine defi- evidence of acidosis, and it is important to test for hyperam-
ciency suggests that urine organic acid analysis should be monemia. Analysis of plasma amino acids and blood acyl-
performed. Patients with many of these disorders may be carnitines is helpful. In virtually all instances, the definitive
asymptomatic at birth and are identified only on the basis diagnosis will come from organic acid analysis of the urine,
of an abnormal newborn screening using tandem mass followed by further enzymatic or molecular tests.
Clinical symptoms Positive family history Unaffected

newborns
Selective metabolic Prenatal testing if applicable Newborn

screening screening
Newborn screening on day
1 in high-risk newborns (e.g.
positive family history for
MSUD)
Plasma amino acids, blood
gases, glucose, lactate, Consider preventative
ammonia, acylcarnitines, treatment where applicable
urinary organic acids. (e.g. reduce natural protein
intake); prevent catabolism
Presumptive positive result /

suspected inborn error of BCAA metabolism
Definitive testing
Make management decisions on the clinical status

and laboratory results available
Start/continue with treatment as required (e.g.
reduce natural protein intake); prevent catabolism;
emergency treatment if needed
Monitoring and follow-up investigations as required
Fig. 7.2 Diagnostic flow chart
128 I. Knerr et al.
7.8 Specimen Collection, Prenatal

Diagnosis and DNA Analysis
7.8.1 Specimen Collection
Tests Sample requirements

Urine 5–10 ml, random, fresh or frozen without preservatives and shipped frozen (packed in
Amino acids dry-ice) or lyophilized and shipped at room temperature with original volume specified
Organic acids or shipped as fresh sample with 1–2 drops of chloroform for preservation
Test urinary amino acids e.g. in patients with hyperammonemia or kidney dysfunction,
Carnitine (free/total)
otherwise plasma sample preferred
Acylglycines
2,4-dinitrophenylhydrazine (DNPH) test Fresh or frozen random
This test has historical importance but has since been replaced
May give false negative results
Usually positive if blood leucine is greater than 800 µmol/l
May be positive in other conditions with oxoacids
False positives with methenamine and radio-opaque contrast material
Ferric chloride test Fresh or frozen random
This test has historical importance but has since been replaced
Turned colors to various abnormal urinary metabolites
Plasma Plasma, 0.5–1.0 ml for each determination from heparinized or EDTA blood (or serum)
Amino acids supernatant from clinical centrifugation (within 20 min), fresh or promptly frozen and
Organic acids shipped frozen (packed with dry-ice) or lyophilized and shipped at room temperature
with original volume specified. For quantitative amino acids: random or semi-fasting
Carnitine (free/total)
conditions (i.e. 4 h)
Acylcarnitine profile Plasma ketone body concentration changes with fasting over time, check glucose
Organic acids (such as methylmalonic acid) simultaneously
ketone bodies
Dried blood spots Drops of whole blood correctly collected on filter paper
Acylcarnitine profile It is important neither to overload nor to dilute the sample
Amino acids Blood spots should dry completely before storage or transportation (at room
temperature)
Organic acids (such as methylmalonic acid)
Cerebrospinal fluid 0.5–1 ml for each investigation (standard plastic lumbar puncture tube), fresh or frozen
Amino acids and shipped frozen (packed with dry-ice). Check plasma amino acids simultaneously
7.8.2 Prenatal Diagnosis
Disorder Materiala Timing, trimester

7.1 BCAA aminotransferase deficiency ?
7.2 Maple syrup disease Molecular analysis, I, II
CV tissue, cultured AFC
7.3 Isovaleric acidemia Molecular analysis, I, II
Amniotic fluid II
7.4 Isolated 3-methylcrotonyl-CoA carboxylase deficiency Molecular analysis, I, II
Amniotic fluid II
7.5 3-Methylglutaconic aciduria type I (3-methylglutaconyl-CoA Molecular analysis, I, II
hydratase deficiency) CV tissue, cultured AFC
Amniotic fluid II
7.6 3-Methylglutaconic aciduria type II (Barth syndrome) Molecular analysis I, II
7.7 MEGDEL syndrome Molecular analysis I, II
7.8 Neonatal mitochondrial encephalocardiomyopathya Molecular analysis I, II
7.9 3-Methylglutaconic aciduria type III (Costeff syndrome)a Molecular analysis I, II
7.10 3-Methylglutaconic aciduria type IVb ?
7.11 3-Hydroxy-3-methylglutaric aciduria Molecular analysis, I, II
Amniotic fluid II
7.12 2-Methylbutyrylglycinuria Molecular analysis I, II
Disorder Materiala Timing, trimester

7.13 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency Molecular analysis I, II
7.14 Beta-ketothiolase deficiency Molecular analysis, I, II
Amniotic fluid II
7.15 Isobutyryl CoA dehydrogenase deficiency Molecular analysis, I, II
7.16 3-Hydroxyisobutyryl-CoA deacylase Molecular analysis deficiency I, II
7.17 3-Hydroxyisobutyrate dehydrogenase deficiencyb Molecular analysis I, II
Amniotic fluid II
7.18 Methylmalonate semialdehyde dehydrogenase deficiencyb Molecular analysis I, II
7.19 Propionic acidemia Molecular analysis, I, II
Amniotic fluid II
7.20 Methylmalonic academia Molecular analysis, I, II
CV tissue, cultured AFC,
Amniotic fluid II
7.21 Malonic aciduria Molecular analysis, I, II
Amniotic fluid II
7.22 Combined methylmalonic and malonic aciduriab Molecular analysis I, II
Amniotic fluid II
a
Prenatal diagnostic tests comprise of direct mutation analysis using chorionic villus samples/amniocytes, enzyme testing in chorionic villus (CV)
tissue or cultured amniocytes, and metabolite analyses in amniotic fluid. Contact specialized laboratory for details
b
Thus far, studies have been limited to patients categorized according to confirmatory enzymatic/molecular test results
7.8.3 DNA Analysis
Disorder Material Methodology

7.1 BCAA aminotransferase deficiency For all entities: For all entities:
7.2 Maple syrup disease WBC, FB or other suitable cells RT-PCR, reverse transcription-
7.3 Isovaleric acidemia or tissue samples may be used polymerase chain reaction/genomic
amplification and sequencing
7.4 Isolated 3-methylcrotonyl-CoA carboxylase
In some cases allele-specific
deficiency
oligonucleotide hybridization or
7.5 3-Methylglutaconic aciduria type I single-stranded conformational
(3-methylglutaconyl-CoA hydratase deficiency) polymorphism analysis may be used
7.6 3-Methylglutaconic aciduria type II
(Barth syndrome)
7.7 MEGDEL syndrome
7.8 Neonatal mitochondrial Encephalomyopathy
7.9 3-Methylglutaconic aciduria type III
(Costeff syndrome)
7.10 3-Methylglutaconic aciduria type IVa, b
7.11 3-Hydroxy-3-methylglutaric aciduria
7.12 2-Methylbutyrylglycinuria
7.13 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase
deficiency
7.14 Beta-ketothiolase deficiency
7.15 Isobutyryl CoA dehydrogenase deficiency
7.16 3-Hydroxyisobutyryl-CoA deacylase deficiency
7.17 3-Hydroxyisobutyrate dehydrogenase deficiencyb
7.18 Methylmalonate semialdehyde dehydrogenase
deficiencyb
7.19 Propionic acidemia
7.20 Methylmalonic academia
7.21 Malonic aciduria
7.22 Combined methylmalonic and malonic aciduriab
a
Very limited evidence
b
Thus far, studies have been limited to patients categorized according to confirmatory enzymatic/molecular test results
130 I. Knerr et al.
7.9 Treatment Summary quantity of conditions to support excretion of organic acids

as carnitine adducts, thus sparing coenzyme-A and preserv-
The mainstay of treatment for all of the disorders is to limit ing the function of the Krebs cycle. Hyperammonemia may
intake of the affected amino acid(s) while preventing catabo- occur, particularly in PA and MMA, but conjugating agents
lism. With severe forms of the disorders, special medical are typically not indicated for therapy as ammonia normal-
foods (devoid of all BCAA, Leu, Ile, Val or Ile/Met/Thr/Val, izes with reversal of the primary metabolic derangement. If
as required for the different conditions) are needed to allow severe, hemodialysis may be indicated. The chronic treat-
for adequate caloric, protein, and other nutrient intake. ment of severe forms of the disorders, particularly of PA and
Milder forms may only require a moderately reduced natural MMA, is also challenging. Late complications must be
protein intake. The amount of natural whole protein toler- anticipated, including neurologic complications, renal fail-
ated is determined by monitoring parameters such as growth, ure (particularly in MMA), or cardiomyopathy. Liver trans-
control of acidosis, excretion of abnormal metabolites, blood plantation and combined liver/kidney transplantation have
amino acid levels, and testing for body protein stores. The been applied in a number of cases and there is evidence of
least restrictive dietary approach should be taken in order to benefit, but concerns arise because of documented cases of
avoid over-treatment and nutritional deficiencies. Emergency neurologic complications (e.g., in MMA).
treatment includes promotion of anabolism using a special
high-calorie diet, transient stop of natural protein intake or Emergency Treatment
of the precursor amino acids, carefully adjusted IV treat- • Disorder 7.1: Branched-Chain Amino Acid
ment including adequate amounts of IV dextrose, correction Transferase Deficiency
of acidosis, detoxification, prevention of brain edema, and – Management may be adapted from tables for disorder
emergency hemodialysis in case of coma/encephalopathy 7.2 (MSUD). Follow-up treatment relies on the correct
with severe hyperammonemia or profound metabolic acido- diagnosis.
sis in classic forms of the disorders. Carnitine is used in a • Disorder 7.2: Maple Syrup Urine Disease
Emergency treatment (includes management of ill neonates) for disorder 7.2

Objective Treatment
1. Correct dehydration Normal saline bolus as clinically indicated. Start IV dextrose 10–12.5 % at 1–1.5 times maintenance;
add NaCl and KCl depending on renal output and serum electrolyte levels. Plan rehydration over a 48 h
period to prevent cerebral edema
2. Correct acidosis Usually corrects with reestablishment of anabolic state. In extremis, give 1–2 mEq/kg of NaBicarbonate
3. Maintain normal plasma sodium (a) Check plasma sodium (aim for 140–145 mEq/l with minimal fluctuation) and plasma osmolality
and osmolality levels. High risk (290–300 mosm/l) regularly; monitor intake and output, body weight; urine osmolality
for SIADH (≤300–400 mosm/l), urine electrolytes and output (2–4 ml/kg/h)
(b) Give 3 % NaCl, dosage carefully calculated to replace deficit if hyponatremic. May also need
furosemide 0.50 (0.25–1) mg/kg per dose every 6–8 h if plasma osmolality falls
(c) Limit free water intake observe for fluid overload due to excessive vasopressin production
(d) Monitor for signs of increased intracranial pressure
4. Correct hyperglycemia (blood Regular insulin drip, e.g., 0.05–0.1 units/kg/h until blood glucose is controlled
glucose >200 mg/dl) induced
by IV fluids
5. Reestablish anabolism (a) BCAA-free formula or IV fluids to provide total caloric intake of 120–150 % of maintenance
or give step 1 sick-day diet by PO or NG/G-tube (see table protocol for intercurrent illness below)a
(b) Add interlipid 2–3 g/kg/day if NPO
(c) Add back natural protein beginning with e.g., 100 mg of leucine per day after 24 h; total protein
including BCAA-free supplements 2.5–3.5 g/kg/day
6. Reduce persistently elevated (a) Hemodialysis (HD) or hemofiltration (HF) in encephalopathic patients if intensified treatment
leucine levels or hyperammonemia (measure 1–6) is insufficient over a period of 2–4 h
or encephalopathy (b) Expect rate of decrease in plasma leucine levels >750 μmol/l per 24 h
a
Isoleucine and valine levels should be high during crisis, at over 400–600 μM, to suppress entry of leucine into the brain. Isoleucine and valine
usually need to be added early (after 24 h) into treatment (e.g., 40–100 mg/kg/day each). Leucine is reintroduced when its plasma level falls close
to the normal range (e.g., total daily dose of 100 mg of leucine per day when plasma level below 400 μmol/l, 200 mg/day when level below
300 μmol/l; provide in total 2.5–3.5 g/kg/day of protein equivalent). Aim at target ratios of approximately 1:2:2 for plasma leucine, isoleucine, and
valine, respectively
Comments/Additions for ketones as indicated by the clinical history and exami-

1. Stop all natural protein sources initially but add back leu- nation. Acute pancreatitis may accompany metabolic
cine restricted source after 24 h to reestablish protein crisis.
anabolism. 3. Patients with the E3 subunit deficiency may experience
2. Monitor laboratory studies including blood glucose, elec- severe lactic acidosis and abnormal blood glucose.
trolytes, pH, blood gases, ammonia, lactate, osmolality, 4. Thiamine 10 mg/kg/day (50–500 mg/day) may be given
plasma amino acids, urinary organic acids, and dipstick until genotype is known.
Protocol for intercurrent illness for disorder 7.2

Treatment Branched-chain amino acid-free special medical food Natural food leucine intake
First 24 h 1.2–1.5 times usual daily amount with additional isoleucine and valinea None
24–48 h 1.2–1.5 times usual daily amount with additional isoleucine and valinea None to half usual dietary intake
>48 h or when well Usual daily amount Gradual increase to usual full dietary intake
a
Additions of isoleucine and valine should be increased during sick days. Goal is to keep levels of isoleucine and valine between 150–350 and
200–400 μM, respectively, when the patient is well and at over 400–600 μM when ill
Comments/Additions Disorder 7.3: Isovaleric Acidemia

1. Families/individuals should start sick-day formula (to Disorder 7.4: 3-Methylcrotonylglycinuria1
decrease leucine intake, increase isoleucine and valine Disorder 7.5: 3-Methylglutaconic Aciduria type I2
intake, and suppress catabolism) with the onset of inter- Disorder 7.11: 3-Hydroxy-3-Methylglutaric Aciduria3
current illness or symptoms related to loss of metabolic
control. Fluids without calories or electrolytes should be 1
Most patients do not become acutely ill.
avoided or intake minimized. 2
Treatment is mainly symptomatic.
2. If the patient is unable to take in oral fluids and has per- 3
Patients with HMGCL (disorder 7.11) usually present with acute
sistent vomiting or the clinical condition deteriorates, hypoketotic or nonketotic hypoglycemia, and dehydration may be
underestimated. Give IV glucose bolus (e.g., 2 ml/kg of dextrose 10 %)
they should proceed urgently to an experienced emer- in a hypoglycemic patient. Add carnitine (e.g., 100 mg/kg/day)
gency care facility. (Dasouki et al. 1987).
Emergency treatment (includes management of ill neonates) for disorders 7.3, 7.4, 7.5, and 7.11
Objective Treatment
1. Treat dehydration Normal saline bolus as clinically indicated. Start IV dextrose 10–12.5 % at 1–1.5 times
maintenance; add NaCl and KCl depending on renal output and serum electrolyte levels
2. Correct acidosis Usually corrects with reestablishment of anabolic state. In extremis, give 1–2 mEq/kg of
NaBicarbonate
3. Correct hyperglycemia (blood glucose Regular insulin drip, e.g., 0.05 units/kg/h, until blood glucose is controlled
>200 mg/dl) induced by IV fluids
4. Reestablish anabolism (a) Leucine-free formula or IV fluids (plus low fat in HMGCL deficiency/disorder 7.11)
with caloric intake 120–150 % of maintenance
(b) Can add intralipids for additional calories except for HMGCL deficiency (disorder 7.11)
5. Metabolite conjugation Carnitine: 100 mg/kg/day in three divided doses IV or PO
Glycine (IVA only): usual daily dose (e.g., 250 mg/kg/day) in sick-day enteral formula or TPN
132 I. Knerr et al.
Comments/Additions 2. Monitor laboratory studies including blood glucose, elec-

1. Stop all natural protein sources initially. Begin to add trolytes, pH, blood gases, ammonia, lactate, plasma
back leucine-restricted protein after 24 h and complete amino acids, urinary organic acids, acylcarnitine profile,
protein after 48 h. liver function tests, CK, and any other laboratory tests
indicated by the clinical history and examination.
Protocol for intercurrent illness: initial measures for disorders 7.3, 7.4, 7.5, 7.11
Treatment Leucine-free special medical fooda Natural food leucine intake L-carnitine (mg/kg/day)
First 24 h 1.2–1.5 times usual daily amount None 100
24–48 h 1.2–1.5 times usual daily amount None to half usual dietary intake 100
>48 h or when well Usual daily amount Gradual increase to usual full dietary intake 100
a
Leucine-free and low fat for HMGCL (disorder 7.11)
Comments/Additions Comments/Additions
1. Families/individuals should start sick-day formula (to MGA types II–IV do not involve defects in the leucine path-
decrease leucine intake and suppress catabolism). Start way but affect mitochondrial functions through various
carnitine if not used routinely. Fluids without calories or pathomechanisms; treatment for patients with these forms of
electrolytes should be avoided or intake minimized. MGA is largely symptomatic.
2. Monitor urine ketones, which will become positive with • 7.11: 3-Hydroxy-3-Methylglutaric Aciduria
loss of metabolic control or inadequate caloric intake – Emergency treatment (includes management of ill
except in patients with HMGCL deficiency, who are neonates)
unable to make ketones. Management may be adapted from tables for disorders 7.4
3. If the patient is unable to take in oral fluids and has persistent (3-methylcrotonylglycinuria) and 7.5 (3-methylglutaconic
vomiting or the clinical condition deteriorates, they should aciduria type I) (see above). Prompt administration of
proceed urgently to an experienced emergency care facility. carbohydrates/IV dextrose is mandatory. Intralipid is
• 7.6: Barth Syndrome contraindicated.
Symptomatic treatment includes cardiac medications as • Isoleucine Catabolic Pathway
required, antibiotic drugs, and subcutaneous G-CSF if – 7.12: 2-Methylbutyrylglycinuria4
needed. Prevent hypoglycemia; monitor for lactic acido- – 7.13: 2-Methyl-3-Hydroxybutyryl-CoA Dehydro-
sis and (mild) hyperammonemia. The value of L-carnitine genase Deficiency
supplements or pantothenic acid has not been proven. – 7.14: Beta-Ketothiolase Deficiency
• 7.7: MEGDEL Syndrome • Valine Catabolic Pathway
Individualize treatment. Prevent hypoglycemia, monitor for – 7.15: Isobutyryl-CoA Dehydrogenase Deficiency4
lactic acidosis. Avoid fasting and ensure adequate intake of – 7.16: 3-Hydroxyisobutyryl-CoA Deacylase
energy (e.g., lipids). Patients may present with severe infec- Deficiency
tions, not only in the neonatal period but also later in life. – 7.17: 3-Hydroxyisobutyrate Dehydrogenase
• 7.8: Neonatal Mitochondrial Encephalo- Deficiency
cardiomyopathy – 7.18: Methylmalonate Semialdehyde Dehydro-
Individualize treatment. Patients may suffer from early onset genase Deficiency
of severe muscular hypotonia and cardiomyopathy along Treatment is similar to the leucine pathway disorders
with hyperammonemia and lactic acidosis requiring inten- except that isoleucine or valine reduced concentration for-
sive care. Fasting intolerance and low glucose tolerance mulae are required. Check carnitine status and add
(e.g., 3–4 mg/kg/min) must be anticipated. Fat tolerance is L-carnitine as required (e.g., 50–100 mg/kg/day). In patients
usually preserved and IV lipids (3.5 g/kg/day) are well toler- with beta-ketothiolase deficiency (disorder 7.14), oral or IV
ated. Data on the usage of conjugating agents such as sodium carbohydrate administration is most effective in suppress-
benzoate/phenylacetate in this condition is lacking. ing ketogenesis; metabolic acidosis should be treated cau-
• 7.9: Costeff Syndrome tiously to prevent hypernatremia and paradoxical CNS
Most patients do not become acutely ill. acidosis.
• 7.10: Methylglutaconic Aciduria Type IV
Individualize treatment. 4
Patients not usually acutely ill.
Emergency treatment (includes management of ill neonates) for disorders 7.19, 7.20
Objective Treatment
1. Treat dehydration Normal saline bolus as clinically indicated. Start IV dextrose 10–12.5 % at 1–1.5 times maintenance;
add NaCl and KCl depending on renal output and serum electrolyte levels. Plan rehydration over a 48 h
period to prevent cerebral edema
3. Correct hyperglycemia Regular insulin drip, e.g., 0.05–0.1 units/kg/h until blood glucose is controlled
(blood glucose >200 mg/dl)
4. Reestablish anabolism (a) If able to feed enterally, use Ile/Met/Thr/Val-free formula to provide total caloric intake of 120–150 %
of maintenance
(b) Adjust IV treatment as required (peripheral/central line)
(c) 20 % fat emulsion, rate 1–3 g/kg/day IV
(d) If NPO, use lowered Ile/Met/Thr/Val-free TPN
6. Metabolite conjugation (a) L-carnitine 100 mg/kg/day IV or PO/NGa
and reduction (b) Add metronidazole PO or NG for intermittent treatment (10 mg/kg/day for 1 week)
(c) Urgently consider HD/HF in encephalopathic patients or if blood ammonia is ≥300–400 μmol/l,
particularly if intensified treatment is insufficient over a period of 2–4 hd
7. Enhance residual enzyme activity Cobalamin-responsive MMA: 1 mg hydroxycobalamin/dose IM dailyb, c
8. Other (a) Treat infections effectively. Note that neutropenia and thrombocytopenia are frequent findings during
metabolic crisis
(b) Monitor for further complications (e.g., cerebral edema, acute pancreatitis)
a
Higher doses of L-carnitine have also been used (200–400 mg/kg/day). It is reasonable to increase the IV dose, e.g., when the patient undergoes
HD/HF
b
Each patient with MMA should be tested for B12 responsiveness by a trial of parenteral hydroxycobalamin in a dose of 1 mg/day. In those who
respond, therapy is continued but the oral route may be used. Cyanocobalamin may be used if hydroxycobalamin is not available
c
In PA, biotin responsiveness is doubtful. Biotin supplementation (10 mg daily) is therefore rarely beneficial
d
N-carbamylglutamate may be started as a bridge, e.g., while waiting for HD/HF. In undiagnosed patients presenting with hyperammonemia,
sodium benzoate/phenylacetate may be given but should be stopped once PA or MMA has been diagnosed (Chapman et al. 2012)
• Disorder 7.19: Propionic Acidemia 2. Laboratory studies including blood gases, pH, ammonia,
• Disorder 7.20: Methylmalonic Acidemia blood glucose, lactate, electrolytes, osmolality, plasma
Comments/Additions amino acids, acylcarnitines, ALT, AST, coagulation
1. Stop all natural protein sources initially, and then, rein- screen, CK, full blood count, urinary ketones and urinary
troduce restricted natural protein by 24 h (0.2–0.5 g/kg/ organic acids, and any other laboratory tests indicated by
day) and begin addition of complete protein by 48 h with the clinical history and examination
escalation to normal intake thereafter. Use additional Ile/ • Disorder 7.21: Malonic Aciduria
Met/Thr/Val-free formula for adequate amino acid • Disorder 7.22: Combined Malonic Aciduria and Methyl-
supply malonic Aciduria
Protocol for intercurrent illness: initial measures for disorders 7.19, 7.20
Treatment Ile/Met/Thr/Val-free special medical food Natural food protein intake
First 24 h 1.2–1.5 times usual daily amount None
24–48 h 1.2–1.5 times usual daily amount None to half usual dietary intake
>48 h or when well Usual daily amount Gradual increase to usual full dietary intake
Emergency treatment (includes management of ill neonates) for disorders 7.21, 7.22
Objective Treatment
1. Treat hypoglycemia Give IV glucose bolus (e.g., 2 ml/kg of dextrose 10 %); start IV dextrose 10–12.5 % at maintenance and add NaCl
and KCl depending on serum electrolyte levels
2. Metabolite conjugation Carnitine: 100 mg/kg/day in three divided doses IV or PO
4. Other (a) Monitor cardiac function
(b) Monitor for further complications (e.g., lactic acidemia, seizures)
There is limited experience in managing this condition
134 I. Knerr et al.
Comments/Additions Chronic Treatment

1. If the patient is feeding orally, increase the amount of • Disorder 7.1: Branched-Chain Amino Acid
calories from carbohydrate relative to fat, introduce MCT Transferase Deficiency
fat, and prevent fasting. – The existence of this disorder in humans remains in
2. Laboratory studies including blood gases, pH, blood glu- question.
cose, lactate, electrolytes, full blood count, osmolality, • Disorder 7.2: Maple Syrup Urine Disease
CK, ALT, AST, acylcarnitines, cholesterol, ketones and
urinary organic acids, and any other laboratory tests indi-
cated by the clinical history and examination.
Standard treatment for disorder 7.2

No. Disease/Symbol Age Medication/diet Dosage Target plasma levels
7.2 MSUD – severe forms All ages Lowered BCAA dieta See table below Leucine 100–300 μM
(MSUD 1A, 1B, 2) Isoleucine and valine Adjusted to blood levels Isoleucine 150–350 μM
supplementsb
Glutamine and alaninec 100–250 mg/kg/day each Valine 200–400 μMh
Glutamine 400–800 μM
Alanine 150–500 μM
NaCld 3–5 mEq/kg/day Within normal limits
Thiaminee 50–300 mg/day
7.2 MSUD – milder forms All ages Reduced natural protein dietf See table below See above (severe forms)
(1A, 2) Multivitamin with minerals As required
Thiaminee 50–300 mg/day
7.2 MSUD – thiamine-responsive All ages Reduced natural protein dietf See table below See above (severe forms)
forms (MSUD 2) Multivitamin with minerals As required
Thiamine 10 mg/kg/day (50–500 mg/day)
7.2 MSUD 3 (combined All ages See above (severe forms)g See above (severe forms)
dehydrogenases deficiency)
a
Special medical food devoid of the branched-chain amino acids and enriched with micronutrients (see table nutritional treatment for patients with
maple syrup urine disease below)
b
Typically as 10 mg/ml solutions
c
Supplements of glutamine and alanine may also be given (100–250 mg/kg/day each). Calculate the amount present in the diet, and add supple-
ments to meet the recommended intake
d
Calculate the amount present in the diet, and add supplements to meet the recommended intake
e
Thiamine given until molecular genotype is known. Not given in patients with the Mennonite mutation Y393N (Morton et al. 2002)
f
Protein intake of approximately 1.5–2.0 g/kg/day in young infants and 0.6–1.5 g/kg/day in older children and adults
g
Attempts at treatment with diet and cofactors have been unsuccessful in some patients in preventing CNS deterioration; usually not thiamine
responsive
h
Target ratios of approximately 1:2:2 for leucine, isoleucine, and valine, respectively
Comments/Additions 2. All patients with MSUD 1A, MSUD 1B, and MSUD 2
1. Intake of whole protein and supplements of individual should be given a trial of thiamine therapy for at least
amino acids are adjusted based on plasma quantitative 3 weeks or until the molecular genotype is known.
amino acids levels and individual needs to meet the target Patients homozygous for the Y393N Mennonite mutation
levels. are not thiamine responsive.
• Disorder 7.3: Isovaleric Acidemia
Standard nutritional treatment for patients with maple syrup urine disease
Total protein requirementa Leucine toleranceb Isoleucine intake Valine intake Energy requirementc
Age (g/kg/day) (mg/kg/day) (mg/kg/day) (mg/kg/day) (kcal/kg/day)
Neonates 2.7–3.5 40–100 30–90 40–95 100–145
Infants 2.5–3.2 35–75 20–70 30–80 80–135
Young children 1.8–2.6 20–65 10–30 20–50 60–130
Older children and adults 1.4–1.8 5–50 5–30 15–30 35–70
Modified from Strauss et al. (2010); Acosta and Yannicelli (2001) and Marriage (2010). These recommendations are only a guide and must be
individualized for each patient, based on the severity of their disorder, actual needs, and blood quantitative amino acid levels
a
Includes protein intake from special medical foods devoid of BCAA plus that from natural whole protein sources
b
Leucine (mg/kcal ratio of 0.5–0.8 for neonates, 0.4–0.6 for infants; ratio of 0.25–0.30 in children and older)
c
Lipids should comprise 40–50 % of total calories. Formula concentrations over 24 kcal/oz may result in loose stools, diarrhea, and dehydration

No. Symbol Age Medication/diet Dosage Target plasma levels
7.3 IVA – severe forms All ages Lowered leucine dieta See tables below Leucine 50–180 μM or normal
range for laboratory
L-carnitine 100 mg/kg/day in three dosesc Normal to high normal range
for free carnitine
Glycine 250 (150–300) mg/kg/day in three dosesd Glycine 200–400 μM
7.3 IVA – mild forms All ages Reduced natural protein dietb See tables below
Multivitamin with minerals As required
L-carnitine 30–100 mg/kg/day in three dosesc Normal free carnitine
Glycine 150–250 mg/kg/day in three dosesd, e Glycine 200–400 μM
a
Special medical food devoid of leucine and enriched with micronutrients may be needed for severe forms of the disorder. Patients with milder
forms of the disorder will only require a reduced natural protein intake. The least restrictive diet sufficient to maintain metabolic control should be
used
b
Natural protein intake of approximately 1.5–2.0 g/kg/day in young infants and 0.6–1.5 g/kg/day in older children and adults. Patients with a mild
IVA form and the missense mutation 932C > T (A282V) usually do not require protein restriction
c
Calculate the amount present in the special medical food or protein-free product, and add supplements to meet the recommended intake
d
Glycine is often omitted from chronic therapy. If used, it is added to the daily special formula as weighed dry powder or 100 mg/ml solution
e
Patients with a mild IVA form may not require glycine supplements
Standard nutritional treatment for patients with isovaleric acidemia

Leucine intakeb from whole natural
Age Protein requirementa (g/kg/day) protein (mg/kg/day) Energy requirementc (kcal/kg/day)
Neonates 2.5–3.5 65–150 95–145
Infants 2.0–3.0 50–140 80–135
Young children 1.5–2.0 40–90 60–130
Older children and adults 1.1–1.8 30–60 35–70
Modified from Acosta and Yannicelli (2001) and Marriage (2010). These recommendations are only a guide and must be individualized for each
patient, based on the severity of their disorder. Patients with milder forms of the disorder will tolerate a higher leucine intake and may only require
a reduced natural protein diet
a
Includes protein intake from special medical food devoid of leucine plus that from natural whole protein sources
b
These figures reflect leucine intake if special medical foods devoid of leucine are used and may be too low for some actively growing infants and
children
c
Formula concentrations over 24 Kcal/oz may result in loose stools, diarrhea, and dehydration
Comments/Additions for some growing infants and children. Many affected

1. Although leucine is the precursor amino acid for the patients are able to tolerate a near-normal leucine intake
disorder, it is the organic acids that are toxic to the and may be treated with a lowered natural protein diet,
patients and not the leucine per se as with MSUD. without selective leucine restriction. The least restric-
Monitoring leucine levels gives an indication as to tive dietary approach allowing metabolic control should
whether there is sufficient intake of natural protein to be used in order to avoid over-treatment and leucine
support growth, losses, and tissue repair. The plasma deficiency.
leucine range of 50–150 μM, however, may be too low • Disorder 7.4: Methylcrotonylglycinuria

7.4 3MCCC1 All ages Lowered leucine dieta See tables for IVA (disorder 7.3) Leucine 50–180 μM or normal
3MCCC2 range for laboratory
L-carnitine 50–100 mg/kg/day in two to three dosesb Normal free carnitine
a
Special medical food devoid of leucine may be needed for severe forms of the disorder. Patients with milder forms of the disorder will only require
a reduced natural protein intake. The least restrictive diet should be used. Glycine is usually not given, though it might be considered in severe
cases (175–250 mg/kg/day) as it increases 3-methylcrotonylglycine excretion
b
Calculate the amount present in the special medical food if used, and add supplements to meet the recommended intake
136 I. Knerr et al.
Comments/Additions 2. Patients are not responsive to biotin therapy.

1. Special medical food devoid of leucine and enriched with 3. Asymptomatic individuals may not need dietary restric-
micronutrients may be needed for severe forms of the dis- tions or L-carnitine but may occasionally need blood and
order. Patients with milder forms of the disorder will only urine monitoring.
require a reduced natural protein intake. The least restric- • Disorder 7.5: 3-Methylglutaconic Aciduria, Type I
tive diet allowing metabolic control should be used.

7.5 3MG1 All ages L-carnitine 100 mg/kg/day in three dosesa Normal free carnitine
Lowered leucine dietb See tables for IVA (disorder 7.3) Leucine 50–180 μM or normal range for laboratory
a
Calculate the amount present in the special medical food if used, and add supplements to this to meet the recommended intake
b
No dietary regimen has been therapeutically proven. Special medical food devoid of leucine may be used for severe forms of the disorder. Patients
with milder forms of the disorder might require a reduced natural protein intake. The least restrictive diet allowing metabolic control should be used
Comments/Additions • Disorder 7.8: Neonatal Mitochondrial

1. See comment 1 for disorder 7.3. Encephalocardiomyopathy
• Disorder 7.6: Barth Syndrome Individualize treatment. Avoid fasting and ensure ade-
Symptomatic treatment includes cardiac medications as quate intake of energy. Monitor for lactic acidosis and
required, prophylactic antibiotic drugs, and subcutaneous hyperammonemia. Frequent feeding and administration
G-CSF if needed (e.g., in patients with symptomatic neu- of fats as a main source of energy might prevent or attenu-
tropenia). Cornstarch supplements at bedtime may be ate a metabolic crisis.
used to prevent hypoglycemia. The value of L-carnitine • Disorder 7.9: Costeff Syndrome
supplements or pantothenic acid has not been proven. Symptomatic treatment.
• Disorder 7.7: MEGDEL Syndrome • Disorder 7.10: Methylglutaconic aciduria type IV
Individualize treatment. Avoid fasting and ensure adequate Individualize treatment.
intake of energy. Patients may present with severe infec- • Disorder 7.11: 3-Hydroxy-3-Methylglutaric Aciduria
tions, not only in the neonatal period but also later in life.

7.11 HMGCL All ages Lowered leucine and fat, See tables for IVA (disorder 7.3) Leucine 50–180 μM or
high-carbohydrate dieta Of note, fat is limited to 20–25 % normal range for laboratory
of total daily caloric intake
L-carnitine 100 mg/kg/day in three doses Normal free carnitine
a
Special medical food devoid of leucine may be needed for severe forms of the disorder. Patients with milder forms of the disorder will only require
a reduced natural protein intake and low-fat diet. Avoid fasting. The least restrictive diet that allows metabolic control should be used
Comments/Additions • Disorder 7.13: 2-Methyl-3-Hydroxybutyryl-CoA

1. See comment 1 for disorder 7.3. Dehydrogenase Deficiency
2. In addition to leucine restriction, daily caloric intake of • Disorder 7.14: Alpha-Methylacetoacetic Aciduria
fat is limited to 20–25 % of total caloric intake per day. • Disorder 7.15: Isobutyryl-CoA Dehydrogenase
Use a leucine-free product that contains carbohydrates Deficiency
and other nutrients but no or very low fat. • Disorder 7.16: 3-Hydroxyisobutyryl-CoA Deacylase
3. Avoid fasting. Overnight drip nasogastric or gastrostomy Deficiency
feedings may be needed. • Disorder 7.17: 3-Hydroxyisobutyrate Dehydrogenase
4. Uncooked cornstarch added to the special metabolic for- Deficiency
mula may be used to prevent hypoglycemia. • Disorder 7.18: Methylmalonate Semialdehyde
• Disorder 7.12: 2-Methylbutyrylglycinuria Dehydrogenase Deficiency
Standard nutritional treatment for patients with disorders limited to isoleucine or valine
Natural protein Total protein Energy requirement
Age Isoleucine intakea Valine intakeb requirementc (g/kg/day) requirement (g/kg/day) (kcal/kg/day)
Neonates 90–150 mg/kg/day 65–110 mg/kg/day 1.5–2.5 2.8–3.5 95–145
Infants 50–115 mg/kg/day 40–90 mg/kg/day 1.2–2.0 2.5–3.2 80–135
Young children 630–1,250 mg/day 600–1,100 mg/day 0.8–1.5 1.8–2.6 60–130
Older children and adults 965–1,900 mg/day 900–2,015 mg/day 0.5–1.4 1.4–1.8 35–70
Modified from Yanicelli (2010). These recommendations are only a guide and must be individualized for each patient, based on the severity of their
disorder, actual needs, and blood quantitative amino acid levels
a
The targets relating to isoleucine curtailment would apply to MBD deficiency (disorder 7.12), MHBD deficiency (disorder 7.13), and in principle
to beta-ketothiolase deficiency (disorder 7.14)
b
The targets relating to valine curtailment would apply to MMSDH deficiency (disorder 7.18), HIBDH deficiency (disorder 7.17), IBD deficiency
(disorder 7.15), and in principle to HIBCH deficiency (disorder 7.16)
c
In addition to the natural protein intake, special medical food (devoid of Ile or Val as appropriate) provides adequate total protein intake. The least
restrictive diet should be used. These are approximate therapeutic strategies, and individual patients’ requirements may vary substantially. Also,
for a given patient, variations must be anticipated in relationship to growth rate, physical activity, intercurrent illness etc
Monitor carnitine status and add L-carnitine as required (e.g., 50–100 mg/kg/day)
In beta-ketothiolase deficiency (disorder 7.14): avoid fasting and high-fat intake; carbohydrate-rich meals and frequent feeding have been effective
to avoid ketonuria
• Disorder 7.19: Propionic Acidemia

• Disorder 7.20: Methylmalonic Acidemia
Standard treatment for disorder 7.19 and 7.20

7.19 PA All ages Lowered Ile/Val/Met/Thr dieta See table below Low normal to normal range of age for
laboratory for Ile/Val/Met/Thrb; normal
ammonia; normal acid–base status
L-carnitine 100 mg/kg/day in three doses Normal to high normal range for free
carnitine
Metronidazole 10 mg/kg/day for
intermittent treatment POc, d
7.20 MMA All ages Lowered Ile/Val/Met/Thr dieta See table below Low normal to normal range of age for
laboratory for Ile/Val/Met/Thrb; normal
plasma ammonia; normal acid–base status
L-carnitine 100 mg/kg/day in three doses Normal to high normal range for free
carnitine
In cobalamin-responsive MMA: 1 mg/day IMe
hydroxycobalamin
Metronidazole 10 mg/kg/day for
intermittent treatment POc
In renal failure: symptomatic therapy As required
This plan must be individualized for each patient, based on the severity and type of their disorder
a
Individualize depending upon tolerance to protein, growth, and nutritional adequacy, guided by parameters in table standard nutritional treatment
for propionic acidemia and methylmalonic acidemia below
b
Provide an adequate intake; prevent deficiencies
c
Short course or intermittent treatment (e.g., 1 week per month; >6 months)
d
In PA, biotin responsiveness is doubtful and supplementation rarely beneficial
e
Each patient with MMA should be tested for B12 responsiveness by a trial of parenteral hydroxycobalamin in a dose of 1 mg/day IM. In those
that respond (decrease in urinary MMA excretion by ≥50 %), therapy is continued but the oral route may be used and the dose may be adjusted.
Cyanocobalamin may be used if hydroxycobalamin is not available
138 I. Knerr et al.
Standard nutritional treatment for propionic acidemia and methylmalonic acidemia

Natural protein Total protein
requirementa requirementb
Age Isoleucine intake Methionine intake Threonine intake Valine intake (g/kg/day) (g/kg/day) Energy requirementc
Neonates 60–110 mg/kg/day 20–50 mg/kg/day 50–125 mg/kg/day 60–105 mg/kg/day 1.2–1.8 2.7–3.5 125–145 kcal/kg/day
Infants 40–90 mg/kg/day 15–40 mg/kg/day 20–75 mg/kg/day 40–80 mg/kg/day 0.8–1.5 2.5–3.2 115–140 kcal/kg/day
Young 485–735 mg/day 275–390 mg/day 415–600 mg/day 550–830 mg/day 0.7–1.2 1.8–2.6 900–1,800 kcal/day
children
Older 630–1,470 mg/day 360–950 mg/day 540–1,455 mg/day 720–2,000 mg/day 0.5–0.8 1.4–1.7 1,500–3,200 kcal/day
children
and adults
Modified from Yanicelli 2010. These recommendations are only a guide and must be individualized for each patient, based on the severity of their
disorder, actual needs, and blood quantitative amino acid levels
a
In addition to the whole protein, specialized formulas (devoid of Ile/Met/Thr/Val) are needed. Of note, individual patients’ requirements may vary
substantially
b
Includes protein intake from special medical foods devoid of Ile/Met/Thr/Val plus that from natural whole protein sources. Total protein recom-
mendation is higher when the majority of protein is supplied by free amino acids (Ile/Met/Thr/Val-free formula). However, patients with hyperam-
monemia or MMA patients with renal impairment require carefully adjusted protein amounts
c
Energy requirement is usually lower during well state compared with unwell state in patients
• Disorder 7.21 Malonic Aciduria

• Disorder 7.22 Combined Malonic Aciduria
and Methylmalonic Aciduria
Standard treatment for disorder 7.21 and 7.22

No. Symbol Age Medication/diet Dosagea, b Target plasma levels
7.21 MA All ages High-carbohydrate and low long-chain triglycerides
Dietary fat may comprise Normal blood glucose,
(LCT) fat diet; medium-chain triglycerides (MCT) fat
30–50 % LCT and 50–70 % normal essential fatty
supplements MCT acids, normal cholesterol
L-carnitine 100 mg/kg/day in three Normal free carnitine
doses
7.22 CMAMMA All ages High-carbohydrate and low long-chain triglycerides Dietary fat may comprise Normal blood glucose,
(LCT) fat diet; medium-chain triglycerides (MCT) fat 30–50 % LCT and 50–70 % normal essential fatty
supplements; moderate restriction of natural protein MCT acids, normal cholesterol
L-carnitine 100 mg/kg/day in two to Normal free carnitine
three doses
Little information published and effectiveness of treatment has yet to be determined
a
There are no established treatment recommendations and dosage may vary considerably
b
Check whether metabolite levels are responsive to (moderate) protein restriction in patients with combined malonic aciduria and methylmalonic
aciduria (disorder 7.22)
Comments/Additions or angiotensin converting enzyme inhibitor or other medi-

1. Avoid fasting to prevent hypoglycemia. cations might be given depending on individual needs.
2. The management of a patient with cardiomyopathy should 3. Cholesterol supplementation should be given in patients
be done in consultation with a cardiologist; a beta-blocker with reduced cholesterol levels.
Experimental Treatment5
Disorder no. Symbol Substance

7.2 MSUD Coenzyme Q10 (5 mg/kg/day; electron carrier of the respiratory chain with antioxidant properties; in addition
to standard therapy during crisis)a
MSUD 1A Phenylbutyrate (500 mg/kg/day; to activate E1α/BCKDC enzyme activity; in addition to standard therapy)b
7.19 PA Citric acid (as K-Na-hydrogen citrate, 80–120 mg/kg/day PO; might improve TCA cycle flux; in addition
to standard therapy during crisis with lactic acidosis)c
a
Knerr et al. (2011)
b
Brunetti-Pierri et al. (2011)
c
Siekmeyer et al. (2013)
5
This excludes organ transplantation (see Mazariegos et al. 2012 and special literature).
Standard Therapy and Emergency Therapy Flow Charts

Disorders 7.1–7.22
Fig. 7.3 Management of patients

with disorders of BCAA
Routine treatment plan
metabolisma
Intercurrent illness
or symptoms of loss
of metabolic control
‘Sick day’ plan
YES
Improvement
NO
Acute medical management
Correct Medical tests

Correct Special Detoxification,
abnormal and supportive care
acidosis hypercaloric diet metabolic drugs
blood glucose as required
Intravenous Persistent
high ammonia, Low/high
hyperalimentation
acidosis or leukocytes
(special CHA/TPN)
encephalopathy or pancytopenia
Hemodialysis or Cultures
hemofiltration Antibiotics
Improvement
YES
a
These are approximate guidelines, and individual patients’
requirements may vary substantially
140 I. Knerr et al.
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Cerebral Organic Acidurias
8
Stefan Kölker, Eduard A. Struys, Marjo S. van der Knaap,
and Cornelis Jakobs
Contents Summary
8.1 Introduction ........................................................................ 143 A group of organic acidurias, including Canavan disease
(N-acetylaspartic aciduria), glutaric aciduria type I, l-2-
8.2 Nomenclature ..................................................................... 145
hydroxylgutaric aciduria and d-2-hydroxyglutaric acid-
8.3 Metabolic Pathways ........................................................... 146 uria types I and II, are characterised by a predominantly or
8.4 Signs and Symptoms .......................................................... 148 even exclusively neurological presentation and have there-
8.5 Reference Values ................................................................ 150
fore been termed ‘cerebral’. Frequent neurological symp-
toms are motor and/or mental retardation or regression,
8.6 Pathological Values ............................................................ 150 extrapyramidal movement disorders and epilepsy. These
8.7 Diagnostic Flow Chart(s) ................................................... 151 symptoms are the result of acute and/or chronic pathological
8.8 Specimen Collection ........................................................... 153 changes in various brain regions including grey matter (cor-
tex, basal ganglia, cerebellum) and white matter (periven-
8.9 Treatment Summary .......................................................... 154
tricular and subcortical). Unlike ‘classic’ organic acidurias
References .................................................................................... 156 (e.g. propionic and methylmalonic aciduria), acute metabolic
decompensations with hyperammonemia, metabolic acido-
sis and elevated concentrations of lactate and ketone bodies
are uncommon for cerebral organic acidurias. Biochemically,
these diseases are characterised by accumulation of charac-
teristic organic acids, mostly dicarboxylic acids, in body flu-
ids. At high concentrations some of these may become
neurotoxic. Since the blood–brain barrier has a low transport
capacity for dicarboxylic acids, cerebral accumulation of
dicarboxylic acids is facilitated. Impairment of brain energy
S. Kölker (*)
Division of Inborn Metabolic Diseases, metabolism is suggested to play a central role in the patho-
Department of General Pediatrics, University Children’s Hospital, physiology of this disease group. Metabolic treatment initi-
Im Neuenheimer Feld 430, Heidelberg 69120, Germany ated in neonatally diagnosed patients with glutaric aciduria
e-mail: stefan.koelker@med.uni-heidelberg.de
type I has significantly improved the neurological outcome,
E.A. Struys whereas current treatment strategies for the other cerebral
Metabolic Unit, Clinical Chemistry, VUmc Medical Center,
organic acidurias are ineffective.
De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands
e-mail: e.struys@vumc.nl
M.S. van der Knaap
Department of Child Neurology, Neuroscience Campus 8.1 Introduction
Amsterdam, VU University Medical Center,
De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands Canavan disease (Van Bogaert-Bertrand disease, N-
e-mail: ms.vanderknaap@vumc.nl
acetylaspartic aciduria) is caused by an autosomal recessively
C. Jakobs inherited deficiency of aspartoacylase (aminoacylase 2)
Department of Clinical Chemistry, PK 1X 014,
which is exclusively expressed in oligodendrocytes. Most
VU University Medical Center Amsterdam,
De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands patients have the infantile form which generally manifests at
e-mail: c.jakobs@vumc.nl 2–4 months of age with head lag, muscular hypotonia and
144 S. Kölker et al.
macrocephaly, progressing to marked developmental delay, glutarylcarnitine as diagnostic parameter. Glutarate and
seizures, optic nerve atrophy, progressive spasticity and opis- 3-hydroxyglutarate concentrations are increased in urine and
thotonic posturing (Matalon et al. 1995). Death usually occurs other body fluids but may be (intermittently) normal in
in a few years. However, the initial symptoms may already patients with a low-excreter phenotype. Therefore, a normal
start at or shortly after birth (neonatal form) or after the age of organic acid analysis result does not unequivocally exclude
5 years (juvenile form). Cranial MRI studies show diffuse or the diagnosis. Suspected diagnosis is confirmed by signifi-
exclusively subcortical involvement of the white matter due cantly decreased glutaryl-CoA dehydrogenase activity in
to spongiform myelinopathy and involvement of thalamus leukocytes or fibroblasts and/or the identification of two
and globus pallidus. Diagnosis can be made by finding ele- disease-causing mutations in the GCDH gene localised on
vated N-acetylaspartate concentrations in urine using GC/MS 19p13.2. Glutarate and 3-hydroxyglutarate concentrations
analysis of organic acids. A decreased aspartoacylase activity are 100–1,000-fold higher in the brain than in plasma which
in cultured skin fibroblasts and/or the identification of two is caused by the very low efflux transport of dicarboxylic
disease-causing mutations in the ASPA gene localised on acids across brain capillary endothelial cells (Sauer et al.
17p13.3 confirms the diagnosis. N-acetylaspartate is formed 2006). At high concentrations, accumulating dicarboxylic
in neurons and hydrolysed to l-aspartate and acetate by oli- acids may become neurotoxic inhibiting energy metabolism
godendrocytes. No effective treatment exists for Canavan dis- (2-oxoglutarate dehydrogenase, dicarboxylate shuttle
ease. Lithium citrate decreases brain N-acetylaspartate between astrocytes and neurons) and activating
concentrations (Assadi et al. 2010) and glyceryl triacetate N-methyl-d-aspartate receptors. The development of prog-
treatment supplies the brain with acetate (Segel et al. 2011). nostic relevant striatal injury can be prevented in the major-
Although this treatment is considered as safe, there is still no ity of children if the diagnosis is made and metabolic
proof for its therapeutic efficacy. Adenoviral transfer of the treatment is started neonatally (Heringer et al. 2010).
ASPA gene to the brain has been initiated but no follow-up Metabolic treatment according to a recently revised guide-
data have been published (Leone et al. 2000). line includes a low lysine diet, carnitine supplementation and
Glutaric aciduria type I (glutaric acidemia type I) is emergency treatment to counteract catabolism (Kölker et al.
caused by an autosomal recessively inherited deficiency of 2011). Patients with a high- and low-excreter phenotype
glutaryl-CoA dehydrogenase, an FAD-dependent mitochon- have the same risk of developing striatal injury and thus
drial matrix enzyme. This enzyme is involved in the cata- receive the same treatment (Kölker et al. 2006).
bolic pathways of lysine, hydroxylysine and tryptophan, l-2-hydroxyglutaric aciduria (L2HGA) is an autosomal
catalysing the oxidative decarboxylation of glutaryl-CoA to recessive inborn error of metabolism, caused by mutations in
crotonyl-CoA. Transient muscular hypotonia and macro- the L2HG dehydrogenase (L2HGDH) gene. The L2HGDH
cephaly is often found in newborns. At this age, cranial MRI gene product, i.e. L2HGDH, is an FAD-dependent
of affected individuals reveals temporal hypoplasia, dilated membrane-bound enzyme responsible for the conversion of
external CSF spaces, subependymal pseudocysts, myelina- l-2-hydroxyglutarate (L2HG) into 2-ketoglutaric acid
tion delay and an immature gyral pattern which all may (2KG). The current opinion is that nonspecific mitochondrial
improve or even resolve in early treated children (Harting formation of L2HG out of 2KG by l-malic dehydrogenase is
et al. 2009). In the time interval between 3 and 36 the sole source of L2HG and that L2HGDH is an enzyme for
(−72) months, however, most untreated patients develop a metabolite repair (Van Schaftingen et al. 2009). L2HGA has
complex movement disorder best described as generalised an insidious onset starting in childhood with developmental
dystonia superimposed on axial hypotonia (Gitiaux et al. delay, macrocephaly, epilepsy and cerebellar ataxia as clini-
2008). These symptoms are the consequence of bilateral cal signs. In a minority of patients, the diagnosis is estab-
striatal injury which may occur acutely during acute enceph- lished in adulthood, but retrospective evaluation of the
alopathic crises precipitated by catabolism or insidiously clinical course reveals an earlier subtle onset (Steenweg et al.
without preceding crises (Kölker et al. 2006). Aside from 2010). MRI reveals disease-specific alterations characterised
striatal injury, MRI may show additional frontal atrophy and by predominantly subcortical cerebral white matter abnor-
subdural haemorrhage. A few adolescent and adult patients malities and abnormalities of the dentate nucleus, globus
with a late-onset form have been reported presenting with pallidus, putamen and caudate nucleus (Steenweg et al.
headaches, vertigo, reduced fine motor skills and white mat- 2009). Metabolic investigations will reveal increased 2HG in
ter abnormalities (Harting et al. 2009). Patients can be identi- the urinary organic acid screening, and subsequent chiral dif-
fied in the first week of life by newborn screening using ferentiation shows the increased excretion of exclusively
8 Cerebral Organic Acidurias 145
L2HG. Apart from the massive increase of L2HG in all body these elevations can be modest. The increase of D2HG in all
fluids, a modest increase of CSF lysine is observed, while body fluids is the sole biochemical alteration in this disease,
plasma lysine levels may be normal. Since the massive and the pathophysiology of the disease is likely to be
increase of L2HG is the major biochemical finding, pathol- explained by this. Currently, there is no treatment. However,
ogy is likely to be explained by the pathologic levels of it can be hypothesised that in individual cases, riboflavin
L2HG; however, lowered (peripheral) 2KG levels might also supplementation might be beneficial.
attribute to the disease. Currently, there is no established d-2-hydroxyglutaric aciduria (D2HGA) type II is the
treatment protocol for L2HGA apart from two anecdotic second form of D2HGA and is caused by a gain-of-func-
reports mentioning positive effects of treatment with ribofla- tion mutation in the isocitrate dehydrogenase 2 (IDH2)
vin and/or FAD. gene (Kranendijk et al. 2010). Heterozygous mutations in
d-2-hydroxyglutaric aciduria (D2HGA) type I is one of IDH2 result in the formation of a neomorph enzyme which
the two subtypes of D2HGA and has an autosomal recessive is able to efficiently convert 2KG into D2HG. This is in
pattern of inheritance. The disease is caused by mutations in contrast with the normal action of IDH2, i.e. the conversion
the D2HG dehydrogenase (D2HGDH) gene, resulting in a of isocitrate into 2KG. D2HGA type II has an autosomal
deficiency of d-2-hydroxyglutarate (D2HG) dehydrogenase dominant trait, and in the vast majority of patients, the
(Struys et al. 2005). This FAD-dependent mitochondrial mutation arose de novo. The degree of D2HG accumulation
enzyme converts D2HG, most likely formed by the action of in D2HGA type II is higher than in type I, despite properly
hydroxyacid:oxoacid transhydrogenase (HOT), into 2KG. functioning D2HG dehydrogenase. Patients affected with
Although several hypothetical metabolic pathways for D2HGA type II suffer from developmental delay, hypoto-
D2HG have been proposed, there is strong evidence that nia and epilepsy, and their clinical presentation is generally
D2HG is directly and exclusively formed out of 2KG (Struys more severe than that of patients with D2HGA type I.
et al. 2004). The disease displays a strong clinical heteroge- Cardiomyopathy is frequently observed in D2HGA type II
neity from severely affected individuals to asymptomatic and absent in type I. The unique underlying mechanism of
individuals. However, frequently reported clinical findings D2HGA type II opens perspectives to specifically inhibit
are developmental delay, hypotonia and epilepsy. Usually, the neomorph enzyme.
patients are first recognised by an increase of 2HG in the A most recent review covering L2HGA and both types of
urinary organic acid screening. In contrast with L2HGA, D2HGA was published by Kranendijk et al. (2012).
8.2 Nomenclature
Gene Chromosomal
No. Disorder Alternative name Abbreviation symbollocalisation Affected protein OMIM no. Subtype
8.1 Canavan disease Van Bogaert-Bertrand CD ASPA 17p13.3 Aspartoacylase 271900 All forms
disease (aminoacylase 2)
8.2 Glutaric aciduria Glutaric acidemia type GA-I GCDH 19p13.2 Glutaryl-CoA 231670 All forms
type I I dehydrogenase
8.3 l-2-hydroxyglutaric l-2-hydroxyglutarate L2HGA L2HGDH 14q21.3 l-2-hydroxyglutarate 236792 All forms
aciduria dehydrogenase dehydrogenase
deficiency
8.4 d-2-hydroxyglutaric d-2-hydroxyglutarate D2HGA D2HGDH 2q37.3 d-2-hydroxyglutarate 600721 All forms
aciduria type I dehydrogenase type I dehydrogenase
deficiency
8.5 d-2-hydroxyglutaric Isocitrate D2HGA IDH2 15q26.1 Isocitrate 613657 All forms
aciduria type II dehydrogenase defect type II dehydrogenase 2
Canavan Disease
Fig. 8.1 Metabolic pathway of Aspartate

Canavan disease.
Acetyl-CoA
N-acetylaspartate is produced in
neurons from l-aspartate and
acetyl-CoA and is transported to
oligodendrocytes where it is CoA
hydrolysed to l-aspartate and
acetyl-CoA by aspartate N-Acetylaspartate
aminoacylase II. Inherited
deficiency of this enzyme results
in accumulation of 8.1
N-acetylaspartate in patients with
Canavan disease
Aspartate
+ Acetyl-CoA
Glutaric Aciduria Type I
Lysine
2-Oxoglutarate
Saccharopine pathway Pipecolic acid pathway
2-Aminoadipic
Hydroxylysine semialdehyde
2-Aminoadipic acid
Tryptophan 2-Oxoadipic acid
3
–
Glutaric acid
Glutaryl-CoA
Glutarylcarnitine
4
Glutaconic acid
Glutaconyl-CoA
3-Hydroxyglutaryl-CoA
4
Crotonyl-CoA 3-Hydroxyglutaric acid
Acetyl-CoA
Fig. 8.2 Metabolic pathway of glutaric aciduria type I. Glutaryl-CoA dependent epilepsy), 2 2-aminoadipate aminotransferase, 3 2-oxogluta-
is formed within the catabolic pathways of lysine, tryptophan and rate dehydrogenase-like complex (enzyme complex contains three
hydroxylysine. The quantitatively major precursor is lysine. Deficient subunits: DHTKD1 is deficient in 2-aminoadipic/2-oxoadipic aciduria;
activity of glutaryl-CoA dehydrogenase (4) results in variable accumu- the E2 subunit is deficient in 2-oxoglutarate dehydrogenase complex
lation of glutaric, 3-hydroxyglutaric and glutaconic acid as well as glu- deficiency; E3 subunit (lipoamide dehydrogenase), which is shared
tarylcarnitine, which are important for the diagnosis and can be with pyruvate dehydrogenase and branched-chain oxoacid dehydroge-
determined in body fluids. Elevated glutaryl-CoA – similarly to homol- nase complexes, is deficient in E3 deficiency, biochemically resembling
ogous succinyl-CoA – results in feedback inhibition of the TCA cycle maple syrup urine disease, 2-oxoglutarate dehydrogenase complex
enzyme 2-oxoglutarate dehydrogenase complex (3). 1 2-aminoadipic deficiency and pyruvate dehydrogenase complex deficiency, 4 glutaryl-
semialdehyde dehydrogenase (enzyme is deficient in pyridoxine- CoA dehydrogenase
l-2-Hydroxyglutaric Aciduria d-2-Hydroxyglutaric Aciduria Type II
NADH + H+ D-2-HGDH
NAD+
L-malDH
TCA TCA
L-2-HG NADPH + H+
cycle cycle
+
FAD 2-KG IDH NADPH
2 mut
2-KG L-2-HGDH
D-2-HG
FADH2
HOT
Fig. 8.3 Metabolic pathway of l-2-hydroxyglutaric aciduria. l-2- Fig. 8.5 Metabolic pathway of d-2-hydroxyglutaric aciduria type II.
hydroxyglutaric acid is formed by a nonspecific conversion of mito- As a consequence of a heterozygous mutation in IDH2, the neomorph
chondrial 2KG into l-2-hydroxyglutaric acid by the action of IDH2 enzyme produces vast amounts of D2HG, which exceed the
NADH-dependent l-malic acid dehydrogenase. L2HG dehydrogenase capacity of D2HG dehydrogenase, an enzyme with a low Km, and as a
corrects for this metabolic imperfection by reconverting L2HG into net result, D2HG accumulates. The neomorph IDH2 enzyme consumes
2KG both 2KG and NADPH, which might lead to their shortages
d-2-Hydroxyglutaric Aciduria Type I
TCA
cycle D-2-HGDH
2-KG
D-2-HG
HOT
Fig. 8.4 Metabolic pathway of d-2-hydroxyglutaric aciduria type I.

d-2-hydroxyglutaric acid is formed out of mitochondrial 2KG by the
action of hydroxy acid-oxoacid transhydrogenase (HOT). D2HG is
reconverted into 2KG by D2HG dehydrogenase. This pathway is
believed to play a role in GABA/GHB homeostasis
Table 8.1 Canavan disease

CNS Decerebrate posture ± ± ±
Dysarthria ± ± ± ± ±
Extrapyramidal movement disorder ± ± ± ± ±
Loss of very early milestones + + + +
Mental retardation + + + + +
Motor retardation + + + + +
Muscular hypotonia ± + + ± ±
Opisthotonous ± ± ± ± ±
Spasticity ± + + +
Ear Deafness ± ± ± ± ±
Eye Blindness + + + +
Nystagmus ± ± ± ± ±
Optic atrophy + + + +
Musculoskeletal Macrocephaly + + + + +
Special laboratory MRI: bilateral subcortical ± + + + +
leukodystrophy + involvement of globus pallidus
N-acetylaspartic acid (CSF, P) ↑ ↑ ↑ ↑
N-acetylaspartic acid (U) ↑ ↑ ↑ ↑
Most patients follow the infantile form which mostly manifests at 2–4 months of age with head lag, muscular hypotonia and macrocephaly, pro-
gressing to marked developmental delay, seizures, optic nerve atrophy, progressive spasticity and opisthotonic posturing (Matalon et al. 1995)
Table 8.2 Glutaric aciduria type I

CNS Abasia ± ± ± ±
Astasia ± ± ±
Ataxia ± ± ±
Chorea ± ± ± ±
Dystonia ± ± ± ±
Encephalopathic crisis, acute ± ±
Headache ± ±
Hypokinesia ± ± ± ±
Hypotonia, axial ± ± ±
Spasticity ± ± ±
Swallowing difficulties ± ± ± ±
Vertigo ± ±
Digestive Feeding difficulties ± ± ± ±
Vomiting ± ± ± ±
Musculoskeletal Macrocephaly ± + ± ± ±
Respiratory Pneumonia ± ± ± ±
Routine laboratory ASAT/ALAT (P) (↑) (↑) (↑) (↑)
Creatine kinase (P) (↑) (↑) (↑) (↑)
Special laboratory 3-Hydroxyglutaric acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
C5DC glutarylcarnitine (P, B) n-↑ n-↑ n-↑ n-↑ n-↑
Glutaconic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
MRI: periventricular white matter and other extrastriatal ± ± + +
MR abnormalities
MRI: striatal atrophy ± ± ± ±
MRI: temporal hypoplasia, dilated external CSF spaces + + ± ± ±
Table 8.3 l-2-hydroxyglutaric aciduria

CNS Ataxia ± + + +
Choreoathetosis ± ± ±
Dystonia ± ± ±
Hypotonia ± ± ± ± ±
Mental retardation ± + + +
Seizures ± ± ± ±
Spasticity ± ± ±
Tremor ± ± ±
Musculoskeletal Macrocephaly ± ± ± ±
Routine laboratory Ammonia (B) n-↑
Lactate (P) n-↑
Protein (CSF) ↑ ↑
Special laboratory l-2-hydroxyglutaric acid (U, P, CSF) ↑ ↑ ↑ ↑ ↑
Lysine (P, CSF) n n-↑ n-↑ n-↑ n-↑
MRI/CT: white matter abnormalities ± + + + +
MRI: globus pallidus and dentate nudeus lesions ± + + + +
Table 8.4 d-2-hydroxyglutaric aciduria type I

Cardiovascular Cardiomyopathy n n n n n
CNS Developmental delay + + + + +
Hypotonia + + + ± ±
Special laboratory D2HG (U, P, CSF) ↑ ↑ ↑ ↑ ↑
Table 8.5 d-2-hydroxyglutaric aciduria type II

CNS Developmental delay + + + + +
Epilepsy + + + + +
Hypotonia + + + ± ±
Special laboratory D2HG (U, P, CSF) ↑ ↑ ↑ ↑ ↑
8.5 Reference Values 8.6 Pathological Values
Disease 8.1 (CD): N-Acetylaspartic Acid (NAA)

N-acetylaspartic acid (NAA)
NAA (U) NAA (P) NAA (CSF) N-acetylaspartic acid (NAA)
Age mmol/mol creatinine μmol/L μmol/L NAA (U) NAA (P) NAA (CSF)
6–36 0.17–0.84 0.25–2.8 Age mmol/mol creatinine μmol/L μmol/L
Gas chromatography/mass spectrometry (preferably with stable isotope 60–10,000 Up to 10
dilution assay) Gas chromatography/mass spectrometry (preferably with stable isotope
dilution assay)
Glutaric acid (GA) Disease 8.2 (GA-I): Glutaric Acid (GA), 3-Hydroxyglutaric
GA (U) GA (P) GA (CSF) Acid (3-OH-GA), Glutarylcarnitine (C5DC)
Age mmol/mol creatinine μmol/L μmol/L Glutaric acid (GA)
< 10 0.5–2.9 0.18–0.63
GA (U) GA (P) GA (CSF)
Gas chromatography/mass spectrometry (preferably with stable isotope
Age mmol/mol creatinine μmol/L μmol/L
dilution assay)
N-10,000 N-200 N-40
3-Hydroxyglutaric acid (3-OH-GA) dilution assay)
3-OH-GA (U) 3-OH-GA (P) 3-OH-GA (CSF)
3-Hydroxyglutaric acid (3-OH-GA)
3-OH-GA (U) 3-OH-GA (P) 3-OH-GA (CSF)
<8 0.2–1.36 < 0.2
dilution assay) N-500 N-30 N-5
dilution assay)
Glutarylcarnitine (C5DC)
Glutarylcarnitine (C5DC)
C5DC (DBS) C5DC (P) C5DC (U)
C5DC (DBS) C5DC (P) C5DC (U)
< cut-offa < cut-offa < cut-offa
> cut-offa > cut-offa > cut-offa
Tandem mass spectrometry
a
Cut-off needs to be set by each lab Tandem mass spectrometry
a
Cut-off needs to be set by each lab
L-2-hydroxyglutaric acid (L2HG) Disease 8.3 (L2HGA)

L2HG (U) L2HG (P) L2HG (CSF) L2HG (U) L2HG (P) L2HG (CSF)
Age mmol/mol creatinine μmol/L μmol/L Age mmol/mol creatinine μmol/L μmol/L
1–19 0.5–1 0.3–2.3 226–4,299 7–84 23–474
Separation and individual quantification of the d- and l-isomer of 2HG Separation and individual quantification of the d- and l-isomer of 2HG
by gas chromatography–mass spectrometry or liquid chromatography- by gas chromatography–mass spectrometry or liquid chromatography-
tandem mass spectrometry tandem mass spectrometry
Disease 8.4 (D2HGA type I)

D-2-hydroxyglutaric acid (D2HG)
D2HG (U) D2HG (P) D2HG (CSF)
103–2,414 26–123 6–18
3–17 0.3–0.9 0.07–0.3
Separation and individual quantification of the d- and l-isomer of 2HG
Separation and individual quantification of the d- and l-isomer of 2HG by gas chromatography–mass spectrometry or liquid chromatography-
by gas chromatography–mass spectrometry or liquid chromatography- tandem mass spectrometry
tandem mass spectrometry
Disease 8.5 (D2HGA type II)

448–11,305 99–757 30–172
Separation and individual quantification of the d- and l-isomer of 2HG
by gas chromatography–mass spectrometry or liquid chromatography-
tandem mass spectrometry
8.7 Diagnostic Flow Chart(s) should be initiated by analysis of N-acetylaspartate (NAA) in

urine. If increased NAA concentrations are found, the diag-
Disease 8.1 (CD) nosis can be confirmed by significantly decreased aspartoac-
If neurological symptoms occur and/or neuroradiological ylase activity in cultured skin fibroblasts and/or the
abnormalities are found (→8.5, Signs and symptoms) which identification of two disease-causing mutations in the
are characteristic for Canavan disease, the diagnostic process ASPA gene.
Disease 8.2 (GA-I)
A.Newborn
screening
Low excretors
No
with known MS/MS
mutations?
Yes
No
C5DC > cut-off?
B. Selective
Yes
screening
No diagnostic
Suspicious signs No
work-up
and symptoms?
recommended
Quantitative
Yes
3-OH-GA and GA
analysis in urine
3-OH-GA No**
(and GA)
Fig. 8.6 Diagnostic flow chart for elevated?
glutaric aciduria type I. A. Newborn
Yes
screening for glutaric aciduria type I
(GA-I) is performed using tandem
mass spectrometry (MS/MS). In Start treatment
low-excreter cohorts with known
mutations, GCDH gene mutation
analysis should be considered as
alternative method (dotted line). Note GCDH gene
mutation analysis
that in these patients, treatment should
be started after the identification of two
disease-causing mutations (*). B.
Selective screening should be started if Two disease- No GCDH enzyme
diagnosis of GA-I is suspected or a causing
analysis
positive family history is known. Note mutations?
that a few patients with a low-excreting Yes

phenotype may show (intermittently)
normal urinary excretion of
3-hydroxyglutaric acid (3-OH-GA) and No
Low or deficient
Stop treatment
glutaric acid (GA). If an individual GCDH activity?
shows normal 3-OH-GA (and GA)
concentrations in urine but presents Yes
with highly suspicious signs and

symptoms for GA-I, it should be GA-I GA-I
decided individually whether established* excluded
diagnostic work-up is continued (**)
Diseases 8.3–8.5 (L2HGA, D2HGA type I and D2HGA

type II)
L2HGDH
L2HGDH gene mutations
2HG L2HG sequencing present?
L2HGA
D2HG
No, except for

Other modest Gain-of- D2HGDH D2HGDH
N
metabolites increases of function gene mutations
increased? CAC mutation in sequencing present?
intermediates IDH2?
Y Y
Y
Secondary
increase in e.g. D2HGA
SSADH def or D2HGA
Type I
GA II Type II
Fig. 8.7 Flowchart for the diseases 8.3, 8.4 and 8.5. The observed L2HG is within the reference range) can be secondary increased in
increase in 2HG in the urinary organic acid screening has to be pursued SSADH deficiency and GA-I. If these options are excluded, molecular
by an enantiomeric separation and individual quantification of both investigation on the two known heterozygous gain-of-function muta-
D2HG and L2HG. In case of an isolated increase of L2HG, subsequent tions will either exclude or confirm mutated IDH2 as the cause of the
molecular investigation of the L2HGDH gene usually reveals mutations D2HG increase. If IDH2 molecular studies are negative, the D2HGDH
confirming the biochemical diagnosis. In case of an isolated increase of gene should be investigated for the presence of mutations
D2HG, the diagnostic algorithm is more complicated. D2HG (whereas
Disease
no. Symbol Test Preconditions Material Handling Pitfalls
8.1 CD Organic acids None Urine Keep frozen Compound has poor recovery in
(NAA) (−20 °C) organic solvent extraction
8.2 GA-I Quantitative 3.5–4 h postprandially, Plasma Keep frozen
amino acids no dietary changes (−20 °C)
prior to the test
Trytophan 3.5–4 h postprandially, Plasma Keep frozen Losses due to inappropriate
no dietary changes (−20 °C) deproteinisation
prior to the test
Organic acids None Urine Keep frozen Reliable identification of
(3-OH-GA, GA) (−20 °C) 3-OH-GA may require the use of
a quantitative GC/MS method;
differential diagnoses of elevated
GA and 3-OH-GA include GA-II
and GA-III, SCHAD deficiency
and ketosis
Carnitine status None, also informs on Plasma serum Keep frozen
adherence to carnitine (−20 °C)
supplementation
Acylcarnitine None Dried blood spots Plasma, keep frozen C5DC may be also elevated in
profile (C5DC) Plasma (−20 °C) GA-II, renal insufficiency, MCAD
deficiency
(pseudoglutarylcarnitinemia)
Enzyme activity None Fibroblasts RT
(GCDH) Leukocytes from Keep frozen
heparinised blood (−20 °C)
8.3 L2HGA Organic acids None Urine Keep frozen For specific quantification of
(−20 °C) L2HG enantiomeric separation,
hyphenated to mass spectrometry
is required
L2HGA L2HG Isolation of cells Fibroblasts, RT, pellets should
dehydrogenase according to specific lymphoblasts, be frozen
activity protocol lymphocytes
8.4 D2HGA Organic acids None Urine Keep frozen For specific quantification of
type I (−20 °C) D2HG enantiomeric separation,
is required
D2HGA D2HG Isolation of cells Fibroblasts, RT, pellets should
type I dehydrogenase according to specific lymphoblasts be frozen
activity protocol
8.5 D2HGA Organic acids None Urine Keep frozen For specific quantification of
type II (−20 °C) D2HG enantiomeric separation,
is required D2HG also
accumulates GA-I and SSADH
D2HGA IDH2 gain-of- Isolation of cells Lymphoblasts RT, pellets should
type II function assay according to specific be frozen
protocol
3-OH-GA 3-hydroxyglutarate, C5DC glutarylcarnitine, GA glutarate, GA-II glutaric aciduria type II, GA-III glutaric aciduria type III, GCDH
glutaryl-CoA dehydrogenase, GC/MS gas chromatography/mass spectrometry, MCAD medium-chain acyl-CoA dehydrogenase, NAA
N-acetylaspartate, SCHAD short-chain 3-hydroxyacyl-CoA, SSADH succinic semialdehyde dehydrogenase deficiency
Prenatal diagnosis table for all disorders (if applicable) and sample requirements
Disease no. Symbol Material Timing trimester Pitfalls
8.1 CD Chorionic villi (accomplished) I Assay of aspartoacylase in amniocytes is not reliable. A
Amniotic fluid, amniocytes II combination of mutation analysis together with the exact
(accomplished) quantitation of N-acetylaspartate in the amniotic fluid is
recommended
8.2 GA-I Chorionic villi I
Amniocytes, amniotic fluid II
8.3 L2HGA Chorionic villi I
8.4 D2HGA type I Chorionic villi I
8.5 D2HGA type II Chorionic villi I Disease is usually caused by a heterozygous de novo
Amniocytes, amniotic fluid II mutation. Somatic and germline mosaicism in the
parents cannot be excluded; thus, the recurrence risk for
the parents is not zero. Therefore, prenatal diagnosis
should always be offered
In case of DNA-based prenatal diagnosis, maternal contamination should be excluded by VNTR marker analysis
Trimester one: only mutation analysis
Trimester two: a combination of metabolite and DNA investigations is recommended
DNA testing table for all disorders (if applicable) and sample requirements
Disease no. Symbol Tissue Methodology
8.1 CD Lymphocytes, fibroblasts Sequencing
8.2 GA-I Lymphocytes, fibroblasts Sequencing
8.3 L2HGA Blood, lymphocytes, fibroblasts, lymphoblasts Sequencing
8.4 D2HGA type I Blood, lymphocytes, fibroblasts, lymphoblasts Sequencing
8.5 D2HGA type II Blood, lymphocytes, fibroblasts, lymphoblasts Sequencing, targeted mutation analysis
8.9 Treatment Summary Emergency Treatment Table for All Disorders of Your
Chapter (If Applicable) and Medication Requirements
Effective metabolic treatment has only been described for (A. Including Box After the Table, with Pitfalls and Important
glutaric aciduria type I (low lysine diet, carnitine supplemen- Information)
tation, emergency treatment). Riboflavin should be consid- Diseases 8.1 and 8.3–8.5
ered as a treatment option for patients with l-2-hydroxyglutaric No emergency treatment is available.
aciduria aiming to activate residual enzyme activity. Disease 8.2 (GA-I)
Treatment of patients’ Canavan disease with lithium citrate, Emergency treatment is thought to be the most effective
lowering brain N-acetylaspartate concentrations, and glyc- component of current treatment strategies to prevent acute
erol triacetate, supplying the brain with acetate, is consid- striatal injury during infectious disease and for other causes
ered as safe; however, it is yet unknown whether it helps to of catabolism in glutaric aciduria type I (Heringer et al.
improve the outcome. Metabolic treatment for d-2- 2010). It must be initiated before the onset of severe neuro-
hydroxyglutaric aciduria type I and II has not yet been logical signs, which already indicate the manifestation of
described. neuronal damage. Therefore, during episodes that are likely
Although effective treatment is only known for some to induce catabolism (e.g. infectious disease) emergency
cerebral organic acidurias, symptomatic and supportive treatment should start without delay. Treatment should con-
treatment is important. This includes adequate supply with sist of frequent high carbohydrates feeds and increased car-
nutrient, minerals and micronutrients, physiotherapy, occu- nitine supplementation, followed by high-dose intravenous
pational therapy and pharmacotherapy of epilepsy and extra- glucose and carnitine (Kölker et al. 2011). All patients with
pyramidal movement disorder among others. The therapeutic glutaric aciduria type I should be supplied with an emer-
concept should be implemented after the assessment of indi- gency card. This concept should be strictly followed for the
vidual needs and, subsequently, monitored and evaluated by first 6 years of life. After this age emergency treatment is
an interdisciplinary team of specialists. individually adjusted.
Emergency treatment at home

A. Oral carbohydratesa Maltodextran
Age (years) % kcal/100 mL KJ/100 mL Volume (mL) per day orally
Up to 0.5 10 40 167 min. 150/kg
0.5–1 12 48 202 120/kg
1–2 15 60 250 100/kg
2–6 20 80 334 1,200–1,500
B. Protein intake
Natural protein Stop for max 24 (−48) h, then reintroduce and increase stepwise until the amount of maintenance treatment is
reached within 48 (−72) h
AAM If tolerated, AAM should be administered according to maintenance therapy
C. Pharmacotherapy
l-carnitine Double oral carnitine supplementations
According to Kölker et al. 2011
a
Solutions should be administered every 2 h day and night. Patients should be reassessed every 2 h. AAM, lysine-free, tryptophan-reduced amino
acids mixtures
Emergency treatment in hospital (according to Kölker et al. 2011)

A. Intravenous infusions
Glucose Age (years) Glucose (g/kg per day IV)
0–1 (12–) 15
1–3 (10–) 12
3–6 (8–) 10
Insulin If persistent hyperglycemia >150 mg/dL (>8 mmol/L) and/or glucosuria occurs, start with 0.05 IE insulin/kg
per h IV and adjust the infusion rate according to serum glucose
B. Protein intake
Natural protein Stop for max 24 (−48) h, then reintroduce and increase stepwise until the amount of maintenance treatment is
reached within 48 (−72) h
AAM If tolerated, AAM should be administered orally or by nasogastric tube according to maintenance therapy
C. Pharmacotherapy
l-carnitine 100 (−200) mg/kg per day IV
AAM, lysine-free, tryptophan-reduced amino acid mixtures
Standard Treatment Table for All Disorders of Your Diseases 8.1, 8.3, 8.4 and 8.5
Chapter (if applicable) and Medication Requirements No specific treatment is available.
(A. Including Box After the Table, with Pitfalls and Important Disease 8.2 (GA-I)
Information)
Disorder no. Symbol Age Medication/diet Dosage Doses/day (n)

8.2 GA-I <6 years Carnitine (50–) 100 mg/kg per day 3
>6 years Carnitine (30–) 50 mg/kg per day 3
Riboflavina 100 mg 2
Treatment of extrapyramidal movement disordersb
Antiepileptic treatmentc
Diet (see below)
a
Riboflavin responsiveness seems to be a very rare occasion in glutaric aciduria type I and no standard protocol exists to test it. There is no proven
clinical benefit of riboflavin supplementation (Kölker et al. 2006)
b
The complex movement disorder of symptomatic patients with glutaric aciduria type I is often difficult to treat. Baclofen and benzodiazepames
as monotherapy or in combination should be used as first-line drug treatment for focal and generalised dystonia. Intrathecal baclofen should be
considered as additional therapy for generalised dystonia and spasticity. Trihexyphenidyl should be considered as second-line treatment for dysto-
nia, particularly in adolescents and adults. Botulinum toxin A injections should be considered as additional therapy for severe focal dystonia
(according to Kölker et al. 2011)
c
Epilepsy is not frequently found in glutaric aciduria type I. Dystonic and epileptic movements, however, may be confused. Indication, prescription
and monitoring of antiepileptic treatment should be performed by a child neurologist or neurologist. Valproate should not be used due to the theo-
retical risk of mitochondrial dysfunction and secondary carnitine depletion
Dietary treatment (low lysine diet, according to Kölker et al. 2011)

Age
Treatment Dosage 0–6 months 7–12 months 1–3 years 4–6 years >6 years
Lysine (from natural protein)a mg/kg per day 100 90 80–60 60–50 Avoid excessive intake of natural
Protein from amino acid mixturesb g/kg per day 1.3–0.8 1.0–0.8 0.8 0.8 protein; use natural protein with
Energy kcal/kg per day 115–80 95–80 95–80 90–80 a low lysine content according to
‘safe levels’
Micronutrientsc % ≥100 ≥100 ≥100 ≥100 ≥100
a
The lysine/protein ratio (i.e., 2–9 mg lysine/100 mg protein) varies in natural food and thus natural protein intake in children on a low lysine diet
is dependent on the natural protein source
b
Lysine-free, tryptophan-reduced amino acid mixtures should be preferably supplemented with minerals and micronutrients. Safe intakes of essen-
tial amino acids are provided from natural protein and lysine-free, tryptophan-reduced amino acid supplements
c
According to dietary recommendations
Gitiaux C, Roze E, Kinugawa K et al (2008) Spectrum of movement

Dangers/Pitfalls disorders associated with glutaric aciduria type 1: a study of 16
patients. Mov Disord 23:2392–2397
1. Dietary treatment needs to be adapted to the indi-
Harting I, Neumaier-Probst E, Seitz A et al (2009) Dynamic changes of
vidual needs, in particular in dystonic patients. striatal and extrastriatal abnormalities in glutaric aciduria type I.
Overtreatment by protein restriction may result in Brain 132:1764–1782
malnutrition with essential nutrients. Heringer J, Boy SPN, Ensenauer R et al (2010) Use of guidelines
improves the neurological outcome in glutaric aciduria type I. Ann
2. Dysphagia is a frequent problem in dystonic
Neurol 68:743–752
patients. Tube feeding (via nasogastric tube or per- Kölker S, Garbade S, Greenberg CR et al (2006) Natural history,
cutaneous gastrostomy) should be considered if outcome, and treatment efficacy in children and adults with
oral food intake is inadequate. glutaryl-CoA dehydrogenase deficiency. Pediatr Res 59:840–847
Kölker S, Christensen E, Leonard JV et al (2011) Diagnosis and
management of glutaric aciduria type I – revised recommendations.
J Inherit Metab Dis 34:677–694
Experimental Treatment for All Disorders of Your Kranendijk M, Struys EA, Van Schaftingen E et al (2010) IDH2
mutations in patients with D-2-hydroxyglutaric aciduria. Science
Chapter (If Applicable) and Medication Requirements 330:336
(A. Including Box After the Table, with Pitfalls and Important Kranendijk M, Struys EA, Salomons GS, Van der Knaap MS, Jakobs C
Information) (2012) Progress in understanding 2-hydroxyglutaric acidurias.
J Inherit Metab Dis 35(4):571–587
Alternative therapies/experimental Leone P, Janson CG, Bilanuk L et al (2000) Aspartoacylase gene
Disease no. Symbol trials transfer to the mammalian central nervous system with therapeutic
implications for Canavan disease. Ann Neurol 48:27–38
8.1 CD Lithium citrate
Matalon R, Michals K, Kaul R (1995) Canavan disease: from spongy
Glycerol triacetate degeneration to molecular analysis. J Pediatr 127:511–517
Gene therapy Sauer SW, Okun JG, Fricker G et al (2006) Intracerebral accumulation
8.2 GA-I Arginine or homoarginine of glutaric and 3-hydroxyglutaric acids secondary to limited flux
supplementation has yet only been across the blood–brain barrier constitutes a biochemical risk factor
studied in GCDH-deficient mice, an for neurodegeneration in glutaryl-CoA dehydrogenase deficiency.
animal model for GA-I, and in a J Neurochem 97:899–910
small number of patients (arginine) Segel R, Anikster Y, Zevin S et al (2011) A safety trial of high glucose
8.3 L2HGA Riboflavin supplementation triacetate for Canavan disease. Mol Genet Metab 103:203–206
8.4 D2HGA type I On the basis that D2HG Steenweg ME, Salomons GS, Yapici Z et al (2009) L-2-Hydroxyglutaric
dehydrogenase is an FAD-dependent aciduria: pattern of MR imaging abnormalities in 56 patients.
enzyme, riboflavin supplementation Radiology 2009(251):856–865
is a therapeutic option Steenweg ME, Jakobs C, Errami A et al (2010) An overview of L-2-
hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a
8.5 D2HGA type II Development of specific inhibitor of
genotype-phenotype study. Hum Mutat 31:380–390
the IDH2 mutant enzyme
Struys EA, Verhoeven NM, Brunengraber H et al (2004) Investigations
by mass isotopomer analysis of the formation of D-2-
hydroxyglutarate by cultured lymphoblasts from two patients with
D-2-hydroxyglutaric aciduria. FEBS Lett 557(1–3):115–120
References Struys EA, Salomons GS, Achouri Y et al (2005) Mutations in the D-2-
hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric
Assadi M, Janson C, Wan DJ et al (2010) Lithium citrate reduces exces- aciduria. Am J Hum Genet 76:358–360
sive intracerebral N-acetylaspartate in Canavan disease. Eur J Van Schaftingen E, Rzem R, Veiga-da-Cunha M (2009) L-2-
Paediatr Neurol 14:354–359 hydroxyglutaric aciduria, a disorder of metabolite repair. J Inherit
Ethylmalonic Encephalopathy
9
Alberto Burlina and Massimo Zeviani
Contents Summary
9.1 Introduction ..................................................................... 158 Ethylmalonic encephalopathy is a severe mitochondrial
disease of early infancy clinically characterised by a com-
9.2 Nomenclature................................................................... 158
bination of developmental delay, progressive pyramidal
9.3 Metabolic Pathway .......................................................... 159 signs and vascular lesions including petechial purpura,
9.4 Signs and Symptoms ....................................................... 160 orthostatic acrocyanosis and chronic hemorrhagic diarrhoea.
9.5 Normal and Pathological Values .................................... 162
Biochemical hallmarks of the disease are persistently high
levels of lactate, and C4–C5-acylcarnitines in blood, mark-
9.6 Diagnosis .......................................................................... 162
edly elevated urinary excretion of methylsuccinic and ethyl-
9.7 Specimen Collection ........................................................ 162 malonic (EMA) acids and defective cytochrome c oxidase
9.8 Treatment ......................................................................... 162 (COX) in the muscles. The corresponding gene was named
ETHE1 and mutation analysis including: nonsense and mis-
Further Reading ............................................................................ 162
sense mutations, frameshift and deletion of single exons or
of the entire gene have been reported. The prognosis of the
disease is poor but combined treatment with metronidazole,
N-acetylcysteine and coenzyme Q10 resulted in disappear-
ance of diarrhoea, petechial showers and acrocyanosis.
Ethylmalonic encephalopathy, first described by Burlina
et al. 1991, is a severe mitochondrial disease due to muta-
tions in the ETHE1 gene (MIM ≠ 608451). Clinically it is
characterised by a combination of symptoms including
developmental delay, progressive pyramidal signs, vascular
lesions including petechial purpura, orthostatic acrocyanosis
and chronic hemorrhagic diarrhoea. The clinical condition is
quite homogeneous but a broad clinical spectrum of severity
has been described. The prognosis is poor with half of the
patients dying within the first 2 years of life from metabolic
decompensation. Brain MRI reveals symmetric patchy sig-
nals in basal ganglia, periventricular white matter and den-
A. Burlina (*) tate nuclei, along with brain and spinal cord malformations.
Division of Metabolic Disorders, Department of Pediatrics, Biochemical hallmarks of the disease are: persistently high
University Hospital, Via Giustiniani 3, Padova, 35128, Italy levels of lactate, C4–C5-acylcarnitines in blood, markedly
e-mail: alberto.burlina@unipd.it
elevated urinary excretion of methylsuccinic and ethylma-
M. Zeviani lonic acids and defective COX in the muscles and brain but
Unit of Molecular Neurogenetics,
not in cultured skin fibroblasts. The pathogenic mechanisms
Fondazione Istituto Neurologico “Carlo Besta”,
Milan, Italy underlying the clinical and biochemical abnormalities are
the main consequence of ETHE1 impairment. The main
Mitochondrial Biology Unit,
Cambridge, UK consequence of ETHE1 loss is the accumulation of hydro-
e-mail: zeviani@istituto-besta.it gen sulphide (H2S), a product of intestinal anaerobes and,
158 A. Burlina and M. Zeviani
in trace amount, tissues. Increased concentration of sulphide and deletion of single exons or of the entire gene have been
in tissues (i.e. colonic mucosae, muscle and brain) causes reported. To date, a total of 70 mutant patients have been identi-
rapid inhibition of COX activity and long-term degrada- fied in our laboratory (Massimo Zeviani unpublished).
tion of COX subunits. In addition, H2S blocks short-chain The ETHE1 protein, a 30 kDa polypeptide located in the
fatty acid oxidation by inhibiting the activity of short-chain mitochondrial matrix, functions in vivo as a homodimeric,
acyl-CoA dehydrogenase (and possibly other beta-oxidation Fe-containing sulphur dioxygenase (SDO) activity, involved
enzymes as well), which explains the accumulation of EMA. in the oxidative detoxification of (harmful) sulphide to (inert)
Furthermore, H2S has vasoactive and vasotoxic effects, sulphate.
which in turn explain the vascular lesions in the skin and, Impaired activity of ETHE1-SDO leads to the accumula-
possibly, other organs. Combined treatment with metronida- tion of H2S in critical tissues, including colonic mucosa,
zole, N-acetylcysteine and coenzyme Q10 resulted in some liver, muscle and brain, up to concentrations that inhibit
neurological improvement, disappearance of diarrhoea, SCAD and COX activities, therefore accounting for EMA
petechial showers and acrocyanosis. Further opportunities aciduria and high levels of C4- and C5-acylcarnitines, EMA
for therapy will include transplantation of hemopoietic stem and lactate, in plasma. The pathway outlined in Fig. 9.1
cells and adeno-associated virus mediated gene therapy. explains these findings.
In addition to H2S, which is a highly unstable, difficult-to-
measure gas, its stable derivative thiosulphate is also present
9.1 Introduction at very high concentrations in tissues and body fluids of
Ethe1-/- mice and EE patients and can well be considered a
Ethylmalonic encephalopathy (EE) described in this chapter specific biomarker of the disease.
is a very rare mitochondrial disorder caused by mutations in Besides COX and SCAD deficiency, other symptoms of
the ETHE1 gene localised to chromosome 19q13. EE are explained by accumulation of H2S, including dam-
Biochemically, EE is characterised by the unusual combina- age of endothelial cells and vasodilation, which account for
tion of severe deficiency of COX, the terminal component of the petechiae and the acrocyanosis. Since a substantial
the mitochondrial respiratory chain, in the muscles and brain, amount of H2S derives from the anaerobic bacterial flora
leading to high levels of lactate in blood and accumulation of residing in the intestinal lumen, COX activity is markedly
ethylmalonate (ethylmalonic acid, EMA) in urines. EMA, a reduced in the luminal colonocytes of Ethe1-/- mice, whereas
dicarboxylic organic acid produced by the carboxylation of it is normal in cryptal colonocytes that are relatively
butyrate, is a hallmark of enzymatic defects of β-oxidation of secluded from the luminal surface. This difference is likely
fatty acids and branched-chain amino acids, for instance to reflect the different exposure of the two cell populations
defects of the short-chain and branched-chain acylCoA to the inhibitory action of exogeneous H2S. Excessive pro-
dehydrogenase (SCAD, BCAD). Like in SCAD and BCAD duction and absorption of H2S, as well as reduced detoxifi-
deficiency, accumulation of C4- and C5-acylcarnitines has cation by colonocytes, is regarded to play an important role
been documented in blood of EE patients. in the mucosal damage of ulcerative colitis. A similar
The corresponding gene was named ETHE1 and mutation mechanism can well account for the severe chronic
analysis including nonsense and missense mutations, frameshift diarrhoea afflicting EE patients.
9.2 Nomenclature
Alternative Gene Chromosomal Affected OMIM

No. Disorder name Abbreviation symbol localisation protein no. Subtype
9.1 Ethylmalonic ETHE1 ETHE1 – 19p13.32 Mitochondrial 602473 Autosomal
encephalopathy deficiency matrix protein recessive
9 Ethylmalonic Encephalopathy 159
NH2
HS OH
cysteine
O
pyruvate
O
pyruvate CH3 OH
H2SO3
O
H2S
SO4
(sulfate)
SOX
(Sulfite Oxidase)
H2SO3
(sulfite)
ETHE1 Rhodanese
H2S2O3
(SDO: Sulfur Dioxygenase) (Thiosulfate Sulfure Transferase)
R -SSH R -SSH
H2S (persulfide)
(persulfide)
e–
S S O2 2H2O
R (Sulfide quinone
matrix Oxidoreductase)
SQR
UQ
inner mitochondrial
e– e–
membrane
UQH2 Cyt c
intermembrane space CIII e– CIV
Fig. 9.1 Metabolic pathway of ethylmalonic encephalopathy. From Ethe1 into sulphite (SO32−). Sulphite is further oxidised to sulphate
different sources (green arrow), through desulphuration and deamina- (SO42−) by sulphite oxidase (SOX) or exploited as a sulphur acceptor to
tion into pyruvate, H2S (red arrow) is initially oxidised, and the sulphur form thiosulphate (S2O32−) by rhodanese. The electrons extracted by
atom is fixed by sulphide–CoQ reductase (SQR) to form a persulphide, SQR are transferred to the mitochondrial respiratory chain via coen-
which is then converted by the sulphur dioxygenase (SDO) activity of zyme Q (Q)

Table 9.1 Ethylmalonic encephalopathy
Cardiovascular Orthostatic acrocyanosis +++ +++
CNS Hypotonia, axial ++ ++
Retardation, psychomotor +++ +++
Seizures ++ ++
Spastic tetraplegia +++ +++
Leigh-like syndrome +++ +++
Dermatological Petechiae +++ +++
Digestive Failure to thrive +++ +++
Hemorrhagic diarrhoea, chronic +++ +++
Routine laboratory Hematuria + +
Lactate (P) ↑↑ ↑↑
Lactic acidosis +++ ++
Special 2-Methylbutyrylglycine (U) ↑↑ ↑↑
laboratory C4 butyryl-carnitine (P, B) ↑↑ ↑↑ ↑↑
C4 isobutyryl-carnitine (P, B) ↑ ↑
C5 2-methylbutyryl-carnitine (P, B) ↑ ↑
C5 isovaleryl-carnitine (P, B) ↑ ↑
C5-DC glutaryl-carnitine (P, B) ↑↑ ++ ++ ++ ++
Ethylmalonic acid (U) ↑↑ ↑↑↑ ↑↑↑
Isovalerylglycine (U) + +
Methylsuccinic acid (U) ↑↑ ↑↑
Thiosulphate (U) +++ +++
Ethylmalonic encephalopathy is characterised by of vascular function, as an endothelium-derived relaxing

psychomotor regression and hypotonia, later evolving into factor and a regulator of endothelial and smooth-muscle cell
spastic tetraparesis, dystonia and eventually global neuro- growth and apoptosis. However, the vasotoxic effects of H2S
logic failure. The encephalopathy is typically accompanied in vivo have not been clearly demonstrated.
by chronic diarrhoea and failure to thrive. Endothelial damage and loss was observed also in the
A clinical trait characteristic of ethylmalonic encepha- gastric antrum and colonic mucosa and submucosa, associ-
lopathy is the presence of orthostatic acrocyanosis and pete- ated with microhaemorrhages. Importantly, damage of small
chial purpura (Fig. 9.2). This phenomenon has been attributed vessels in critical organs, notably brain and colon, supports a
to the vasoactive and vasotoxic effects of H2S. Increasing common pathogenic mechanism in Ethe1- patients, centred
evidence supports an important role for H2S in the regulation on the accumulation of hydrogen sulphide to toxic levels.
Fig. 9.2 The clinical hallmarks

of the ethylmalonic
encephalopathy: the widespread
lesions of the small blood vessels
causing showers of petechiae,
easy bruising and orthostatic
acrocyanosis (black arrows)
Cerebral abnormalities manifest early after birth, with nonvascular areas, such as striatum and brainstem, and
hypotonia, delayed development and, later, spasticity. underlined the disappearance of these lesions over time, pos-
Generalised seizures are associated with abnormal focal dis- sibly due to brain maturation. Recently, analysis of a brain
charges on the electroencephalogram. Neurologic deteriora- from an EE patient showed widespread luminal micro-
tion accelerates following intercurrent infectious illness, and thrombi, acute microhaemorrhages and focal perivascular
most patients die in the early years of life. haemosiderin-laden macrophages, the latter being consistent
The neuroradiological pattern in EE patients is nonspecific with previous bleedings. As a consequence, the brain showed
with symmetrical necrotic lesions characterised by high sig- features of both acute and chronic ischaemic damage, consis-
nals on T2-weighted images in the deep grey matter structures tent with abnormal signal intensity lesions, on repeated MRI.
(see Fig. 54.11). These hyperdensities were deemed to be the Brain vascular lesions seem to be a specific neuropathologi-
result of infarcts related to the toxicity of accumulating cal feature of EE due to Ethe1 mutations, as compared to
organic acids (like EMA) or the consequence of vascular other forms of ethylmalonic aciduria and to disorders caused
changes seen in this disorder. Brain damage was observed in by primary respiratory chain defects such as Leigh’s disease.
9.5 Normal and Pathological Values
Metabolite Normal Pathological values

C4-acylcarnitine (B) 0.07–0.50 μmol/l >3.5 μmol/l
C5-acylcarnitine (B) 0.03–0.50 μmol/l >1.0 μmol/l
Ethylmalonic (U) <10 μmol/mmol creat >60 μmol/mmol creat
Isovalerylglycine (U) <0.9 μmol/mmol creat >2 μmol/mmol creat
2-Methylsuccinic (U) <3 μmol/mmol creat >10 μmol/mmol creat
2-Methylbutyrylglycine (U) ≤0.1 μmol/mmol creat >2 μmol/mmol creat
Thiosulphate (U) 100–560 μmol/mg creat >1,500 μmol/mg creat
9.6 Diagnosis ascorbic acid, vitamin E and glycine supplementations all

have been tried without benefit, although individual patients
The main biochemical features of ethylmalonic were reported to show a slight improvement in motor func-
encephalopathy are increased urinary ethylmalonic and tion, cognitive behaviour and chronic mucoid diarrhoea after
methylsuccinic acids associated with abnormal excretion treatment with riboflavin and/or coenzyme Q10.
of C4 and C5 (n-butyryl-, isobutyryl-, isovaleryl- and A diet low in branched-chain or sulphur-containing
2-methylbutyryl-) acylglycines and acylcarnitines, as well (methionine) amino acids, failed to show any benefit and
as severe lactic acidosis. Initial laboratory studies in the may in fact be harmful.
investigation of ethylmalonic aciduria should include Combined exposure to metronidazole, a nitroimidazole
blood glucose, lactate, ammonia, electrolytes and blood anti-infective compound that can buffer the excess of free
gases, a complete blood count, a blood acylcarnitine pro- H2S, and N-acetylcysteine that acts as one of the physiologi-
file and quantitative urine organic acid analysis by GC-MS. cal acceptors of the sulphur atom of H2S operated by sul-
Secondary COX-deficiency in the muscles has been phide–CoQ reductase, has been effective in improving the
described in several patients. Mutation analysis of the main symptoms in a pilot study on EE patients, including
ETHE1 gene can now provide the definitive diagnosis, marked attenuation or disappearance of the vascular lesions
including prenatal diagnosis. and diarrhoea, as well as amelioration of some neurological
Ethylmalonic encephalopathy should be included in the abnormalities.
differential diagnosis of persistent ethylmalonic aciduria, Recently further opportunities for therapy have been
which also includes short-chain acyl-CoA dehydrogenase proposed. For instance adeno-associated virus mediated
deficiency, defects of the mitochondrial electron-transfer fla- gene targeting could be exploited to express recombinant
voprotein pathway or glutaric aciduria type II and some Ethe1 in the liver, the filter organ that receives blood from
forms of respiratory chain deficiency. the gastrointestinal tract, the major source of exogenous
H2S. A complementary strategy, based on bone marrow
transplantation could determine the widespread diffusion
9.7 Specimen Collection of an Ethe1-proficent tissue to promoting the clearance of
toxic H2S from circulating body fluids, thus reducing the
exposure of critical organs such as brain and skeletal
Test Specimen Storage muscle.
C4-acylcarnitine Blood spot, plasma Keep frozen (−20°)
C5-acylcarnitine Blood spot, plasma Keep frozen (−20°) Dosage Route and
Ethylmalonic Urine Keep frozen (−20°) Disorder Medication (mg/kg/day) frequency
Isovalerylglycine Urine Keep frozen (−20°) ETHE1 def. Riboflavin 3–20 Os–2
2-Methylsuccinic Urine Keep frozen (−20°) Ubiquinone (CoQ10) 5 Os–2
2-Methylbutyrylglycine Urine Keep frozen (−20°) Metronidazole 25–50 Os–1
Thiosulphate Urine Keep frozen (−20°) N-acetylcysteine 50–100 Os–2
9.8 Treatment
Further Reading
The prognosis of ethylmalonic encephalopathy is poor, being Barth M, Ottolenghi C, Hubert L et al (2010) Multiple sources of meta-
usually lethal in early childhood. No effective treatment of bolic disturbance in ETHE1-related ethylmalonic encephalopathy. J
ethylmalonic encephalopathy is known. Riboflavin, carnitine, Inherit Metab Dis 33(Suppl 3):443–53
Burlina A, Zacchello F, Dionisi-Vici C et al (1991) New clinical phenotype Ozand PT, Rashed M, Millington DS et al (1994) Ethylmalonic
of branched-chain acyl-CoA oxidation defect. Lancet 338:1522–1523 aciduria: an organic acidemia with CNS involvement and vascu-
Burlina AB, Dionisi-Vici C, Bennett MJ, Gibson KM et al (1994) A lopathy. Brain Dev 16:12–22
new syndrome with ethylmalonic aciduria and normal fatty acid Pigeon N, Campeau P, Cyr D, Lemieux B, Clarke JTR (2009) Clinical
oxidation in fibroblasts. J Pediatr 124:79–86 heterogeneity in ethylmalonic encephalopathy. J Child Neurol
Di Meo I, Fagiolari G, Prelle A, Viscomi C, Zeviani M, Tiranti V (2011) 24:991–996
Chronic exposure to sulfide causes accelerated degradation of cyto- Tiranti V, D’Adamo P, Briem E, Ferrari G, Mineri R, Lamantea E,
chrome c oxidase in ethylmalonic encephalopathy. Antioxid Redox Mandel H, Balestri P, Garcia-Silva MT, Vollmer B, Rinaldo P, Hahn
Signal 15:353–362 SH, Leonard J, Rahman S, Dionisi-Vici C, Garavaglia B, Gasparini
Drousiotou A, DiMeo I, Mineri R, Georgiou T, Stylianidou G, Tiranti V P, Zeviani M (2004) Ethylmalonic encephalopathy is caused by
(2011) Ethylmalonic encephalopathy: application of improved bio- mutations in ETHE1, a gene encoding a mitochondrial matrix pro-
chemical and molecular diagnostic approaches. Clin Genet tein. Am J Hum Genet 74:239–252
79:385–390 Tiranti V, Briem E, Lamantea E, Mineri R, Papaleo E, De Gioia L,
Dweikat I, Naser E, Damsah N, Libdeh BA, Bakri I (2012) Ethylmalonic Forlani F, Rinaldo P, Dickson P, Abu-Libdeh B, Cindro-Heberle L,
encephalopathy associated with crescentic glomerulonephritis. Owaidha M, Jack RM, Christensen E, Burlina A, Zeviani M (2006)
Metab Brain Dis 27(4):613–616 ETHE1 mutations are specific to ethylmalonic encephalopathy. J
García-Silva MT, Campos Y, Ribes A et al (1994) Encephalopathy, pete- Med Genet 43:340–346
chiae, and acrocyanosis with ethylmalonic aciduria associated with Tiranti V, Viscomi C, Hildebrandt T, Di Meo I, Mineri R, Tiveron C,
muscle cytochrome c oxidase deficiency. J Pediatr 125:843–844 Levitt MD, Prelle A, Fagiolari G, Rimoldi M, Zeviani M (2009)
García-Silva MT, Ribes A, Campos Y, Garavaglia B, Arenas J (1997) Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide
Syndrome of encephalopathy, petechiae, and ethylmalonic aciduria. toxicity in ethylmalonic encephalopathy. Nat Med 15:200–205
Pediatr Neurol 17:165–170 Viscomi C, Burlina AB, Dweikat I, Savoiardo M, Lamperti C,
Giordano C, Viscomi C, Orlandi M, Papoff P, Spalice A, Burlina A, Di Hildebrandt T, Tiranti V, Zeviani M (2010) Combined treatment
Meo I, Tiranti V, Leuzzi V, d’Amati G, Zeviani M (2012) with oral metronidazole and N-acetylcysteine is effective in ethyl-
Morphologic evidence of diffuse vascular damage in human and in malonic encephalopathy. Nat Med 16:869–871
the experimental model of ethylmalonic encephalopathy. J Inherit Walsh DJ, Sills ES, Lambert DM, Gregersen N, Malone FD, Walsh AP
Metab Dis 35:451–458 (2010) Novel ETHE1 mutation in a carrier couple having prior off-
Grosso S, Mostardini R, Farnetani MA, Molinelli M, Berardi R, spring affected with ethylmalonic encephalopathy: genetic analysis,
Dionisi-Vici C, Rizzo C, Morgese G, Balestri P (2002) Ethylmalonic clinical management and reproductive outcome. Mol Med Report
encephalopathy: further clinical and neuroradiological characteriza- 3:223–226
tion. J Neurol 249:1446–1450 Wang R (2011) Signaling pathways for the vascular effects of hydrogen
McGowan KA, Nyhan WL, Barshop BA, Naviaux RK, Yu A, Haas RH, sulfide. Curr Opin Nephrol Hypertens 20:107–112
Townsend JJ (2004) The role of methionine in ethylmalonic enceph- Zafeiriou DI, Augoustides-Savvopoulou P, Haas D, Smet J, Triantafyllou
alopathy with petechiae. Arch Neurol 61:570–574 P, Vargiami E, Tamiolaki M, Gombakis N, van Coster R, Sewell
Nowaczyk MJ, Blaser SI, Clarke JT (1998) Central nervous system AC, Vianey-Saban C, Gregersen N (2007) Ethylmalonic encepha-
malformations in ethylmalonic encephalopathy. Am J Med Genet lopathy: clinical and biochemical observations. Neuropediatrics
75:292–296 38:78–82
Part III
Vitamins, Cofactors, and Metals
Disorders of Folate Metabolism
and Transport 10
Fernando Scaglia and Nenad Blau
Contents Summary
10.1 Introduction ..................................................................... 168 Folates play an essential role in one-carbon methyl transfer
reactions, mediating several biological processes including
10.2 Nomenclature................................................................... 169
DNA synthesis, regulation of gene expression through meth-
10.3 Metabolic Pathway .......................................................... 170 ylation reactions, embryonic central nervous system develop-
10.4 Signs and Symptoms ....................................................... 171 ment, synthesis and breakdown of amino acids, and synthesis
of thymidines, purines, and neurotransmitters (Blount et al.
10.5 Reference Values.............................................................. 173
1997; Linhart et al. 2009; Ghoshal et al. 2006; Pogribny
10.6 Pathological Values.......................................................... 174 et al. 2008; Fournier et al. 2002). In mammals, folates are
10.7 Specimen Collection ........................................................ 174 mostly derived from exogenous sources as folate is stored
10.8 Prenatal Diagnosis and DNA Testing ............................ 175 in the liver for few months. The biologically active folic
acid derivative is 5,6,7,8-tetrahydrofolate (THF). Dietary
10.9 Treatment ......................................................................... 175
folate is absorbed in the intestine. In the cytoplasm, inter-
References ...................................................................................... 177 conversion of 5,10-methylene-THF and 5,10-methenyl-THF,
interconversion of 5,10-methenyl-THF and 10-formyl-THF,
and reaction of THF with formate to synthesize 10-formyl-
THF are mediated by the MTHFD1 gene that encodes a
trifunctional protein. Metabolism of 5,10-methylene-THF
to 5-methyl-THF in the liver is catalyzed by methylene-
THF reductase (MTHFR) (Fig. 10.1). 5-methyl-THF is
then widely distributed in the bloodstream. The transport of
5-methyl-THF inside the cells is mediated by different trans-
port systems that include the proton-coupled folate trans-
porter (PCFT), the reduced folate carrier 1 (RFC1), and the
two GPI-anchored receptors, folate receptor alpha (FRα) and
beta (FRβ) (Matherly and Goldman 2003). The physiologi-
cal form of folate, 5-methyl-THF is actively transported to
the central nervous system by FRα-mediated endocytosis in
F. Scaglia
choroid epithelial cells, reaching a higher concentration in
Department of Molecular and Human Genetics,
Baylor College of Medicine, Suite 1560, the cerebrospinal fluid when compared to the serum. FRα is
36701 Fannin Street, Houston, TX 77030, USA a high-affinity low-capacity receptor that functions at a nano-
e-mail: fscaglia@bcm.tmc.edu molar range of extracellular folate concentrations (Weitman
N. Blau (*) et al. 1992). Thus far, seven different inherited disorders of
Division of Inborn Metabolic Diseases, folate metabolism are known which lead to folate deficiency
Department of General Pediatrics, University Children’s Hospital,
including hereditary folate malabsorption, folate recep-
Im Neuenheimer Feld 430, Heidelberg 69120, Germany
tor alpha deficiency, methylenetetrahydrofolate reductase
Division of Metabolism,
deficiency, methenyltetrahydrofolate synthetase deficiency,
Zürich, Switzerland dihydrofolate reductase deficiency, and methylenetetrahy-
e-mail: nenad.blau@med.uni-heidelberg.de drofolate dehydrogenase deficiency (Watkins and Rosenblatt
168 F. Scaglia and N. Blau
2012; Watkins et al. 2011). Furthermore, in some cases, an Methylenetetrahydrofolate reductase (MTHFR) defi-
additional disorder, namely, cerebral folate deficiency (CFD) ciency is an autosomal recessive condition caused by
caused by FOLR1 autoantibodies has also been described mutations in the MTHFR gene that maps to 1p36.22 (Goyette
(Ramaekers and Blau 2004). et al. 1994). This condition represents the most common of
the known inborn errors of folate metabolism with more than
100 subjects identified. MethyleneTHF reductase mediates
10.1 Introduction the conversion of 5,10-methylene-THF to 5-methyl-THF.
Biochemically, patients with this condition have hyperho-
Hereditary folate malabsorption is an autosomal recessive mocysteinemia with low or low-normal methionine levels,
condition where infants present at 2–6 months of age with reflecting decreased activity of methionine synthase. Clinical
clinical features suggestive of megaloblastic anemia, failure features of this disorder include developmental delay, intel-
to thrive, diarrhea, frequent infections, mucositis, and neu- lectual disability, neurological and psychiatric features,
rological features including epilepsy, developmental delay, thrombotic events, and serious disease leading to death
and intellectual disability due to a defect in the uptake of (Strauss et al. 2007) (see Table 10.3). Severe MTHFR defi-
folates from the intestine and the blood–brain barrier of the ciency should be a diagnostic consideration in infantile epi-
choroid plexus. Folate levels on both serum and cerebrospi- leptic encephalopathy (Prasad et al. 2011). Microcephaly and
nal fluid are low (see Table 10.1). This condition is caused developmental regression become noticeable following the
by mutations in the SLC46A1 gene that maps to 17q11.2 and onset of seizures. Patients with this condition do not exhibit
encodes the PCFT (Qiu et al. 2006). Several mutations have megaloblastic anemia. Other subjects may present later in
been reported including missense, nonsense, and splice site childhood with developmental delay and other neurological
mutations and it has been demonstrated that they affect folate manifestations. The striking changes of demyelination do not
transport in an in vitro system (Qiu et al. 2006). The therapy necessarily reflect an effect from the vascular lesions, sug-
of this condition has involved the parenteral administration gesting the participation of other factors such as inadequate
of folate. However, oral therapy has been successfully imple- methionine synthesis, a deficiency of S-adenosylmethionine
mented in some cases (Watkins and Rosenblatt 2012). or accumulation of toxic intermediates from the elevated lev-
Folate receptor alpha deficiency is an autosomal reces- els of homocysteine (Prasad et al. 2011). Among different
sive condition caused by loss of function mutations in the therapeutic strategies, the best outcome has been seen with
FOLR1 gene encoding the folate receptor alpha that leads betaine-homocysteine methyltransferase that converts homo-
to impaired transport of folate to the CNS (see Table 10.2). cysteine to methionine without requiring folate when started
The patients initially described exhibit null mutations prenatally or soon after birth (Strauss et al. 2007).
causing severe cerebral folate deficiency (CFD) (Steinfeld Dihydrofolate reductase (DHFR) deficiency is a recently
et al. 2009). These patients have an initial period of nor- described inborn error of folate metabolism (Cario et al. 2011;
mal development and they eventually exhibit psychomotor Banka et al. 2011) (see Table 10.4). DHFR is a key enzyme in
decline, progressive movement disturbance, white matter folate metabolism and an important target of several pharma-
disease, and epilepsy (Steinfeld et al. 2009; Cario et al. cological compounds. Three individuals from two families
2009). Myoclonic-astatic seizures have been described in were described, who presented with megaloblastic anemia
the most affected patients and the epilepsy in these patients and/or pancytopenia, severe CFD, and cerebral tetrahydro-
seems to be drug refractory (Steinfeld et al. 2009; Cario biopterin deficiency due to a germline missense mutation in
et al. 2009) although a case associated with progressive DHFR, leading to profound enzymatic deficiency. Cerebral
myoclonic epilepsy has responded to high dose of folinic folate levels, anemia, and pancytopenia were corrected by
acid supplementation (Perez-Duenas et al. 2011). These treatment with folinic acid. These individuals presented in
patients have almost undetectable levels of cerebrospinal the first weeks of life with epilepsy, developmental delay,
fluid (CSF) 5-methyl-THF. MR-based in vivo metabolite and cerebral and cerebellar atrophy (Banka et al. 2011).
analysis indicates depletion of white matter choline and ino- Individuals from a third family presented at 2–11 years and
sitol. Folinic acid therapy restores glial choline and inositol two exhibited atypical childhood absence epilepsy (Cario
contents, CSF 5-methyl-THF and ameliorates symptoms in et al. 2011). All of these subjects were homozygous for
these patients. Folate receptor alpha deficiency caused by a mutations in the DHFR gene that maps to 5q14.1. Therapy
homozygous p.(Arg204*) mutation in the FOLR1 gene has with folinic acid has corrected the biochemical abnormali-
been described in a patient with congenital deafness, laby- ties. However, in some cases, the epilepsy became persistent
rinthine aplasia, microtia, and microdontia (LAMM) syn- when the treatment with folinic acid was not given in a con-
drome (Dill et al. 2011). Folinic acid treatment helped to sistent basis (Cario et al. 2011).
regain consciousness but the epilepsy was controlled with Mutations in the MTHFD1 gene mapping to 14q23.3
pyridoxal 5′-phosphate. have been identified by exome sequencing in an infant
10 Disorders of Folate Metabolism and Transport 169
who presented at 2 months of age with megaloblastic ane- were normal and there were no signs of anemia. MTHFS
mia, atypical hemolytic uremic syndrome, and severe com- converts 5-formyl-THF to 5,10-methenyl-THF, thus making
bined immunodeficiency (see Table 10.5). The MTHFD1 folinic acid not a therapeutic option.
gene encodes the trifunctional enzyme that mediates the CFD may be associated with autistic features in a subset
5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF of children with developmental regression, intellectual dis-
cyclohydrolase, and 10-formyl-THF synthetase reactions ability, epilepsy, dyskinesia, and autism (Moretti et al. 2008)
(Watkins et al. 2011). Homocysteine and methylmalonic acid (see Table 10.7). In some cases, CFD may be associated with
were increased. In vitro studies demonstrated decreased syn- the presence of blocking autoantibodies to folate receptor
thesis of methylcobalamin, required for the activity of methi- alpha (Ramaekers and Blau 2004). These patients may pres-
onine synthase. Serum folate was normal (Keller et al. 2013). ent in early childhood with generalized tonic-clonic seizures
A single patient with MTHFS gene mutation and with and develop intractable epilepsy that is refractory to treat-
frequent seizures and autistic features, unresponsive to ment with folinic acid (Bonkowsky et al. 2008). However,
folinic acid administration, was reported with very low CSF some of them may present in adolescence with schizophrenic
5MTHF (see Table 10.6). All other biochemical parameters symptoms and worsening catatonia (Ho et al. 2010).
10.2 Nomenclature

10.1 Hereditary folate Proton-coupled folate HFM SLC46A1 17q11.2 SLC46A1 transporter 229050 All forms
malabsorption transporter (PCFT)
deficiency
10.2 Folate receptor alpha Cerebral folate CFD FOLR1 11q13.4 FOLR1 transporter 613068 All forms
10.3 Methylenetetrahydrofolate Homocystinuria due MTHFR MTHFR 1p36.22 5,10-methylenetetrahydrofolate 236250 All forms
reductase deficiency to deficiency of MTHFR reductase
activity
10.4 Dihydrofolate reductase Megaloblastic anemia DHFRD DHFR 5q14.1 Dihydrofolate reductase 126060 All forms
deficiency due to DHFR deficiency
10.5 5,10-methylene- Trifunctional MTHFD1 MTHFD1 14q23.3 5,10-methylene- 172460 All forms
tetrahydrofolate dehydrogenase / tetrahydrofolate
dehydrogenase deficiency cyclohydrolase/ dehydrogenase/5,10-methenyl-
synthetase tetrahydrofolate
cyclohydrolase/formyl-
tetrahydrofolate synthase
10.6 5,10-methenyltetrahydrofolate 5-formyltetrahydrofolate MTHFS MTHFS 15q25.1 5,10-methenyltetrahydrofolate 604197 All forms
synthetase deficiency cycloligase synthetase
10.7 Cerebral folate deficiency Cerebral folate CFD – – Nongenetic – Idiopathic
due to FOLR1 autoantibodies deficiency
LIVER
FAICAR
dTMP
AICARTF THF CO2+THF
AICAR FTHFDH TS
FTHFI dUMP
10-Formyl-THF 5-Formyl-THF
FGAR 10.6 10.5

MTHFCH
THF 5,10-Methylene-THF
GARTF Folic acid
GAR BRAIN
10.3
5,10-Methenyl-THF 10.4
NH4+ FTHFS
10.2
FITHFCH DHF 5-Methyl-THF
5-Formimino-THF Formate 10.7
10.4
Folic
acid INTESTINE
THF
10.1
B12
Glu FIGLU
MS hCys
His Met
SHMT
Ser Gly
Fig. 10.1 Metabolic pathway; functions in purines, pyrimidines, formyl-5′-phosphoribosylglycinamide, GAR 5′-phosphoribosylglycin-
and amino acid metabolism and transport of folates in human body; amide, hCys homocysteine, THF tetrahydrofolate. Enzymes: AICART
and inherited metabolic disorders (in red) resulting in cerebral folate 5′-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase,
deficiency. Metabolites: AICAR 5′-phosphoribosyl-5-aminoimid- FITHFCH formimino-THF cyclodeaminase, FTHFDH formyl-THF
azole-4-carboxamide, DHF dihydrofolate, dTMP deoxythymidine dehydrogenase, FTHFI formyl-THF isomerase, FTHFS 10-formyl-
monophosphate, dUMP deoxyuridine monophosphate, FAICAR THF synthase, GARTF 5′-phosphoribosylglycinamide transformylase,
formyl-5′-phosphoribosyl-5-aminoimidazole-4-carboxamide, FGAR MTHFCH methenyl-THF cyclohydrolase
Table 10.1 Hereditary folate malabsorption

Cardiovascular Heart failure ±
CNS Mental retardation ±
Movement disorder ±
Neurologic deterioration + +
Neuropathy, peripheral ±
Seizures ± ±
Digestive Diarrhea ± ±
Failure to thrive ± ±
Stomatitis ± ±
Hematological Anemia, megaloblastic + +
Pancytopenia ± ±
Other Frequent infections + +
Routine laboratory Immunoglobulins ↓-n ↓-n
Special laboratory 5-Methyl-THF (CSF) ↓ ↓
Cobalamins (P) n
FIGLU (U) n-↑
Folates (S) ↓ ↓
Homocysteine (P) n-↑ n-↑
Table 10.2 Folate receptor alpha deficiency

CNS Ataxia + ++ +
Autistic spectrum disorder ± ± ±
Behavior, aggressive ± ± ±
Cerebral and cerebellar atrophy ± ± ±
Developmental regression + + ++
Gait disturbance ± ± ±
Hypertonia + ++ +
Hypomyelination (MRS) + + +
Movement disorder ±
Seizures + ++ ++
Seizures, myoclonic-astatic + ++ ++
Seizures, tonic-clonic ± ± ±
Tendon reflexes, increased ± ± ±
Tremor ± ± ±
Musculoskeletal Microcephaly ±
Routine laboratory EEG: abnormal + + +
Special laboratory 5-Methyl-THF (CSF) ↓↓↓ ↓↓↓ ↓-n
Choline (MRS) ↓ ↓ ↓
Inositol (MRS) ↓ ↓ ↓
Table 10.3 Methylenetetrahydrofolate reductase deficiency

CNS Apnea ± ± ± ±
Developmental regression + + + +
Gait disturbance ± ± ±
Hydrocephalus +
Mental retardation ± ± ±
Neuropathy, peripheral +
Psychiatric symptoms ±
Seizures, myoclonic, generalized + ± ± ±
tonic-clonic, and infantile spasms
Digestive Feeding difficulties +
Hematological Thromboembolic episodes ±
Musculoskeletal Microcephaly ± ± ±
Muscle weakness ± ± ±
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) ↓-n ↓-n ↓-n
5-Methyl-THF (CSF) ↓ ↓ ↓
Homocysteine (P) ↑ ↑ ↑
Homocysteine-cysteine (P) ↑ ↑ ↑
Homovanillic acid, HVA (CSF) ↓-n ↓-n ↓-n
Methionine (P) ↓-n ↓-n ↓-n
Table 10.4 Dihydrofolate reductase deficiency

CNS Epilepsy + +
Eye Eye movements, abnormal, ± ±
oculogyric crisis
Hematological Anemia, megaloblastic ++ ++
Pancytopenia + +
Routine laboratory Hemoglobin (B) ↓ ↓
LDH (P) ↑ ↑
Special laboratory 5-Hydroxyindoleacetic acid, ↓-n ↓-n
5HIAA (CSF)
5MTHF (CSF) ↓↓↓ ↓↓↓
BH4 (CSF) ↓-n ↓-n
FIGLU (U) n-↑ n-↑
Folic acid (B) ↓↓ ↓↓
Homovanillic acid, HVA (CSF) ↓-n ↓-n
Methylmalonic acid (U) n-↑ n-↑
Orotic acid (U) n-↑ n-↑
Table 10.5 5,10-methylene-tetrahydrofolate dehydrogenase deficiency

Hematological Anemia, megaloblastic +
Hemolytic uremic syndrome +
(atypical)
Immunodeficiency, +
severe combined (SCID)
Special laboratory Homocysteine, total (P) ↑
Methylcobalamin synthesis (FB) ↓
Methylmalonic acid (P) ↑
Folate (S) n
Table 10.6 5,10-methenyl-tetrahydrofolate synthetase deficiency

CNS IQ n mental retardation mental retardation
Seizures + + +
Speech delay + + +
Other Recurrent infections + +
Special laboratory 5MTHF (CSF) ↓↓
Table 10.7 Cerebral folate deficiency due to FOLR1 autoantibodies

CNS Ataxia + + +
Brain MRI: cerebellar ± ± ±
hypoplasia/cortical atrophy
Demyelination ± ± ±
Dyskinesia ± ± ±
Epilepsy ± ± ±
Retardation, psychomotor + + +
Sleep disturbances + + ±
Spastic paraplegia ± ± ±
Unrest + +
Ear Hearing loss ± ± ±
Eye Vision, impaired ± ± ±
Musculoskeletal Microcephaly + ± ±
Special laboratory 5MTHF (CSF) ↓ ↓ ↓
FOLR1 blocking ↑ ↑ ↑
autoantibodies (S)
Age 5MTHFa (CSF) nmol/L 5MTHFa (S) nmol/L

0–0.49 64–182 38–90
0.5–1.99 64–182 36–96
2–4.99 63–111 22–71
5–10.99 41–117 14–77
11–15.99 41–117 12–63
>16 41–90 10–39
a
HPLC with EC or fluorimetric detection
5MTHF 5MTHF (S) hCys (P) FIGLU (U) Clb (P) Inositol Choline 5HIAA,
(CSF) (MRS) (MRS) HVA (CSF)
10.1 ↓ ↓ n-↑ n-↑ n – – –
10.2 ↓↓↓ n – – – ↓ ↓ –
10.3 ↓↓ – ↑ – – – – ↓-n
10.4 ↓↓↓ ↓↓ – n-↑ – – – ↓-n
10.5 ↓↓ ↓ ↑ – – – – –
10.6 ↓↓ – – – – – – –
10.7 ↓ n – – – – – n
Test Precondition Material Handling Pitfalls

Cobalamins Plasma or serum Frozen (−20 °C)
Homocysteine Plasma Frozen (−20 °C) Immediate separation
Methionine from plasma essential
FIGLU Urine Frozen (−20 °C)
Folates Serum Frozen (−20 °C)
5MTHF CSF Frozen (−80 °C)
HVA, 5HIAA CSF Frozen (−80 °C)
BH4 CSF Frozen (−80 °C)
Add DTE +
Autoantibodies Serum Frozen (−20 °C)
blocking FOLR1
10.8 Prenatal Diagnosis and DNA Testing 10.9 Treatment
No data available for prenatal diagnosis. DNA testing Drugs and dosages to be used in acute/chronic presentations
possible for disorders 10.1–10.5 of suspected disorders of folate transport and metabolism
Disorder Folinic acid Betaine Hydroxocobalamin Methionine Vitamin B6

10.1 Hereditary folate Folinic acid parenterally – – – –
malabsorption (intravenous in acute
presentation and intramuscular
for chronic treatment) 4 mg/
day
10.2 Folate receptor Folinic acid orally 2–5 mg/kg/ – – – –
alpha deficiency day
100 mg intravenously or once
a week have been used
concomitantly with 5 mg per
kilo per day of oral folinic
acid
Preliminary results indicate
that intrathecal administration
may be more efficient than
oral administration. However,
no doses have been reported
(Grapp et al. 2012)
10.3 Methylene-THF Folinic acid orally 15 mg/day Betaine orally Hydroxocobalamin Methionine orally Vitamin B6
reductase deficiency 100–250 mg/kg/day in 0.5–1 mg orally 40–50 mg/kg/day orally 100–
neonates and infants. In or 1 mg IM monthly 250 mg/day
children and adults, 6–9 g/
day in three divided doses
with a maximum daily
dose of up to 20 g/day
10.4 Dihydrofolate Folinic acid orally 1 mg/kg/ – – –
reductase deficiency day
10.5 Methylene-THF L-methyl-THF 3 mg – – –
dehydrogenase compounded with 35 mg of
deficiency pyridoxal 5′-phosphate and
2 mg of methylcobalamin
given orally once a day
10.6 Methenyl-THF L-methyl-THF 3 mg
synthetase deficiency compounded with 35 mg of
pyridoxal 5′-phosphate and
2 mg of methylcobalamin
given orally once a day
10.7 Cerebral folate Folinic acid orally with a – – –
deficiency due to starting dose of 0.5 mg/kg/day
FOLR1 autoantibodies and increased to 1 mg/kg/day
Folic acid should not be used to treat these disorders since it is not a transport of folate across the choroid plexus. In addition, folate has to
physiologic form of folate and binds in an irreversible way to folate be converted by a series of enzymatic reactions to the biological active
receptor alpha, inhibiting the binding of 5-methylTHF and blocking the 5MTHF
Hereditary Folate Malabsorption. Folic acid should not be 5-formyl-THF is a racemic and stable form of folate found in
used to treat hereditary folate malabsorption. Folic acid is the human tissues. It can be used in oral formulation. The active
most common pharmacologic form of folate and very stable. L isomer (isovorin) may also be used for oral administra-
However, it is not a physiologic form of folate. It binds in tion. Most patients benefit from this treatment and respond
an irreversibly way to folate receptors and may block these within 2–6 months of therapy with reduced frequency of
receptors from transporting folate across the choroid plexus seizures, almost complete normalization of neurological sta-
(Kamen and Smith 2004). 5-formyl-tetrahydrofolate (5-for- tus, improved motor skills, and increase of the CSF 5MTHF
myl-THF), also known as folinic acid, is a racemic and stable concentration in the CSF (Steinfeld et al. 2009; Cario et al.
form of folate that is found in human tissues and can be used 2009; Grapp et al. 2012). In addition, after 6 months of treat-
in oral and parenteral formulations. The active L isomer may ment, the white matter choline and inositol signals normal-
also be used for parenteral administration. ized and signs of remyelination were observed on brain
The active isomer, L-5-methyltetrahydrofolate (L-5MTHF), MRI (Steinfeld et al. 2009). In one additional case of folate
is the principal folate in the diet, the form that is absorbed receptor alpha deficiency, a trial with 30 mg/day of folinic
in the intestine and circulating in the blood. It is avail- acid at the age of 26 months controlled the seizures com-
able commercially but only for oral and not parenteral pletely in 1 month and ameliorated the myoclonic jerks and
supplementation. choreic movements presented by the patient (Perez-Duenas
No controlled studies have been conducted in order to et al. 2010). In few patients, alternative routes of folinic acid
provide guidelines for optimal treatment. The required dose supplementation (intravenous and/or intrathecal) have been
of reduced folate seems to vary among patients. The dose of tried and the results indicate better treatment efficacy (Grapp
reduced folate needed to improve the neurological features is et al. 2012).
much higher than that needed to improve the hematological Methylenetetrahydrofolate Reductase Deficiency. The
problems. Dosing should be done and guided by its effect aim of the treatment is the decrease of homocysteine lev-
on reaching normal age ranges for trough CSF 5MTHF els by using the compound betaine. Betaine is a substrate
concentrations. of the enzyme betaine-homocysteine S-methyltransferase
Oral doses of 150–200 mg of 5-formyl-THF have been (BHMT) that catalyzes remethylation of homocysteine to
associated with good clinical outcomes (Geller et al. 2002). methionine in kidney and liver causing hypermethioninemia
An appropriate starting dose of a reduced folate in an infant in these patients. Betaine is administered orally and treat-
could be 50 mg or 10–15 mg/kg given daily as a single dose. ment is begun with 100 mg/kg/day. Doses may range from
Appropriate dosing of a reduced folate should be tailored to 100 to 250 mg/kg/day in neonates and infants. In children
achieve a normal trough CSF 5MTHF concentration for age. and adults doses can range to 6–9 g/day in two to six divided
The parenteral dose used to achieve adequate blood doses and the total daily dose can be increased up to 20 g/day
folate levels is lower than the oral dose. The hematological (Prasad et al. 2011). Since the expression of BHMT is low in
abnormalities have corrected with intramuscular injections the central nervous system, it is not surprising to find out that
of 1.0 mg/day of 5-formyl-THF. Nevertheless, the nor- the best therapeutic outcome with betaine has been observed
malization of CSF 5MTHF levels requires higher reduced when started prenatally or soon after birth with restoration of
folate doses. Dosing should be adjusted in each individual plasma SAM levels and attainment of normal brain growth
to achieve a CSF 5MTHF level normal for age (Grapp et al. and development (Strauss et al. 2007). Frequent dosing of
2012). orally administered betaine (six divided doses) normalized
Folate Receptor Alpha Deficiency. Since this is a rare total plasma homocysteine levels in one infant with MTHFR
autosomal recessive disorder, no controlled studies have deficiency (Ucar et al. 2010). Supplementation with methio-
been conducted in order to provide guidelines for optimal nine (40–50 mg/kg/day), folinic acid (5–30 mg/day), vita-
treatment. It has been presumed that alternative or residual min B6 (100–250 mg/day), and vitamin B12 (0.5–1 mg/day
transport pathways into the central nervous system can be orally) may be contemplated based on the fact that they play
used by increasing the plasma 5MTHF concentrations. Thus, a role as cofactors in different enzymatic reactions of the
most patients have been treated with folinic acid at oral doses folate utilization pathway that ultimately leads to the synthe-
of 2–5 mg per kg/body weight daily. Folic acid should not be sis of 5MTHF (Schiff et al. 2011).
used to treat folate receptor alpha deficiency since, as previ- Dihydrofolate Reductase Deficiency. Therapy with 1 mg/
ously stated, it is not a physiologic form of folate and binds kg per day of folinic acid corrects the biochemical abnor-
in an irreversible way to folate receptor alpha, inhibiting the malities including cerebral 5MTHF levels, total red blood
binding of 5MTHF to this receptor and blocking the trans- cell folate levels, anemia, and pancytopenia (Cario et al.
port of folate across the choroid plexus (Kamen and Smith 2011). The treatment can also improve school performance
2004). In addition, folate has to be converted by a series and control the epilepsy; however, irregular treatment
of enzymatic reactions to the biological active 5MTHF. with folinic acid is associated with recurrence of epilepsy
(Cario et al. 2011). In another report, treatment with 10–30 mg with cerebral folate deficiency (CFD) and congenital deafness with
of daily folinic acid corrected the cerebral 5MTHF level and labyrinthine aplasia, microtia and microdontia (LAMM). Mol Genet
Metab 104:362–368
improved the anemia and the seizure control (Banka et al. Fournier I, Ploye F, Cottet-Emard JM, Brun J, Claustrat B (2002)
2011). Folate deficiency alters melatonin secretion in rats. J Nutr
Methylene-THF Dehydrogenase Deficiency. No treatment 132:2781–2784
was initially reported in the only patient identified with this Geller J, Kronn D, Jayabose S, Sandoval C (2002) Hereditary folate
malabsorption: family report and review of the literature. Medicine
particular inborn error of metabolism (Watkins et al. 2011). (Baltimore) 81:51–68
However, the treatment was subsequently reported as con- Ghoshal K, Li X, Datta J, Bai S, Pogribny I, Pogribny M, Huang Y,
sisting of 3 mg of L-methyl-THF compounded with 35 mg of Young D, Jacob ST (2006) A folate- and methyl-deficient diet alters
pyridoxal 5′-phosphate and 2 mg of methylcobalamin given the expression of DNA methyltransferases and methyl CpG binding
proteins involved in epigenetic gene silencing in livers of F344 rats.
orally once a day. This treatment provided partial immune J Nutr 136:1522–1527
reconstitution (Keller et al. 2013). Goyette P, Sumner JS, Milos R, Duncan AM, Rosenblatt DS, Matthews
Methenyl-THF Synthetase Deficiency. No treatment has RG, Rozen R (1994) Human methylenetetrahydrofolate reductase:
been reported so far. isolation of cDNA mapping and mutation identification. Nat Genet
7:551
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Autoantibodies. Folinic acid is used with a starting dose of Steinfeld R (2012) Molecular characterization of folate receptor 1
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Vitamin B6-Dependent
and Responsive Disorders 11
Barbara Plecko, Eduard A. Struys, and Cornelis Jakobs
Contents Summary
11.1 Introduction ..................................................................... 179 The importance of vitamin B6 is evident by its role as the
most abundant cofactor in human metabolism. A total of six
11.2 Nomenclature ................................................................... 181
different B6 vitamers follow a complex pathway of absorp-
11.3 Metabolic Pathways ........................................................ 182 tion and transformation into the final active cofactor, pyri-
11.4 Signs and Symptoms ....................................................... 185 doxal 5′-phosphate (PLP), which catalyses over 100
reactions, mainly in amino acid and neurotransmitter metab-
11.5 Reference and Pathologic Values ................................... 186
olism. Over recent years a number of genetic defects have
11.6 Diagnostic Algorithm ...................................................... 187 been identified as the underlying cause of vitamin B6-
11.7 Specimen Collection ........................................................ 188 dependent epilepsies that need to be considered particularly
11.8 Prenatal Diagnosis ........................................................... 188 in neonatal, therapy-resistant seizures of unclear aetiology.
With diagnostic delay these disorders can be fatal or may
11.9 DNA Testing ..................................................................... 188
lead to irreversible brain damage. Therefore, a standardised
11.10 Treatment ......................................................................... 188 vitamin B6 trial should be part of a protocol for neonatal
References ..................................................................................... 189 seizures in every institution caring for the critically ill new-
born. The underlying mechanisms of vitamin B6-dependent
epilepsies can be assigned to either reduced production/
availability of PLP or to inactivation of PLP by accumulating
compounds and formation of a Knoevenagel product. The
disorders can be distinguished by specific biomarkers in
urine, plasma or CSF and confirmed by molecular testing.
Affected patients need a lifelong oral treatment with
pyridoxine or pyridoxal 5′-phosphate and withdrawal will
inevitably lead to recurrence of seizure. Due to autosomal
recessive inheritance, recurrence risk for all disorders dis-
cussed here is 25 % and intrauterine treatment with vitamin
B6 administered to the mother from early pregnancy may be
B. Plecko (*) considered in forthcoming pregnancies. Prenatal testing is
Department of Pediatric Neurology,
available by molecular analysis.
Children’s Hospital, University of Zürich,
Steinwiesstrasse 75, Zürich 8032, Switzerland
e-mail: barbara.plecko@kispi.uzh.ch
E.A. Struys 11.1 Introduction
Metabolic Unit, Clinical Chemistry, VUmc Medical Center,
De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands Vitamin B6 is a water-soluble vitamin derived from various
e-mail: e.struys@vumc.nl
animal and plant food sources as well as intestinal bacterial
C. Jakobs flora in six different vitamers: pyridoxal, pyridoxine-
Department Clinical Chemistry, PK 1X 014,
glucoside, pyridoxamine and their 5′-phosphorylated esters.
VU University Medical Center Amsterdam,
De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands Pyridoxal 5′-phosphate is the only active cofactor and catal-
e-mail: c.jakobs@vumc.nl yses over 100 enzymatic reactions, mainly in amino acid
180 B. Plecko et al.
metabolism (e.g. the glycine cleavage system, serine dehy- refractory to common anticonvulsants with a high tendency
dratase, threonine dehydratase) as well as in neurotransmit- towards status epilepticus. Partial response to phenobarbitone
ter metabolism (e.g. the glutamate decarboxylase and is possible. Cranial imaging may be normal or may show some
aromatic acid decarboxylase/AADC). The demand of grey and white matter atrophy, hydrocephalus, thin corpus cal-
vitamin B6 is 0.1–0.3 mg/day during infancy and around losum mega cisterna magna and also cortical dysplasia. About
1.2–1.4 mg/day in adulthood. As cellular uptake and trans- 30 % of PDE patients suffer from birth asphyxia or may have
port across the blood–brain barrier is only possible for free signs of unclear encephalopathy. Abdominal distension with
bases of vitamin B6, phosphorylated vitamers undergo bile-stained vomiting, elevated lactate or hypoglycaemia may
hydrolysation by intestinal phosphatases and tissue non-spe- be confusing confounders. Atypical presentations with onset
cific alkaline phosphatase (TNSALP), respectively. The lat- of seizures up to 3 years of age, initial response to common
ter is attached to the cellular membrane by a PIGV anchor anticonvulsants or response to extremely low dose of pyridox-
system. Within cells pyridoxine, pyridoxamine and pyri- ine have been observed. Most cases of PDE are caused by
doxal undergo rephosphorylation by pyridoxal kinase with antiquitin deficiency (Mills et al. 2006; Plecko et al. 2007).
consecutive oxidation of pyridoxine 5′-phosphate and pyri- The antiquitin gene is located on 5q31 and encodes alpha-
doxamine 5′-phosphate into pyridoxal 5′-phosphate (PLP) aminoadipic semialdehyde dehydrogenase, an enzyme
by pyridox(am)ine 5′-phosphate oxidase (PNPO) (Fig. 11.1). involved in the L-lysine degradation pathway (Plecko et al.
Though the liver seems to play an important role in the for- 2000) (Fig. 11.2). The accumulating compound alpha-amino-
mation of PLP, expression of PNPO has also been shown in adipic semialdehyde (AASA) is in equilibrium with L-Δ1-
human intestinal and muscle cells, as well as in neurons. piperideine-6-carboxylate (P6C). The latter inactivates
The role of vitamin B6 in epileptogenesis has been well pyridoxal 5′-phosphate by a so-called Knoevenagel condensa-
recognised by the occurrence of seizures in nutritional defi- tion, leading to profound cerebral PLP deficiency (Footitt
ciency as well as in genetic disorders. Nutritional vitamin B6 et al. 2011). Though the majority of PDE cases are caused by
deficiency is rare nowadays, eventually seen in children with antiquitin deficiency, linkage studies support genetic heteroge-
severe chronic disease or on high dosages of the tuberculo- neity (Bennett et al. 2005). It may therefore become feasible,
static isoniazid and cured by adequate vitamin supply. to distinguish antiquitin deficiency from PDE of unclear aeti-
In contrast, genetically determined vitamin B6-dependent ology. Diagnosis is confirmed by molecular analysis of the
epilepsies are caused by inborn errors of metabolism antiquitin also known as ALDH7A1 gene.
(Clayton 2006). To date, four different disorders are known In 2009, folinic acid-responsive seizures were found to be
that can be assigned to two different mechanisms: inactiva- allelic to antiquitin deficiency (Gallagher et al. 2009). The effect
tion of PLP in pyridoxine-dependent epilepsy (PDE) due to of folinic acid is not understood to date and may be important in
antiquitin deficiency, and in hyperprolinaemia type II, and to the interruption of initial status epilepticus, if the initial response
reduced synthesis or availability of PLP in pyridoxal to pyridoxine is limited. Inheritance of PDE is autosomal reces-
5′-phosphate-dependent epilepsy and congenital hypophos- sive with a 25 % recurrence risk for PDE in forthcoming preg-
phatasia (Clayton and Plecko 2008; Plecko and Stöckler nancies. Outcome of patients with PDE is variable, and about
2009). Affected patients have a lifelong need for pharmaco- 70 % have some degree of cognitive impairment (Plecko et al.
logical doses of vitamin B6 with recurrence of seizures upon 2005; Mills et al. 2010; Bok et al. 2012). Normal IQ has been
withdrawal. Aside from hyperprolinaemia type II, which has described in single patients despite prolonged status epilepticus
a somewhat later onset, pyridoxine as well as pyridoxal (Plecko et al. 2005). Patients, who never received pyridoxine
5′-phosphate-dependent epilepsies usually manifest in the experience high mortality, as shown by a considerable rate of
neonatal period. Patients with congenital hypophosphatasia deceased siblings diagnosed retrospectively as well as some
may manifest seizures before the life-determining impact of patients who had been on folinic acid monotherapy, before the
bone hypomineralisation becomes obvious. Specific bio- underlying genetic defect of folinic acid-responsive seizures as
markers serve in the differentiation of the four disorders and an allelic condition had been identified.
exceed the diagnostic value of a vitamin B6 trial, which at Hyperprolinaemia Type II (HP II). This inborn error has
first may give ambiguous response. While administration of first been described in Irish travellers, with primary gener-
PLP would serve all four disorders, pyridoxine is ineffective alised seizures in late infancy or childhood in 50 % of
in most cases of PNPO deficiency. In neonates with therapy- affected individuals. Mental retardation may be present, but
resistant seizures, a standardised vitamin B6 trial and diag- several individuals were reported with normal school perfor-
nostic work-up should be performed in parallel, as diagnostic mance (Flynn et al. 1989). In many patients seizures are trig-
delay leads to irreversible brain damage. gered by fever and are relatively benign and may be controlled
Pyridoxine-Dependent Epilepsy (PDE). Patients with PDE by common anticonvulsants. Thus, several patients with this
usually present with seizures soon after birth (Baxter 2003; autosomal recessive disorder may remain undiagnosed. Until
Gospe 2006; Stockler et al. 2011). Seizures are myoclonic, now only single case reports have been described outside the
tonic or clonic with variable EEG patterns. They are typically Irish Traveller Community.
11 Vitamin B6-Dependent and Responsive Disorders 181
HP II is caused by defective Δ1-pyrroline 5-carboxylate before the first administration of PLP. Recent studies have
dehydrogenase, leading to increased utilisation of PLP. In shown, that low PLP concentrations in CSF are unspecific and
analogy to the pathophysiologic mechanism in PDE, L-D1- do not allow distinction between antiquitin and PNPO defi-
pyrroline-5-carboxylate (P5C) inactivates PLP by a ciency. Diagnosis has to be confirmed by molecular analysis
Knoevenagel condensation (Farrant et al. 2001). Patients of the PNPO gene, prenatal diagnosis is possible.
show high proline on plasma amino acid analysis as well as A variety of secondary biochemical findings has been
elevated P5C in urine, plasma and CSF. described in patients with PNPO as well as antiquitin defi-
Pyridoxal 5′-Phosphate (PLP)-Dependent Epilepsy ciency. These include lactic acidosis, elevated threonine and
(PNPO) Deficiency. The clinical presentation is indistin- glycine in plasma or CSF and abnormal metabolites of bio-
guishable from PDE except for a higher rate of prematurity. genic amines in CSF, as low homovanillic acid, low hydroxy-
Again, about a third of patients had perinatal distress with indole acetic acid, increased 3-methoxytyrosine and high
low APGAR scores, requiring primary intubation in some. vanillactate in urine. These secondary findings are all
All patients reported to date presented with neonatal myo- explained by impaired function of PLP-dependent enzymes
clonic seizures up to 2 weeks of age, sometimes accompa- in amino acid or neurotransmitter metabolism. They are nev-
nied by roving eye movements and most often severely ertheless inconsistent and non-specific and may be found in
abnormal EEG (Mills et al. 2005; Hoffmann et al. 2007). all circumstances of (cerebral) PLP depletion.
Cranial imaging may be normal or show marked white mat- Congenital Hypophosphatasia. The main impact of con-
ter changes. Patients with PNPO deficiency may suffer from genital hypophosphatasia is poor bone mineralisation and
systemic comorbidity as anaemia, coagulopathy, hypogly- early death due to respiratory insufficiency. Single patients
caemia and lactic acidosis, renal dysfunction as well as fail- with the severe, congenital form of this disease may present
ure to thrive. Many patients had deceased siblings with a with therapy-resistant seizures from birth before skeletal
neonatal epileptic encephalopathy and burst suppression signs become more prominent (Balasubramaniam et al.
EEG, falsely assigned to Ohtahara syndrome. Prognosis 2010). EEG is usually severely abnormal and may show a
relies on early initiation of specific treatment. burst suppression pattern. While seizures are resistant to
Pyridoxal 5′-phosphate-dependent epilepsy is caused by common anticonvulsants, they are easily controlled by oral
autosomal recessive mutations in the PNPO gene (Mills or i.v. administration of pyridoxine.
et al. 2005). This gene encodes pyridox(am)ine 5′-phos- The autosomal recessive defect of tissue non-specific
phate oxidase, an enzyme needed for the oxidation of pyri- alkaline phosphatase (TNSALP) leads to reduced intracel-
doxine and pyridoxamine into the only active vitamin B6 lular uptake and availability of PLP, while plasma levels of
cofactor, which is pyridoxal 5′-phosphate (PLP). This PLP are (extremely) high. TNSALP is needed for calcium
enzyme is expressed in liver but most probably also within uptake and bone mineralisation and lack of TNSALP leads
brain cells and may also play a role in intracellular recycling to increased serum calcium, low serum phosphate levels and
and trafficking of PLP. severe osteomalacia.
In contrast to PDE, patients with PNPO deficiency suffer Diagnosis is established by markedly low alkaline phos-
from systemic PLP deficiency, which may explain the broader phatase in serum and confirmed by molecular analysis of the
organ involvement and very high mortality in untreated ALPL gene.
patients. Diagnosis of PNPO deficiency is established by mea-
surement of low PLP levels in plasma and CSF (Footitt et al.
2013). Unlike in PDE, diagnostic samples have to be saved 11.2 Nomenclature
Chromosomal Affected
No. Disorder Alternative name Abbreviation Gene symbol localisation protein OMIM no. Subtype
11.1 Pyridoxine Alpha-amino adipic AASADHD ALDH7A1 5q31 Alpha-amino 266100 All forms
dependent semialdehyde (AASA) adipic
epilepsy (PDE) dehydrogenase semialdehyde
11.2 Pyridox(am)ine Pyridoxal 5′-phosphate PNPO PNPO 17q21.32 Pyridox(am) 610090 All forms
5′-phosphate (PLP)-dependent deficiency ine
oxidase deficiency seizures 5′-phosphate
oxidase
11.3 Hyperprolinaemia Pyrroline-5-carboxylate P5CDH ALDH4A1 1p36.13 Pyrroline-5- 239510 All forms
type II dehydrogenase carboxylate
11.4 Congenital Phosphoethanolaminuria HOPS ALPL 1p36.12 Alkaline 241500 All forms
hypophosphatasia phosphatase
Vitamin B6 Metabolism
Pyridoxal-P Pyridoxamine-P Pyridoxine-glucoside

Diet
IP IP
Absorption Pyridoxal Pyridoxamine Pyridoxine
PK PK PK
Hepatic
Pyridoxamine-P Pyridoxine-P
Metabolism
PNPO
Blood Pyridoxal-P
Cell membrane TNSALP PIGV anchor
Pyridoxal Pyridoxamine Pyridoxine
PK PK PK
Cell Pyridoxamine-P Pyridoxine-P
PNPO
Pyridoxal-P
Fig. 11.1 Vitamin B6 is absorbed in different vitamers that are dephos- across the blood–brain barrier, phosphorylated vitamers (mainly PLP)
phorylated by intestinal alkaline phosphatases (IP). In the liver they are are hydrolysed by tissue non-specific alkaline phosphatase (TNSALP).
then rephosphorylated to their 5′-phosphate esters by phosphate kinase Within the (brain) cell, rephosphorylation of PL as the major source and
(PK) and pyridox(am)ine is consequently converted into pyridoxal oxidation of pyridox(am)ine by PNPO provides PLP as the only active
5′-phosphate (PLP) by pyridoxamine 5′-phosphate oxidase (PNPO) cofactor for intracellular enzyme reactions. Pyridoxal 5′-phosphate
and partly released into circulation. For cellular uptake or transport exhibits feedback inhibition upon PNPO activity
Lysine Degradation Pathway
L-lysine
H 2N COOH
NH2
H2N COOH
2-keto 6-amino caproic acid
O
HOOC
COOH
piperideine-2-carboxylate N COOH N
H NH2
saccharopine
HOOC
N COOH
pipecolic acid
O COOH
N COOH
piperideine-6-carboxylate NH2
alpha aminoadipic semialdehyde
COOH
HOOC
NH2
alpha aminoadipic acid
Fig. 11.2 Lysine degradation pathway. Antiquitin deficiency leads to Piperideine-6-carboxylate inactivates PLP by a Knoevenagel condensa-
accumulation of alpha-aminoadipic semialdehyde, piperideine- tion, leading to cerebral PLP deficiency
6-carboxylate and is accompanied by elevated pipecolic acid.
Hyperprolinaemia Type II
Fig. 11.3 Catabolism of L-proline. In hyperprolinaemia l-proline

type II, deficiency of Δ1-pyrroline-5-carboxylate (P5C)
dehydrogenase leads to accumulation of glutamic acid γ
semialdehyde and P5C. P5C inactivates PLP by a
N COOH
Knoevenagel condensation
glutamic semialdehyde
P5C NH2
spontaneous
O COOH
N COOH
P5C dehydrogenase
NH2 glutamic acid
HOOC COOH
Scheme of All Four Defects Causing Vitamin

B6-Responsive Seizures
Diet
Pyridoxal (5‘-P) Pyridoxamine (5‘-P) Pyridoxine Glucoside
Liver PNPO PNPO

Fig. 11.4 Scheme illustrating all
four inborn errors leading to
vitamin B6-dependent epilepsy:
Two inborn errors within vitamin PLP
B6 metabolism leading to reduced L-Proline L-Lysine
PIGV TNSAP
synthesis of pyridoxal 5′-phosphate
(PLP): pyridox(am)ine
5′-phosphate oxidase (PNPO)
deficiency and tissue non-specific PL
alkaline phosphatase (TNSALP)
deficiency in congenital P5C PL P6C
Pi n
hypophosphatasia, and two inborn na tio
ctiv c tiva
ati
on ina
errors leading to inactivation of PLP
PLP
PLP by a Knoevenagel P5C-Dehydrogenase AASA
condensation, hyperprolinaemia Dehydrogenase
type II (P5C dehydrogenase
deficiency) and antiquitin (alpha-
AASA dehydrogenase) deficiency
Table 11.1 Pyridoxine-dependent epilepsy (PDE)

CNS Agenesis/hypogenesis, corpus callosum +-n +-n +-n +-n +-n
Developmental delay n n-↑ n-↑ n-↑ n-↑
Hypotonia n-+ n-+ n-+ n-+ n-+
Megacisterna magna n-+ n-+ n-+ n-+ n-+
Seizures, pharmacoresistant +++ ++ ++ n n
Digestive Intestinal pseudo obstruction n-+(++) n n n n
Vomiting n-+(++) n-+ n n n
Other Low APGAR scores +(++)-n n n n n
Routine Glucose (P) ↓-n n n n n
laboratory Lactate (P) n-↑ n n n n
Special Alpha-aminosemialdehyde pretreatment (U, P, CSF) ↑↑↑ ↑↑↑ ↑↑↑
laboratory Alpha-aminosemialdehyde under B6 treatment (U, P, CSF) ↑↑ ↑(↑↑) ↑ ↑ ↑
PA pretreatment (U, P, CSF) ↑↑↑ ↑↑↑ ↑↑↑
PA under B6 treatment (CSF) ↑↑↑ ↑↑ ↑ ↑
PA under B6 treatment (P) ↑↑ ↑ ↑ n-↑ n-↑
PA under B6 treatment (U) ↑↑↑ n-↑ n n n
PLP (CSF) ↓↓↓ ↓↓↓
Table 11.2 Pyridox(am)ine 5′-phosphate oxidase deficiency

CNS Developmental delay ++ + - +++ + - +++
Hypotonia ++ ++ ±
Seizures, pharmacoresistant +++ ++ ++
Digestive Vomiting n-+(++) n-+ n n n
Other Low APGAR scores +(++)-n n n n n
Prematurity +++ − − − −
Routine laboratory Glucose (P) ↓-n n n n n
Lactate (P) n-↑ n n n n
Special laboratory 3-methoxytyrosine (CSF) ↑↑ ↑↑
5-hydroxyindoleacetic acid, 5-HIAA (CSF) ↓ ↓
Homovanillic acid, HVA (CSF) ↓-n ↓-n
PLP (CSF) ↓↓ ↓↓
Threonine (CSF) n-↑ n-↑
Threonine (P) n-↑ n-↑
Vanillinlactic acid (U) ± ±
Table 11.3 Hyperprolinaemia type II

CNS Mental retardation n ± ± ± ±
Seizures, febrile n ± ± n n
Seizures, pharmacoresistant n ± ± ± ±
Special laboratory Hydroxyproline (P) n n n n n
Hydroxyproline (U) n-↑↑↑ n-↑↑↑ n-↑↑↑ n-↑↑↑ n-↑↑↑
P5C (U) + + + + +
Proline (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Proline (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 11.4 Congenital hypophosphatasia

CNS Seizures n-+++ n-+++ n n n
Respiratory Respiratory failure ++ ++ + n n
Other Skeletal hypomineralisation +++ +++ ++ ++ ++
Routine laboratory Alkaline phosphatase (P) ↓↓↓ ↓↓↓ ↓↓ ↓↓ ↓↓
Ca++ (P) ↑↑ ↑↑ ↑↑ ↑-↑↑ ↑-↑↑
Phosphate (P) ↓↓ ↓↓ ↓ ↓ ↓
Special laboratory PLP (P) ↑↑↑ ↑↑↑ ↑↑ ↑↑ ↑↑
11.5 Reference and Pathologic Values
Pyridoxine-Dependent Epilepsy
Reference values
Alpha-AASA (U) Alpha-AASA (P, CSF) Pipecolic acid (P) Pipecolic acid (CSF)
Age mmol/mol creatinine μmol/L μmol/L μmol/L
<0.5 year <2 <0.1 <1 week 0.55–10.8 All ages: 0.009–0.12
>0.5 <1 year <1 <0.1 >1 week 0.54–2.46
>1 year <0.5 <0.1
Pathological values
Alpha-AASA (U) Alpha-AASA (P, CSF) Pipecolic acid (P) Pipecolic acid (CSF)
Age mmol/mol creatinine μmol/L μmol/L μmol/L
<0.5 year >3–90 All ages: All ages: All ages:
>0.5 <1 year >2–40 0.5–25 (P) 2.7–18 1.4–14
>1 year >2–20 1–15 (CSF)
Hyperprolinaemia Type II
Reference values
Proline Hydroxyproline P5C-glycine conjugate (U)
All ages 50–400 (P) (μM) All ages Trace-90 (P) (μM) Not detectable
Age Age
0–6 months Trace-190 (U) 0–6 months Trace-400 (U)
>6 months Trace-18 (U) >6 months Trace-50 (U)
>1 year Trace-10(U)
Plasma values in μM; urinary values in mmol/mol creatinine
Pathological values
Proline Hydroxyproline P5C-glycine conjugate (U)
All ages 500–3,700 (P) All ages 1–46 (P) Present
2,100–40,125 (U) 84–3,769 (U)
Plasma values in μM; urinary values in mmol/mol creatinine
Pyridoxal 5′-Phosphate (PLP)-Dependent Epilepsy
Reference values
PLP (CSF) HVA (CSF) 5-HIAA (CSF) 3-methoxytyrosine (3-OMD) Threonine (CSF) Threonine (P)
nM nM nM nM μM μM VLA (U)
49–89 300–1,100 200–600 <300 10–45 70–220 Nd
Please note that the reference values are age dependent, and those depicted here of informative nature
Pathological values
PLP (CSF) HVA (CSF) 5-HIAA (CSF) 3-methoxytyrosine (3-OMD) Threonine (CSF) Threonine (P)
nM nM nM nM μM μM VLA (U)
<20 150–500 117–193 885–5,600 34–242 53–484 Present
Congenital Hypophosphatasia
Reference values
Serum alkaline phosphatase PLP (P)
(I.U.) nM
80–340 10–100
Pathological values
Serum alkaline phosphatase PLP (P)
(I.U.) nM
<30 200–20,000
11.6 Diagnostic Algorithm
neonatal seizures of unknown etiology
Diagnostic Algorithm for Neonatal Seizures

neonatal seizures of unknown etiology
“resistance” to first line drug (eg. phb, benzodiazepins)
save serum, urine, evt. CSF (freeze at -80°)
pyridoxine, 100 mg iv. followed by 30 mg/kg/day in 2-3 SD over 1-3 days
if ineffective add on of folinic acid 3-5 mg/kg/day in 1-2 SD
Switch to PLP 30 to 60 mg/kg/day in 3-4 SD over 3 days

(may need adjustment for break through seizures)
if seizures stop, continue pyridoxine or PLP until results are available
Fig. 11.5 Diagnostic algorithm for neonatal seizures

11.7 Specimen Collection 11.10 Treatment
Urine is the best material to test for AASA. As AASA and Established Treatment Options
P6C are quite unstable, samples should be frozen immedi- A standardised vitamin B6 trial should be performed in every
ately and kept at −20 °C until analysis (Struys et al. 2012). neonate with therapy-resistant seizures of unclear aetiology.
AASA and P6C may be secondarily elevated in patients While PLP is effective in all four inborn errors discussed in this
with molybdenum cofactor deficiency and isolated sulfite chapter, pyridoxine would only benefit three, while it is ineffec-
oxidase deficiency, most probably due to inhibition of the tive in most cases of PNPO deficiency. Only recently a new
alpha-aminoadipic semialdehyde dehydrogenase by sul- subgroup of patients harbouring pyridoxine responsive PNPO
fite toxicity. Cerebrospinal fluid specimens should be free mutations has been described (Plecko et al. 2013). The compa-
of blood contamination. In those with blood contamina- rably higher incidence of antiquitin deficiency and availability
tion, a centrifugal step at low speed can be performed to of pyridoxine as a licensed drug are in favour of pyridoxine for
pellet the erythrocytes. For measurement of vitamin B6 first line use. PLP is available as a chemical compound for oral
vitamers, blood samples should be centrifuged immedi- application. Figure 11.5 illustrates a diagnostic and treatment
ately and plasma as well as CSF samples be protected algorithm for neonatal seizures. As severe apnea may occur in
from light and stored at −80° until analysis (Albersen responders along a first vitamin B6 trial, resuscitation equip-
et al. 2012). ment should be at hand. In any case, determination of respec-
tive biomarkers should be performed in parallel and will
differentiate among the different disorders. In the presence of
11.8 Prenatal Diagnosis diagnostic biomarkers, a diagnostic vitamin B6 withdrawal is
contraindicated and treatment with pharmacological doses of
For all entities discussed in this chapter prenatal diagnosis is pyridoxine or PLP has to be continued lifelong. As long-term
feasible by molecular analysis of the respective gene in cho- administration of PLP has lead to liver fibrosis in single patients
rionic villi or amniotic fluid and should be accompanied by with PNPO deficiency, pyridoxine may be the preferred vita-
genetic counseling. mer wherever possible. Future studies on vitamer concentra-
tions in CSF may help to optimise dosages of pyridoxine or
PLP in the individual patient.
11.9 DNA Testing In the classical case of neonatal PDE, administration of
pyridoxine, 50–100 mg i.v. or p.o. results in cessation of sei-
DNA testing for all entities discussed in this chapter is avail- zures within minutes, though some patients may show
able in specialised laboratories and some commercial institu- ambiguous response (Mills et al. 2010). Pyridoxine mono-
tions. In antiquitin deficiency patients may harbour deletions therapy should be tempted, once a clear pyridoxine effect has
and may need MLPA analysis in the presence of normal been observed. Dosages of 30 mg/kg/day up to a maximum
sequencing of the ALDH7A1 gene. of 200–300 mg of pyridoxine per day, divided into two to
SELECTIVE SCREENING FOR VITAMIN B6

DEPENDENT SEIZURES
URINE PLASMA CSF Gene
PLPdep.E. (vanillactate) (PLP level) PLP level, AA PNPO

Fig. 11.6 Selective screening for AA
vitamin B6 dependent seizures. Plp
dep. E pyridoxalphosphate
dependent epilepsy, AA Cong. HP Alk Phosph, Ca, Ph, ALPL
aminoacids, HP hypophosphatasis,
AASA alpha aminoadipic
semialdehyde, PA pipecolic acid, ALDH7A1 AASA, P6C (PA) AASA, PA (AASA, PA) ALDH7A1
L-Δ1 piperideine-6-carboxylate HP
(peak X)
II hyperprolinemia type II, P5C Δ1
pyrolline-5-carboxylate. Red ovals
mark the best biomarker/specimen HP II AA, P5C AA, P5C (AA, P5C) ALDH4A1
for the respective entity
three single doses (SD) seem to establish seizure freedom in Baxter P (2003) Pyridoxine-dependent seizures: a clinical and bio-
most patients and prevent from sensory neuropathy observed chemical conundrum. Biochim Biophys Acta 1647:36–41
Bennett CL, Huynh HM, Chance PF, Glass IA, Gospe SM Jr (2005)
with dosages as high as 1 g/day. In patients with break- Genetic heterogeneity for autosomal recessive pyridoxine-
through seizures during infectious illness, the dosage should dependent seizures. Neurogenetics 6:143–149
be doubled during subsequent episodes of fever (Stockler Bok LA, Been JV, Struys EA, Jakobs C, Rijper EA, Willemsen MA
et al. 2011). This treatment algorithm can also be applied for (2010) Antenatal treatment in two Dutch families with pyridoxine-
dependent seizures. Eur J Pediatr 169(3):297–303
HPII and congenital hypophosphatasia. Bok LA, Halbertsma FJ, Houterman S, Wevers RA, Vreeswijk C,
Add-on of folinic acid 3–5 mg/kg/day may have addi- Jakobs C, Struys EA, Van Der Hoeven JH, Sival DA, Willemsen
tional benefit in neonates with antiquitin deficiency and MA (2012) Long-term outcome in pyridoxine-dependent epilepsy.
incomplete pyridoxine response (Gallagher et al. 2009; Dev Med Child Neurol 54:849–854
Clayton PT (2006) B6-responsive disorders: a model of vitamin depen-
Stockler et al. 2011) though the underlying mechanisms are dency. J Inherit Metab Dis 29:317–326, Review
still not understood. Clayton P, Plecko B (2008) Pyridoxine- and pyridoxalphosphate-
Patients with PNPO deficiency are more difficult to treat dependent epilepsies. In: 40th European Metabolic Group meet-
and will need 30–60 mg/kg/day of PLP given in 4–6 SD. As ing, Heidelberg. Milupa Metabolics GmbH, Friedrichsdorf,
Germany. pp 31–40. ISBN 978-3-9811868-1-9
PLP is rapidly oxidised, oral suspension should be prepared Farrant RD, Walker V, Mills G, Mellor JM, Langley GJ (2001) Pyridoxal
immediately before application (Peter Clayton 2012, per- phosphate de-activation by pyrroline-5-carboxylic acid. J Biol
sonal communication). Chem 276:15107–15116
Flynn MP, Martin MC, Moore PT, Stafford JA, Fleming GA, Phang JM
(1989) Type II hyperprolinaemia in a pedigree of Irish travellers
Experimental Therapies (nomads). Arch Dis Child 64:1699–1707
In antiquitin deficiency, a first report on lysine-restricted diet Footitt EJ, Heales SJ, Mills PB, Allen GF, Oppenheim M, Clayton PT
in addition to pyridoxine treatment has shown a decline of (2011) Pyridoxal 5’-phosphate in cerebrospinal fluid; factors affect-
biomarkers but lack of normalisation of AASA in CSF (van ing concentration. J Inherit Metab Dis
Footitt EJ, Clayton PT, Mills K, Heales SJ, Neergheen V, Oppenheim
Karnebeek et al. 2012). M, Mills PB (2013) Measurement of plasma B(6) vitamer profiles in
Intrauterine treatment with pyridoxine, 100 mg/day given children with inborn errors of vitamin B6 metabolism using an
from early pregnancy, may benefit offsprings of forthcoming LC-MS/MS method. J Inherit Metab Dis 36(1):139–145
pregnancies in families with antiquitin deficiency (Bok et al. Gallagher RC, Van Hove JL, Scharer G, Hyland K, Plecko B, Waters PJ,
Mercimek-Mahmutoglu S, Stockler-Ipsiroglu S, Salomons GS,
2010). In these offsprings, molecular and biochemical con- Rosenberg EH, Struys EA, Jakobs C (2009) Folinic acid-responsive
firmation should be sought immediately after birth to prevent seizures are caused by α-amino adipic semialdehyde dehydrogenase
potential neuronal pyridoxine toxicity in unaffected new- deficiency and are genetically identical to pyridoxine-dependent
borns (Hartmann et al. 2011). epilepsy. Ann Neurol 65:550–556
Gospe S (2006) Pyridoxine dependent seizures: new genetic and bio-
The rational would also argue for administration of PLP chemical clues to help with diagnosis and treatment. Curr Opin
in pregnancies at risk for PNPO deficiency, but no data have Neurol 19:148–153
been published so far. Hartmann H, Fingerhut M, Jakobs C, Plecko B (2011) Status epilepti-
As PNPO is a riboflavin-dependent enzyme, mutations cus in a neonate treated with pyridoxine because of a familial recur-
rence risk for antiquitin deficiency: pyridoxine toxicity? Dev Med
with residual activity may benefit from the administration of Child Neurol 53:1150–1153
riboflavin in addition to PLP, but so far no clinical experience Hoffmann GF, Schmitt B, Windfuhr M, Wagner N, Strehl H, Bagci S,
on this hypothesis has been reported. Franz AR, Mills PB, Clayton PT, Baumgartner MR, Steinmann B,
For congenital hypophosphatasia, recombinant enzyme Bast T, Wolf NI, Zschocke J (2007) Pyridoxal 5′-phosphate may be
curative in early-onset epileptic encephalopathy. J Inherit Metab Dis
replacement therapy may become available in near future. 30:96–99
Mills PB, Surtees RA, Champion MP, Beesley CE, Dalton N, Scambler
PJ, Heales SJ, Briddon A, Scheimberg I, Hoffmann GF, Zschocke J,
Clayton PT (2005) Neonatal epileptic encephalopathy caused by
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NM (2012) Vitamin B6 vitamer concentrations in cerebrospinal Clayton PT (2006) Mutations in the antiquitin (ALDH7A1) gene in
fluid differ between preterm and term newborn infants. Pediatrics patients with pyridoxine-dependent seizures. Nat Med 12:307–309
130(1):e191–e198 Mills PB, Footitt EJ, Mills KA, Tuschl K, Aylett S, Varadkar S,
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presenting as neonatal epileptic encephalopathy with abnormal neu- MG, Hughes MI, Struys EA, Jakobs C, Zuberi SM, Clayton PT
rotransmitter metabolism secondary to reduced co-factor pyridoxal- (2010) Genotypic and phenotypic spectrum of pyridoxine-
5′-phosphate availability. J Inherit Metab Dis 33(Suppl 3):25–33 dependent epilepsy (ALDH7A1 deficiency). Brain 133:2148–2159
Plecko B, Stöckler S (2009) Vitamin B6 dependent seizures. Can J Stockler S, Plecko B, Gospe SM, Coulter-Mackie M, Connolly M,
Neurol Sci 36(Suppl 2):73–77 van Karnebeek C, Mercimek-Mahmutoglu S, Hartmann H,
Plecko B, Stöckler-Ipsiroglu S, Paschke E, Erwa W, Struys EA, Jakobs C Scharer G, Struys E, Tein I, Jakobs C, Clayton P, Van Hove JL
(2000) Pipecolic acid elevation in plasma and cerebrospinal fluid of two (2011) Pyridoxine dependent epilepsy and antiquitin deficiency:
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Plecko B, Hikel C, Korenke GC, Schmitt B, Baumgartner M, Baumeister diagnosis, treatment and follow-up. Mol Genet Metab
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Molybdenum Cofactor Disorders
12
Günter Schwarz and Alex Veldman
Contents Summary
12.1 Introduction.................................................................... 192 Molybdenum (Mo) cofactor deficiency (MoCD) is
characterized by neonatal seizures, high-pitch crying,
12.2 Nomenclature ................................................................. 193
convulsions, and abnormal EEG and MRI findings accom-
12.3 Metabolic Pathway ........................................................ 193 panied by rapidly progressing neurodegeneration. In the
12.4 Signs and Symptoms ...................................................... 197 absence of treatment, patients usually die within the first
12.5 Reference and Pathological Values............................... 199
years of life and show no neurodevelopmental improve-
ment. The molecular cause of the disease is mainly due to
12.6 Diagnosis/Diagnostic Flowchart ................................... 200 the loss of sulfite oxidase activity, one out of four molyb-
12.7 Specimen Collection....................................................... 200 denum cofactor-dependent enzymes. Sulfite oxidase
12.8 Prenatal Diagnosis Table for All Disorders catalyzes the terminal step in the oxidative degradation
and Sample Requirement .............................................. 200 of cysteine; a loss of activity results in the accumulation
12.9 DNA Testing Table for All Disorders and Sample of toxic sulfite, which in turn triggers the alteration of
Requirement ................................................................... 200 secondary-related metabolites such as S-sulfocysteine,
12.10 Treatment Summary...................................................... 200
thiosulfate, taurine, hypotaurine, and cystine. Xanthine
oxidoreductase catalyzes the catabolism of purines from
References .................................................................................... 201 hypoxanthine to xanthine and further to uric acid, which
is reduced in patients while xanthine and to a lesser
extent hypoxanthine accumulate. The molybdenum cofac-
tor (Moco) is synthesized by a three-step biosynthetic
pathway, which involves gene products of the MOCS1,
MOCS2, MOCS3, and GEPH loci. Depending on the
mutation, type A, B, and C deficiencies are known. While
MoCD types A and B are clinically indistinguishable,
MoCD type C has a more severe neurological presentation
due to the loss of synaptic inhibition, which is dependent
on GEPHYRIN function. Dietary restriction (low cyste-
ine and methionine) has been reported in some case, how-
ever, disease improvement was marginal. A first causative
therapy has been established for MoCD type A patients
and is based on the treatment with cyclic pyranopterin
G. Schwarz (*) monophosphate, the first intermediate in the molybdenum
Department of Chemistry, cofactor pathway. Given the high neurotoxicity of sul-
Institute of Biochemistry, University of Cologne,
Zülpicher Str. 47, Köln 50674, Germany
fite and its related compounds, early diagnosis has been
e-mail: gschwarz@uni-koeln.de shown to be the key determinant in the treatment outcome.
A. Veldman
Patients that were treated shortly after birth and have not
Colbourne Pharmaceuticals GmbH, been exposed to extensive anticonvulsive therapy showed
Viktoriaweg 7, Niederkassel 53859, Germany best clinical and neurodevelopmental outcome.
192 G. Schwarz and A. Veldman
12.1 Introduction similarities to isolated SO deficiency, the major cause of

neurodegeneration is sulfite accumulation. Besides a gen-
In men, four molybdenum enzymes are known, each of them eral cellular toxicity of sulfite, the formation of secondary
catalyzes either catabolic or detoxifying reactions in the body metabolites, such as S-sulfocysteine (SCC), or the depletion
(Schwarz et al. 2009). Sulfite oxidase (SO) is localized in the of metabolites, such as cystine, is believed to contribute to
intermembrane space of mitochondria and couples sulfite neuronal dysfunction (Tan et al. 2005).
oxidation to the reduction of cytochrome c. Together with the Mutations in patients with MoCD have been identified in
recently identified mitochondrial amidoxime-reducing com- three of the four Moco synthetic genes (Reiss et al. 1998a, b),
ponent (Havemeyer et al. 2006), which is believed to function and prenatal diagnosis in carrier families has been established
in the reduction of N-hydroxylated prodrugs, they form a (Reiss et al. 1999a). Today more than 64 disease-causing
family of molybdenum enzyme characterized by a conserved mutations are known in MOCS1, MOCS2, and GEPH (Reiss
protein-derived cysteine coordinating the molybdenum cofac- and Hahnewald 2011). In nearly all cases, disease-causing
tor (Hille 1996). SO deficiency (see 3.6) has been reported in mutations result in a complete loss of enzyme function due to
more than 30 cases so far and is very similar to MoCD (for frameshifts, splice site and nonsense mutations, or missense
details, see below). The other family of molybdenum enzymes mutations affecting highly conserved or invariant residues.
is formed by xanthine oxidoreductase (XO) and aldehyde Two thirds of all MoCD patients carry mutations (>40) in the
oxidase, catalyzing the hydroxylations of purine or other het- Mocs1 gene (Reiss and Hahnewald 2011). Approximately one
erocyclic substrates, respectively (Schwarz et al. 2009). Their third of MoCD patients are affected in the second step of
molybdenum center requires the addition of a terminal sulfido Moco synthesis; 23 disease-causing mutations have been
ligand, which is dependent on the Moco sulfurylase (MCSU) found in both open reading frames affecting encoded by the
(Ichida et al. 2001). Consequently, a mutation in MCSU MOCS2 gene. Interestingly, no MoCD patient with a MOCS3
causes a deficiency of both enzymes, a disease named xan- mutation has been found so far, which might be due to an addi-
thinuria type II (see 41.9.1). While an isolated deficiency in tional function of MOCS3 in tRNA thiolation (Chowdhury
aldehyde oxidase has not been reported in men, deficiencies et al. 2012). In most MoCD cases, homozygous mutations are
in XO are well known and cause xanthinuria type I (see 41.9). found due to the high consanguinity of heterozygous parents,
Moco deficiency (MoCD) is characterized by the simultane- while compound heterozygous cases are the minority.
ous loss of all Mo-enzyme activities due to a mutational block Only two cases with GEPH mutations have been described
in the biosynthesis of Moco, which can be divided into three so far. One patient was the last of three affected infants that all
major steps based on the two intermediates: cyclic pyranop- died in the neonatal period (day 12, 29, and 3, respectively),
terin monophosphate (cPMP) (Santamaria-Araujo et al. 2004), with symptoms of MoCD (Reiss et al. 2001). Genetic analy-
previously named precursor Z (Wuebbens and Rajagopalan sis revealed an early stop codon resulting in a total loss of
1993), and the metal-binding pterin or molybdopterin (MPT) GEPHYRIN expression. As a result, both functions of
(Johnson et al. 1984) (Scheme 1). Each of the steps involves the GEPYHRIN, Moco synthesis as well as receptor clustering,
action of one or more proteins producing additional reaction were impaired. A second case with a missense mutation in
intermediates such as pyranopterin triphosphate (Mehta et al. GEPH affected the catalytic site of GEPHYRIN E-domain
2013), thio-pyranopterin phosphate (Wuebbens and (Reiss et al. 2011). Given that the binding site of both glycine
Rajagopalan 2003), and adenylylated MPT (Kuper et al. 2004) (Schrader et al. 2004) and GABA receptors (Maric et al.
(Scheme 1). Four genes have been found to encode for proteins 2011) is distinct from Moco synthesis (Llamas et al. 2006), an
essential for Moco biosynthesis in men. MOCS1 and MOCS2 isolated MoCD can be assumed in this case, as confirmed by
each produce two or more proteins, being essential for steps typical symptoms of MoCD.
one and two in Moco synthesis, respectively. MOCS3 is essen- The vast majority of MoCD patients present a very severe
tial for the activation of MOCS2 proteins and GEPH encodes neurological phenotype. Only a handful cases have been
for a multifunctional protein functioning in Moco synthesis as reported with mild forms of the disease. To our knowledge,
well as glycine and GABA type A receptor clustering. A fifth only one mild presentation was reported for MoCD type A
gene (MCSU) is needed for the sulfuration of Moco. deficiency (Arenas et al. 2009) affecting the splicing of
Human MoCD is a rare autosomal recessive disorder, MOCS1 exon 9 probably affecting the mitochondrial matura-
which mostly affects neonates and is characterized by pro- tion and/or targeting of MOCS1AB translation products thus
gressive brain injury leading to early childhood death. More leading to a reduced but not completely absent cPMP-synthetic
than 100 cases with MoCD have been reported (Johnson activity (G. Schwarz 2013). All other mild cases carry either
and Duran 2001; Reiss and Hahnewald 2011), which nearly mutations in MOCS2 thus showing only partially impaired
all share a predominant deterioration of the central nervous molybdopterin synthesis (Johnson et al. 2001) (G. Schwarz
system as main disease feature mimicking hypoxic-isch- 2013) or mutations in the SUOX gene (Barbot et al. 1995; Del
emic encephalopathy (Vijayakumar et al. 2011). Given the Rizzo et al. 2013; Touati et al. 2000).
12 Molybdenum Cofactor Disorders 193
Two MoCD animal models are currently available. The Mo-enzyme activities following repeated intrahepatic injec-
gephyrin knockout mouse develops both symptoms of tions of purified cPMP (Schwarz et al. 2004). Improvement
impaired synaptic inhibition and MoCD (Feng et al. 1998). of treated animals was directly correlated with cPMP injec-
Geph–/– neonates appeared externally normal and failed to tions as withdrawal of cPMP caused death within 10–14 days
suckle, and following mild tactile stimuli, they retained rigid following the final injection, and subsequent reduction in
with a hyperextended posture and exhibited apnea; animals Mo-enzyme activities was observed. Treated animals showed
usually died within 12 h after birth due to respiratory failure. normal behavior and were fertile.
Due to the high prevalence of MoCD type A, a Mocs1 knock- Given the positive results in mocs1–/– mice, a first treat-
out mouse was generated (Lee et al. 2002). Mocs1-/- mice ment attempt was initiated in a MoCD type A patient.
display a severe phenotype characterized by a retarded Treatment was started at day 36 of life with a starting dose of
growth, abnormal behavior, lack of feeding, and death within 80 μg/kg body weight per intravenous infusion (Veldman
the first 11 days of life (Lee et al. 2002). As observed in et al. 2010). Urinary markers of SO and XO deficiency
humans, biochemical characterization of mocs1−/− mice returned within days to almost normal readings. Clinically,
revealed elevated urinary sulfite and xanthine levels, uric the patient became more alert a few days after treatment was
acid was undetectable, and Mo-enzyme activities were started; convulsions and twitching disappeared as docu-
absent. mented by an electroencephalogram showing the return of
A substitution therapy was established in mocs1–/– mice rhythmic elements and markedly reduced epileptiform dis-
showing a phenotypic normalization and reconstitution of charges (Veldman et al. 2010).
12.2 Nomenclature
Gene Chromosomal Affected

No. Disorder Alternative name Abbreviation symbol localization protein OMIM no. Subtype
12.1 Molybdenum MoCo deficiency, MoCD type A MOCS1 6p21.3 MOCS1A, 603707 All forms
cofactor complementation group MOCS1AB
deficiency A A
12.2 Molybdenum MoCo deficiency, MoCD type B MOCS2 603708 MOCS2A, 603708 All forms
cofactor complementation group MOCS2B
deficiency B B
12.3 Molybdenum MoCo deficiency, MoCD type C GEPH 14q23.3 GEPHYRIN 603930 All forms
cofactor complementation group
deficiency C C
12.3 Metabolic Pathway proteins (Gray and Nicholls 2000). These proteins only
harbor MOCS1B activity due to the truncation of function-
Moco Biosynthesis ally important C-terminal residues within the C-terminus
Proteins encoded by the MOCS1 gene catalyze the first of MOCS1A (Hänzelmann et al. 2002). MOCS1A is
step in Moco biosynthesis (Fig. 12.1), the conversion of believed to initiate the transformation of 5′-GTP by
GTP into cPMP. MOCS1 was first reported to produce a abstracting the 3′ proton from the ribose resulting in the
bicistronic transcript encoding for two open reading formation of pyranopterin triphosphate (Mehta et al.
frames, MOCS1A and MOCS1B (Reiss et al. 1998b); 2013), which is subsequently converted into cPMP by the
however, later studies demonstrated that this transcript activity of MOCS1AB.
only encodes for functional MOCS1A, which catalyzes The second step in Moco biosynthesis (Fig. 12.1) is cata-
the ring opening reaction of GTP (Hänzelmann et al. lyzed by MPT synthase, which converts cPMP into MPT
2002). MOCS1A is believed to use the same kind of chem- and is encoded by the bicistronic MOCS2 gene. The two
istry as shown for its bacterial orthologue, MoaA, an overlapping open reading frames (MOCS2A and MOCS2B)
S-adenosylmethionine (SAM)-dependent radical enzyme are translated by a ribosomal leaky scanning mechanism
containing two [4Fe-4S] clusters (Hänzelmann and producing both subunits in an approximately equimolar
Schindelin 2004). Alternative splicing of MOCS1 was ratio (Stallmeyer et al. 1999). MOCS3 encodes for the Moco
found to produce two other types of transcripts with a sin- sulfurase (Matthies et al. 2004), which is required for the
gle open reading frame encoding for MOCS1AB fusion ATP-dependent thiolation of MOCS2A.
Fig. 12.1 Moco biosynthesis, O

Mo enzymes, and deficiencies. HN
NH
Intermediates of Moco synthesis
are cyclic pyranopterin mono- H2N N N
GTP
phosphate (cPMP), metal-binding OH OH OH
pterin (MPT), and adenylylated P P P O

HO O O O
MPT (MPT-AMP). Moco O O O
undergoes an additional
MOCS1A OH OH
sulfuration prior to its incorpora-
tion into xanthine oxidase and MOCS1AB 12.1
aldehyde oxidase. In the absence
of this function, xanthinuria type O
H HO OH
II is developed (41.9.1). Proteins N O O cPMP
involved in Moco synthesis are HN P O
shown. Note that the individual O

H2N N N O
domains of GEPHYRIN catalyze H H
two subsequent steps in Moco
MOCS2A 12.2
synthesis. Deficiencies in MOCS2B
MOCS1, MOCS2, and GEPH
cause MoCD type A (12.1), type O SH
B (12.2), and type C (12.3), H
N SH MPT
respectively. Isolated deficiencies HN
O-
in SO and XO cause 3.6 and H2N N N O
O
P 3.6
41.9. For details, see the relevant H -O O
chapters Sulfite
GEPHYRIN 12.3
(G domain) Sulfite oxidase
NH2 MPT-AMP Sulfate
N
O SH N R=N-OH
H
N SH Amidoxime reducing
HN N N
OH OH component
O O O R-NH
H2N N N O P P
H O
O O
GEPHYRIN 41.9
(E domain) 12.3 OH OH
41.9.1 Xanthine
O O
O- Xanthine oxidase
O S Mo
H
S Moco Uric acid
N
HN
R-H
O O-
H2N N N O P
H Aldehyde oxidase
O- O
R-OH
The third and final step in Moco synthesis (Fig. 12.1) Cysteine Catabolism, Sulfite Toxicity, and Altered
involves the adenylylation of MPT (forming MPT-AMP) and Metabolites
subsequent molybdate-dependent hydrolysis of MPT-AMP MoCD is clinically very similar to the less prevalent
resulting in Moco. Both reactions are dependent on isolated SO deficiency implicating sulfite toxicity as a major
GEPHYRIN, which encodes for a multi-domain cytosolic underlying cause for neurodegeneration in MoCD patients.
protein composed of an N-terminal G-domain (GEPH-G, SO, which is localized in the mitochondrial intermembrane
MPT-AMP synthesis), a central C-domain, and a C-terminal space, oxidizes sulfite to sulfate; thus, no accumulation of
E-domain (GEPH-E, molybdenum insertion, Scheme 1). sulfite in the cytosol or extracellular compartments is seen
Besides Moco biosynthesis, GEPHYRIN functions as cyto- (Scheme 2). In MoCD and SO deficiency, sulfite accumu-
solic membrane-associated receptor-clustering protein, lates first in mitochondria where it has been shown to increase
being essential for the formation of inhibitory synapses reactive oxygen species (Zhang et al. 2004). Sulfite also
(Fritschy et al. 2008). The GEPH gene is highly mosaic with decreases ATP synthesis in mitochondria when respiring on
27 exons distributed over 760 kb of genomic DNA on chro- glutamate, while other respiratory substrates such as malate,
mosome 14q32. At least six of these exons are subject to α-ketoglutarate, and succinate did not show any sulfite vul-
alternative splicing producing more than ten different splice nerability (Zhang et al. 2004). The mechanism underlying
variants (Rees et al. 2003). the inhibition of ATP synthesis reduction has been related to
glutamate dehydrogenase inhibition by sulfite, which in the Jahoor et al. 1999), and its depletion will have a major impact
brain, due to the fact that glutamate dehydrogenase operates on cell viability. Despite the dramatically reduced levels of
in the direction of oxidative deamination, will lead to a plasma cystine in MoCD, no information is available regard-
decrease in the availability of α-ketoglutarate and other tri- ing GSH concentrations in affected patients, which together
carboxylic acids, resulting in an overall decrease in ATP syn- with SSC, cystine, and glutamate levels in cerebrospinal
thesis. Inhibition of glutamate dehydrogenase may also fluid may further contribute to the understanding of the
affect the metabolism of other neurotransmitters, such as underlying neurodegeneration in MoCD.
GABA, contributing to the accelerated injury in neuronal Besides oxidative cysteine catabolism, which usually con-
rather than nonneuronal tissue, as observed in MoCD. tributes to 80–90 % of sulfur elimination, there is also a non-
Sulfite is a strong reductant and will therefore reduce oxidative degradation, which involves the contribution of one
disulfide bridges, primarily in membrane and extracellular of the three enzymes, cystathionine γ-lyase (CSE), cystathio-
proteins thus affecting their folding, stability, and function. nine β-synthase (CBS), and 3-mercaptopyruvate sulfurtrans-
Probably first, sulfite reacts with cystine leading to the for- ferase (MSPT, Fig. 12.2). All three enzymes have been shown
mation of the secondary metabolite S-sulfocysteine (SSC, to be involved in cysteine-dependent production of hydrogen
Fig. 12.2) (Rupar et al. 1996). SSC is very abundant in sulfide (Kamoun 2004), which was considered to be toxic as
MoCD patients, and its excretion in urine is detectable reported in several poisoning cases (Nam et al. 2004), while
shortly after birth and increases with age (Veldman et al. other studies suggest a functional role as neural messenger
2010), which supports the view that sulfite is cleared during (Baranano et al. 2001). Hydrogen sulfide is further oxidized
maternal gestation (Belaidi et al. 2012; Reiss et al. 2005). to thiosulfate in mitochondria by three sequential enzymatic
SSC is structurally similar to glutamate, is able to bind to reactions (Fig. 12.2) (Hildebrandt and Grieshaber 2008).
NMDA receptors, and therefore postulated as main agent First, the mitochondrial membrane flavoprotein-quinone oxi-
responsible for seizure development and subsequent brain doreductase (SQR) converts sulfide to a protein-bound per-
damage in MoCD (Gorman and Griffiths 1994; Olney et al. sulfide and transfers two electrons to the ubiquinone pool
1975). In fact, early studies in rats demonstrated that subcu- (Kabil and Banerjee 2010). Next, the persulfide is handed
taneous administration of SSC induces the same type of over to a sulfur dioxygenase, which converts the persulfide
brain damage that glutamate and other excitatory amino molecule to sulfite using molecular oxygen. Finally, a sulfur
acids are known to cause (Olney et al. 1975). In contrast to transferase adds a second persulfide molecule from SQR to
the neurotransmitter glutamate, whose release in the extra- sulfite yielding the final product thiosulfate (Hildebrandt and
cellular compartment is highly controlled by vesicular fusion Grieshaber 2008). The relative contribution of the nonoxida-
and cellular reuptake, SSC is continuously produced by sul- tive pathway in cysteine catabolism is usually low and insen-
fite accumulation. In the absence of a specific transporter, sitive to cysteine dietary intake (Bagley and Stipanuk 1994),
SSC is assumed to persist in the extracellular compartment while nonoxidative cysteine catabolism is increased in case of
and/or inhibits the uptake of glutamate (Dunlop et al. 1991) MoCD or SO deficiency as observed by high levels of thiosul-
thus potentiating its excitotoxic effect leading to neuronal fate excretion. A similar disbalance can be seen if CDO activ-
death. Besides SSC, taurine is also elevated in MoCD ity is lost as observed in the CDO-/- mice (Ueki et al. 2011),
(Belaidi and Schwarz 2013) and known to be neuroprotec- showing to some degree similar symptoms to another animal
tive by playing an important role in glutamate and GABA model, Ethe1−/− mice, that accumulate both sulfide and thio-
signaling (El Idrissi and Trenkner 1999); however, this posi- sulfate (Tiranti et al. 2009).
tive effect seems to be erased by SSC toxicity. MoCD patients have been reported to present homocyste-
SSC formation goes hand in hand with cystine depletion inemia (Graf et al. 1998; Sass et al. 2003) suggesting a gen-
(Barbot et al. 1995; Johnson and Duran 2001). Cystine is the eral reduction in methionine as well as S-adenosylmethionine
major transport form of cysteine in plasma (Fig. 12.2). In the levels. The molecular cause of these upstream alterations in
brain, cystine is taken up into glial cells, where it is reduced MoCD are not understood, but might reflect a general “leak”
and incorporated into glutathione (GSH), the major antioxi- of cysteine due to SSC accumulation, which subsequently
dant in neuronal tissue and most abundant low molecular alters the methionine-cysteine balance. Furthermore, recent
weight thiol in animal cells (0.5–10 mM) (Wu et al. 2004). studies demonstrated a sulfite-dependent reduction of pyri-
An increase in cysteine supply via oral or intravenous admin- doxal 5′-phosphate (PLP) in cerebrospinal fluid (CSF)
istration enhances GSH synthesis and prevents GSH defi- (Footitt et al. 2011). Sulfite is one of the nucleophiles known
ciency in humans (Townsend et al. 2003). Thus, cysteine is to be able to react with PLP. As a result of reduced PLP lev-
generally considered to be the limiting amino acid for GSH els, the excretion of other metabolites such as α-aminoadipic
synthesis in humans, rats, and pigs (Chung et al. 1990; semialdehyde (Mills et al. 2012).
Methionine
MAT ATP
Transsulfuration pathway Tetra

S-Adenosylmethionine
Betaine hydrofolate
BHMT MT MS
CH3
Dimethylglycine 5-Methyl
tetrahydofolat
S-Adenosylhomocysteine
SAHH
Adenosine
Homocysteine
Glutathione CBS Ser

Gly
GS Cystathionine
γ-Glutamyl
cysteine α-KB
CSE
Glu
NH3
GCS
Cysteine Cystine
CDO
Homo-
Cysteine cysteine KG
sulfinic acid O2
AAT Ser
Cysta- CBS
CSD KG AAT thionine Glu CSE
CO2 Glu
Pyr.
β-Sulfinyl H2S 3-Mercapto
H 2S
Hypotaurine pyruvate pyruvate
MPST
SQR SQR
Pyr.
RSSH
SO3 2–
SDO
ST
SO H2O
Taurine SO4 2– SO3 2– S2O3 2– S-sulfocysteine
SO
Oxidative cysteine catabolism Non-oxidative cysteine catabolism
Fig. 12.2 Cysteine catabolism and S-containing metabolites that are S-adenosylhomocysteine hydrolase, BHMT betaine-homocysteine methyl-
altered in MoCD and SO deficiency. Transsulfuration pathway, oxidative transferase, MT methionine synthase, CBS cystathionine β-synthase, CSE
and nonoxidate cysteine catabolism are summarized with the involved cystathionine γ-lyase (cystathionase), GCS γ-glutamylcysteine synthetase,
enzymes and metabolites. Changes in MoCD are highlighted in blue with GS glutathione synthetase, CDO cysteine dioxygenase, CSD cysteinesul-
corresponding arrows indicating an increase or decrease in concentration finate decarboxylase, AAT aspartate aminotransferase, SO sulfite oxidase,
in comparison to control individuals. Enzyme abbreviations are as follows: MPST 3-mercaptopyruvate sulfurtransferase, SQR quinone oxidoreduc-
MAT methionine-S-adenosyl transferase, MT methyltransferase, SAHH tase, SDO sulfur dioxygenase, and ST sulfur transferase

Table 12.1 Molybdenum cofactor deficiency A
Cardiovascular Hypotension ++
CNS Apnea ± ±
Consciousness disturbance ++ +
Hypotonia, muscular +++ +
Orobulbar dysfunction +++ +++ +++ +++
Seizures, tonic-clonic +++ +++ +++ ++
Spastic tetraplegia + ++ +++
Twitching ++ ++ ++ ++
Digestive Feeding difficulties +++ +++ +++ +++
Eye Blindness, cortical + + +
Lens dislocation + + +
Musculoskeletal Dysmorphic features ± + + ++
Microcephaly ± ++ ++ ++
Renal Nephrolithiasis + ++ ++
Other Life-threatening illness +++ +++ ++ +
Routine laboratory Uric acid (P) ↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Special laboratory Cyclic pyranopterin monophosphate; cPMP n n n n
(U)
Cystine (P) ↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Homocysteine (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
PLP (P) ↓ ↓ ↓ ↓ ↓
Sulfocysteine (P, U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Sulfite (U) +++ +++ +++ +++
Taurine (P, U) ↑ ↑↑ ↑↑ ↑↑ ↑↑
Urothione (U) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Xanthine (P, U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 12.2 Molybdenum cofactor deficiency B

CNS Apnea ± ±
Hypotonia, muscular +++ +
Special laboratory Cyclic pyranopterin monophosphate; ↑↑ ↑↑ ↑↑ ↑↑
cPMP (U)
Cystine (P) ↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Homocysteine (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
PLP (P) ↓ ↓ ↓ ↓ ↓
Sulfite (U) +++ +++ +++ +++
Taurine (P, U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Urothione (U) ↓↓ ↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Xanthine (P, U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 12.3 Molybdenum cofactor deficiency C

System Symptom Neonatal Infancyb Childhoodb Adolescenceb Adulthoodb
CNS Apnea ± ±
Hyperekplexiaa +++
Hyperreflexiaa +++
Hypotonia, muscular +++ + + + +
Special laboratory Cyclic pyranopterin monophosphate; n-↑ n-↑ n-↑ n-↑
cPMP (U)
Cystine (P) ↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Homocysteine (P) ↓-↑ ↓↓ ↓↓ ↓↓ ↓↓
PLP (P) ↓ ↓ ↓ ↓ ↓
Sulfite (U) +++ +++ +++ +++
Taurine (P, U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Urothione (U) n-↑ n-↑ n-↑ n-↑ n-↑
Xanthine (P, U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
a
Only for patients with GEPH deletions and splice mutations or deletions. Patients with missense mutation, which do not affect the receptor cluster
function of GEPHYRIN, do not present those symptoms
b
Patients with GEPH deletions and splice mutations or deletions will not reach this age. Patients with missense mutation, which do not affect the
receptor cluster function of GEPHYRIN, do not present those symptoms
The first human patient with MoCD was described in sented by feeding difficulties accompanied by intractable
1978 (Duran et al. 1978) and was presented in his neonatal seizures with a predominant opisthotonus and an exagger-
period with initial feeding difficulties, therapy-resistant sei- ated startle reaction (Reiss and Hahnewald 2011). Disease
zures, high-pitch crying followed by severe neurological progression is accompanied by psychomotor retardation due
abnormalities, lens dislocation of the eyes, and major dys- to progressive cerebral atrophy and ventricular dilatation,
morphic features of the head. At the time of identification, which are typical in brain MRI of patients. In addition, major
the chemical nature of Moco was not known, neither its bio- radiological features of the disease include global cerebral
synthesis. Based on the identified alterations in the biomark- edema, cystic encephalomalacia, cortical and white matter
ers of two Mo-dependent enzymes, XO and SO, a defect in atrophy, focal or bilateral changes within the globi pallidi
either molybdenum metabolism or transport has been pro- and subthalamic regions, and dysgenesis of the corpus cal-
posed (Duran et al. 1978). Since then, more than 100 cases losum and ventriculomegaly (Arslanoglu et al. 2001; Bayram
with MoCD have been reported (Johnson and Duran 2001; et al. 2013; Vijayakumar et al. 2011). Patients that survive
Reiss and Hahnewald 2011), which nearly all share a pre- the neonatal period show essentially no neuronal develop-
dominant deterioration of the central nervous system as main ment, are unable to make coordinated movements, require
disease feature mimicking hypoxic-ischemic encephalopa- tube feeding and show no signs of communication with their
thy (Vijayakumar et al. 2011). In general, first symptoms are environment, and usually die within their first years of life
observed within the first days of life, which are initially pre- (Johnson and Duran 2001).
12.5 Reference and Pathological Values
Purines in urine and plasma (HPLC-UV and LC-MS/MS)

Urine Plasma
0–1 year 1–5 years 5–16 years 0–100 years
Compound rangea (n = 16) (n = 47) (n = 27) >16 years (n = 26) Pathological valuesb (n = 00)
Uric acidc 820–1,026 527–790 326–436 222–287 <100 220–230
Hypoxanthine 1–71.9 1–88.1 1–14.1 1–14.0 >100 0–10.0
Xanthine 0–63.4 0–54.7 0–21.7 0.3–10.7 >70 0–7.0
a
μmol/mmol creatinine
b
In MoCD, xanthinuria type I and II
c
95 % Cl
See also disorder 41.9
Sulfite-related metabolites in urine and plasma

Urine Plasma
0–1 year 1–5 years 5–16 years 0–100 years
Compound rangea (n = 16) (n = 47) (n = 27) >16 years (n = 26) Pathological valuesb (n = 100)
Total homocysteinec 3.3–8.3 4.7–10.3 <15 <15 <1 0.2–3.7
S-sulfocysteined 0–18 0–25 >50 0–3
Thiosulfatee 0–55 <100 <9
Cystinef 57–200 μmol/g <10 23–58
Taurineg 30–100 400–2,000 35–245
PLPh 26–69 8–136 8–41 0–5
a
b
In MoCD and SO deficiency
c
See also disorder 3.6
d
Belaidi et al. (2012)
e
Plasma: Bamforth et al. (1990); Urine: van Gennip et al. (1991)
f
Reynolds and Harkness (1991)
g
Plasma: Arslanoglu et al. (2001); Urine: Belaidi and Schwarz (2013)
h
CSF: Footitt et al. (2011)
12.6 Diagnosis/Diagnostic Flowchart Test Sample requirement

Sulfite dipstick Fresh urine, bedside dipstick test
S-sulfocysteine in urine Urine, frozen 1 ml
Neonate or infant with S-sulfocysteine in plasma EDTA blood, 1 ml
seizures and/or HIE
like clinical picture
Uric acid in urine Urine
Uric acid in plasma EDTA blood, 1 ml
Xanthine in urine Urine
Xanthine in plasma EDTA blood, 1 ml
Sulphite dipstick
positive AND/OR UA
Condition other than
in plasma low AND/ No
MoCD
OR SSC in plasma or
urine elevated
12.8 Prenatal Diagnosis Table for All
Disorders and Sample Requirement
Timing/
Yes Disorder Material trimester
All MoCD types Chorionic villus cells I
Cultured amniocytes II
Neonate with Amniotic fluid (might be positive III
suspected MoCD for SSC, unclear diagnostic
accuracy)
Urine negative for Urine positive for 12.9 DNA Testing Table for All Disorders
compound Z AND/
OR response to
compound Z AND/
OR no response to
and Sample Requirement
cPMP cPMP
In MoCD patients, mutations have been found in MOCS1,

MOCS2, and GEPH; no mutations were found in the MOCS3
Yes Yes gene. Given the highest frequency of MOCS1 mutations,
analysis of this gene is suggested before testing for the
MOCS2 gene. Only if no mutation has been found in either
Neonate with Neonate with
suspected MoCD suspected MoCD of the two MOCS genes, sequence analysis of GEPH is sug-
Type A Type A gested. Primers and protocols have been published previ-
ously by Reiss et al. (2001, 1998b, 1999b).

Genetic confirmation
Up to today, there is no approved specific therapy for MoCD

available, and the prognosis remains poor with most affected
patients dying in infancy and early childhood. Some few
long-term survivors with a delayed onset of symptoms have
Fig. 12.3 Flowchart to diagnose MoCD
been described (Johnson and Duran 2001).
Treatment in all age groups is mainly focused on seizure
control, which is notoriously difficult, and other symptom-
atic and supportive approaches. In the early neonatal period,
prolonged apnea, probably a result of status epilepticus, can
12.7 Specimen Collection make mechanical ventilation necessary, and some few
patients might need mild ionotropic support for low blood
Specimen collection as early as possible in those cases in pressure. Nasogastric feeding tubes or percutaneous gastros-
which a treatment attempt with cPMP (currently as experi- tomy tubes for feeding are almost always needed. Later in
mental drug) is undertaken, specimen to be collected prior to life, botulinum toxin injections can become indicated in the
the first dose of cPMP. treatment of spastic quadriplegia.
Some reports on sulfur-restricted diet document marginal Requirements for cPMP treatment
to moderate improvement in seizure control. Pyridoxine sup- Parameter Result
plementation has been described to improve seizure control 1. Clinical presentation Neonatal seizures
in selected patients (Boles et al. 1993; Del Rizzo et al. 2013; 2a. Either sulfite Positive in two independent
Touati et al. 2000). dipsticks
Recently, replacement therapy with cyclic pyranopterin 2b. Or uric acid Low
monophosphate (cPMP) has been described in patients with Additional SO-related parameters are supportive but not strictly
required:
MoCD type A since this subtype of MoCD features a muta-
SSC (can replace sulfite) High
tional block in the Moco biosynthesis upstream of cPMP Thiosulfate High
(Veldman et al. 2010). Preliminary data indicate that if this Cystine Low
therapy is initiated within the first week of life, significant Taurine High
improvement in neurodevelopmental outcome is possible Homocysteine Low
(Hitzert et al. 2012). cPMP is currently available in Additional XO-related parameters are supportive but not strictly
Compasionate Use programs only, but clinical trials are required:
expected to commence in the very near future. Reference to Xanthine (can replace uric High
www.clinicaltrials.gov is recommended for most recent acid)
Hypoxanthine High
updates on this development.
Age <10 days
MoCD is characterized by the enormous neurotoxicity
Seizures <5 days
of sulfite and related metabolites. This and secondary neu-
Status epilepticus <2 days
ronal injury by continuous seizures and status epilepticus
together with high-dose anticonvulsive therapy is believed
to be major cause of rapidly progressing neurodegenera- Following treatment initiation additional analyses are
tion. As a result, very early diagnosis and start of treatment required to justify continuation.
is required to preserve significant neurocognitive function.
With cPMP emerging as a potential causative therapy at Requirements for continuation of cPMP treatment
least for a subset of patients, sulfite removal by hemofiltra- Biochemical and clinical
tion has been tried as a bridge to therapy; results of the effi- parameter Result
cacy and safety of this intervention are pending (J. Aschner Genetic analysis Mutation in MOCS1
et al. 2012, Vanderbuilt University, personal communica- Normalization of biomarkers:
tion). Also, in patients in whom prenatal diagnosis is avail- Sulfite Disappearance within 3–5 days
SSC Normalization within 3–10 days
able, early delivery to prevent the risk of in utero brain
Uric acid Normalization within 5–14 days
damage by elevated sulfite levels in late pregnancy might be
Xanthine Normalization within 5–14 days
considered in individual cases.
MRI No major brain atrophy or cystic
Emergency Treatment Table for All Disorders and malformation
Medication Requirements
In the event, cPMP therapy will be an option, hemofiltra-
tion may be considered as emergency treatment option.
During hemofiltration, careful monitoring of anticonvulsant
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279:18733–18741
Vitamin B12 Disorders
13
Matthias R. Baumgartner and Brian Fowler
Summary
Contents
Vitamin B12 (Cbl) is needed for just two metabolic reactions
13.1 Introduction ..................................................................... 205 in man, the methylation of homocysteine to methionine
13.2 Nomenclature ................................................................... 207 (cofactor methyl-Cbl) and the conversion of methylmalonyl-
13.3 Metabolic Pathway .......................................................... 208 CoA to succinyl-CoA (cofactor adenosyl-Cbl). A complex
sequence of processes is required to convert dietary Cbl to its
13.4 Signs and Symptoms ....................................................... 209
cofactors and to correctly deliver them to the target enzymes.
13.5 Diagnosis .......................................................................... 212 A wide range of acquired or hereditary disorders of absorp-
13.6 Reference Values.............................................................. 213 tion, transport and intracellular processing of Cbl are known
13.7 Pathological Values.......................................................... 214
resulting in combined methylmalonic aciduria and homocys-
tinuria or each in isolation. Fifteen distinct genetic disorders
13.8 Diagnostic Flow Chart .................................................... 215
have been identified involving carrier proteins, receptors,
13.9 Specimen Collection ........................................................ 215 membrane proteins, molecular chaperones and enzymes, and
13.10 Prenatal Diagnosis ........................................................... 216 the genes have been characterised for each of these, although
the exact function of some of the proteins remains to be elu-
13.11 Treatment ......................................................................... 216
cidated. Main clinical hallmarks of these disorders are hae-
References ..................................................................................... 217 matological and neurological disease of varying severity.
Diagnosis centres on measurement of the two precursors of
the Cbl enzymes, methylmalonic acid and homocysteine,
together with measurements of other parameters such as
total vitamin B12, holotranscobalamin, 3-hydroxypropionic
acid and methylcitric acid, C3-acylcarnitine and methionine.
Absorption and transport defects respond well to treatment
with Cbl. Intracellular processing defects also respond to
Cbl, but biochemical and clinical abnormalities may not
fully resolve and long-term outcome can be poor. Prenatal
diagnosis can be reliably performed in those disorders where
this is indicated.
13.1 Introduction
Vitamin B12 (cobalamin, Cbl) is a complex molecule with a

cobalt-containing corrin ring. It is derived almost exclusively
from animal sources, secondary to production by microor-
M.R. Baumgartner (*) • B. Fowler ganisms. Cbl is needed for just two metabolic reactions in
Division for Metabolic Diseases, University Children’s Hospital,
man, methylation of homocysteine to methionine and con-
Steinwiesstrasse 75, Zürich 8032, Switzerland
e-mail: matthias.baumgartner@kispi.uzh.ch; version of methylmalonyl-CoA to succinyl-CoA (Fig. 13.1).
brian.fowler@kispi.uzh.ch Deficiency of Cbl, inability to absorb Cbl normally or
206 M.R. Baumgartner and B. Fowler
inability to process Cbl to its two active cofactors can result cblC disease is the most frequent inborn error of Cbl metabo-
in elevated circulating and urinary levels of the precursors lism with over 400 patients known worldwide. The gene
methylmalonic acid (MMA) and homocysteine which are responsible for cblC, designated MMACHC for MMAuria
useful diagnostic markers for Cbl disorders. cblC type with homocystinuria, is thought to be a porter that
In children, causes of Cbl deficiency fall into three cate- carries out targeted delivery of Cbl from the lysosome to
gories: (1) dietary deficiency, (2) abnormal absorption and other Cbl-related proteins but also functions as a decyanase
transport and inborn errors of cellular uptake and (3) intra- and dealkylase (Lerner-Ellis et al. 2006; Banerjee et al.
cellular processing of cobalamins. While the first category is 2009). The cblD defect has only recently been elucidated,
an acquired form of Cbl deficiency, the two latter categories over 30 years after the first description of the complementa-
represent mostly inherited disorders (Watkins and Rosenblatt tion group in two siblings. Only 19 patients are known
2011). worldwide (Coelho et al. 2008; Stucki et al. 2012, plus per-
Abnormal Cbl absorption and transport may result from sonal experience). The cblD defect is remarkable in that
hereditary causes such as absent or abnormal intrinsic factor mutations in the same gene (MMADHC for MMAuria, cblD
(IF), defective ileal receptors or deficient transport into cells or type, with homocystinuria) cause three distinct biochemical
from a wide range of nonhereditary absorption disorders such phenotypes: (1) deficient synthesis of both Cbl coenzymes
as surgery involving the stomach or terminal ileum, decreased causing combined MMAuria and homocystinuria (cblD-
Cbl release from food protein, competition for Cbl in the ileum MMA/HC) associated with truncating mutations in the mid-
or loss of the ileal absorptive surface. Cbl malabsorption (or dle part of the gene, (2) deficiency of MeCbl synthesis
juvenile Cbl deficiency, JCD) is a potentially fatal condition causing isolated homocystinuria (cblD-HC) associated with
that is mostly hereditary in nature in children since the classic missense mutations in the C-terminal part and (3) deficient
adult form of autoimmune pernicious anaemia is rare in early synthesis of AdoCbl causing isolated MMAuria (cblD-
life. Two main forms of inherited JCD exist: intrinsic factor MMA), associated with truncating mutations in the
deficiency (IFD) and Imerslund-Najman-Gräsbeck syndrome N-terminal region (Fig. 13.1).
(IGS), also known as juvenile/hereditary megaloblastic anae- The cblD-HC, cblE and cblG defects are associated with
mia, MGA1 or juvenile pernicious anaemia with proteinuria. deficient methionine synthesis associated with elevated tHcy,
IFD is caused by mutations in the gastric IF gene (GIF, Tanner low methionine and SAM in plasma with normal MMA. The
et al. 2005). In IGS, deleterious mutations in the genes encod- cblE defect is caused by deficiency of methionine synthase
ing the ileal Cbl receptor cubilin (CUBN, Aminoff et al. 1999) reductase (MTRR), which is required for the activation of the
or its facilitator amnionless (AMN, Tanner et al. 2003) cause methionine synthase apoenzyme by reductive methylation
malabsorption of Cbl. Both proteins are involved in intestinal (Fig. 13.1). The cblG defect is caused by deficient activity of
absorption and renal tubular reabsorption and form the het- the methionine synthase apoenzyme (MTR) itself. The genes
erodimer named CUBAM that accomplishes the internalisa- for both defects have been described with a single missense
tion of the IF-Cbl complex (Fyfe et al. 2004). mutation in MTR accounting for a large proportion of mutant
Conversion of Cbl to its active cofactors, methylcobalamin alleles (Rosenblatt et al. 2011).
(MeCbl) and adenosylcobalamin (AdoCbl), requires a series MMAurias are a heterogeneous group of inborn errors
of biochemical modifications that have been classified as Cbl of metabolism biochemically characterised by the
complementation groups A–J, each of which represents a dis- accumulation of MMA in body fluids and tissues. They
tinct autosomal recessive genetic disease (Fig. 13.1). result from deficiency of the mitochondrial enzyme
Patients belonging to the complementation groups cblF, methylmalonyl-CoA mutase (MCM, encoded by MUT) or
cblJ, cblC and one form of cblD have combined deficiencies by a defect in the synthesis of its cofactor AdoCbl (cblA,
of AdoCbl and MeCbl synthesis and are thus characterised cblB and cblD-MMA) (Fig. 13.1). MCM deficiencies
by both methylmalonic aciduria (MMAuria) and elevated are further subdivided into defects without (mut0) and
total homocysteine (tHcy) which is often associated with low with residual activity (mut-). The conversion of
plasma methionine and S-adenosylmethionine (SAM). The L-methylmalonyl-CoA to succinyl-CoA, catalysed by
cblF defect is a rare disorder described in less than 20 unre- MCM, links the final catabolic pathways of branched-chain
lated families. The gene responsible for the defect (LMBRD1) amino acids, odd-chain fatty acids and cholesterol to the
encodes a lysosomal membrane protein that is thought to act Krebs cycle (Fowler et al. 2008). In the final step of AdoCbl
as a lysosomal exporter for Cbl (Rutsch et al. 2009). The cblJ synthesis in the mitochondrion, adenosyltransferase
defect has recently been described in two unrelated patients encoded by the cblB locus (MMAB) converts cob(II)alamin
with the biochemical phenotype of cblF. The gene responsible to AdoCbl and transfers the product directly to MCM, in a
for the defect, ABCD4, encodes a presumed ABC transporter process gated by the protein encoded by the cblA locus
that may interact with the cblF protein (Coelho et al. 2012). (MMAA) (Banerjee et al. 2009).
13
13.2 Nomenclature
Chromosomal
No. Disorder Alternative name Abbreviation Gene symbol localisation Affected protein OMIM no. Subtype
13.1 Intrinsic factor deficiency
Congenital pernicious anaemia IFD GIF 11q13 Transcobalamin III 261000 All forms
13.2.1 Cubilin deficiency Imerslund-Najman-Gräsbeck IGS CUBN 10p12.1 Cubilin 261100 All forms
syndrome (IGS) due to CUBN
Vitamin B12 Disorders
13.2.2 Amnionless deficiency Imerslund-Najman-Gräsbeck IGS AMN 14q32.32 Amnionless 261100 All forms
syndrome due to AMN
13.3 Haptocorrin deficiency TC I (TCN1) deficiency HCD TCN1 11q12.1 B12-binding alpha-globulin 193090 All forms
13.4 Transcobalamin deficiency TC II deficiency TCD TCN2 22q12.2 Vitamin B12-binding protein 2 ? 275350 All forms
13.5 Transcobalamin receptor TCR/CD320 defect TCR CD320 19p13.2 CD32 receptor 613646 All forms
defect
13.6 Adenosylcobalamin Methylmalonic aciduria, cblA cblA MMAA 4q31.21 ? 251100 All forms
synthesis defect – cblA type
13.7 Adenosylcobalamin Methylmalonic aciduria, cblB cblB MMAB 12q24.11 Adenosyltransferase 251110 All forms
synthesis defect – cblB type
13.8 Adenosylcobalamin Methylmalonic aciduria cblD-MMA MMADHC 2q32.2 ? 277410 All forms
synthesis cblD-MMA type
defect – cblD-MMA
13.9 Methylcobalamin Homocystinuria, cblD-HC type cblD-HC MMADHC 2q32.2 ? 277410 All forms
synthesis
defect – cblD-HC
13.10 Methionine synthase Methylcobalamin deficiency, cblG MTR 1q43 Methionine synthase, 250940 All forms
deficiency – cblG cblG type 5-methyltetrahydrofolate-
homocysteine methyltransferase
13.11 Methionine synthase Methylcobalamin deficiency, cblE MTRR 5p15.31 Methionine synthase reductase, 236270 All forms
reductase cblE type 5-methyltetrahydrofolate-
deficiency – cblE homocysteine methyltransferase
reductase
13.12 Adenosylcobalamin and Methylmalonic aciduria and cblC MMACHC 1p34.1 ? 277400 All forms
methylcobalamin synthesis homocystinuria, cblC type
defect – cblC
13.13 Adenosylcobalamin and Methylmalonic aciduria and cblD-MMA/HC MMADHC 2q32.2 ? 277410 All forms
methylcobalamin synthesis homocystinuria, cblD type
defect – cblD-MMA/HC
13.14 Adenosylcobalamin and Methylmalonic aciduria and cblF LMBRD1 6q13 Lysosomal export of cobalamin? 277380 All forms
methylcobalamin synthesis homocystinuria, cblF type
defect – cblF
13.15 Adenosylcobalamin and Methylmalonic aciduria and cblJ ABCD4 14q24 Lysosomal export of cobalamin? ? All forms
methylcobalamin synthesis homocystinuria, cblJ type
defect – cblJ
207
13.3 Metabolic Pathway terminal ileum via specific receptor-mediated endocytosis.

Free Cbl then enters the portal circulation bound to transco-
Cobalamin and its metabolism are shown in Fig. 13.1. Cbl is balamin II (TC II) and is transported to tissues where the
released from food proteins in the acidity of the stomach and Cbl-TC II complex binds to the TC receptor and is inter-
binds to haptocorrin (HC), produced in saliva and in the nalised (Watkins and Rosenblatt 2011). In the cell, conver-
stomach. In the duodenum, pancreatic enzymes degrade the sion of Cbl to its active cofactors, methylcobalamin (MeCbl)
Cbl-HC complexes releasing free Cbl which forms a com- and adenosylcobalamin (AdoCbl), requires a series of bio-
plex with intrinsic factor (IF) from gastric parietal cells. The chemical modifications that have been classified as Cbl com-
Cbl-IF factor complex is taken up by epithelial cells in the plementation groups A–J (Fig. 13.1).
Dietary Cbl
HC
Gastric secretion
Cbl-HC IF
Pancreatic protease
Cbl-IF
Absorption and transport
CUBN, AMN
Ileal mucosal cell

HC/TC
Distribution via blood
Cbl-HC Cbl-TC
TCblR
Cbl Lysosome
cblF, cblJ Target cell

Cbl
cblC
Cbl
cblD
cblD-MMA
cblD-Hcy
Mitochondrion
Intracellular metabolism
Cbl
Cbl
cblE
cblA cblB
Adenosylcobalamin Methylcobalamin
Methylmalonyl-CoA Succinyl-CoA Homocysteine Methionine
MUT cblG
MTHF THF
Methylmalonic acid
Fig. 13.1 Cobalamin and its metabolism. For details see text. Cbl holotranscobalamin, MMA methylmalonic acid, Hcy homocysteine,
cobalamin, HC haptocorrin (= transcobalamin 1), IF intrinsic THF tetrahydrofolate, MTHF methyltetrahydrofolate
factor, CUBN cubilin, AMN amnionless, TC transcobalamin 2, Cbl-TC
13 Vitamin B12 Disorders 209
13.4 Signs and Symptoms developmental regression, irritability, apathy, anorexia and
refusal of solid foods (Whitehead 2006). Clinically,
The characteristic hallmark of Cbl disorders is haematologic Imerslund-Najman-Gräsbeck syndrome (IGS) and intrinsic
abnormalities with megaloblastic anaemia, hypersegmenta- factor deficiency (IFD) overlap considerably; mild, Cbl-
tion of neutrophils and (pan)cytopenia. However, these resistant proteinuria (not obligatory) in IGS may help to dis-
symptoms may only manifest late or not at all. In most disor- tinguish the two disorders. While patients with IGS and IFD
ders, the phenotypes encompass a spectrum ranging from usually present between age 1 and 10 (Tables 13.1, 13.2.1
early-onset to late-onset clinical forms. The clinical presen- and 13.2.2), those with transcobalamin (TC) deficiency pres-
tation and characteristic laboratory findings of each disease ent much earlier, mainly in the first weeks to months of life
are summarised in Tables 13.1, 13.2.1 and 13.2.2, 13.3, 13.4, (Table 13.3). Although haptocorrin deficiency has been asso-
13.5, 13.6, 13.7 and 13.8, 13.9, 13.10 and 13.11, 13.12, ciated with low serum Cbl levels, there is no evidence that
13.13, 13.14 and 13.15. these low levels result in clinical manifestation of Cbl defi-
ciency (Table 13.4) (Watkins and Rosenblatt 2011). Five
Absorption and Transport Disorders patients with defects in the TC receptor have come to medi-
In disorders of Cbl absorption and transport, the symptoms cal attention as the result of positive newborn screening
are usually indistinguishable from those caused by dietary results for MMAuria. Homocysteine was also elevated in
deficiency of vitamin B12. Unspecific clinical signs and most patients (Table 13.5). So far, all five patients have
symptoms may already arise in the first months of life remained asymptomatic, and the long-term effects of this
including failure to thrive (weight and head circumference), condition are yet to be unravelled (Quadros et al. 2010).
Tables 13.1, 13.2.1 and 13.2.2 Intrinsic factor deficiency (13.1) and Imerslund-Najman-Gräsbeck syndrome (13.2.1, 13.2.2)
System Symptom Neonatala Infancy Childhood Adolescence Adulthoodb
CNS Apathy ± ± ±
Deep tendon reflexes ↓-n ↓-n ↓-n
Developmental regression ± ± ±
Hypotonia ± ± ±
Irritability ± ±
Movement disorder ± ± ±
Psychosis, dementia ± ±
Seizures ± ± ±
Digestive Anorexia ± ± ±
Failure to thrive ± ± ±
Haematological (Pan)cytopenia ± ± ±
Anaemia, megaloblastic ± + ±
Routine laboratory Neutrophils, hypersegmented ± + ±
Proteins, total (U)c n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Homocysteine, total (P) ↑ ↑ ↑
Homocysteine (U) ↑ ↑ ↑
Methylmalonic acid (P) ↑ ↑ ↑
Methylmalonic acid (U) ↑ ↑ ↑
Vitamin B12 (S) ↓ ↓↓ ↓
Holotranscobalamin (P)d ↓ ↓ ↓
a
Neonatal presentation extremely rare
b
Solid data on adult patients not available
c
Mild Cbl-resistant proteinuria may be present in IGS (not obligatory) and help to distinguish from IFD
d
Following oral vitamin B12 load (Bor et al. 2005)
Table 13.3 Haptocorrin deficiency

Other No consistent clinical picture + +
Special laboratory Holotranscobalamin (P) n n
Homocysteine, total (P) n n
Vitamin B12 (S) ↓ ↓
Table 13.4 Transcobalamin deficiency

System Symptom Neonatal Infancy Childhood Adolescence Adulthooda
CNS Apathy ± + +
Deep tendon reflexes ↓-n ↓-n
Developmental delay ± ± ±
Hypotonia ± ± ±
Neurologic dysfunction ± ± ±
Digestive Diarrhoea, chronic ± ++ +
Failure to thrive ± ++ +
Haematological (Pan)cytopenia ± + +
Anaemia, megaloblastic ± ++ + + ±
Routine laboratory Neutrophils, hypersegmented ± ± ± ±
Special laboratory Holotranscobalamin (P) ↓ ↓ ↓
Homocysteine, total (P) ↑ ↑
Homocysteine (U) ↑ ↑
Methylmalonic acid (P) ↑ ↑ ↑
Methylmalonic acid (U) ↑ ↑ ↑
Vitamin B12 (S) ↓-n ↓-n ↓-n
a
Solid data on adult patients not available
Table 13.5 Transcobalamin receptor defect

System Symptom Neonatal Infancy Childhooda Adolescencea Adulthooda
Other No consistent clinical + +
picture
Special laboratory Homocysteine, total (P) (↑) (↑)
Methylmalonic acid (U) ↑ ↑
a
Newly discovered disorder; no patients beyond infancy have been described
Intracellular Disorders patients are symptomatic in the first year of life, but isolated
Although disorders of intracellular Cbl metabolism share cases with later onset and minimal findings have also been
many clinical features with the forms of Cbl deficiency due reported (Vilaseca et al. 2003).
to abnormal absorption and transport, some clinical Patients with combined deficiency of MeCbl and AdoCbl
manifestations are unique. Symptoms are generally more synthesis (cblC, cblD, CblF, cblJ) usually present within the
severe, and in most cases there is only a partial or no response first year of life with poor feeding, failure to thrive, develop-
to parenteral hydroxo-Cbl treatment (Rosenblatt et al. 2011). mental delay, megaloblastic anaemia and (pan)cytopenia
The majority of patients with isolated MMAuria (cblA, (Tables 13.12, 13.13, 13.14 and 13.15). In cblF and cblJ, low
cblB, cblD-MMA and mut) present during the newborn birth weight, minor facial abnormalities and congenital heart
period or infancy with metabolic crises, often precipitated by defects have been reported as well. In cblC patients, at least
catabolic stress, e.g. induced by febrile illness. Symptoms two distinct phenotypes differentiated by age of onset have
include vomiting, dehydration, tachypnea, lethargy, failure to been delineated and related to specific mutations in MMACHC
thrive, developmental delay, hypotonia and encephalopathy (Lerner-Ellis et al. 2009). Early-onset patients present in the
(Tables 13.6, 13.7 and 13.8). Long-term complications first year of life with systemic, neurologic, haematologic and
include chronic renal failure, developmental delay, meta- characteristic ophthalmologic symptoms including visual
bolic stroke, extrapyramidal movement disorder and optic impairment, nystagmus and retinopathy (Gerth et al. 2008).
neuropathy (Hörster et al. 2007; Traber et al. 2011). Patients These patients may develop multisystem pathology, such as
with mut0 and cblB defects tend to have earlier onset of renal failure, hepatic dysfunction, cardiomyopathy, intersti-
symptoms and a higher frequency of complications and tial pneumonia or haemolytic uremic syndrome (Rosenblatt
deaths than those with mut- and cblA defects. et al. 2011). In contrast, a few cblC patients come to clinical
The clinical features of the isolated homocystinuria defects attention after the first year of life and may present as late as
(cblE, cblG, cblD-HC) include poor feeding and vomiting in the fourth decade (Thauvin-Robinet et al. 2008 and
with failure to thrive, megaloblastic anaemia and neurologi- Rosenblatt et al 1997). Clinical findings in this group are
cal disease including developmental delay, cerebral atrophy, mainly neurologic and include gait abnormalities, confusion,
hypotonia or hypertonia, ataxia, neonatal seizures, nystagmus disorientation, psychosis and dementia. Macrocytic anaemia
and visual disturbances (Tables 13.9, 13.10 and 13.11). Most is only found in about a third of these patients.
Tables 13.6, 13.7 and 13.8 Adenosylcobalamin synthesis defect: cblA (13.6), cblB (13.7), and cblD-MMA (13.8)
Cardiovascular Cardiomyopathy ±
CNS Encephalopathic crisis, acute ± ± ± ± ±
Extrapyramidal movement disorder ± ± ± ±
Mental retardation ± ± ± ±
Digestive Failure to thrive ± ± ±
Pancreatitis ± ± ± ±
Vomiting ++ + ± ± ±
Eye Optic neuropathy + +
Haematological (Pan)cytopenia ± ± ± ± ±
Renal Renal failure, chronic ± ± + ++
Other Dehydration ++ + ± ± ±
Life-threatening illness ++ + + ± ±
Routine Ammonia (B) ↑↑ n-↑ n-↑ n-↑ n-↑
laboratory Anion gap + + + ± ±
Metabolic acidosis +++ ++ ± ± ±
Recurrent episodes of ketosis and ++ + + ± ±
acidosis
Special 3-Hydroxypropionic acid (U) ↑ ↑ ↑ ↑ ↑
laboratory C3 propionylcarnitine (P, B) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Carnitine, free (DBS, P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Homocysteine, total (P) n n n n n
Methylcitric acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Methylmalonic acid (P, U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
MRI: basal ganglia lesions ± ± ± ±
Tables 13.9, 13.10 and 13.11 Methylcobalamin synthesis defect: cblD-HC (13.9), CblG (13.10), and CblE (13.11)
CNS Ataxia ± ± ± ±
Cerebral atrophy ± ± ± ±
Developmental delay ± + + ±
Myelopathy n n n ± ±
Neurological symptoms ± ++ + + +
Psychiatric symptoms n n n ± ±
Digestive Failure to thrive ± + + ±
Eye Nystagmus ± ± ± ±
Vision, impaired ± ± ± ±
Haematological Anaemia, megaloblastic ± ++ + + ±
Special laboratory Homocysteine, total (P) ↑ ↑↑ ↑↑ ↑↑ ↑↑
Homocysteine (U) ↑ ↑ ↑ ↑ ↑
Methionine (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Methylmalonic acid (P, U)a n n n n n
S-Adenosylmethionine (P, CSF) ↓ ↓ ↓ ↓
a
Elevated MMA in isolated cases with cblE
Tables 13.12, 13.13, 13.14 and 13.15 Adenosylcobalamin and methylcobalamin synthesis defect: (13.12) cblC, (13.13) cblD-MMA/HC,
(13.14) cblF, and (13.15) cblJ
Cardiovascular Cardiac, anomalies/malformations ± ±
Cardiomyopathy ± ±
CNS Cerebral atrophy ± ± ± ±
Dementia ± ±
Extrapyramidal signs ± ± ± ±
Hypotonia + ++ + + +
Myelopathy n n ± ± ±
Neurologic dysfunction + ++ ++ ++ ++
Psychiatric symptoms ± ±
Digestive Failure to thrive ± ++ +
Liver dysfunction ± ±
Eye Maculopathy/retinopathy ± ± ± ±
Nystagmus ± ± ± ±
Vision, impaired ± ± ± ±
Haematological Anaemia, megaloblastic ± + + + ±
Musculoskeletal Dysmorphic features ± ±
Renal Haemolytic uraemic syndrome ± ± ±
Other Life-threatening illness + + ± ± ±
Low birth weight ±
Routine laboratory Neutrophils, hypersegmented ± ± ± ± ±
Special laboratory 3-Hydroxypropionic acid (U) ↑ ↑ ↑ ↑ ↑
C3 propionylcarnitine (P, B) ↑ ↑ ↑ ↑ ↑
Homocysteine, total (P) ↑ ↑ ↑ ↑ ↑
Homocysteine (U) ↑ ↑ ↑ ↑ ↑
Methionine (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Methylmalonic acid (P, U) ↑ ↑ ↑ ↑ ↑
S-Adenosylmethionine (P, CSF) ↓ ↓ ↓ ↓
a
Combined deficiencies in adulthood only reported in cblC deficiency to date
13.5 Diagnosis may be normal in patients with TC deficiency and inborn

errors of intracellular Cbl metabolism. In these disorders,
Haematologic investigations and measurement of serum Cbl measurement of the two metabolic markers MMA and tHcy
can be helpful. Cbl levels below 125 pmol/L are almost which are sensitive indicators of Cbl deficiency is indicated.
always indicative of Cbl deficiency. However, symptomatic Limitations are the specificity of tHcy which is also increased
patients with low-normal Cbl that are responsive to Cbl treat- in folate deficiency and the complexity and cost of the assay
ment have been reported. Accumulating evidence indicates for MMA. Further studies are usually required to determine
that plasma measurements of the biologically active Cbl the cause of Cbl deficiency, e.g. tests for investigating Cbl
holotranscobalamin may be superior to serum Cbl, and these absorption or enzymatic and genetic complementation stud-
are being introduced increasingly into the clinical setting ies in defects of Cbl metabolism. These tests should be per-
(Nexo and Hoffmann-Lücke 2011). However, both markers formed in laboratories with specific expertise.
Infant Child Adolescent Adult

Analyte <1 year 1–12 years 12–18 years >18 years
Serum holotranscobalamin (pmol/l) 37–170
Plasma propionylcarnitine (C3) (μmol/l) <0.55
Blood propionylcarnitine (C3) (μmol/l) <3.2
Plasma quantitative amino acids (μmol/l)
Total homocysteinea 3.3–8.3 4.7–10.3 <15
Methionine 11–31 11–30 16–23 14–34
S-Adenosylmethionine (nmol/l) 29.6–118
Urinary quantitative amino acids (mmol/mol creatinine)
Homocyst(e)ine 0.2–3.7
Plasma/urinary quantitative organic acids (μmol/l/mmol/mol creatinine)
Methylmalonic acid (P) <0.3
Methylmalonic acid (U) <7.8 <4.6 <3.3
Methylcitric acid (U) <25 <18.8 <8.6
3-Hydroxypropionic acid (U) <21 <23 <9
Amino acids in CSF (μmol/l)
Methionine 2.05–4.7 1.6–2.9
S-Adenosylmethionine (nmol/l) 189–243 61–201
a
Cave preanalytics
214
Megaloblastic Vitamin Holotranscobalamin MMA C3 propionylcarnitine MMA Methylcitric 3-Hydroxypropionic Homocysteine Homocyst(e) Methionine
Disorder anaemia B12 (S) (S) (P) (B) (U) acid (U) acid (U) (P) ine (U) (P)
13.1 IFD + ↓↓ ↑-↑↑ n-↑ ↑-↑↑ n-↑ n-↑ ↑↑ ↑↑ n-↓
13.2.1 + ↓↓ ↑-↑↑ n-↑ ↑-↑↑ n-↑ n-↑ ↑↑ ↑↑ n-↓
IGS
13.2.2 + ↓↓ ↑-↑↑ n-↑ ↑-↑↑ n-↑ n-↑ ↑↑ ↑↑ n-↓
AMN
13.3 HCD n ↓ n n
13.4 TCD + n-↓ ↑-↑↑ n-↑ ↑-↑↑ n-↑ n-↑ ↑↑ ↑↑ n-↓
13.5 TCR ↓↓ ↑ n-↑
13.6 cblA n n n ↑↑ ↑-↑↑ ↑↑ ↑-↑↑ ↑-↑↑ n n n
13.7 cblB n n n ↑↑↑ ↑↑ ↑↑↑ ↑↑ ↑↑ n n n
13.8 n n n ↑-↑↑ ↑ ↑-↑↑ ↑ ↑ n n n
cblD-
MMA
13.9 + n n n n n n n ↑↑ ↑↑ ↓
cblD-HC
13.10 + n n n n n n n ↑↑ ↑↑ ↓
cblG
13.11 + n n n n n n n ↑↑ ↑↑ ↓
cblE
13.12 + n n ↑-↑↑ ↑ ↑-↑↑ ↑ ↑ ↑↑↑ ↑↑↑ ↓↓
cblC
13.13 + n n ↑-↑↑ ↑ ↑-↑↑ ↑ ↑ ↑↑ ↑↑ ↓
cblD-
MMA/HC
13.14 + n n ↑-↑↑ ↑ ↑-↑↑ ↑ ↑ ↑↑ ↑↑ ↓
cblF
13.15 cblJ (+) n n ↑-↑↑ ↑ ↑-↑↑ ↑ ↑ ↑↑ ↑↑ ↓
M.R. Baumgartner and B. Fowler
Suspicion of vitamin B12-disorder / ↑ C3 on newborn screening
Nutritional history
vitamin B121/ methylmalonic acid2 / homocysteine3
↑ tHcy & ↑ MMA ↑ MMA, tHcy normal ↑ tHcy, MMA normal
Loading test with parenteral OH-Cbl Methionine
Normalization of Incomplete Persistent

MMA & tHcy response4 MMAuria Normal - ↑ ↓ - Normal
± related
metabolites
Dietary history
Nutritional Investigate
Negative Positive B12 deficiency CBS-deficiency
Propionate incorporation5 Propionate incorporation5 Methionine synthesis5

Holo-
Methionine synthesis, Enzymes ± OH-Cbl, Enzymes ± OH-Cbl, Enzymes
transcobalamin
Complementation analysis Complementation analysis Complementation analysis
Normal Low
Absorption Transcobalamin Intracellular defect of Intracellular defect of Intracellular defect of

defects deficiency Ado- / MeCbl-synthesis AdoCbl-synthesis MeCbl-synthesis
Mutation analysis of specific gene
1serum vitamin B12; 2plasma or urinary methylmalonic acid (MMA); 3plasma total homocysteine (tHcy); 4cblF may show complete response
5functional/enzymatic studies in cultured fibroblasts
Fig. 13.2
13.9 Specimen Collection 13.9 cblD-HC Cultured fibroblasts:

13.10 cblG Methionine synthesis from formate/
MeTHF
Overview on methods and required samples for enzyme and mutation 13.11 cblE Cobalamin coenzyme synthesis
analysis 13.12 cblC Cultured fibroblasts:
Metabolite Sample 13.13 cblD-MMA/HC Propionate fixation
Methylmalonic acid Plasma 13.14 cblF Methionine synthesis from formate/
Total homocysteine Plasmaa MeTHF
Holo-transcobalaminb Plasma or serum 13.5 cblJ Cobalamin coenzyme synthesis
S-Adenosylmethionine Plasmac Mutation analysis
CSF All disorders DNA analysis possible
Disorder Enzyme assaysd: method a
Immediate separation of plasma essential
b
13.6 cblA Cultured fibroblasts: propionate fixation, Stable parameter (Nexo and Hoffmann-Lücke 2011)
c
methylmalonyl-CoA mutase EDTA blood on ice, immediate separation of plasma and
13.7 cblB Lymphocytes: methylmalonyl-CoA deproteinization
d
mutase In general, enzymatic workup as a first step is advised. In some cases
13.8 cblD-MMA
(common mutation or small gene), mutation analysis as first step
13.10 Prenatal Diagnosis therapy is 1,000 μg of parenteral cyano-Cbl or hydroxo-Cbl

daily for 1 week, followed by 100 μg cyano-Cbl weekly for
1 month and monthly thereafter. This therapy is believed to
Tests recommended and sample
replete and sustain body Cbl stores.
Disorder requirements
Treatment of diseases caused by defects of intracellular
13.6 cblA CVS DNAa
13.7 cblB Cultured amniocytes – enzymatic assays
processing of Cbl usually requires very large doses of
13.8 cblD-MMA Cell-free amniotic fluid – MMA, cofactor, i.e. 1,000 μg or even more of parenteral hydroxo-
methylcitrate Cbl two to three times weekly (Watkins and Rosenblatt
13.9 cblD-HC CVS DNAa 2011). In patients with combined MMA and Hcy (cblC,
13.10 cblG Cultured amniocytes – enzymatic assays cblD, cblF, cblJ) and in those with isolated Hcy (cblE,
13.11 cblE Cell-free amniotic fluid – tHcy cblG, cblD-HC), treatment with parenteral hydroxo-Cbl,
13.12 cblC CVS DNAa betaine, folate and theoretically methionine usually cor-
13.13 cblD-MMA/HC Cultured amniocytes – enzymatic assays rects the haematologic abnormalities and improves but usu-
13.14 cblF Cell-free amniotic fluid – tHcy, MMA, ally does not completely correct the biochemical
methylcitrate
abnormalities. Moreover, neurological symptoms respond
13.15 cblJ
only partially, and severe neurological deficits often persist.
CVS chorionic villous sampling
a
First choice if disease causing mutations and their parental origin
Treatment of isolated MMAurias consists of dietary restric-
confirmed tion of protein, carnitine supplementation and, in Cbl-
responsive patients, parenteral hydroxo-Cbl 2× weekly.
While most patients with cblA will respond even to oral
hydroxo-Cbl, only about one third of cblB patients and
13.11 Treatment even fewer patients with mut will do so (Matsui et al. 1983).
Thus, it is important to reliably classify these patients and
In patients with defects of Cbl absorption and transport, identify those who genuinely benefit from hydroxo-Cbl
treatment with relatively small amounts of Cbl, administered treatment using a standardised test (Fowler et al. 2008).
by injection to circumvent the barrier of the intestinal
mucosa, generally results in rapid resolution of biochemical Emergency and Standard Treatment Tables and
abnormalities and symptoms of deficiency. The conventional Medication Requirements
Drugs and dosages to be used in acute presentation of suspected disorders of vitamin B12 metabolism
Hydroxo-Cbl Folic or folinic acid Sodium benzoate (to be
(CN-Cbl less (never give without L-arginine hydrochloride (to given IV in glucose
Disorder effective) Cbl) L-carnitine be given IV in glucose 10 %) 10 %)
All disorders 1–5 mg/day Folic acid: 15 mg/kg/ 50–100 mg/kg/daya – –
i.m., i.v. or s.c. day (i.v. in three
doses)
Folinic acid: 3 mg/kg/
day (i.v. in one dose)
Disorders with Same Optional 50–200 mg/kg/day i.v. 250 mg/kg (1.2 mmol/kg) as 250 mg/kg as bolus
MMA only bolus in 90–120 min, then in 90–120 min,
(cblA, cblB, maintenance 250 mg/kg/day then maintenance
cblD-MMA)b (1.2 mmol/kg/day) 250–500 mg/kg/dayc
> 20 kg bw: 5.5 g/m2/
day
a
Optional
b
For more details see methylmalonyl-CoA mutase deficiency in Chap. 7 (disorder 7.18)
c
If on haemodialysis/haemodiafiltration, doses should be increased to 350 mg/kg/day (maintenance dose)
Dosages of drugs to be used for long-term treatment

Hydroxo-Cbl
(Cyano-Cbl Folinic/folic acid
generally less (never give without
Disorder effective) Cbl) L-Carnitine Diet Betaine
13.1 IFD 1 mg i.m. or s.c. Folic acid up to – – –
13.2.1 and every 1–3 months 10 mg 4× daily
13.2.2 IGS
13.3 HCD – – – – –
13.4 TCD 0.5–1 mg p.o. 2× Folic acid up to – – –
weekly or 1 mg i. 10 mg 4× daily
m. 1× weekly
13.5 TCR ? – – – –
13.6 cblA 1 mg i.m. or s.c. – 50–100 mg/kg/day Protein restriction and –
13.7 cblB 1–7× weeklyb supplementation of precursor-
13.8 free amino acid mixtured
cblD-MMAa
13.9 cblD-HC 1 mg i.m. or s.c. Folinic acid 15 mg/ – – Up to 250 mg/kg/day
13.10 cblG daily dayd
13.11 cblE
13.12 cblC 1 mg i.m. or s.c. Folinic acid 15 mg/ 50 mg/kg/dayd Moderate protein restrictiond Up to 250 mg/kg/d
13.13 cblD dailyc dayd
13.14 cblF
13.15 cblJ
a
See also methylmalonyl-CoA mutase deficiency in Chap. 7 (disorder 7.18)
b
Cbl responsiveness should be carefully tested starting with 1–2 mg OHCbl i.m. or s.c. for several days followed by titrating cobalamin dosage
against methylmalonate excretion and clinical status in cobalamin-responsive patients
c
In cblC up to 20 mg OHCbl daily may be needed; the OHCbl should be titrated to control the patients’ methylmalonic acidemia and homocystin-
uria (Carillo-Carrasco et al. 2009)
d
Optional
Experimental Treatment Coelho D, Suormala T, Stucki M et al (2008) Gene identification for the
In the cblC defect, prenatal diagnosis and treatment of the cblD defect of vitamin B12 metabolism. N Engl J Med 358:1454–1464
Coelho D, Kim JC, Miousse IR et al (2012) Mutations in ABCD4 cause a
affected foetus by administration of hydroxo-Cbl i.m. in the new inborn error of vitamin B12 metabolism. Nat Genet 44:1152–1155
mother have been reported (Huemer et al. 2005). Furthermore, Fowler B, Leonard JV, Baumgartner MR (2008) Causes of and diagnos-
attempts to treat this disorder with antioxidants and creatine tic approach to methylmalonic acidurias. J Inherit Metab Dis
have been made. In the cblA and cblB defects, transplanta- 31:350–360
Fyfe JC, Madsen M, Hojrup P et al (2004) The functional cobalamin
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in methylmalonic acidurias is influenced by the underlying defect
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Biotin Disorders
14
Bruce A. Barshop
Contents Summary
14.1 Introduction.................................................................... 219 Disorders in the processing of biotin present with deficien-
14.2 Nomenclature ................................................................. 220 cies of the biotin-dependent carboxylases, i.e., multiple car-
boxylase deficiency. The biochemical and clinical
14.3 Metabolic Pathways ....................................................... 220
abnormalities reflect those observed in individual, isolated
14.4 Signs and Symptoms ...................................................... 221 defects of three mitochondrial carboxylases: methylcrotonyl-
14.5 Diagnosis ......................................................................... 222 CoA carboxylase, propionyl-CoA carboxylase, and pyruvate
14.6 Reference and Pathological Values ............................... 223
carboxylase. Multiple carboxylase deficiency is caused by
defects in holocarboxylase synthetase or in biotinidase.
14.7 Specimen Collection....................................................... 223 Treatment of biotinidase deficiency with biotin supplementa-
14.8 Prenatal Diagnosis ......................................................... 223 tion is highly effective in reversing the abnormalities, and
14.9 Treatment........................................................................ 223 that is usually also the case for the treatment of holocarbox-
ylase deficiency, though there may be some variability of
14.10 Follow-Up and Monitoring............................................ 224
response.
References .................................................................................... 224
14.1 Introduction
Biotin is a vitamin cofactor used in carboxylation reactions

which fix CO2 from bicarbonate to substrates to generate car-
boxylic acid moieties; in fact each biotin-dependent reaction
in human metabolism converts a carboxylic acid (or CoA
ester) to a dicarboxylic acid (or CoA ester). In its active form,
and the form most often ingested, biotin is covalently bound
to lysine on the carboxylase holoenzyme. Utilization of bio-
tin requires its transfer to the correct lysine residue of the car-
boxylase apoenzymes to generate the active holoenzymes, a
reaction carried out by holocarboxylase synthetase (HCS).
Recovery of biotin, from endogenous carboxylase proteins
as they are proteolyzed and from most protein-bound dietary
sources, requires cleavage of the lysine conjugate (biocytin),
B.A. Barshop a reaction catalyzed by the enzyme biotinidase. Deficiency
Department of Pediatrics, UCSD School of Medicine,
La Jolla, CA 92093-0830, USA of biotinidase or HCS causes a functional defect in all the
e-mail: bbarshop@ucsd.edu carboxylases, termed multiple carboxylase defect (MCD).
220 B.A. Barshop
14.2 Nomenclature

No. Disorder Name Abbreviation symbol localization Affected protein OMIM no. Subtype
14.1 Biotinidase Late-onset BTD BTD 3p25.1 Biotinidase 253260 All forms
deficiency multiple deficiency
carboxylase
deficiency
14.2 Holocarboxylase Infantile-onset HCSD HLCS 21q22.13 Holocarboxylase 253270 All forms
synthetase multiple synthetase
deficiency carboxylase
deficiency
Fig. 14.1 Free Biotin
Transport
Lysine NH NH
H H
S COOH
Biotinidase Biotin
O (free pool)
NH NH Apocarboxylases
H H (ACC, MCC, PCC, PC)
H
N COOH
S
O NH2
Holocarboxylase
Proteolysis Synthetase
Dietary and other

protein-bound biotin Holocarboxylases
Bbiotinylated
(ACC, MCC, PCC, PC)
Hi stones
Leucine
Propionyl-CoA
Malonyl-CoA Methylmalonyi-CoA Oxaloacetate

3-Methylcrotonyi-CoA
Acetyl-CoA Pyruvate
3-Methylglutaconyi-CoA
Fatty acid Leucine Propionate Gluconeogenesis,
synthesis catabolism metabolism Anaplerosis
HCS is a complex enzyme which activates biotin to form There are four biotin-dependent enzymes in human
D-biotinyl-5΄-adenylate and then catalyzes the attachment of metabolism. Acetyl-CoA carboxylase (ACC) is used to gen-
the biotin to an active site ε-amino group of a lysine residue erate malonyl-CoA which is important in initiation of the
of the newly synthesized apocarboxylase enzyme. The cova- synthesis of fatty acids and regulation of their oxidation.
lent binding to biotin conveys enzymatic activity and ACC is present in two isomers, one of which (ACC1) is
holocarboxylase status to the apocarboxylase protein. cytoplasmic and the other of which (ACC2) is associated
14 Biotin Disorders 221
with the endomembrane system (including primarily the with treatment and while compensated, the neurological
cytosolic side of the outer mitochondrial membrane). There function is otherwise expected to be normal, and the neuro-
are as yet no know disorders due to defects in ACC, but the logical examination may be normal despite a hyperammone-
other three carboxylases, which are all localized to the mito- mic episode (Dabbagh et al. 1994). Muscular hypotonia and
chondrial matrix, each have a disease state associated with hypertonia have been described, as have more severe forms
its deficiency. These include methylcrotonyl-CoA carboxyl- of dystonia and movement disorders, including athetosis and
ase (2 subunits, MCCC1 and MCCC2), propionyl-CoA opisthotonus. There may be electroencephalographic abnor-
carboxylase (2 subunits, PCCA and PCCB), and pyruvate malities and abnormal findings on cranial computed tomog-
carboxylase (PC). raphy or magnetic resonance imaging, particularly involving
The first patient ascertained with MCD had HCSD and the white matter. Subependymal cysts were observed in one
was described in 1971 (Gompertz et al. 1971) as having an infant and reported to disappear following 6 months of treat-
abnormality of leucine metabolism due to identification of ment (Squires et al. 1997), and subependymal cysts were
3-methylcrotonylglycine and 3-hydroxyisovaleric acid in also observed in 7 Samoan infants who had severe disease,
the urine. A defect was found in 3-methylcrotonyl-CoA car- incomplete responsiveness to biotin, and early life-
boxylase (Gompertz et al. 1973). When methylcitric and threatening metabolic events (Wilson et al. 2005), as well as
hydroxypropionic acids were also found to be increased in a Samoan infant in an earlier series, who had a poor derma-
the same patient in 1977 (Sweetman et al. 1977), enzymatic tological response to biotin (Sweetman et al. 1982).
analysis revealed defective activity of propionyl-CoA car- The biochemical hallmark of this disease is the excretion
boxylase (Weyler et al. 1977) in addition. The third mito- of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine,
chondrial carboxylase, pyruvate carboxylase, was also plus elevated quantities of lactic acid in blood and urine. The
shown to be defective in activity (Saunders et al. 1979), and first clinical chemical clue to the disease may be the discov-
the disorder was then renamed multiple carboxylase ery of lactic acidemia. Organic acid analysis during an acute
deficiency. acidosis also reveals methylcitric and 3-hydroxypropionic
acids and may also include tiglylglycine (Sweetman et al.
1982). The excretion of 3-hydroxyisovaleric acid is almost
14.4 Signs and Symptoms always greater than that of 3-methylcrotonylglycine and may
be as high as 200 times normal (Sweetman et al. 1977).
Holocarboxylase Synthetase Deficiency. Patients with HCS Biotinidase Deficiency. Biotinidase deficiency presents
deficiency generally present in the first days or months of life with a median age of 3 months (Wolf et al. 1983a), but it may
with overwhelming illness identical to those of propionic present in the second decade of life (Wolf et al. 1998). In
acidemia or other classic organic acidemia. The age of onset earlier literature biotinidase deficiency was referred to as the
of clinical symptoms generally has generally been before later infantile form of multiple carboxylase deficiency (Wolf
6 weeks of life (Burri et al. 1985), but it is clear that patients et al. 1983a, b) to distinguish it from the usual neonatal pre-
with an abnormal holocarboxylase synthetase can present at sentation of holocarboxylase synthetase deficiency. It is also
any age from 1 day to 6 years of age (Suormala et al. 1997; possible for adults with profound biotinidase deficiency to
Sherwood et al. 1982). remain asymptomatic, although those individuals would be
In the acute episode of illness, the infant has massive predicted to be at ongoing risk for symptoms to arise at times
ketosis and metabolic acidosis with an anion gap. There may of intercurrent infection or other stress. Such individuals
be tachypnea or Kussmaul breathing, and blood ammonia have been ascertained because of abnormal results on new-
may be elevated. The episode may progress to dehydration born screening of their babies (Wolf et al. 1997).
and deep coma, and a number of patients have died of this The cutaneous lesions tend to be patchy (Bartlett et al.
disease; the initial episode may be lethal within hours of 1980; Thoene et al. 1981), in contrast to the total body erup-
birth (Sweetman et al. 1982). Cutaneous features are an inte- tion seen in holocarboxylase synthetase deficiency, or there
gral part of the untreated disease, though some patients have may be severe generalized involvement of the skin with red-
died before the development of skin lesions, and now patients ness and desquamation. Concomitant mucocutaneous candi-
are being treated before the development of cutaneous diasis is common. The alopecia may be progressive to
lesions. An erythematous eruption may involve the entire alopecia totalis but is usually less than total.
body, with bright red, scaly, or desquamative lesions. Neurological manifestations of biotinidase deficiency
Complicating monilial infection is common. Varying degrees tend to be indolent and significant. Ataxia is a prominent fea-
of alopecia are seen, including alopecia totalis, with absence ture and may interfere with walking (Thoene et al. 1981).
of eyelashes, eyebrows and lanugo, as well as the hair of the Seizures are common and may be the only obvious symptom
head. There may be persistent vomiting and failure to thrive. (Salbert et al. 1993); they may be general or myoclonic or
Neurological abnormalities appear to be related to the effects may present as infantile spasms. There may be developmen-
of the initial or subsequent episodes of illness, which might tal delay and neurodevelopmental regression. Stridorous
include decreased brain perfusion and hyperammonemia; breathing and apnea have been reported in some patients,
222 B.A. Barshop
Table 14.1 Biotinidase deficiency

Cardiovascular Valvulitis, mitral ± ++ ++, * * *
CNS Ataxia ± + ++, * * *
Bulbar dysfunction ± ± +, * * *
Developmental delay ± + ++, * * *
Seizures ± + ++, * * *
Dermatological Alopecia ± + ++, * * *
Skin rash ± + ++, * * *
Digestive Glossitis ± ++ +, * * *
Stomatitis ± ++ +, * * *
Ear Hearing loss − + ++, * * *
Eye Corneal erosion − + ++, * * *
Optic atrophy − ± +, * * *
Respiratory Stridor, inspiratory ± ± ±, * * *
Routine laboratory Lactate (P) ± + ++, * * *
Special laboratory 3-Hydroxypropionic acid (U) ± ++ +++, * * *
3-Methylcrotonylglycine (U) ± ++ +++, * * *
C5-OH-Carnitine (P, B) ± ++ +++, * * *
Methylcitric acid (U) ± ++ +++, * * *
*Expression of symptoms highly depends upon adequacy of and compliance with treatment
Table 14.2 Holocarboxylase synthetase deficiency

Cardiovascular Valvulitis, mitral ++ +++ +++, * * *
CNS Ataxia ± ++ ++, * * *
Bulbar dysfunction ± ± +, * * *
Developmental delay + ++ +++, * * *
Seizures + ++ +++, * * *
Dermatological Alopecia ++ +++ +++, * * *
Alopecia ++ +++ +++, * * *
Skin rash ++ +++ +++, * * *
Routine laboratory Lactate (P) + +++ +++, * * *
Special laboratory 3-Hydroxypropionic acid (U) + ++ +++, * * *
3-Methylcrotonylglycine (U) + ++ +++, * * *
C5-OH-Carnitine (P, B) + ++ +++, * * *
Methylcitric acid (U) + ++ +++, * * *
*Expression of symptoms highly depends upon adequacy of and compliance with treatment
may be a presenting sign, and may be expected to resolve 14.5 Diagnosis

with biotin treatment. The untreated disease may be fatal
(Wolf et al. 1983a; Sander et al. 1980). Biotinidase deficiency is diagnosed by enzyme assay. It is
Visual and auditory neurosensory abnormalities have generally conducted as a fluorometric assay and routinely
been reported in a considerable number of patients, often as performed on bloodspots in newborn screening programs
late complications. Loss of visual function is associated with worldwide (Heard et al. 1986); serum or plasma samples
optic atrophy and appears to be encountered only in patients may be assayed also, and that is usually done for confir-
for whom diagnosis and treatment is delayed (Wolf 2011). mation. The enzyme defect can be demonstrated whether
Neurosensory hearing loss seems to follow the same pattern the patient takes biotin or not, and the objective is to
(Wolf 2011). Many of the neurological features of disease make the diagnosis prior to other biochemical changes.
disappear in response to treatment with biotin, as do the cuta- In the event of delayed treatment, the first biochemical
neous and metabolic features; but sensorineural abnormali- changes generally include elevation of blood lactate and
ties involving the optic and auditory nerves are among those urine 3-hydroxyisovalerate and 3-methylcrotonylglycine.
which are not reversible once they have appeared. The diagnostic findings of holocarboxylase synthetase
deficiency are similar in terms of organic acid changes, 14.8 Prenatal Diagnosis
but enzymatic confirmation is more complicated. The
assay may involve formation of acid-precipitable radio- Though feasibility of prenatal diagnosis of biotinidase defi-
label from H14CO2 in the presence of apocarboxylases ciency by enzymatic assay activity in amniocytes was dem-
prepared from biotin-deficient rats (Burri et al. 1981), but onstrated as early as 1984 (Secor McVoy et al. 1984),
that is a difficult assay to validate to clinical standards. prenatal diagnosis is rarely undertaken, probably because
Presumptive diagnosis may be made in fibroblast cultures outcome is expected to be favorable with treatment. Prenatal
which are grown in biotin-depleted media to check the testing of amniocytes and chorionic villi has yielded evi-
activities of carboxylases (Feigenbaum et al. 2009). A dence of normal fetuses and heterozygotes (Pomponio et al.
practical solution may be to use p67, a peptide comprising 1998; Chalmers et al. 1994). If disease-causing mutations
the 67 C-terminal amino acids of propionyl-CoA carbox- have been identified in the family, it is recommended to use
ylase, as the substrate (Rios-Avila et al. 2011). Molecular DNA-based methods if prenatal diagnosis is desired (Wolf
methodology to document DNA mutations (Morrone 1993). In holocarboxylase synthetase deficiency, amniotic
et al. 2002; Pindolia et al. 2010) is increasingly practical fluid at 16 weeks gestation showed methylcitrate and
as a primary diagnostic step, and molecular studies are 3-hydroxyisovalerate to be only slightly elevated, but enzyme
recommended to confirm an enzymatic diagnosis. assay was diagnostic in amniocytes (Suormala et al. 1998)
and may also be applied to chorionic villi (Thuy et al. 1999).
Given the complexity of the enzyme assay for HCS, molecu-
14.6 Reference and Pathological Values lar analysis affords advantages (Malvagia et al. 2005).
Urine organic acids reference values

Compound Range (mml/mol creatinine) 14.9 Treatment
Lactic acid 0–197
3-Hydroxyisovalerate 0–58 Supplementation with pharmacologic doses of biotin is the
3-Hydroxypropionate 0–24 cornerstone of therapy. Doses of 5–20 mg daily are generally
Propionylglycine 0–2 used. In most cases, patients respond quickly and most
Pyruvate 0–22 symptoms are reversible; when dosage is inadequate or is
3-Methylcrotonylglycine 0–2 stopped due to error or noncompliance, symptoms may reap-
Methylcitrate 0–5 pear. Currently, dosage of biotin in the range of 5–10 mg per
day appears to be adequate and effective during childhood
(Wolf 2010). Usually the dose is not changed, so with growth,
Urine organic acids pathological values the body mass-normalized dosage tends to decrease. To
Compound Range (mml/mol creatinine) assure that the dosage is adequate and that the patient is in
Lactic acid Varies (generally >300, often >1,000) compliance, some have monitored urinary organic acids
3-Hydroxyisovalerate Varies (generally >100) (3-hydroxyisovleric and 3-methylcrotonylglycine) and/or
3-Hydroxypropionate Varies (generally >40) plasma acylcarnitines (C5OH-carnitine) (Wolf 2010). There
Propionylglycine Varies (generally >10) is some concern arising from anecdotal reports of several
Pyruvate Varies (generally >40) females with biotinidase deficiency who have begun to lose
3-Methylcrotonylglycine Varies (generally >10) hair when entering puberty, with reversal when the dose is
Methylcitrate Varies (generally >15) increased to 15–20 mg from 10 mg.
Supplementation must be in the form of free biotin in bio-
tinidase deficiency (as opposed to some formulations mar-
keted in the health food industry which are derived from
yeast extracts and are composed of biocytin). The dosage is
determined only by the body’s requirements for biotin, since
Samples for biochemical diagnosis of biotin disorders
the supplemented biotin does not interact directly with the
Metabolite Enzyme Molecular biotinidase enzyme. In HCSD, however, there is a direct
No. Disorder analysis (OAs) assay characterization interaction at the biotin binding site of the enzyme, so the
14.1 Biotinidase U S, P, F B
effectiveness may be determined by the concentration of bio-
deficiency
14.2 HCS U F B
tin. A few cases have been encountered which have been
deficiency incompletely responsive or unresponsive to biotin (Wilson
Abbreviations: U urine, P plasma, S serum, B blood, F fibroblast, HCS et al. 2005; Feigenbaum et al. 2009; Morrone et al. 2002;
holocarboxylase synthetase, OAs organic acids Santer et al. 2003; Sakamoto et al. 1999) and those have
224 B.A. Barshop
proven to correspond to mutations outside of the biotin bind- Burri BJ, Sweetman L, Nyhan WL (1981) Mutant holocarboxyl-
ing domain (exons 4–8), resulting in decreased Vmax. ase synthetase: evidence for the enzyme defect in early infantile
biotin-responsive multiple carboxylase deficiency. J Clin Invest
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As opposed to HCSD, the rationale is not clear for boxylase synthetase in patients with biotin-responsive multiple car-
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Thiamine Disorders
15
Frédéric Sedel
Contents Summary
15.1 Introduction ...................................................................... 227 Thiamine is a water-soluble vitamin acting, in the mitochon-
dria, as a cofactor for energy metabolism and, in the cyto-
15.2 Nomenclature.................................................................... 229
plasm, in the pentose phosphate biosynthetic pathway. Its
15.3 Metabolic Pathways ......................................................... 229 transport through the plasma membrane requires two trans-
15.4 Signs and Symptoms Table .............................................. 230 porters with overlapping functions: THTR1 encoded by
15.5 Diagnostic Flowchart ....................................................... 230
SLC19A2 and THTR2 encoded by SLC19A3. Thiamine is
transformed into its active form, thiamine pyrophosphate
15.6 Reference and Pathological Values ................................. 230 (TPP), by a kinase encoded by the TPK1 gene. Then it may
15.7 Prenatal and DNA Testing ............................................... 231 enter the mitochondria through a TPP transporter encoded
15.8 Treatment Summary ........................................................ 231 by SLC25A19. Mutations in SLC19A2 cause thiamine-
responsive megaloblastic anemia (TRMA) characterized by
References .................................................................................... 231
megaloblastic anemia, progressive deafness, and diabetes
mellitus eventually associated with optic neuropathy.
Mutations in SLC19A3 cause biotin-/thiamine-responsive
basal ganglia disease characterized by episodes of severe
Leigh-like encephalopathy often triggered by fever that
respond to a combination of biotin and thiamine. Mutations
in SLC25A19 may cause early microcephaly with death in
infancy (also called Amish microcephaly) or a later-onset
bilateral striatal necrosis with progressive peripheral neu-
ropathy. Recently, mutations in the TPK1 gene have been
associated with recurrent encephalopathy with mild lactic
acidosis.
15.1 Introduction
Thiamine is a water-soluble aromatic vitamin that has to be

taken up from nutrition by many eukaryotes. In mammalian
systems, its active form, thiamine pyrophosphate, serves as
cofactor for aldehyde transfers, which include the oxidative
decarboxylation of α-keto acids such as pyruvic,
α-ketoglutaric, and branched-chain α-keto acids as well as
F. Sedel transketolase reaction.
Département de Neurologie, Unité neuro-métabolique et centre Thiamine requirement is directly related to both total
de référence des maladies lysosomales, GRC13UPMC,
caloric intake and the proportion of calories provided as car-
Université Pierre et Marie Curie-Paris 6,
AP-HP, Hôpital de la Salpêtrière, Paris, 75013, France bohydrates, and the recommended dose of thiamine for an
e-mail: frederic.sedel@psl.aphp.fr average, healthy adult is 1.4 mg/day or 0.5 mg of thiamine
228 F. Sedel
per 1,000 kcal consumed (Sechi and Serra 2007). Vitamin in toddlers. The diabetes mellitus is non-type I in nature,
B1 is active in the form of thiamine pyrophosphate (TPP) but with age of onset from infancy to adolescence. Optic atrophy
is absorbed in the form of either thiamine or thiamine mono- seems common (Lagarde et al. 2004). Cardiovascular abnor-
phosphate in the small intestine. Its intestinal transport is malities, including sudden death, stroke, high-output heart
rate-limited: a single dose of oral thiamine over 6 mg cannot failure, paroxysmal atrial tachycardia, atrial standstill, and
be absorbed (Tanphaichitr 2001). At the blood–brain barrier, congenital heart defects, have been reported. Significant
transport occurs by both passive and active mechanisms, neurologic deficit including stroke and focal or generalized
which allows a rapid correction of brain thiamine deficiency, epilepsy has been reported in 27 % of individuals with
mainly by passive diffusion, as happens after parenteral TRMA (Shaw-Smith et al. 2012). Even without thiamine
administration of the vitamin (Tanphaichitr 2001). supplementation, serum thiamine concentrations are normal;
In the brain, as in most other mammalian tissues, TPP is there is no evidence of acidosis or aciduria.
the most abundant thiamine derivative, amounting to Mutations in SLC19A3 cause biotin–thiamine-responsive
80–90 % of total thiamine. However, five other forms may be basal ganglia disease (MIM 607483), a subacute Leigh-like
present in various proportions: free thiamine and thiamine encephalopathy. Although the disease is a panethnic condi-
monophosphate generally account for 5–15 % of total thia- tion, it is most prevalent in Saudi Arabia and usually occurs
mine and have no known physiological function. The three in preschool- and school-aged children (Dabbagh et al. 1994;
remaining derivatives are thiamine triphosphate (ThTP) Ozand et al. 1998; Debs et al. 2010; Zeng et al. 2005; Tabarki
(Makarchikov et al. 2003) and the recently discovered ade- et al. 2013). The typical clinical presentation of patients with
nosine thiamine triphosphate (AThTP) (Bettendorff et al. BBGD is recurrent subacute episodes of encephalopathy,
2007) and adenosine thiamine diphosphate (AThDP) often triggered by febrile illness or mild trauma and charac-
(Frédérich et al. 2009). They are minor components, gener- terized by confusion, seizures, dystonia, external ophthal-
ally amounting to less than 1 % of total thiamine under nor- moplegia, and dysphagia eventually leading to coma and
mal physiological conditions, but they seem more likely to even death unless treatment with biotin and/or thiamine is
play specific roles as intracellular messengers or metabolic provided to the patient. Brain magnetic resonance imaging
regulators. (MRI) shows characteristic bilateral lesions of the caudate
In humans there are two isoforms of thiamine transporters nuclei and putamen. Other neuroimaging features, including
in the plasma membrane encoded by SLC19A2 and vasogenic edema, infra- and supratentorial cortex, and brain-
SLC19A3 (Ganapathy et al. 2004). The SLC19A2 gene stem involvement, have been reported (Debs et al. 2010;
encodes the human thiamine transporter 1 (hTHTR1) pro- Tabarki et al. 2013). Patients display no biological or clinical
tein, whereas human thiamine transporter 2 (hTHTR2) pro- signs of biotin deficiency and biotinidase and holocarboxylase
tein is encoded by the SLC19A3 gene. All SLC19 family synthetase activities are normal. Beside the “classical pheno-
genes are expressed ubiquitously, although at variable levels type,” several phenotypes with different levels of severity
in different tissues which probably explains different clinical have been linked to SLC19A3 mutations such as Wernicke’s-
presentations in SLC19A2- and SLC19A3-related diseases. like encephalopathy responding to thiamine alone (Kono
As soon as thiamine enters the cell, it is pyrophosphorylated et al. 2009) and severe early-onset encephalopathy with
in the cytosol by the thiamine pyrophosphokinase (TPK) to infantile spasms, generalized epilepsy, severe psychomotor
form the enzymatically active TPP. TPP is either bound to retardation, and spastic tetraparesis (Yamada et al. 2010;
the cytosolic thiamine-dependent enzyme transketolase from Gerards et al. 2013). In dogs, SLC19A3 mutations have
the pentose phosphate cycle or transported into mitochondria recently been involved in Alaskan Husky Encephalopathy
by means of the mitochondrial thiamine pyrophosphate car- (Vernau et al. 2013).
rier encoded by SLC25A19. There it serves as a cofactor for Defects of the mitochondrial thiamine pyrophosphate
pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, transporter SLC25A19, originally described as a deoxynu-
and branched-chain α-keto acid dehydrogenase. cleotide carrier, result in severe encephalopathy with micro-
Mutations in SLC19A2 have been identified as a cause of cephaly. The disease was originally discovered in the Amish
thiamine-responsive megaloblastic anemia syndrome population in North America. Small head circumference is
(TRMA or Rogers syndrome) that associates with megalo- already evident at the 20th gestational week; the patients do
blastic anemia, thrombocytopenia, diabetes mellitus, and not acquire any developmental milestones and die in infancy.
sensorineural deafness (Oishi and Diaz 2012; Diaz et al. In addition, a milder clinical presentation has been reported
1999; Labay et al. 1999). TRMA is exceedingly rare outside in four patients with episodes of flaccid paralysis and
of consanguineous pairings or isolated populations and encephalopathy associated with bilateral striatal necrosis and
approximately 40 pedigrees are known in various ethnici- chronic progressive polyneuropathy (MIM 607196) (Spiegel
ties. Onset of megaloblastic anemia is between infancy and et al. 2009).
adolescence. A clinical diagnosis of TRMA should be con- More recently, mutations in TPK1 have been found in five
sidered in individuals with megaloblastic anemia with nor- affected individuals from three families presenting with a
mal vitamin B12/folic acid levels. Progressive sensorineural variable degree of psychomotor retardation, progressive dys-
hearing loss has generally occurred early and can be detected tonia, and lactic acidosis. All affected individuals had several
15 Thiamine Disorders 229
crises, triggered by infections, and in four of five affected Besides the genetic defects within thiamine metabolism,
individuals, there was a clearly progressive course of the dis- thiamine deficiency is well known from nutritional deficits
ease (Mayr et al. 2011). where it causes beriberi or Wernicke’s encephalopathy.
15.2 Nomenclature
Chromosomal Affected
No. Disorder Alternative name Abbreviation Gene symbol localization protein OMIM no. Subtype
15.1 Thiamine-responsive THTR1 THTR1 SLC19A2 1q23.3 THTR1 603941 All forms
megaloblastic anemia deficiency transporter
syndrome (SLC19A2)
15.2 Wernicke’s-like SLC19A3 SLC19A3 2q36.3 Reduced folate 606152 All forms
encephalopathy and family of
BRBG (SLC19A3) micronutrient
transporter
15.3 Bilateral striatal Mitochondrial SLC25A19 SLC25A19 Amish 606521 All forms
necrosis (SLC25A19) thiamine microcephaly
pyrophosphate
carrier deficiency
Thiamine monophosphate (TMP)
plasma THTR1 THTR2

membrane (SLC19A2) (SLC19A3)
Thiamine pyrophosphate
Thiamine
(TPP)
5
TPP tr
Glucose 1-P
(SLC25A19)
Mitochondrion
Pentose Citric acid cycle
phosphate Glucose 6-P TPP
pathway NADP+
Pyruvate
NADPH Succinate
2 Oxaloacetate 1
DNA Ribose 5-P Ketoglutatare
Acetyl-CoA
4
Glyceraldehyde 3 Isocitrate citrate
phosphate
Serine dihydroxyacetone glycerol 3-phosphate

phosphate (DHAP)
sphingolipids plasmalogens phospholipids Branched chain acylCoAs
Branched chain aminoacids
Fig. 15.1 Thiamine enters the cell through THTR1 and THTR2 recep- drates and amino acids. In the cytosol, TPP is a cofactor of the transke-
tors encoded by SLC19A2 and SLC19A3 genes, respectively. It is then tolase (4) in the pentose phosphate pathway. This pathway leads to the
converted into thiamine pyrophosphate (TPP) by the thiamine phospho- production of NAPDH, an important cofactor in biosynthetic pathways,
kinase enzyme encoded by TPK1 (5). TPP enters the mitochondria as well as important intermediary molecules such as ribose 5-phosphate
through the TPP transporter (TPP tr) encoded by the SLC25A19 gene. needed for the synthesis of nucleic acids and GA3P for the synthesis of
In the mitochondria, TPP serves as a cofactor for ketoglutarate-(2), complex lipids including plasmalogens, sphingolipids, and phospholip-
pyruvate-(1) and branched-chain α-keto acid (3)-dehydrogenases that ids (Adapted from Lamari et al. (2013))
are involved in the mitochondrial energy production from carbohy-
230 F. Sedel
15.4 Signs and Symptoms Table
Table 15.1 Thiamine-responsive megaloblastic anemia syndrome (SLC19A2)

Cardiovascular Cardiomyopathy ± ± ± ±
CNS Epilepsy + + + +
Ear Deafness ± +++ +++ ++
Endocrine Diabetes mellitus ++ ++ +++ +++
Eye Optic neuropathy ± + + +
Hematologic Anemia, megaloblastic ++ +++ +++ ++
Thrombocytopenia ± ++ ++ ++
Routine laboratory Lactate (P) n n n n n
Thiamine in plasma n n n n n
Table 15.2 Wernicke’s-like encephalopathy and BRBG (SLC19A3)

CNS Basal ganglia lesions +++ +++ +++ +++
Dystonia ± ± ++ ++ ++
Encephalopathic crisis, acute +++ +++ +++ +++
Epilepsy, intractable ++ ± ± ±
Focal epilepsy ++ ++ ++
Routine laboratory Hemoglobin (B) n n n n
Lactate (P) n n n n
Pyruvate (P) n n n n n
Special laboratory Alanine (P) n n n n n
MRI: basal ganglia lesions +++ +++ +++ +++
Thiamine in plasma n n n
Table 15.3 Bilateral striatal necrosis (SLC25A19)

CNS Basal ganglia lesions +++ +++ +++
Dystonia ++ ++ ++
Encephalopathy acute, precipitated by infection +++ +++ +++
polyneuropathy +++ +++ +++
Routine laboratory Lactate (CSF) (↑) (↑) (↑)
15.5 Diagnostic Flowchart anemia, thrombocytopenia, hearing loss, diabetes).

SLC25A19 should be suspected in patients with Leigh syn-
The diagnosis of thiamine metabolism disorders relies on drome and progressive polyneuropathy as well as in patients
molecular analysis. No specific biochemical abnormalities with severe encephalopathy and microcephaly.
are found. Only patients with SLC25A19 mutations may dis-
play high blood lactate. Depending on the clinical context,
specific analyses can be recommended. SLC19A3 has to be 15.6 Reference and Pathological Values
sequenced in patients with Leigh syndrome and normal bio-
chemical tests in blood and CSF. SLC19A2 analysis is pre- All tests are usually normal including dosage of thiamine
dicted in patients with Rogers syndrome (megaloblastic (normal range 20–50 ng/ml).
15 Thiamine Disorders 231
15.7 Prenatal and DNA Testing Patients with SLC25A19 have not been reported to
respond to either biotin or thiamine. As a consequence,
Prenatal diagnosis can be proposed to siblings. DNA testing treatment is supportive only. This is also true for patients
is diagnostic in these disorders without any specific with TPK1 mutations.
biochemical marker.
References
Bettendorff L, Wirtzfeld B, Makarchikov AF et al (2007) Discovery of
a natural thiamine adenine nucleotide. Nat Chem Biol 3:211–212
In patients with TRMA, the anemia is corrected with thia- Borgna-Pignatti C, Azzalli M, Pedretti S (2009) Thiamine-responsive
mine (25–75 mg/day). However, the red cells remain mac- megaloblastic anemia syndrome: long term follow-up. J Pediatr
rocytic. The anemia can recur when thiamine is withdrawn. 155:295–297
Dabbagh O, Brismar J, Gascon GG, Ozand PT (1994) The clinical
High-dose thiamine supplementation may improve or spectrum of biotin-treatable encephalopathies in Saudi Arabia.
delay onset of diabetes mellitus (Valerio et al. 1998). Brain Dev 16:S72–S80
Hearing loss is irreversible and may not be prevented by Debs R, Depienne C, Rastetter A et al (2010) Biotin-responsive basal
thiamine treatment (Borgna-Pignatti et al. 2009). Whether ganglia disease (BBGD) in Europeans with novel SLC19A3 muta-
tions. Arch Neurol 67:126–130
treatment with thiamine from birth, or even prenatally, Diaz GA, Banikazemi M, Oishi K et al (1999) Mutations in a new gene
could reduce the hearing defect is a matter of conjecture. encoding a thiamine transporter cause thiamine-responsive megalo-
Management focuses on lifelong use of pharmacologic blastic anaemia syndrome. Nat Genet 22:309–312
doses (25–75 mg/day) of thiamine (vitamin B1) in affected Frédérich M, Delvaux D, Gigliobianco T et al (2009) Thiaminylated
adenine nucleotides. Chemical synthesis, structural characterization
individuals. The following tests are recommended to moni- and natural occurrence. FEBS J 276:3256–3268
tor the treatment and should be performed at least yearly: Ganapathy V, Smith SB, Prasad PD (2004) SLC19: the folate/thiamine
hematologic tests, CBC and reticulocyte count; assessment transporter family. Pflugers Arch 447:641–646
for glucose intolerance, fasting serum glucose concentra- Gerards M, Kamps R, van Oevelen J, Boesten I, Jongen E, de Koning B,
Scholte HR, de Angst I, Schoonderwoerd K, Sefiani A, Ratbi I,
tion; hearing test; ophthalmologic evaluation; and cardiac Coppieters W, Karim L, de Coo R, van den Bosch B, Smeets H
evaluation (Oishi and Diaz 2012). (2013) Exome sequencing reveals a novel Moroccan founder muta-
In patients with SLC19A3 mutations, early administration in SLC19A3 as a new cause of early-childhood fatal Leigh
tion of biotin and thiamine results in partial or complete syndrome. Brain 136:882–890
Kono S, Miyajima H, Yoshida K, Togawa A, Shirakawa K, Suzuki H
improvement within days. Treatment given later in the disor- (2009) Mutations in a thiamine-transporter gene and Wernicke’s-
der or the lack of treatment may result in death or neurologic like encephalopathy. N Engl J Med 360:1792–1794
sequelae including dystonia, quadriparesis, epilepsy, or mild Labay V, Raz T, Baron D et al (1999) Mutations in SLC19A2 cause
mental retardation. The optimal dose of biotin in this disease thiamine-responsive megaloblastic anaemia associated with diabe-
tes mellitus and deafness. Nat Genet 22:300–304
remains debated. Ozand et al. (1998) reported high doses of Lagarde WH, Underwood LE, Moats-Staats BM, Calikoglu AS (2004)
biotin: 5–10 mg/kg/day. Tabarki used lower doses (2–3 mg/ Novel mutation in the SLC19A2 gene in an African-American
kg/day) in addition to thiamine (100–300 mg/day) with the female with thiamine-responsive megaloblastic anemia syndrome.
same efficacy. Kono et al. (2009) used thiamine alone (100– Am J Med Genet A 125A:299–305
Lamari F, Mochel F, Sedel F, Saudubray JM (2013) Disorders of phos-
300 mg/day), while thiamine alone did not work in patients pholipids, sphingolipids and fatty acids biosynthesis: toward a new
published by Ozand et al. (1998). A few patients did not category of inherited metabolic diseases. J Inherit Metab Dis 36:
improve with high doses of biotin but did improve only after 411–425
the addition of thiamine suggesting that biotin and thiamine Makarchikov AF, Lakaye B, Gulyai IE et al (2003) Thiamine
triphosphate and thiamine triphosphatase activities: from bacteria to
may act synergistically (Debs et al. 2010). Overall, it is sug- mammals. Cell Mol Life Sci 60:1477–1488
gested to use a combination of high doses of biotin (3–10 mg/ Mayr JA, Freisinger P, Schlachter K et al (2011) Thiamine pyrophos-
kg/day) and thiamine during encephalopathic episodes and phokinase deficiency in encephalopathic children with defects in the
lower doses of biotin alone (2–3 mg/kg/day) for long-term pyruvate oxidation pathway. Am J Hum Genet 89:806–812
Oishi K, Diaz GA (2012) Thiamine-responsive megaloblastic anemia
treatment. Indeed, in the report from Ozand et al. (1998), no syndrome. In: Pagon RA, Bird TD, Dolan CR, Stephens K, Adam
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a month. Patients with SLC19A3 require regular clinical Ozand PT, Gascon GG, Al Essa M et al (1998) Biotin-responsive basal
ganglia disease: a novel entity. Brain 121:1267–1279
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Riboflavin and CoQ Disorders
16
Rita Horvath and Anne Lombès
Contents Summary
16.1 Introduction ..................................................................... 234 Riboflavin, or vitamin B2, is the precursor of flavin adenine
dinucleotide (FAD) and flavin mononucleotide (FMN),
16.2 Nomenclature................................................................... 235
which are essential cofactors of numerous dehydroge-
16.3 Metabolic Pathways ........................................................ 236 nases. The most common form of riboflavin disorder is its
16.4 Signs and Symptoms Tables for Each Disorder ........... 237 deficiency due to insufficient dietary intake. Riboflavin-
responsive inborn errors of metabolism were shown to be
16.5 Diagnosis .......................................................................... 239
due to the riboflavin chaperone-like function, which stabi-
16.6 Reference Values ............................................................. 240 lises mutant FAD-containing dehydrogenases, most often
16.7 Pathological Values Table ............................................... 240 ETFDH. Recessive mutations of SLC52A2 and SLC52A3
16.8 Diagnostic Flow Chart .................................................... 241 encoding the human riboflavin transporters RFVT2 and
RFVT3 cause Brown-Vialetto-Van Laere and Fazio-Londe
16.9 Specimen Collection ........................................................ 242
syndromes, while haploinsufficiency of SLC52A1 has been
16.10 Prenatal Diagnosis Table proposed to cause persistent riboflavin deficiency.
and Sample Requirements.............................................. 242
Ubiquinone (coenzyme Q10, CoQ10) is a lipid-soluble
16.11 DNA Testing Table and Sample Requirements ............ 242 component of the cell membranes, where it functions as
16.12 Treatment ......................................................................... 242 a mobile electron and proton carrier but also participates
in other cellular processes as a potent antioxidant and
References ...................................................................................... 243
by influencing pyrimidine metabolism. Five major clini-
cal phenotypes of CoQ10 deficiency have been described
(encephalomyopathy, multisystem infantile variant, cer-
ebellar form, Leigh syndrome, isolated myopathy) before
the identification of disease genes. An increasing number
of molecular defects in the CoQ10 biosynthetic pathways
have been identified in different clinical variants (PDSS1,
PDSS2, COQ2, COQ6, COQ9, CABC1/ADCK3). Despite
this, the number of reported patients is still low, and the
absence of clear genotype-phenotype correlations makes
the genetic diagnosis chellenging. In addition to primary
R. Horvath (*)
CoQ10 deficiencies, where the mutation impairs a pro-
Institute of Genetic Medicine, tein directly involved in CoQ10 biosynthesis, secondary
Newcastle University, Central Parkway, CoQ10 deficiencies include disease not due to deficient
Newcastle upon Tyne, NE1 3BZ, UK synthesis of CoQ10 but associated with decreased
e-mail: rita.horvath@ncl.ac.uk
CoQ10 levels, which may be contributing to the clinical
A. Lombès symptoms.
APHP, Biochimie Métabolique, GH Pitié-Salpêtrière,
Inserm UMRS 1016 Institut Cochin, CNRS UMR 8104,
Because of the beneficial effect of CoQ10 and riboflavin
Université Paris Descartes, Paris 75014, France supplementation, early recognition of riboflavin and CoQ10
e-mail: anne.lombes@inserm.fr diseases is important.
234 R. Horvath and A. Lombès
16.1 Introduction Five major clinical phenotypes of CoQ10 deficiency

have been described, which may overlap: (1) an encepha-
Riboflavin, or vitamin B2, is the precursor of flavin adenine lomyopathic form with exercise intolerance, mitochon-
dinucleotide (FAD) and flavin mononucleotide (FMN), drial myopathy, myoglobinuria, seizures and ataxia
which are essential cofactors of numerous dehydrogenases (Ogasahara et al. 1989); (2) a multisystem infantile variant
involved in β-oxidation, branched-chain amino acid catabo- with encephalopathy, cardiomyopathy, ataxia, optic nerve
lism, mitochondrial electron transport chain or the tricarbox- atrophy, deafness and nephrotic syndrome resulting in
ylic acid cycle. renal failure (Rotig et al. 2000); (3) a predominantly cer-
The most common form of riboflavin disorder is its ebellar form with ataxia and cerebellar atrophy (Lamperti
deficiency due to insufficient dietary intake causing anae- et al. 2003); (4) Leigh syndrome with growth retardation,
mia, cheilosis and stomatitis. Severe cases of riboflavin ataxia and deafness (Maldergem et al. 2002); and (5) an
deficiency resemble multiple acyl-CoA dehydrogenation isolated myopathic phenotype (Lalani et al. 2005; Horvath
defect (MADD). They have essentially been obtained in et al. 2006). Recent studies identified mutations in sev-
animal models but may be encountered in humans (Chiong eral nuclear genes encoding CoQ10 biosynthesis enzymes
et al. 2007). (PDSS1, PDSS2, COQ2, COQ9, CABC1, COQ6) (Quinzii
Riboflavin-responsive inborn errors of β-oxidation et al. 2005, 2006; López et al. 2006; Mollet et al. 2007,
were recognised almost 30 years ago (Gregersen 1985). 2008; Lagier-Tourenne et al. 2008; Duncan et al. 2009;
Most were MADD later shown to be caused by muta- Heeringa et al. 2011) and in genes outside that pathway
tions in the genes encoding the FAD-containing dehy- showing the secondary nature of some CoQ10 defects.
drogenases ETF and ETFDH (Olsen et al. 2007). In these The number of reported patients with identified mutations
cases the beneficial effect of riboflavin supplementation remains low, rendering it difficult to assign genotype-phe-
appeared due to the chaperone-like function of the cofac- notype correlations.
tor that stabilises the intramitochondrial mature enzyme The encephalomyopathic form was the first disease asso-
(Henriques et al. 2009) and not to a primary defect of ciated with CoQ10 deficiency in 1989 (Ogasahara et al.
riboflavin metabolism. 1989). Two sisters were reported with recurrent myoglobin-
Primary mutations of riboflavin transporters have been uria, epilepsy, mental retardation, mitochondrial myopathy,
recently identified. Mutations in SLC52A2 and SLC52A3 fluctuating creatine kinase (CK) elevation and lactic acido-
have been shown to be responsible for the recessive Brown- sis. Several similar patients have been reported (Quinzii and
Vialetto-Van Laere and Fazio-Londe syndromes (Green Hirano 2010; Auré and Benoist 2004). One case was caused
et al. 2010; Bosch et al. 2010). The progressive neurodegen- by mutations in the CABC1/ADCK3 gene (Mollet et al.
eration of these syndromes, dominated by pontobulbar 2008).
palsy, clearly differs from the acute metabolic presentation The causal mutations have been identified in several
of MADD (Green et al. 2010). However, careful metabolic multisystemic infantile variants of CoQ10 deficiency.
screening of patients disclosed abnormal plasma acylcarni- Mutations in the CoQ2 gene have been found in infan-
tines and urinary organic acids, which demonstrated the tile multisystem disease, including glomerulopathy with
defect of short-chain and medium-chain acyl-CoA dehydro- nephrotic syndrome, associated with a beneficial effect
genases (Bosch et al. 2010). The beneficial effect of ribofla- of CoQ10 supplementation (Quinzii et al. 2006; Mollet
vin supplementation was shown, but its long-term efficacy et al. 2007; Diomedi-Camassei et al. 2007). Mutations in
on neurodegeneration remains to be demonstrated. A het- the PDSS2 gene also cause infantile nephrotic syndrome
erozygous splice mutation in SLC52A1 has been hypothe- and Leigh syndrome (López et al. 2006), while mutations
sized to cause persistent riboflavin deficiency in a mother in the PDSS1 gene cause a multisystem disease including
who gave birth to a child with transient MADD (Chiong deafness, encephaloneuropathy, obesity, livedo reticularis
et al. 2007; Ho et al. 2011). and cardiac valvulopathy (Mollet et al. 2007). Mutations in
Ubiquinone (coenzyme Q10, CoQ10) deficiencies are a COQ6 have been identified in 13 patients from 7 families
heterogeneous group of autosomal recessive conditions with infantile steroid-resistant nephrotic syndrome, deaf-
affecting both children and adults. CoQ10 is a lipid-soluble ness and additional symptoms such as ataxia, epilepsy,
component of the cell membranes, where it functions as a muscle weakness and developmental delay in some cases
mobile electron carrier but also participates in other cellular (Heeringa et al. 2011). The causative mutations have not
processes as a potent antioxidant and by influencing pyrimi- yet been found for the first variant described by Rotig et al.
dine metabolism (Quinzii and Hirano 2010; Rahman et al. (Rotig et al. 2000) in 3 siblings with optic atrophy, nystag-
2012). It contains a benzoquinone ring and an isoprenoid mus, sensorineural hearing loss, ataxia, dystonia, muscle
chain. The relative role of these diverse CoQ10 functions in weakness and nephropathy (personal communication,
the disease symptoms is still unknown. Agnes Rötig).
16 Riboflavin and CoQ Disorders 235
Mutations in the CABC1/ADCK3 gene have been identified Secondary muscle CoQ10 deficiency has been
in 16 patients (9 families) with childhood-onset ataxia, often demonstrated in several instances such as mutations of the
associated with intolerance to exercise, psychomotor retarda- APTX gene, causing ataxia oculomotor apraxia (AOA1)
tion or epilepsy (Lagier-Tourenne et al. 2008; Mollet et al. 2008; (Quinzii et al. 2005; Le Ber et al. 2007), and mutations of the
Gerards et al. 2010). The causal mutations remain elusive in ETFDH or less frequently the ETFA and ETFB genes caus-
several groups of patients with cerebellar ataxia, seizures, pyra- ing mild, predominantly myopathic forms of multiple acyl-
midal signs and muscle CoQ10 deficiency (Lamperti et al. CoA dehydrogenase deficiency (glutaric aciduria type II)
2003; Musumeci et al. 2001; Gironi et al. 2004). No mutation (Gempel et al. 2007), primary mtDNA disease (mostly due
has yet been reported in the family with Leigh syndrome, to mtDNA mutations; Sacconi et al. 2010) or mtDNA deple-
ataxia and deafness and lactic acidosis (Maldergem et al. tion in one patient (Montero et al. 2009) and in the cardiofa-
2002). ciocutaneous syndrome (Aeby et al. 2007).
16.2 Nomenclature
Chromosomal
16.1 Brown-Vialetto-Van BVVLS SLC52A1 17p13.2 Riboflavin transporter 2 211530 All forms
Laere syndrome SLC52A2 8q24.3
16.2 Fazio-Londe Allelic with SLC52A3 20p13 Riboflavin transporter 2 211500 All forms
syndrome BVVLS
16.3 Prenyl diphosphate Decaprenyl diphosphate synthase PDSS1 PDSS1 10p12.1 Prenyl diphosphate 607429, All forms
synthase, subunit 1 (DPS) deficiency synthase 607426
(PDSS1) deficiency
16.4 Prenyl diphosphate Decaprenyl diphosphate synthase PDSS2 PDSS2 6q21 Prenyl diphosphate 610564, All forms
synthase, subunit 2 (DPS) deficiency synthase 607426
(PDSS2) deficiency
16.5 CoQ2 deficiency Mitochondrial COQ2 COQ2 4q21–q22 4-Hydroxybenzoate- 609825, All forms
4-hydroxybenzoate- polyprenyltransferase 607426
polyprenyltransferase deficiency
16.6 CoQ6 deficiency Early-onset steroid-resistant COQ6 COQ6 14q24.3 CoQ6 monooxygenase No All forms
nephrosis with sensorineural deficiency number
deafness yet
16.7 CoQ9 deficiency Primary coenzyme Q10 deficiency COQ9 COQ9 16q13 Coenzyme Q10 612837, All forms
607426
16.8 CABC1/ADCK3 Chaperone activity of BC1 CABC1/ ADCK3 1p42.2 AARF domain- 606980, All forms
deficiency complex-like, CABC1 deficiency ADCK3/ containing kinase 3 607426
COQ8
16.9 Ataxia oculomotor Aprataxin (APTX) deficiency: AOA1 APTX 9p13.3 Aprataxin 606350 All forms
apraxia 1 (AOA1) secondary coenzyme Q10
deficiency
16.10 Myopathic form of ETFDH ETFDH 4q32–qter ETFDH 231675 All forms
CoQ10 deficiency
(ETFDH)
We can differentiate primary and secondary riboflavin riboflavin level may nevertheless significantly contribute
or CoQ10 deficiencies (Fig. 16.1 Rahman et al. 2012). In to the clinical symptoms. It however depends on the pres-
primary deficiencies the mutation impairs a protein directly ence of irreversible damage before treatment and, espe-
involved in biosynthesis or transport of riboflavin or cially for CoQ10, on the bioavailability of the molecule in
CoQ10. In secondary defects, the decreased CoQ10 or the target tissues.

Pyruvate Acetyl-CoA Mevalonic Acid Farnesyl-PP
PDSS1
PDHC PDSS2
Para- Decaprenyl-PP
Acetyl COA hydroxybenzoate
COO–
PPO 10
Decaprenyl diphosphate
OH
PHB
Krebs cycle
Decaprenyl-PHB
pABA
COO– (Yah1 and Arh1)
PPO
in yeast
H+ NADH FAD OH H+ H+ ADP ADP
10
Decaprenyl-
PAB
Matrix
COX I
Inner ND1 ND4 e– e– e–
e– A8
membrane Cyt b Cyt c COX II
ND2 ND4L
e–
CoQ10 COX III
ND3 ND5 ND6 A6
Meo OH CH3
Meo
OH 10
CoQ10
Complex I Complex II Complex III Complex IV Complex V
Outer
membrane
Fig. 16.1 Schematic representation of the coenzyme Q10 biosynthetic pathway (Modified from the 176th ENMC workshop report, Rahman et al.
(2012))
16.4 Signs and Symptoms Tables

for Each Disorder
Table 16.1 Brown-Vialetto-Van Laere syndrome

CNS Pontobulbar palsy + ++ ++ ++
Ear Deafness, sensorineural + + + + +
Musculoskeletal Muscle weakness + +++ ++ +
Respiratory Respiratory insufficiency + +++ ++ +
Special laboratory C4–C18 acylcarnitines (P,B) n-↑ n-↑ n-↑ n-↑ n-↑
Dicarboxylic acids (U) n-↑ n-↑ n-↑ n-↑ n-↑
Table 16.2 Fazio-Londe syndrome

CNS Pontobulbar palsy + ++ ++ ++
Musculoskeletal Muscle weakness + +++ ++ +
Respiratory Respiratory insufficiency + +++ ++ +
Table 16.3 Prenyl diphosphate synthase, subunit 1 (PDSS1) deficiency

Cardiovascular Cardiomyopathy − − − + +
CNS Mental retardation − − + + +
Neuropathy, peripheral − − + + +
Digestive Obesity − − + + +
Ear Deafness − + + + +
Eye Optic atrophy − − + + +
Musculoskeletal Macrocephaly − − + + +
Table 16.4 Prenyl diphosphate synthase, subunit 2 (PDSS2) deficiency

CNS Epilepsy + +++ Unknown Unknown Unknown
Leigh syndrome + +++ Unknown Unknown Unknown
Musculoskeletal Muscle weakness +++ +++ Unknown Unknown Unknown
Renal Nephrotic syndrome + +++ Unknown Unknown Unknown
Table 16.5 CoQ2 deficiency

CNS Epilepsy + +++, death Unknown Unknown Unknown
Regression, psychomotor − − ++
Stroke-like episodes − − ++
Musculoskeletal Muscle weakness − +++ +++
Renal Nephrotic syndrome + + ++
Other Severe multisystem disease +++ Unknown Unknown Unknown Unknown

CNS Ataxia − − ±
Epilepsy − − ±
Renal Nephrotic syndrome − ± ++ ++

Autonomic system Hypothermia +++ See above See above See above
Cardiovascular Cardiomyopathy − ++ See above See above See above
CNS Epilepsy + +++ See above See above See above
Regression, psychomotor − +++ See above See above See above
Digestive Feeding difficulties ++ ++ See above See above See above
Renal Renal tubulopathy + +++ See above See above See above
Routine laboratory Lactic acidosis +++ +++ See above See above See above
Table 16.8 CABC1/ADCK3 deficiency

CNS Ataxia − + ++ ++ ++
Cognitive dysfunction − ± ± ± ±
Dystonia − ± ± ± ±
Epilepsy − + + + +
Pyramidal signs − ± ± ± ±
Musculoskeletal Muscle weakness − + + + +
Routine Laboratory Lactate (P) n-↑ n-↑ n-↑ n-↑ n-↑
Table 16.9 Ataxia oculomotor apraxia 1 (AOA1)

CNS Ataxia − − + ++ +++
Cognitive dysfunction − − ± ± ±
Neuropathy, peripheral − − + ++ +++
Oculomotor apraxia − − + ++ +++
Routine laboratory Albumin (S) − − + + +
Table 16.10 Myopathic form of CoQ10 deficiency (ETFDH)

CNS Encephalopathy, episodic − − ± ± ±
Digestive Liver dysfunction − − ± episodic ± episodic ± episodic
Vomiting − − ± episodic ± episodic ± episodic
Musculoskeletal Muscle weakness ± + ++ +++ +++
Routine laboratory Creatine kinase (P) n-↑ n-↑ n-↑ n-↑ n-↑
Riboflavin Deficiency of blood mononuclear cells has been suggested as an alter-

The Brown-Vialetto-Van Laere and Fazio-Londe syndromes native tissue for CoQ10 evaluation; however, its sensitivity
are now considered a single disease entity (Bosch et al. and limitations are currently being evaluated (Rahman et al.
2010; Dipti et al. 2005). They share progressive pontobulbar 2012). The gold standard method in the diagnosis of CoQ10
palsy, which is the hallmark of the disease. Early recognition deficiency is its measurement in skeletal muscle. Fibroblast
appears essential as supplementation has been proposed to and myoblast measurements can also be useful, but they
possibly improve the disease outcome in primary as well as appear less reliable than muscle.
secondary riboflavin defects (Olsen et al. 2007; Bosch et al. Biochemical measurement of the respiratory chain
2010) (Table 16.1, 16.2). enzymes may show a typical pattern of CoQ10 defect, which
Riboflavin is most commonly evaluated by the glutathi- includes decreased activities of combined complexes I + III
one reductase activity in circulating red blood cells (Hoey [NADH-cytochrome c reductase] or complexes II + III [suc-
et al. 2009). Plasma riboflavin, FAD and FMN levels may cinate cytochrome c oxidoreductase] that rely on endogenous
also be measured by high-performance liquid chromatogra- CoQ10 levels and normal activities of the individual com-
phy (Capo-Chichi et al. 2000). plexes I, II and III. However, mild CoQ10 deficiencies may
Evaluation of blood acylcarnitines and urine organic acids be encountered with normal respiratory chain activities, and
is essential in the diagnosis. Urine organic acids are most the recommendation is to directly measure the muscle
often abnormal during acute crises encountered with second- CoQ10 content by high-performance liquid chromatography
ary riboflavin defect. The observed anomalies depend on the in all cases where deficiency is clinically suspected.
primary enzymatic defect. Blood acylcarnitine profile is
abnormal even without acute crises and is therefore most Other Potential Biomarkers
useful for diagnosis. Acylcarnitines show moderate accumu- Blood lactate may be increased, but normal lactate does not
lation of short- and medium-chain acylcarnitines in the exclude CoQ10 deficiency. Low serum albumin is character-
Brown-Vialetto-Van Laere and Fazio-Londe syndromes and istic for APTX deficiency. Patients with MADD may be diag-
more severe alterations in secondary riboflavin deficiency nosed by tandem mass spectroscopy showing the increase
depending on the primary enzymatic defect. of multiple acylcarnitines in blood or by organic acid chro-
matography showing the increase of glutaric acid in urine.
CoQ10 Deficiency Serum CK may reach high levels in acute phases and may be
The clinical recognition of CoQ10 deficiency is difficult used to monitor disease severity in the follow-up of patients
because of extreme clinical variability. However, there are with MADD. Histology of skeletal muscle may reveal lipid
some recognizable clinical phenotypes (Table 16.3–16.10). accumulation and mitochondrial abnormalities, such as cyto-
If the clinical symptoms suggest the diagnosis of a CoQ10 chrome c oxidase-negative fibres, succinate dehydrogenase-
deficiency disease, the following laboratory investigations hyperreactive fibres and subsarcolemmal mitochondrial
can be useful in the diagnosis. proliferation. However, typical ragged red fibres are rare in
CoQ10 deficiencies.
16.5 Diagnosis Biosynthesis Assays

Some special assays of CoQ10 biosynthesis in cultured
The concentration of plasma CoQ10 may not accurately skin fibroblasts can be used to confirm a defect in the
reflect the level in tissues as it appears to depend upon the CoQ10 biosynthetic pathway. Typically 3H-mevalonate and
14
concentration of lipoproteins and dietary intake. Assessment C-hydroxybenzoate are used as radiolabeled substrates
in cell culture, while 3H-decaprenyl diphosphate is used in may be possible in the future using tandem mass spectrom-
homogenised fibroblasts (López et al. 2006; Quinzii et al. etry methods to identify accumulation of abnormal metabo-
2006). Multiple steps in the CoQ10 biosynthetic pathway lites (Rahman et al. 2012).
cannot be distinguished using the available assays, but this
Reference values are dependent upon the method used. The listed values should only be used as a guide
Cerebrospinal
Compound Serum/blood (mmol/l) Urine (mmol/mol creat) fluid (CSF) (mmol/l)
Lactate <2 mmol/l <0.1 <1.8
Creatine kinase (CK) <190 U/l N/A N/A
Albumin 35–55 g/l No proteinuria N/A
Acylcarnitines Normal profile Normal profile N/A
Organic acids Normal profile Normal profile N/A
Reference values in skeletal muscle (each laboratory has their own values)
Compound Skeletal muscle
Histology N/A
RC biochemistry Complex I, II/III, I/III, IV values differ between laboratories
CoQ10 measurement HPLC or TMS methods, different reference range in each laboratory
HPLC high-performance liquid chromatography, TMS tandem mass spectroscopy, RC respiratory chain
16.7 Pathological Values Table
Biochemical screening in blood (serum, plasma, dry blood spot)

Compound Blood (serum, plasma, dry blood spot)
Lactate >2 mmol/l
Creatine kinase (CK) >190 U/l
Serum albumin Low <35 g/l
Acylcarnitines Increased short-/medium-/long-/very-long-chain acylcarnitines
Organic acids Increased glutaric acid or nonspecific increase of other organic acids
Biochemical screening in urine

Compound Urine
Acylcarnitines Multiple acylcarnitines increased
Organic acids Glutaric aciduria
Histology and biochemistry of skeletal muscle

Compound Skeletal muscle
Histology Lipid accumulation, cytochrome c oxidase-defective fibres, mitochondrial
proliferation with increased succinate dehydrogenase stain
RC biochemistry Decreased activities of CI/III and CII/III (rescued by supplementation of CoQ)
CoQ10 measurement Decreased CoQ10 in skeletal muscle or fibroblasts, myoblasts
Investigation of Suspected Coenzyme Q10 Deficiency
Clinical suspicion of coenzyme Q10 deficiency: infantile- Suspected mitochondrial disease but
onset encephalopathy and nephropathy; juvenile-onset clinical phenotype not suggestive of
cerebellar ataxia and atrophy; steroid-resistant nephrotic Coenzyme Q10 deficiency
syndrome, encephalomyopathy with recurrent myoglobinuria
and ragged-red fibres; or myopathy with lipid storage)
Blood for WBC CoQ10

measurement
Low PMN Normal PMN Muscle biopsy

CoQ10 level CoQ10 level
Histochemistry
Ragged red fibres and/or Other histological

increased lipid content of appearances
CoQ10 level and muscle fibres
biosynthesis assays in
cultured skin fibroblasts
Respiratory chain
enzyme assays
Decreased ↓CII+III and/or

fibroblast CoQ10 ↓CI+III
level and
biosynthesis Other respiratory
Measure
chain enzyme
muscle CoQ10
defect(s)
level
if clinical suspicion
Targeted
sequence of No defect found
relevant If normal, consider
Low muscle alternative diagnoses
CoQ10
CoQ10 level
biosynthesis
gene(s)* Normal
sequence Targeted sequence of relevant CoQ10
biosynthesis gene(s)*
Sequence all known CoQ10
biosynthesis gene Normal
sequence
*Sequence ADCKS and APTX if ataxia present;

ETFDH if mypathy present; CoQ2 if steroid
resistant nephrotic syndrome present; CoQ2, Mutation identified
PDSS2, PDSS1 and CoQ9 if infantile
onset multisystemic disease present.
Fig. 16.2 Diagnostic flow chart (From the 176th ENMC workshop report Rahman et al. (2012))
16.9 Specimen Collection et al. 2010). The very small number of patients and the
presence of irreversible neurological damage before treat-
In order to ensure that the investigations provide a definitive ment clearly hamper the appraisal of supplementation
diagnosis, it is important that patients under investigation efficacy.
should not be on CoQ10 supplementation. If they are already Riboflavin efficacy has been well established in the
on this therapy, it should be stopped for at least 1 month “riboflavin-responsive MADD” (Olsen et al. 2007; Gempel
before performing the muscle biopsy. Muscle specimen has et al. 2007), but it remains to be evaluated with the progres-
to be frozen within 1 h of biopsy for CoQ10 measurement. sive neurodegeneration of Brown-Vialetto-Van Laere and
Requirements for the measurement of the respiratory chain Fazio-Londe syndromes.
enzymes are listed in the chapter concerning mitochondrial Limited bioavailability of exogenous CoQ10 and
disease. unknown molecular defects underlie the controversy about
The measurement of acylcarnitines in MADD may give CoQ10 supplementation. Beneficial impact of CoQ10 sup-
fluctuating results but detects some abnormalities in many plementation has, however, been repeatedly observed with
cases. If the test is negative and there is a strong clinical sus- renal manifestations in COQ2 and COQ6 mutations and with
picion of MADD, it is prodent to repeat it after 12 h of fast- metabolic alterations in patients with multisystemic presen-
ing, which triggers the production of abnormal metabolites. tation (Quinzii and Hirano 2010; Rahman et al. 2012;
It is important that the test sample reaches the laboratory Heeringa et al. 2011). The neurological improvement seems
within the shortest time (optimally within 1 day); however, dry more questionable, but has been reported in a few studies
blood spots (Guthrie cards) can be used after storage extended. (Quinzii et al. 2005; Klopstock et al. 2011).
If the transport time exceeds 1 h, the serum has to be separated The use of CoQ10 analogues may also show variability,
and can be kept frozen at −20° until the test is performed. as idebenone supplementation resulted in significant worsen-
ing in some patients (Auré and Benoist 2004).
Current research using both cellular and animal models
16.10 Prenatal Diagnosis Table and Sample targets the development of different CoQ10 analogues with
Requirements improved efficacy. The combination of these two strategies
will hopefully expand our understanding toward translation
Prenatal diagnosis in fetal chorionic villus sampling of CoQ10 supplementation into clinical trials.
(CVS) is possible if the primary nuclear genetic defect
has been identified within the family. All known genetic Emergency Treatment Table (If Applicable) and
defects thus far are inherited in an autosomal recessive Medication Requirements
manner. If both parents carry a heterozygous mutation, Patients with riboflavin or CoQ10 deficiency disorders
there is a 25 % risk for an affected child. Heterozygous may require symptomatic emergency treatment for acute
carriers of mutations in any of the CoQ10 deficiency metabolic crises or severe epilepsy. There are no specific
genes are asymptomatic. Carrier testing of at-risk family antiepileptic medications recommended for this group
members is possible if the disease-causing mutation in the of disorders. Metabolic crises can be triggered by infec-
family has been identified. tions, pregnancy/delivery, hormonal changes, surgery,
stress or alcohol. Intravenous glucose administration
and oral riboflavin or CoQ10 supplementation are sug-
16.11 DNA Testing Table and Sample gested in crisis situations and can result in rapid recovery
Requirements (Rahman et al. 2012).
Standard Treatment Table and Medication Requirements

Molecular genetic testing of the riboflavin or CoQ10
There are no uniformly determined standard treatments for
deficiency-related nuclear genes (SLC52A2, SLC52A3,
riboflavin or CoQ10 genetic deficiencies. The recommended
PDSS1, PDSS2, COQ2, COQ9, CABC1/ADCK3) can be
dose with oral CoQ10 in primary deficiencies is 10–30
performed in genomic DNA (from any tissue).
mg/kg/day in children and 1,000–3,000 mg/day in adults.
Usually lower doses have been used in secondary deficien-
cies (200–500 mg/day).
16.12 Treatment The recommended dose of riboflavin supplementation is
100–200 mg/day, with maximal dose of 400 mg/day.
Supplementation with oral riboflavin (10 mg/kg body
weight/day) or CoQ10 (10–30 mg/kg body weight/day Experimental Treatments
in children and 1,000–3,000 mg/day in adults) should be Apart from acute metabolic crises, it seems that riboflavin
considered. The response to therapy appears, however, and CoQ10 can block the chronic progression of disease,
variable (Bosch et al. 2010; Rahman et al. 2012; López but cannot reverse established damage, which indicates that
their supplementation should be started as early as possible. Gempel K, Topaloglu H, Talim B, Schneiderat P, Schoser BG, Hans
Therefore efforts should be focused on prompt diagnosis. VH, Pálmafy B, Kale G, Tokatli A, Quinzii C, Hirano M, Naini A,
DiMauro S, Prokisch H, Lochmüller H, Horvath R (2007) The myo-
There are tissue-specific differences in response to CoQ10 pathic form of coenzyme Q10 deficiency is caused by mutations in
supplementation need to be specifically addressed. Transport the electron-transferring-flavoprotein dehydrogenase (ETFDH)
of CoQ10 is insufficient to some organs (Bentinger et al. gene. Brain 130:2037–2044
2003). In addition some CoQ10 analogues seem to only be Gerards M, van den Bosch B, Calis C, Schoonderwoerd K, van Engelen
K, Tijssen M, de Coo R, van der Kooi A, Smeets H (2010) Nonsense
effective in selected conditions (idebenone in LHON, ubi- mutations in CABC1/ADCK3 cause progressive cerebellar ataxia
quinone in ataxic form). and atrophy. Mitochondrion 10:510–515
Functional studies in human primary cells of patients with Gironi M, Lamperti C, Nemni R, Moggio M, Comi G, Guerini FR,
CoQ10 deficiency have revealed important insights. Cells Ferrante P, Canal N, Naini A, Bresolin N, DiMauro S (2004) Late-
onset cerebellar ataxia with hypogonadism and muscle coenzyme
with mutations in COQ9, COQ2 and PDSS2 reveal a pro- Q10 deficiency. Neurology 62:818–820
longed pharmacokinetics of CoQ10 to reach the mitochon- Green P, Wiseman M, Crow YJ, Houlden H, Riphagen S, Lin JP,
drial respiratory chain. This may explain the delayed clinical Raymond FL, Childs AM, Sheridan E, Edwards S, Josifova DJ
response in some patients after oral supplementation with (2010) Brown-Vialetto-Van Laere syndrome, a ponto-bulbar palsy
with deafness, is caused by mutations in c20orf54. Am J Hum Genet
CoQ10. Additionally it was shown that short-tail ubiquinone 86:485–489
analogues cannot substitute for CoQ10 deficiency in the Gregersen N (1985) Riboflavin-responsive defects of beta-oxidation. J
respiratory chain in human fibroblasts, indicating the impor- Inherit Metab Dis 8:65–69
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Part IV
Energy Metabolism
Mitochondrial Fatty Acid Oxidation
Disorders 17
Ute Spiekerkoetter and Marinus Duran
Contents Summary
17.1 Introduction .................................................................... 248 Mitochondrial fatty acid oxidation disorders have been
included in newborn screening programs worldwide since
17.2 Metabolic Markers ......................................................... 248
the implementation of tandem mass spectrometry-based
17.3 Nomenclature .................................................................. 250 screening. Disease-specific acylcarnitine profiles pinpoint at
17.4 Metabolic Pathway ......................................................... 251 the respective enzyme defect; however, the diagnosis has
17.5 Signs and Symptoms ...................................................... 252
invariably to be confirmed by enzyme assay and/or molecu-
lar analysis. Metabolic profiles can be normal in the anabolic
17.6 Reference Values ............................................................. 258 state and, consequently, newborn screening may miss the
17.7 Pathological Values ......................................................... 258 diagnosis when performed outside the catabolic state on
17.8 Diagnostic Tools and Flowchart .................................... 259 days 2 and 3 of life. Mitochondrial fatty acid oxidation
disorders comprise four groups: (1) disorders of the entry of
17.9 Specimen Collection ....................................................... 261
long-chain fatty acids into mitochondria, (2) intramitochon-
17.10 Prenatal Diagnosis of Fatty Acid drial β-oxidation defects of long-chain fatty acids affecting
Oxidation Disorders ....................................................... 261
membrane-bound enzymes, (3) β-oxidation defects of short-
17.11 DNA Analysis and Prevalent Mutations ....................... 262 and medium-chain fatty acids affecting enzymes of the mito-
17.12 Treatment ........................................................................ 262 chondrial matrix, and (4) disorders of impaired electron
transfer to the respiratory chain from mitochondrial
References .................................................................................... 264
β-oxidation. The main pathogenic mechanisms of fatty acid
oxidation defects include toxic effects by accumulating acyl-
carnitine and acyl-CoA species and energy deficiency due to
impaired fatty acid oxidation and ketone body formation.
The respective disorders present with heterogeneous pheno-
types. Before newborn screening (NBS) was introduced, the
most common clinical presentations were hypoketotic hypo-
glycemia and sudden death, usually precipitated by an infec-
tion or fasting in the neonatal period or early childhood. In
addition, severe cardiomyopathy and arrhythmias were lead-
ing clinical signs in the first months of life. Older children or
adults may present with exercise- or illness-induced rhabdo-
myolysis. Patients can remain asymptomatic throughout life
U. Spiekerkoetter (*)
Department of General Pediatrics, Adolescent Medicine if they have mild defects and are not exposed to metabolic
and Neonatology, University Children’s Hospital, stress. Correlation of genotype and/or residual enzyme activ-
Albert-Ludwigs-University Freiburg, Mathildenstraße 1, ity with disease phenotype has been reported for some
Freiburg 79106, Germany
defects, whereas in others additional disease modifiers are
e-mail: ute.spiekerkoetter@uniklinik-freiburg.de
suspected. With newborn screening, disease incidence has
M. Duran
significantly increased and the proportion of milder pheno-
Laboratory Genetic Metabolic Diseases, Academic Medical
Center, Meibergdreef 9, Amsterdam 1105AZ, The Netherlands types has grown. Newborn screening greatly reduces the
e-mail: m.duran@amc.uva.nl morbidity and mortality, though it does not eliminate early
248 U. Spiekerkoetter and M. Duran
neonatal death in severe phenotypes. With respect to the degree of hypoglycemia. Hyperammonemia occurs in some
heterogeneous clinical presentation, treatment needs to be severe defects, and lactic acidemia is particularly seen in
tailored to the severity of the respective phenotype and the LCHAD and MTP deficiencies and in MAD deficiency.
specific disorder. Rhabdomyolysis is accompanied by highly elevated creatine
kinase (CK) up to 100,000 U/l or higher. Hepatomegaly and
steatosis resulting from endogenous lipolysis are reflected by
17.1 Introduction increased liver transaminases (AST, ALT). CK and transami-
nases are very sensitive markers to indicate metabolic
Fat is an important source of energy and the body’s principal derangement in long-chain fatty acid oxidation defects. The
fuel store. In postnatal life fatty acids are used in preference toxic effects of accumulating long-chain acylcarnitines and
to glucose by the heart. They are also the main fuel for skel- acyl-CoA esters are likely responsible for arrhythmias in
etal muscle during sustained exercise. these disorders that are especially severe in disorders of the
Mitochondrial fatty acid oxidation involves four pro- mitochondrial trifunctional protein (MTP and LCHAD defi-
cesses: (1) entry of fatty acids into mitochondria (the carni- ciencies) and CACT deficiency (Corr et al. 1989). The devel-
tine cycle), (2) mitochondrial β-oxidation via a spiral opment of retinopathy and peripheral neuropathy in LCHAD
pathway utilizing membrane-bound enzymes, (3) mitochon- and MTP deficiencies is associated with the accumulation of
drial β-oxidation of chain-shortened fatty acids using matrix long-chain hydroxyacylcarnitines.
enzymes, and (4) electron transfer to the respiratory chain. The disease-specific acylcarnitine profile is used for new-
The clinical presentation of these disorders is heteroge- born screening with tandem mass spectrometry on days 2
neous. Since implementation of newborn screening for fatty and 3 of life; postponing the blood sampling may yield false-
acid oxidation disorders (FAODs), milder phenotypes and negative results later on. Secondary carnitine deficiency may
asymptomatic patients with different genotypes are identi- result in an otherwise normal acylcarnitine profile. Organic
fied. Before newborn screening, FAODs have three charac- acid analysis in urine has previously been an important tool
teristic clinical presentations: for the diagnosis of fatty acid oxidation defects and the
1. Acute hypoketotic hypoglycemia and encephalopathy assessment of dicarboxylic aciduria is highly diagnostic;
accompanied by hepatomegaly and liver dysfunction pre- however, nowadays acylcarnitine analysis is the primary
cipitated by fasting or an infection. This is often described as diagnostic tool, invariably followed by enzyme (or molecu-
a hepatic presentation or “Reye-like symptoms.” Presentation lar) analysis, since milder defects often present with a nor-
generally occurs in the first weeks and months of life. mal organic acid profile in urine.
2. Cardiomyopathy (usually hypertrophic, but also dilated in Carnitine transporter Deficiency. The organic cation
later stages), arrhythmias, or conduction defects. The car- carnitine transporter OCTN2 is responsible for the carni-
diac phenotype generally occurs in the first weeks and tine uptake across the plasma membrane, particularly in
months of life. heart, muscle, and kidney. Defects of the transporter lead to
3. Myopathy, presenting either with weakness, with pain, or primary systemic carnitine deficiency with increased renal
with acute rhabdomyolysis, precipitated by exercise or loss of carnitine and extremely low plasma carnitine con-
infection. The myopathic phenotype mainly occurs after centrations. Parallel measurement of carnitine in blood and
infancy and in later childhood or adolescence. urine is the first diagnostic step. On newborn screening,
A number of patients will never develop symptoms, either low carnitine concentrations may also be found in new-
because they have a mild defect or because they are not borns of vegan mothers or mothers with an undiagnosed
exposed to the necessary environmental stress; it is not yet OCTN2 deficiency. Asymptomatic adults with very low
clear how many of the NBS-diagnosed patients will remain plasma carnitine concentrations have been identified.
asymptomatic throughout life. Therefore, carnitine measurement in the maternal plasma
and urine has to be part of the diagnostic workup of a low
carnitine on NBS.
17.2 Metabolic Markers Carnitine Palmitoyltransferase I (CPT I) Deficiency.
Three different genetic isoforms of CPT I have been found in
Fasting hypoglycemia is primarily due to increased periph- liver and kidney (CPT IA), muscle and heart (CPT IB), and
eral glucose consumption with a concomitant decreased pro- brain (CPT IC). Only CPT IA deficiency has been identified
duction of ketones (Herrema et al. 2008); consequently the in humans presenting in infancy with hypoketotic hypogly-
hypoglycemia is hypoketotic. Small amounts of ketone bod- cemia and hepatopathy, induced by fasting or illness.
ies can be synthesized, particularly in medium- or short- Affected patients have an extremely high level of free carni-
chain FAODs or if there is high residual enzyme activity, but tine in their blood spot with a concomitant decrease of the
the plasma concentrations are lower than expected for the long-chain acylcarnitines.
17 Mitochondrial Fatty Acid Oxidation Disorders 249
Carnitine-Acylcarnitine Translocase (CACT) Deficiency. approximately 1:10,000 in Europe, Australia, and the USA.
Most patients have presented in the neonatal period and died Affected patients only present clinically if exposed to an
in infancy. This phenotype is characterized by severe ven- appropriate environmental stress such as prolonged fasting
tricular arrhythmias that are most likely triggered by accu- or an infection with vomiting. Even before the era of new-
mulating toxic fatty acid metabolites during catabolism. The born screening, many patients (affected siblings of symp-
acylcarnitine profile resembles that found in carnitine palmi- tomatic patients) remained asymptomatic (Wilcken et al.
toyltransferase II deficiency. 2007). Blood C 8-carnitine is a highly specific marker, but
Carnitine Palmitoyltransferase II (CPT II) Deficiency. may be only moderately elevated in mild phenotypes. The
The most common form of this disorder presents with recur- excretion of hexanoylglycine in urine is pathognomonic of
rent episodes of rhabdomyolysis and myoglobinuria precipi- MCADD; however, in milder phenotypes it is not detectable.
tated by prolonged exercise, mainly in adolescents or young Residual enzyme activity correlates with the genotype and
adults. The plasma CK often normalizes between episodes, the expected clinical phenotype. Compound heterozygous
but it may also remain moderately elevated, associated with individuals with the c199T > C mutation on one allele pres-
chronic muscle weakness. Severe neonatal onset CPT II defi- ent with residual activities in the range of heterozygotes,
ciency is lethal and is associated with congenital malforma- doubting a clinical relevance of this mutation.
tions, principally renal cysts and neuronal migration defects Short-Chain Acyl-CoA Dehydrogenase (SCAD)
(North et al. 1995). The adult myopathic form of CPT II defi- Deficiency. There are two polymorphisms in the SCAD gene
ciency is biochemically difficult to diagnose since acylcarni- (c.625G > A and c.511C > T). In northern Europe, 6 % of the
tine profiles may be normal, even during metabolic general population has one of these variants on both alleles.
derangement. Biochemically the diagnosis relies on the finding of increased
Very Long-Chain Acyl-CoA Dehydrogenase (VLCAD) blood C4-carnitine and/or urine ethylmalonic acid. The clini-
Deficiency. VLCAD deficiency is the second most common cal significance of SCAD deficiency is unclear. It may confer
fatty acid oxidation disorder in Europe and the USA, with a susceptibility to disease together with a second mitochon-
prevalence between 1:50,000 and 1:100,000. This is much drial impairment (van Maldegem et al. 2006).
higher than was assumed from the number of clinically man- Short-Chain 3-Hydroxyacyl-CoA Dehydrogenase
ifesting patients. Neonatal screening has identified milder (SCHAD) Deficiency. SCHAD deficiency is associated with
phenotypes with different genotypes and higher residual hypoglycemia due to hyperinsulinism, in contrast to other
activities (Spiekerkoetter et al. 2010). A large number of FAODs. SCHAD has a second role independent of
patients are asymptomatic at diagnosis and during follow-up β-oxidation, binding and inhibiting glutamate dehydroge-
over up to 10 years. nase (GDH): SCHAD mutations that prevent GDH binding
Mitochondrial Trifunctional Protein (MTP), LKAT, and lead to increased GDH activity and insulin secretion, partic-
LCHAD Deficiencies. Isolated LKAT deficiency is rare ularly in response to leucine (Li et al. 2010). Increased
and has only been reported in one patient. Patients with iso- plasma 3-hydroxy-C4-carnitine and urine 3-hydroxyglutaric
lated LCHAD deficiency or general MTP deficiency irre- acid are the accepted diagnostic parameters.
spective of the localization of the mutation in the HADHA or Electron Transfer Defects (Multiple Acyl-CoA
HADHB gene present with similar heterogeneous pheno- Dehydrogenase Deficiency, MADD). This disorder results
types (Spiekerkoetter et al. 2004). Importantly, this is the from defects of the electron transfer flavoprotein (ETF) or the
only FAOD associated with retinopathy and peripheral neu- ETF ubiquinone oxidoreductase (ETF:QO). Consequently,
ropathy (Tyni et al. 1998). In neuromyopathic phenotypes, all acyl-CoA dehydrogenases will have a decreased activity
the acylcarnitine profile is often normal, even during an epi- in addition to glutaryl-CoA dehydrogenase and d-2-
sode of rhabdomyolysis. Mothers who are heterozygous for hydroxyglutarate dehydrogenase. The biochemical diagnosis
LCHAD or MTP deficiency have an increased risk of HELLP is based on multiple elevated blood acylcarnitines and abnor-
syndrome (hemolysis, elevated liver enzymes, and low plate- malities of urine organic acids. Severely affected patients
lets) and acute fatty liver of pregnancy (AFLP) during preg- present in the first days of life and generally also exhibit con-
nancies when they are carrying an affected fetus (Wilcken genital anomalies and facial dysmorphism. The malforma-
et al. 1993). tions resemble those seen in CPT II deficiency and this
Acyl-CoA Dehydrogenase 9 (ACAD9) Deficiency. phenotype is also mainly lethal in the neonatal period. The
ACAD9 is homologous to VLCAD and has dehydrogenase riboflavin-responsive phenotype has a good prognosis with
activity towards various long-chain acyl-CoA esters in vitro. riboflavin treatment. Mutations in the gene coding for
Its physiological role is in the assembly of the mitochondrial ETF:QO have been identified in these patients (Olsen et al.
respiratory chain complex I (Nouws et al. 2010). 2007). Only recently, patients with a riboflavin transporter
Medium-Chain Acyl-CoA Dehydrogenase (MCAD) defect have been described also presenting as mild MAD
Deficiency. MCAD deficiency presents with an incidence of deficiency and responding to riboflavin treatment.
17.3 Nomenclature
250
Chromosomal OMIM
No. Disorder Alternative name Abbreviation Gene symbol localization Affected protein no. Subtype
17.1.1 Carnitine transporter deficiency Carnitine uptake defect OCTN2 SLC22A5 5q31 Organic cation carnitine 212140 All forms
transporter 2
17.1.2 Carnitine palmitoyltransferase I Carnitine palmitoyl-CoA CPT I CPT Ia 11q22.23 Carnitine palmitoyltransferase 255120 All forms
deficiency transferase I deficiency
17.1.3 Carnitine acylcarnitine translocase CACT SLC25A20 3p21.31 Carnitine acylcarnitine translocase 212138 All forms
deficiency (30 patients)
17.1.4 Carnitine palmitoyltransferase II Carnitine palmitoyl-CoA CPT II CPT II 1p32 Carnitine palmitoyltransferase II 255110 All forms
deficiency transferase II deficiency
17.2.1 Very long-chain acyl CoA VLCAD ACADVL 17p13 Very long-chain acyl-CoA 201475 All forms
17.2.2 Mitochondrial trifunctional protein mTFP, MTP HADHA, 2p23 Long-chain enoyl-CoA hydratase, HADHA All forms
deficiency HADHB long-chain 3-hydroxyacyl-CoA 600890;
dehydrogenase, 3-ketoacyl-CoA HADHB
thiolase 143450
17.2.3 Isolated deficiency of long-chain LCHAD HADHA 2p23 Long-chain 3 hydroxyacyl-CoA 600890 All forms
3-hydroxyacyl-CoA dehydrogenase
dehydrogenase
17.2.4 Isolated deficiency of long-chain LKAT HADHB 2p23 Long-chain 3-ketoacyl-CoA 143450 All forms
3-ketoacyl CoA thiolase patient thiolase
17.2.5 Acyl-CoA dehydrogenase 9 Complex I assembly ACAD9 ACAD9 3q26 Acyl-CoA dehydrogenase 9 611126 All forms
deficiency disorder
17.3.1 Medium-chain acyl CoA MCAD ACADM 1p31 Medium-chain acyl CoA 201450 All forms
17.3.2 Short-chain acyl CoA Ethylmalonic aciduria SCAD ACADS 12q22-qter Short-chain acyl CoA 201470 All forms
17.3.3 Short-chain 3-hydroxyacyl-CoA SCHAD HADH, 4q22–q26 Short-chain 3-hydroxyacyl-CoA 231530 All forms
dehydrogenase deficiency (13 SCHAD dehydrogenase
patients)
17.4.1 Multiple acyl-CoA dehydrogenase Glutaric aciduria type II, ETF, MAD ETFA, ETFB ETFA 15q23-q25, Electron transfer flavoprotein ETFA All forms
(ETF) deficiency Electron transfer defect ETFB 19q13.3 231680
(ETF) ETFB
130410
17.4.2 Multiple acyl-CoA dehydrogenase Glutaric aciduria type II, ETF-DH, ETFDH 4q32-qter ETF-ubiquinone oxidoreductase 231675 All forms
(ETF-DH) deficiency ETF-ubiquinone ETF-QO, MAD
oxidoreductase deficiency
17.4.3 Riboflavin-responsive multiple Glutaric aciduria type II, ETF-DH, ETFDHa 4q32-qter ETF-ubiquinone oxidoreductase 231675 All forms
acyl-CoA dehydrogenase Electron transfer defect ETF-QO, MAD
deficiency (ETF)
a
Also affection of other genes proposed
U. Spiekerkoetter and M. Duran
17.4 Metabolic Pathway CoA dehydrogenase (LCHAD) in the α-subunit and the
long-chain 3 ketoacyl-CoA thiolase (LKAT) in the β-subunit.
Fatty acids are activated to coenzyme A (CoA) esters in the Each turn of the β-oxidation spiral involving four steps short-
cytoplasm, but long-chain fatty acids need to be esterified ens the acyl-CoA by two carbons. These include two dehydro-
with carnitine in order to cross the inner mitochondrial mem- genation reactions, linked respectively to flavin adenine
brane. Medium- and short-chain fatty acids can enter the dinucleotide (FAD) and nicotinamide adenine dinucleotide
mitochondria independent of carnitine. Carnitine mainly (NAD). The β-oxidation is catalyzed by enzymes of different
derives from dietary meat or is provided by hepatic endoge- chain length specificities. Medium- and short-chain enzymes
nous synthesis. In vegans endogenous production is enhanced. such as medium-chain acyl-CoA dehydrogenase (MCAD),
By the high-affinity organic cation carnitine transporter 2 short-chain acyl-CoA dehydrogenase (SCAD), enoyl-CoA
(OCTN2), carnitine is shuttled into the cytosol. At the outer hydratase or crotonase, and short-chain 3-hydroxyacyl-CoA
mitochondrial membrane, the formation of acylcarnitine dehydrogenase (SCHAD) are located in the mitochondrial
esters is catalyzed by carnitine palmitoyltransferase I (CPT I). matrix. The liberated electrons are passed to the respiratory
The carnitine esters are then shuttled across the inner mem- chain either directly (from NADH to complex I) or via two
brane by the carnitine acylcarnitine translocase (CACT). transfer proteins (from FADH2 to ubiquinone). Each action of
Carnitine palmitoyltransferase II (CPT II) is attached to the an acyl-CoA dehydrogenase produces two electrons which
inner mitochondrial membrane and catalyzes the transfer back are transferred to the electron transfer flavoprotein (ETF) and
to CoA esters. The enzymes for the β-oxidation of long-chain subsequently to ETF-dehydrogenase (ETF-DH, ETF-QO).
substrates (C18-C14 fatty acids) are also membrane-bound Electron transfer plays a role in amino acid and choline
and comprise the very long-chain acyl-CoA dehydrogenase metabolism in addition to mitochondrial β-oxidation. Acetyl-
(VLCAD) and three enzymes in the mitochondrial trifunc- CoA residues released by β-oxidation can be either oxidized
tional protein complex (mTFP or MTP). This complex is in the Krebs cycle or, utilized to synthesize ketone bodies in
composed of two subunits, harboring the long-chain enoyl- the liver, Flavin adenine dinucleotide (FAD), which is derived
CoA hydratase (LCEH) and the long-chain 3-hydroxyacyl- from riboflavin, acts as a cofactor in these reactions.
Fatty acid
Long-chain Carnitine Plasma membrane

Fatty acid Transporter
Transporter
Carnitine
Fatty acid Acyl-CoA
CPT I
Acylarnitine + CoA Carnitine Outer mitochondrial membrane
Inner mitochondrial membrane

CACT
Respiratory
Carnitine chain
CPT II V
C18 Acylarnitine
Acyl-CoA
CoA
Carnitine
C16-C14 IV
VLCAD
FADH2 FAD+
Cyt c
FAD+
1
C12 MCAD/ III
FADH2 SCAD
Enoyl-CoA
1
CoQ
Acetyl-CoA FADH2
2 4
2 NAD+
3 ETF / II
NADH+H+ ETF-QO FAD+
SCHAD
3
LCHAD 4 NADH+H+
Acetyl-CoA I
NAD+ NADH+H+
Trifunctional protein NAD+
Fig. 17.1 Transport of fatty acids into the mitochondrion, mitochondrial β-oxidation, and electron transfer (Modified according to Rinaldo et al. (2002))
Table 17.1.1 Carnitine transporter deficiency

Cardiovascular Cardiomyopathy ± ± ± n n
Digestive Liver dysfunction ± ± ± n n
Musculoskeletal Rhabdomyolysis n ± ± ±
Skeletal myopathy/muscular hypotonia ± ± ± ± ±
Asymptomatic ± ± ± ± ±
Ketones during hypoglycemia ↓ ↓ ↓
Transaminases (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Carnitine, free (P) ↓ ↓ ↓ ↓ ↓
Carnitine, free (DBS) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Dicarboxylic acids (U) n-↑ n-↑ n-↑
Long-chain acylcarnitines (DBS, P) ↓ ↓ ↓ ↓ ↓
Table 17.1.2 Carnitine palmitoyltransferase 1 deficiency

Renal Renal tubular acidosis ± ± ± ±
Routine laboratory Glucose (P) ↓-n ↓-n ↓-n
Special laboratory Carnitine, free (P) n-↑ n-↑ n-↑ n-↑ n-↑
Carnitine, free (DBS) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Long-chain acylcarnitines (DBS, P) ↓ ↓ ↓ ↓ ↓
Table 17.1.3 Carnitine acylcarnitine translocase deficiency (30 patients)

Cardiovascular Cardiac arrhythmias, sudden death +++ +++ Many did not survive
Cardiomyopathy +++ +++ Many did not survive
CNS Coma, lethargy ± ± ±
Digestive Liver dysfunction ± ± ±
Musculoskeletal Skeletal myopathy/muscular hypotonia ± ± ±
Other Lethality of severe phenotypes, high +++ +++ Many did not survive
Routine laboratory Creatine kinase (P) ↑↑ ↑↑ ↑↑↑
Lactate (P) ↑ ↑ ↑
Transaminases (P) ↑ ↑ ↑
Special laboratory C16–C18 acylcarnitines ↑ ↑ ↑
Carnitine, free (DBS, P) ↓-n ↓-n ↓-n
Dicarboxylic acids (U) n-↑ n-↑ n-↑
Table 17.1.4 Carnitine palmitoyltransferase 2 deficiency

CNS Coma, lethargy ± ± ± n n
Musculoskeletal Rhabdomyolysis, exercise induced n +++ +++ +++
Skeletal myopathy/muscular ± ± ± ± ±
hypotonia
Other Malformations (kidney, brain) + Generally lethal
Routine laboratory Creatine kinase (P) n-↑ n-↑ n-↑↑↑ n-↑↑↑ n-↑↑↑
Transaminases (P) n-↑ n-↑ n-↑↑↑ n-↑↑↑ n-↑↑↑
Special laboratory C16–C18 acylcarnitines n-↑ n-↑ n-↑ n-↑ n-↑
Table 17.2.1 Very long-chain acyl-CoA dehydrogenase deficiency

Musculoskeletal Rhabdomyolysis, exercise induced n ± ++ ++ ++
Routine laboratory Creatine kinase (P) n-↑ n-↑ n-↑↑ n-↑↑ n-↑↑
Transaminases (P) n-↑ n-↑ n-↑↑ n-↑↑ n-↑↑
Special laboratory C14:1 tetradecenoyl-carnitine (DBS, P) (n)-↑ (n)-↑ (n)-↑ n-↑ n-↑
Dicarboxylic acids (U) n-↑ n-↑ n-↑ n-↑ n
Table 17.2.2 Mitochondrial trifunctional protein deficiency

Cardiovascular Cardiac arrhythmias n – ↑↑↑, n – ↑↑↑,
often lethal often lethal
Cardiomyopathy ± ± ± n n
Intrauterine cardiomyopathy Lethal
Neuropathy, peripheral n n ± + ++
Digestive Liver dysfunction ± ± ± ± ±
Eye Pigmentary retinopathy n ± ± ± ±
Skeletal myopathy/muscular hypotonia + + + + +
Other Intrauterine growth retardation +
Maternal HELLP syndrome ±
Routine laboratory Ammonia (B) (↑) (↑) (↑) n n
Creatine kinase (P) n-↑ n-↑ n-↑↑ n-↑↑ n-↑↑
Lactic acidosis ± ± n n n
Special laboratory C16–C18 hydroxyacylcarnitines (DBS, P) n-↑ n-↑ n-↑ n-↑ n-↑
Dicarboxylic and 3-hydroxydicarboxylic acids (U) n-↑ n-↑ n-↑ n-↑ n
Table 17.2.3 Isolated deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase

Cardiovascular Cardiac arrhythmia ± ±
Cardiomyopathy ± ± ± n n
Neuropathy, peripheral n n ± ± ±
Eye Pigmentary retinopathy n ± ± ± ±
Skeletal myopathy/muscular hypotonia + + + + +
Other Intrauterine growth retardation +
Maternal HELLP syndrome ±
Routine laboratory Ammonia (B) (↑) (↑) (↑) n n-↑
Creatine kinase (P) n-↑ n-↑ n-↑↑ n-↑↑ n-↑↑
Lactic acidosis ± ± n n n
Special laboratory C16–C18 hydroxyacylcarnitines (DBS, P) n-↑ n-↑ n-↑ n-↑ n-↑
Dicarboxylic and 3-hydroxydicarboxylic acids (U) n-↑ n-↑ n-↑ n-↑ n
Table 17.2.4 Isolated deficiency of long-chain 3-ketoacyl-CoA thiolase (1 patient)

Cardiovascular Cardiac arrhythmia
Cardiomyopathy + +
Digestive Liver dysfunction + +
Musculoskeletal Skeletal myopathy/muscular hypotonia + +
Respiratory Pulmonary edema + +
Other Lethality, high + +
Routine laboratory Creatine kinase (P) n n
Glucose (P)
Ketones during hypoglycemia
Lactic acidosis ++ +
Transaminases (P) ↑ ↑
Special laboratory C16–C18 hydroxyacylcarnitines (DBS, P) ↑ ↑
Carnitine, free (DBS, P) n n
Dicarboxylic and 3-hydroxydicarboxylic acids (U) ↑ ↑
Table 17.2.5 Acyl-CoA dehydrogenase 9 deficiency

Cardiovascular Cardiomyopathy, dilated + + n n
CNS Encephalopathy n +, lethal (2 patients)
Neurologic dysfunction + +
Digestive Failure to thrive + +
Liver dysfunction + +
Liver failure + +
Reye-like +
Ear Hearing loss + +
Musculoskeletal Exercise intolerance n + +
Rhabdomyolysis + +
Skeletal myopathy/muscular hypotonia + +
Other Complex I assembly disorder + +
Routine laboratory Ammonia (B) (↑) (↑) (↑)
Beta-hydroxybutyrate (U) ↑
Creatine kinase (P) ↑ ↑
Glucose (P) ↓ ↓
Lactic acidosis + +
Transaminases (P) ↑↑ ↑↑
Special laboratory Carnitine, free (DBS, P) ↓ ↓
Long-chain acylcarnitines (DBS, P) ↑ ↑
Table 17.3.1 Medium-chain acyl-CoA dehydrogenase deficiency

Special laboratory Carnitine, free (DBS, P) ↓-n ↓-n ↓-n ↓-n ↓-n
Hexanoylglycine (U) ± ± ± ± ±
Octanoylcarnitine (C8) (DBS, P) ↑ ↑ ↑ ↑ ↑
Table 17.3.2 Short-chain acyl CoA dehydrogenase deficiency

CNS Behavioral disorder ± ± ±
Developmental delay ± ± ± ±
Epilepsy ± ±
Hypotonia ± ± ±
Musculoskeletal Dysmorphic features ± ± ± ± ±
Exercise intolerance ± ± ±
Other Predisposition for symptomatic disease (together + + + + +
with a second mitochondrial affection)
Special laboratory Butyrylcarnitine (C4) (DBS, P) ↑ ↑ ↑ ↑ ↑
Ethylmalonic acid (U) ↑↑ ↑↑ ↑↑ ↑ n-↑
Table 17.3.3 Short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (13 patients)

CNS Mental retardation n + + +
Seizures + +
Digestive Liver failure, Reye-like + +
Musculoskeletal Microcephaly +
Hyperinsulinism + +
Hypoketotic hypoglycemia + +
Special laboratory (3-Hydroxy) dicarboxylic acids (U) (↑) (↑) (↑)
3-hydroxyglutarate (U) ↑ ↑ ↑
Hydroxybutyrylcarnitine (C4 OH) (P, DBS) ↑ ↑ ↑
Table 17.4.1 Multiple acyl-CoA dehydrogenase deficiency (ETF)

Vomiting, cyclic ± ± ± ±
Other Congenital anomalies (kidney, brain) +, lethal
Metabolic acidosis + + ±, episodic ±, episodic ±, episodic
Special laboratory C4–C18 Acylcarnitines (DBS, P) ↑ ↑ ↑ ↑ ↑
(C5–C10) dicarboxylic acids (U) ↑ ↑ ↑ ↑ ↑
Ethylmalonic acid (U) ↑ ↑ ↑ ↑ ↑
D-2-Hydroxyglutaric acid (U) ↑ ↑ ↑ ↑ ↑
Table 17.4.2 Multiple acyl-CoA dehydrogenase deficiency (ETFDH)

Vomiting, cyclic ± ± ± ±
Other Congenital anomalies (kidney, brain) +, lethal
Metabolic acidosis + + ±, episodic ±, episodic ±, episodic
Special laboratory C4–C18 acylcarnitines (DBS, P) ↑ ↑ ↑ ↑ ↑
(C5–C10) dicarboxylic acids (U) ↑ ↑ ↑ ↑ ↑
Ethylmalonic acid (U) ↑ ↑ ↑ ↑ ↑
D-2-Hydroxyglutaric acid (U) ↑ ↑ ↑ ↑ ↑
Table 17.4.3 Riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency

CNS Encephalopathy acute, precipitated by infection n ± ± ±
Speech disorder n n ± ± ±
Digestive Vomiting, cyclic n ± ± ±
Musculoskeletal Muscle weakness n ± ± ± ±
Rhabdomyolysis, episodic n n ± ±
Special laboratory C4–C18 Acylcarnitines (DBS, P) + + +
C5–C10 dicarboxylic acids (U) + + +
Carnitine, free (DBS, P) ↓ ↓ ↓
Hexanoylglycine/other short-chain glycine conjugates (U) + + +
Respiratory chain impairment (especially complex I) + + +
17.6 Reference Values C12-C16 unsaturated carnitine esters (Costa et al. 1999).
Reference values are given in Chap. 51 of this book dealing
Values of carnitine and acylcarnitines in dried blood spots and with the analysis of acylcarnitines in various body fluids.
plasma differ (de Sain-van der Velden et al. 2013). The values Organic acid analysis in urine usually reveals a disease-
also differ according to age, and in newborns there are refer- specific profile that is not found in healthy individuals.
ence values for the different days of life. Catabolism increases Fasting significantly increases the excretion of disease-
acylcarnitine concentrations also in healthy individuals, specific organic acids. The normal fasting urine may display
especially C2-carnitine, 3-hydroxybutyrylcarnitine, and the a profile of organic acids as shown in the table below.
Table 17.6 Fasting urine organic acids in healthy controls (mmol/mol creatinine)
Substance Fasting level Substance Fasting level
3-Hydroxybutyrate 160–6,460 Acetylglycine 2.5–47.6
Ethylmalonate <10 Isobutyrylglycine 0.003–1.5
Methylsuccinate <3 Butyrylglycine 0.01–0.12
Glutarate <10 Isovalerylglycine 0.2–0.9
Adipate 74–610 Hexanoylglycine 0.2–0.8
Suberate 19–230 Phenylpropionylglycine 0.002–0.15
Decanedioate 90–310 Suberylglycine 0.02–0.52
Dodecanedioate 10–200
3-Hydroxyadipate 10–100
3-Hydroxysuberate 5–50
3-Hydroxydecanedioate 10–200
3-Hydroxydodecanedioate 10–200
17.7 Pathological Values with abnormal acylcarnitine profiles suggesting a FAOD dur-
ing severe catabolism. For VLCAD deficiency a false-posi-
An extremely wide variation in pathological values can be tive rate of 1:2,600 is reported on newborn screening
observed. In addition, patients can be missed at any time when (Spiekerkoetter et al. 2010).
the blood sample is taken at a moment when fatty acid oxida- Reference pathological values are summarized by
tion is not requested such as postprandial blood samples. In McHugh et al. and represent the 5th and 90th percentiles of
general, adult and adolescent patients have a less prominent pathological values taken from a large worldwide cohort of
acylcarnitine profile than the neonatal/infantile presentations. patients identified by newborn screening (McHugh et al.
Care should be taken in the interpretation of ketotic samples, 2011). The table below summarizes pathological values
since values can also be increased in healthy individuals. when diagnosing clinically selected patients with FAO
Pathological values of acylcarnitines in newborn blood defects and exemplifies the variation of the levels of the clin-
spot samples generally decrease in the first days of life and ically important acylcarnitines as well as that of free carni-
may normalize. Therefore, it will be inevitable that some tine. Any patient with a primary defect of fatty acid beta
isolated patients may be missed if screened too late. oxidation may have free carnitine levels as low as those
Importantly, also healthy newborns and children may present observed in the carnitine transporter defect OCTN2.
Table 17.7 Pathological plasma acylcarnitine concentrations in the various defects of mitochondrial fatty acid beta-oxidation
OCTN2 CPT I CACT CPT II VLCAD MTP MCAD SCAD SCHAD MAD
Condition 17.1.1 17.1.2 17.1.3 17.1.4 17.2.1 17.2.2/3/4 17.3.1 17.3.2 17.3.3 17.4.1/2/3
Free carnitine 0–5 46–230 4–58 1–13 7–18 3–54 15–34 34–44 4–23
C2-carnitine
C4-carnitine .6–4.2 0.25 .12–15
C4OH-carnitine 0.3–1.1
C5-carnitine .09–5.5
C5DC-carnitine .11–1.15
C6-carnitine .12–2.1 .22–3.5
C6DC-carnitine
C8-carnitine 1.1–26 .25–8
C8DC-carnitine
C10:1-carnitine .26–2.2 –.41
C10-carnitine .10–.58 .65–5.9
C10DC-carnitine
C12:1-carnitine .05–.07
C12-carnitine .12–.19 .41–2.2
C12OH-carnitine
C14:2-carnitine .22–.47
C14:1-carnitine .05–1.7 .65–19 .03–1.7 .24–3
C14-carnitine .13–5.0 .2–.4
C14OH-carnitine .01–.34
C16:1-carnitine .06–.9 .09–9.1
C16-carnitine <.10 .3–11 .25–14.4 .12–5.9
C16:1OH-carnitine .01–.88
C16OH-carnitine .03–1.8
C18:2-carnitine <.03 .08–1.7 .05–2.1 .08–.64
C18:1-carnitine <.07 .4–7 .18–1.1 .18–.1.9
C18-carnitine <.02 .1–2 .06–6.0 .13–.95
C18:2OH-carnitine .02–0.72
C18:1OH-carnitine .02–2.4
C18OH-carnitine .02–.67
An extremely wide variation can be observed, implicating that patients can be missed at any time when the blood sample is taken at a time when
fatty acid oxidation is not requested such as postprandial blood samples. No entry in the table means that the corresponding acylcarnitine is gener-
ally normal in the relevant condition. Care should be taken in the interpretation of ketotic samples
17.8 Diagnostic Tools and Flowchart is very low, abnormal acylcarnitines may be hard to detect.
Abnormalities may be masked by intravenous glucose or
The investigation of a suspected FAOD starts by looking for dietary treatment, such as the use of medium-chain triglycer-
abnormal acylcarnitines. Its diagnostic specificity can be ides (MCT) in long-chain FAODs. An interpretation is espe-
increased by measuring the ratios of different acylcarnitines. cially difficult for samples obtained terminally or postmortem:
Severe CPT II and CACT deficiencies have indistinguishable these often show multiple raised acylcarnitine species, resem-
acylcarnitine profiles, as do LCHAD and MTP deficiencies. bling MAD deficiency. The acylcarnitine profile can be com-
For CPT I deficiency, carnitine concentrations in dried blood pletely normal in patients with high residual enzyme activity,
spots are higher than in plasma, and diagnosis may be missed such as mild VLCAD or MTP deficiencies. Abnormalities
with measurement in plasma. may, however, be detectable in samples collected after
The clinical circumstances have a major effect on the acyl- overnight fasting, exercise, or loading with carnitine.
carnitine profile. Abnormalities are usually more marked dur- If acylcarnitine analysis suggests a specific diagnosis,
ing catabolism, but if the plasma-free carnitine concentration it has to be confirmed by enzyme assays and/or mutation
analysis. If the metabolite results are nonspecific or if they ketonuria can also be seen in some respiratory chain defects.
are normal despite a strong clinical suspicion, it may be The analysis of acylcarnitines in the urine will disclose the
helpful to measure acylcarnitine production in vitro or flux presence of dicarboxylic acid carnitine esters that may have
through the pathway. additional diagnostic significance. Defects of LCHAD and
Quantitative acylcarnitine profiling may indicate the site MTP deficiencies will show unusual hydroxydicarboxylic
of a defect if this is not clear from metabolite results. acids in addition, whereas MCADD is characterized by an
Acylcarnitines are analyzed by MS/MS after incubating abnormal excretion of several acylglycines such as hexan-
fibroblasts or lymphocytes with fatty acids, labeled with sta- oylglycine, suberylglycine, and phenylpropionylglycine.
ble isotopes (2H or 13C) (Ventura et al. 1999). This tech- The same acylglycines but also short branched-chain acyl-
nique can identify most FAODs except carnitine transporter carnitines will appear in MADD, in general accompanied by
deficiency. CACT and severe CPT II deficiencies cannot be a variety of dicarboxylic acids such as ethylmalonic acid,
distinguished and respiratory chain defects sometimes mimic glutaric acid, and d-2-hydroxyglutaric acid.
FAODs (Sim et al. 2002). All mitochondrial FAODs show an autosomal recessive
Fatty acid oxidation flux is measured by incubating cells pattern of inheritance. There is molecular heterogeneity in all
with radiolabeled fatty acids and collecting the oxidation these disorders, but prevalent mutations have been identified.
products (Olpin et al. 1997). This is useful in evaluating the The relationship between genotype and phenotype varies in
severity of a disorder, but acylcarnitine profiling yields more the different FAODs. In CPT II and VLCAD deficiencies,
diagnostic information. Severity of a disorder can also be nonsense mutations on both alleles are generally associated
best analyzed by direct enzyme activity measurement. with severe early-onset disease, whereas adult-onset rhabdo-
myolysis is associated with conservative missense mutations.
Urinary Organic Acids and Acylglycines In MCAD deficiency, the c.199T > C mutation is associated
For many FAODs, urine organic acid analysis is normal with significant residual activity and appears to be benign.
when patients are well. During fasting or illness, however, Enzyme assays are generally performed on cultured fibro-
medium-chain (and sometimes long-chain) dicarboxylic blasts or lymphocytes (Wanders et al. 2010) and are available
acids are elevated with little or no increase in ketone bodies. for all defects. Enzymology in lymphocytes allows a rapid
Dicarboxylic acids are formed whenever plasma-free fatty confirmation of diagnosis. Moreover, for VLCAD and
acid concentrations are increased, by β-oxidation in peroxi- MCAD deficiencies, residual activity may allow some pre-
somes and ω-oxidation in microsomes, but normally they are dictions with respect to the expected severity of the defect
accompanied by ketonuria. Dicarboxylic aciduria without (Hoffmann et al. 2012).
Clinical signs suggestive for a defect of fatty acid oxidation Newborn screening for a fatty acid oxidation disorder
Carnitine / Acylcarnitine analysis in

blood (preferably during catabolism)
Abnormal result on
Abnormal Normal first screening (days
result result 2 or 3 of life)
With definite In vitro acylcarnitíne profiling

clinical suspicion, flux studies
Enzymology or molecular confirmation (in case of prevalent mutations)
Fig. 17.2 Diagnostic flowchart

Test Material Handling Transport Pitfalls

Acylcarnitines Plasma, dried Room Normal mail During anabolism eventually normal values, different
blood spot (DBS) temperature(DBS) profile with MCT diet, long-chain acylcarnitine
Frozen (plasma) accumulation also in healthy individuals during catabolism
Carnitine Plasma, dried Room Normal mail Blood and tissue concentrations do not correlate; in CPT I
blood spots temperature(DBS) deficiency carnitine is lower in plasma than in DBS; it may
(DBS) Frozen (plasma) be missed on analysis in plasma
Dicarboxylic Spot urine Frozen for longer Normal mail MCT diet and glucose infusion change the profile
acids storage
Free fatty acids Plasma Frozen Frozen Determine before the next food intake
Enzyme assays EDTA plasma Room temperature Has to reach the Poor quality of the lymphocytes (due to transport
in lymphocytes (2 ml) laboratory within 48 h conditions, low temperature, etc.) may result in lower
after withdrawal, residual enzyme activities
room temperature
Enzyme assays Skin biopsy Room temperature, In culture medium Fibroblast culture must grow 4–8 weeks until enzyme
in fibroblasts in sterile 0.9 % assays can be performed
sodium chloride,
fibroblast culture
Molecular EDTA blood, Room temperature Normal mail Delineation of only one mutation does not rule out
analysis dried blood spots deficiency of the enzyme
17.10 Prenatal Diagnosis of Fatty Acid defect is known in the index case. All enzymes of fatty acid
Oxidation Disorders oxidation are expressed in chorionic villus biopsies and amnio-
cytes. Prenatal diagnosis is, therefore, also possible using
Prenatal diagnosis is available for all fatty oxidation disorders. enzyme assays. Chorionic villus biopsy can be performed at
Mutation analysis is the preferred technique, if the molecular 11 + 0 gestational weeks, amniotic fluid test at 14 + 0 weeks.
Deficiency of Prenatal diagnosis suggested Remarks

OCTN2 − Very favorable clinical outcome
CPT I − Very favorable outcome
CACT + Majority of patients die in the neonatal period due to severe cardiac arrhythmias
CPT II + Suggested for severe neonatal phenotypes with congenital anomalies
− Myopathic phenotypes
VLCAD − Very favorable clinical outcome, many asymptomatic “patients,” skeletal myopathy
needs to be discussed with the parents
mTFP/MTP + Suggested for severe phenotypes, neonatal phenotypes generally lethal, irreversible
(+) neuropathy/retinopathy needs to be discussed with the parents in milder phenotypes
LCHAD ± Irreversible retinopathy/neuropathy needs to be discussed with the parents
LKAT + Only 1 patient so far, died in the neonatal period
ACAD 9 + Global mitochondrial dysfunction
MCAD − Very favorable clinical outcome since screening
SCAD − Only predisposition for disease
SCHAD ± Phenotypes of different severity and response to treatment
MAD + Suggested for severe neonatal/infantile phenotypes with/without congenital anomalies
− Myopathic phenotypes
Riboflavin-responsive − Very favorable clinical outcome with riboflavin supplementation
MAD
17.11 DNA Analysis and Prevalent The pathogenicity of other sequence variants is sometimes
Mutations hard to assess. Moreover, standard sequencing may miss
some mutations, such as large deletions and those in introns
Molecular studies are used to confirm the diagnosis as that affect splicing.
an alternative to enzymology. This is satisfactory for Where enzymology is the confirmatory test, mutation anal-
well-defined mutations, such as the prevalent mutations ysis may also be undertaken to define the disorder more pre-
seen in MCAD, LCHAD, and CPT II deficiencies. cisely, allow carrier testing, and simplify prenatal diagnosis.
Prevalent mutations (Caucasian population)

Deficiency of Mutation Amino acid change Remarks
OCTN2 No prevalent mutation
CPT I c.1436C > T (Inuit p.P479L 70 % of babies homozygous for c.1436C > T in the Inuit population
population ) (Canada and Greenland)
CACT No prevalent mutation
CPT II c.338C > T p.S113L Allele frequency (60 %)
VLCAD c.848T > C p.V243A Suggestive of mild VLCADD
mTFP/MTP No prevalent mutations Many deletions and splice site mutations in the HADHA gene, most
compound heterozygotes for c.1528G > C, and a second HADHA
mutation have MTP deficiency
LCHAD c.1528G > C p.E474Q Heterogeneous presentations despite homozygosity for c.1528G > C
LKAT No prevalent mutation Only 1 patient reported
ACAD 9 No prevalent mutation
MCAD c.985A > G p.K329E Classical MCADD, before screening: 80 % homozygosity
c.199G > C p.Y67H Asymptomatic/(mild) variant, allele frequency: 6 % of mutant alleles
in screened population, never found in a clinically diagnosed patient
SCAD c.625G > A p.G209S Polymorphism with predisposition for disease, 625G > A: allele
frequency: 22 %
c.511C > T p.171W 511C > T: allele frequency, 3 %
SCHAD No prevalent mutation
MAD No prevalent mutation
Riboflavin-responsive No prevalent mutation Mutations in the ETF-DH gene
MAD Possibly also other genes affected
17.12 Treatment myopathic VLCAD deficiency. Studies in VLCAD-deficient

mice also suggest that MCT should be given according to the
Prolonged fasting should be avoided in all FAODs in order to energy demand, since MCT are otherwise elongated and
prevent acute metabolic decompensation. Frequent, regular stored as saturated long-chain fatty acids.
feeds are recommended, especially during the first year of life, Carnitine treatment is undisputedly effective in patients with
but subsequently overnight fasting will be tolerated in most dis- carnitine transporter deficiency. With a dose of 100 mg/kg/day,
orders. Prolonged overnight fasting should be postponed until plasma concentrations may reach the lower normal range, but
later childhood/adolescence especially in MTP and LCHAD muscle carnitine concentrations remain less than 5 % of normal
deficiencies in order to reduce the risk of retinopathy and neu- (Stanley et al. 1991). The value of carnitine supplementation
ropathy as a consequence of accumulating toxic metabolites. in other FAODs is controversial. Plasma-free carnitine con-
Dietary fat restriction is not indicated in MCAD deficiency centrations are often low, particularly after an acute illness, but
and mild long-chain FAODs recently identified by newborn tissue concentrations have seldom been measured. Carnitine
screening. Long-chain fat, however, needs to be restricted in treatment may even be harmful in long-chain FAODs, as it
severe long-chain FAODs and substituted by medium-chain increases the concentrations of long-chain acylcarnitines,
triglycerides (MCT). The amount of dietary MCT depends on which are potentially arrhythmogenic (Primassin et al. 2008).
the severity of the phenotype. MTP and LCHAD deficiencies Patients with riboflavin-dependent MAD deficiency
require dietary modification that is stricter (Spiekerkoetter respond to treatment with riboflavin (100 mg/day). In mod-
et al. 2009). For symptomatic infants special MCT formulas erately severe MAD deficiency, 3-hydroxybutyrate has been
exist. Anecdotal evidence suggests that a bolus of MCT used with success in a few patients for the treatment of car-
before exercise can prevent rhabdomyolysis in patients with diomyopathy (Van Hove et al. 2003).
Bezafibrates (PPAR-α and PPAR-δ agonists) may be Triheptanoin (C7 odd-chain fatty acid) has been
promising in the treatment of patients with myopathic CPT II substituted for MCT in a few patients with long-chain
or VLCAD deficiencies (Gobin-Limballe et al. 2007; FAODs (Roe et al. 2002), but has not found a widespread
Bonnefont et al. 2009). application so far.
Emergency Treatment
Deficiency of Emergency treatment Pharmacological emergency treatment

OCTN2 Glucose i.v., oral glucose monomers (to avoid hypoglycemia) reach l-carnitine i.v. (100–300 mg/kg/day)
anabolism
CPT I Glucose i.v., oral glucose monomers (to avoid hypoglycemia) reach No carnitine, d,l-3-hydroxybutyrate in
CACT anabolism: case of severe cardiomyopathy
CPT II <3 years: 10–12 mg/kg/min Pharmacological treatment of
3–10 years: 8–10 mg/kg/min arrhythmias
VLCAD
>10 years: 5–8 mg/kg/min
mTFP/MTP Oral MCT, i.v. MCT generally not available (special preparations)
LCHAD In case of severe decompensation reach anabolism with use of insulin
LKAT ACAD 9: cave lactate
ACAD 9
MCAD Glucose i.v., oral glucose monomers (to avoid hypoglycemia) reach
anabolism
No MCT
SCAD Glucose i.v. in case of hypoglycemia
SCHAD Glucose i.v. Glucagon, somatostatin diazoxide
(treatment of hyperinsulinism)
MAD Glucose i.v., oral glucose monomers (to avoid hypoglycemia) reach d,l-3-hydroxybutyrate in case of severe
anabolism cardiomyopathy
Riboflavin-responsive MAD Glucose i.v., oral glucose monomers (to avoid hypoglycemia) Riboflavin (100–300 mg/day)
Standard Treatment
Pharmacological
Deficiency of Dietary treatment treatment
OCTN2 Normal diet, regular meals, avoid catabolism l-carnitine p.o.
(100–300 mg/kg/day)
CPT I Normal diet, rarely MCT supplementation in severe phenotypes No general carnitine
CACT MCT modified diet, strictly avoid catabolism (cave arrhythmias!) supplementation
CPT II Severe phenotypes:
VLCAD Carbohydrate-enriched diet (65–75 % of total calories), fat restriction (25 % of total
calories, 10–15 % MCT, 4 % essential fatty acids, up to 10 % LCT)
Regular meals
After 1st year of life: glucose polymers at night
Myopathic phenotypes:
Fat-reduced or normal diet
MCT prior to exercise
Regular meals, normal overnight fasting tolerance
Asymptomatic patients: no dietary interventions
mTFP/MTP Strict long-chain fat reduction
LCHAD MCT supplementation (see CPT II, VLCAD)
LKAT Docosahexaenoic acid (200–400 mg/kg/day) supplementation to prevent
retinopathy is controversial
ACAD 9 No recommendations available
MCAD No dietary intervention, regular meals
SCAD No dietary intervention, regular meals
SCHAD No dietary intervention, regular meals Glucagon, somatostatin,
diazoxide (treatment of
hyperinsulinism)
MAD Fat-reduced and less protein-reduced diet or normal diet, regular meals
Riboflavin-responsive MAD Normal diet, regular meals Riboflavin
(100–300 mg/day)
Experimental Treatment
Treatment Mechanism of treatment Current status Disorders

Bezafibrate PPAR-α and PPAR-δ agonists, stimulate Successful in isolated patients, clinical Myopathic CPT II and VLCAD
residual enzyme activity in milder phenotypes trials going on deficiencies
Triheptanoin Odd-chain C7 fatty acid, anaplerotic action by Successful in isolated patients, clinical Myopathic CPT II and VLCAD
provision of propionyl-CoA trials going on deficiencies
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Roe CR, Sweetman L, Roe DS et al (2002) Treatment of cardiomyopa-
Bonnefont JP, Bastin J, Behin A et al (2009) Bezafibrate for an
thy and rhabdomyolysis in long-chain fat oxidation disorders using
inborn mitochondrial beta-oxidation defect. N Engl J Med 360:
an anaplerotic odd-chain triglyceride. J Clin Invest 110:259–269
838–840
Sim KG, Carpenter K, Hammond J et al (2002) Acylcarnitine profiles
Corr PB, Creer MH, Yamada KA et al (1989) Prophylaxis of early
in fibroblasts from patients with respiratory chain defects can
ventricular fibrillation by inhibition of acylcarnitine accumulation.
resemble those from patients with mitochondrial fatty acid beta-
J Clin Invest 83:927–936
oxidation disorders. Metabolism 51:366–371
Costa CC, de Almeida IT, Jakobs C, Poll-The BT, Duran M (1999)
Spiekerkoetter U, Khuchua Z, Yue Z et al (2004) General mitochondrial
Dynamic changes of plasma acylcarnitine levels induced by fast-
trifunctional protein (TFP) deficiency as a result of either alpha- or
ing and sunflower oil challenge test in children. Pediatr Res 46:
beta-subunit mutations exhibits similar phenotypes because muta-
440–444
tions in either subunit alter TFP complex expression and subunit
de Sain-van der Velden MGM, Diekman EF, Jans JJ et al (2013)
turnover. Pediatr Res 55:190–196
Differences between acylcarnitine profiles in plasma and bloodspots.
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Mol Genet Metab 110:116–121
mendations in long-chain fatty acid oxidation defects: consensus
Gobin-Limballe S, Djouadi F, Aubey F et al (2007) Genetic basis for
from a workshop. J Inherit Metab Dis 32:498–505
correction of very-long-chain acyl-coenzyme A dehydrogenase
Spiekerkoetter U, Haussmann U, Mueller M et al (2010) Tandem mass
deficiency by bezafibrate in patient fibroblasts: toward a genotype-
spectrometry screening for very long-chain acyl-CoA dehydroge-
based therapy. Am J Hum Genet 81:1133–1143
nase deficiency: the value of second-tier enzyme testing. J Pediatr
Herrema H, Derks TG, van Dijk TH et al (2008) Disturbed hepatic
157:668–673
carbohydrate management during high metabolic demand in
Stanley CA, DeLeeuw S, Coates PM et al (1991) Chronic cardiomy-
medium-chain acyl-CoA dehydrogenase (MCAD)-deficient mice.
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Hepatology 47:1894–1904
carnitine uptake. Ann Neurol 30:709–716
Hoffmann L, Haussmann U, Mueller M et al (2012) VLCAD enzyme
Tyni T, Kivela T, Lappi M et al (1998) Ophthalmologic findings in
activity determinations in newborns identified by screening: a
long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency caused
valuable tool for risk assessment. J Inherit Metab Dis 35:269–277
by the G1528C mutation: a new type of hereditary metabolic
Li C, Chen P, Palladino A et al (2010) Mechanism of hyperinsulin-
chorioretinopathy. Ophthalmology 105:810–824
ism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency
Van Hove JL, Grunewald S, Jaeken J et al (2003) D, L-3-hydroxybutyrate
involves activation of glutamate dehydrogenase. J Biol Chem
treatment of multiple acyl-CoA dehydrogenase deficiency (MADD).
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van Maldegem BT, Duran M, Wanders RJ et al (2006) Clinical, bio-
of cut-off target ranges in newborn screening of metabolic disorders
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Genet Med 13:230–254
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replenishing low free carnitine. Pediatr Res 63:632–637
Disorders of Carbohydrate Metabolism
and Glucose Transport 18
René Santer, Joerg Klepper, and G. Peter A. Smit
Contents 18.7.8 Glycogen Storage Diseases: Mainly Affecting Muscle .... 299
18.7.9 GSD-IIa ............................................................................ 300
18.1 Introduction .................................................................... 266
18.1.1 Glucose Transport Disorders............................................ 266 References ..................................................................................... 300
18.1.2 Disorders of Monosaccharide Conversion into Glucose .. 266 Further Reading ........................................................................... 300
18.1.3 Glycogen Storage Disorders ............................................ 267
18.2 Nomenclature.................................................................. 268
18.3 Metabolic Pathways ....................................................... 270
Summary
18.4 Signs and Symptoms ...................................................... 274
Carbohydrates are one of the most important sources of energy
18.5 Diagnosis ......................................................................... 286 in the human organism. Within the body, glucose is the most
18.5.1 Reference and Pathological Values .................................. 286
18.5.2 Specimen Collection ........................................................ 287
abundant monosaccharide which can be stored as glycogen,
18.5.3 Provocation and Loading Tests ........................................ 288 a branched polymer, in liver and muscle. Inborn errors of
18.5.4 Metabolites, Enzymatic and Genetic Tests ...................... 290 metabolism may affect the uptake, distribution and reabsorp-
18.6 Prenatal Diagnosis.......................................................... 292 tion of monosaccharides in different organs, a process which
is meticulously regulated by a system of transporter proteins.
18.7 Treatment ........................................................................ 292
18.7.1 Intestinal Glucose-Galactose Malabsorption ................... 293 Congenital disorders may impair the conversion of other
18.7.2 Renal Glucosuria .............................................................. 293 monosaccharides (fructose, galactose) into glucose. They
18.7.3 Glucose Transporter-1 Deficiency.................................... 293 can further affect glycogen formation, glycogen breakdown
18.7.4 Fanconi-Bickel Syndrome................................................ 295 (glycogenolysis), glucose metabolism to acetyl-CoA (gly-
18.7.5 Disorders of Galactose Metabolism ................................. 295
18.7.6 Disorders of Fructose Metabolism ................................... 296 colysis) and de novo synthesis of glucose from glucoplastic
18.7.7 Glycogen Storage Diseases: Mainly Affecting Liver ...... 297 amino acids or from lactate (gluconeogenesis).
Glucose transport disorders present with a very variable
clinical picture depending on which of the organ- and
substrate-specific transporters are affected. This group of
disorders includes intestinal glucose-galactose malabsorp-
tion, renal glucosuria, glucose transporter-1 deficiency of the
blood-brain barrier and Fanconi-Bickel syndrome (a disease
with hepatic glycogen storage and massive renal tubular mal-
R. Santer (*)
Department of Pediatrics, University Medical Center Hamburg absorption of glucose and other substances).
Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany Disorders of fructose and galactose metabolism affect dif-
e-mail: r.santer@uke.de ferent organs because of the accumulation of toxic interme-
J. Klepper diates. After these monosaccharides have been introduced
Kinderklinik, Klinikum Aschaffenburg, Am Hasenkopf, 63739 with the diet, symptoms secondary to impaired liver function
Aschaffenburg, Germany
are frequently the first signs.
e-mail: joerg.klepper@klinikum-aschaffenburg.de
Glycogen storage diseases can be divided into those
G.P.A. Smit
mainly presenting with hepatic manifestations (hepatomegaly,
Department of Metabolic Diseases, Beatrix Children’s Hospital,
Hanzeplein 1, 9713 GZ Groningen, The Netherlands hypoglycaemia) and those with muscular presentations (exer-
e-mail: g.p.a.smit@umcg.nl tion intolerance, rhabdomyolysis). Some show a combination
266 R. Santer et al.
of these symptoms, cardiomyopathy may be an additional characterised by the fact that glucose transport is coupled to
feature, and further accompanying symptoms, e.g. haemo- sodium transport. Since the driving force for this type of
lytic anaemia, may be observed depending on the tissue dis- transport is the electrochemical gradient for sodium (pro-
tribution of the affected protein. duced by the cellular Na+/K+-ATPase system), glucose can
Management of most carbohydrate disorders requires be (‘actively’) transported against its own gradient. The sec-
symptomatic measures, supportive care and a multidisci- ond family, the GLUT (glucose transporter) proteins, medi-
plinary approach. In some conditions, dietetic treatment is ates the so-called facilitative diffusion along an existing
possible which may have its basis in an exclusion of certain glucose gradient, and in the past members of this family have
monosaccharides. A ketogenic diet supplying non- been regarded to function as ‘passive’ transporters. However,
carbohydrate energy sources may have dramatic effects in GLUT proteins are hormonally and substrate regulated; thus,
glucose transporter-1 deficiency, and frequent meals or the they play a pivotal role in the regulation of carbohydrate
administration of slowly absorbed carbohydrates are suc- metabolism. For example, one of the key functions of insulin
cessful means to avoid hypoglycaemia in disorders with is the translocation of GLUT4-bearing vesicles to the cell
impaired endogenous glucose production. Outcome, how- membrane of muscle cells and adipocytes, allowing glucose
ever, is very variable and mainly depends on the underlying influx.
type of disorder. A causal treatment is not available for most The genes of the growing number of members of both the
types of carbohydrate disorders. Enzyme replacement ther- SGLT and the GLUT family have been identified during
apy (with as yet unsatisfactory results) is only available for recent years. This was a key step in the study of the function
lysosomal α-glucosidase deficiency (GSD-IIa). In some of these proteins and of their tissue-specific expression and
patients with severe types of disorders or an unfavourable in the identification of genetic defects. Knowledge of the
course, organ transplantation (liver, kidney, heart) is the only tissue-specific expression of monosaccharide transporter
option for long-term survival. proteins helps to define the clinical picture of the different
disease entities. Many tissues carry more than one type of
transporter and many members of the two monosaccharide
18.1 Introduction transporter families are expressed in more than one tissue.
Therefore, congenital disorders of different isoforms of
This chapter deals with disorders of carbohydrate metabo- monosaccharide transporters may present with a complex
lism which can be divided into glucose transport disorders, clinical picture and may involve different organ systems.
disorders of fructose and galactose metabolism and disorders Well-characterised disease entities are congenital intestinal
that affect glycogen metabolism including diseases that glucose-galactose malabsorption (GGM, SGLT1 defect), the
impair glycogen degradation but also glycogen formation. glucose transporter-1 deficiency syndrome (GLUT1-D) and
Most of them are inherited according to an autosomal- the Fanconi-Bickel syndrome (FBS, GLUT2 defect). In con-
recessive trait. The clinical presentations can be mild or trast, due to the high solubility of glucose, renal glucosuria
severe, in some of them even life-threatening. The clinical due to SGLT2 deficiency (SGLT2-D) is rarely accompanied
features are very variable and can be understood when look- by specific clinical symptoms, and like some of the amino
ing at pathophysiological mechanisms of the different groups acid transporter defects, it can be classified as a ‘non-
of disorders. disease’. The diagnosis has, however, a clinical significance
as it prevents the repeated investigation for diabetes mellitus
in these patients.
18.1.1 Glucose Transport Disorders
The cellular uptake and release of glucose and other mono- 18.1.2 Disorders of Monosaccharide
saccharides is a protein-mediated process. Such proteins are Conversion into Glucose
embedded into the cell membrane and can be regarded as
hydrophilic pores within the hydrophobic lipid bilayer. There are three known disorders of galactose metabolism:
Monosaccharide transporters are substrate- and stereospe- galactokinase deficiency (GALK-D), galactose-1-phosphate
cific. Their action is saturable and, like for an enzymatic uridyltransferase deficiency (galactosaemia, GALT-D) and
reaction, kinetic characteristics can be described by affinity uridine diphosphate galactose-4-epimerase deficiency
constants. The number of transporter protein molecules (GALE-D). Among these disorders, galactosaemia is the
determines maximal transport velocity (vmax). most common and the most severe. Several partial defects of
Two classes of monosaccharide transporters exist. The transferase deficiency have been reported of which the best
SGLT (sodium-dependent glucose transporter) family is known is the Duarte-2 variant.
18 Disorders of Carbohydrate Metabolism and Glucose Transport 267
Isolated Hepatomegaly –
(age 4 -18 months)
Further symptoms and laboratory findings
Disorder of Disorder of Disorder of Disorder of

glycogenolysis and gluconeogenesis glycogenolysis glycogenolysis glycogen synthesis
(symptomatic) hypoglycemia and severe tubulopathy (with an abnormal glycogen)
with poor fasting tolerance (2-4 hrs) moderate propensity to hypoglycemia moderate propensity to hypoglycemia
absence of ketosis, and only after longer fasting periods, and only after longer fasting periods, absence of hypoglycemia,
hyperventilation due to lactic acidosis, fasting ketosis fasting ketosis progressive liver dysfunction,
kidneys enlarged on ultrasound (foetor acetonaemicus,ketostix + -+++ ) plus:glucosuria, aminoaciduria, cirrhosis
phosphaturia, proteinuria, polyuria
signs of myopathy ??
(CK elevation ??)
+ –
GSD type I GSD type III GSD type VI, IX FBS GSD type IV
Fig. 18.1 Flowchart for clinical differentiation of hepatic glycogen storage disorders
All three disorders can be identified by newborn 18.1.3 Glycogen Storage Disorders
screening programmes based upon enzymatic investiga-
tions or detection of increased amounts of galactose and Glycogen storage disorders result from enzymatic blocks or
galactose-1-phosphate in dried blood spots. The clinical impaired transporter function in the pathway of glycogen deg-
manifestations of classical galactosaemia occur when radation, but defects of glycogen synthesis in liver and muscle
galactose is introduced with the diet and the accumulation have also been termed glycogenoses (GSD-0, GSD-IV).
of metabolic intermediates is responsible for organ dam- Simply speaking, glycogen storage disorders with cytosolic
age. Galactitol is responsible for cataract formation, while glycogen accumulation may present as a disease of liver only
galactose-1-phosphate (together with phosphate depletion) (GSD-I, GSD-III, GSD-VI and GSD-IX), of liver and kidney
is a key factor for hepatic, renal and central nervous (FBS, GSD-I) and with liver involvement and (cardio)myopa-
manifestations. thy (GSD-III, GSD-IX) or myopathy without liver involve-
Four disorders affecting fructose metabolism are known: ment (GSD-V, GSD-VII, rare muscular types). Lysosomal
fructokinase deficiency (FK-D), fructose-1-phosphate aldolase storage of glycogen results in a multisystem disorder generally
deficiency (hereditary fructose intolerance, HFI), fructose-1,6- presenting with cardiac and/or muscular problems (GSD-II).
biphosphatase deficiency (FBP-D) and D-glyceric acidaemia. GSD-I, GSD-III, GSD-VI and GSD-IX are similar in
While FK-D is another non-disease, severe symptoms in HFI physical appearance and are usually detected during infancy
occur after the ingestion of fructose and are the consequence or childhood because of failure to thrive and marked hepa-
of accumulation of fructose-1-phosphate with secondary tomegaly (and, generally, without splenomegaly or cho-
effects mainly on liver cell metabolism, phosphate depletion lestasis) (Fig. 18.1). Hypoglycaemia is another leading sign
and protein glycosylation. FBP-D is a disorder of gluconeo- in hepatic GSDs which is most severe in GSD-I where both
genesis; symptoms may occur in the fasted state and with- glycogenolysis and gluconeogenesis are affected.
out fructose ingestion, although fructose may enhance these Classical muscle GSDs involve skeletal muscle and
effects. D-glyceric acidaemia (glycerate kinase deficiency, present with muscle weakness, exertion intolerance and/or
GLYCTK-D) is associated with a large variety of symptoms, myoglobinuria but are usually not diagnosed before late
mainly of neurological type, which can be explained by the childhood and adolescence. Rare muscle GSDs with defects
fact that GLYCTK-D impairs several metabolic pathways in the glycolysis pathway of muscle may, however, present
including fructose and serine metabolism. with a more complex clinical picture.
18.2 Nomenclature
268
Chromosomal
No. Disorder Alternative name Abbreviation Gene symbol localisation Affected protein OMIM no.
18.1 Intestinal glucose-galactose SGLT1 deficiency GGM SGLT1 (SLC5A1) 22q12.3 Sodium-dependent 606824
malabsorption glucose(-galactose)
transporter-1
18.2 Renal glucosuria SGLT2 deficiency SGLT2-D SGLT2 (SLC5A2) 16p11.2 Sodium-dependent glucose 233100
transporter-2
18.3 Glucose transporter-1 GLUT1 deficiency GLUT1-D GLUT1 (SLC2A1) 1p34.2 Glucose transporter-1 606777, 612126, 601042,
deficiency 614847
18.4 Fanconi-Bickel syndrome GLUT2 deficiency FBS GLUT2 (SLC2A2) 3q26.2 Glucose transporter-2 227810
18.5 Arterial tortuosity syndrome GLUT10 deficiency GLUT10-D GLUT10 20q13.12 Glucose transporter-10 208050
(SLC2A10)
18.6 Galactokinase deficiency GALK-D GALK 17q24 Galactokinase 230200
18.7 Galactosaemia Galactose-1-phosphate GALT-D GALT 9p13.3 Galactose-1-phosphate 230400
uridyltransferase uridyltransferase
deficiency
18.8 Uridine diphosphate GALE-D GALE 1p36-p35 Uridine diphosphate 230350
galactose-4-epimerase galactose-4-epimerase
deficiency
18.9 Essential fructosuria Fructokinase deficiency FK-D KHK 2p23.3 Fructokinase 229800
18.10 Hereditary fructose Fructose-1-phosphate HFI ALDOB 9q31.1 Fructose-1-phosphate 229600
intolerance aldolase deficiency aldolase
18.11 Fructose-1,6-bisphosphatase FBP-D FBP1 9q22.32 Fructose-1,6-bisphosphatase 229700
deficiency
18.12 Glycerate kinase deficiency D-glyceric acidaemia GLYCTK-D GLYCTK 3p21.1 Glycerate kinase 610516
18.13 Glycogen storage disease Liver glycogen synthase GSD-0a GYS2 12p12.1 Liver glycogen synthase 240600
type 0a deficiency
18.14 Glycogen storage disease Muscle glycogen synthase GSD-0b GYS1 19q13.33 Muscle glycogen synthase 611556
type 0b deficiency
18.15 Glycogen storage disease Muscle glycogenin GSD-XV GYG1 3q24 Glycogenin-1 613507
type XV deficiency
18.16 Glycogen storage disease Glycogen branching GSD-IV GBE1 3p12.2 Glycogen branching enzyme 232500
type IV enzyme deficiency
18.17 Glycogen storage disease Phosphoglucomutase 1 GSD-XIV Phosphoglucomutase 1 612934, 614921
type XIV deficiency
18.18 Glycogen storage disease Glucose-6-phosphatase GSD-Ia G6PC 17q21.31 Glucose-6-phosphatase 232200
type Ia deficiency
18.19 Glycogen storage disease Glucose-6-phosphate GSD-I non-a G6PT1 (SLC37A4) 11q23.3 Glucose-6-phosphate 232220
type I non-a translocase deficiency translocase
18.20 Glycogen storage disease Lysosomal alpha-1,4- GSD-IIa GAA 17q25.3 Alpha-1,4-glucosidase 232300
type IIa glucosidase deficiency
R. Santer et al.
18.21 Glycogen storage disease LAMP2 deficiency GSD-IIb LAMP2 Xq24 Lysosome-associated 300257
type IIb membrane protein-2
18.22 Glycogen storage disease Amylo-1,6-glucosidase GSD-III AGL 1p21.2 Amylo-1,6-glucosidase 232400
type III (debrancher) deficiency
18.23 Glycogen storage disease Muscle phosphorylase GSD-V PYGM 11q13.1 Muscle glycogen 232600
type V deficiency phosphorylase
18.24 Glycogen storage disease Liver glycogen GSD-VI PYGL 14q22.1 Liver glycogen 232700
type VI phosphorylase deficiency phosphorylase
18.25 Glycogen storage disease Liver phosphorylase GSD-IXa–c PHKA2, PHKB, Xp22.13, Liver phosphorylase kinase 306000, 261750, 613027
type IXa–c kinase deficiency PHKG2 16q12.1, 16p11.2
18.26 Glycogen storage disease Muscle phosphorylase GSD-IXd PHKA1 Xq13.1-q13.2 Muscle phosphorylase kinase 300559
type IXd kinase deficiency
18.27 Constitutional AMP- AMPK-A PRKAG2 7q36.1 AMP-activated protein 600858, 261740, 194200
activated protein kinase kinase
activation
18.28 Glycogen storage disease Muscle GSD-VII PFKM 12q13.11 Phosphofructokinase 232800
type VII phosphofructokinase
deficiency
18.29 Glycogen storage disease Aldolase A deficiency ALDOA-D ALDOA 16p11.2 Aldolase A 611881
type XII
18.30 Muscle phosphoglycerate PGK-D PGK1 Xq21.1 Phosphoglycerate kinase 300653
kinase deficiency
18 Disorders of Carbohydrate Metabolism and Glucose Transport
18.31 Glycogen storage disease Muscle phosphoglycerate GSD-X PGAM2 7p13 Phosphoglycerate mutase-2 261670
type X mutase deficiency
18.32 Glycogen storage disease Beta-enolase deficiency GSD-XIII ENO3 17p13.2 Beta-enolase 612932
type XIII
18.33 Glycogen storage disease Lactate dehydrogenase A LDHA-D LDHA 11p15.1 LDHA 612933
type XI deficiency
269
18.3 Metabolic Pathways chapter because of its relation to fructose metabolism;

other disorders of gluconeogenesis, however, are covered
Metabolic pathways of carbohydrate metabolism can be in other parts of this book.
divided into (1) the cellular uptake and release of glucose Disorders of organ-specific glucose transport are system-
(and other monosaccharides) by different organs, (2) the atically depicted in Fig. 18.2, and galactose and fructose
intracellular pathways linking the metabolism of other metabolism are presented in Figs. 18.3 and 18.4, respec-
monosaccharides to glucose metabolism, (3) the break- tively. The major steps of glycogen breakdown are shown in
down and synthesis of glycogen to and from glucose, Fig. 18.5, while individual steps with associated defects of
respectively, and (4) the steps of utilisation of glucose. glycogenolysis and disorders of glycolysis are presented in
Fructose-1,6-biphosphatase deficiency is found in this Fig. 18.6.
Glia cells Neurons
GLYCOGEN Glc Glc
Glc CSF GLYCOGEN
Hepatocytes 18.3 GLUT3 Glc

Glucose
Endothelial cells
Muscle cells
from (“Blood Brain Barrier”)
Adipocytes
food 18.4
GLUT4
Glc
Glc
BLOOD 18.2 18.4
Enterocytes
18.4 GLYCOGEN
18.1
Glc
18.4 18.5
Glc Endothelial cells Renal tubulus cells
18.1
b cells
Insulin
secretion URINE
“active“ transporters: “passive“ transporters: vesicular transport:
SGLT1, SGLT2 GLUT1, GLUT2, GLUT3, GLUT4, GLUT10
Fig. 18.2 Cellular uptake and release of glucose and transcellular while rectangular symbols stand for facilitative, the so-called passive
transport mediated by specific transporter proteins. Transport across transporters (encoded by genes of the SLC2 family). Known defects are
cell membranes is indicated by arrows. Transporter proteins are shown by blue bars and the respective number. CSF cerebrospinal fluid
depicted by different symbols. Round symbols represent sodium- (Modified from Santer and Klepper (2012))
dependent active transporters (encoded by genes of the SLC5 family),
Lactose ( Lactose )
Glycogen
complex complex
carbohydrates carbohydrates
18.8
UDP-Gal UDP-Glc
18. 1 18. 4 18.6

Galactose Galactose Gal-1-P
18.7
Galactitol Glc-1-P
18. 1 18. 4 Galactonat
Glucose Glucose
Glc-6-P Glc Glc
Enterocytes (Liver) Cell
Fig. 18.3 Metabolic pathway of galactose metabolism
Glucose Glucose Glc-1-P Glc-6-P Glc Glc
Frc-6-P
? 18.9 18.11
Fructose Fructose Frc-1-P Frc-1,6-P2
Frukt -18.10
Sorbitol Sorbitol
Glyceraldehyde Dihydroxyacetone-P Glyceraldehyde-3-P
D-Glycerate
18.12
Sucrose 2-P-Glycerate Pyr
Enterocytes (Liver) Cell
Fig. 18.4 Metabolic pathway of fructose metabolism

18.25 (L)
18.26 (M)
18.27 Glycogen
Phosphorylase-b-Kinase
1–6
1–4 ... ... ... O

Phosphorylase b Phosphorylase a
18.23 (M)
18.24 (L)
+ Limit dextrin
4-alpha-Glucanotransferase,
Amylo-1,6-Glucosidase +
18.22 (L,M)
+
Phosphoglucomutase
18.17 (L,M)
Glucose-6-Phosphatase system
18.18 (L)
18.19 (L)
Glucose
Fig. 18.5 Principal steps in glycogen degradation. Note that the scheme is a summary of pathways in different tissues. Tissue-specific isoforms
of enzymes (L liver, M muscle) may result in different disease entities with tissue-related signs and symptoms
Lysosome
18.16
18.15 Glycogen
18.14 18.23 18.20

18.13 18.24 Limit Dextrin 18.21
18.25
UDP-Glc 18.22
18.26
18.27
Glc
Glc-1-P ?
18.17
Glc Glc-6-P Glc-6-P Glc Glc Glc

18.3 18.19 18.18
18.4
eR
Frc-6-P
18.11 18.28
Frc-1,6-DiP
18.29
Glyceraldehyde-3-P
1,3-DiP-Glycerate
18.30
3-P-Glycerate Cytosol
18.31
2-P-Glycerate
18.32
Phosphoenolpyruvate
18.33
Pyr Lac
Krebs
Cycle
Fig. 18.6 Metabolic pathways of glycogen degradation. Note that the scheme is a summary of pathways in different tissues. eR endoplasmic
reticulum
Table 18.1 Intestinal glucose-galactose malabsorption

Digestive Diarrhoea, profuse, +++ +++ ++ ++ ++
osmotica
Other Dehydrationa +++ +++ ++ ++ ++
Hypovolaemic shocka + + + + +
Routine laboratory Glucose (U) ↑ ↑ ↑ ↑ ↑
Reducing sugars (stool)a ↑ ↑ ↑ ↑ ↑
Sodium (P)a n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Fructose loading test, p.o. n n n n n
Galactose loading test, + + + + +
p.o.
Glucose loading test, p.o. + + + + +
Glucose, galactose ↓ ↓ ↓ ↓ ↓
uptake by enterocytes
a
Disease features occur on a glucose- and/or galactose-containing diet
Table 18.2 Renal glucosuria

Routine laboratory Glucose (P) n n n n n
Glucose (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Special laboratory Amino acids (U) n-↑ n-↑ n-↑ n-↑ n-↑
Table 18.3 Glucose transporter-1 deficiency

Dystonia ± ± ± ±
Hypotonia, muscular ± ± ± ±
Microcephaly ± ± ± ±
Movement disorder, ± ± ± ±
complex and
paroxysmal
Haematological Anaemia, haemolytic ± ± ± ±
Routine laboratory Glucose (CSF)a ↓ ↓ ↓ ↓ ↓
Glucose ratio (CSF/P) ↓ ↓ ↓ ↓ ↓
Lactate (CSF) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory Glucose uptake (RBC) ↓ ↓ ↓ ↓ ↓
GLUT1 in RBC ↓ ↓ ↓ ↓ ↓
(Western blot)
a
Do not misinterpret the results of random postictal lumbar punctures that can give false-high blood glucose concentrations and an abnormal ratio.
In case of a clinical suspicion, perform determination of CSF/plasma glucose ratio in samples drawn after at least 4 h of fasting with sample for
blood glucose drawn first
Table 18.4 Fanconi-Bickel syndromea

Digestive Hepatomegaly n ± ++ + +
Loose stools ± ±
Malabsorption ± ±
Eye Cataractb ±
Musculoskeletal Rickets ± ± ±
Short stature n ++ +++ +++ +++
Renal Nephromegaly ± ± ± ± ±
Renal failure, ± ±
hyperfiltration
Renal tubulopathy, + + + + +
generalised
Routine laboratory ASAT/ALAT (P) n-↑ n-↑ n-↑ n-↑ n-↑
Calcium (U) ↑ ↑ ↑ ↑ ↑
Cholesterol (P) ↑ ↑ ↑ ↑ ↑
Glucose (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Glucose, fasted (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Glucose, fed (P) n-↑ n-↑ n-↑ n-↑ n-↑
Phosphate (U) ↑ ↑ ↑ ↑ ↑
Triglycerides (P) ↑ ↑ ↑ ↑ ↑
Uric acid (P) ↑ ↑ ↑ ↑ ↑
Special laboratory Amino acids (U) ↑ ↑ ↑ ↑ ↑
Galactitol (U)b n-↑ n-↑ n-↑ n-↑ n-↑
Galactose (P)b n-↑ n-↑ n-↑ n-↑ n-↑
Galactose (U)b n-↑ n-↑ n-↑ n-↑ n-↑
Glycogen (liver) ↑ ↑ ↑ ↑ ↑
Glycogenolytic enzymes n n n n n
(all tissues)
a
From older classifications also known as GSD type XI
b
On a galactose-containing diet
Table 18.5 Arterial tortuosity syndrome

System Symptoma Neonatal Infancy Childhood Adolescence Adulthood
Cardiovascular Hypertension, arterial + + + + +
Pulmonary stenoses + + + + +
Tortuosity and elongation of + + + + +
large- and medium-size arteries
CNS Stroke, ischaemic + + + +
Dermatological Cutis laxa + + + + +
Teleangiectases + + + +
Musculoskeletal Arachnodactyly + + + + +
Hernia, diaphragmatic + + + + +
Joint laxity + + + + +
Other Facial stigmata + + + + +
Pectus excavatum/carinatum + + + + +
a
Recently demonstrated to be secondary to impaired mitochondrial import of dehydroascorbic acid and altered collagen metabolism rather than a
consequence of disturbed glucose transport (see Lee et al. 2010)
Table 18.6 Galactokinase deficiency

CNS Pseudotumor cerebria + +
Eye Cataracta + + + + +
Routine laboratory Reducing substances (U)a + + + + +
Special laboratory Galactitol (U)a ↑ ↑ ↑ ↑ ↑
Galactokinase (RBC,FB) ↓ ↓ ↓ ↓ ↓
Galactose (P,U)a ↑ ↑ ↑ ↑ ↑
a
Features occur on a galactose-containing diet
Table 18.7 Galactosaemia

CNS Brain oedemaa +
Digestive Anorexiaa + +
Hepatomegalya + +
Liver cirrhosisa + +
Liver failurea + +
Vomitinga + +
Eye Cataracta + +
Genitourinary Hypergonadotropic + +
hypogonadism (females)
Ovarian failure + +
Haematological Anaemia, haemolytica + +
Other E. coli sepsis +
Early deatha + +
Routine laboratory ASAT/ALAT (P)a ↑ ↑ n-↑ n-↑ n-↑
Bilirubin (P)a ↑ ↑ n-↑ n-↑ n-↑
Calcium (U)a ↑ ↑ n-↑ n-↑ n-↑
Coagulation factors (P)a ↓ ↓ ↓-n ↓-n ↓-n
Glucose (U)a ↑ ↑ n-↑ n-↑ n-↑
Phosphate (U)a ↑ ↑ n-↑ n-↑ n-↑
Proteins, total (U)a ↑ ↑ n-↑ n-↑ n-↑
Reducing substances (U)a + + + + +
Special laboratory Amino acids (U)a ↑ ↑ n-↑ n-↑ n-↑
Galactitol (U)a ↑ ↑ ↑ ↑ ↑
Galactose (P,U)a ↑ ↑ ↑ ↑ ↑
Galactose-1-phosphate (RBC) ↑ ↑ ↑ ↑ ↑
Galactose-1-phosphate ↓ ↓ ↓ ↓ ↓
uridyltransferase (RBC)
Partial Gal-1-P-uridyltransferase deficiency (e.g. in individuals with the Duarte-2 variant) is usually asymptomatic
a
Features occur on a galactose-containing diet
Table 18.8 Uridine diphosphate galactose-4-epimerase deficiency

Digestive Anorexiaa + +
Hepatomegalya + +
Liver cirrhosisa + +
Liver failurea + +
Vomitinga + +
Eye Cataracta + +
Other Early deatha + +
Routine ASAT/ALAT (P)a ↑ ↑ n-↑ n-↑ n-↑
laboratory Bilirubin (P)a ↑ ↑ n-↑ n-↑ n-↑
Calcium (U)a ↑ ↑ n-↑ n-↑ n-↑
Coagulation factors (P)a ↓ ↓ ↓-n ↓-n ↓-n
Glucose (U)a ↑ ↑ n-↑ n-↑ n-↑
Phosphate (U)a ↑ ↑ n-↑ n-↑ n-↑
Proteins, total (U)a ↑ ↑ n-↑ n-↑ n-↑
Reducing substances (U)a + + + + +
Special Amino acids (U)a ↑ ↑ n-↑ n-↑ n-↑
laboratory Galactose (P,U)a n-↑ n-↑ n-↑ n-↑ n-↑
Galactose-1-phosphate (RBC)a ↑ ↑ n-↑ n-↑ n-↑
UDP-Gal epimerase (RBC) ↓ ↓ ↓ ↓ ↓
Patients with the mild form are clinically asymptomatic but may be detected in neonatal screening programmes and show abnormal laboratory
results
a
Disease features occur following dietary galactose intake
Table 18.9 Essential fructosuria

Other No clinical significance
Routine Glucose (U) n n n n n
laboratory Reducing substances (U)a + + + + +
Special Ketohexokinase (liver) ↓ ↓ ↓ ↓ ↓
laboratory
a
Disease features occur following dietary fructose intake
Table 18.10 Hereditary fructose intolerance

Digestive Abdominal paina + + ± ±
Failure to thrivea + + ± ±
Feeding habits, abnormala + +
Hepatomegalya + + ± ±
Liver cirrhosisa ± ± ±
Liver failurea + + ± ±
Vomitinga + + ± ±
Renal Renal tubulopathya + + ± ±
Other No dental cariesb + + +
Routine ASAT/ALAT (P)a ↑ ↑ n-↑ n-↑
laboratory Bilirubin (P)a ↑ ↑ n-↑ n-↑
Coagulation factors (P)a ↓ ↓ ↓ ↓-n
Glucose (P)a ↓-n ↓-n ↓-n ↓-n
Phosphate (P)a ↓-n ↓-n ↓-n ↓-n
Reducing substances (U)a ± ± ± ±
Uric acid (P)a ↑ ↑ n-↑ n-↑
Special Fructose-1-phosphate aldolase (liver) ↓ ↓ ↓ ↓ ↓
laboratory Glycerol (U)a ↑ ↑ ↑ ↑
a
Disease features occur following dietary fructose intake
b
On Frc/Sac restriction
Table 18.11 Fructose-1,6-bisphosphatase deficiency

Respiratory Tachypnea + + +
Routine Glucose (P)a ↓ ↓ ↓ ↓-n ↓-n
laboratory Ketones (U,P)a ↑ ↑ ↑
Lactate (P)a ↑ ↑ ↑ ↑
Phosphate (P)a ↓-n ↓-n ↓-n n n
Triglycerides (P)a,b ↑ ↑ ↑ n-↑ n-↑
Uric acid (U,P)a n-↑ n-↑ n-↑ n n
Special Alanine (P)a ↑ ↑ ↑ ↑
laboratory Fructose-1,6-bisphosphatase (liver) ↓ ↓ ↓ ↓ ↓
Glycerol (U)a n-↑ n-↑ n-↑
a
Disease features occur in the fasted state and can be exaggerated by fructose, sorbitol or glycerol intake
b
Pseudo(!)hypertriglyceridemia
Table 18.12 Glycerate kinase deficiency

CNS Encephalopathy ± ± ±
Hypotonia, muscular ± ± ±
Seizures ± ± ±
Speech delay ± ± ±
Other Early death ± ± ±
Routine Metabolic acidosis ± ± ± ±
laboratory
Special D-glycerate (P,U,CSF) ↑ ↑ ↑ ↑
laboratory D-glycerate kinase (liver) ↓ ↓ ↓ ↓
Glycine (P, U, CSF) n-↑ n-↑ n-↑ n-↑ n-↑
Table 18.13 Glycogen storage disease type 0a

Routine Glucose, fasted (P) ↓ ↓ ↓ ↓-n ↓-n
laboratory Glucose, fed (P) n-↑ n-↑ n-↑ n-↑ n-↑
Ketones, fasted (U,P) ↑ ↑ ↑ n-↑ n-↑
Lactate, fed (P) ↑ ↑ ↑ n-↑ n-↑
Special Glycogen (liver) ↓-n ↓-n ↓-n ↓-n ↓-n
laboratory Glycogen synthase (liver) ↓ ↓ ↓ ↓ ↓
Table 18.14 Glycogen storage disease type 0b

Cardiovascular Cardiac arrest, sudden + + +
Cardiomyopathy, hypertrophic + + +
Syncope + + +
CNS Mental retardation + + +
Seizures + + +
Musculoskeletal Exertion intolerance + + +
Muscle weakness + + +
Special Glycogen (muscle) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
laboratory Glycogen synthase (muscle) ↓ ↓ ↓ ↓ ↓
Table 18.15 Glycogen storage disease type XV

Cardiovascular Cardiomyopathy, + +
hypertrophic
Ventricular arrhythmia + +
Musculoskeletal Exertion intolerance ± ± + +
Muscle weakness ± ± + +
Special laboratory Glycogen (muscle) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 18.16 Glycogen storage disease type IV

Cardiovascular Cardiomyopathy ± ±
Digestive Hepatopathy ± + +
Liver cirrhosis n + +
Haematological Splenomegaly n + +
Musculoskeletal Hypotonia, muscular + +
Muscle weakness + +
Muscular atrophy + +
Other Early death + +
Failure to thrive + +
Routine laboratory ASAT/ALAT (P) n-↑ ↑ ↑
Bilirubin (P) n-↑ ↑ ↑
Coagulation factors (P) ↓-n ↓ ↓
Special laboratory Branching enzyme (L, M, FB, ↓ ↓ ↓
WBC, RBC)
Glycogen (liver) ↓-n ↓-n
Glycogen, abnormal + + +
(amylopectin) (liver)
a
Very heterogenous disorder: in addition to the classic hepatic manifestation, there is a more benign nonprogressive hepatic form. Rare neuromus-
cular forms may present prenatally and neonatally with hydrops fetalis and an arthrogryposis-like picture, and there are also childhood and adult
neuromuscular forms, the latter being termed ‘adult polyglucosan body disease’
Table 18.17 Glycogen storage disease type XIV

Musculoskeletal Exertion intolerancea ± +
Muscle crampsa ± +
Muscle weaknessa ± +
Routine laboratory Ammonia (B)b n-↑
Ammonia rise in forearm ↑
exercise test (B)
Creatine kinase (P) n-↑ ↑
Lactate rise in forearm exercise ↑
test (P)
Special laboratory Glycogen (muscle) n-↑ ↑
Myoglobin (U)a n-↑
Phosphoglucomutase (FB,M) ↓ ↓ ↓ ↓ ↓
a
Symptoms usually induced by strenuous exertion
b
Only very recently, a second manifestation of PGM1 deficiency has been described which has features both of a hepatic and muscular glycogen
storage disorder and of a congenital disorder of glycosylation with a TIEF type I/II pattern. It has been termed PGM1-CDG or CDG It. Patients
present with facial stigmata, cleft palate, hepatopathy, dilated cardiomyopathy or thrombosis (see Pérez et al. 2013)
Table 18.18 Glycogen storage disease type Ia

Digestive Diarrhoea ± ± ± ± ±
Hepatomegaly n ± ++ + +
Liver adenoma, carcinoma ±
Pancreatitis ± ± ±
Haematological Bleeding tendency ± ± ± ± ±
Musculoskeletal Osteopenia ± ± ± + +
Short stature n ± + + +
Renal Glomerulosclerosis ± +
Hyperfiltration ± +
Renal enlargement + + + + +
Other Adiposity (doll-like facies) n ± + + +
Glucose, fasted (P) ↓ ↓ ↓ ↓ ↓
Ketones, fasted (U,P) ↓ ↓ ↓ ↓ ↓
Lactate, fasted (P,U) ↑ ↑ ↑ ↑ ↑
Uric acid (U,P) ↑ ↑ ↑ ↑ ↑
Special laboratory Biotinidase (P) n-↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑ n-↑
Glucose-6-phosphatase (liver) ↓ ↓ ↓ ↓ ↓
Glycogen (liver) n-↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 18.19 Glycogen storage disease type I non-a

Hepatomegaly n ± ++ + +
Inflammatory bowel disease + + + + +
Liver carcinoma, adenomata n ± ++ + +
Oral ulcerations + + + + +
Pancreatitis ±
Haematological Bleeding tendency ± ± ± ± ±
Leukocyte function impaired + + + + +
Musculoskeletal Osteopenia ± ± ± ± ±
Renal Glomerulosclerosis ± +
Hyperfiltration ± +
Recurrent infections + + + + +
Routine laboratory Glucose, fasted (P) ↓ ↓ ↓ ↓ ↓
Neutrophil count ↓ ↓ ↓ ↓ ↓
Special laboratory Glucose-6-phosphatase (liver, frozen) n n n n n
Glucose-6-phosphatase (liver, unfrozen) ↓ ↓ ↓ ↓ ↓
Glycogen (liver) n-↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 18.20 Glycogen storage disease type IIa

Cardiovascular Cardiac failure + + +b
Cardiac preexcitation syndrome + +
Cardiomyopathy, hypertrophic and dilated ++ ++
CNS Hypotonia, muscular ++ ++ ++b
Mental retardation + +b
Neuropathy, peripheral + +
Digestive Hepatomegaly ± ±
Ear Hearing loss, sensorineural +b
Haematological Vacuolated lymphocytes ↑ ↑
Macroglossia + +
Routine laboratory ASAT/ALAT (P) ↑ ↑
EEG: abnormal + +
Special laboratory Alpha-1,4-glucosidase (muscle, FB) ↓ ↓ ↑
Glucotetrasaccharides (U) ↑ ↑
Glycogen (all tissues) ↑ ↑ ↑
Myocytes, vacuolated ↑
a
Refers to the infantile type (Pompe’s disease). A juvenile and adult-onset type is characterised by muscular problems (with an abnormal EMG)
with walking difficulties, early muscle fatigue and with normal cardiac and mental function
b
Described in patients with the infantile type with prolonged survival on enzyme replacement therapy
Table 18.21 Glycogen storage disease type IIb

Cardiovascular Cardiac failure ++ ++
Cardiac preexcitation syndrome + + +
Cardiomyopathy, hypertrophic and dilated ++ ++
CNS Hypotonia, muscular + + +
Mental retardation + ++ ++
Eye Lens changes + + +
Loss of central vision + + +
Loss of peripheral retinal pigment + + +
Myopia + + +
Musculoskeletal Muscle cramps + + +
Routine laboratory ASAT/ALAT (P) ↑ ↑ ↑
Creatine kinase (P) ↑ ↑ ↑
EEG: abnormal + + +
Special laboratory Alpha-1,4-glucosidase (muscle, FB) n n n n n
Glycogen (heart, muscle) ↑ ↑ ↑
Vacuolated myocytes + + +
a
Refers to the symptoms in hemizygous males. Heterozygous females can also be affected but generally milder and at a higher age
Table 18.22 Glycogen storage disease type III

Cardiovascular Cardiomyopathya + + + +
Digestive Hepatomegaly n ± ++ + +
Liver adenoma, carcinoma ±
Liver fibrosis/biliary cirrhosis ± ± ±
Musculoskeletal Exertion intolerance ± +
Muscle weakness + ++
Osteopenia +
Routine laboratory ASAT/ALAT (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Creatine kinase (P)a n-↑ n-↑ n-↑ n-↑ n-↑
Glucose, fasted (P) ↓ ↓ ↓ ↓-n ↓-n
Ketones, fasted (U,P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Lactate, fasted (P,U) n n n n n
Uric acid (U,P) n n n n n
Special laboratory Amylo-1,6-glucosidase (WBC, liver) ↓ ↓ ↓ ↓ ↓
Biotinidase (P) n-↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑ n-↑
Glycogen (liver) n-↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
a
Not found in GSD-IIIb patients
Table 18.23 Glycogen storage disease type V

Musculoskeletal ‘Second wind’ phenomenon ± +
Exertion intolerance ± +
Muscle crampsa ± +
Muscle paina ± +
Muscle weaknessa ± +
Routine laboratory Ammonia rise in forearm exercise test (B) n n
Lactate rise in forearm exercise test (P) ↓ ↓
Uric acid (P) ↑ ↑
Special laboratory Glycogen (muscle) ↑ ↑ ↑ ↑ ↑
Myoglobin (U)a n-↑ n-↑
Phosphorylase (muscle) ↓ ↓ ↓ ↓ ↓
a
Induced or exaggerated by exertion
Table 18.24 Glycogen storage disease type VI

Digestive Hepatomegaly ± ± ± n n
Musculoskeletal Short stature n ± + + n
Other Adiposity (doll-like facies) n ± + + n
Cholesterol (P) n-↑ n-↑ n-↑ n-↑ n-↑
Triglycerides (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Biotinidase (P) n-↑ n-↑ n-↑ n-↑ n-↑
Glycogen (liver) n-↑↑ ↑↑ ↑↑ n-↑ n-↑
Glycogen phosphorylase (liver) ↓ ↓ ↓ ↓ ↓
Table 18.25 Glycogen storage disease type IXa–c

Digestive Hepatomegaly ± ± ± n n
Musculoskeletal Short stature n ± + + n
Other Adiposity (doll-like facies) n ± + + n
Cholesterol (P) n-↑ n-↑ n-↑ n-↑ n-↑
Triglycerides (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Biotinidase (P) n-↑ n-↑ n-↑ n-↑ n-↑
Glycogen (liver) n-↑↑ ↑↑ ↑↑ n-↑ n-↑
Phosphorylase kinase (blood cellsb, liver) ↓ ↓ ↓ ↓ ↓
a
Refers to the most common type IXa ( = X-linked liver glycogenosis, XLG) caused by defects of the hepatic α-unit. Defects of the liver γ-unit
may present with more severe hepatic problems and the development of fibrosis and cirrhosis as additional signs. A defect of the ubiquitous
β-subunit can cause mild muscle weakness as a further symptom
b
Note that there are two subtypes of type IXa, IXa-1 (XLG-1) and IXa-2 (XLG-2), of which the latter cannot be detected enzymatically in blood
cells but in liver only
Table 18.26 Glycogen storage disease type IXd

System Symptoma,b Neonatal Infancy Childhood Adolescence Adulthood
Musculoskeletal ‘Second wind’ phenomenon ± ± +
Exertion intolerance ± ± +
Muscle cramps ± ± +
Muscle pain ± ± +
Muscle weakness ± ± +
Lactate rise in forearm exercise test (P) ↓ ↓
Uric acid (P) ↑ ↑
Special laboratory Glycogen (muscle) ↑ ↑ ↑ ↑ ↑
Myoglobin (U) n-↑ n-↑
Phosphorylase kinase (muscle) ↓ ↓ ↓ ↓ ↓
a
X-linked disorder; symptoms only observed in males
b
More variable age at onset (childhood to adult); most patients have mild adult-onset of symptoms
Table 18.27 Constitutional AMP-activated protein kinase activation

Cardiovascular Cardiac preexcitation syndrome + + + + +
Cardiomyopathy, hypertrophic + + + + +
Heart block (variable types) + + + + +
Glucose (P) ↓ ↓
Special laboratory Glycogen (heart) ↑↑b ↑↑b ↑ ↑ ↑
Glycogen (muscle) n-↑ n-↑ n-↑ n-↑ n-↑
Phosphorylase kinase (heart) ↓b ↓b
Protein kinase, AMP-activated (heart, muscle) ↑↑b ↑↑b ↑ ↑ ↑
a
Dominant disorder, clinical heterogeneity
b
Severe neonatal type (‘fatal congenital heart glycogenosis’)
Table 18.28 Glycogen storage disease type VII

Digestive Gallstones n ± + +
Jaundice n + + +
Haematological Anaemia, haemolytic n + + +
RBC life span ↓ ↓ ↓ ↓
Musculoskeletal ‘Second wind’ phenomenon ± ± + +
Exertion intoleranceb ++a + + +
Muscle crampsb ++a + + +
Muscle painb ++a + + +
Muscle weaknessb ++a + + +
Routine laboratory Ammonia rise in forearm n n n
exercise test (B)
Bilirubin (P) n ↑ ↑ ↑
Creatine kinase (P)b ↑ ↑ ↑ ↑
Lactate rise in forearm exercise ↓ ↓ ↓
test (P)
Reticulocytes (B) n ↑ ↑ ↑
Uric acid (P) ↑ ↑ ↑ ↑
Special laboratory 2,3-diphosphoglycerate (RBC) ↓ ↓ ↓ ↓
Glycogen (muscle) ↑ ↑ ↑ ↑ ↑
Myoglobin (U)b ± ± ± ±
Phosphofructokinase (blood ↑ ↑ ↑ ↑ ↑
cells, FB, muscle)
a
A severe infantile type with rapidly progressive course (limb weakness, seizures, cortical blindness, corneal clouding and early death) has been
rarely reported
b
Table 18.29 Glycogen storage disease type XII

CNS Mental retardation ± ± ± ±
Haematological Anaemia, haemolytic + + + +
Musculoskeletal Dysmorphic features ± ± ± ±
Muscle weakness ± ± ± ±
Short stature ± ± ± ±
Routine laboratory Bilirubin (P) ↑ ↑ ↑ ↑
Creatine kinase (P)b n-↑ n-↑ n-↑ n-↑
Reticulocytes (B) ↑ ↑ ↑ ↑
Special laboratory Aldolase A (RBC) ↓ ↓ ↓ ↓
Glycogen (liver) n-↑ n-↑ n-↑ n-↑
Glycogen (muscle) n-↑ n-↑ n-↑ n-↑
a
Very rare, single cases with very variable symptoms
b
Table 18.30 Muscle phosphoglycerate kinase deficiency

CNS Motor retardation ± ± ± ±
Eye Retinal dystrophy ± ± ± ±
Haematological Anaemia, haemolytic ± ± ± ±
RBC life span ↓-n ↓-n ↓-n ↓-n
Musculoskeletal Exertion intoleranceb ± ± ± ±
Muscle crampsb ± ± ± ±
Muscle painb ± ± ± ±
Muscle weaknessb ± ± ± ±
Routine laboratory Bilirubin (P) n-↑ n-↑ n-↑ n-↑
Creatine kinase (P)b n-↑ n-↑ n-↑ n-↑
Reticulocytes (B) n-↑ n-↑ n-↑ n-↑
Special laboratory Myoglobin (U)b n-↑ n-↑ n-↑ n-↑
Phosphoglycerate kinase ↓ ↓ ↓ ↓
(blood cells, muscle)
a
X-linked disorder, symptoms refer to hemizygous males; heterozygous females may exhibit a variable degree of enzyme deficiency and a more
heterogenous clinical presentation
b
Table 18.31 Glycogen storage disease type X

Musculoskeletal Exertion intoleranceb + +
Muscle crampsb + +
Muscle painb + +
Muscle weaknessb + +
Routine laboratory Creatine kinase (P)b ↑ ↑
Special laboratory Glycogen (muscle) n-↑ n-↑
Myoglobin (U)b ↑ ↑
Phosphoglycerate mutase (muscle) ↓ ↓
a
Autosomal-recessive disorder, symptoms refer to homozygotes; heterozygotes may exhibit a variable degree of enzyme deficiency and may pres-
ent with a more heterogenous clinical picture
b
Table 18.32 Glycogen storage disease type XIII

System Symptoma,b Neonatal Infancy Childhood Adolescence Adulthood
Musculoskeletal Exertion +
intoleranceb
Muscle crampsb +
Muscle painb +
Muscle weaknessb +
Routine laboratory Creatine kinase (P)b ↑
Special laboratory Beta-enolase ↓
(muscle)
Glycogen (muscle) ↑
Myoglobin (U)b ↑
a
Single case reported
b
Table 18.33 Glycogen storage disease type XI

Dermatological Skin rash ± ±
Genitourinary Uterine muscle stiffnessa +
Musculoskeletal Exertion intoleranceb + +
Muscle crampsb + +
Muscle painb + +
Muscle weaknessb + +
Creatine kinase (P)b ↑ ↑
Lactate (P)b ↑ ↑
Lactate rise in forearm exercise test (P)b ↑ ↑
Special laboratory Glycogen (muscle) n-↑ n-↑
Lactate dehydrogenase (RBC, muscle) ↓ ↓
Myoglobin (U)b ↑ ↑
a
In pregnancy
b
18.5 Diagnosis specific reference values and can be presented as a percent-

age of the average of normal controls. For example,
The reference values for metabolites whose concentrations diagnostic values of monosaccharide uptake by enterocytes
are altered in congenital disorders of carbohydrate metabo- in SGLT1 deficiency (an autosomal-recessive condition)
lism are shown in Table 18.34. In general, characteristic usually fall below 10 % of normal; diagnostic values of glu-
clinical findings together with first results of metabolite cose uptake by erythrocytes in GLUT1 deficiency (an auto-
analyses are a first clue to diagnosis. Loading or provocation somal-dominant condition) have been reported to be 46 ± 8%
tests can be helpful in certain conditions (Table 18.35). of controls.
Special laboratory tests to complete the diagnosis should be Since functional transporter studies are laborious and
performed in laboratories with specific expertise. These pro- enzymatic studies may be invasive if a liver or muscle biopsy
cedures include both functional studies in tissues expressing is necessary, a primary molecular genetic approach may be
the affected protein (a transporter or an enzyme) and molec- warranted in certain conditions. In that case, a working
ular genetic analysis of the relevant gene (usually in DNA hypothesis has to be formulated on the basis of clinical and
samples from peripheral blood, but DNA can be obtained non-invasive paraclinical investigations. This is an option,
also from other sources, such as fibroblasts). Due to variable particularly in disorders in which the affected gene is small
assay conditions, results of these transport or enzymatic or when few prevalent mutations are responsible for the
tests have to be interpreted in comparison to laboratory- cases of a population (see Table 18.36).
18.5.1 Reference and Pathological Values

Table 18.34 Reference and pathological values
Metabolite Age group Normal values Pathological values
Glucose (P) Fasting 1 day 2.2–3.3 mmol/l 40–60 mg/dl
>1 day 2.8–5.0 mmol/l 50–90 mg/dl
Child 3.3–5.5 mmol/l 60–100 mg/dl
Adult 3.9–5.8 mmol/l 70–105 mg/dl
1 h after Adult 6.6–9.4 mmol/l 120–170 mg/dl
ingestion
Glucose (CSF) Fasting Child 1.7–3.7 mmol/l 32–68 mg/dl
Glucose (CSF/P Child 0.65–0.85 <0.5
ratio)
Glucose (U) <0.8 mmol/l <15 mg/dl >0.8 mmol/l >15 mg/dl
0.13–0.32 g/1.73 m2/day >0.32 g/1.73 m2/day
Reducing Negative Positive
substances (stool)
Galactose (P) Fasting 0–0.03 mmol/l 0–0.5 mg/dl
After milk Newborn <1.1 mmol/l <20 mg/dl >1.1 mmol/l >20 mg/dl
ingestion

Metabolite Age group Normal values Pathological values
Child <0.5 mmol/l <9 mg/dl >0.5 mmol/l >9 mg/dl
Adult <0.24 mmol/l <4.3 mg/dl >0.24 mmol/l >4.3 mg/dl
Galactose-1- <0.17 μmol/g Hb >1.70 μmol/g Hb
phosphate (RBC)
Galactose (U) <377 mmol/mol crea >631 mmol/mol crea
Galactitol (U) 2–5 mmol/mol crea >30 mmol/mol crea
Fructose (B) <0.16 mmol/l >0.16 mmol/l
Fructose (U) 10–14 mmol/mol creat >20 mmol/mol crea
Sorbitol (U) 2–26 mmol/mol crea
D-Glycerate (B) n.d. 0.19–0.32 mmol/l
D-Glycerate (U) n.d. >5.0 mmol/mol crea
Lactic acid (P) Fasting Child 0.6–1.8 mmol/l >3.0 mmol/l
Lactic acid (CSF) Child 1.1–2.6 mmol/l >3.0 mmol/l
Lactic acid (U) Child <200 mmol/mol crea >300 mmol/mol crea
Biotinidase (P) 7.0–10.6 mU/ml >10.6 mU/ml
Glycogen (L) <5.0 g/100 g wet tissue >5.0 g/100 g wet tissue
Glycogen (M) <1.0 g/100 g wet tissue >1.0 g/100 g wet tissue
n.d. not detectable
18.5.2 Specimen Collection
Table 18.35 Specimen collection

Test Material Preconditions Handling Pitfalls
Metabolites
Glucose P Fasted/non-fasted NaF tubes Bacterial contaminationa
Glucose CSF Fasted (!)b Bacterial contamination,a
pleocytosis
Glucose U Bacterial contaminationa
Reducing substances U Bacterial contamination,a
medications
Reducing substances Stool Bacterial contaminationa
Galactose P Fasted/non-fasted Perchloric acid extract
Galactose U Bacterial contaminationa
Gal-1-P RBC Washed
Galactitol U Bacterial contaminationa
Sorbitol U Bacterial contaminationa
Lactic acid P Fasted/non-fasted Perchloric acid extract or Hypoxiaa
NaF tubes
a
Lactic acid CSF Fasted (!) Perchloric acid extract or
NaF tubes
Glycogen Liver Freeze immediately, store
at −70 °Cc
Glycogen Muscle Freeze immediately, store
at −70 °Cc
Enzymes
Galactokinase RBC Washed, frozenc
GALT RBC Washed, ship at room
temperaturec
UDP-Gal-4-epimerase RBC Washed, frozenc
Frc-1-P aldolase (AldoB) Liver Freeze immediately, store
at −70 °Cc
Frc-1,6-P biphosphatase Liver Freeze immediately, store
at −70 °Cc
D-glycerate kinase Liver Freeze immediately, store
at −70 °Cc
(continued)

Liver GSD enzymes Liver Freeze immediately, store
at −70 °Cc
Muscle GSD enzymes Muscle Freeze immediately, store
at −70 °Cc
Transporter assays
GLUT1 RBC Assay in unfrozen EDTA
samplec
SGLT1 Intestinal biopsy Assay in unfrozen tissuec
Glucose-6-P-translocase Liver Assay in unfrozen tissuec
a
Always store samples at –20 °C if immediate processing is not possible
b
Always use fasted samples and a drawn plasma sample for determination of CSF/P ratio just before the lumbar puncture
c
Since assay conditions may vary with time and with different reference laboratories, you are encouraged to contact these laboratories before
obtaining specimens
18.5.3 Provocation and Loading Tests
Table 18.36 Provocation and loading tests

Fasting test
Indication
Assessment of fasting intolerance and recurrent episodes of hypoglycaemia of unknown origin and determination of metabolic profile during
hypoglycaemia
Stable clinical condition. Plan maximal fasting time according to anamnestic tolerance
During test measure glucose (P), blood gases, lactate (P) and ketones (U) every hour starting with first meal that is missed
Terminate test if hypoglycaemia (<45 mg/dl, <2.5 mM) or clinical symptoms occur after diagnostic blood samples were drawn for glucose (P),
blood gases, lactate (P), amino acids (alanine) (P), acylcarnitines (dried blood spots), organic acids (U), ketone bodies (U), (FFA (P),
3-hydroxybutyrate (P)), insulin (P), cortisol (P), growth hormone (P) and others
Caution
Hypoglycaemia from dangerous causes has to be excluded prior to a fasting test. For example, accumulation of metabolites from disorders of
fatty acid metabolism may lead to life-threatening complications which means that fatty acid oxidation defects have to be excluded by
determination of acylcarnitines prior to such a test. In general, fasting tests should only be performed in specialised centres experienced with
potential differential diagnoses
10 % i.v. glucose solution should be immediately available as needed in symptomatic patients (see Table 18.38)
Interpretation
Glucose (P) <45 mg/dl (2.5 mM) is suggestive of an abnormal response particularly when combined with typical changes of other metabolites
such as ketones or lactate (see Tables)
Oral glucose loading test
Indication
1. To test for impaired intestinal uptake in SGLT1-D
2. To test for glucose intolerance (decreased hepatic uptake in FBS and GSD-0a, decreased β-cell insulin secretion in FBS)
Fasting state: 4 to 12 h (depending on age)
Glucose dose: 2 g/kg as 10 % solution or as an equivalent amount of oligosaccharides (maximum dose 50 g) given by mouth within 5–7 min
Criteria
ad 1. Clinical signs, abdominal pain, diarrhoea, reducing substances in stool, H2 breath test and glucose (P) determination at baseline and every
30 min for 3 h
ad 2. Glucose (P) and lactic acid (P) determinations at baseline and every 30 min for 3 h
Caution:
Hypovolaemia may develop in SGLT1-D
Interpretation
Pathological increase of H2 (breath test) and diminished increase of glucose (P) in SGLT1-D. Glucose (P) low or normal and lactate (P) high or
normal at baseline, exaggerated increase of glucose (P) and decrease of lactate(P) after glucose load in FBS. Exaggerated increase of glucose
(P) and increased lactate (P) after glucose in GSD-0a
Oral galactose loading test
Indication
1. To test for impaired intestinal uptake in SGLT1-D
2. To test for galactose intolerance (decreased hepatic uptake) in FBS

Galactose dose: 1 g/kg as 20 % solution given by mouth within 5–7 min
Criteria
ad 1. Clinical signs, abdominal pain, diarrhoea, reducing substances in stool, H2 breath test and glucose (P) determination at baseline and every
30 min for 3 h
ad 2. Glucose (P), galactose (P) and lactic acid (P) determinations at baseline and every 30 min for 3 h
Caution
Hypovolaemia may develop in SGLT1-D
Not indicated in galactosaemia patients who may be detected in screening programmes or by typical metabolites and enzymatic studies on a
galactose-containing diet
Interpretation
Pathological increase of H2 (breath test) and diminished increase of galactose (P) in SGLT1-D. Glucose low or normal and lactate high or
normal at baseline, exaggerated increase of galactose after galactose load in FBS
Oral fructose loading test
Indication
While dangerous and obsolete in the diagnosis of HFI, the oral fructose loading test serves as a reference test in the diagnosis of SGLT1-D
Fructose dose: 1 g/kg as 20 % solution given by mouth within 5–7 min
Criteria
Clinical signs, abdominal pain, diarrhoea, reducing substances in stool, H2 breath test and glucose (P) determination at baseline and every
30 min for 2 h
Interpretation
No increase of H2 (breath test) and normal increase of glucose (P) in SGLT1-D
Intravenous fructose loading test
Indication
Diagnosis of HFI and FBP-D (FK-D) in questionable cases. (Before this test, think of less dangerous ways of diagnosis. Should only be
performed in experienced centres)
Preparation: 2 weeks before the test, a diet without sucrose and fructose is prescribed
Fasting state: overnight or 6 h
Fructose dose: 0.25 g/kg as 10 % solution given over 1–3 min
Blood samples: baseline, 10, 20, 30, 40, 50, 60 and 90 min
Determinations: glucose, (fructose), lactic acid, phosphate and uric acid (P)
Caution
Watch for hypoglycaemia between 20 and 50 min
10 % i.v. glucose solution should be immediately available as needed in symptomatic patients (see Table 18.38)
Nausea and abdominal pain are frequent in affected individuals
Interpretation
A decrease of glucose and phosphate and an increase of uric acid are typical for HFI. A decrease of glucose and an increase of lactate are
characteristic for FBP-D. In FK-D a blunted increase of glucose and a rise in fructose can be detected. Oral fructose loading test is not advocated
in the case of suspicion of HFI since oral administration of fructose may cause severe gastrointestinal symptoms and long-lasting hypoglycaemia
Indications
In patients suspected of having GLYCTK-D
Fasting state: overnight or 6 h
Fructose dose: 1.0 g/kg as 10 % solution given over 5–10 min
Following the load, approximately 4 % of the test dose is excreted as D-glycerate in a 24-h urine. A loading test with 200–300 mg/kg of the
amino acid L-serine may be equally effective
Intravenous glucagon test
Indication
Differentiation of hypoglycaemia. Today less important after development of enzymatic and molecular genetic studies. Helpful in questionable cases
Application of glucagon 0.03 mg/kg (slowly i.v.) during spontaneous or induced hypoglycaemia.
Baseline glucose (P) <60 mg/dl (<3.5 mM), glucose (P) and lactate (P) at 15, 25, 35, 45, 60 min
Caution
Nausea and vomiting, if given too fast
Interpretation
Normally, glucose (P) rises by >25 mg/dl (>1.4 mM). Insufficient response indicates depleted glycogen stores, inability to convert glycogen to
glucose or, at the end of a fasting test, impaired gluconeogenesis. Lactate (P) may rise in disorders of gluconeogenesis
(continued)

Semi-ischaemic forearm test
Indications
Diagnosis of muscular GSDs, e.g. types V and VII
Possible in children older than about 8 years. Fasting is not necessary. A sphygmomanometer placed around the upper arm is inflated at
mid-systolic-diastolic blood pressure level. After baseline blood sampling, a hand manometer is squeezed for 2 min in a frequency of 1/s
Immediately after exercise the cuff is released
Blood samples: baseline and every 2 min for 20 min and at 30 and 60 min
Determinations: ammonia (P) and lactate (P)
Cautions
Muscle cramp, stop exercise immediately
Interpretation
No increase of lactate (P) occurs in muscle GSDs (except GSD-XI, LDH deficiency); in contrast ammonia (P) increases
18.5.4 Metabolites, Enzymatic and Genetic disorders a definite diagnosis is possible by transporter stud-
Tests ies or measurement of enzymatic activity in biopsy samples.
However, affected proteins are not always expressed in tis-
Table 18.37 refers to the question how to arrive at a definite sues that are easily available. Molecular genetic studies from
diagnosis with minimal invasiveness and calculated costs. DNA samples from peripheral blood are possible for all dis-
Metabolites (in easily accessible samples) can be helpful in orders. However, efforts will depend on the size of the
some cases; a classification as a GSD is possible by determi- respective gene and the existence of common mutations in
nation of glycogen content in liver or muscle tissue. In all the population under study
Table 18.37 Metabolites, Enzymatic and Genetic Tests

Metabolite analysis
(from peripheral Enzyme/transport assay (from
blood) peripheral blood) Molecular characterisation (from peripheral blood)
No. Disorder Possible Possible Possible (Gene size) Common mutations
18.1 GGM + (15 exons)
18.2 SGLT2-D (+) + (14 exons)
18.3 GLUT1-D (+) (+) Transporter prot in + (10 exons)
RBC
18.4 FBS (+) + (11 exons)
18.5 GLUT10-D + (5 exons)
18.6 GALK-D (+) + Enzyme act in RBC + (8 exons)
p.P28T in Eastern European Roma
p.A198V in Japanese and Koreans
18.7 GALT-D (+) + Enzyme act in RBC + (10 exons)
p.Q188R in Caucasians
p.K285N in Caucasians
18.8 GALE-D (+) + Enzyme act in RBC + (11 exons)
18.9 FK-D (+) + (7 exons)
18.10 HFI + (9 exons)
p.A150P in Caucasians
p.A175D in Caucasians
p.N335K on the Balkans
IVS1+1G>C in Hispanics and
Afro-Americans
18.11 FBP-D + (7 exons)
c.960insG in Japan
18.12 GLYCTK-D (+) + (6 exons)
18.13 GSD-0a + (16 exons)
18.14 GSD-0b + (16 exons)
18.15 GSD-XV + (8 exons)

Metabolite analysis
(from peripheral Enzyme/transport assay (from
blood) peripheral blood) Molecular characterisation (from peripheral blood)
No. Disorder Possible Possible Possible (Gene size) Common mutations
18.16 GSD-IV + Enzyme act in + (16 exons)
WBC, RBC
18.17 GSD-XIV + (12 exons)
18.18 GSD-Ia + (5 exons)
p.R83C in Caucasians, Turks,
Ashkenazi Jews
p.Q347X in Caucasians
c.648G>T [p.=] in Japanese, Chinese
c.380insTA in Hispanics
18.19 GSD-I non-a + (9 exons)
p.W118R in Japanese
p.G339C in Caucasians
c.1042_3delCT in Caucasians
18.20 GSD-IIa + Enzyme act in + (20 exons)
WBC, IVS1-13T>G in Caucasiansa
dried blood spots c.525delT in Caucasians
ex18del in Caucasians
p.D645E in Chinese and Taiwanese
18.21 GSD-IIb + (10 exons)
IVS5+1G>A (?)b in Japanese,
Caucasians
18.22 GSD-III + Enzyme act in + (35 exons)
WBC p.R408X in Norwegians and Faroese
c.4455delT in North African Jews
c.16C>T [p.R6X]c
c.17_18delAGc
18.23 GSD-V + (20 exons)
p.R50X in Japanese and Caucasians
p.G205S in Japanese and Spanish
p.L542T in Japanese
18.24 GSD-VI + (20 exons)
18.25 GSD-IXa–c +d Enzyme act in + (33 exons + 33 exons + 10 exons)
RBC, WBCc
18.26 GSD-IXd + (31 exons)
18.27 AMPK-A + (12 exonse)
p.R302Q (?)b,f
p.R531Q (?)b,g
18.28 GSD-VII + Enzyme act in RBC + (24 exons)
IVS5+1G>A in Ashkenazi Jews
18.29 ALDOA-D + Enzyme act in RBC + (12 exons)
18.30 PGK-D + Enzyme act in RBC + (11 exons)
18.31 GSD-X + (3 exons)
p.W78X in Afro-Americans (?)b
18.32 GSD-XIII + (12 exons)
18.33 GSD-XI + Enzyme act in RBC + (7 exons)
ex6 del20bp in Japanese (?)b
a
With an adult-type variant of the disease
b
Question mark refers to a small number of cases
c
In GSD-IIIb patients
d
In types IXb and IXc but only in 50 % of GSD-IXa cases ( = GSD-IXa-1 = XLG-1)
e
For PRKAG2b
f
In lethal congenital glycogen storage disease of the heart
g
In hypertrophic cardiomyopathy and WPW syndrome
18.6 Prenatal Diagnosis 18.7 Treatment
In principle, prenatal diagnosis is possible for all disorders Treatment depends on the underlying disorder. In many of
by molecular genetic techniques with DNA samples from them with hypoglycaemia as a symptom of acute presenta-
chorionic villi or amniotic fluid cells. For some disorders, tion, a rapid normalisation of blood glucose is important (see
prenatal enzymatic studies in amniotic fluid cells have been Table 18.38). Long-term treatment aims and measures are
reported. summarised in Table 18.39; specific treatment in certain
groups of disorders are given below.
Table 18.38 Acute treatment of hypoglycaemia in patients with a disorder affecting endogenous glucose production
Immediate bolus (within 5–10 min) Followed by continuous glucose infusiona
Age mg Glc/kg b.w. ml (Glc 10 %)/kg b.w. mg glucose/kg b.w. per min
0–1 year 500 5 7–9
1–6 years 400 4 6–8
6–12 years 350 3.5 5–7
Adolescents 300 3 4–6
Adults 250 2.5 2–4
a
Increase amount of glucose in case of fever by 10–30 %
Table 18.39 Long-term treatment of disorders of carbohydrate metabolism

No. Disorder Principle Measuresa
18.1 GGM Avoid non-absorbable monosaccharides Glc- and Gal-free diet for life
18.2 SGLT2-D No specific Tx necessary
18.3 GLUT1-D Improve energy supply to the brain Avoid fasting, low-CH, high-fat (modified
Use ketone bodies (transported by MCT1 at the BBB) as an Atkins) diet, ketogenic diet
alternative substrate
18.4 FBS Compensate for impaired hepatic Glc uptake and release Continuous enteral supply of slowly released CH
to stabilise blood Glc and suppress
gluconeogenesis
Compensate for impaired hepatic Gal uptake Gal restriction
Compensate for generalised impairment of proximal renal Symptomatic Tx of renal Fanconi syndrome
tubular cells secondary to Glc and glycogen overload
18.5 GLUT10-D No specific Tx available
18.6 GALK-D Prevent accumulation of toxic galactitol in eye lens Gal-restricted diet for life
18.7 GALT-D Prevent accumulation of toxic Gal-1-P Gal-restricted diet for life
18.8 GALE-D Prevent accumulation of toxic Gal-1-P Gal-restricted diet for life
18.9 FK-D No specific Tx necessary
18.10 HFI Prevent accumulation of toxic Frc-1-P Frc-restricted diet for life
18.11 FBP-D Circumvent impaired gluconeogenesis Avoid fasting,
Avoid additional inhibition of glycogenolytic enzymes by Frc-restricted diet for life
accumulating Frc-1-P
18.12 GLYCTK-D Frc restriction (?)
18.13 GSD-0a Provide exogenous CH that can compensate for diminished Avoid fasting, frequent CH-rich feeds,
glycogen stores (L) continuous enteral supply of slowly released CH
18.14 GSD-0b Compensate for diminished glycogen stores (M)
18.15 GSD-XV Compensate for diminished glycogen stores (M)
18.16 GSD-IV Treat cirrhosis induced by abnormal glycogen (L) Symptomatic Tx (… liver Tpx)
18.17 GSD-XIV Compensate for diminished access to glycogen (M)
18.18 GSD-Ia Provide exogenous CH that can be metabolised despite Avoid fasting, frequent CH-rich feeds,
impairment of glycogenolysis and gluconeogenesis (L) continuous enteral supply of slowly released CH,
Gal and Frc restriction
Correct primary defect L-Tpx (?)
Treat adenoma complications (anaemia, tumour) L-Tpx
Treat renal complications Symptomatic Tx (… kidney Tpx)

No. Disorder Principle Measuresa
18.19 GSD-I non-a See 18.18 plus See 18.18 plus
Prophylaxis and Tx of bacterial infections Antibiotic prophylaxis, G-CSF
18.20 GSD-IIa Replace missing enzyme in lysosomes Enzyme replacement therapy
18.21 GSD-IIb No specific Tx available, Symptomatic Tx (…
heart Tpx)
18.22 GSD-III Provide exogenous CH that can be metabolised despite Avoid fasting, frequent CH-rich feeds,
impairment of glycogenolysis (L) continuous enteral supply of slowly released CH
Treat cirrhosis Symptomatic Tx (… liver Tpx)
18.23 GSD-V Compensate for diminished access to glycogen (M)
18.24 GSD-VI Provide exogenous CH that can be metabolised despite Avoid fasting, frequent CH-rich feeds,
18.25 GSD-IXa–c Provide exogenous CH that can be metabolised despite Avoid fasting, frequent CH-rich feeds,
Treat cirrhosisb Symptomatic Tx (… liver Tpx)b
18.26 GSD-IXd Compensate for diminished access to glycogen (M)
18.27 AMPK-A No specific Tx available, Symptomatic Tx (…
heart Tpx)
18.28 GSD-VII Compensate for diminished access to glycogen (M)
18.29 ALDOA-D Compensate for diminished access to glycogen (M)
18.30 PGK-D Compensate for diminished access to glycogen (M)
18.31 GSD-X Compensate for diminished access to glycogen (M)
18.32 GSD-XIII Compensate for diminished access to glycogen (M)
18.33 GSD-XI Compensate for diminished access to glycogen (M)
(L) in liver, (M) in muscle, Tx treatment, CH carbohydrates, BBB blood-brain barrier, Tpx transplantation, G-CSF granulocyte colony-stimulating
factor
a
See below for more details
b
Only in GSD-IXc
18.7.1 Intestinal Glucose-Galactose 18.7.2 Renal Glucosuria

Malabsorption
Treatment: Allow free access to fluid. Caloric intake should
Treatment: Hypertonic dehydration frequently requires compensate for renal losses in the severe cases. No specific
intravenous fluid therapy for which glucose-containing solu- treatment is necessary.
tions with electrolytes can be used. Long-term enteral nutri- Follow-up: Only the severe recessive types need system-
tion has to avoid both glucose and galactose as monomers, as atic follow-up.
well as disaccharides and polymers of these sugars.
Specialised commercial infant formulas containing fat and
protein but free of a carbohydrate component can be used. 18.7.3 Glucose Transporter-1 Deficiency
Fructose should be added according to the dietary allow-
ances for carbohydrates to meet caloric needs. Small amounts Treatment aims at the prevention of low cerebrospinal fluid
of fructose are tolerated later in life. High fluid intake is rec- (CSF) glucose concentration (hypoglycorrhachia) and the
ommended to prevent renal stone formation, which has provision of other substrates for brain metabolism. An effec-
repeatedly been reported in this condition. tive treatment is available by means of a modified Atkins diet
Follow-up: Clinical monitoring (nutrient intake, growth, or a ketogenic diet, both providing ketones as an alternative
nutritional status, general health) and biochemical and para- fuel for the brain. Ketones enter the brain via the facilitative
clinical monitoring (haemoglobin, total protein, general MCT1 transporter. The diet needs to be introduced in a clini-
parameters of liver and kidney function, plasma osmolarity cal setting and requires an experienced paediatrician and a
and electrolytes, urinary glucose, renal ultrasound) should dietician. While a modified Atkins diet may be sufficient in
be planned depending on age, severity of initial decompen- milder cases with an isolated movement disorder, a 3:1
sation and compliance. ratio (fat vs. non-fat intake in grams) using long-chain
triglycerides is usually sufficient for seizure control in infan- Experimental treatment: In individual patients acetazol-
tile cases. Fluids and calories are not restricted. Supplements amide has shown good responses in movement disorders
(multivitamins, calcium and often carnitine) are required. caused by GLUT1-D; α-lipoic acid, an antioxidant, has been
Certain anticonvulsive drugs (phenobarbital, chloral hydrate, shown to increase glucose transport in cultured muscle cells,
valproate, topiramate) interfere with the diet, while others but in vivo data is not yet available. Oral triheptanoic acid, an
(methylxanthines, ethanol) have been shown to impair artificial C7-ketone body, potentially providing additional
GLUT1 function in vitro. fuel for brain energy metabolism, is currently on clinical trial.
Pitfalls/Dangers of a Ketogenic Diet
1. Incorrect calculations: Note that the ratio of the ketogenic diet is defined in grams, not in calories or percentages! A 3:1 ratio means
that for 3 g of ingested fat, only 1 g of protein and carbohydrates is allowed. Thus, on a 3:1 ketogenic diet, 87 % of kilocalories per
day are supplied by fat. Percentages of protein and carbohydrates vary due to age-dependent protein requirements
2. Non-compliance: Assess ketones in blood and urine. If ketones are inappropriately low, intensify dietary instructions and be aware
that many medications have a high carbohydrate content
3. Unawareness of contraindications: Exclude β-oxidation defects. Initiate the diet with moderate fasting as inpatient to exclude
ketolysis and ketogenesis defects
Table 18.40 Nutritional requirements on a ketogenic diet

Fat requirements Protein requirementsa Carbohydrates Energy demandb
Age (g/kg b.w. per day) (g/kg b.w. per day) (g/kg b.w. per day) (g/kg b.w. per day)
0–4 months 9.0 2.2 0.8 93
4–12 months 9.0 1.6 1.4 91
1–3 years 8.7 1.2 1.7 90
4–6 years 7.8 1.1 1.5 80
7–9 years 7.0 1.0 1.3 72
10–12 years 5.8 1.0 0.9 60
13–15 years 5.0 1.0 0.7 52
Adults 5.0 1.0 0.7 52
a
Recommendations from the German Society for Nutrition (DGE; 1991)
b
German-Austrian-Swiss (DACH) recommendations (2000)
Table 18.41 Investigations on introduction and follow-up of a ketogenic diet

On admission Initiation of diet On discharge Follow-up every 2–3(–6) months
Clinicala Clinicala Clinicala Clinicala
Paraclinicalb Paraclinicalb Paraclinicalb Paraclinicalb
Glucose (P), OHB, BGA Glucose (P), OHB, BGA Glucose (P), OHB, BGA Glucose (P), OHB, BGA
Electrolytes Every 4–6 h (bedside) Electrolytes Electrolytes
Liver/kidney parameters Ketones in urine Liver/kidney parameters Liver/kidney parameters
Blood count, CRP EEG Blood count, CRP
Carnitine Abdominal sonography Carnitine
Blood lipids Blood lipids
Essential fatty acids Essential fatty acids
Drug monitoring Drug monitoring
EEG, EKG EEG (seizure control?)
Abdominal sonography EKG (long QT?)
Abdominal sonography
(nephrolithiasis?)
a
Nutrient intake, growth, nutritional status, somatic findings
b
OHB hydroxybutyrate, BGA blood gas analysis
Table 18.42 Monitoring of ketosis on a ketogenic diet

Sample Ketone body Test Target value
Urine Acetoacetate Test strips 80 (++)–160 (+++) mg/dl
Urine Hydroxybutyrate Test strips >2 mmol/l
Blood Total ketone bodies Enzymatic 3–5 mmol/l
18.7.4 Fanconi-Bickel Syndrome lactose-/galactose-free feeding regimen should be initiated

without delay, even before the diagnosis has been confirmed
Treatment: Patients with FBS show signs of a hepatic GSD with enzymatically or by DNA analysis. In classical galactosae-
impaired glycogenolysis and gluconeogenesis. Therefore, treat- mia supportive care depends on the severity of liver, renal
ment should be similar to GSD-I (see below) with frequent feeds and central nervous system disease and comprises intrave-
and the use of slowly absorbed carbohydrates. Blood glucose nous fluids, plasma and vitamin K. Initiate treatment with
concentrations should be in a range, so that gluconeogenesis is broad-spectrum antibiotics without delay if suspicion of sep-
suppressed in order to avoid glycogen accumulation. This can be sis arises, since, in the event of acute metabolic derangement,
accomplished by a constant supply of slow release carbohydrates patients are at risk for infections due to the compromised
(frequent feeds, cornstarch, nasogastric oligosaccharide drip response of the immune system.
feeding). In contrast to GSD-I, there is no evidence that a fruc- Long-term treatment requires a galactose-restricted
tose-/saccharose-free diet is beneficial to FBS patients. Likewise, diet for life in all three conditions. There is increasing evi-
there are patients with FBS that have ingested high amounts of dence that small amounts of galactose are acceptable in
galactose/lactose without developing cataracts. Therefore, galac- GALK-D and GALT-D as long as it is in the range of
tose restriction is not generally recommended, but galactose and endogenous galactose production. In the severe forms of
galactose-1-phosphate levels should be monitored. GALE-D, some dietary galactose (1–2 g/day) and also
Due to the propensity to hypoglycaemia of FBS patients, N-acetyl-galactosamine intake is even necessary for the
the use of insulin for impaired glucose tolerance has to be biosynthesis of complex carbohydrates and galactolipids.
considered with extreme caution and only after dietary mea- Excess, however, should be avoided, as this leads to accu-
sures have failed. mulation of galactose-1-phosphate. Sufficient calcium
There is no specific treatment for the Fanconi-type intake should be guaranteed to protect patients from
nephropathy. Symptomatic treatment is recommended to osteoporosis.
compensate for losses of water, sodium, potassium, calcium, Ovarian dysfunction with hypergonadotropic hypogonad-
phosphate, vitamin D and bicarbonate. Maintenance therapy ism is observed in almost all female patients with GALT-D.
should be monitored by urinary calcium excretion. Carnitine Start ethinyl oestradiol therapy from age 12–13 years, when
supplementation should only be performed at low plasma gonadotropin levels are high and oestradiol levels are low
levels or when signs and symptoms of a secondary mito- (first 6 months, 2 μg daily; 6–12 months, 2–5 μg daily; 12–24
chondrial disorder are observed. months, 5 μg daily; 24–36 months, 10 μg daily; after 3 years,
followed by an oral contraceptive preparation containing
ethinyl oestradiol and a progestogen daily for 21 days, 7 days
18.7.5 Disorders of Galactose Metabolism abstinence).
Treatment: If a disorder of galactose metabolism is

suspected in a newborn, dietary treatment consisting of a
Table 18.43 Follow-up in patients with disorders of galactose and fructose metabolism
Disorder Investigations Frequency
GALK-D Galactose (U) <18 years: annually
Ophthalmological investigation >18 years: biannually
GALT-D Clinicala,b
Ophthalmological investigations <1 year: 3 monthly
Paraclinical 1–4 years: 4 monthly
Liver/kidney parameters 4–18 years: 6 monthlyc
Gal-1-P (RBC) [aim <0.5 μmol/g haemoglobin] >18 years: annuallyc
Galactitol (U)
LH, FSH, oestradiol (starting around 8 years)
Pelvic ultrasound (in females)
X-ray (left hand for bone age)
Bone mineral density assessment
GALE-D Clinicala,b
Ophthalmological investigations <1 year : 3 monthlyd
Paraclinical 1–4 years: 4 monthlyd
Liver/kidney parameters 4–18 years: 6 monthlyd
Gal-1-P (RBC) >18 years: annuallyd
UDP-Gal (RBC)
Galactitol (U)
X-ray (left hand for bone age)
Bone mineral density assessment
FK-D None
HFI Clinicala
Paraclinical <12 years: 6 monthly
Liver/kidney parameters 12–18 years: annually
CDTe >18 years: biannually
Liver ultrasound
FBP-D Clinicala
Paraclinical
Liver parameters
Glucose (P), Lactate (P) during infections
GLYCTK-D Clinicala,b Depending on symptoms
Paraclinical
a
Nutrient intake, growth, nutritional status, somatic findings (liver size)
b
Neurological, psychological, cognitive functions, developmental investigations
c
Follow-up of female GALT-D patients on hormone treatment should be done on a more regular interval
d
In milder forms of GALE-D, follow-up can be less extensive
e
CDT, carbohydrate-deficient transferrin (abnormal glycosylation of transferrin)
18.7.6 Disorders of Fructose Metabolism For the maintenance of normal blood glucose concentra-
tions, patients with FBP-D depend on glycogen breakdown
Treatment: If HFI or FBP-D is suspected, dietary treatment and on exogenous glucose from intestinal absorption.
consisting of a sucrose-, fructose- and sorbitol-free feeding Especially in young children, the relative amount of hepatic
regimen should be initiated without delay, even before the glycogen is limited. Only after a short period of fasting,
diagnosis has been confirmed enzymatically or by DNA patients may develop hypoglycaemia accompanied by accu-
analysis. Supportive care depends on the severity of liver and mulation of lactate. The most important aim of the dietary
renal disease and comprises intravenous fluids, plasma and treatment is therefore maintenance of normoglycaemia by
vitamin K. avoidance of fasting. In FBP-D, fructose intake should there-
On long-term, fructose tolerance in HFI is very variable. fore be limited during periods of acute illness since accumu-
At least in infancy, fructose should be maximally restricted lating fructose-1-phosphate inhibits liver phosphorylase.
and intake should not be determined by subjective tolerance. Certain amounts of fructose, particularly when taken with
other carbohydrates, are generally tolerated in non-catabolic Intensive dietary treatment with carbohydrates slowly
periods. absorbed from the intestine and dietary protein enrichment
Since D-glycerate kinase catalyses the conversion of may also ameliorate secondary myogenic symptoms in the
D-glyceric acid to 2-phosphoglycerate, an intermediary group of hepatic GSDs by counteracting increased gluconeo-
reaction found in several metabolic pathways, including the genesis and avoiding a drain from muscle protein.
degradation of serine, as well as the breakdown of fructose, Dietary treatment is based on frequent feedings during
symptomatic patients with D-glyceric acidaemia may also daytime depending on GSD type and individual fasting toler-
benefit from dietary fructose and sucrose restriction. ance. For type I patients, the entire endogenous glucose pro-
In patients on a fructose-restricted diet, vitamin C and duction has to be replaced by enteral feedings. The numbers
folic acid should be supplemented. given in the right column of Table 18.38 can serve as an ori-
entation for a dietary prescription and have to be adapted
individually. Fasting tolerance during daytime can be pro-
18.7.7 Glycogen Storage Diseases: Mainly longed by using uncooked cornstarch from which glucose is
Affecting Liver only slowly released to the blood stream. During the night
and at a younger age (especially in GSD-I and GSD-III),
Treatment: Patients with defects in hepatic glycogenolysis continuous gastric drip feeding may be necessary for 8–12 h.
(FBS, GSD-I, GSD-III, GSD-VI, GSD-IXa–c) but also with Alternatively but only at a later age, uncooked cornstarch
diminished hepatic glycogen formation (GSD-0a) may may be given during the night at 4- to 6-h intervals, in late
develop hypoglycaemia after only a short period of fasting. adolescence or adulthood at 6- to 8-h intervals. There are
This holds especially true for younger patients and patients encouraging developments on novel starch preparations with
with GSD-I. A guideline for acute treatment of hypoglycae- a long-lasting effect on blood glucose concentration.
mia is given in Table 18.38. Hypoglycaemia may be accom- In general, it is not necessary to replace breast milk in
panied by metabolic acidosis caused by accumulation of infants, except for those with GSD-I who may benefit from
lactate (GSD-I) or ketones (GSD-III, GSD-VI, GSD-IXa–c, glucose-enriched lactose-/sucrose-free feedings previ-
GSD-0, FBS). While lactic acidaemia will improve upon ous to breastfeeding or an oligosaccharide-based formula.
glucose administration in GSD-I patients, application of an Theoretically, uncooked cornstarch should not be started in
excess of glucose in GSD-0a patients may lead to hypergly- children less than 1 year of age, as pancreatic amylase activ-
caemia and/or lactacidaemia. ity is insufficiently mature in these children. For continuous
The aim of long-term treatment in hepatic GSDs is the sta- gastric drip feeding, a glucose/glucose polymer solution
bilisation of blood glucose with concentrations at which glu- should be used; formulas enriched with maltodextrin are not
coneogenesis is suppressed. No consensus exists about the recommended due to their high energy content.
extent of avoiding lactate production in GSD-I from galac-
tose, fructose and saccharose. Moderate hyperlactacidaemia Table 18.44 Dietary manipulations in hepatic glycogen storage
may prevent cerebral symptoms if blood glucose concentra- diseases
tion is low, as lactate may serve as an alternative fuel for the Disorder
brain. On the other hand, some evidence exists that avoiding GSD-0a Frequent feeds, prevention of fastinga
lactate production from endogenous sources or from galac- Protein-enriched diet (20 %)
tose and fructose intake may favour long-term outcome. GSD-Ia Frequent feeds, prevention of fastinga
Since glycogenolysis is impaired in hepatic GSDs (with GSD-I non-a High carbohydrate intake (55–65 % of energy)
the additional affection of gluconeogenesis in GSD-I), these Moderate fat restriction (20–30 %)
patients depend on exogenous glucose from intestinal Predominantly polyunsaturated fatty acids
absorption for the maintenance of a normal blood glucose Moderate protein restriction (10–15 %)
concentration. After a short period of fasting, especially Sodium restriction
younger patients and patients with GSD-I may develop Galactose/fructose restriction
hypoglycaemia. Therefore, the most important aim of dietary GSD-III Frequent feeds, prevention of fastinga
treatment is avoidance of fasting. Intensive dietary treatment Carbohydrate-enriched diet (50–55 % of energy)
induces catch-up growth, reduces liver size and ameliorates Moderate fat restriction (20–30 %)
secondary biochemical abnormalities. In GSD-I, lifelong Predominantly polyunsaturated fatty acids
Protein enriched diet (20 %)b
dietary treatment is necessary. In GSD-III, dietary treatment
All Frequent feeds, prevention of fastinga
is often less demanding and in general and in types VI and a
Depending on GSD type, individual fasting tolerance and age, consider
IX, therapy is markedly easier with only about 50 % of
frequent snacks or cornstarch during daytime and continuous gastric
patients being prone to hypoglycaemia. In the latter types, drip feeding and use of cornstarch during the night (see text)
dietary treatment is generally limited to younger children. b
In type IIIa with muscle involvement
In adolescent patients with GSD-I, in analogy to protein- In GSD-I non-a, prophylactic oral antibiotic treatment
uric insulin-dependent diabetic mellitus, reduction of protein may be beneficial in patients with neutropenia, but the effect
intake should be considered. Furthermore, a reduction of has not been systematically studied. Co-trimoxazole has
sodium intake may enhance the beneficial effects of been recommended in symptomatic patients or those with a
angiotensin-converting enzyme inhibitors. neutrophil count below 500/nl. In mild cases of inflamma-
Renoprotective treatment with an ACE inhibitor should tory bowel disease, conservative treatment with 5-aminosali-
be started in GSD-I patients as soon as microalbuminuria cylic acid might be of benefit. No controlled trials are
persists and before hypertension develops. Angiotensin II available to support G-CSF therapy in GSD-I non-a.
antagonists may elicit comparable results; however, clinical Therefore, the use of G-CSF should be restricted to the fol-
experience in GSD-I is more limited. lowing indications: (1) persistent neutrophil count <200/nl,
In order to prevent urate nephropathy in patients with (2) a life-threatening infection requiring intravenous antibi-
hepatic glycogenosis, allopurinol (10 mg/kg/day in three otics and (3) serious complaints of inflammatory bowel dis-
doses, max 900 mg/day) should be started if serum uric acid ease, including oral or perianal infections or severe diarrhoea.
concentration exceeds the upper normal level for age despite GSD-I non-a patients seem to respond to low doses of
optimal dietary treatment. As uric acid is regarded to be a G-CSF: a starting dose of 2.5 μg/kg sc daily or every other
potent radical scavenger (with a possible protective role day is therefore recommended. Determine neutrophil count
against premature atherosclerosis), the targeted uric acid frequently and adjust the dose in steps of 2.5–5 μg/kg/day
concentration is in the high normal range. (maximum 25 μg/kg) to maintain neutrophils >1,000/nl.
Supplementation of vitamins should commence when WHO Fibrates are indicated if triglyceride concentration in
recommendations are not met. Furthermore, supplementation of patients with hepatic GSDs remain above 10.0 mmol/l
calcium and vitamin D is important when intake of milk and despite maximal dietary efforts. Fish oil treatment of hyper-
milk-derived products is limited (GSD-I). Special attention is lipidaemia has a limited and temporary effect; use of
also needed regarding vitamin B1 intake as increased metabo- medium-chain triglycerides has been advocated, but remains
lism of carbohydrates needs sufficient vitamin B1. controversial. Statins may be indicated in adult GSD-I
If GSD-I patients show persistence of a deficit of bases patients with massive hypercholesterolaemia.
(base excess <−5 mmol/l) or a low blood bicarbonate con- Treatment with growth hormone in patients with the
centration (<20 mmol/l) despite intensive dietary treatment, hepatic GSDs and growth retardation is not advocated, since
it is recommended to correct lactacidaemia with (sodium) the effect on final height is negligible.
bicarbonate or (potassium) citrate. In addition to correcting Liver transplantation is considered with increasing fre-
acidosis, this will result in alkalisation of the urine which quency in patients with hepatic GSDs. It can be an option in
reduces the risk for urolithiasis and nephrocalcinosis. Citrate GSD-Ia to correct the basic defect in liver. Also patients with
(initial dosage 1–2 mEq/kg/day in three to four doses) is pre- hepcidin-producing adenoma and severe anaemia (GSD-I),
ferred since it also corrects hypocitraturia, another risk factor proven or suspected hepatocellular carcinoma within an ade-
for urolithiasis. Hypocitraturia is more often seen with noma (mainly GSD-I) or cirrhosis (GSD-III, GSD-IV, GSD-
increasing age of the patients. IXc) may benefit from this procedure.
Table 18.45 Follow-up in patients with hepatic glycogen storage diseases

Disorder Investigations Frequency /remarks
FBS See GSD-I plus
Ophthalmological examination (cataract?)
Glucose (P) (postprandial target conc <10.0 mM) Depending on age, symptoms and medication
Gal, Gal-1-P
Carnitine

Disorder Investigations Frequency /remarks
GSD-I Clinicala,b,c,* 0–3 years: 2 monthly
3–20 years: 3 monthly
>18 years: 6 monthly
Paraclinical Depending on age, symptoms and medication
Haematological parameters*
Electrolytes
Blood gases, lactate
Lactate (U) (target conc <0.6 mmol/l or < 0.06 mmol/mmol crea)
Liver parameters*
Kidney function (creatinine, GFR)
Uric acid (P)*
Calcium, phosphate, AP, PTH, vitamin D
Cholesterol, triglycerides, amylase, lipase
Home blood glucose profiled,*
(Preprandial target conc >3.5–4.0 mM)
Microalbumin (U), protein (U)
α-fetoprotein*
Ultrasound (liver*, pancreas, ovaries)
Bone density
EEGc
GSD-I non-a See GSD-I plus
CRP, ESR Depending on age, symptoms and medication
Faecal α1-antitrypsin
Total blood count with differential
Bone marrow
GSD-IIIa See GSD-I plus
Creatine kinase (P) Depending on age, symptoms and medication (in
EKG, echocardiography general less often than in GSD-I)
Muscle function
Lung function
GSD-VI, See GSD-I Depending on age, symptoms and medication (in
GSD-IXa–c general markedly less often than in GSD-I) and
restricted to investigations marked by *
a
Nutrient intake, growth, sexual maturation, nutritional status, somatic findings (liver size)
b
Neurological, psychological, cognitive functions, developmental investigations on demand
c
Particular in cases with frequent hypoglycaemia
d
Consider subcutaneous continuous monitoring
18.7.8 Glycogen Storage Diseases: Mainly aerobic exertion tolerance in patients with GSD-V. However, it
Affecting Muscle may also lead to overweight and to increased insulin secretion,
potentially inhibiting the use of fatty acids. The benefits of using
Treatment: Patients with GSD-0b, GSD-XV, GSD-XIV, uncooked cornstarch in these patients to guarantee a constant
GSD-III, GSD-V, GSD-IXb and GSD-IXd, GSD-VII, glucose source from blood to muscle as oxidative substrate for
ALDOA-D, PGK-D, GSD-X, GSD-XIII and LDHA-D glycolysis needs further investigation. Supplementation with
may have primary muscle weakness; in GSD-V and GSD- branched-chain amino acids, vitamin B6 (50–100 mg/kg/day)
VII and the rare types GSD-XIV, ALDOA-D, PGK-D, and creatine (10–20 g/day) has been recommended.
GSD-X, GSD-XIII and LDHA-D, severe rhabdomyolysis Patients with GSD-VII depend on fatty acid oxidation in
which sometimes results in acute renal failure may addi- muscle as the main energy substrate. Therefore, in these
tionally occur after (short-term) intensive exertion. These patients a surplus of dietary carbohydrates should be avoided,
patients benefit from regular physical exercise at a sub- since it would enhance the metabolic muscle problems by
maximal level. decreasing the availability of fatty acids for oxidation. The
A carbohydrate-enriched diet (55–65 % of energy) and diet should be rich in fat (30–40 %) and proteins (15–20 %
sucrose, ribose or glucose ingestion before exercise may improve of energy).
18.7.9 GSD-IIa References
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Mitochondrial GLUT10 facilitates dehydroascorbic acid import and
cular) symptoms is taken by GSD-IIa, a lysosomal disorder.
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still are important to maintain range of motion and assist in of glycosylation type without central nervous system involvement
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Enzyme replacement therapy (ERT) with alglucosidase-α
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In: Fernandes J, Saudubray JM, van den Berghe G, Walter JH
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Bosch AM, Grootenhuis MA, Bakker HD, Heijmans HS, Wijburg FA,
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Pyruvate Carboxylase and Pyruvate
Dehydrogenase Deficiency 19
Linda De Meirleir
Contents Summary
19.1 Introduction ..................................................................... 303 Pyruvate carboxylase and pyruvate dehydrogenase deficiency
are the most common disorders in pyruvate metabolism and
19.2 Nomenclature................................................................... 305
almost always affect the central nervous system. The severity
19.3 Metabolic Pathway .......................................................... 306 and the clinical phenotypes vary, with a range from over-
19.4 Signs and Symptoms ....................................................... 307 whelming neonatal lactic acidosis and early death to milder
19.5 Reference Values ............................................................. 309
presentations. In the differential diagnosis, there are always
the respiratory chain defects (see Chap. 22 on mitochondrial
19.6 Pathological Values/Differential Diagnosis ................... 309 disorders).
19.7 Diagnostic Flow Chart .................................................... 310 Diagnosis depends on biochemical analysis in plasma,
19.8 Specimen Collection ........................................................ 310 urine, and CSF followed by enzymatic analysis in various
tissues and confirmation by DNA analysis.
19.9 Diagnostics ....................................................................... 310
Pyruvate carboxylase (PC) deficiency constitutes a defect
19.10 Prenatal Diagnosis of PC Deficiency ............................. 310 both in the Krebs cycle and in gluconeogenesis and generally
19.11 Treatment ......................................................................... 310 presents with severe neurologic dysfunction and lactic acido-
Further Reading ............................................................................ 311
sis more frequently than with fasting hypoglycemia. There
are three different phenotypes with wide spectrum in
severity.
Pyruvate dehydrogenase deficiency (PDH) is most com-
mon due to deficiency in the X-linked PDHE1alpha, but also
defects in the other subunits of PDH complex have been
described. The clinical picture is PDHE1alpha is usually dif-
ferent in boys and girls. Neonatal lactic acidosis and Leigh’s
encephalopathy occur more frequently in boys; girls can
present with severe seizures and microcephaly. The diagno-
sis is suspected when lactate and pyruvate are elevated, with
a normal pyruvate to lactate ratio.
Further confirmation is done biochemically on fibroblasts,
lymphocytes, or muscle, and the different genes can be
investigated. A prenatal diagnosis is then possible. Ketogenic
diet might help in some patients.
19.1 Introduction
Pyruvate carboxylase is a biotin-containing nuclear-encoded

L. De Meirleir
mitochondrial enzyme, responsible for the carboxylation of
Department of Pediatric Neurology, VZ-Brussel,
Laarbeeklaan 101, Brussels 13-1090, Belgium pyruvate to oxaloacetate. The reaction covers for the replen-
e-mail: linda.demeirleir@uzbrussel.be ishment of pools of intermediates of the citric acid cycle, a
304 L. De Meirleir
process called anaplerosis. Pyruvate carboxylase is also first presentation, neonatal lactic acidosis is often associated
involved in several metabolic pathways that depend on the with brain dysgenesis, such as agenesis of the corpus callo-
availability of oxaloacetate such as gluconeogenesis, glyco- sum. In Leigh’s encephalopathy, the initial presentation is
gen synthesis, lipogenesis, glycerogenesis, and synthesis of usually within the first 5 years of life and includes respira-
amino acids and neurotransmitters. tory disturbances or episodic weakness and ataxia with
Pyruvate Carboxylase Deficiency absence of tendon reflexes. Respiratory disturbances may
Pyruvate carboxylase is associated with metabolic acido- lead to apnea, dependence on assisted ventilation, or sudden
sis, failure to thrive, and developmental delay. Three pheno- unexpected death. Intermittent dystonic posturing of the
types are associated with pyruvate carboxylase deficiency. In lower limbs occurs frequently. A moderate to severe devel-
type A infantile North American phenotype, patients are opmental delay becomes evident within the next years.
severely ill between 2 and 5 months of age with progressive A very small subset of male patients is initially much less
hypotonia. Episodes of acute vomiting, dehydration, tachy- severely affected, with intermittent episodic ataxia after
pnea, facial pallor, cold cyanotic extremities, and metabolic carbohydrate-rich meals, progressing slowly over years into
acidosis, characteristically precipitated by metabolic or a mild Leigh’s encephalopathy. A number of patients have
infectious stress, occur. Clinical examination includes pyra- developed an acute peripheral neuropathy during infancy or
midal tract signs, ataxia, and nystagmus. All patients are an acute episodic ataxia.
severely mentally retarded and most of them have In females with PDHE1α deficiency, there is a more uni-
convulsions. form clinical presentation, although with variable severity.
In type B, patients (French phenotype) become acutely ill This includes dysmorphic features, microcephaly, moderate
3–48 h after birth with hypothermia, hypotonia, lethargy, and to severe mental retardation, and spastic di- or quadriplegia,
vomiting. There is a rapid deterioration with rigidity, hypoki- resembling nonprogressive encephalopathy. Dysmorphism
nesia, and tremor (resembling infantile Parkinson) and comprises a narrow head with frontal bossing, wide nasal
abnormal ocular movements. Most die in the neonatal period. bridge, upturned nose, long philtrum, and flared nostrils.
Some survive but remain unresponsive and severely hypo- Seizures are encountered in almost all female patients. These
tonic, often with death due to respiratory infection before the appear within the first 6 months of life and are diagnosed as
age of 5 months. infantile spasms (flexor and extensor) or severe myoclonic
Type C is a more benign phenotype, only been reported in seizures. Brain MRI frequently reveals severe cortical/sub-
a few patients. The clinical course consists of acute episodes cortical atrophy, dilated ventricles, and partial to complete
of lactic acidosis and ketoacidosis, usually responding rap- corpus callosum agenesis. Severe neonatal lactic acidosis
idly to hydration and bicarbonate therapy. Despite the impor- can be present.
tant enzymatic deficiency on testing in fibroblasts, the PDHE1beta (19.2.2)
patients have a nearly normal cognitive and neuromotor Few cases have been reported and patients present with
development. early-onset lactic acidosis and severe developmental delay.
Pyruvate Dehydrogenase Deficiency A moderate clinical course with slowly progressive neuro-
PDHC is composed of three components: E1; E2, dihy- logical features reflecting basal ganglia and brain stem
drolipoamide acyltransferase; and E3, dihydrolipoamide involvement associated with typical findings of Leigh syn-
dehydrogenase. E1 utilizes thiamine pyrophosphate and is drome has also been reported.
composed of two different subunits, E1α and E1β. The E1 PDHE2 (19.2.3; Dihydrolipoamide Transacetylase)
reaction results in decarboxylation of the specific α- keto- A few cases of PDHE2 (dihydrolipoamide transacetylase)
acid. For the PDHC, the E1 component is the rate-limiting deficiency have been reported recently with developmental
step and is regulated by phosphorylation/dephosphorylation delay and lactic acidosis.
catalyzed by two enzymes, E1 kinase (inactivation) and E1 PDH E3 (19.2.4)
phosphatase (activation). E2 is a transacetylase that utilizes Since this enzyme is common to all the 2-ketoacid dehy-
lipoic acid. E3 is a flavoprotein common to all three drogenases, E3 deficiency results in multiple 2-ketoacid
2-ketoacid dehydrogenases. Another important structural dehydrogenase deficiency and should be thought of as a
component of the PDHC is E3BP, E3 binding protein, for- combined PDHC and TCA cycle defect. E3 deficiency
merly protein X. This component has its role in attaching E3 presents with severe and progressive hypotonia and failure to
subunits to the core of E2. thrive, starting in the first months of life. Metabolic decom-
PDH E1alpha (19.2.1) pensations are triggered by infections. Progressively hypoto-
The most common features of PDHE1α deficiency are nia, psychomotor retardation, microcephaly, and spasticity
delayed development, hypotonia, seizures, and ataxia. occur. Some patients develop a typical picture of Leigh’s
In males, there are three presentations: neonatal lactic aci- encephalopathy. A Reye-like picture with liver involvement
dosis, Leigh’s encephalopathy, and intermittent ataxia. In the and myopathy with myoglobinuria without mental retarda-
19 Pyruvate Carboxylase and Pyruvate Dehydrogenase Deficiency 305
tion is seen in the Ashkenazi Jewish population. Increased development, and prolonged survival. Often more slowly
blood lactate and pyruvate, elevated plasma alanine, gluta- progressive, it also comprises early-onset neonatal lactic aci-
mate, glutamine, and branched-chain amino acids (leucine, dosis associated with subependymal cysts and thin corpus
isoleucine, and valine) and increased urinary lactic, pyruvic, callosum.
2-ketoglutaric, and branched-chain 2-hydroxy and 2-keto E1-Phosphatase (19.2.6)
acids are characteristic. E1-phosphatase deficiency has been identified in two
Pyruvate Dehydrogenase E3X (19.2.5) brothers with hypotonia, feeding difficulties, and delayed
The main clinical manifestations of E3BP (formerly pro- psychomotor development. A lethal infantile phenotype has
tein X) deficiency are hypotonia, delayed psychomotor also been described.
19.2 Nomenclature
Alternative Gene Chromosomal Affected OMIM
No. Disorder name Abbreviation symbol localization protein no. Subtype
19.1 Pyruvate carboxylase PCD PC 11q13.4–q13.5 Pyruvate 266150 All
deficiency carboxylase forms
19.2.1 Pyruvate dehydrogenase Pyruvate dehydrogenase PDH PDHA1 Xp22.2–p22.1 Pyruvate 312170 All
complex deficiency E1a E1α subunit deficiency dehydrogenase forms
E1α subunit
19.2.2 Pyruvate dehydrogenase Pyruvate dehydrogenase PDH PDHB 3p13–q23 Pyruvate 179060 All
complex deficiency E1b E1β subunit deficiency dehydrogenase forms
E1β subunit
19.2.3 Pyruvate dehydrogenase Dihydrolipoyl PDHC E2 PDHDD 11q23.1 Dihydrolipoyl 245348 All
complex deficiency E2 transacetylase deficiency transacetylase forms
19.2.4 Pyruvate dehydrogenase Dihydrolipoyl PDHC E3 DLD 7q31.1 Dihydrolipoyl 248600 All
complex deficiency E3 dehydrogenase dehydrogenase forms
deficiency
19.2.5 Pyruvate dehydrogenase Pyruvate dehydrogenase E3BP PDHX 11p13 Pyruvate 245349 All
complex deficiency E3 X E3 binding protein dehydrogenase E3 forms
deficiency binding protein
19.2.6 Pyruvate dehydrogenase Pyruvate dehydrogenase PDHPD PDP1 8q22.1 Pyruvate 608782 All
complex deficiency PDHP phosphatase deficiency dehydrogenase forms
phosphatase
306 L. De Meirleir

Fig. 19.1 Overview of glucose, Glucose
pyruvate/lactate, fatty acid, and
amino acid oxidation by the
tricarboxylic acid cycle. A
P-enolpyruvate
aconitase, CS citrate synthase,
F fumarase, ID isocitrate
dehydrogenase, KDHC α-or
2-ketoglutarate dehydrogenase PEPCK Fatty acids
Lactate
complex, MD malate Pyruvate
alanine
dehydrogenase, PC pyruvate
carboxylase, PDHC pyruvate
dehydrogenase complex, PEPCK CO2
CO2
phosphoenolpyruvate
carboxykinase, SD succinate Pc
PDHC
dehydrogenase, ST succinyl Acetyl CoA
coenzyme A transferase
CS
Citrate
Oxyloacetate A
Aspartate
MD
Aconitate
Malate
A
F
Isocitrate
Fumarate CO2
ID
SD α-Ketoglutarate
Succinate
KDHC
ST
Succinyl CoA Glutamate


Table 19.1 Pyruvate carboxylase deficiency
CNS Developmental delay +++ +++ +++
Hypokinesia ++ +
Hypotonia +++ +++ +++
Parkinsonism, hypokinetic ++ n n
features
Pyramidal signs ++ ++ ++
Digestive Failure to thrive +++ +++ +++
Vomiting +++ +++ ++
Routine laboratory Ammonia (B) n-↑ n-↑ n-↑
Glucose (P) ↓-n ↓-n ↓-n ↓-n
Ketones (U,P) ↑ ↑ ↑
Lactate/pyruvate ratio ↑↑ n n
Pyruvate (P) ↑↑ ↑ ↑
Special laboratory Alanine (P) ↑ ↑ ↑
Citrulline (P) ↑ ↑ ↑
Table 19.2.1 Pyruvate dehydrogenase complex deficiency E1a

CNS Agenesis, corpus callosum + + + + +
Developmental delay +++ +++ +++
Leigh syndrome ± ++ ++ ++ ++
Neuropathy, peripheral + ++ ++ ++
Microcephaly + + + + +
Routine Glucose (P) n n n n n
laboratory Ketones (U,P) ↑ ↑ ↑ ↑ ↑
Lactate/pyruvate ratio n n n n n
Lactic acidosis ++ ++ ++ ++ ++
Pyruvate (P) ↑↑ ↑↑ ↑↑ ↑↑ ++
Special laboratory Alanine (P) ↑ ↑ ↑ ↑ ↑
Table 19.2.2 Pyruvate dehydrogenase complex deficiency E1b

CNS Agenesis/hypogenesis, + + (↑)
corpus callosum
Leigh syndrome ± ++ ++ ++ ++
Musculoskeletal Microcephaly + + + + +
Routine laboratory Glucose (P) n n n n
Pyruvate (P) ↑↑ ↑↑ ↑↑ ↑↑ ++
308 L. De Meirleir
Table 19.2.3 Pyruvate dehydrogenase complex deficiency E2

CNS Developmental delay +++ +++ +++
Dystonia +++ +++ +++
Routine laboratory Glucose (P) n n n n
Pyruvate (P) ↑↑ ↑↑ ↑↑ ↑↑ ++
Table 19.2.4 Pyruvate dehydrogenase complex deficiency E3

CNS Agenesis/hypogenesis, + + +
corpus callosum
Developmental delay ++ ++ ++
Hypotonia, progressive ++ ++ ++
generalized
Leigh syndrome ± ++ ++
Spasticity ± ++ ++
Musculoskeletal Microcephaly + ++ ++
Myoglobinuria ↑ ↑
Myopathy n n n ↑ ↑
Routine laboratory Lactate (U) ↑ ↑ ↑ ↑ ↑
Special laboratory 2-Oxoglutaric acid (U) ↑ ↑ ↑ ↑ ↑
Isoleucine (P) ↑ ↑ ↑ ↑ ↑
Leucine (P) ↑ ↑ ↑ ↑ ↑
Valine (P) ↑ ↑ ↑ ↑ ↑
Table 19.2.5 Pyruvate dehydrogenase complex deficiency E3 X

CNS Agenesis, corpus callosum + + + + +
Seizures + +
Musculoskeletal Microcephaly + + + + +
Ketones (U,P) ↑ ↑ ↑ ↑ ↑
Pyruvate (P) ↑↑ ↑↑ ↑↑ ↑↑ ++
Table 19.2.6 Pyruvate dehydrogenase complex deficiency PDHP

CNS Developmental delay +++ +++ +++ +++
Ketones (U,P) ↑ ↑ ↑ ↑ ↑
Pyruvate (P) ↑↑ ↑↑ ↑↑ ↑↑ ++

Lactate blood L/P ratio blood Serum ammonia Plasma amino acids 3-OH-butyrate/acetate plasma
0.5–2 mmol/L <25 <30 days Alanine Ratio 0.8–1
17–91 mmol/L 179–378 mmol/L
1–12 m Proline
15–71 mmol/L 125–279 mmol/L
1–14 years Citrulline
14–65 mmol/L 11–35 mmol/L
Lysine
121–286 mmol/L
Glutamine
347–786 mmol/L
19.6 Pathological Values/Differential Hypoglycemia and ketosis

Diagnosis Type C
Episodic metabolic acidosis with normal citrulline plasma
Type A concentrations and elevated lysine and proline plasma
Normal lactate/pyruvate ratio concentration
Lactate 2–10 mmol/L; increased alanine and proline; normal Lactate 2–5 mmol/L
citrulline and lysine Lactate/pyruvate ratio normal
Hypoglycemia; ketosis Ketosis during metabolic stress
Type B CSF
Increased lactate/pyruvate ratio; increased acetoacetate to Elevated lactate and pyruvate
3-OH-butyrate ratio; elevated concentrations of citrul- Marked reduced glutamine
line (100–400 μM, normal <40), lysine, and ammo- Elevated glutamic acid and proline
nia (100–600 μM, normal <60); low concentration of Urine
glutamine; lactate >10 mmol/L; pyruvate in type B Organic acid profile shows, besides large amounts of
0.14–0.90 mmol/L; normal range 0.04–0.13, resulting lactate, pyruvate, and 3-OH-butyrate, an increase of
in increased lactate/pyruvate ratio >20 α-ketoglutarate
310 L. De Meirleir
Fig. 19.2 Increased lactate
Urine Acids Organic Increased pyruvate Decreased or normal

acids abnormal normal L/P ratio pyruvate,increased L/P
ratio
Dicarboxylic aciduria
acyl carnitines
Fatty acid oxidation Hypoglycemia Normoglycemia Decreased 3- Muscle biopsy,

defects ± ketones hydroxybutyrate respiratory chain
acetoacetate ratio complexes or
Hypoglycemia mitochondrial
myopathy
PDHC
Biotinidase G6Pase PC E1 E3 PDH PC

Multiple F1,6 Type E2 Phosphatase Type B
Carboxylae Dphase A X
HMGCoA lyase PEPCK
Propionic acidemia
Methylmaloni
acidemia
Other organic acidurias
19.8 Specimen Collection 19.10 Prenatal Diagnosis of PC Deficiency
During acute presentation blood lactate, pyruvate, ketones, Measurement of PC or PDH activity in cultured amniotic
glucose fluid cells or direct measurement in chorionic villi biopsy
Organic acids and amino acids in urine specimens or DNA analysis, when the familial mutations
CSF for lactate and pyruvate are known
19.9 Diagnostics 19.11 Treatment
Cultured fibroblasts or liver Emergency Treatments

PC enzyme assay fibroblast PC enzyme activity <5 % of con- Patients should be promptly treated with intravenous 10 %
trols in type B and 5–23 % in type A and <10 % in type C; glucose and may require bicarbonate to correct acidosis
similar in lymphocytes Standard Treatment
Assays can also be performed in postmortem liver, in which Treat acute acidotic episodes
the activity of PC is 10-fold higher than in fibroblasts but Neither biotin, thiamine, dichloroacetate, nor a high-fat or
must be interpreted with caution because of rapid post- high-carbohydrate diet has been shown to provide clinical
mortem degradation of the enzyme benefit
DNA testing is possible The general prognosis for individuals with PDHC deficiency
Total PDH activity, subunits, and DCA activation can be mea- is poor, and treatment is not very effective. Best strategy
sured using spectrophotometric methods on cultured fibro- for treating PDHC deficiency is the use of a ketogenic
blasts, lymphocytes, and muscle. Western blot for E3BP diet. Thiamine has been given in variable doses (500–
DNA testing is possible; most frequently PDHE1alpha on X 2,000 mg/day), with lowering of blood lactate and clinical
chromosome improvement in some patients
DL-lipoic acid has been tried, but its efficacy remains Head R, Brown R, Zolkipli Z et al (2005) Clinical and genetic spectrum
controversial in PDHE3 of pyruvate dehydrogenase deficiency: dihydrolipoamide acetyl-
transferase (E2) deficiency. Ann Neurol 58:234–241
Experimental Treatment Hong YS, Korman SH, Lee J et al (2003) Identification of a common
An orthotopic hepatic transplantation reversed ketoacidosis mutation (Gly194Cys) in both Arab Moslem and Ashkenazi Jewish
and renal tubular abnormalities, and decreased lactic aci- patients with dihydrolipoamide dehydrogenase (E3) deficiency:
demia in a patient with a severe phenotype possible beneficial effect of vitamin therapy. J Inherit Metab Dis
26:816–818
A patient with the French phenotype was started on early Lissens W, De Meirleir L, Seneca S et al (2000) Mutations in the
treatment with triheptanoin in order to restore anaplero- X-linked pyruvate dehydrogenase (E1) α sububit gene (PDHA1) in
sis. Although there was a clinical improvement without patients with a pyruvate dehydrogenase complex deficiency. Hum
evidence of neurodegeneration, the patient died during an Mutat 15:209–219
Maj M, Mackay N, Levandovskyi V et al (2005) Pyruvate dehydrogenase
episode of acute decompensation at 8 months of age phosphatase deficiency: identification of the first mutation in two
brothers and restoration of activity by protein complementation. J
Clin Endocrinol Metab 90:4101–4107
Michotte A, De Meirleir L, Lissens W et al (1993) Neuropathological
Further Reading findings of a patient with pyruvate dehydrogenase E1 alpha defi-
ciency presenting as a cerebral lactic acidosis. Acta Neuropathol
Barnerias C, Saudubray JM, Touati G et al (2010) Pyruvate dehydroge- (Berl) 85:674–678
nase complex deficiency: four neurological phenotypes with differ- Monnot S, Serre V, Chadefaux-Vekemans B et al (2009) Structural
ing pathogenesis. Dev Med Child Neurol 52(2):e1–e9 insights on pathogenic effects of novel mutations causing pyruvate
Brown RM, Head RA, Boubriak II et al (2004) Mutations in the gene carboxylase deficiency. Hum Mutat 30:734–740
for the E1β subunit: a novel cause of pyruvate dehydrogenase defi- Quintana E, Gort L, Busquets C et al (2009) Mutational study in the
ciency. Hum Genet 115:123–127 PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase
Brown RM, Head R, Morris AA, Raiman JA et al (2006) Pyruvate complex deficiency. Clin Gen 77:274–282
dehydrogenase E3 binding protein (protein X) deficiency. Dev med Robinson BH, Taylor J, Sherwood WG (1980) The genetic heterogene-
Child Neurol 48:756–760 ity of lactic acidosis: occurrence of recognizable inborn errors of
De Meirleir L, Specola N, Seneca S, Lissens W (1998) Pyruvate dehy- metabolism in a pediatric population with lactic acidosis. Pediatr
drogenase E1alpha deficiency in a family: different clinical presen- Res 14:956–962
tation in two siblings. J Inherit Metab Dis 21:224–226 Robinson BH, Oei J, Sherwood WG et al (1984) The molecular basis
Debray FG, Lambert M, Gagne R et al (2008) Pyruvate dehydrogenase for the two different clinical presentations of classical pyruvate car-
deficiency presenting as intermittent isolated acute ataxia. boxylase deficiency. Am J Hum Genet 36:283–294
Neuropediatrics 39:20–23 Robinson BH, MacKay N, Chun K, Ling M (1996) Disorders of pyru-
Elpeleg ON, Ruitenbeek W, Jakobs C et al (1995) Congenital lacticaci- vate carboxylase and the pyruvate dehydrogenase complex. J Inherit
demia caused by lipoamide dehydrogenase deficiency with favor- Metab Dis 19:452–462
able outcome. J Pediatr 126:72–74 Saudubray JM, Marsac C, Charpentier C et al (1976) Neonatal congeni-
Garcia-Gazorla A, Rabier D, Touati G et al (2006) Pyruvate carboxyl- tal lactic acidosis with pyruvate carboxylase deficiency in two sib-
ase deficiency: metabolic characteristics and new neurological lings. Acta Paediatr Scand 65:717–724
aspects. Ann Neurol 59:121–127 Van Coster RN, Fernhoff PM, De Vivo DC (1991) Pyruvate carboxyl-
Grafakou O, Oexle K, van den Heuvel L et al (2003) Leigh syndrome ase deficiency: a benign variant with normal development. Pediatr
due to compound heterozygosity of dihydrolipoamide dehydroge- Res 30:1–4
nase gene mutations. Description of the first E3 splice site mutation. Willemsen M, Rodenburg RJT, Teszas A et al (2006) Females with PDHA1
Eur J Pediatr 162:714–718 gene mutations: a diagnostic challenge. Mitochondrion 6:155–159
Disorders of the Krebs Cycle
20
Eva Morava and Rosalba Carrozzo
Contents Summary
20.1 Introduction ..................................................................... 314 This chapter focuses on two classic Krebs cycle disorders
(2-oxoglutaric aciduria and fumarase deficiency) and two
20.2 Nomenclature................................................................... 315
recently discovered disorders of the Krebs cycle, severely
20.3 Metabolic Pathways ........................................................ 315 affecting mitochondrial function and mitochondrial mainte-
20.4 Signs and Symptoms ....................................................... 316 nance (succinyl-CoA synthetase –SCS – deficiencies, char-
acterized by mutations in SUCLA2 and SUCLG1 genes).
Fumarase deficiency and 2-oxoglutaric aciduria are rare dis-
20.6 Pathological Values ......................................................... 319 orders with global developmental delay and severe neuro-
20.7 Diagnostic Flowchart ...................................................... 319 logic problems in infants. Patients with oxoglutaric aciduria
20.8 Specimen Collection and Pitfalls ................................... 320 have a variable severity of neurological involvement and
metabolic acidosis and develop severe microcephaly and
20.9 Prenatal Diagnosis........................................................... 321
mental retardation. A special form (DOOR syndrome) occurs
20.10 Treatment and Prognosis ................................................ 321 with sensorineural deafness and osteodystrophy. Patients
20.11 Alternative Therapies ..................................................... 321 with fumarase deficiency present with either a fulminant
References ...................................................................................... 321
course associated with fatal outcome within the first 2 years
of life or a subacute encephalopathy with profound speech
delay without metabolic crises. SUCLA2 and SUCLG1
defects have the clinical presentation of mitochondrial deple-
tion syndromes with profound hypotonia, progressive dysto-
nia, and muscular atrophy, in addition to severe sensorineural
hearing impairment, which has been specifically associated
with SUCLA2 defect. The most important clues for the diag-
nosis in all these disorders rely in urine organic analysis.
2-oxoglutaric aciduria leads to chronic metabolic acidosis
and variable urinary excretion of 2-oxoglutarate, while
fumarase deficiency occurs with an increased excretion of
fumarate associated with succinate and lactate excretion
with eventual 2-oxoglutaric aciduria. A normal excretion of
E. Morava (*)
Department of Pediatrics,
fumaric acid and a relative high fumarase rest activity do not
Hayward Genetics Center, rule out fumarase deficiency. In questionable cases mutation
Tulane University Medical Center, analysis is needed to confirm the diagnosis. In SCS defects
1430 Tulane Ave, mild methylmalonic aciduria with abnormal urine carnitine-
New Orleans, LA 70112, USA
e-mail: emoravakozicz@tulane.edu
ester profile is associated with only subtle abnormalities of
the Krebs cycle intermediates. Due to the recognizable pat-
R. Carrozzo
Molecular Genetic, Bambino Gesù Children’s Hospital,
tern of dystonia/±deafness syndrome and mild methylmalo-
Piazza S. Onofrio 4, Rome, 00165, Italy nic aciduria in SCS defects, direct genetic testing is a possible
e-mail: rosalba.carrozzo@opbg.net approach in the diagnosis of SUCLA2 and SUCLG1 defects.
314 E. Morava and R. Carrozzo
Carrier screening in fumarase deficiency is important due to other allele. Other mutations in the FH gene are private,
the possible increased risk for certain malignancies. although, interestingly, most mutations are clustered at the
C-terminus (Piceuad et al. 2011).
Most heterozygous relatives of patients with fumarase
20.1 Introduction defect are normal. However, the finding of cutaneous leio-
myomata without uterine fibroids in the mother of an affected
The Krebs cycle disorders 2-oxoglutaric aciduria and fuma- child, a report of a mother with uterine myomas, and the death
rase deficiency are clinically similar disorders of hypotonia, of the mother of an affected child from renal cell carcinoma
spasticity, developmental delay, and extrapyramidal symp- in a third family raised the possibility of increased risk for
toms of variable severity. This very rare disorder presents tumor genesis in carriers for fumarate hydratase deficiency.
with episodes of acute metabolic acidosis and eventually Mutations on SUCLA2 and SUCLG1 genes are associated
hypoketotic hypoglycemia in stress situations. The disorder to unique disorders due to the involvement of both the citric
has episodes of severe disarrangements, related mostly to acid cycle, due to abnormal succinate metabolism, and the
acute, intercurrent infections. Lactic acidosis can be signifi- mitochondrial DNA (mtDNA) maintenance, manifesting
cant in acute episodes, and the disorder might imitate a mito- with low mtDNA content.
chondrial disorder both based on the clinical and metabolic Succinyl-CoA synthetase (SCS), which catalyzes the
presentation. Most cases are lethal in the first 10 years of life. reversible conversion of succinyl-CoA and ADP or GDP to
The 2-KGD complex is composed of three separate succinate and ATP or GTP, is part of the Krebs cycle. Deficient
enzymes: E1, E2, and E3. It has been postulated that in most SCS activity has been associated with mutations in two out of
patients, the E2 component could be responsible for the the three subunits making up the enzyme: SUCLA2 and
“classic metabolic” presentation of the defect (Bonnefont SUCLG1, respectively. These genes encode the β-subunit of
et al. 1992; Guffon et al. 1993). In occasional cases the dis- the ADP-forming SCS and the α-subunit of SCS. In multicel-
ease appears only after infancy, with progressive extrapyra- lular eucaryotes, two different isoforms exist, consisting of
midal and psychiatric symptoms (Kohlshutter et al. 1982). the common α-subunit and a different β-subunit, one specific
There are two distinct phenotypes in 2-oxoglutaric acid- for ATP (A-SCS) and the other specific for GTP (G-SCS)
uria, an encephalopathic form related to E2 subunit deficiency (Johnson et al. 1998). Therefore, the β-subunits determine the
and a clinically recognizable form of deafness, onycho- substrate specificity of the two forms of the enzyme. Both
osteodystrophy, thumbs and sensorineural deafness, also enzymes are located in the mitochondrial matrix, where
labeled as DOOR syndrome, due to a defect of the E1 subunit A-SCS, and probably also G-SCS, participates in the citric
of the enzyme complex (Surendan et al. 2002). The diagnosis acid cycle. Several studies have indicated that SCS forms a
might remain uncertain in some cases, due to relatively high complex with nucleoside diphosphate kinase (NDPK), which
residual activity of the 2-oxoglutarate dehydrogenase in is important for the salvage of deoxyribonucleotides for
homogenates of cultured skin fibroblasts which can be found mtDNA synthesis and for its integrity (Kadrmas et al. 1991;
in some patients with a proven E2 subunit deficiency. Kavanaugh-Black et al. 1994; Kowluru et al. 2002).
Fumarase deficiency has been described in approximately Mutations in SUCLA2, the gene encoding the β-subunit of
44 cases with severe infantile encephalopathy (De Meirleir ADP-forming SCS (Elpeleg et al. 2005), have been reported in
et al. 2006; Zinn et al. 1986; Zeman et al. 2000). The disorder is several ethnicities and are associated with a distinct clinical
lethal in one-third of the patients in early childhood and leads phenotype consisting of a Leigh-like syndrome, dystonia, deaf-
to severe mental retardation. Fumarase deficiency has been ness in association with lactic acidosis, variable degree of defi-
reported with intrauterine complications such as brain malfor- ciency of the respiratory chain enzyme complexes,
mations and intrauterine growth retardation. Some of the chil- mild-to-moderate mtDNA depletion, and mild elevation of
dren are dysmorphic (Mandrin et al. 2006). Severe visual methylmalonic acid in body fluids (Carrozzo et al. 2007;
disturbance, Dandy-Walker malformation, and polymicrogyria Ostergaard et al. 2007a, b; Morava et al. 2009). A founder
have been observed in a few cases. The oldest reported patient effect has been found underlying the common mutation in the
survived to the second decade of life (Allegri et al 2010). Faroese population.
The human fumarate hydratase (FH) gene consists of 10 Patients with mutations in SUCLG1, the gene cod-
exons encoding 510 amino acids. The first exon (exon 0) ing for the α-subunit of SCS, might present with either a
encodes a mitochondrial localization signal peptide of 43 severe, fatal form of mitochondrial encephalomyopathy
amino acids. (Ostergaard et al. 2007a, b) or a somewhat milder clinical
The 3-bp AAA duplication (c.1431_1433dupAAA) cod- syndrome highly reminiscent of that seen in children with
ing for an additional lysine amino acid is detected in approx- mutations in SUCLA2 (Ostergaard et al. 2009; Rouzier et al.
imately one-third of cases and is the most frequent abnormal 2010). SUCLG1 mutations that lead to complete absence of
allele in patients. All affected individuals with this allele are SUCLG1 protein are responsible for a very severe disorder
compound heterozygous with a different mutation on the with antenatal manifestations, whereas a SUCLA2-like
20 Disorders of the Krebs Cycle 315
phenotype is found in patients with residual SUCLG1 pro- a degradation of SUCLA2 when its heterodimer partner,
tein. Furthermore, it has been shown that in the absence of SUCLG1, is absent. Moreover, Ostergaard and coauthors
SUCLG1 protein, no SUCLA2 protein is found in fibro- noted in this patient phenotypic similarities to fumarase
blasts by western blot analysis. This result is consistent with deficiency (606812).
20.2 Nomenclature
Chromosomal OMIM
20.1 2-oxoglutaric Amish lethal OGDH, DLST 7p14-p13 2-Oxoglutarate 203740 All forms
aciduria microcephaly dehydrogenase
20.2 Fumarase Fumaric aciduria FH1 1q42.1 Fumarase 136850 All forms
deficiency
20.3 SCS deficiencies Encephalomyopathy with SCS SUCLA2 13q12-q13 Succinate-CoA ligase 612073 All forms
SUCLA2 methylmalonic aciduria β-subunit deficiency
20.4 SCS deficiency SCS-SUCLG1 SUCLG1 Succinate-CoA ligase 245400 All forms
SUCLG1 α-subunit deficiency
Pyruvate Lactate
Acetyl-CoA
Citrate
Fumarate α−Ketoglutarate
Succinate Succinyl CoA
ATP ADP Methylmalonyl CoA Propionyl CoA
Succinyl carnitine Methylmalonic acid Methylcitrate

ester (C4DC) Methylmalonyl carnitine 3-Hydroxpropionate
ester (C4DC) Propionylcarnitine (C3)
Fig. 20.1 Relevant metabolic pathways illustrating the metabolic effects of succinyl-CoA synthetase deficiency
Table 20.1 2-Oxoglutaric aciduria

CNS Choreoathetosis ± ± ±
Dystonia ± ++
Hypotonia, axial + + +
Neurological symptoms ++ ++ ++
Pyramidal signs ± +
Retardation, psychomotor ± ++ ++
Digestive Failure to thrive + + +
Liver dysfunction + + +
Ear Deafness, sensorineural ± ± ±
Musculoskeletal Dystrophic thumbs ± ± ±
Osteodystrophy ± ± ±
Routine laboratory ASAT/ALAT (P) ± ± ±
Glucose (P) ↓ ↓ ↓
Ketones (U, P) ↓ ↓ ↓
Lactate (P) ↑↑ ↑↑ ↑↑
Lactate/pyruvate ratio ↑ ↑ ↑
Lactic acidosis + + +
Special laboratory 2-Oxoglutaric acid (U) ↑↑ ↑↑ ↑↑
Table 20.2 Fumarase deficiency

CNS Altered consciousness ++ ++ ++
Athetosis ++ ++ ++
Autism ± ±
Cerebral palsy + ++ +++
Dystonia ± ± ±
Hypotonia +++ ++ +
Irritability +++ +++ +++
Neurological symptoms +++ +++ +++
Pyramidal signs + ++ ++
Regression, motor ± ++ ++
Retardation, psychomotor +++ +++ +++
Seizures ++ ++ ++
Speech delay +++ +++
Digestive Failure to thrive ++ ++ +
Feeding difficulties +++ +++ +++
Gastroesophageal reflux ++ ++
Hepatosplenomegaly ± ± ±
Malnutrition, chronic ++ ++ ++
Eye Optic atrophy ± ± ±
Vision, impaired ± ± ±
Hematological Neutropenia ± ± ±
Musculoskeletal Coarse facial features ± ± ±
Dysmorphic features ± ± ±
Hypertelorism ± ± ±
Macrocephaly ± ± ±
Other Course, episodic + + +
Fetal hydrops ±
Sudden death ++ ++ +
Routine laboratory ASAT/ALAT (P) ± ± ±
Bilirubin (P) ± ±
EEG: abnormal ± ± ±
Lactate (CSF) ± ± ±
Lactate (P) + + +
Lactic acidosis ± ± ±
Metabolic acidosis + + +
Special laboratory 2-Oxoglutaric acid (U) ± ± ±
Fumaric acid (U) ↑↑ ↑↑ ↑↑
Succinic acid (U) ± ± ±
Table 20.3 SCS deficiencies SUCLA2

CNS Choreoathetosis ± ± ± ± ±
Dystonia − ± + ++ ++
Hypotonia, axial + ++ ++ + +
Leigh syndrome − ± + ++ ++
Neurological symptoms ++ ++ ++ ++ ++
Neuropathy, peripheral − − ± ± ±
Pyramidal signs − ± + ++ ++
Retardation, psychomotor ± ++ ++ ++ ++
Digestive Failure to thrive + + + + +
Liver dysfunction ± ± ± + +
Ear Deafness, sensorineural ± ++ ++ ++ ++
Routine laboratory ASAT/ALAT (P) ± ± ± ± ±
Lactate (P) ↑ ↑ ↑ ↑ ↑
Lactate/pyruvate ratio ↑ ↑ ↑ ↑ ↑
Special laboratory C4-DC methylmalonyl-carnitine (U) ↑ ↑ ↑ ↑ ↑
C4-DC succinyl-carnitine (U) ↑ ↑ ↑ ↑ ↑
Methylmalonic acid (U) ↑ ↑ ↑ ↑ ↑
Table 20.4 SCS deficiency SUCLG1

Cardiovascular Congenital heart defects ± ± ± ± ±
Dystonia − ± + ++ ++
Hypotonia, axial + ++ ++ + +
Leigh syndrome − ± + ++ ++
Neurological symptoms ++ ++ ++ ++ ++
Neuropathy, peripheral − − ± ± ±
Pyramidal signs − ± + ++ ++
Retardation, psychomotor ± ++ ++ ++ ++
Digestive Failure to thrive ++ ++ ++ ++ ++
Feeding difficulties ++ ++ ++ ++ ++
Liver dysfunction ± ± ± + +
Ear Deafness, sensorineural ± ± ± ± ±
Routine laboratory ASAT/ALAT (P) ± ± ± ± ±
Lactate (P) ↑ ↑ ↑ ↑ ↑
Lactate/pyruvate ratio ↑ ↑ ↑ ↑ ↑
Special laboratory C4-DC methylmalonyl-carnitine (U) ↑ ↑ ↑ ↑ ↑
C4-DC succinyl-carnitine (U) ↑ ↑ ↑ ↑ ↑
Methylmalonic acid (U) ↑ ↑ ↑ ↑ ↑

Metabolite Normal range Sample Disorder Definition Sample
Fumaric acid (FA) 0–20 umol/mmol creat Urine 20.1 2-oxoglutaric 2OGA 5–1,700 umol/mmol creat Urine
Succinic acid (SA) 0–50 umol/mmol creat Urine aciduria LA 2.5–8.0 mmol/l Blood
2-oxoglutaric acid (2OGA) 0–50 umol/mmol creat Urine 20.2 Fumarase FA 20–3,828 umol/mmol creat Urine
Methylmalonic acid (MMA) 0–5.0 umol/mmol creat Urine deficiency SA 50–800 umol/mmol creat Urine
Lactic acid (LA) 0.5–2.1 mmol/l Blood 2OGA 5–800 umol/mmol creat Urine
Lactic acid (LA) 0.4–1.4 mmol/l CSF LA 2.1–3.0 mmol/l Blood
Glucose 3.4–6.4 mmol/l Blood 20.3 SUCLA2 MMA 16–80 umol/mmol creat Urine
deficiency
20.3 SUCLG1 MMA 24–2,400 umol/mmol creat Urine
deficiency
20.7 Diagnostic Flowchart
Urine organic acids analysis

Lactate measurement in blood
Glucose measurement in blood
Ketone measurement in blood
Lactate measurement in CSF
Urine carnitine profile
Elevated urine fumarate, Elevated blood lactate and

Krebs-cycle intermediates, Elevated blood lactate, and urine methylmalonic acid
mild lactate elevation in blood urine 2- oxoglutarate, acidosis, (mild elevation), abnormal
and/or CSF low ketones and hypoglycemia urine carnitine profile
Fumarase enzyme activity 2-oxoglutarate dehydrogenase

Respiratory chain enzymes
measurement in fibroblasts enzyme activity in fibroblasts
measurement in muscle
FH mutation analysis Western blot of E1/E2 subunits SUCLA2 and SUCLG1

mutation analysis
Fig. 20.2 Diagnostic flow chart. Note: direct sequencing can be performed without functional studies based on the clinical/metabolic phenotype
20.8 Specimen Collection and Pitfalls in patients with SUCLA2 mutations (Carrozzo et al. 2007;
Morava et al. 2009), but without neurosensorial deafness in
The diagnosis of 2-oxoglutaric aciduria and fumarase defi- the vast majority of the cases, mutations in SUCLG1 were
ciency is established by combining the clinical features, the discovered.
metabolic findings, and the biochemical enzyme activity and Metabolic Findings in Plasma and Cerebral Spinal Fluid
should be validated by molecular genetic testing. Increased plasma concentrations of 2-oxogutarate and
Metabolic Findings in Urine significant elevation of lactate with increased L/P ratio
The massive excretion of 2-oxoglutaric acid and fumaric and hypoketosis have been detected in 2-oxoglutaric acid-
acid in urine is, respectively, diagnostic in almost all patients. uria. Increased plasma concentrations of fumarate, lac-
The excretion of 2-oxoglutaric or fumaric acid in the two tate, and pyruvic acid have been detected in fumarase
different disorders is, respectively, 15- to 1,000-fold elevated deficiency. These metabolites are also abnormally ele-
when compared to the normal control range. 2-oxoglutaric vated in the cerebrospinal fluid (CSF), especially lactic
acid might be elevated in fumarase deficiency, but less pro- acid. In one patient with fumarase deficiency, addition-
nounced. Unfortunately fumarate is not at all times present ally, two succinylpurine derivatives were found in CSF.
in excessive amounts in the urine in children with fumarase These two metabolites, 5-aminoimidazole-N-succinyl-
deficiency, and the concentration is not always correspond- carboxamide ribotide (SAICAR) and adenylosuccinate, have
ing with the measured residual fumarase activity (Ottolenghi a potential neurotoxic effect.
et al. 2011). High residual activity has been found in some of Mutations in SUCLA2 and SUCLG1 are associated with
the patients with 2-oxoglutaric aciduria as well. The pres- mild methylmalonic acidemia with normal homocysteine
ence of Krebs cycle intermediates in fumarase deficiency is and the increased C4-dicarboxylic-carnitine level in plasma
probably due to secondary enzymatic inhibition of succinate and even more in urine. As expected in a TCA cycle defect,
dehydrogenase (SDH). Other metabolites have been detected variable lactic acidemia and consistent lactate increase in
as well in the urine, without a consistent pattern. CSF are accompanying characteristics in patients with these
Mild methylmalonic aciduria, Leigh-like encephalomy- defects. TCA cycle intermediates were found increased to a
opathy, dystonia, and deafness characterize the group of variable extent in the urine of the patients. The increase of
patients with SUCLA2 gene mutations, in whom the key methylmalonic acid in body fluids that is considerably less
diagnostic features are represented by mild methylmalonic pronounced in patients with SCS defect than in classical
acid excretion combined with an abnormal profile of carni- MMA (Deodato et al. 2006) may be explained by the
tine esters (specifically succinyl-carnitine). Most of the impaired conversion of succinyl-CoA to succinate, resulting
patients present with early onset hypotonia, severe develop- in metabolite accumulation proximal to the enzymatic block.
mental delay or early neurological deterioration, slowly pro- Enzyme Measurements
gressive dystonic/athetoid movements, and neurosensorial The activity of 2-oxoglutarate dehydrogenase in homog-
deafness. In the course of the first year of life, no abnormali- enates of cultured skin fibroblasts can be as high as 25 % of
ties or mild cerebral atrophy with enlarged subarachnoid control values in patients. Additional Western blot analysis
spaces and widening of the ventricular system is observed. of the E1 and E2 subunits might be needed to confirm the
Later on basal ganglia involvement becomes obvious. At first diagnosis.
putamen and caudate seem to be specifically affected, but The residual enzyme activity of fumarate hydratase defi-
after infancy also the other parts of the basal ganglia show ciency shows no clear correlation with either the metabolic
abnormalities on the MRI. Unlike classical MMA (Trinh derangement or the clinical severity. The enzyme activity
et al. 2001), the globus pallidus can be spared or seems to be can be measured in fibroblasts, lymphoblasts, and white
the least affected. In the end stage of the disease, as observed blood cells. Different tissues show different residual activi-
postmortem in one case, only a very small residual volume ties, and although most patients show an activity below
of the nucleus caudatus remained intact. The increase of 10 %, the FH activity can range from undetectable to 35 %
methylmalonic acid in body fluids is considerably less pro- compared to the activity measured in healthy controls.
nounced in these patients compared to classical MMA Enzyme measurements therefore might be insufficient to
patients (Deodato et al. 2006). Abnormal activity of respira- confirm the diagnosis, since the FH activity in healthy
tory chain enzymes and mtDNA depletion, together with heterozygous parents ranges from 22 to 74 % of controls.
methylmalonic aciduria, are the biochemical hallmarks of The SCS-A activity in muscle mitochondria from a
this disorder. SUCLA2-mutated patient has been demonstrated to be
In addition, in a subgroup of patients, all with mild reduced compared to controls (Carrozzo et al. 2007), as well
methylmalonic aciduria, Leigh-like encephalomyopathy, as the SUCLA2 and SUCLG1 gene products measured
and dystonia, clinical features closely resembling those seen through Western blotting in muscle homogenate and in
fibroblasts mitochondria, respectively (Carrozzo et al. 2007; multiple cutaneous and uterine leiomyomas (MCUL) or
Ostergaard et al. 2007a, b). papillary renal cell carcinoma with leiomyomatosis (HLRCC)
(Tomlinson et al. 2000).
20.9 Prenatal Diagnosis Disorder Specific treatment

2-oxoglutaric aciduria Bicarbonate supplementation
Frequent feedings
Molecular genetic testing is possible in fumarase deficiency
Avoidance of high CH intake
and can be started with targeted mutation analysis of FH for
Fumarase deficiency None
the common 3-bp AAA duplication (c.1431_1433dupAAA),
SUCLA2 and SUCLG1 defects Protein restriction was not
as the first genetic screening test. Due to compound het- proved to be effective
erozygosity of the mutation, further analysis of the whole
FH gene is required to fully confirm the genetic defect.
Prenatal diagnosis and preimplantation genetic diagnosis
for at-risk pregnancies require prior identification of the 20.11 Alternative Therapies
disease-causing mutations in the family, although prenatal
diagnosis of fumarase deficiency has even been successfully Disorder Alternative therapies
performed by enzyme activity measurements (Coughlin 2-oxoglutaric aciduria Not successful
Fumarase deficiency Not successful
et al. 1998; Manning et al. 2000). In SUCLA2 and SUCLG1
SUCLA2 and SUCLG1 defects Anaplerosis
defects, prenatal diagnosis is also possible in chorionic sam-
ples by sequence analysis in at-risk pregnancies.
Disorder Prenatal diagnosis

Fumarase deficiency Mutation analysis in chorionic sample References
FH enzyme measurement in amniocytes
SUCLA2 Mutation analysis in chorionic sample Allegri G, Fernandes MJ, Scalco FB et al (2010) Fumaric aciduria: an
and SUCLG1 defects overview and the first Brazilian case report. J Inherit Metab Dis
33:411–419
Bonnefont JP, Chretien D, Rustin P et al (1992) Alpha-ketoglutarate
dehydrogenase deficiency presenting as congenital lactic acidosis. J
Pediatr 121:255–258
Carrozzo R, Dionisi-Vici C, Steuerwald U et al (2007) SUCLA2 muta-
20.10 Treatment and Prognosis tions are associated with mild methylmalonic aciduria, Leigh-like
encephalomyopathy, dystonia and deafness. Brain 130:862–874
So far there is no curative treatment, and there are no known Coughlin EM, Christensen E, Kunz PL et al (1998) Molecular analysis
distinctive clinical symptoms and biochemical or genetic and prenatal diagnosis of human fumarase deficiency. Mol Genet
Metab 63:254–262
findings that can be used for prognosis assessment and pre-
De Meirleir L, Hansikova H, Zeman J et al (2006) Fumarate hydratase
diction of the course of 2-oxoglutaric aciduria and fumarase deficiency – a rare cause of development delay and seizures. J
deficiency. In 2-oxoglutaric aciduria, bicarbonate supple- Inherit Metab Dis 29:106–108
mentation might be needed due to the chronic metabolic aci- Deodato F, Boenzi S, Santorelli FM, Dionisi-Vici C (2006)
Methylmalonic and propionic aciduria. Am J Med Genet C Semin
dosis, and dietary intervention should be applied in patients
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with recurrent hypoketotic hypoglycemia. The outcome of Elpeleg O, Miller C, Hershkovitz E et al (2005) Deficiency of the ADP-
both disorders appears to depend on the progression of the forming succinyl-CoA synthase activity is associated with encepha-
central nervous system symptoms and how effectively these lomyopathy and mitochondrial DNA depletion. Am J Hum Genet
76:1081–1086
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Guffon N, Lopez-Mediavilla C, Dumoulin R et al (1993) 2-ketoglutarate
within the first year of life to survival to puberty or adult- dehydrogenase deficiency, a rare cause of primary hyperlactatae-
hood. The oldest patient with fumarase deficiency described mia: report of a new case. J Inherit Metab Dis 16:821–830
was 25 years old at time of the case report. Johnson JD, Muhonen WW, Lambeth DO (1998) Characterization of
the ATP- and GTP-specific succinyl-CoA synthetases in pigeon.
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to adulthood. Some of the SUCLG1 patients have a neonatal, Kadrmas EF, Ray PD, Lambeth DO (1991) Apparent ATP-linked suc-
fatal outcome. cinate thiokinase activity and its relation to nucleoside diphosphate
kinase in mitochondrial matrix preparations from rabbit. Biochim
If the fumarate hydratase-causing mutations are known
Biophys Acta 1074:339–346
in the family, it is appropriate to consider offering molecular Kavanaugh-Black A, Connolly DM, Chugani SA, Chakrabarty AM
genetic testing to relatives who might be at risk of developing (1994) Characterization of nucleoside-diphosphate kinase from
Pseudomonas aeruginosa: complex formation with succinyl-CoA encephalomyopathic form, with methylmalonic aciduria. Eur J
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Hyperinsulinism
21
Khalid Hussain and Pascale De Lonlay

21.1 Introduction ..................................................................... 323
21.1.1 Transient Hyperinsulinism ................................................ 323 Hyperinsulinism of infancy (HI) is the most common cause
21.1.2 Congenital Hyperinsulinism ............................................. 323 of recurrent and severe hypoglycaemia in the neonatal and
21.2 Nomenclature................................................................... 325 infancy periods. The biochemical basis of this disorder is
characterised by the excessive and inappropriate secretion of
21.3 Signs and Symptoms ....................................................... 325
insulin in relation to the prevailing blood glucose concentra-
21.4 Diagnosis .......................................................................... 328 tion. The inappropriate insulin secretion leads to severe and
21.5 Diagnostic Flow Chart .................................................... 329 recurrent hypoglycaemia. HI can be either persistent or
21.6 Pathophysiology of CHI.................................................. 330 transient.
21.6.1 Histology ........................................................................... 331
21.7 Treatment ......................................................................... 331
21.7.1 Immediate Management .................................................... 331 21.1.1 Transient Hyperinsulinism
21.8 Surgical Management of CHI ........................................ 333
21.8.1 Medical Management of Diazoxide The transient form of HI is associated with maternal diabetes
Unresponsive Diffuse CHI ................................................ 333 mellitus (gestational or insulin dependent), intrauterine
References ...................................................................................... 334 growth retardation, perinatal asphyxia and erythroblastosis
fetalis, after the maternal administration of some drugs such
as sulphonylureas and after intravenous maternal glucose
infusions during labour. The transient form may also be
“idiopathic” with no risk factor for hyperinsulinism (Yap
et al. 2004). Some patients with intrauterine growth retarda-
tion and asphyxia have a protracted form of HI which
resolves over several months and may require treatment with
diazoxide (Hoe et al. 2006). The mechanism(s) causing tran-
sient HI in these conditions is not clear.
21.1.2 Congenital Hyperinsulinism
K. Hussain (*) Congenital hyperinsulinism (CHI) is the most common

Department of Endocrinology, Great Ormond Street, Children’s cause of severe persistent hypoglycaemia in neonates and
Hospital, London, WC1N 1EH, UK infants during their first year of life. It has previously mas-
e-mail: khalid.hussain@ucl.ac.uk
queraded under a variety of different descriptive names
P. De Lonlay including “idiopathic hypoglycaemia of infancy”, leucine-
Departments of Pediatrics, Hôpital Necker Enfants Malades,
Université Paris-Descartes, Faculté de Médecine,
sensitive hypoglycaemia, neonatal insulinoma, microadeno-
Paris Descarte, 75743 Paris, France matosis, focal hyperplasia, nesidioblastosis and persistent
e-mail: pascale.delonlay@nck.aphp.fr hyperinsulinaemic hypoglycaemia of infancy (PHHI).
324 K. Hussain and P. De Lonlay
The hyperinsulinism causes hypoglycaemia primarily as 2009a). The most common syndrome associated with HI is
a result of increased utilisation of glucose together with a Beckwith-Wiedemann syndrome (BWS). This syndrome is
decreased rate of endogenous glucose production. These characterised by prenatal and/or postnatal overgrowth, mac-
effects are entirely due to inappropriate secretion of insulin. roglossia, anterior abdominal wall defects, organomegaly,
Both sporadic and familial variants of CHI are recognised, hemihypertrophy, ear lobe creases, helical pits and renal
with sporadic forms being relatively uncommon (incidence tract abnormalities. HI is observed in about 50 % of patients
1/40,000 live births) and familial forms being common in with BWS, and in the vast majority of patients with BWS,
communities with high rates of consanguinity; in these com- the HI is usually transient and resolves spontaneously in a
munities the incidence may be as high as 1 in 2,500 live few days (Munns and Batch 2001). However, a small num-
births (Bruining 1990; Mathew et al. 1988). ber of patients (5 % of cases) have persistent HI requiring
Clinical presentation HI most commonly presents in the medical therapy or even subtotal pancreatectomy (Munns
newborn, but it can also present during infancy and child- and Batch 2001).
hood. The clinical presentation of hypoglycaemia is most Some types of hyperinsulinism are elicited only after
severe in the newborn and may be quite subtle in the infancy provocation testing. For example, in patients who have the
and childhood periods. The hypoglycaemia is usually refrac- hyperinsulinism and hyperammonaemia syndrome (who
tory to oral feeds, and normoglycaemia can only be main- also have fasting hypoglycaemia), protein/leucine loading
tained by giving large volumes of concentrated dextrose precipitates hypoglycaemia (Hsu et al. 2001). Similarly in
infusions (Aynsley-Green et al. 2000). Hypoglycaemic those patients with exercise-induced hyperinsulinism, exer-
symptoms may vary from being non-specific (such as poor cise provocation testing will cause hypoglycaemia after the
feeding, lethargy and irritability) to being severe (such as exercise test (Meissner et al. 2005).
apnoea, seizures or coma). As a result of the fetal hyperinsu- An insulinoma is a rare cause of hyperinsulinism and
linaemia, newborns with CHI are typically macrosomic; must be considered in older children or adolescents pre-
however, the absence of macrosomia does not exclude CHI. senting with HI. Insulinomas may be a part of multiple
Fetal hyperinsulinaemia also accounts for the hypertrophic endocrine neoplasia syndrome type 1 (MEN1), and hence a
cardiomyopathy and hepatomegaly (increased storage of family history may provide a diagnostic clue in the familial
glucose as glycogen) that is commonly observed in patients cases (Grant 2005). Munchausen by proxy can present as
with CHI. factitious HI due to administration of insulin or antidiabetic
Most CHI patients present with isolated hypoglycae- drugs such as sulphonylureas. In some cases, this has led to
mia. However, a large number of developmental syndromes misdiagnosis and consequent pancreatectomy (Giurgea
may present in the newborn period with HI (Kapoor et al. et al. 2005).
21 Hyperinsulinism 325
21.2 Nomenclature

No. Disorder Alternative name Abbreviation symbol localisation Affected protein no. Subtype
21.1 Hyperinsulinism of Persistent HHF1 ABCC8 11p15.1 ATP-binding cassette, 256450 Diffuse and
infancy hyperinsulinaemic subfamily C, (member focal form
hypoglycaemia of 8 sulfonylurea receptor)
infancy (PHHI)
21.2 Hyperinsulinism of Persistent HHF2 KCNJ11 11p15.1 Kir6.2 subunit of the 601820 Diffuse and
infancy 2 hyperinsulinaemic inwardly rectifying focal form
hypoglycaemia of potassium channel
infancy (PHHI)
21.3 Hyperinsulinism- Hyperinsulinaemic GLUD1 GLUD1 10q23.3 Glutamate 606762 All forms
hyperammonaemia hypoglycaemia-6 dehydrogenase-1
syndrome
21.4 Hyperinsulinaemic HHF3 GCK 7p15-p13 Glucokinase 602485 All forms
hypoglycaemia-3 (hexokinase-4)
21.5 Hyperinsulinaemic Short-chain l-3- HHF4 HADH 4q22-q26 Short-chain l-3- 609975 All forms
hypoglycaemia-4 hydroxyacyl-CoA hydroxyacyl-CoA
deficiency (see also
17.3.3)
21.6 Hepatocyte nuclear HNF4A HNF4 a 20q12-q13.1 Hepatocyte nuclear 600281 All forms
factor 4A loss-of- factor 4A
function mutations
21.7 Hyperinsulinaemic Monocarboxylate HHF7 SLC16A1 1p12 Monocarboxylate 610021 All forms
hypoglycaemia-7, transporter transporter 1
exercise induced gain-of-function
21.8 Uncoupling protein UCP2 UCP2 11q13 Uncoupling protein 2 601693 All forms
2 deficiency
21.9 HNF1A deficiency HNF1A HNF1A 12q24.31 HNF1A 142410 All forms
21.10 Hyperinsulinaemic HHF5 INSR 19p13.2 Insulin receptor 609968 Autosomal
hypoglycaemia-5 dominant
Table 21.1 Hyperinsulinism of infancy

CNS Convulsions ++ ++ + +
Endocrine Hyperinsulinism ++ ++ ++ +
Other Macrosomia ±
Routine Free fatty acids (S) ↓↓↓ ↓↓↓ ↓↓ ↓↓
laboratory Glucose (P) ↓ ↓ ↓ ↓
Hypoglycaemia, hypoketotic +++ +++ ++ +
Ketones during hypoglycaemia ↓↓↓ ↓↓↓ ↓↓ ↓
Table 21.2 Hyperinsulinism of infancy 2

CNS Convulsions ++ ++ + +
Endocrine Hyperinsulinism ++ ++ ++ +
Other Macrosomia ±
Routine laboratory Free fatty acids (S) ↓↓↓ ↓↓↓ ↓↓ ↓↓
Glucose (P) ↓ ↓ ↓ ↓
Hypoglycaemia, hypoketotic +++ +++ ++ +
Ketones during hypoglycaemia ↓↓↓ ↓↓↓ ↓↓ ↓
Table 21.3 Hyperinsulinism-hyperammonaemia syndrome

CNS Convulsions ± ± ±
Epilepsy, generalised + ++ ++
Endocrine Hyperinsulinism + ++ ++
Leucine sensitivity ++ ++ ++ ++ ++
Routine laboratory EEG: abnormal + + +
Free fatty acids (S) ↓ ↓ ↓ ↓
Glucose (P) ↓ ↓ ↓ ↓ ↓
Hypoglycaemia ++ ++ ++ +
Hypoketotic hypoglycaemia ++ ++ ++ +
Ketones (U,P) ↓ ↓ ↓ ↓
Special laboratory Alpha-ketoglutarate (U) ± ± ± ± ±
Ammonia, fasting (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 21.4 Hyperinsulinaemic hypoglycaemia-3

CNS Epilepsy ± ± ± ±
Endocrine Hyperinsulinism ++ ++ ++ ++
MODY + + ++
Routine laboratory Free fatty acids (S) ↓ ↓ ↓ ↓ ↓
Glucose (P) ↓ ↓ ↓ ↓ ↓
Hypoglycaemia ++ ++ ++ ++
Hypoketotic hypoglycaemia ++ ++ ++ ++
Ketones during hypoglycaemia ↓ ↓ ↓ ↓

CNS Mental retardation n + + +
Seizures + +
Digestive Liver failure, Reye-like + +
Endocrine Hyperinsulinism + +
Protein sensitivity + ++ ++ ++
Musculoskeletal Microcephaly +
Hypoketotic hypoglycaemia + +
Special laboratory 3-Hydroxydicarboxylic acid (U) (↑) (↑) (↑)
3-Hydroxyglutarate (U) ↑ ↑ ↑
C4-OH hydroxybutyrylcarnitine (P, DB) ↑ ↑ ↑
Medium-chain dicarboxylic acids (U) (↑) (↑) (↑)
Table 21.6 Hepatocyte nuclear factor 4A loss-of-function mutations

Endocrine Diabetes mellitus type 2 + ++ +++
Hyperinsulinism + + +
Other Macrosomia +++
Routine laboratory Free fatty acids (S) ↓ ↓ ↓
Hypoglycaemia + + +
Hypoketotic hypoglycaemia + + +
Ketones during hypoglycaemia ↓ ↓ ↓
Table 21.7 Hyperinsulinaemic hypoglycaemia-7, exercise induced

Endocrine Hyperinsulinism ++ +++
Routine laboratory Free fatty acids (S) ↓ ↓↓
Glucose (P) ↓ ↓↓
Hypoglycaemia (exercise induced) ++ ++
Ketones (U,P) ↓ ↓
Ketones during hypoglycaemia ↓ ↓↓
Special laboratory Pyruvate test +++ +++
Table 21.8 Uncoupling protein 2 deficiency

Endocrine Hyperinsulinism ↓ ↓ ↓
Routine laboratory Free fatty acids (S) ↓ ↓ ↓
Hypoketotic hypoglycaemia + + +
Ketones during hypoglycaemia ↓ ↓ ↓
Table 21.9 HNF1A deficiency

MODY + ++ +++
Routine laboratory Free fatty acids (S) ↓ ↓
Glucose (P) ↓ ↓
Ketones during hypoglycaemia ↓ ↓

CNS Seizures + + +
Dermatological Hyperpigmentation ±
Routine laboratory Free fatty acids (S) ↓ ↓
Glucose (P) ↓ ↓
Ketones during ↓ ↓
hypoglycaemia
21.4 Diagnosis HI will require a formal exercise test and/or a pyruvate

load to demonstrate post-exercise-induced hypoglycaemia
The early diagnosis of HI is fundamentally important for (Meissner et al. 2005; Otonkoski et al. 2003). Patients with
preventing hypoglycaemic brain injury; hence clinicians HI/HA syndrome will have hypoglycaemia after having a
should have a low threshold for recognising patients with HI. load with protein/leucine, but they usually present high lev-
Any patient with recurrent or persistent hypoglycaemia can els of ammonia leading to the diagnosis (Hsu et al. 2001;
potentially have hyperinsulinism. HI is the only cause of Stanley et al. 1998).
hypoglycaemia which persists despite continuous adminis- Some cases of HI may be subtle and these will require
tration of glucose. Thus, a powerful clue to the dysregulated more detailed evaluation. In some patients a positive glycae-
insulin secretion is the calculation of the intravenous glucose mic response (rise in the blood glucose concentration of
infusion rate required to maintain normoglycaemia. An >1.5 mmol/l) following an intramuscular/intravenous injec-
intravenous glucose infusion rate of >8 mg/kg/min (normal tion of glucagon at the time of hypoglycaemia provides sup-
is 4–6 mg/kg/min) is virtually diagnostic of hyperinsulinism portive evidence (Finegold et al. 1980). A glycaemic
(Aynsley-Green et al. 2000). In milder forms of HI, it will be response to a subcutaneous dose of octreotide may also aid
important to establish the duration of fasting and whether the diagnosis along with decreased serum levels of insulin
hypoglycaemia is precipitated by meals (protein sensitivity) growth factor-binding protein-1 (IGFBP-1) as insulin sup-
or by exercise (Hsu et al. 2001; Meissner et al. 2005). presses the transcription of the IGFBP-1 gene (Levitt Katz
Typically newborns with HI have markedly reduced fasting et al. 1997).
tolerance (less than 1 h).
In HI there is an inappropriate concentration of serum Diagnostic biochemical features of hyperinsulinaemic
insulin (and/or c-peptide) for the level of blood glucose hypoglycaemia
Glucose infusion rate >8 mg/kg/min
(spontaneous or provoked). The metabolic effect of this
Laboratory blood glucose <3 mmol/l with:
inappropriate insulin secretion is reflected by the inappropri-
Detectable serum insulin/C-peptide
ately low levels of serum ketone bodies and fatty acids dur-
Suppressed/low serum ketone bodies
ing the hypoglycaemic episode (Aynsley-Green et al. 2000).
Suppressed/low serum fatty acids
There is no correlation between the serum insulin concentra- Blood ammonia level and urine alpha-ketoglutarate may be
tion and the severity of the hypoglycaemia. In some difficult increased [HI/HA syndrome (21.3)]
cases the diagnosis of HI should not be based on an isolated Leucine-sensitive hypoglycaemia [HI/HA syndrome (21.3)]
serum insulin concentration but on the clinical presentation Raised plasma hydroxybutyrylcarnitine and urinary
and the biochemical profile of insulin action (low beta- 3-hydroxyglutarate [HADH deficiency (21.5)]
hydroxybutyrate and fatty acid concentrations). An elevated Exercise-induced hyperinsulinaemic hypoglycaemia [SLC16A1
mutation (21.7)]
serum ammonia concentration in a patient with HH is sug-
Supportive evidence (when diagnosis is in doubt)
gestive of the hyperinsulinism and hyperammonaemia (HI/
Positive glycaemic (>1.5 mmol/l) response to intramuscular/
HA) syndrome (Stanley et al. 1998). Raised plasma hydroxy- intravenous glucagon
butyrylcarnitine and urinary 3-hydroxyglutarate are diagnos- Positive glycaemic response to a subcutaneous/intravenous
tic of a rare type of HI (hydroxyacyl-coenzyme A dose of octreotide
dehydrogenase (HADH) deficiency) (Clayton et al. 2001). Low levels of serum IGFBP1
The diagnosis of some forms of HI will require provo- Suppressed branched-chain amino acids (leucine, isoleucine
cation testing. For example, patients with exercise-induced and valine)
Initial stabilisation of glucose level (i.v. glucose / glucagon)
Assess response to diazoxide
Diazoxide unresponsive
Diazoxide responsive II paternal ABCC8/KCNJ11
mutation found
Rapid mutational analysis of ABCC8 and KCNJ11 genes

Assess fasting tolerance and discharge
1. Paternal ABCC8/KCNJ11gene mutations Genetically confirmed diffuse disease

(highly indicative of focal lesion) (Homozygous/ compound heterozygous
Specific mutational 2. No mutations identified (less likely focal lesion) for ABCC8/ KCNJ11 mutations)
analysis in persisting
hyperinsulinism
18FDOPA-PET/CT High calorie diet/frequent feeds

Octreotide therapy
Near total pancreatectomy
Focal Disease Diffuse Disease
Surgical resection of focal lesion

(laparoscopic if possible potentially curing
patient) Follow up:
Growth and Development
Neurological
Genetic counseling
Post near total pancreatectomy:
Diabetes mellitus management
Assess pancreatic exocrine function
Fig. 21.1
21.6 Pathophysiology of CHI secretion. Activating mutations cause an increased affinity of

glucokinase for glucose with increased rates of glycolysis at
The pancreatic β-cell adenosine triphosphate-sensitive low blood glucose concentrations (Glaser et al. 1998). This
potassium channels (KATP channels) play a pivotal role in increases insulin secretion independent of blood glucose con-
glucose-stimulated insulin secretion. These channels are centration. The age of onset and severity of symptoms can
hetero-octameric complexes comprising of four inwardly vary markedly within families, from mild hypoglycaemia sen-
rectifying potassium channel (Kir6.2) subunits and four of sitive to medical treatment to severe hypoglycaemia requiring
the sulphonylurea receptor 1 (SUR1) subunits (Clement subtotal pancreatectomy (Cuesta-Muñoz et al. 2004).
et al. 1997). The channels couple glucose metabolism to 21.5 Loss-of-Function Mutations in the HADH Gene
membrane electrical activity and insulin release in pancreatic Recessively inherited mutations in the HADH gene
β-cells (Ashcroft et al. 1984). Glucose metabolism leads to (601609) which encodes the enzyme hydroxyacyl-coenzyme
an increase in the intracellular ratio of ATP/ADP within the A dehydrogenase (HADH) (previously known as short-chain
β-cell causing closure of the channels; this results in cell l-3-hydroxyacyl-CoA dehydrogenase (SCHAD)) have been
membrane depolarisation, Ca2+ influx via voltage-gated cal- described in a few patients with CHI (Clayton et al. 2001;
cium channels and insulin exocytosis. Molven et al. 2004). HADH catalyses the penultimate step in
21.1–21.2 Mutations in ABCC8/KCNJ11 Genes fatty acid β-oxidation in the mitochondria. Patients typically
The SUR1 and Kir6.2 proteins are encoded by the ABCC8 present with raised plasma hydroxybutyrylcarnitine and uri-
(600509) and KCNJ11 (600937) genes, respectively. The nary 3-hydroxyglutarate levels. Protein sensitivity has been
most common known cause of CHI is loss-of-function muta- demonstrated in patients with HADH mutations, and this has
tions in these two genes (Thomas et al. 1995, 1996). These been confirmed in the HADH knockout mouse (Kapoor et al.
mutations either impair the ability of MgADP to stimulate 2009b; Li et al. 2010). However, the precise mechanism of
channel activity or affect the expression of the KATP channels dysregulated insulin secretion in patients with a HADH defi-
at the surface membrane with continuous depolarisation of ciency is not understood.
the β-cell membrane and dysregulated insulin secretion 21.6 Mutations in HNF4A Gene
(Kane et al. 1996). The majority of KATP channel mutations Heterozygous loss-of-function mutations in the hepato-
have been known to act recessively as diffuse form. cyte nuclear factor 4A (600281, HNF4A) gene can cause
Focal disease results from paternal uniparental disomy transient or persistent CHI (Pearson et al. 2007; Kapoor et al.
(UPD) encompassing chromosome 11p15.5-11p15.1 2008). These HNF4A mutations are also associated with
within a single pancreatic β-cell which unmasks a pater- increased birth weight and macrosomia and some patients
nally inherited KATP channel mutation at 11p15.1 (Hojlund have neonatal HI. The severity of hypoglycaemic episodes
et al. 2004). Autosomal dominantly inherited mutations in varies from diet-controlled neonatal hypoglycaemia to per-
ABCC8/KCNJ11 can lead to mild or severe forms of CHI sistent hyperinsulinism requiring diazoxide treatment for a
(Pinney et al. 2008; Flanagan et al. 2011). number of years. The mechanism by which HNF4A muta-
21.3 Activating Mutations in the GLUD1 Gene tions cause CHI is not known.
Heterozygous mutations in the GLUD1 gene (138130), 21.7 Mutations in the SLC16A1 Gene
which encodes the enzyme glutamate dehydrogenase (GDH), Heterozygous gain-of-function mutations in the SLC16A1
have been identified in patients with HI/HA syndrome with gene (610021) which encodes the monocarboxylate trans-
plasma ammonium levels being persistently raised to three to porter (MCT-1) causing physical exercise-induced hyperin-
eight times the upper limit of normal (Stanley 2004). HI/HA sulinism (EIHI). Patients with EIHI resulting from a
syndrome typically causes protein-sensitive hypoglycaemia, in SLC16A1 mutation have increased expression of the pyru-
addition to the fasting hypoglycaemia (Hsu et al. 2001). GDH is vate and lactate transporter, MCT-1, within the pancreatic
an intra-mitochondrial enzyme involved in protein (leucine)- β-cell (Otonkoski et al. 2007). Usually, reduced expression
mediated insulin release. In the β-cell leucine stimulates the of MCT-1 renders the β-cell unresponsive to acute changes
release of insulin by allosterically activating GDH which results in the extracellular concentrations of lactate or pyruvate.
in increased oxidation of glutamate. GLUD1 mutations that Activating mutation in the promoter region of SLC16A1
impair inhibitory control of the enzyme by its allosteric inhibitor induces increased expression of MCT1 in the β-cell (where
GTP result in increased enzyme activity and ultimately increased this gene is not usually transcribed) allowing pyruvate uptake
insulin secretion. The mechanism by which GLUD1 mutations and pyruvate-stimulated insulin release despite ensuing
cause hyperammonaemia remains to be determined. hypoglycaemia (Otonkoski et al. 2007). Patients with an
21.4 Activating Mutations in the GCK Gene SLC16A1 mutation usually present with symptoms of severe
Heterozygous activating mutations in the glucoki- hypoglycaemia following strenuous exercise.
nase gene (138079, GCK) lead to CHI. Glucokinase is the 21.8 Mutations in the UCP2 Gene
rate-limiting enzyme for glucose metabolism in the β-cell The mitochondrial uncoupling protein 2 (UCP2) is a nega-
and hence is pivotal in regulating glucose-induced insulin tive regulator of insulin secretion, by decreasing ATP/ADP
ratio in β-cells and or modulating reactive oxygen species pro- that a higher threshold of blood glucose concentration is used
duction. A few patients have been described with mild HI due to intervene and blood glucose concentrations are maintained
to loss-of-function mutations in the UCP2 gene (González- within the normal range (3.5–6 mmol/l) (Aynsley-Green et al.
Barroso et al. 2008). 2000). This often requires the insertion of a central venous
21.9 Mutations in the HNF1A Gene catheter to deliver concentrated solutions of glucose intrave-
Inactivating mutations in hepatocyte nuclear factor 1A nously. A combination of oral feeds with a glucose polymer
(142410, HNF1A) causes the monogenic form of maturity- (such as Maxijul or Polycal) and intravenous fluids can be
onset diabetes of youth (MODY)-3. Two patients with hyper- used to provide the carbohydrates.
insulinaemic hypoglycaemia in infancy which improved In an emergency situation where venous access is difficult
with age and carrying missense mutations of HNF1A have to obtain, intramuscular glucagon (0.5–1 mg) can be admin-
been recently described (Stanescu et al. 2012). istered in order to temporarily improve blood glucose con-
21.10 Mutations in the Insulin Receptor Gene centrations (Aynsley-Green et al. 2000). Glucagon causes
Heterozygosity for a point mutation in the insulin receptor immediate release of glycogen stores from the liver and also
gene (147670) has been reported in all affected members of has actions on gluconeogenesis, ketogenesis and lipolysis.
a third-generation Danish family with autosomal dominant However, glucagon in high doses causes paradoxical insulin
hyperinsulinaemic hypoglycaemia (Hojlund et al. 2004). secretion, so patients receiving a glucagon bolus should have
intravenous glucose infusion to prevent rebound hypogly-
caemia. It can also be administered (alone or in combination
21.6.1 Histology with octreotide) as an intravenous or subcutaneous infusion
to stabilise blood glucose concentrations in the acute man-
Two main histological subtypes have been described in agement of infants with HI (Aynsley-Green et al. 2000).
patients with CHI (Rahier et al. 2000). Focal pancreatic
lesions appear as small regions of islet adenomatosis mea- 21.7.1.1 Medical Therapy
suring 2–10 mm which are characterised by β-cells with Diazoxide
enlarged nuclei surrounded by normal tissue. In contrast dif- Mechanism of action: Diazoxide is a ligand of the KATP
fuse pancreatic disease affects all the β-cells within the islets which will activate intact KATP channels reversing glucose-
of Langerhans. The histological form of CHI can be a guide induced channel closure.
as to the mode of inheritance. Diffuse disease can be familial Pharmacodynamics/kinetics: Diazoxide is structurally
or sporadic and can result from recessively inherited or dom- related to the thiazide diuretics but has an antidiuretic action
inantly acting mutations in the genes previously described, producing fluid retention. It is readily absorbed from the gas-
while focal disease is always sporadic. trointestinal tract and more than 95 % of the drug is bound to
Focal disease results from paternal uniparental disomy albumin (Pruitt et al. 1973). Diazoxide is partially metabo-
(UPD) encompassing chromosome 11p15.5-11p15.1 within lised by oxidation and sulphate conjugation and is excreted
a single pancreatic β-cell which unmasks a paternally inher- by glomerular filtration as unchanged drug and metabolites.
ited KATP channel mutation at 11p15.1 (Damaj et al. 2008). In adults the plasma half-life of diazoxide is estimated to be
Paternal UPD at 11p15.5 causes altered expression of a num- about 20–45 h, whereas in children the half-life is thought to
ber of imprinted genes, including the maternally expressed be considerably shorter, 9.5–20 h (Pruitt et al. 1973), but
tumour suppressor genes H19 and CDKN1C, and the pater- there is no data on neonates. The concentration of diazoxide
nally expressed growth factor IGF2, likely leading to clonal required in the blood for the hyperglycaemic action in neo-
expansion of the single cell and dysregulated insulin secre- nates and children is not known, although in adults a peak
tion from the resulting focal lesion (De Lonlay et al. 1997). blood level of 16 μg/ml can cause hyperglycaemia within 4 h
A few patients have been reported to have “atypical diffuse after oral administration of a single dose of 10 mg/kg/day.
histology” (Hussain et al. 2008). Dosage: 5–20 mg/kg/day orally in two to three divided
doses.
Adverse effects: Fluid retention and hypertrichosis are
21.7 Treatment common side effects. The fluid retention is mostly observed
in the neonatal period and may cause cardiac failure, hence
21.7.1 Immediate Management the concurrent use of a thiazide diuretic to prevent fluid
retention. Hypertrichosis (excess hair growth which may
The early diagnosis and immediate meticulous management involve vellus hair and/or pigmented terminal hair espe-
are the cornerstones for preventing brain injury in patients with cially on the eyebrows, eyelashes, back and arms) is a
HI. Once the diagnosis is established, the priority is to main- major cosmetic side effect. This is reversible and disap-
tain normoglycaemia (3.5–6 mmol/l). Given the biochemical pears when the diazoxide is stopped. Other side effects
basis (hypoketotic) of the hypoglycaemia, it is recommended include increased uric acid levels, ketoacidosis and
hyperosmolar coma, neutropenia, eosinophilia, thrombocy- nausea is not mediated by effects on the brain but due to the
topenia and allergic reactions. delay in gastric emptying caused by glucagon as shown in
Interactions with other drugs: Significant interactions of adults. There has been one case report of a 35-week gestation
diazoxide have been reported with diuretics, phenytoin, war- infant who developed severe hyponatraemia and thrombocy-
farin, chlorpromazine and aspirin (Petro et al. 1976; Sellers topenia after continuous infusion of glucagon for the treat-
and Koch-Weser 1970; Aynsley-Green and Illig 1975; ment of intractable hypoglycaemia (Belik et al. 2001).
Newman and Brodows 1983). The interactions with phenyt- Another possible side effect of glucagon therapy may be ery-
oin, warfarin and chlorpromazine involve either displace- thema necrolyticum migrans, which was reported in two
ment from albumin-binding sites or increase/decrease neonates with persistent hyperinsulinaemic hypoglycaemia
metabolism by induction of liver enzymes. The mechanism (Wald et al. 2002).
of hyperglycaemia due to thiazide diuretics such as chloro- Intramuscular glucagon injection is the treatment of
thiazide involves activation of potassium channels in the choice in situations where intravenous access is not accessi-
β-cell membrane and calcium influx (Sandström 1993), but ble in patients with hyperinsulinaemic hypoglycaemia (1 mg
the mechanism is unclear in the case of loop diuretics such as dose). Higher doses of glucagon (>20 μg/kg/h) can cause
furosemide. insulin secretion, which leads to worsening of the hypogly-
caemia in patients with hyperinsulinism (Kawai et al. 1995).
Nifedipine A few patients with CHI have been managed on long-term
Mechanism of action: Calcium channel antagonist. therapy with glucagon (Mohnike et al. 2008).
Dose: 0.25–2.5 mg/kg/day divided into three doses.
Side effects: No major side effects have been reported Octreotide
when nifedipine has been used in patients with hyperinsu- Background: Octreotide is the acetate salt of a cyclic octa-
linaemic hypoglycaemia. Overall the response to nifedip- peptide. It is a long-acting octapeptide with pharmacologic
ine has been disappointing. Children who have properties mimicking those of the natural hormone
hyperinsulinism as a result of an abnormality in the two somatostatin.
components in the KATP channel usually fail to respond to Indication: The short- and long-term management of
nifedipine. Despite this, there have been several reports of hyperinsulinaemic hypoglycaemia.
nifedipine-responsive forms of HI (Baş et al. 1999; Shanbag Mechanism of action: Octreotide is one of the many octa-
et al. 2002). peptide and hexapeptide somatostatin analogues that, unlike
somatostatin, show a high degree of affinity for somatosta-
Glucagon tin receptors (sstr) 2 and 3 and little or no binding to sstr1
Mechanism of action: Glucagon activates adenylate cyclase (Patel 1999). Somatostatin and its analogues can inhibit
via the G-protein-coupled receptor (Gs). Activated adenylate insulin secretion by activation of sstr 5, which is mediated
cyclase phosphorylates cAMP-dependent protein kinase by stimulation of the Gi/Go protein (Ribalet and Eddlestone
PKA that triggers a cascade of events. These include activa- 1995). Subcutaneous or intravenous octreotide inhibits first-
tion of phosphorylase kinase (release of glucose from stored phase insulin secretion and attenuates insulin responses to
glycogen), deactivation of pyruvate kinase effectively creat- activated Gs-protein-coupled receptors (such as the
ing an excess of phosphoenolpyruvate (gluconeogenesis) glucagon-like peptide-1R). In pancreatic β-cells, activation
and the transcription of phosphoenolpyruvate carboxykinase of sstr 5 inhibits calcium mobilisation and acetylcholine
(PEPCK) which catalyses the reaction from oxaloacetate to activity and decreases insulin gene promoter activity, result-
phosphoenolpyruvate (gluconeogenesis). In summary gluca- ing in reduced insulin biosynthesis. Somatostatin also
gon stimulates glycogenolysis, gluconeogenesis, lipolysis, exhibits an effect on insulin secretion distal from the inhibi-
protein degradation, amino acid catabolism and ketogenesis. tion of Ca2+ mobilisation and adenylate cyclase inhibition
Onset of action of glucagon is within 10–15 min. (Renstrom et al. 1996). It has been suggested that the β-cell
Dose: 1–20 μg/kg/h (subcutaneous or intravenous con- sstr is coupled to the KATP channel, but the effect of this is
tinuous infusion). not considered to be relevant physiologically, as somatosta-
Interactions: Glucagon stimulates the synthesis and tin is still capable of reducing insulin secretion in the pres-
release of growth hormone, insulin and pancreatic soma- ence of sulfonylureas.
tostatin. Amino acids, cortisol, infections, stress, adrenergic Pharmacodynamics/kinetics: The activity of octreotide is
stimulators and acetylcholine all stimulate glucagon similar to that of somatostatin. Octreotide, however, has a
secretion. longer half-life; greater selectivity for inhibiting glucagon,
Side effects: A common side effect of glucagon is a brief growth hormone, and insulin release; and a lower incidence
period of nausea and vomiting (Ranganath et al. 1999). The of rebound hypersecretion following discontinuation.
Octreotide is administered by subcutaneous injection and is heterozygous mutations in ABCC8 and KCNJ11). Patients
rapidly absorbed, with peak concentrations of 5.2 ng/ml with a paternal mutation in ABCC8 and KCNJ11 (or those
occurring around 25 min after a 100 μg dose. Distribution with no mutations in these genes) potentially have focal dis-
occurs rapidly, with approximately 65 % of a dose bound to ease and thus will require further imaging studies with
lipoprotein and albumin in a concentration-dependent man- 18F-DOPA-PET scan for precise preoperative localisation
ner. The apparent half-life of octreotide is approximately of the focal lesion (Otonkoski et al. 2006). Patients with
1.7 h, which is significantly greater than somatostatin half- genetically confirmed diffuse disease do not require further
life of 1–3 min. The effects of octreotide are variable but can imaging studies. However, it is important to be aware that
last for up to 12 h. mutational analysis may not be definitive in some cases. The
Dose: 5–35 μg/kg/day. uptake of the positron emitting tracer 18F-DOPA-PET is
Side effects: Local reactions at the site of injection include increased in β-cells with a high rate of insulin synthesis and
pain, sensation of stinging, tingling and burning as well as secretion compared to unaffected areas allowing visualisa-
redness and swelling. Gastrointestinal side effects include tion of the focal lesion. The sensitivity for detecting focal
anorexia, nausea, abdominal pain, bloating, flatulence, loose lesions varies between 88 and 94 % with a specificity of
stools and diarrhoea. Octreotide causes the inhibition of the 100 % (Hardy et al. 2007). Figure gives an overview of the
release of several hormones including growth hormone, management of patients with CHI.
serotonin, gastrin, vasoactive intestinal polypeptide (VIP),
secretin, motilin, pancreatic polypeptide, ACTH and thyroid-
stimulating hormone (TSH). The suppression of GH (includ- 21.8 Surgical Management of CHI
ing insulin-like growth factors) and thyroid hormones may
lead to stunting of growth. Octreotide can decrease gallblad- The focal form of the disease requires a limited pancreatec-
der contractility and bile secretion, leading to steatorrhea, tomy, whereas diffuse disease will require a near-total pan-
cholestasis, hepatic dysfunction and cholelithiasis. Blood createctomy (Fékété et al. 2004). The operation is
flow to the splanchnic circulation is decreased by octreotide; traditionally carried out with the open approach and is asso-
hence it must be used cautiously in babies at risk of necrotis- ciated with peri- and post-operative complications. The use
ing enterocolitis. Resistance to octreotide therapy can occur of laparoscopy represents a new approach to the diagnosis
even at high doses. and management of infants with CHI (Bax and van der Zee
2007). Near-total pancreatectomy is associated with a high
Long-Acting Octreotide incidence of diabetes mellitus and pancreatic exocrine insuf-
A monthly single dose of intramuscular injection of long- ficiency and hence reserved for those severe cases where all
acting SST analogues, either lanreotide acetate or long- medical therapy has failed.
acting release (LAR) octreotide, may in the future replace
the daily three or four injections of subcutaneous octreotide
injections. Two patients with CHI have been described with 21.8.1 Medical Management of Diazoxide
once-monthly injection of lanreotide acetate where the CHI Unresponsive Diffuse CHI
was well controlled (Modan-Moses et al. 2011).
Some infants with confirmed diffuse disease (genetically/by
Diazoxide Unresponsive CHI 18F-DOPA-PET scanning) who fail to respond to diazoxide
The management of patients who are unresponsive to may be managed with long-term subcutaneous octreotide
first-line treatment with diazoxide needs further investiga- injections in combination with frequent feeding (Glaser et al.
tions. In patients that are unresponsive to diazoxide, it is 1989). It is used in the short- and long-term management of
essential to differentiate focal from diffuse disease as the some patients with CHI. In the short term (with and without
surgical approaches are radically different (Fékété et al. glucagon), it is used to stabilise patients pending further
2004). The precise preoperative localisation and limited investigations. Octreotide has been successively used in the
surgical removal of the focal domain “cure” the patient. In long-term management of some CHI patients in combination
contrast, patients with diffuse disease may require a near- with frequent feeding (Glaser et al. 1989). The principle of
total pancreatectomy which will have life-long implica- this treatment is based on the fact that the hypoglycaemia in
tions (high risk of diabetes mellitus, pancreatic exocrine some patients gradually gets milder over time. A gastros-
insufficiency). tomy is recommend in some patients as this will allow the
Rapid genetic analysis for mutations in ABCC8 and delivery of bolus and continuous overnight feeds. A long-
KCNJ11 allows identification of the majority of patients acting octreotide formulation has now been described in two
with diffuse disease (homozygous or compound patients (Modan-Moses et al. 2011).
Summary of the dietary, medical and surgical management of different forms of HI

Type of HI Diet Medical Surgical
21.1–21.2 High calorie D/C/N/G/O Near-total pancreatectomy
Diffuse form (if no response to medical therapy)
21.1–21.2 High calorie D/C/N/G/O Limited pancreatectomy
Focal form
21.3 Protein restriction/high calorie D No surgery
21.4–21.5 High calorie D No surgery
21.6–21.8
21.9–21.10 N/A D/avoidance of exercise No surgery
21.7
Key: D diazoxide, C chlorothiazide, N nifedipine, G glucagon, O octreotide
Summary of medical therapy for HI

Medication Route of administration Dose Mechanism of action Side effects
Diazoxide Oral 5–20 mg/kg/day divided Agonist of the KATP channel Fluid retention, hypertrichosis,
into two to three doses hyperuricaemia, eosinophilia,
leukopenia, rarely hypotension
Chlorothiazide (used in Oral 7–10 mg/kg/day divided Activation of the KATP Hyponatraemia, hypokalaemia
conjunction with diazoxide) into two doses channel
Nifedipine Oral 0.25–2.5 mg/kg/day Calcium channel blocker Hypotension
divided into three doses
Glucagon SC/IV continuous 1–20 μg/kg/h Increases glycogenolysis Nausea, vomiting, paradoxical
infusion or IM injection and gluconeogenesis insulin secretion, skin rashes
Octreotide SC/IV continuous 5–35 μg/kg/day Multiple actions in the β-cell Suppression of GH, TSH,
infusion or 6–8 hourly (see text) ACTH, glucagon, diarrhoea,
SC injections steatorrhoea, cholelithiasis,
abdominal distension, growth
suppression, tolerance
Key: SC subcutaneous, IV intravenous, IM intramuscular, GH growth hormone, TSH thyroid-stimulating hormone, ACTH adrenocorticotropic
hormone
Clement JP 4th, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten

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Mitochondrial Oxidative
Phosphorylation Disorders 22
Paul de Laat, Richard Rodenburg, and Jan Smeitink
Contents Summary
22.1 Introduction ................................................................... 338 Mitochondrial disorders belong to the most frequently
22.1.1 Physiology of the Oxidative Phosphorylation................. 338 encountered inborn errors of metabolism. Most affected
22.1.2 Mitochondrial Function .................................................. 338 mitochondrial disease patients harbor a defect in the oxi-
22.1.3 Mitochondrial DNA ........................................................ 338 dative phosphorylation system (OXPHOS) of which the
22.1.4 Mitochondrial Dysfunction ............................................. 338
22.1.5 Clinical Signs and Symptoms ......................................... 339 incidence is estimated to be over 1:5,000 live births.
22.1.6 Genotypic Conformation of Mitochondrial OXPHOS is the final step in the aerobe production of
Disorders ......................................................................... 339 adenosine triphosphate (ATP). Defects in OXPHOS can
22.2 Nomenclature ................................................................ 340 be caused by a mutation in either mitochondrial DNA
(mtDNA) or nuclear DNA (nDNA). Maternal inheritance,
22.3 Metabolic Pathway........................................................ 343
heteroplasmy, and mitotic segregation are characteristics
22.4 Signs and Symptoms ..................................................... 344 that are unique for mtDNA; understanding of these charac-
22.5 Reference Values ........................................................... 355 teristics is of the utmost importance to understand disorders
22.6 Pathologic Values .......................................................... 355 caused by mtDNA mutations.
Most patients with mitochondrial disorders present with a
22.7 Diagnostic Flow Chart .................................................. 356
multisystem disorder. The organs requiring the most energy,
22.8 Specimen Collection ...................................................... 357 such as the brain, retina, heart, kidney, and skeletal muscle,
22.9 Prenatal Diagnosis......................................................... 358 are most commonly and severely affected. Onset of disease
can be at any age and the symptoms are almost always pro-
22.10 Treatment ....................................................................... 358
22.10.1 Coenzyme Q10................................................................ 358 gressive. A definitive diagnosis solely based on signs and
22.10.2 Multiorgan Treatment ..................................................... 358 symptoms is very unlikely. Lactate, pyruvate and alanine in
22.10.3 Supportive Care............................................................... 358 blood and cerebrospinal fluid (CSF), imaging studies of the
22.10.4 Preventive Care ............................................................... 358
brain and heart, and histological and biochemical analysis of
22.10.5 Emergency Treatment ..................................................... 359
22.10.6 Standard Treatment ......................................................... 359 muscle are used for diagnosis of mitochondrial disorders.
22.10.7 Experimental Treatment .................................................. 359 Elevated concentration of FGF-21 might give additive sup-
Further Reading ............................................................................ 359 port in the decision-making process towards performing a
muscle biopsy.
Except for isolated coenzyme Q10 deficiency, no
cure is available for mitochondrial disease. Managing of
these patients is restricted to preventing complications,
minimizing disability, and providing genetic counsel-
ing. For diagnosis and treatment of their disease, patients
P. de Laat • R. Rodenburg • J. Smeitink (*)
with a mitochondrial dysfunction should be referred to a
Nijmegen Centre for Mitochondrial Disorders, specialized center.
Radboud University Medical Centre,
Geert Grooteplein 10, 9101,
6500 HB Nijmegen, The Netherlands
e-mail: paul.delaat@radboudumc.nl;
richard.rodenburg@radboudumc.nl;
jan.smeitink@radboudumc.nl
338 P. de Laat et al.
22.1 Introduction mtDNA, under the control of nuclear-encoded products,

involves the unwinding of the double-stranded mtDNA and
22.1.1 Physiology of the Oxidative the polymerization of new mtDNA strands. Factors involved
Phosphorylation include the polymerase gamma (POLG), the helicase
Twinkle, and the single-stranded binding proteins.
The condensation of energy in the form of ATP by
mitochondria is a stepwise process, of which the oxidative
phosphorylation (OXPHOS) constitutes the final step. The 22.1.3 Mitochondrial DNA
metabolic pathways preceeding the oxidative phosphoryla-
tion are fatty acid oxidation (Chap. 17), glycolysis (Chap. Three important features of mtDNA are as follows: (a)
18), pyruvate dehydrogenase (Chap. 19), and Krebs cycle Maternal inheritance: mtDNA is exclusively maternally
(Chap. 21). During the aerobic conversion of substrates, such inherited. The reproductive part of sperm contains almost no
as glucose and fatty acids, into ATP, the formation of the mitochondria, and these paternal mitochondria are actively
reduction equivalents NADH and FADH2 takes place in the degraded after fertilization of an oocyt. All mitochondria in
tricarboxylic acid cycle and the beta-oxidation. These reduc- a fertilized oocyt are therefore from maternal origin. It can
tion equivalents are oxidized by the respiratory chain com- be assumed that if an mtDNA mutation segregates in a fam-
plexes I and II, respectively. The electrons that are released ily, all relatives in the maternal line are (dormant) carriers of
during this conversion flow through the electron transport the mutation. (b) Heteroplasmy: Human cells contain over
chain via coenzyme Q10 and cytochrome c and finally to 100–1,000 mitochondria each, and every mitochondrion
molecular oxygen, which results in the formation of water. contains one to ten copies of mtDNA. When all mtDNA cop-
These subsequent conversions are catalyzed by the respira- ies are the same, there is a homoplasmic genotype. However,
tory chain enzymes complex I, II, III, and IV. The oxida- when there are two different kinds of mtDNA, there is a het-
tion-reduction reactions are accompanied by the pumping of eroplasmic genotype. The amount of mutated mtDNA is
protons from the mitochondrial matrix to the cellular envi- indicated in a percentage; this can range from 0 to 100 %. (c)
ronment by complex I, III, and IV. This results in a proton Mitotic segregation: Heteroplasmy can vary in different tis-
gradient (and membrane potential) over the inner mitochon- sues of one patient, and it can change in time during a carri-
drial membrane. This proton gradient is used by complex V er’s lifetime. This variation in heteroplasmy between tissues
(ATP synthase) to convert ADP and Pi into ATP. This results and in time complicates the clear demonstration of a rela-
in an increase in the ATP/ADP ratio within the mitochondrial tionship between the severity of the disease and the degree of
matrix, which is balanced by the export of ATP by the ade- heteroplasmy. In some mutations there are clear threshold
nine nucleotide transporter (ANT), which is accompanied by levels of heteroplasmy; below these levels, the carriers do
equimolar amounts of import of ADP. not express the phenotype. In other mutations there is much
variation in the genotype-phenotype relation. Overall, there
is a more severe phenotype in patients with a higher hetero-
22.1.2 Mitochondrial Function plasmy percentage.
Primary mitochondrial function is dependent of both nuclear

DNA (nDNA) and mitochondrial DNA (mtDNA). mtDNA 22.1.4 Mitochondrial Dysfunction
encodes 37 genes, 22 code for tRNA’s, two for rRNA’s, and
13 code for subunits of the OXPHOS complexes I, III, IV, Mitochondrial dysfunction can be a result of a primary defect
and V. Mitochondria are not self-supporting entities, but in the OXPHOS system, caused by a mutation in nDNA or
require imported nuclear-encoded proteins and other mtDNA. Secondary causes of mitochondrial dysfunction
imported molecules in order to function properly. In addition include aging, cellular stress, and inactivity. Also other
to the OXPHOS genes, the mtDNA also encodes the RNA inherited metabolic diseases (e.g., creatine biosynthesis
molecules required for the translation of these mtDNA- defects and organic acidurias) can cause secondary mito-
encoded OXPHOS genes. The mtDNA-encoded ribosomal chondrial dysfunction. As this chapter is about mitochon-
RNAs, together with nuclear-encoded ribosomal proteins, drial disorders, we will focus on primary mitochondrial
form mitoribosomes. The mtDNA-encoded tRNAs are dysfunction. The incidence of primary mitochondrial dys-
charged with amino acids by their corresponding (nuclear function, based on an OXPHOS defects, is over 1:5,000 of
encoded) aminoacyl tRNA synthetases. The mitoribosomes all live births. Mutations in mtDNA theoretically cause iso-
and charged tRNAs, with the help of nuclear-encoded translated complex deficiencies of the complexes that contain
lation initiation and elongation factors, synthesize the 13 mitochondrial-encoded subunits, complex I, III, IV, and V. A
polypeptides of the OXPHOS system. Replication of mutation in one of the mitochondrial-encoded tRNAs or
22 Mitochondrial Oxidative Phosphorylation Disorders 339
rRNAs causes a multi-complex deficiency of the complexes histochemical evidence of mitochondrial disease,
that contain mitochondrial-encoded subunits. Mutations in biochemistry, and molecular genetic testing. Family history
nuclear genes encoding proteins involved in the OXPHOS is of utmost importance as it can give indication on maternal
system may cause both single and multi-complex deficien- inheritance. Maternal inheritance could be suspected based
cies of all complexes. on a maternal history, symptoms of the abovementioned
organ systems presenting in maternal relatives.
22.1.5 Clinical Signs and Symptoms

22.1.6 Genotypic Conformation of
Mitochondrial dysfunction causes a wide variety of symp- Mitochondrial Disorders
toms, in many different organ systems, as shown in the tables
of the individual mitochondrial disorders. The disease course Several mitochondrial syndromes are known (22.1–22.18).
is often progressive, although the rate of deterioration is If a patient presents with one of these syndromes, a mito-
highly variable between different patients. chondrial disease is highly likely, and diagnostics and man-
Onset of symptoms can be at any age. The organs requir- agement can be adjusted to the known clinical course of the
ing the most energy, such as the brain, retina, heart, kidney, disease. Mostly, patients do not present with the whole spec-
and skeletal muscle, are most commonly and severely trum of symptoms described. However, in the case of a mito-
affected. A mitochondrial disease should therefore be sus- chondrial DNA (mtDNA) mutation, additional clinical
pected in patients with progressive multisystem involvement, symptoms fitting the syndrome can be found in maternal
but mitochondrial disorders may present with “any symptom relatives, such as the patient with maternally inherited diabe-
of any organ at any age.” In general, symptoms present in tes and deafness who lost his mother in an early stroke and
mitochondrial disorders can also be seen in a variety of other other neurologic problems. Symptoms do not have to limit
diseases. Signs and symptoms where mitochondrial disor- exactly to the original syndrome description, but may involve
ders should be first and foremost on the differential diagnosis other organ systems.
include combinations of encephalopathy and hepatopathy, The clinical presentation of a patient with a mitochondrial
myoclonic epilepsy, developmental regression, stroke-like disorder is in most cases not as obvious as in the mitochon-
episodes, ophthalmoplegia, optic nerve atrophy, retinal drial syndromes. The patient’s signs and symptoms should in
degeneration, early onset hearing loss, and ataxia. these cases trigger a physician to perform additional testing
In many patients, diagnosis cannot be made on clinical leading to the right diagnosis.
signs and symptoms alone. A more structured approach is High-throughput sequencing technologies are becoming
needed, including blood and/or cerebrospinal fluid (CSF) available for diagnostic application, and these techniques
lactate, pyruvate and alanine concentrations, neuroimaging, will improve the diagnostic route of rare genetic diseases,
cardiac evaluation and muscle biopsy for histological and such as mitochondrial disorders.
22.2 Nomenclature
22.1 Leigh syndrome Infantile subacute LS Both nuclear Several different 256000 All
necrotizing and proteins can be forms
encephalopathy mitochondrial affected
22.2 Leigh syndrome with LSFC LRP130 2p21-p16 LRPPRC 220111 All
French-Canadian forms
ethnicity
22.3 Lethal infantile LIMM MT-TT 551000 All
mitochondrial myopathy forms
22.4 Pearson syndrome Pearson marrow- ? Large mtDNA ? 557000 All
pancreas syndrome deletions forms
22.5 Myoclonic epilepsy MERRF m.8344A>G MT-TK 545000 All
associated with ragged forms
red fibers
22.6 Neuropathy ataxia and NARP m.8993T>G ATPase6 551500 All
retinitis pigmentosa forms
22.7 Maternally inherited Leber optic atrophy – m.14459T>A, MTND6, 500001 All
mitochondrial dystonia and dystonia and several MTND4, forms
others MTND3,
MTND1
22.8 Mitochondrial myopathy, MELAS 3243A>G, MT-TL1 540000 All
encephalopathy, lactic m.3271T>C forms
acidosis, and stroke-like
episodes
22.9 Maternally inherited Ballinger-Wallace MIDD m.3243A>G, MT-TL1, MT-TE, 520000 All
deafness and diabetes syndrome m.14709T>C, MT-TK forms
m.8396A>G
22.10 Mitochondrial myopathy ? m.14709T>C MT-TE 500002 All
with diabetes mellitus forms
22.11 Mohr-Tranebjaerg Dystonia deafness MTS TIMM8A Xq22.1 TIMM8A 304700 All
syndrome syndrome forms
22.12 Growth retardation, Fellman syndrome GRACILE BCS1L 2q33 BCS1L 603358 All
aminoaciduria, forms
cholestasis, iron overload,
lactic acidosis, and early
death syndrome
22.13 Kearns-Sayre syndrome KSS Large mtDNA ? 530000 All
deletions forms
22.14 Progressive external PEO Several different 157640, All
ophthalmoplegia protein can be 258450 forms
affected
22.15 Childhood-onset Juvenile optic atrophy OPA1 OPA1 3q28-q29 OPA1 165500 All
autosomal dominant optic forms
atrophy
22.16 Optic atrophy 1 and ? OPA1 3q28-q29 OPA1 125250 All
deafness forms
22.17 Leber hereditary optic LHON mt.11887G>A, MTND4, 535000 All
neuropathy, LHON mt.3460G>A, MTND1, forms
mt.14484T>C MTND6
22.18 Sensory ataxic SANDO POLG 15q25 Polymerase 607459 All
neuropathy, dysarthria gamma (POLG) forms
and ophthalmoparesis
22.19 Complex I deficiency CI def. Multiple Multiple 252010
All
forms
22.20 Complex II deficiency CII def. 5p15, SDHA, SDHAF1 252011 All
19q12-q13.2 forms
22.21 Complex III deficiency CIII def. BCS1L 2q33 BCS1L 124000 All
forms

22.22 Complex IV deficiency CIV def. Multiple Multiple 220110 All
forms
22.23 ATP synthase deficiency Complex V deficiency ? Multiple Multiple 500003, All
551500, forms
604273
22.24 Combined oxidative Early fatal progressive COXPD1 GFM1 3q25.1-q26.2 Elongation factor 609060 All
phosphorylation defect 1 hepatoencephalopathy G1 (EFG1) forms
22.25 Combined oxidative Corpus callosum COXPD2 MRPS16 10q22.1 MRPS16 610498 All
phosphorylation defect 2 agenesis with forms
dysmorphism and
fatal lactic acidosis
22.26 Combined oxidative Encephalomyopathy, COXPD3 TSFM 12q13-q14 TSFM 610505 All
phosphorylation defect 3 respiratory failure and forms
lactic acidosis
22.27 Combined oxidative COXPD4 TUFM 16p11.2 Elongation factor 610678 All
phosphorylation defect 4 Tu (TUFM) forms
22.28 Combined oxidative COXPD5 MRPS22 3q23 MRPS22 611719 All
phosphorylation defect 5 forms
22.29 Combined oxidative X-linked COXPD6 AIFM1 Xq25-q26 AIFM1 300816 All
phosphorylation defect 6 mitochondrial forms
myopathy
22.30 Combined oxidative COXPD7 C12ORF65 12q24.31 C12ORF65 613559 All
phosphorylation defect 7 forms
22.31 Mitochondrial depletion Mitochondrial MTDPS1 TYMP 22q13.32-qter TYMP 603041 All
syndrome 1 neurogastrointestinal forms
encephalopathy
syndrome (MNGIE)
22.32 Mitochondrial depletion TK2-related MTDPS2 TK2 16q22 Thymidine kinase 609560 All
syndrome 2 mitochondrial DNA (TK2) forms
depletion myopathy
22.33 Mitochondrial depletion MTDPS3 DGUOK 2p13 DGUOK 251880
All
syndrome 3 forms
22.34 Mitochondrial depletion Alpers-Huttenlocher MTDPS4A POLG 15q25 Polymerase 203700 All
syndrome 4A syndrome gamma (POLG) forms
22.35 Mitochondrial depletion Encephalomyopathy MTDPS5 SUCLG1 13q12.2-q13 Succinate-CoA 612073 All
syndrome 5 with methylmalonic ligase (SUCLG1) forms
aciduria
22.36 Mitochondrial depletion Encephalomyopathy MTDPS8A RRM2B 8q23.1 RRM2B 612075 All
syndrome 8A with renal forms
tubulopathy
22.37 Mitochondrial depletion Fatal infantile lactic MTDPS9 SUCLG1 2p11.2 Succinate-CoA 245400 All
syndrome 9 acidosis ligase (SUCLG1) forms
22.38 Mitochondrial phosphate – SLC25A3 12q23 SLC25A3 610773 All
carrier deficiency forms
22.39 Global cerebral Mitochondrial GCH SLC25A12 2q24 SLC25A12 612949 All
hypomyelination aspartate glutamate forms
carrier 1 deficiency
22.40 Early infantile epileptic Mitochondrial EIEE3 SLC25A22 11p15.5 SLC25A22 609304 All
encephalopathy 3 glutamate carrier 1 forms
deficiency
22.41 Pyridoxine-refractory – SLC25A38, 3p22.1, SLC25A38, 205950 All
sideroblastic anemia GLRX5 14q32.13 GLRX5 forms
22.42 Myopathy lactic acidosis MLASA PUS1, 12q24.33, PUS1, YARS2 600462, All
and sideroblastic anemia YARS2 12p11.21 613561 forms
22.43 Acute infantile liver – TRMU 22q13 TRMU 613070 All
failure forms
22.44 Leukoencephalopathy LBSL DARS2 1q25.1 DARS2 611105 All
with brain stem and forms
spinal cord involvement
and lactate elevation

22.45 Mitochondrial myopathy COX m.14674T>C MT-TE 500009 All
with reversible forms
cytochrome c oxidase
(COX) deficiency
22.46 Hereditary myopathy HML ISCU 12q24.1 ISCU 255125 All
with lactic acidosis forms
22.47 ACAD9 deficiency – ACAD9 3q26 Acyl-CoA 611126 All
dehydrogenase-9 forms
(ACAD9)
22.48 Coenzyme Q10 Ubiquinone – Multiple Multiple 607426 All
deficiency deficiency forms
22.49 Uncoupling protein UCP def. 4q28-q31, Uncoupling 601665, All
deficiency 11q13, 11q13.4, protein 1, 2, and 607447 forms
3
22.50 Aminoglycoside-induced Streptomycin – m.1555A>G, MTRNR1, 580000 All
deafness ototoxicity m.7444G>A MTCO1 forms
Matrix 2 IMM IMS

NADH 3
Complex I (NADH:ubiquinone
e- oxidoreductase)
NAD+
-Rotenone sensitive
1 Fuel oxidation - Contains FMN
e-
- 45 subunits, 7 mtDNA encoded
H+ H+ 9
Succinate e-
e- 5 e-
Complex II (succinate-
Fumarate CoQ oxidoreductase)
-Contains FAD
-4 subunits, all nuclear
encoded 4
CoQ
5 e-
Various electron transfer
flavoproteins
- All nuclear encoded
e-
e-
6 Complex III, CoQ-cytochrome c
Organic acid oxidation (fatty oxidoreductase
acids, lysine) -Antimycin a sensitive
-Contains cytochrome b, c1
-11 subunits, 1 mtDNA encoded
H+ H+ 9
e- 7
e- Cyt c
8
Complex IV, cytochrome c
oxidase
-Cyanide sensitive
- Contains cytochrome aa3
- 13 subunits, 3 mtDNA
encoded
ADP + Pi H+ H+ 9
2H+ e-
½ O2 O2
11 Free H2O
ATP energy 10 Complex V, ATP synthase
H+ H+
-Oligomycin sensitive
- 14 subunits, 2 mtDNA encoded
Fig. 22.1 Oxidative phosphorylation (OXPHOS) system in mamma- electrons lose free energy at each transfer step and in complexes I, III,
lian mitochondria. Electrons (e−) from carbon oxidations (step 1 and and IV. The energy is harnessed and coupled to the movement of H+
dotted lines) are transferred via NADH (step 2) into OXPHOS complex (step 9 and dashed lines) from the mitochondrial matrix to the inter-
I (step 3), which is embedded in the inner mitochondrial membrane membrane space (IMS). The proton gradient thus generated is used for
(IMM), then transported to coenzyme Q (CoQ) (step 4). Some electrons the production of ATP by complex V (step 10 and 11). Except for com-
from organic-acid oxidations are transferred, via other flavin-contain- plex II, all complexes contain some proteins encoded by the mitochon-
ing enzyme complexes (step 5), directly to CoQ. CoQ delivers electrons drial genome and others encoded by the nuclear genome. The number
via complex III (step 6) and cytochrome c (Cyt c) to the final electron of subunits for each complex is indicated (FMN flavin mononucleotide,
acceptor complex IV (step 8). Here, oxygen is reduced to water. The mt mitochondrial)
Table 22.1 Leigh syndrome

CNS Ataxia ++ ++ ++ ++
Dystonia ++ ++ ++ ++
Neuropathy, peripheral + + + +
Regression, psychomotor ++ ++ + +
Seizures ++ ++ ++ ++
Digestive Vomiting ++ ++ ++ ++
Eye Ophthalmoplegia ++ ++ ++ ++
Musculoskeletal Muscle weakness ++ ++ ++ ++
Myopathy + + + +
Other Death +++ +++ + +
Routine laboratory Lactate (P) n-↑↑↑ n-↑↑↑ n-↑↑↑ n-↑↑↑
Special laboratory MRI: Leigh-like lesions ++ ++ ++ ++
Table 22.2 Leigh syndrome with French-Canadian ethnicity

CNS Ataxia + + +
Developmental delay ++ ++ ++
Hypotonia ++ ++ ++
Musculoskeletal Facial dysmorphism, mild + + + +
Other Death ++ +++ +
Routine laboratory Lactate (P) ↑↑↑ ↑↑↑ ↑↑↑
Special laboratory MRI: Leigh-like lesions ++ ++ ++
Table 22.3 Lethal infantile mitochondrial myopathy

Other Death ++ ++
Routine laboratory Lactate (P) ↑↑↑ ↑↑↑
Special laboratory Respiratory chain ↓↓↓ ↓↓↓
activity (M)
Table 22.4 Pearson syndrome

CNS Hypertonia + + +
Hypotonia + + +
Kearns-Sayre syndrome, evolution to + ++ ++
Digestive Liver steatosis + + +
Pancreatic dysfunction, exocrine ++ ++ ++
Endocrine Pancreatic dysfunction, endocrine ++ ++ ++
Hematological Anemia, hypoplastic macrocytic ++ ++ ++
Anemia, sideroblastic ++ ++ ++
Leukopenia + + +
Thrombocytopenia + + +
Vacuolization of hematopoietic precursors ++ ++ ++
Renal Renal tubulopathy, proximal + + +
Other Death + +++ +++
Failure to thrive ++ +
Low birth weight ++
Routine laboratory Lactate (P) ↑↑ ↑↑ ↑↑
Lactate (U) ↑ ↑ ↑
Lactate/pyruvate ratio ↑↑ ↑↑ ↑↑
Table 22.5 Myoclonic epilepsy associated with ragged red fibers

Cardiovascular Cardiomyopathy + +
CNS Ataxia + +
Dementia + +
Epilepsy, generalized + ++
Epilepsy, myoclonic + +++
Musculoskeletal Myopathy + ++
Table 22.6 Neuropathy ataxia and retinitis pigmentosa

CNS Ataxia + ++
Epilepsy + +
Mental retardation + +
Neuropathy + ++
Eye Blindness +
Pigmentary retinopathy + ++
Musculoskeletal Myopathy + ++
Table 22.7 Maternally inherited mitochondrial dystonia

CNS Bulbar dysfunction + +
Hypokinesia + ++ ++ ++
Eye Loss of central vision + +++
Optic atrophy + +
Musculoskeletal Short stature + + +
Special laboratory MRI: bilateral striatal necrosis + + + +
Table 22.8 Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes
CNS Encephalopathy + ++ ++ ++
Epilepsy + ++ ++ ++
Headache, migraine like ++ ++ ++
Stroke-like episodes + ++ ++ ++
Digestive Vomiting + + + +
Ear Deafness, sensorineural + ++ ++
Eye Blindness + + +
Macular dystrophy + +
Musculoskeletal Myopathy ++ ++ ++ ++ ++
Short stature + + +
Renal Focal segmental glomerulosclerosis + ++
Renal Fanconi syndrome ± ± ± ±
Routine laboratory Lactic acidosis ± + ++ ++ ++
Table 22.9 Maternally inherited deafness and diabetes

Cardiovascular Cardiomyopathy ± ±
Ear Deafness, sensorineural ± + +++
Endocrine Diabetes mellitus ± ± ++
Eye Macular dystrophy ±
Ptosis of eyelid ±
Musculoskeletal Myopathy +
Renal Focal segmental glomerulosclerosis ± ±
Table 22.10 Mitochondrial myopathy with diabetes mellitus

CNS Ataxia +
Neuropathy +
Endocrine Diabetes mellitus ++ ++
Eye Ophthalmoplegia +
Musculoskeletal Myopathy ++ ++
Table 22.11 Mohr-Tranebjaerg syndrome

CNS Dystonia + ++ ++
Mental retardation + + ++
Ear Deafness, sensorineural ± + ++ +++
Eye Blindness ± ± +
Vision, impaired + + + +
Table 22.12 Growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis, and early death syndrome
Digestive Hemosiderosis ++ ++
Renal Renal Fanconi syndrome ++ ++
Other Death +++ +++
Intrauterine growth retardation +++
Table 22.13 Kearns-Sayre syndrome

Cardiovascular Cardiomyopathy + ++ ++
Conduction deficits + + +
CNS Ataxia + +
Mental retardation +
Eye Ophthalmoparesis +
Ophthalmoplegia + ++ +++
Pigmentary retinopathy + ++ ++
Musculoskeletal Myopathy + ++ ++
Short stature + ++ ++
Other Onset before 20 years + + +
Routine laboratory Protein (CSF) ↑
Table 22.14 Progressive external ophthalmoplegia

Cardiovascular Cardiomyopathy ±
CNS Ataxia +
Depression +
Neuropathy, sensory axonal +
Parkinsonism +
Digestive Gastrointestinal dysmotility +
Ear Hearing loss +
Endocrine Hypogonadism ± +
Eye Cataract ± ± +
Ophthalmoplegia, progressive external ++ ++
Musculoskeletal Exercise intolerance ++ ++
Muscle weakness, progressive +
Table 22.15 Childhood-onset autosomal dominant optic atrophy

Eye Color vision deficit + + +
Scotomata + + +
Temporal optic nerve pallor + + +
Vision loss, onset +++ ++ +
Table 22.16 Optic atrophy 1 and deafness

CNS Ataxia ±
Ear Deafness, sensorineural ± + + + ++
Optic atrophy + ++ ++ ++
Ptosis of eyelid +
Table 22.17 Leber hereditary optic neuropathy, LHON

Cardiovascular Conduction deficits + +
CNS Ataxia ± ±
Neuropathy, sensory ± ±
Eye Loss of central vision ++ +++
Optic atrophy + +
Table 22.18 Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis

CNS Dysarthria ++
Neuropathy, sensory axonal +++
Seizures +
Ear Hearing loss +
Eye Ophthalmoparesis ++
Musculoskeletal Myopathy ++
Table 22.19 Complex I deficiency

Cardiovascular Cardiomyopathy + + + + +
CNS Encephalopathy ++ ++ ++ ++ ++
Epilepsy ++ ++ ++ ++ ++
Leigh syndrome ++ +++ +++ ++
Leukodystrophy, progressive ++ ++ ++ ++ ++
Retardation, psychomotor ++ +++ +++ +++ +++
Eye Leber hereditary optic neuropathy + ++ ++
Musculoskeletal Myopathy ++ ++ ++ ++ ++
Other Failure to thrive ++ +++ +++
Routine laboratory Lactate (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Table 22.20 Complex II deficiency

Cardiovascular Cardiomyopathy, hypertrophic + + + +
CNS Dementia + + +
Encephalopathy + + + +
Kearns-Sayre syndrome +
Leigh syndrome + +
Musculoskeletal Myopathy + + + +
Short stature + + + +
Table 22.21 Complex III deficiency

CNS Developmental delay + ++ ++
Encephalopathy + + +
Hypotonia ++ ++ ++
Digestive Liver dysfunction ± ± ±
Renal Renal tubulopathy ± ± ±
Other Death + +++ ++
Failure to thrive ++ +++ ++
Routine laboratory Glucose (P) ↓ ↓ ↓
Lactate (P) ↑↑ ↑↑ ↑↑
Table 22.22 Complex IV deficiency

Cardiovascular Cardiomyopathy, hypertrophic + + + +
CNS Epilepsy + + +
Leigh syndrome + +
Retardation, psychomotor + + + +
Digestive Liver failure + + + +
Musculoskeletal Muscle weakness + + + + +
Myopathy + + + +
Renal Renal failure, chronic + + + +
Other Failure to thrive + + +
Routine laboratory Lactate (P) ↑ ↑ ↑ ↑
Table 22.23 ATP synthase deficiency

Cardiovascular Cardiomegaly ++ + +
CNS Epilepsy + + + + +
Hypotonia + + + + +
Musculoskeletal Facial dysmorphism + + + + +
Other Failure to thrive ++ ++ ++
Intrauterine growth retardation ++
Routine laboratory Lactate (P) ↑↑↑ ↑↑↑ ↑↑ ↑ ↑
Special laboratory 3-Methylglutaconic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Table 22.24 Combined oxidative phosphorylation defect 1

CNS Encephalopathy ++ ++
Minimal spontaneous movements + +
Retardation, psychomotor ++ ++
Digestive Liver failure, progressive ++ ++
Eye Ophthalmoparesis ++ ++
Other Death +++ +++

CNS Agenesis, corpus callosum ++
Hypotonia ++
Minimal spontaneous movements ++
Musculoskeletal Facial dysmorphism ±
Other Death +++
Routine laboratory Lactate (P) ↑↑↑

Seizures ++ ++
Musculoskeletal Muscle weakness ++ ++
Rhabdomyolysis ++ ++
Other Death +++
Routine laboratory Creatine kinase (P) ↑↑ ↑↑
Lactate (P) ↑↑ ↑↑

Hypotonia, axial ++ ++
Regression, psychomotor + +++
Spasticity, limbs ++ ++
Digestive Liver dysfunction + +
Respiratory Respiratory failure ++ +
Other Death ++ ++
Routine laboratory Lactate (P) ↑↑ ↑↑

Cardiovascular Cardiomyopathy, hypertrophic ++
CNS Hypotonia ++
Dermatological Edema, generalized ++
Digestive Ascites +
Renal Renal tubulopathy ++
Routine laboratory Ammonia (B) ↑↑
Lactate (P) ↑↑↑

CNS Areflexia ++ ++
Hypotonia ++ ++
Neurologic deterioration + +++
Neuropathy ++ ++
Regression, psychomotor ++ ++
Seizures + ++
Other Death ++ ++
Routine laboratory Lactate (P) ↑ ↑

CNS Ataxia + + + +
Leigh syndrome + ± ±
Neurologic deterioration + ++ +++ +++
Regression, psychomotor + +++ ++ ++
Eye Nystagmus + ++ ++ ++
Ophthalmoplegia ++ ++ ++
Optic atrophy ++ +++ +++
Other Death + + +
Failure to thrive ++ ++
Table 22.31 Mitochondrial depletion syndrome 1

CNS Leukoencephalopathy, diffuse +++
Neuropathy, peripheral ++
Digestive Abdominal pain +
Cachexia ++
Diarrhea +
Gastrointestinal dysmotility +++
Intestinal pseudo obstruction +
Vomiting +
Ear Deafness, sensorineural +
Eye Ophthalmoplegia, progressive external ++
Ptosis of eyelid ++
Routine laboratory Lactate (P) ↑↑

CNS Hypotonia, progressive generalized ++ +++ +++
Eye Ophthalmoplegia, progressive external + ++ ++ ++ ++
Musculoskeletal Motor skills, delayed + ++ ++
Muscle weakness ++ +++ +++ +++ +++
Respiratory Failure ± ± ±
Routine laboratory Lactate (P) ↑↑↑ ↑↑ ↑↑ ↑ ↑
Special laboratory Amino acids, all ± ± ±

CNS Encephalopathy + ++
Hypotonia ++ ++ ++
Digestive Hepatosplenomegaly + +++ +++
Liver failure, fatal +++ ++
Liver failure, progressive + +++ +++
Eye Nystagmus + + +
Other Failure to thrive ++ ++ ++
Routine laboratory Glucose (P) ↓ ↓ ↓
Lactate (P) ↑ ↑ ↑
Lactate/pyruvate ratio ↑ ↑ ↑
Table 22.34 Mitochondrial depletion syndrome 4A

CNS Ataxia ++ ++
Developmental delay ++ ++
Epilepsy, intractable +++ +++
Hypotonia ++ ++
Mental retardation ++ ++
Retardation, psychomotor +++ +++
Digestive Liver failure, progressive +++ +++
Vomiting + +
Eye Vision, impaired + +
Other Death ++ ++
Routine laboratory Lactate (P) ↑ ↑
Special laboratory 3-Methylglutaconic acid (U) ↑ ↑

CNS Dystonia + + + +
Encephalopathy ++ ++ ++ ++
Hypotonia ++ ++ ++ ++
Movement disorder, ++ ++ ++ ++
hyperkinetic-dystonic
Retardation, psychomotor ++ +++ +++ +++
Seizures + + + +
Ear Deafness, sensorineural ++ ++ ++ ++
Eye Ophthalmoplegia ++ ++ ++ ++
Renal Renal tubulopathy ++ ++ ++ ++
Routine laboratory Lactate (CSF) ↑ ↑ ↑ ↑
Lactate (P) ↑ ↑ ↑ ↑
Special laboratory 3-Methylglutaconic acid (U) ↑ ↑ ↑ ↑
Methylmalonic acid (U) ↑ ↑ ↑ ↑
Table 22.36 Mitochondrial depletion syndrome 8A

CNS Hypotonia +++ +++
Neurologic deterioration +++ +++
Seizures + +
Digestive Feeding difficulties ++ ++
Eye Vision, impaired + +
Renal Renal tubulopathy, proximal +++ +++
Other Death ++ +++
Failure to thrive ++ ++

Encephalopathy, necrotizing ++ ++
Hypotonia ++ ++ + +
Neurologic deterioration ++ ++ +++ +++
Retardation, psychomotor +++ +++ +++ +++
Other Death + + + +
Routine laboratory Lactate (P) ↑ ↑ ↑ ↑
Special laboratory Methylmalonic acid (U) ↑ ↑ ↑ ↑
Table 22.38 Mitochondrial phosphate carrier deficiency

Cardiovascular Cardiomyopathy, hypertrophic +++ +++
CNS Hypotonia +++ +++
Other Death +++
Failure to thrive +++ +++
Table 22.39 Global cerebral hypomyelination

CNS Hypotonia ++ ++ ++
Spasticity +++
Tendon reflexes, increased ++
Special laboratory MRI: cerebral hypomyelination ++ ++ ++
MRS: decrease N-acetylaspartate ++ ++ ++
Table 22.40 Early infantile epileptic encephalopathy 3

CNS Cerebral atrophy ++ ++ ++ ++
Encephalopathy + ++ +++ +++
Hypotonia ++ + + +
Retardation, psychomotor ++ +++ +++ +++
Seizures, intractable ++ ++ ++ ++
Other Death + + + +
Table 22.41 Pyridoxine-refractory sideroblastic anemia

Digestive Hepatosplenomegaly + + + +
Hematological Anemia, microcytic hypochromic ++ ++ ++ ++ ++
Anemia, sideroblastic ++ ++ ++ ++ ++
Routine laboratory Ferritin (S) ↑ ↑ ↑ ↑
Table 22.42 Myopathy lactic acidosis and sideroblastic anemia

Cardiovascular Cardiomyopathy, hypertrophic + + +
Hematological Anemia, sideroblastic ++ ++ ++
Exercise intolerance + + +
Myopathy ++ ++ ++
Table 22.43 Acute infantile liver failure

Digestive Hepatosplenomegaly ++ +++ n n
Jaundice + + n n
Liver failure +++ +++ ± n n
Pancreatic failure + + n n
Vomiting ++ ++ n n
Hematological Coagulopathy ++ ++ n n
Table 22.44 Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation
CNS Ataxia ++ ++ ++
Dorsal column dysfunction ++ ++ ++
Leukoencephalopathy + ++ ++
Retardation, psychomotor ± ± ±
Spasticity + + +
Routine laboratory Lactate (P) ↑ ↑ ↑
Special laboratory MRS: selective involvement ++ ++ ++
of brain stem and spinal tracts
Table 22.45 Mitochondrial myopathy with reversible cytochrome c oxidase (COX) deficiency
CNS Hypotonia ++ ++ + n n
Tendon reflexes, decreased + + + n n
Digestive Liver dysfunction + + + n n
Macroglossia + + + n n
Musculoskeletal Myopathy ++ ++ + ± ±
Routine laboratory Creatine kinase (P) ↑ ↑ ↑ n n
Lactate (P) ↑↑ ↑↑ ↑ n n
Table 22.46 Hereditary myopathy with lactic acidosis

Musculoskeletal Exercise intolerance + + +
Muscle cramps + + +
Myopathy ++ +++ +++
Table 22.47 ACAD9 deficiency

Cardiovascular Cardiomyopathy ++ ++
CNS Encephalopathy + + +
Stroke, cerebellar + + +
Digestive Liver failure + + +
Musculoskeletal Muscle weakness + + +
Special laboratory Long-chain acylcarnitines ↑ ↑ ↑
Table 22.48 Coenzyme Q10 deficiency

Cardiovascular Cardiomyopathy + + + +
CNS Ataxia + + + +
Cerebral atrophy + + + +
Encephalopathy + + + +
Leigh syndrome + + +
Musculoskeletal Myopathy + + + +
Renal Renal failure, chronic + + + +
Other Favorable response to coenzyme ++ ++ ++ ++
Q10 supplementation
Table 22.49 Uncoupling protein deficiency

Endocrine Diabetes mellitus type 2 + + +
Other Susceptibility to obesity + + + +
Table 22.50 Aminoglycoside-induced deafness

(after aminoglycosides)

Compound Serum/blood (μmol/l) Urine (mmol/mol creat) Cerebrospinal fluid (μmol/l)
Lactate 450–1,800 (B) <270 (<2 months) 1,100–1,700
<200 (2 months–2 years)
<85 (>2 years)
Pyruvate 60–100 (B) 80–140
Lactate/pyruvate ratio <15 (B) <15
Alanine 150–450 (P, S) 70–250 (<6 months) 16–41 (<1 year)
35–165 (6 months–1 year) 13–31 (1–3 years)
25–130 (1 year–7 years) 13–31 (>3 years)
20–70 (>7 years)
Acetoacetate 5–50 (P)
3-Hydroxybutyrate 15–90 (B)
3-Hydroxybutyrate/acetoacetate ratio <1.0 (B)
Ammonia 10–50 (P)
Creatine kinase <200 (M)(S, U/l)
<170 (F)(S, U/l)
Protein (total) 450–1,100 (<1 month, mg/l)
160–650 (>1 month, mg/l)
Ethylmalonic acid <20
3-Methylglutaconic acid <20
22.6 Pathologic Values

Compound Serum/blood (μmol/l) Urine (mmol/mol creat) Cerebrospinal fluid (μmol/l)
Lactate >2,000 (B) >350 (<2 months) >2,000
>300 (2 months–2 years)
>130 (>2 years)
Pyruvate >130 (B) >200
Lactate/pyruvate ratio >17 (B) >17
Alanine >450 (P, S) >300 (<6 months)
>150 (6 months–7 years)
>100 (>7 years)
3-Hydroxybutyrate + acetoacetate ratio Postprandial increase (B)
Ammonia >100 (P)
Creatine kinase >200 (M)(S, U/l)
>170 (F)(S, U/l)
Protein (total) >1,300 (<1 month, mg/l)
>650 (>1 month, mg/l)
Ethylmalonic acid >25
3-Methylglutaconic acid >25
B blood, S serum, P plasma, M male, F female, U/l units/l
Suspicion of a Mitochondrial Disorder
Test for common mtDNA

Clear maternal inheritance Yes mutations in blood and urine.
Characteristic clinical syndrome? Nuclear sequencing:
Leigh Syndrome, Pearson Syndrome, POLG1, POLG2, PEO1,
MERRF, NARP, MELAS, ANT1, TYMP, OPA1
MIDD,GRACILE, KSS, CPEO, LHON,
MNGIE
No Negative result, further investigion
- Abnormal lactate in glucose loading test

Lactate ↑, Pyruvate ↑, Alanine ↑
No - Abnormal change in ketone bodies after meal
MRI, CT or MRS aberrations
- Abnormalities in 31P NMR studies
No
Yes
Yes
Muscle biopsy (in specialized centre) Patient is very strongly

Biochemistry and Histochemistry Yes suspected on clinical grounds
Normal Abnormal No
No known mitochondrial disorder Mitochondrial disorder established Mitochondrial disorder unlikely
Red ragged
Single defect Multiple defects
fibers
Yes
Nuclear gene sequencing: Southern blot

mtDNA sequencing - CI, CIII, CIV, CV assembly factors Depletions?
- Structural subunits
- CoQ biosynthetic enzymes
Yes No
Nuclear gene sequencing: Nuclear gene sequencing of

TK2, SUCLG1, SUCLA2, mt protein synthesis machine:
RRM2B, POLG1, PEO1, PUS1, EFG1, EFTu, EFTs,
DGUOK, MPV17 MRPS16, RARS2, DARS2
Fig. 22.2
22.8 Specimen Collection or cell is preferable in each individual case, thereby causing
the patient as little inconvenience as possible. Tissue-specific
In order to provide the most optimal opportunity to reach a expression of mitochondrial deficiencies renders fibroblasts
definite diagnosis, biochemical examination of tissue speci- and lymphocytes less universally appropriate than skeletal
mens is usually required, with the exception of a small num- muscle, although fibroblast analysis does have an added
ber of well-defined mitochondrial syndromes that can be diagnostic value when combined with a muscle sample
tested genetically. As a rule, the patient under investigation examination.
should not be on vitamin therapy, in order to avoid masking In the case of unexpected death, blood and urine speci-
of possible enzyme deficiencies. mens should be collected immediately after death. They
For most biochemical determinations, one should ask the should be snap frozen (not fixed) immediately after collec-
diagnostic center for information about specific requirements tion using liquid nitrogen and stored for possible additional
as to the practice of collecting and transporting material. studies. For enzymatic purposes tissues must be removed
Especially in the case of enzyme analysis in tissues or cells, within 1–2 h after death and should be frozen immediately in
one must consult the diagnostic laboratory in advance about liquid nitrogen. Skin biopsy can be performed as late as 48 h
the conditions for removal, preparation, storage (usually at after death and must be collected at room temperature in cell
–70 °C), and transport of the specimens. If fresh tissue is to be culture medium, after which the biopsy can be transported at
studied, samples have to be collected in a special, ice-cold but room temperature to the cell culture laboratory.
not frozen buffer and immediately transported to the labora- Because few reference values are generally available for
tory and should be examined biochemically within 2 h after neonates, it is recommended to perform a muscle biopsy
collection. In this fresh material, in which mitochondrial beyond the first month of life, unless a life-threatening situa-
integrity has not been compromised, mitochondrial flux mea- tion exists.
surements, such as oxygen consumption, substrate oxidation, MtDNA analysis can, in principle, be performed in all
and ATP production can be measured, in addition to the anal- types of tissues or cells available. However, heteroplasmy
ysis of individual mitochondrial enzymes. In frozen tissue levels vary from tissue to tissue. Therefore, it is recommended
samples, only the latter tests can be performed. to perform mtDNA studies in biopsies from affected tissue
The physician should inform the laboratory about the (usually skeletal muscle). However, some mutations can also
clinical findings to ensure an adequate analysis and data be tested noninvasively, such as LHON mutations in blood
interpretation. It is important to discuss which type of tissue and the MELAS/MIDD m.3243A>G mutation in urine.
Test Material Storage Pitfalls

Lactate B, CSF, U −20 °C Prevent glycolysis
Pyruvate B, CSF −20 °C Prevent glycolysis and LDH activity
Amino acids S, P, U, CSF −20 °C –
Blood gases B No storage allowed –
Creatine kinase S −20 °C –
Acetoacetate B −20 °C Feeding state is important
3-Hydroxybutyrate B −20 °C Feeding state is important
Carnitine S, M, L, FB −20 °C –
Organic acids U −20 °C –
Screening Material Storage Pitfalls

Amino acids U −70 °C Antiepileptic drugs, antibiotic artifacts
Pyruvate dehydrogenase M, FB, L, CV −70 °C
2-Oxoglutarate dehydrogenase M, FB, L −70 °C
Fumarase M, FB, L −70 °C
Respiratory chain enzymes M, FB, L, CV −70 °C
ATPase M, FB −70 °C
ATP/ADP translocator M, FB, L −70 °C
Substrate oxidations M, FB, FL No storage allowed Maintain at 0 °C
ATP production M, FB No storage allowed Maintain at 0 °C
Oxygen consumption M, FB, BrF No storage allowed Maintain at 0 °C
Coupling state M, FB No storage allowed Maintain at 0 °C
DNA
Respiratory chain enzymes M, FB, B −20 °C
M muscle (fresh or frozen), L liver (fresh or frozen), MF fresh muscle required, LF fresh liver required, BrF brain (fresh), FB fibroblasts, CV
chorionic villi
22.9 Prenatal Diagnosis early, such as cardiomyopathy and kidney failure should be
screened. In patients with KSS and minor conduction defects,
At present prenatal diagnosis, at the enzyme level, in mito- an implanted cardiac defibrillator (ICD) is indicated immedi-
chondrial disorders can be performed in families in which ately. In patients with severe lactic acidosis (>7 mmol/l),
the proband is suffering (or has suffered) from a complex I, sodium bicarbonate is given to prevent deleterious effects of
complex II, succinate: cytochrome c oxidoreductase, com- acidosis. The goal of sodium bicarbonate is to normalize
plex IV, or pyruvate dehydrogenase deficiency (or combina- blood pH.
tions of these enzymes), at least in our center. A prerequisite
for prenatal diagnosis at the enzyme level is that mtDNA
mutations must have been excluded in the proband. In case 22.10.3 Supportive Care
an mtDNA mutation has been identified, other options for
prenatal diagnosis, such as preimplantation genetic diagno- Since patients with mitochondrial disorders mostly pres-
sis (PGD), might still be possible. Prenatal diagnosis at the ent a combination of multiple symptoms, it is important to
enzyme level is preferably performed in native chorionic focus on the relief of the symptoms or adjust his/her environ-
villi because they can be obtained earlier in pregnancy as ment to his/her needs. The help of a team of rehabilitation
compared with amniocytes. Moreover, it is not necessary to specialists, occupational therapist, orthopedists, and speech
cultivate chorionic villi in contrast with amniocytes, thus therapists may be inevitable for these handicapped patients.
reducing the time of the diagnostic procedure considerably. With the help of the rehabilitation specialist, wheelchairs,
In case the investigation of chorionic villi yields no conclu- splints, adjusted furniture, etc. can be obtained. Tube feed-
sive result, amniocytes can also be investigated, although in ing, either or not via a gastrostomy, is indicated in feeding
practice this is hardly ever necessary. In case causative muta- difficulties to maintain an adequate intake of vitamins and
tions in the nuclear DNA have been identified, prenatal diag- energy. Thickened fluids or nil by mouth can be necessary
nosis is possible using conventional prenatal genetic testing for patients with recurrent aspiration pneumonia. In patients
for all nuclear genetic defects. Because of the expanding with gastrointestinal dysmotility, osmotic laxatives as well as
possibilities of molecular genetic testing of suspected mito- anti-reflux medication should be used accessibly. Deafness
chondrial patients, nuclear DNA-based prenatal diagnosis is can be relieved using hearing aids or cochlear implants in
expected to increase in the future. However, for those fami- patients with severe deafness. Ptosis can be alleviated by
lies in which it has not (yet) been possible to identify the surgical correction, improving the quality of life of patients.
causative genetic defect, the measurement of enzyme activi- Diplopia in ophthalmoparesis can be corrected by fixation
ties in chorionic villi provides an alternative possibility for of the eye muscles. Retinal pigmentary changes are usually
prenatal diagnostic testing. asymptomatic but require protective sunglasses. Nocturnal
respiration assistance can prevent headaches and tiredness
during the day in patients with respiratory abnormalities or
22.10 Treatment severe muscle weakness.
22.10.1 Coenzyme Q10

22.10.4 Preventive Care
Patients with coenzyme Q (CoQ) deficiency clinically improve
on high supplementation of CoQ. However, while muscle It is recommended to perform exercise regularly adjusted to
abnormalities generally improve, cerebral symptoms are only the patients’ capacity, since disuse leads to secondary mito-
partially ameliorated, possibly since coenzyme does not pen- chondrial dysfunction and increased handicap in daily life.
etrate the blood-brain barrier. Unfortunately, apart from CoQ However, muscle pain and exhaustion are major side effects,
deficiency, no treatment for mitochondrial diseases is avail- and exercise should be actively encouraged, possibly with a
able. Many attempts to treat mitochondrial disease are slowly physiotherapist.
progressing from cell to animal experiments. A multivitamin We also recommend a balanced diet, with adequate vita-
cocktail, although not evidence based, can be administered to min and adequate energy intake. It is recommended to have
any patient for a restricted period of time (6 months). a dietician advising the minimal intake, without risking obe-
sity since it will limit mobility.
Generally, it is important to actively treat conditions lead-
22.10.2 Multiorgan Treatment ing to increased energy demand early, such as fever and epi-
lepsy. Also, intravenous fluids and glucose are indicated in
During clinical follow-up, all organs should be screened intercurrent illnesses with inadequate energy intake. One
since every tissue may be affected by mitochondrial dysfunc- should avoid excess glucose intake and restrict to normal
tion. Especially conditions which can be prevented if treated daily needs.
Since many drugs interfere with mitochondrial processes, mitochondrial dysfunction. Examples include sodium
it is important to avoid such medicine in patients with valproate, certain anesthetics, and metformin.
22.10.5 Emergency Treatment

Medication Indication Dose (route)
Sodium bicarbonate Severe lactic acidosis (lactate >7 mmol/l) (0.33 mmol/kg) × base deficit
l-Arginine Acute stroke-like episode 150–500 mg/kg/day (IV)
22.10.6 Standard Treatment

Ubiquinone (coenzyme Q10) Isolated ubiquinone deficiency 60–250 mg/day (oral)
22.10.7 Experimental Treatment

Ubiquinone (coenzyme Q10) All mitochondrial disorders 60–250 mg/day (oral)
Vitamins
Thiamin (B1) All mitochondrial disorders Up to 900 mg/day (oral)
Riboflavin (B2) All mitochondrial disorders 10–100 mg/day (oral)
Metabolic supplements
Succinate Complex I def, KKS, and MELAS 6 g/day (oral)
Creatine Mitochondrial myopathies Up to 10 g/day (oral)
Carnitine Secondary carnitine deficiency Up to 3 g/day (oral)
Lipoic acid CPEO 600 mg/day (oral)
Dichloroacetate Lactic acidosis 25 mg/kg/day (oral)
l-Arginine m.3243A>G/MELAS stroke-like episodes 150–300 mg/kg/day (oral)
Koopman WJ, Willems PH, Smeitink JA (2012) Monogenic mitochon-

Further Reading drial disorders. N Engl J Med 366:1132–1141
McFarland R, Taylor RW, Turnbull DM (2010) A neurological perspec-
DiMauro S, Rustin P (2009) A critical approach to the therapy of mito- tive on mitochondrial disease. Lancet Neurol 9:829–840
chondrial respiratory chain and oxidative phosphorylation diseases. Smeitink JAM, van den Heuvel L, DiMauro S (2001) The genetics and
Biochim Biophys Acta 1792:1159–1167 pathology of oxidative phosphorylation. Nat Rev Genet 2:
DiMauro S, Schon EA (2008) Mitochondrial disorders in the nervous 342–352
system. Annu Rev Neurosci 31:91–123 Wolf NI, Smeitink JA (2002) Mitochondrial disorders: a proposal for
Koene S, Smeitink J (2009) Mitochondrial medicine: entering the era of consensus diagnostic criteria in infants and children. Neurology
treatment. J Inter Med 265:193–209 59:1402–1405
Disorders of Ketone Body Metabolism
23
Jörn Oliver Sass and Sarah C. Grünert
Contents Summary
23.1 Introduction ...................................................................... 361 Ketone body utilisation is of special importance in times of
fasting/starvation or increased energy demand. However, both
23.2 Nomenclature.................................................................... 362
formation and utilisation of ketone bodies (ketogenesis and
23.3 Metabolic Pathways ......................................................... 363 ketolysis) can be impeded by inborn errors of metabolism. In
23.4 Signs and Symptoms ........................................................ 366 case of genetic deficiency of mitochondrial 3-hydroxy-
3-methylglutaryl-coenzyme A synthase (mHMGS) or of
3-hydroxy-3-methylglutaryl-coenzyme A lyase (HMGL), the
23.6 Pathological Values .......................................................... 369 formation of ketone bodies is impaired. If one of the enzymes
23.7 Specimens for the Biochemical Diagnosis of ketolysis is affected, namely, succinyl-CoA:3-oxoacid
of Inborn Errors of Ketone Body Metabolism .............. 369 CoA transferase (SCOT) or methylacetoacetyl-CoA thiolase
23.8 Prenatal Diagnosis ............................................................ 369 (MAT, “β-ketothiolase”), ketones accumulate and a life-
23.9 Treatment .......................................................................... 370
threatening ketoacidosis may result. Since treatment options
allow to minimise the risk for metabolic decompensations,
References .................................................................................... 371
awareness of those diseases is important, as is information on
how to treat and to prevent clinical manifestations.
23.1 Introduction
Ketone bodies acetoacetate and d-3-hydroxy-n-butyric acid

are mainly formed in the liver. They are derived from fatty
acids and ketogenic amino acids (Mitchell and Fukao 2001;
Sass 2012). In contrast to free fatty acids, acetoacetate and
d-3-hydroxy-n-butyric acid can be utilised by the brain.
After prolonged fasting they can account for approximately
two-thirds of the brain’s energy supply.
During ketone body synthesis (ketogenesis), mHMGS
catalyses the formation of 3-hydroxy-3-methylglutaryl-
coenzyme A (HMG-CoA) from acetyl-CoA and acetoacetyl-
CoA (Fig. 23.1). This is the rate-limiting step of ketogenesis.
J.O. Sass (*)
Division of Clinical Chemistry and Biochemistry, HMG-CoA, which can also result from leucine degradation,
University Children’s Hospital Zürich, is converted by HMGL to acetyl-CoA and acetoacetate.
Steinwiesstr. 75, 8032 Zürich, Switzerland The interconversion of acetoacetate with d-3-hydroxy-n-
e-mail: joernoliver.sass@kispi.uzh.ch
butyric acid is catalysed by d-3-hydroxy-n-butyrate
S.C. Grünert dehydrogenase and essentially reflects the oxidation status of
Zentrum für Kinder- und Jugendmedizin,
the mitochondrial matrix (Mitchell et al. 1995). Acetone,
Universitätsklinikum Freiburg, Mathildenstr. 1,
79106 Freiburg, Germany which may give ketotic patients a typical odour, is formed by
e-mail: sarah.gruenert@uniklinik-freiburg.de nonenzymatic decarboxylation of acetoacetate (Fig. 23.2).
362 J.O. Sass and S.C. Grünert
Ketone body utilisation (ketolysis) occurs in extrahepatic Cytosolic acetoacetyl-CoA thiolase also catalyses the
tissues. Its first and rate-limiting step requires SCOT which thiolytic cleavage of acetoacetyl-CoA. It has been consid-
activates acetoacetate to acetoacetyl-CoA (Fig. 23.3). The ered the first enzyme of cholesterol synthesis. In two girls
enzyme MAT then catalyses the formation of two acetyl- deficiency of cytosolic acetoacetyl-CoA thiolase has been
CoA molecules per molecule acetoacetyl-CoA. assumed (De Groot et al. 1977; Bennett et al. 1984). One of
Genetic deficiency of mHMGS typically presents as the patients, with a developmental delay, showed a pro-
hypoketotic hypoglycaemia, while the lack of HMGL nounced metabolic reaction on a ketogenic diet, the other
activity not only affects ketone body formation but also had persistent ketonuria as the only abnormal laboratory
results in the accumulation of leucine metabolites finding. However, at that time optimised enzyme assays were
(Table 23.1). SCOT deficiency is characterised by ketotic not available and the metabolic disorder has never been con-
episodes or even permanent ketosis, but presents no spe- firmed on the gene level. Therefore, this supposed metabolic
cific metabolite abnormalities. Since MAT is not only disease will not be addressed further here. However, it may
involved in ketone body metabolism but also in the catabo- be appropriate to consider detailed studies of cholesterol and
lism of the amino acid isoleucine, it may yield a character- ketone body metabolism in future patient with severe neuro-
istic, isoleucine-derived metabolite pattern in addition to logic symptoms and persistent ketonuria not explained oth-
hyperketosis. erwise (Mitchell and Fukao 2001).
23.2 Nomenclature
No. Disorder Alternative name Abbreviation symbol localisation Affected protein no. Subtype
23.1 3-Hydroxy- Mitochondrial mHMGS HMGCS2 1p12 3-Hydroxy- 605911 All forms
3-methylglutaryl- 3-hydroxy-3- deficiency 3-methylglutaryl-
CoA synthase methylglutaryl-CoA CoA synthase 2
deficiency synthase deficiency
23.2 3-Hydroxy-3- Hydroxymethyl- HMGL HMGCL 1p36.11 3-Hydroxy- 246450 All forms
methylglutaryl- glutaryl-CoA lyase deficiency 3-methylglutaryl-
CoA lyase deficiency CoA lyase
deficiency
23.3 Succinyl-CoA: Succinyl-CoA: SCOT OXCT1 5p13.1 Succinyl-CoA: 245050 All forms
3-oxoacid CoA 3-ketoacid CoA deficiency 3-oxoacid CoA
transferase transferase deficiency transferase
deficiency
23.4 Methylacetoacetyl- Beta-ketothiolase MAT ACAT1 11q22.3 Methylacetoacetyl- 203750 All forms
CoA thiolase deficiency deficiency CoA thiolase
deficiency
23.5 Cytosolic CT deficiency ACAT2 6q25.3 Cytosolic 100678 All forms
acetoacetyl-CoA acetoacetyl-CoA
thiolase deficiency thiolase
23 Disorders of Ketone Body Metabolism 363
Fatty acids
O O
MAT O
H3C – C – S – CoA H3C – C – CH2 – C
β-Oxidation
cycle Acetyl-CoA Acetoacetyl-CoA S – CoA
mHMGS
OH
O O
β-Hydroxy-β-
Tricarboxylic
C – CH2 – C – CH2 – C methylglutaryl-CoA
acid cycle
O S –CoA
CH3
HMGCL Leucine
O O OH
O O
3HBD
H3C – C – S – CoA H3C – C – CH2 – C H3C – C – CH2 – C
O O
Acetyl-CoA Acetoacetate H
β - Hydroxy -
butyrate
Fig. 23.1 Ketone body synthesis (ketogenesis)
NAD+ NADH CO2

OH O O
O O
H3C – C – CH2 – C H3C – C – CH2 – C H3C – C – CH3
3HBD
Fig. 23.2 Ketone bodies (3HBD O O
d-3-hydroxy-n-butyrate H
dehydrogenase) β-Hydroxybutyrate Acetoacetate Acetone
Succinyl-CoA Succinate
O O
O O
H3C – C – CH2 – C H3C – C – CH2 – C
O SCOT
Acetoacetate Acetoacetyl-CoA S – CoA
3HBD
OH
O MAT
HSCoA
H3C – C – CH2 – C
O
H
O O
β - Hydroxybutyrate
H3C – C – S – CoA + H3C – C – S – CoA
Acetyl-CoA Acetyl-CoA
Fig. 23.3 Ketone body utilisation (ketolysis)

Fig. 23.4 Cytosolic acetoacetyl-CoA thiolase (CT; cHMGS cytosolic O

O
3-hydroxy-3-methylglutaryl-coenzyme A synthase)
H3C – C – S – CoA + H3C – C – S – CoA
Acetyl-CoA Acetyl-CoA
CT
O
O
H3C – C – CH2 – C
S – CoA
Acetoacetyl-CoA
cHMGS
OH
O O
C – CH2 – C – CH2 – C
S – CoA
O
CH3
β-Hydroxy-β-methylglutaryl-CoA
Cholesterol synthesis
Fig. 23.5 Accumulation of Alloisoleucine

Isoleucine
isoleucine metabolites in
deficiency of methylacetoacetyl-
CoA thiolase (MAT)
2-Methylbutyryl-CoA
Tiglylglycine
Tiglyl-CoA
Tiglylcarnitine
2-Methyl-3-
hydroxybutyrate 2-Methyl-3-
2-Methyl-3- hydroxybutyryl-CoA
hydroxybutyrylcarnitine 2-Ethylhydracrylic acid
2-Methyl-
acetoacetate 2-Methylacetoacetyl-CoA
2-Butanone
MAT
Propionyl-CoA Acetyl-CoA
Table 23.1 3-Hydroxy-3-methylglutaryl-CoA synthase deficiency

CNS Lethargy, coma + +
Mental development n n n n n
Seizures +
Digestive Diarrhoea + +
Liver dysfunction + +
Vomiting + +
Routine laboratory ASAT/ALAT (P) ↑ ↑
Free fatty acids (S) ↑ ↑
Glucose (P, B) ↓ ↓
Ketones (U, P, B) ↓ ↓
Special laboratory Acetoacetate (U, B) ↓ ↓
3-Hydroxy-n-butyric acid (U, B) ↓ ↓
Free carnitine (DBS, P) n (−↓) n
C2 acylcarnitine (DBS,P) (↑)? (↑)?
Medium-chain and long-chain acylcarnitines n n
Crotonylglycine (U) (↑)? (↑)?
4-hydroxy-6-methyl-2-pyrone (U) (↑)? (↑)?
23.4 Signs and Symptoms acid and ammonia are typically unremarkable. Since leu-
cine enters the pathway at the subsequent step only
Deficiency of mHMGS (Table 23.1) typically presents with (Fig. 23.5), mHMGS deficiency affects conversion of fatty
hypoketotic hypoglycaemia, hepatomegaly and encepha- acids to ketone bodies, but not amino acid catabolism.
lopathy, much resembling disorders of fatty acid oxidation Hence, there are no established metabolite markers pointing
(Duran 2003). During a metabolic crisis, urine organic acids to this inborn error of metabolism. Crotonylglycine has
can reveal dicarboxylic aciduria without ketonuria. Elevated been considered a candidate in this regard based on special
free fatty acids may be found in serum, while levels of lactic investigations in urine of a single patient (Kouremenos et al.
Table 23.2 3-Hydroxy-3-methylglutaryl-CoA lyase deficiency

Characteristic clinical Hepatomegaly + + +
findings Pancreatitis ±
Episodic vomitting + + + + +
Cadiovascular Dilated cardiomyopathy ± ± ± ±
CNS Cerebral infarction/ stroke-like encephalopathy ±
White matter lesions + + + + +
Hypoketotic hypoglycemia + + + + +
Ketones (U, B, P) ↓ ↓ ↓ ↓ ↓
Ammonia (P, B) n-↑ n-↑ n-↑ n-↑ n-↑
Acidosis + + + + +
Glucose (P, B) ↓-n ↓-n ↓-n ↓-n ↓-n
Lactate (P) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Special laboratory C6DC acylcarnitine (B, P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
C5OH acylcarnitine (B, P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Hydroxy-3-methylglutaric acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Methylcrotonylglycine (U) n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑
Sebacic acid (U) n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑
Suberic acid (U) n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑
Adipic acid (U) n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑
Glutaric acid (U) n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ ↑
2010). Similarly, raised plasma acetylcarnitine (acylcarni- cific metabolite changes pointing to SCOT deficiency.
tine C2) has been suggested as a marker if associated with Although permanent/postprandial ketosis/ketonuria may
hypoketotic hypoglycaemia, hepatomegaly and dicarbox- lead to the suspicion of SCOT deficiency, it is not a sensitive
ylic aciduria (Aledo et al. 2006). More recently, 4-hydroxy- marker (Fukao et al. 2004; Fukao et al. 2011b).
6-methyl-2-pyrone has been brought into focus as another Differential diagnoses in patients with severe ketonuria
key metabolite for mHMGS deficiency (Zschocke and include diabetes mellitus type I, endocrine disorders, gly-
Hoffmann 2011). However, these metabolites warrant con- cogen storage disease type 0 and idiopathic ketotic hypo-
firmation in a higher number of affected individuals and glycaemia. Elevated ketone body levels as a physiological
further validation. consequence of enhanced ketogenesis (e.g., following
Deficiency of HMGL (Table 23.2) presents with episodes heavy physical exercise) will normally not result in a keto-
of hypoketotic hypoglycaemia and metabolic acidosis, usu- acidotic decompensation, while type I diabetes mellitus may
ally early in life, although diagnosis may sometimes be present in such a way, albeit usually associated with poly-
delayed until adulthood (Gibson et al. 2003; Bischof et al. uria, polydipsia, weight loss and hyperglycaemia. While
2004). Cerebral infarction and pancreatitis are among the SCOT deficiency is typically not associated with major
reported complications (Muroi et al. 2000). abnormalities in blood glucose, ketotic hypoglycaemia
HMGL deficiency affects not only the synthesis of may have endocrine causes such as adrenocortical insuffi-
ketone bodies from fatty acids but also the catabolism ciency, growth hormone deficiency and panhypopituitarism
of the amino acid leucine. Therefore, both the pattern of (Mitchell et al. 1995). In contrast to patients with SCOT
urinary organic acids and blood acylcarnitines may show deficiency, individuals with glycogen storage disease type
characteristic abnormalities. The detection of acetoacetate in 0, who also present without hepatomegaly and who suf-
urinary organic acids of patients with this disorder of keto- fer from hypoglycaemic-hyperketotic episodes, may show
genesis may be explained by degradation of accumulating postprandial increase in lactate and alanine (Weinstein et al.
3-hydroxy-3-methylglutaric acid during analysis. 2006). Another differential diagnosis with a ketoacidotic
Accumulation of ketone bodies due to insufficient ketoly- presentation during the first 8 or 9 years of life is idiopathic
sis because of SCOT deficiency may result in life-threatening ketotic hypoglycaemia. It usually represents an extreme
ketoacidosis (Table 23.3) (Duran 2003). There are no spe- constellation of a high ratio of brain to muscle mass, which
Table 23.3 Succinyl-CoA: 3-oxoacid CoA transferase deficiency

Lethargy, coma (during ketoacidotic episodes) + + +
Characteristic clinical findings Tachypnoea (during acute ketoacidotic episodes) + + +
Hepatomegaly, hepatopathy n n n n n
Cadiovascular Cardiomyopathy/cardiomegaly ±
CNS Mental development n n n n n
Routine laboratory Acidosis + + +
Ketones (U, P, B) ↑ ↑ ↑
Glucose (P, B) ± ± ± ± ±
Lactate (P, U) n n n n n
Ammonia (P, B) n n n
Free fatty acids (S) n n n
Special laboratory 3-Hydroxy-n-butyric acid (U, B) ↑ ↑ ↑
Dicarboxylic acids (U) n-↑↑ n-↑↑ n-↑↑ n-↑↑ n-↑↑
Free carnitine n n n
Acylcarnitines n
Acylglycines (U) n n n
Table 23.4 Methylacetoacetyl-CoA thiolase deficiency

Characteristic clinical findings Lethargy, coma (during ketoacidotic episodes) + + +
Tachypnoea (during acute ketoacidotic episodes) + + + + +
Failure to thrive ± ± ± ±
CNS Mental development ± ± ± ± ±
Putaminal findings in MRI ± ±
Free fatty acids n n n
Routine laboratory Ammonia (B, P) n-↑ n-↑ n-↑ n-↑ n-↑
Acidosis ± ± ±
Ketones (U, P, B) n-↑ n-↑ n-↑ n-↑ n-↑
Anion gap ± ± ± ± ±
C5:1 acylcarnitine n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory C5OH acylcarnitine n-↑ n-↑ n-↑ n-↑ n-↑
Tiglylglycine n-↑ n-↑ n-↑ n-↑ n-↑
2-Ethylhydracrylic acid n-↑ n-↑ n-↑ n-↑ n-↑
3-Hydroxy-2-methylbutyric acid ↑ ↑ ↑ ↑ ↑
2-Methylacetoacetate n-↑ n-↑ n-↑ n-↑ n-↑
Glycine n-↑ n-↑ n-↑ n-↑ n-↑
3-Hydroxy-n-butyric acid (U, B) n-↑ n-↑ n-↑
can usually be adequately managed by glucose administra- 2-methylbutyric acid and usually, but not always,
tion, because ketonuria usually precedes hypoglycaemia by tiglylglycine (Søvik 1993; Fukao et al. 2011a). If rather
hours. Idiopathic ketotic hypoglycaemia essentially repre- labile 2-methylacetoacetate is not detected, this may reflect
sents a diagnosis made by exclusion. inadequate preanalytical conditions. However, it may also
In contrast to SCOT deficiency, MAT deficiency (less suggest that a defect of the enzyme catalysing the preced-
precisely called “β-ketothiolase deficiency”) represents not ing step in isoleucine catabolism, HSD10 disease
only a disorder of ketolysis but also a defect of isoleucine (2-methyl-3-hydroxybutyryl CoA dehydrogenase defi-
catabolism (Fig. 23.5) (Nyhan and Gibson 2003). ciency, OMIM 300256), needs to be included into the dif-
Consequently, it is more easily identified based on the pat- ferential diagnosis (Ofman et al. 2003). Although the
tern of urinary organic acids comprising 3-hydroxyacylcarnitines C5:1 and C5-OH are sometimes used for
newborn screening for MAT deficiency, this is not reliable, more easily accomplished in samples obtained during a met-
their blood levels can be unremarkable in metabolically abolic decompensation.
stable patients.
23.7 Specimens for the Biochemical

23.5 Reference Values Diagnosis of Inborn Errors
of Ketone Body Metabolism
In view of the many variables affecting ketone body concen-
trations physiologically, it is difficult to provide general ref- Confirmation of suspected deficiencies of SCOT, MAT and
erence values. The concentrations of ketone bodies fluctuate HMGL is usually approached by enzyme activity tests.
widely. For details on 3-hydroxy-n-butyrate and free fatty Exceptions are patients resulting from populations known to
acids under fasting conditions, see Bonnefont et al. (1990) host specific mutations, e.g. HMGL deficiency in popula-
and Morris et al. (1996). tions of Saudi Arabia and Portugal/Spain and MAT defi-
Using organic acid analysis according to Lehnert (1994) ciency in Vietnamese.
(applying methylation as the derivatisation procedure), the In contrast, enzyme activity testing for mHMGS defi-
following reference values have been obtained based on 42 ciency would require liver tissue and is therefore usually
metabolically stable control individuals (mean ± standard replaced by mutation analysis.
deviation): 3-hydroxy-2-methylbutyric acid, 10.7 ± 7.6 mmol/
mol creatinine; tiglylglycine, 24.6 ± 14.6 mmol/mol creati- Enzyme Mutation
No. Disorder Metabolites assay analysis
nine (Fukao et al. 2011a).
23.1 3-Hydroxy-3- U, DBS, Liver B
Notably, it is known from quality assessment programmes methylglutaryl-CoA P/S
that quantitative organic acid data vary largely between labo- synthase deficiency
ratories, and laboratories are encouraged to establish their 23.2 3-Hydroxy-3- U, DBS, LYM, FB B
own reference ranges. However, organic acid analysis is methylglutaryl-CoA P/S
lyase deficiency
mostly performed on a qualitative/semiquantitative basis
23.3 Succinyl-CoA:3- U LYM, FB B
based on recognition of diagnostic signal patterns. oxoacid CoA
transferase deficiency
23.4 Methylacetoacetyl- U, DBS, FB, B
23.6 Pathological Values CoA thiolase P/S LYMa
deficiency
During metabolic decompensations, levels of diagnostic Abbreviations: U urine, DBS dried blood spots, P/S plasma/serum, LYM
(immortalised) lymphocytes, F fibroblasts, B blood
metabolites may be elevated 100 fold and more. In mHMGCS a
Lymphocytes not recommended
deficiency the organic acids of a metabolically decompen-
sated patient may resemble the dicarboxylic aciduria Methylacetoacetate, which helps to distinguish MAT defi-
observed in individuals in ketotic states but without concom- ciency from HSD10 disease, is prone to degradation, thus
itant ketonuria. However, in metabolically stable conditions, making the shipment of urine in frozen state advantageous.
defects in ketogenesis or ketolysis may result in unremark-
able metabolite profiles, and thus escape detection which is
Characteristic abnormalities in laboratory values
Once the diagnosis is known, disorders of ketone body
No. Disorder Metabolites
metabolism are amenable to preventive measures including
23.1 3-Hydroxy-3- Ketones ↓
dietary treatment. However, if prenatal testing is requested, it
methylglutaryl-CoA Glucose ↓
synthase deficiency appears technically feasible.
C2 acylcarnitine n-↑↑
23.2 3-Hydroxy-3- Ketones ↓
methylglutaryl-CoA lyase Glucose n-↓ Enzyme Mutation
deficiency Leucine metabolites ↑-↑↑↑ No. Disorder Metabolite assay analysis Source
23.3 Succinyl-CoA:3-oxoacid Ketones n-↑↑↑ 23.1 3-Hydroxy-3- Xa A, CV
CoA transferase methylglutaryl-
Glucose ±
deficiency CoA synthase
No specific metabolite deficiency
abnormalities beyond ketosis
23.2 3-Hydroxy-3- X X X AFC,
23.4 Methylacetoacetyl-CoA Ketones n-↑↑↑ methylglutaryl- A, CV
thiolase deficiency Glucose ± CoA lyase
Isoleucine metabolites ↑↑ deficiency
Enzyme Mutation are rarely necessary, although haemodiafiltration may play

No. Disorder Metabolite assay analysis Source a crucial role in HMG-CoA synthase deficiency (Sass et al.
23.3 Succinyl-CoA: X Xa A, CVb 2013). After diagnosis, treatment with frequent carbohydrate-
3-oxoacid CoA
rich meals during mild infections and intercurrent illnesses is
transferase
deficiency advisable to prevent metabolic decompensation (Dixon and
23.4 Methylacetoacetyl- X X A Leonard 1992). Deficiency of l-carnitine should be compen-
CoA thiolase sated, if present.
deficiency
Abbreviations: A amniocytes, AFC amniotic fluid, CV chorionic villi
a
Not known whether actually done, but can in principle be performed by Treatment when metabolically stable (maintenance therapy)
DNA analysis Nr. Disorder Management
b
For DNA analysis only
23.1 3-Hydroxy-3- Avoid fasting and extreme fat intake
methylglutaryl-CoA Regular feeding in daytime
synthase deficiencyConsider additional bedtime snack in
young children and supplementation
23.9 Treatment with l-carnitine
23.2 3-Hydroxy-3- Avoid fasting and extreme fat intake
The mainstay of therapy in deficiencies of HMG-CoA syn- methylglutaryl-CoA Regular feeding in daytime
lyase deficiency
thase/lyase, SCOT and MAT is a rather preventative mea- Consider additional bedtime snack in
sure: ketogenesis has to be obviated to the extent possible. young children and mild protein
restriction (individualised, about 1.5 g/
Therefore, prolonged fasting has to be avoided and kg body weight/day) and compensate
increased energy requirements have to be met (Mitchell deficiency of free carnitine, if present
and Fukao 2001). During acute decompensations, hypo- 23.3 Succinyl-CoA: Avoid fasting and extreme fat intake
glycaemia, if present, needs to be corrected immediately 3-oxoacid CoA Regular feeding in daytime
followed by constant high-rate intravenous glucose infu- transferase Consider additional bedtime snack in
deficiency young children
sion (5–8 mg/kg body weight/min) with adequate electro-
lytes. Carbohydrates have not only antilipolytic effects but 23.4 Methylacetoacetyl- Avoid fasting and extreme fat intake
CoA thiolase Regular feeding in daytime
may also be useful due to antiproteolytic effects in HMGL, deficiency Consider additional bedtime snack in
SCOT and MAT deficiencies. In the acute phase with keto-
young children and mild protein
acidosis and dehydration, fluid therapy is mandatory (Søvik restriction (individualised, about 1.5 g/
1993). The treatment of metabolic acidosis by bicarbonate is kg body weight/day) and compensate
controversial but alkalinisation may be beneficial in severe deficiency of free carnitine, if present
ketoacidosis (pH < 7.1) (Mitchell and Fukao 2001). Invasive
methods such as dialysis have been shown to be effective but
Likewise, in periods of well-being the avoidance of fast-
Management of acute events
ing is of crucial importance in all disorders of ketogenesis
and ketolysis. This can usually be achieved with a normal
Nr. Disorder Management
meal frequency. In young children a bedtime snack or the
23.1 3-Hydroxy- Immediate correction of
3-methylglutaryl- hypoglycaemia, if present, followed by administration of uncooked cornstarch at bedtime or mid-
CoA synthase constant high-rate glucose infusion night might be necessary. A special diet is usually not
deficiency Consider haemodiafiltration and required; however, extremes of dietary fat intake should be
administration of l-carnitine avoided. A ketogenic diet is clearly contraindicated. Protein
23.2 3-Hydroxy- Immediate correction of restriction may be useful in HMGCL and MAT deficiencies
3-methylglutaryl- hypoglycaemia, if present, followed by
(Søvik 1993) and possibly in SCOT deficiencies while a high
CoA lyase constant high-rate glucose infusion
deficiency Consider administration of l-carnitine
protein intake may – at least in theory – be beneficial in
23.3 Succinyl-CoA: Immediate correction of mHMGS deficiency by compensating for the metabolic
3-oxoacid CoA hypoglycaemia, if present, followed by block and providing a source of ketone bodies. Most paediat-
transferase constant high-rate glucose infusion ric patients with HMGCL or MAT deficiency are stable on a
deficiency Consider alkalinisation if acidosis is protein intake of about 1.5 g/kg/day. However, protein intake
very pronounced (pH < 7.10)
needs to be individualised (Mitchell et al. 1995). In patients
23.4 Methylacetoacetyl- Immediate correction of
CoA thiolase hypoglycaemia, if present, followed by
with low levels of carnitine, l-carnitine supplementation
deficiency constant high-rate glucose infusion should be considered and may help to eliminate accumulat-
Consider alkalinisation if acidosis is ing tiglyl-CoA (Dasouki et al. 1987; Aramaki et al. 1991).
very pronounced (pH < 7.10) and However, large-scale treatment and outcome studies have not
administration of l-carnitine been accomplished in those rare diseases so far.
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Gibson KM, Elpeleg ON, Morton DH, Wappner RS (2003) Disorders
Aledo R, Mir C, Dalton RN, Turner C, Pié J, Hegardt FG, Casals
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N, Champion MP (2006) Refining the diagnosis of mitochon-
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Aramaki S, Lehotay D, Sweetman L, Nyhan WL, Winter SC, Middleton
infant urine using comprehensive two-dimensional gas chromatog-
B (1991) Urinary excretion of 2-methylacetoacetate, 2-methyl-3-
raphy: application to the diagnosis of organic acidurias and bio-
hydroxybutyrate and tiglylglycine after isoleucine loading in the
marker discovery. J Chromatogr A 1217:104–111
diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency. J Inherit
Lehnert W (1994) Long-term results of selective screening for inborn
errors of metabolism. Eur J Pediatr 153:S9–S13
Bennett MJ, Hosking GP, Smith MF, Gray RGF, Middleton B (1984) Mitchell GA, Fukao T (2001) Inborn errors of ketone body catabolism.
Biochemical investigations on a patient with a defect in cytosolic In: Scriver CR, Beaudet al, Sly WS, Valle D (eds) Metabolic and
acetoacetyl-CoA thiolase, associated with mental retardation. molecular bases of inherited disease, 8th edn. McGraw-Hill,
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80–85 and pancreatitis: possible complications of patients with 3-hydroxy-
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3-Hydroxy-3-methylglutaric aciduria: response to carnitine therapy 23:636–637
and fat and leucine restriction. J Inherit Metab Dis 10:142–146 Nyhan WL, Gibson KM (2003) Disorders of valine-isoleucine metabo-
De Groot CJ, Luit-De Haan G, Hulstaert CE, Hoomes FA (1977) lism. In: Blau N, Duran M, Blaskovics M, Gibson KM (eds)
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Clinical and molecular characterization of five patients with Stuttgart
Part V
Organelles
Peroxisomal Disorders
24
Bwee Tien Poll-The and Ronald J.A. Wanders
Contents Summary
24.1 Introduction ....................................................................... 376 Peroxisomes contain a set of substrate-specific metabolic
pathways, including the oxidation of a range of fatty acids,
24.2 Nomenclature..................................................................... 377
the biosynthesis of a special group of phospholipids (plas-
24.3 Metabolic Pathways .......................................................... 378 malogens) and the production of bile acids. Clinically, a dys-
24.4 Signs and Symptoms ......................................................... 383 function of peroxisomes results in most cases in neurologic
24.5 Diagnostic Flow Charts..................................................... 389 symptoms of varying extent, from severe to milder clinical
forms. Ocular and hearing symptoms, dysmorphisms, liver
24.6 Specimen Collection .......................................................... 393
disease and skeletal involvement are variably associated with
24.7 Prenatal Diagnosis ............................................................. 393 these different disorders. Peroxisomal disorders are divided
24.8 Treatment ........................................................................... 393 into two major categories. The first are disorders resulting
from a failure to form normal peroxisomes, giving rise to
24.9 Follow-Up and Monitoring ............................................... 394
multiple metabolic abnormalities. This group is named per-
References ..................................................................................... 396 oxisome biogenesis disorders (PBD) that can be divided into
two subtypes, including (1) the Zellweger spectrum disorders
and (2) rhizomelic chondrodysplasia punctata (RCDP) type 1.
The second category includes the disorders, which result from
the deficiency of a peroxisomal enzyme or transporter.
In single peroxisomal enzyme disorders, the basic problem is
at the level of one of the enzymes or transporters involved in
each of the different metabolic pathways contained in peroxi-
somes. Clinically, these disorders can be as severe as those in
which peroxisomal biogenesis is defective. This is the case for
d-bifunctional protein deficiency, peroxisomal acyl-CoA oxi-
dase 1 deficiency and the RCDP types 2 and 3. The most com-
mon single peroxisomal enzyme disorder is the X-linked
adrenoleukodystrophy (ALD)/adrenomyeloneuropathy (AMN)
complex that consists of a spectrum of phenotypes varying in
the age of onset and severity of clinical presentation.
The diagnosis of a peroxisomal disorder can be determined
by a battery of biochemical assays in blood and/or urine and
B.T. Poll-The (*)
Department of Pediatrics/Pediatric Neurology, should be confirmed in cultured fibroblasts. Prenatal diagnosis
Academic Medical Center, University of Amsterdam, is possible either by biochemical testing or by molecular anal-
Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands ysis. Management of most peroxisomal disorders is focused
e-mail: b.t.pollthe@amc.uva.nl
on supportive care and multidisciplinary treatment of a variety
R.J.A. Wanders of systemic complications. Specific treatment is essential in a
Lab Genetic Metabolic Diseases,
few single enzyme disorders. Bone marrow/haematopoietic
Academic Medical Center, University of Amsterdam,
Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands stem cell transplantation is an option for boys suffering from
e-mail: r.j.wanders@amc.uva.nl the cerebral form of X-ALD, at least when in the early stages.
376 B.T. Poll-The and R.J.A. Wanders
24.1 Introduction of l-pipecolic acid, a metabolite of l-lysine, via l-pipecolate

oxidase; (6) the oxidation of glutaryl-CoA via glutaryl-CoA
Peroxisomes play an indispensable role in human metabo- oxidase; (7) fatty acid chain elongation; and (8) the break-
lism as exemplified by the devastating consequences of the down of hydrogen peroxide via catalase, among other
absence of peroxisomes in Zellweger patients. The most functions.
important functions of peroxisomes so far identified include The disorders described in this chapter represent a broad
(1) the ß-oxidation of a range of fatty acids and fatty acid group of peroxisomal diseases caused by a peroxisome bio-
derivatives; (2) the biosynthesis of a special group of phos- genesis defect or by a defect in a specific peroxisomal
pholipids called ether phospholipids; (3) the α-oxidation of enzyme or transporter involved in one of the metabolic path-
3-methyl-branched fatty acids like phytanic acid; (4) the ways contained in peroxisomes (see Table 24.1). In most
detoxification of glyoxylate via the peroxisomal enzyme ala- cases, this results in neurologic dysfunction of varying extent
nine glyoxylate aminotransferase (AGT); (5) the oxidation and a multitude of other clinical manifestations.
24
24.2 Nomenclature
Chromosomal
No Disorder Alternative name Abbreviation Gene symbol localisation Affected protein OMIM no
24.1 Zellweger spectrum disorders Peroxisome biogenesis disorders ZSD Different PEX genes Multiple loci Different peroxins
Peroxisome assembly disorders PAD
Zellweger syndrome ZS 214100/214110
Neonatal adrenoleukodystrophy NALD 202370
Peroxisomal Disorders
Infantile Refsum disease IRD 266510

24.2 Peroxisomal acyl-CoA oxidase 1 Straight-chain acyl-CoA oxidase SCOX-deficiency ACOX 17q25 Peroxisomal acyl-CoA 264470
deficiency deficiency ACOX1- deficiency oxidase 1, SCOX
24.3 D-bifunctional protein deficiency DBP deficiency DBPD HSD17B4 5q2 D-bifunctional protein 261515
24.4 Alpha-methylacyl-CoA racemase AMACR deficiency Racemase deficiency AMACR 5q13.2-q11.1 AMACR 604489
deficiency
24.5 Rhizomelic chondrodysplasia RCDP type 1 RCDP1 PEX7 6q22.q24 Pex7p 215100
punctata type 1
24.6 Rhizomelic chondrodysplasia RCDP type 2 RCDP2 GNPAT 1q42.1-42.3 Dihydroxyacetone 602744
punctata type 2 phosphate acyltransferase
24.7 Rhizomelic chondrodysplasia RCDP type 3 RCDP3 AGPS 2q33 Alkyl-DHAP synthase 600121
punctata type 3
24.8 X-linked adrenoleukodystrophy and XALD and AMN ABCD1 Xq28 ALD protein 300100
adrenomyeloneuropathy
24.9 Refsum disease (classic, adult) Heredopathia atactica CRD /ARD PHYH/PEX7 10p15- Phytanoyl-CoA 266500
polyneuritiformis p14/10pter. hydroxylase
p11.2
24.10 Dynamin-like protein 1 DLP1 deficiency DLP1 Dynamin-like protein 1
24.11 Peroxisomal bile acid-CoA:amino BAAT deficiency BAAT deficiency BAAT Bile acid-CoA: amino
acid transferase (BAAT) deficiency acid N-acyl transferase
377
24.3 Metabolic Pathways THCA). The latter two compounds are intermediates in the
formation of the primary bile acids cholate and chenodeoxy-
Peroxisomal Fatty Acid ß-Oxidation cholate from cholesterol in the liver. Another major function
The most important substrates handled by the peroxisomal of the peroxisomal ß-oxidation system concerns the “biosyn-
fatty acid oxidation system from the perspective of peroxi- thesis” of polyunsaturated fatty acids including docosahexae-
somal disorders are (1) very-long-chain fatty acids (VLCFAs), noic acid (C22:6ω3). Recent studies have clearly established
notably hexacosanoic acid (C26:0); (2) pristanic acid that the formation of docosahexaenoic acid (C22:6ω3) from
(2,6,10,14-tetramethylpentadecanoic acid), as derived from linolenic acid (C18:3ω3) involves the active participation of
dietary sources either directly or indirectly from phytanic the peroxisomal ß-oxidation system at the level of the conver-
acid; and (3) di- and trihydroxycholestanoic acid (DHCA and sion of C24:6ω3 to C22:6ω3 (see Fig. 24.1).
ENDOGENOUS
SYNTHESIS
DIET
PHYTANIC ACID LINOLENIC ACID CHOLESTEROL
VLCFA PRISTANIC ACID C24:6w3 DHCA THCA

(e.g. C26:0)
PEROXISOMAL β-OXIDATION
acetyl-CoA’s propionyl-CoA C22:6w3 chenodeoxy choloyl-CoA

+ medium/long-chain + acetyl-CoA DHA choloyl-CoA
acyl-CoA’s + 4,8-dimethyl-
nonanoyl-CoA
taurine/glycine
tauro/glycocheno
MITOCHONDRIAL β-OXIDATION deoxycholate
tauro/glycocholate
+ KREBS CYCLE
CO2 + H2O
bile
Fig. 24.1 Schematic representation of the role of the peroxisomal ß-oxidation system in the oxidation of C26:0, pristanic acid, di-and trihydroxy-
cholestanoic acid and C24:0
24 Peroxisomal Disorders 379
With respect to the enzymes involved in the ß-oxidation of all the ß-oxidation of all these compounds. It now turns out,
these compounds, it was long thought that only a single set of however, that peroxisomes harbour two acyl-CoA oxidases, one
enzymes including acyl-CoA oxidase, l-bifunctional protein for straight-chain fatty acids like C26:0 and one for branched-
with enoyl-CoA hydratase and 3-hydroxyacyl-CoA chain fatty acids (pristanic acid, DHCA and THCA), two bifunc-
dehydrogenase activity and peroxisomal thiolase would catalyse tional enzymes and two peroxisomal thiolases (see Fig. 24.2).
ER
3
Fig. 24.2 Enzymology of the

peroxisomal fatty acid ß-oxidation C26:0 C26:0-CoA pristanic acid pristanoyl-CoA THCA THC-CoA
system. Human peroxisomes
contain two acyl-CoA oxidases, one
specific for straight-chain fatty acids 1 2
ALDP ALDP ? ?
like C26:0, therefore called
straight-chain acyl-CoA oxidase
(SCOX or ACOX1) and a second XALD
one, catalysing the dehydrogenation (24.8)
of α-methyl branched-chain fatty THC-CoA
C26:0-CoA pristanoyl-CoA
acids like pristanoyl-CoA and di-
and trihydroxycholestanoyl-CoA.
The latter enzyme is called
O2 O2
branched-chain acyl-CoA oxidase 4 acyl-CoA oxidase
(BCOX or ACOX2). The deficiency (24.2) 5
enoyl-CoA esters of C26:0, pristanic H2O2 H2O2
acid and DHCA and THCA are all
handled by a single bifunctional
enzyme harboring both enoyl-CoA
hydratase and 3-hydroxyacyl-CoA Δ2-C26:1-CoA Δ2-pristenoyl-CoA Δ24-THC-CoA
dehydrogenase activity (see text
under Sect. 24.3). The newly
identified bifunctional protein which
forms and dehydrogenates
d-3-hydroxy-acyl-CoA esters rather
than l-3-hydroxyacyl-CoAs, is the
single enzyme involved in the
oxidation of C26:0, pristanic acid NAD
and DHCA and THCA. With
NADH
6 D-BP deficiency (24.3)
respect to the two peroxisomal
thiolases, pTH1 and pTH2 (=SCPx),
it is clear now that pTH2 is involved
in the oxidation of α-methyl fatty
acids. Current evidence holds that
pTH1 and pTH2 are both involved
in C26:0 ß-oxidation which explains
why a deficiency of either pTH1 or
3-keto-C26:0-CoA 3-keto-pristanoyl-CoA 24-keto-THC-CoA
pTH2 is not associated with a defect
in C26:0 ß-oxidation. The
obligatory involvement of the
CoASH CoASH
X-linked adrenoleukodystrophy
protein (ALDP) in C26:0 7 RCDP Type 1 8 8
acetyl-CoA (24.5)
ß-oxidation is shown. ALDP is a C3-CoA
so-called half ABC-transporter
which can either form homo- or
heterodimers. The protein is C24:0-CoA 4,8,12-trimethyl choloyl-CoA
localized in the peroxisomal tridecanoyl-CoA
membrane and probably transports
C26:0-CoA esters across the
peroxisomal membrane
Ether Phospholipid Biosynthesis phosphoglycerides) with an α,ß-unsaturated ether bond are also
Ether phospholipids are a special class of phospholipids which known by their trivial name plasmalogens. Platelet-activating
differ from the regular, more well-known diacyl phospholipids factor (PAF; 1-O-alkyl-2-acetylglycerophosphocholine) is
in one major aspect which is the occurrence of an ether linkage the best known ether phospholipid. The alcohol moiety in
rather than an ester linkage at the sn-1 position of the glyc- plasmalogens is usually of the ethanolamine or choline type.
erol backbone. Two groups of ether phospholipids can Figure 24.3 depicts the enzymology of the ether phospholipid
be distinguished with either an 1-O-alkyl or an 1-O-alk-1′- biosynthetic pathway with part of the enzymes localised in
enyl-linkage. The latter phospholipids (1-O-alk-1′-enyl-2-acyl- peroxisomes and another part in the endoplasmic reticulum.
PEROXISOMES ENDOPLASMIC
RETICULUM
acyl-CoA alkyl-DHAP
Fig. 24.3 Pathway for the

DHAP
* RCDP-1 (24.5)
NAD(P)H
synthesis of ether-linked DHAPAT 1 3

phospholipids and especially RCDP-2 (24.6)
plasmalogens (1-O-alk-1”- CoASH NAD(P)+
enyl-2-acylphosphoglycerides)
which are the primary end
products of etherphospholipid
biosynthesis in humans. acyl-DHAP alkyl-G3P
Biosynthesis starts in the
peroxisome with the production
of dihydroxyacetonephosphate
long-chain alcohol acyl-CoA
(DHAP) to acyl-DHAP via the
peroxisomal enzyme
* RCDP-1 (24.5)
Alkyl-DHAP synthase 2 4
dihydroxyacetonephosphate RCDP-3 (24.7)
acyltransferase (DHAPAT) fatty acid CoASH
followed by the introduction of
the typical etherbond by the
enzyme alkyl-DHAP synthase.
Both these enzymes are strictly
alkyl-DHAP 1-alkyl-2-acyl-G3P
peroxisomal. The third step is
catalyzed by the enzyme alkyl/
acyl-DHAP:NAD(P)H
oxidoreductase which has a NAD(P)H H2O
bimodal distribution in both
Alkyl/acyl-DHAP :
peroxisomes and endoplasmic 3 5
NAD(P)H oxidoreductase
reticulum. The product
alkylglycerol-3-phosphate NAD(P)+
(alkylG3P) then undergoes
acylation to form 1-alkyl-2-acyl-
G3P followed by hydrolysis to
produce 1-alkyl-2-acyl-glycerol alkyl-G3P 1-alkyl-2-acylglycerol
which can then be converted into
the different 1-alkyl-2-acyl-
phosphoglycerides and
1-O-alk-1’-enyl-2-
acylphosphoglycerides
(plasmalogens). The asterisk in
the figure indicates that the PLASMALOGENS
enzyme is also deficient in the (1-O-ALK-1’ -ENYL-2ACYL
disorders of peroxisome PHOSPHOGLYCERIDES)
biogenesis
Fatty Acid α-Oxidation The mechanism involved in the oxidative decarboxylation of

3-Methyl-branched fatty acids need to undergo oxidative 3-methyl fatty acids like phytanic acid has long remained
decarboxylation via a process called α-oxidation to release mysterious but has now been resolved (Wanders et al. 2000).
the terminal carboxylgroup as CO2. One of the most impor- Figure 24.4 depicts the enzymology of the pathway.
tant physiological 3-methyl-branched fatty acids is phytanic Current evidence holds that the complete pathway from
acid (3,7,11,15-tetramethylhexadecanoic acid), long known phytanoyl-CoA to pristanic acid, pristanoyl-CoA and then on
to accumulate in Refsum disease as well as in other disorders. to 4,8-dimethylnonanoyl-CoA is intraperoxisomal (Fig. 24.4).
CoASH, ATP AMP, PPi

phytanic acid phytanoyl-CoA
1
? PEROXISOMAL
MEMBRANE
phytanoyl-CoA
2-ketoglutarate, O2
phytanoyl-CoA hydroxylase 2 M.Refsum 24.9
succinate, CO2
2-OH-phytanoyl-CoA
H2O
2-OH-phytanoyl-CoA lyase 3
formate + CoASH formyl-CoA
Fig. 24.4 Structure and pristanal

enzymology of the peroxisomal NAD(P)+
phytanic acid α-oxidation system.
Phytanic acid first undergoes pristanal dehydrogenase 4
activation to phytanoyl-CoA,
probably by the long-chain NAD(P)H
acyl-CoA synthetase (LACS) pristanic acid
present at the outer aspect of the
peroxisomal membrane and then CoASH, ATP
enters the peroxisomal matrix,
pristanoyl-CoA synthetase 5
probably via metabolite carrier
protein. Once inside peroxisomes, AMP, PPi
phytanoyl-CoA undergoes
hydroxylation by the enzyme pristanoyl-CoA
phytanoyl-CoA hydroxylase
deficient in adult Refsum disease. ß-oxidation
The same enzyme is also deficient 3 cycles
in RCDP type 1 (but not in type 2
and 3) since the hydroxylase is a
PTS2-protein (see text). 4,8-dimethylnonanoyl-CoA
2-Hydroxyphytanoyl-CoA then carnitine
undergoes cleavage to pristanal
COT 6
followed by its subsequent
oxidation to pristanic acid. After CoASH
activation to its CoA-esters,
pristanoyl-CoA undergoes 3 4,8-dimethylnonanoyl-carnitine
cycles of ß-oxidation in the
peroxisome to produce
4,8-dimethylnonanyl-CoA which ?
leaves the peroxisome in the form
of a carnitine ester to undergo full
oxidation in mitochondria To mitochondria for full oxidation
Biogenesis of Peroxisomes depicts the current model for peroxisome biogenesis as

Peroxisomal proteins are synthesised on free polyribo- favoured by Gould and Valle (2000). It should be noted that
somes. This applies to both peroxisomal matrix as well as recent evidence holds that peroxisomes start their life in a
membrane proteins. The principal features of peroxisomal special subcompartment of the endoplasmic reticulum which
biogenesis are as follows: (1) peroxisomal matrix and suggests that peroxisomes are not truly autonomous organ-
membrane proteins are synthesised on free polyribosomes; elles like mitochondria but in fact semi-autonomous (Tabak
(2) the newly synthesised proteins are posttranslationally et al. 2003).
imported from the cytosol into pre-existing peroxisomes; Figure 24.2 shows the organisation of the peroxisomal
and (3) import of new polypeptides expands the peroxisomal ß-oxidation machinery involved in the oxidation of very-
compartment making them grow until they reach a critical long-chain fatty acids like C26:0, pristanic acid and DHCA
size which results either in division of peroxisomes into and THCA. Peroxisomes contain two acyl-CoA oxidases,
two daughter peroxisomes or in budding from the peroxi- two bifunctional proteins and two thiolases. Current knowl-
somal reticulum followed by subsequent growth. The fact edge holds (Wanders and Waterham 2006, for review) that
that proteins destined for peroxisomes are synthesised on the oxidation of the CoA-esters of C26:0 on the one hand
free polyribosomes implies that these proteins must possess and pristanic acid and DHCA and THCA on the other hand
specific signals to direct them to peroxisomes. Two of such is catalysed by the two different acyl-CoA oxidases, respec-
peroxisome targeting signals (PTS) have been identified. tively (see Fig. 24.2). In line with this notion, pristanic acid,
The first PTS involves a carboxyterminal tripeptide of the DHCA and THCA are completely normal in patients with
sequence serine-lysine-leucine (SKL) or a variant thereof straight-chain acyl-CoA oxidase (SCOX/ACOX1 = enzyme
and many peroxisomal proteins contain such a PTS1. The 1 in Fig. 24.2) deficiency. The situation with respect to
second peroxisome targeting signal (PTS2) has been found the subsequent steps in peroxisomal ß-oxidation has long
in far less peroxisomal proteins and involves a stretch of 9 remained an enigma but recent data have resolved this.
amino acids of which amino acids 1, 2, 8 and 9 are essential. It turns out that it is not the originally discovered bifunc-
The following consensus has been found for the PTS2-motif: tional protein which forms and dehydrogenates l-3-
(R/K)-(L/V/I)-XXXXX-(H/Q)-L/A) in which X may be any hydroxyacyl-CoA esters but the more recently discovered
amino acid. In mammals, the PTS2 has only been identified d-bifunctional protein which forms and dehydrogenates
in peroxisomal thiolase 1, phytanoyl-CoA hydroxylase and d-3-hydroxyacyl-CoA’s which is involved in the ß-oxida-
alkyl-DHAP synthase. Peroxisomal membrane proteins lack tion of C26:0, pristanic acid and the two cholestanoic acids
either a PTS1 or PTS2 signal which implies the existence DHCA and THCA (Fig. 24.2). The role of the two thiolases
of additional signals. The identification of the PTS1 and in peroxisomal ß-oxidation has also been largely resolved,
PTS2 targeting signals was soon followed by the discovery especially with respect to the role of peroxisomal thiolase
of a growing list of so-called PEX genes coding for proteins 2 which is the suggested name for the 58 kDa protein with
playing an indispensable role in peroxisome biogenesis. sterol carrier protein (SCP) and thiolase activity (SCPx).
These genes were first identified in different yeast species. Studies in mutant mice have shown that this enzyme plays
In the last few years, however, most of the corresponding an indispensable role in the oxidation of fatty acids with an
human homologues have been identified as well, which has α-methyl side chain as in pristanic acid, DHCA and THCA.
allowed molecular analysis in patients affected by a disorder The same is true for patients with human SCPx deficiency
of peroxisome biogenesis (see later). The proteins encoded which has only been described in a single patient so far
by the various PEX genes are called peroxins, and virtu- (Ferdinandusse et al. 2006a, 2006b). Most likely, pTH1
ally all of them are localised in the peroxisomal membrane and pTH2 are both involved in C26:0 ß-oxidation as con-
playing a role in the complicated machinery required for the cluded from the fact that C26:0 ß-oxidation is normal in
transmembrane transport of peroxisomal proteins. Pex5p both pTH1-deficient fibroblasts as well as in pTH2-(SCPx)-
and Pex7p are exceptions since they are largely cytosolic deficient fibroblasts (Wanders et al. 2000). Figures 24.3
proteins. Pex5p is the receptor which picks up proteins con- and 24.4 depict the enzymology of the ether phospholipid
taining a PTS1 motif and directs them to the peroxisome, biosynthesis and phytanic acid α-oxidation systems, respec-
whereas Pex7p does the same for PTS2 proteins. Figure 24.5 tively (see legends for detailed information).
PEX5 PEX7
1. Matrix proteins
PTS1 protein
bound by PTS
receptors
PEX5 PTS2 protein
Cytosol
PEX7
2. Transport to peroxisomes 5. Receptor recycling
4. Receptor-ligand
dissociation & ligand
translocation
3. Receptor docking
1
10
14 2
6
13 12
26
Peroxisome
matrix
PTS1 protein
PTS2 protein
Fig. 24.5 Current model for peroxisome biogenesis. See text
Tables 24.1, 24.2 and 24.3 Zellweger spectrum disorders (24.1), Peroxisomal acyl-CoA oxidase 1 deficiency (24.2) and d-BP deficiency (24.3)
CNS Ataxia ± ± ±
Developmental delay + + + +
Mental retardation + ± ± ±
Muscular hypotonia ± ± ± ± ±
Neuropathy, peripheral ± ± ±
Spastic pareses ± ± ±
Digestive Diarrhoea ± ± ± ±
Jaundice ± ± -- -- --
Portal hypertension ± ± ± ±
Ear Deafness, sensorineural + + + +
(continued)
Tables 24.1, 24.2 and 24.3 (continued)

Eye Cataract, retinitis pigmentosa, ± ± ± ± ±
glaucoma
Diminished visual activity + + + +
Musculoskeletal Club foot ± ± ± ± ±
Dysmorphic features ± ± ± ± ±
Epiphyseal and periarticular ± ± – – –
calcific stippling
Fontanel enlarged + + ± – –
Osteopenia + + + +
Other BAEP diminished response ± ± ± ±
ERG diminished response ± ± ± ±
Failure to thrive ± ± ± ± ±
MRI: cerebral neocortical ± ± ± ± ±
dysplasia
MRI: cerebral white matter ± ± ± ± ±
involvement
Ultrasonography: renal cysts ± ± – – –
Routine ASAT/ALAT (P) n-↑ n-↑ n-↑ n-↑ n-↑
Laboratory Bilirubin – total/direct (P) n-↑ n-↑ – – –
Cholesterol (P)
Coagulation factors (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory Adrenocortical reserve ↓-n ↓-n ↓-n ↓-n
Bile acid intermediates (P) n-↑ n-↑ n-↑ n-↑ n-↑
Docosahexaenoic acid (S, RBC) ↓ ↓-n ↓-n ↓-n ↓-n
Fat-soluble vitamins (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Phytanic acid (S) n-↑ n-↑ n-↑ n-↑ n-↑
Pipecolic acid (S, U) n-↑ n-↑ n-↑ n-↑ n-↑
Plasmalogens (RBC) ↓-n ↓-n ↓-n ↓-n ↓-n
Pristanic acid (S) n-↑ n-↑ n-↑ n-↑ n-↑
VLCFA ↑ ↑ ↑ ↑ n-↑
Table 24.4 Alpha-methylacyl-CoA racemase deficiency

CNS Intellectual disability + (+) (+)
Neuropathy, sensorimotor ±
Seizures (+)
Spastic paraparesis (+)
Stroke-like episodes ±
Digestive Diarrhoea + + -
Liver failure ±
Musculoskeletal Rhabdomyolysis ±
Routine laboratory Alkaline phosphatase (P) ↑
Amino acids (U) ↑
ASAT/ALAT (P) ↑
Phosphate (U) ↑
Special laboratory C26:0 fatty acid (P) n n
C27-bile acid (P) ↑ ↑ ↑
Phytanic acid (P) ↑ (↑)
Pristanic acid (P) ↑ ↑
Tables 24.5, 24.6 and 24.7 Rhizomelic chondrodysplasia punctata type 1 (PEX deficiency) (24.5), type 2 (DHAPAT deficiency) (24.6) and
type 3 (alkyl-DHAP synthase deficiency) (24.7)
Cardiovascular Congenital heart defects ± ± ± ± ±
Severe mental deficiency + + + +
Spastic pareses - ± ± ± ±
Dermatological Ichthyosis ± ± ± ±
Ear Deafness, sensorineural ± ± ± ±
Eye Cataract + + + + +
Musculoskeletal Cervical stenosis + + + + +
Contractures + + + + +
Coronal clefts of thoracic ± ± ± + +
and lumbar vertebral bodies
Dysmorphic features + + + + +
Epiphyseal and periarticular + + ± ± ±
calcific stippling
Growth retardation ± + + + +
Joint contractures + + + + +
Metaphyseal and epiphyseal + + + + +
dysplasia
Microcephaly ± + + +
Shortening of long bones, + + + + +
disproportionately affecting
humeri and femora
Skeletal dysplasia + + + + +
Respiratory Increased rate of infections: + + + +
pneumonia, otitis
Special laboratory Bile acid intermediates (P) n n n n n
Phytanic acid (S) n-↑ n-↑ n-↑ n-↑ n-↑
Plasmalogens (RBC) ↓ ↓ ↓ ↓ ↓
Pristanic acid (S) ↓-n ↓-n ↓-n ↓-n ↓-n
VLCFA n n n n n
Table 24.8 X-linked adrenoleukodystrophy and adrenomyeloneuropathy

CNS Behaviour difficulties ± ± ±
Dementia ± ± ±
EEG, VEP, BAER abnormalities ± ± ±
Epilepsy ± ± ±
Peripheral nerve involvement ± ± ±
Signs of leukodystrophy ± ± ±
Spastic pareses ± ± ±
Sphincter control problems ± ± ±
Ear Perceptive hearing loss ± ± ±
Endocrine Sexual dysfunction ± ±
Eye Vision loss ± ± ±
Special laboratory Adrenal insufficiency ± ± ±
Endocrine gonadal failure ± ± ±
VLCFA ↑ ↑ ↑ ↑ ↑
Table 24.9 Refsum disease (classic, adult)

Cardiovascular Cardiac, anomalies/arrhythmias ± ± ±
CNS Anosmia ± ± ±
Ataxia ± ± ±
Paresis/atrophy/neuropathy ± ± ±
Sensory disturbances ± ± ±
Dermatological Ichthyosis ± ± ±
Ear Deafness ± ± ±
Eye Loss of night vision ± ± ±
Progressive retinitis pigmentosa ± ± +
Musculoskeletal Skeletal malformations ± ±
Special laboratory Phytanic acid (P) ↑ ↑ ↑
Pristanic acid (P) n n n
Table 24.10 Peroxisomal and mitochondrial fission defect

CNS Hypotonia ++
Tendon reflexes ↓↓
Eye Poor visual fixation +
Musculoskeletal Dysmorphic features +
Other Death +
Special laboratory VLCFA ↑
Table 24.11 Peroxisomal bile acid-CoA amino acid transferase (BAAT) deficiency
Dermatological Itching ± ± ±
Digestive Failure to thrive ± ±
Jaundice ± ± ± ±
Steatorrhoea ± ±
Musculoskeletal Rickets ± ±
Prothrombin ratio n-↑
Transaminases (P) n-↑ n-↑
Special laboratory Vitamin A (P) ↓-n ↓-n ↓-n
Vitamin D (P) ↓-n ↓-n ↓-n
Vitamin E (P) ↓-n ↓-n ↓-n
See also Poll-The and Gärtner (2012). children are significantly impaired with profound hypoto-
nia, seizures, retinal degeneration and impaired hearing.
Zellweger Spectrum Disorders (24.1) There is usually calcified stippling of the epiphyses and
Zellweger syndrome (ZS) is generally considered to be the small renal cysts. Brain abnormalities in Zellweger syn-
prototype of the group of peroxisomal disorders. ZS drome include cortical dysplasia and neuronal heterotopia
belongs to the “multiple congenital anomaly” disease cate- but also regressive changes. There is dysmyelination rather
gory and is dominated by 1. the typical craniofacial features than demyelination. In Zellweger syndrome peroxisome
including a high forehead, large anterior fontanel, hypo- biogenesis is defective due to mutations in one of the many
plastic supraorbital ridges, epicanthal folds and deformed PEX genes. Apart from Zellweger syndrome with its
ear lobes and 2. profound neurological abnormalities. ZS neonatal onset and early fatal course, the disorders of
peroxisome biogenesis also include neonatal adrenoleuko- racemase (AMACR) deficiency. The clinical presentation of
dystrophy (NALD) with a less progressive course and leu- disorders 2, 3, and 4 resembles that of the disorders of per-
kodystrophy and infantile Refsum disease (IRD) with the oxisome biogenesis, whereas the clinical presentation of
least severe course. Since the clinical features between X-linked ALD/AMN patients is completely different which
Zellweger syndrome, NALD and IRD are strongly overlap- explains why it is better to discuss this entity separately.
ping, it is often difficult to assign a particular patient to one
of these clinical subtypes. These clinical subtypes are still Peroxisomal Acyl-CoA Oxidase1 Deficiency (24.2)
useful when counselling their families, but one should not Peroxisomal acyl-CoA oxidase1 deficiency was first
place too much emphasis on assigning a patient to one of described in 1988 by Poll-The et al. under the name pseudo-
these categories. Late-onset cerebral white matter/leuko- neonatal adrenoleukodystrophy since the two patients
dystrophy has been described in patients with a Zellweger described had the typical signs and symptoms described for
spectrum phenotype, either following IRD or following neonatal adrenoleukodystrophy by Kelley et al. (1986).
normal early development, and in the absence of distinct However, peroxisomes were present in the patients’ liver
external features (Barth et al. 2001), some patients with a albeit of enlarged size. In addition THCA and DHCA were
ZSD can also present with an isolated peripheral neuropa- completely normal.
thy or present with progressive ataxia and neuropathy
(Régal et al. 2010; Sevin et al. 2011). d-Bifunctional Protein Deficiency (d-BP; 24.3)
When the group of patients suffering from a disorder of The second peroxisomal ß-oxidation disorder, was only
peroxisome biogenesis is taken together, roughly three pro- delineated recently (Suzuki et al. 1997; Van Grunsven et al.
files can be distinguished which should warrant detailed bio- 1998; Ferdinandusse et al. 2006a, 2006b). The majority of
chemical investigations. These include: patients show severe clinical abnormalities including hypo-
1. A neonatal profile, essentially involving (a) severe mus- tonia, craniofacial dysmorphia, neonatal seizures, hepato-
cular hypotonia with resultant poor feeding, (b) seizures, megaly and developmental delay. Most patients with d-BP
(c) hepatic dysfunction including mixed hyperbilirubi- deficiency die in the first year of life. A remarkable observa-
naemic jaundice and (d) dysmorphic signs tion is that patients with d-BP deficiency may show disor-
2. A childhood profile, essentially involving (a) retinopathy dered neuronal migration as in ZS.
often leading to early blindness, (b) sensorineural deaf- Using an exome sequencing approach, Pierce et al. (2010)
ness, (c) hepatic dysfunction that may involve symptoms reported mutations in HSD17B4 in two sisters affected by
of vitamin K-responsive coagulopathy, (d) developmental Perrault syndrome, a condition characterised by ovarian dys-
delay, (e) (often) failure to thrive, (f) dysmorphic signs genesis and hearing loss in females and hearing loss in males.
and (g) adrenal insufficiency The lack of mutations in eight other Perrault families pro-
3. A milder profile, essentially involving (a) cerebellar vides evidence for a genetic heterogenecity of this syndrome
ataxia, (b) neuropathy, (c) sensorineural deafness, (d) (Jenkinson et al. 2011).
retinopathy, (e) cholestatic liver disease during infancy Peroxisomal thiolase deficiency has so far been described
and (f) adrenal insufficency. in only a single patient and remains ill defined (Ferdinandusse
Bony changes in the neonatal and childhood group involve et al. 2006a, 2006b).
a large fontanel which only closes after the first birthday,
osteopaenia of long bones and, in approximately half of the α-Methylacyl-CoA Racemase (AMACR) Deficiency (24.4)
cases, calcified stippling in the epiphyseal and periarticular We identified a peroxisomal disorder in two patients suffering
regions of large joints, especially the patellar region. Cerebral from an adult-onset sensorimotor neuropathy (Ferdinandusse
abnormalities in the neonatal and childhood groups essen- et al. 2000). The enzyme involved catalyses the interconver-
tially comprise two sets of abnormalities: (1) neocortical sion of (2R)- and-(2S)-stereoisomers of α-methyl-branched-
dysgenesis, due to neuronal migration failure arising during chain fatty acids like pristanic acid, THCA and DHCA,
the embryo-fetal period, and (2) metabolic changes involving whereas this enzyme is not involved in VLCFA ß-oxidation,
the process of myelination and, in some cases, leading to which explains the accumulation of THCA, DHCA and pris-
later demyelination. tanic acid but not C26:0 in these patients.
The clinical picture has some resemblance to that of
The Peroxisomal ß-Oxidation Disorders Refsum disease with sensorimotor neuropathy. Other
Five defined disorders of peroxisomal ß-oxidation have been patients presented fulminant liver failure very early in life
identified including (1) X-linked adrenoleukodystrophy/ (Setchell et al. 2003) or stroke-like episodes and/or recur-
adrenomyeloneuropathy, (2) peroxisomal acyl-CoA oxidase rent rhabdomyolysis (Kapina et al. 2010), cerebellar ataxia
1 deficiency, (3) d-bifunctional protein deficiency, (4) per- (Dick et al. 2011) or relapsing encephalopathy (Thompson
oxisomal thiolase 2 deficiency and (5.) 2-methylacyl-CoA et al. 2008).
Rhizomelic Chondrodysplasia Punctata (RCDP) ter disturbances and sexual dysfunction; and (c) “Addison
Spectrum Types 1, 2 and 3 (24.5, 24.6 and 24.7) disease only” which presents with primary adrenocortical
The third group of disorders that can be treated as a rela- insufficiency with an onset usually before 7.5 years and in
tively distinct entity includes rhizomelic chondrodysplasia adulthood without evidence of neurologic abnormality.
and its variants. The classic prototype of this disorder Virtually all patients with X-ALD who reach adulthood
involves severe growth failure, proximal shortening of develop AMN, usually between the third and fourth decade.
extremities, contractures, spasticity, absence of significant All symptoms are progressive, and 10–20 % of males with
development and cataracts. The clinical picture is highly AMN may develop progressive brain involvement as in the
specific and a presumptive diagnosis of RCDP can be made childhood cerebral form. Many (>50 %) female carriers
on clinical grounds. After the age of 1–3 years, there is reso- develop neurologic symptoms that resemble AMN but have
lution of the punctate calcifications leaving abnormal epiph- later onset and milder disease than the affected males.
yses and flared and irregular metaphyses (Gilbert et al. 1976; Despite the large variability in clinical expression, all
Bams-Mengerink et al. 2006). The finding of a deficiency of patients affected seem to suffer from defects in the same
erythrocyte plasmalogens confirms the diagnosis. gene, which encodes a peroxisomal membrane protein
Furthermore, plasma phytanic acid levels are usually belonging to the ABC superfamily of transporters but with
increased due to the fact that phytanic acid α-oxidation is unknown function. Biochemical diagnosis is based on mea-
deficient in RCDP next to the deficient activities of DHAPAT surement of VLCFA in plasma, which is reliable except in
and alkyl-DHAP synthase (see Fig. 24.3), and the aberrant some exceptional cases. Additional studies in fibroblasts,
molecular weight of peroxisomal thiolase (44 kDa instead of including immunological analysis of the ALD protein and
41 kDa). mutation analysis, should be performed.
Patients with the clinical picture of classical RCDP but
with isolated deficiencies of DHAPAT and alkyl-DHAP syn- Refsum Disease (24.9)
thase have also been described (see Gould et al. 2001; Characteristic manifestations of the disease include anosmia
Wanders et al. 2001a for review), emphasising the role of and early-onset retinitis pigmentosa with variable combina-
plasmalogen deficiency in determining the RCDP phenotype. tions of cerebellar ataxia, polyneuropathy and an elevated
In these patients phytanic acid is normal, which would sug- CSF protein level. Less constant features include sensorineu-
gest that distinction between classical RCDP and the two ral hearing loss, ichthyosis, skeletal malformations and car-
variants can best be made on the basis of plasma phytanic diac abnormalities. The clinical picture of Refsum disease is
acid levels. This may lead to erroneous conclusions, however, often that of a slowly developing retinopathy and progressive
the reason being that phytanic acid levels may be normal in peripheral neuropathy manifested by severe motor weakness
young patients affected by classical RCDP since phytanic and muscular wasting, especially of the lower extremities.
acid is derived from dietary sources only. Furthermore, some The clinical course is variable. Exacerbations may occur
patients have been described with a milder form of classical with acute illness, fasting, rapid weight loss and when preg-
RCDP, with normal plasma phytanic acid levels despite a nant. Onset has occasionally been detected in early child-
deficiency of phytanic acid α-oxidation in fibroblasts as part hood but not until the fifth decade in others.
of the classical tetrad of four abnormalities. Most patients have clear-cut manifestations before 20
Milder phenotypes, showing absence of significant short- years of age. Patients with Refsum disease may die suddenly
ening of the long bones, milder mental and growth deficiency probably from cardiac arrhythmias and heart failure caused
and cataracts are known (Smeitink et al. 1992; Barth et al. by cardiomyopathy (Wierzbicki et al. 2002; Wanders et al.
1996). Such cases have been found both in the group of mul- 2001b).
tiple peroxisomal abnormalities (RCDP type 1) and with iso-
lated DHAPAT deficiency (RCDP type 2). Dynamin-Like Protein 1 Deficiency (24.10)
The combination of microcephaly, abnormal brain gyral pat-
X-Linked Adrenoleukodystrophy (X-ALD)/ tern, optic atrophy, hypotonia, absence of development and
Adrenomyeloneuropathy (AMN) (24.8) persistent lactic acidaemia with a mildly elevated plasma
X-ALD/AMN comprises a group of disorders with cerebral, concentration of VLCFA has been reported in an infant girl
spinal long-tract and adrenal cortex symptoms in affected who died at the age of 37 days. The gene involved encodes
males, and to some extent also in female heterozygotes (see dynamin-like protein 1 (DLP1). Immunocytochemical stud-
review Moser et al. 2001; Smith et al. 1999). Three main phe- ies in cultured fibroblasts suggested a defect in mitochon-
notypes are seen in affected males: (a) the childhood cerebral drial and peroxisomal fission (Waterham et al. 2007).
form that manifests between ages 3 and 12 years (it initially
resembles attention deficit disorder or hyperactivity progress- Peroxisomal Bile Acid-CoA: Amino Acid Acyltransferase
ing to total disability or death usually within 2 years); (b) Deficiency (BAAT) Deficiency (24.11)
adrenomyeloneuropathy (AMN) which manifests most com- Recent studies, notably by Pellicoro et al. (2007), have shown
monly in the late twenties as progressive paraparesis, sphinc- that the enzyme bile acid-CoA:amino acid N-acyltransferase
(BAAT) is exclusively localised in peroxisomes and not in the gene. This is done in only few laboratories in the world
cytosol nor in the cytosol and in peroxisomes as believed until including our own (see Ebberink et al. (2010) for recent
recently. As a consequence, BAAT deficiency should be review and update). If a POD has been established, the nature
included in the list of peroxisomal disorders. Although only of the true enzymatic defect needs to be determined using
few patients have been described, the overall picture is that of a direct enzyme assays for acyl-CoA oxidase, d-bifunctional
patient presenting variable growth failure, mild or no jaundice, protein and the peroxisomal thiolases (Wanders et al. 2001a)
coagulopathy and increased amounts of unconjugated serum followed by molecular analyses (Ferdinandusse et al. 2006a,
bile acids (Carlton et al. 2003). 2006b, 2007).
Rhizomelic Chondrodysplasia Punctata (RCDP)
Spectrum, Classical and Variant Phenotypes (Fig. 24.7). If
24.5 Diagnostic Flow Charts a patient is suspected to suffer from RCDP, either the classi-
cal or variant phenotype, erythrocyte plasmalogens should be
From a clinical diagnostic point of view, the group of peroxi- determined immediately using the simple method described
somal disorders can be subdivided into four distinct catego- by Bjorkhem et al. (1986) or any other established method
ries including (1) the peroxisome biogenesis disorders including the one described by Vreken et al. The finding of
(PBDs) with generalised peroxisomal dysfunction and the deficient plasmalogens implies that one is dealing with a
single peroxisomal ß-oxidation disorders (PODs), (2) the peroxisomal form of RCDP, either type 1, 2 or 3. This can
(non)rhizomelic chondrodysplasia punctata (RCDP) group, be resolved by detailed studies in fibroblasts which involve
(3) the X-linked adrenoleukodystrophy/adrenomyeloneu- a series of enzymatic studies including determination of
ropathy complex and (4) the remaining disorders which DHAPAT and alkyl-DHAP synthase activities, immunob-
share very little with the disorders under groups 1, 2 and 3 lot analysis, phytanic acid α-oxidation, phytanoyl-CoA
and should be treated individually. hydroxylase activity measurement, and de novo plasmalo-
gen biosynthesis, a.o. Finally, mutation analysis needs to be
Peroxisome Biogenesis Disorders (PBDs) and the done to identify the true molecular basis (Motley et al. 2002;
Peroxisomal ß-Oxidation Disorders (PODs) (Fig. 24.6). Braverman et al. 2002; Ofman et al. 1998; De Vet et al. 1998).
When a patient is suspected to suffer from a PDB or POD, X-linked adrenoleukodystrophy/adrenomyeloneurop-
the first analysis to be made is analysis of very-long- athy complex (Figs. 24.8 and 24.9)
chain fatty acids. If abnormal, it is clear that the patient is Two diagnostic flow charts need to be considered depend-
either affected by a PDB or POD. If normal, a PBD or ing whether one is dealing with a male or female patient. (a)
POD is virtually excluded although exceptions to this rule If a male patient is suspected to suffer from some form of
have been published (Roosewich et al. 2006; Soorani- X-ALD/AMN, very-long-chain fatty acids should be anal-
Lunsing et al. 2005; Ferdinandusse et al. 2006a, 2006b). ysed, which is unequivocal in all cases with one or two pos-
Usually, if VLCFAs have been found to be abnormal, we sible exceptions reported in literature. If abnormal, X-ALD/
immediately ask for fibroblasts since a detailed analysis AMN is virtually established. This should be followed up by
in fibroblasts is absolutely essential to ascertain with full DNA analyses in lymphocytes prepared from the same sam-
certainty whether peroxisome biogenesis is primarily ple. We usually do fibroblast studies as well to ascertain that
affected or whether one is dealing with a peroxisomal C26:0 ß-oxidation is, indeed, deficient in the patient’s cells.
single ß-oxidation disorder. Alternatively, transformed Furthermore, this allows us to do immunofluorescence
lymphoblasts may be used for that purpose (Grønberg microscopy analysis to establish whether the ALD protein is
et al. 2010). present or not. Remarkably, ALDP is absent in about 75 % of
In the meantime plasmalogen levels should be measured X-ALD/AMN patients. If ALDP is absent, this can be of
in erythrocytes, using the method described by Bjorkhem great help in carrier detection (see below). (b) Females: if a
et al. (1986) or another established method like the one female is suspected to suffer from X-ALD/AMN in its het-
described by Vreken et al. (2000). If plasmalogens are erozygous form, two situations must be distinguished. The
decreased, a peroxisome biogenesis defect is established. If simplest scenario is when the lady is from a known X-ALD/
plasmalogens are normal, however, it probably is a POD but AMN family. In that case, VLCFA analysis can be done, but
it may also be a peroxisome biogenesis defect simply because it is much better to do DNA analysis right away. If the lady
in milder PBD forms, notably infantile Refsum disease has no family background of X-ALD/AMN, we suggest to
patients, plasmalogens may be completely normal. perform the complete diagnostic programme including (1)
In addition to the measurement of plasmalogens, we mea- VLCFA analysis in plasma/serum, (2) VLCFA- and ALDP-
sure the levels of the peroxisomal metabolites di- and trihy- immunofluorescence analysis in fibroblasts and ultimately
droxy/cholestanoic acid, phytanic acid and pristanic acid which (3) DNA analysis. Especially, immunofluorescence micros-
may also help to reach to correct diagnosis (see Fig. 24.6). copy analysis may be helpful here since a mosaic pattern of
If a PBD has been established, complementation analysis ALDP-positive and ALDP-negative cells immediately points
needs to be done followed by analysis of the relevant PEX to a female heterozygote.
CLINICAL SUSPICION FOR A PEROXISOME

BIOGENESIS DISORDER (PBD) OR A PEROXISOMAL FATTY
ACID β-OXIDATION DISORDER (POD)
Neonatal onset profile Infantile onset profile

- Neonatal hypotonia - Retinopathy
- Neonatal seizures - Sensorineural deafness
- Liver dysfunction - Liver dysfunction
- External dysmorphia - Developmental delay
- External dysmorphia
- Failure to thrive
normal VLCFA-analysis
Definitely
not
abnormal
a PBD or POD
deficient PLASMALOGEN-ANALYSIS normal
Definitely Probably POD

PBD, not POD PBD not excluded
Full study in plasma which includes: 1. DHCA and THCA

2. Phytanic acid and 3. Pristanic acid plus full studies in fibroblasts
PBD POD
Complementation analysis
Complementation analysis
+ enzyme activity analysis
Complementation group
1. Acyl-CoA oxidase deficiency Unknown
2. D-bifunctional protein deficiency defect
Molecular analysis
of relevant PEX gene
Molecular analysis
Fig. 24.6 Flow chart for the differential diagnosis of patients suffering from a peroxisome biogenesis disorder (PBD) or a peroxisomal ß-oxida-
tion disorder (POD)
Fig. 24.7 Flow chart for the

differential diagnosis of
rhizomelic chondrodysplasia
punctata (RCDP) type 1, 2 or 3 CHONDRODYSPLASIA PUNCTATA, RHIZOMELIC FORM :
CLASSICAL OR VARIANT PRESENTATION
Non-classical presentations of
Classical rhizomelic (R)CDP including atypical bone
chondrodysplasia punctata dysplasia with cataract
and mental retardation
PLASMALOGENS IN ERYTHROCYTES
Normal Deficient
PHYTANIC ACID IN PLASMA OR SERUM
Peroxisomal form
of (R)CDP type 1, Phytanic acid Phytanic acid
2 or 3 excluded elevated normal
Probably
RCDP type 1
RCDP type 2 or 3
Full enzymatic and molecular study in fibroblasts

X-LINKED ADRENOLEUKODYSTROPHY /
ADRENOMYELONEUROPATHY COMPLEX :
CLINICAL SUSPICION
(MALES)
NORMAL VLCFA-analysis
ABNORMAL
X-ALD/AMN
X-ALD/AMN
excluded
FIBROBLAST-STUDIES
1. VLCFA metabolism
2. ALDP-analysis
DNA-analysis
Fig. 24.8 Flow chart for the diagnosis of X-linked adrenoleukodystrophy/adrenomyeloneuropathy (males)
X-LINKED ADRENOLEUKODYSTROPHY /
ADRENOMYELONEUROPATHY COMPLEX :
CLINICAL SUSPICION
(FEMALES)
(a) (b)
KNOWN X-ALD/AMN NO X-ALD/AMN

family family background
VLCFA-analysis
Plasma & Fibroblast NORMAL ABNORMAL

studies
Heterozygosity
Fibroblast studies for ALD/AMN
virtually established
DNA-analysis DNA-analysis
Fig. 24.9 Flow chart for the diagnosis of X-linked adrenoleukodystrophy/adrenomyeloneuropathy (females)
24.6 Specimen Collection (Tables 24.1, and reliable technique is immunoblot analysis of peroxisomal
24.2, 24.3, 24.4, 24.5, 24.6, 24.7, acyl-CoA oxidase and peroxisomal thiolase, which is used for
24.8, 24.9, 24.10 and 24.11) the prenatal diagnosis of the disorders of peroxisome biogen-
esis, rhizomelic chondrodysplasia punctata, acyl-CoA oxidase
Very-long-chain fatty acids (VLCFAs), trihydroxycholesta- deficiency and thiolase deficiency. Results are especially good
noic acid (THCA), phytanic acid and pristanic acid can all be in chorionic villous tissue, thus eliminating the risk of material
measured in plasma/serum (1 ml). From the same blood overgrowth, which may occur upon culturing cells. We have
sample, erythrocytes, platelets and/or leukocytes can be pre- recently switched predominantly to molecular analysis if the
pared for determination of plasmalogens and dihydroxyace- molecular defect has been established in the index patient.
tonephosphate acyltransferase (DHAPAT), respectively.
24.8 Treatment
For patients with abnormal peroxisome biogenesis, the pos-
Prenatal diagnostic methods have been developed for all sibilities for treatment are very poor.
peroxisomal disorders and the feasibility of the methods In Zellweger spectrum disorders, the focus is on symp-
has been established through the years. An especially powerful tomatic and supportive therapy and may include cataract
removal in infancy, glasses, hearing aids, gastrostomy to pro- 24.2 Peroxisomal Acyl-CoA Oxidase 1 Deficiency; 24.3
vide adequate calories, fat-soluble vitamin (e.g. K and E) d-Bifunctional Protein Deficiency; 24.4 Alpha-
supplementation, hyperoxaluria treatment, antiepileptic Methylacyl-CoA Racemase Deficiency (Tables 24.12 and
drugs and preventing an Addison crisis. Recently, a double- 24.13)
blind placebo-controlled trial with DHA supplementation Follow-up as in Zellweger spectrum disorders, except for
showed no benefit over a 1-year period in the two outcome hyperoxaluria and adrenal function.
measures used, growth and visual function (Paker et al.
2010). Anecdotal evidence suggests that ZSD patients may 24.5–24.7 Rhizomelic Chondrodysplasia Punctata Types
benefit from primary bile acid therapy (Setchell et al. 1992). 1–3 (Tables 24.14 and 24.15)
The effectiveness of a diet low in phytanic acid has never Recommendations for medical follow-up include growth
been proven with certainty. parameters, full skeletal survey and orthopaedic care, vision
The supportive and symptomatic treatment for the RCDP testing (or evaluation of visual acuity), developmental
spectrum may include cataract extraction in order to restore
some vision, physical therapy to improve contractures and
Table 24.12 Supportive treatment of ZSD, DBP, acyl-CoA oxidase 1
antiepileptic drugs. Dietary restriction of phytanic acid to deficiency
avoid the consequences of phytanic acid accumulation over- System Problem Intervention
time may be beneficial in milder forms of RCDP. CNS Seizures Anticonvulsent medication
Alkylglycerol supplementation has not shown dramatic clin- Behaviour problems Behaviour management
ical benefit in RCDP patients (personal results). Developmental delay Appropriate educational
In X-linked ALD patients, corticosteroid replacement support and intervention
therapy is essential for those with adrenal insufficiency. Drooling Botox injections, none
Allogeneic haematopoietic stem cell transplantation with a Eye Retinal dystrophy Glasses, none
HLA-matched donor or cord blood can stabilise or even Cataract Removal in infancy
reverse cerebral demyelination when the procedure is per- Ear Deafness Hearing aids
formed at a very early age (Miller et al. 2011). Autologous Dental Enamel abnormality Appropriate intervention
Caries Oral hygiene
haematopoietic stem cell gene therapy with lentiviral vector
Digestive Hepato-splenomegaly None
may prove a valuable alternative (Cartier et al. 2009).
Swallowing problems Pureed diet, freqment meals;
Lorenzo’s oil (a mixture of oleic and erucic acid) allows nor- Gastrostomy
malisation of plasma VLCFA but unfortunately has no cura- Loose stools Surveillance of
tive or preventive effects, at least in symptomatic patients malabsorption, elemental
(Aubourg et al. 1993). There is a retrospective study suggest- formulas may be better
tolerated
ing that it may be beneficial in delaying the onset of neuro-
logical symptoms if administered to asymptomatic patients
(Moser et al. 2005). Table. 24.13 Therapeutic options for Zellweger spectrum disorders
In classical Refsum disease dietary restriction of phytanic
Defect/deficiency Medication/diet Dosage
acid intake, with or without plasma pheresis, helps to resolve Vitamin A Vitamin A 2,500 U/day
the ichthyosis and to arrest the progression of the neuropa- Vitamin E Alpha-tocopherol 50 mg/day
thy. An adequate caloric intake prevents mobilisation of phy- acetate
tanic acid into the plasma. Vitamin K Vitamin K Up to 1 mg/day
(phytomenadione) or more
Docosahexaenoic acid Docasahaexanoic acid 100 mg/kg/day
24.9 Follow-Up and Monitoring Plasmalogens Ether lipids 50 mg/kg/day
(batylalcohol)
Accumulation of bile Cholic acid 10–15 mg/kg/day
24.1 Zellweger Spectrum Disorders (Tables 24.12 and acids (di- and
24.13) trihydroxycholestanoic
To establish the extent of disease in a patient affected by a acids)
Zellweger spectrum disorder, systematic evaluation should Phytanic acid Phytanic acid reduced Reduce following
accumulation diet with adequate products: cow’s
be tailored to disease severity. The following are recom-
caloric intake milk products,
mended: growth and nutrition, hearing, comprehensive certain fatty fish
ophthalmology assessment, liver function and clotting fac- Hyperoxaluria Sufficient fluid intake
tors, development, neurologic function, monitoring for and citrate
hyperoxaluria and endocrine evaluation of adrenal Adrenal insufficiency Adrenal hormone
function. replacement
Table 24.14 Supportive treatment of Rhizomelic chondrodysplasia Table 24.16 Supportive treatment of X-ALD/AMN males
punctata spectrum System Problem Intervention
System Problem Intervention Nervous Behaviour problems Behaviour management,
CNS Seizures Anticonvulsent system psychiatric consultation
medication Dementing General supportive care,
Developmental delay Appropriate educational psychological support
support and intervention Myeloneuropathy (adult Physical therapy,
Eye Cataract Removal in infancy man) treatment of spasticity
Digestive Swallowing problems Gastrostomy neuropathic pain and
Cardio-vascular Congenital heart Appropriate support and bladder issues
disease intervention –Paraparesis
Skeletal Contractures Physical therapy –Sphincter disturbances Management of
Respiratory Respiratory tract Appropriate support and –Sexual dysfunction complications
infections intervention Digestive Insufficient nutritional
Skin Dry skin, ichthyosis Hydrating creams gastrostomy intake
Table 24.17 Therapeutic options for X-ALD/AMN males

Table 24.15 Therapeutic options for Rhizomelic chondrodysplasia
punctata spectrum Defect/deficiency Medication/diet Dosage
Defect/deficiency Medication/diet Dosage Adrenal Adrenal hormone replacement
insufficiency
Plasmalogens Ether lipids 50 mg/kg/day
(batyl alcohol) VLCFA Lorenzo’s oila
accumulation
Phytanic acid Phytanic acid Reduce following products:
accumulation reduced diet cow’s milk products Neurologic Hematopoietic stem cell or bone
dysfunction marrow transplantsb
Autologous hematopoietic stem cell
assessment, cardiac examination and assessments for seizure gene therapy (under investigation)
control. a
Lorenzo’s oil is a mixture of oleic and erucic acid and it was associated
with a reduced risk of developing MRI abnormalities if administered to
asymptomatic patients (boys); Moser et al. (2005)
24.8 X-Linked Adrenoleukodystrophy and b
Recommended only for boys or adolescents with evidence of brain
Adrenomyeloneuropathy (Tables 24.16 and 24.17) involvement by MRI but minimal neuropsychologic findings and clini-
In presymptomatic boys with X-ALD under 15 years of age, cal neurologic abnormalities (Miller et al. 2011)
clinical, neurological and neuroradiological assessment
should be performed every 6 months from 3 until 10 years of brain MRI should be monitored closely for signs of progres-
age and yearly thereafter. Endocrinological evaluation sion of the disease before HSCT is undertaken.
should include assessment of adrenal function by ACTH Patients with AMN should be monitored for adrenal
stimulation testing. Neurological function should be evalu- insufficiency and steroid replacement therapy should be ini-
ated by assessing vision, hearing, speech, gait, fine motor tiated as necessary. This is effective, but often neglected, and
skills and the patient’s daily activities. several patients have died in adrenal crisis. Performance of
Neuropsychological assessment is valuable in the early brain MRI is recommended yearly or every other year to
detection of patients with risk of progressive cerebral indentify early the 20–30 % of AMN patients who also
involvement. Neuropsychological function may be develop inflammatory brain involvement and who may be
assessed by the use of the Wechsler Intelligence Scale for candidates for HSCT.
Children or the Wechsler Preschool and Primary Scale of For affected females with X-ALD, the extent of disease
Intelligence. should be monitored as for men with AMN: progressive gait
Newly recognised brain lesions must be interpreted in the disorders, abnormalities of sphincter control and sensory dis-
context of other evidence of progressive disease, including turbances mainly affecting the legs. Adrenal function is usu-
expanding lesions on follow-up MRI, typical gadolinium ally normal. Since women with X-ALD very rarely develop
enhancement, characteristic changes in MR spectra, progres- cerebral involvement and adrenocortical insufficiency, peri-
sive neuropsychological impairment or new neurological odic evaluation of adrenal function and brain MRI is not
symptoms. Haematopoietic stem cell transplantation (HSCT) mandatory.
is recommended for patients whose cognitive abilities exceed
a verbal or performance IQ of 80. If MRI abnormalities 24.9 Refsum Disease (Tables 24.18 and 24.19)
increase over a 3- to 6-month period, HSCT should be con- The following evaluations are recommended: ophthalmol-
sidered even in the absence of neurological or neuropsycho- ogy, anosmia testing, neurologic examinations, audiology
logical deficits. Boys without evidence of abnormality on and cardiology.
Table 24.18 Supportive treatment of Refsum disease Bams-Mengerink AM, Majoie CBLM, Duran M et al (2006) MRI of
System Problem Intervention the brain and cervical spinal cord in rhizomelic chondrodysplasia
punctata. Neurology 66:798–803
Eye Retinitis pigmentosa Appropriate support and
Barth PG, Wanders RJ, Schutgens RB et al (1996) Variant rhizomelic
Cataract in some intervention
chondrodysplasia punctata (RCDP) with normal plasma phytanic
Ear Deafness Hearing aids acid: clinico-biochemical delineation of a subtype and complemen-
Smell Anosmia Appropriate support tation studies. Am J Med Genet 62:164–168
Cardio-vascular Arrhytmia, Anti-arrhytmic and Barth PG, Gootjes J, Bode H et al (2001) Late onset white matter dis-
cardiomyopathy cardiogenic supportive ease in peroxisome biogenesis disorder. Neurology 57:1949–1955
drug Bjorkhem I, Sisfontes L, Bostrom B et al (1986) Simple diagnosis of
Skin Ichthyosis Hydrating creams the Zellweger syndrome by gas–liquid chromatography of dimeth-
ylacetals. J Lipid Res 27:786–791
Braverman N, Chen L, Lin P et al (2002) Mutation analysis of PEX7 in 60
probands with rhizomelic chondrodysplasia punctata and functional
Table 24.19 Therapeutic options for Refsum disease correlations of genotype with phenotype. Hum Mutat 20:284–297
Defect/deficiency Medication/diet Dosage Carlton VE, Harris BZ, Puffenberger EG (2003) Complex inheritance
of familial hypercholanemia with associated mutations in TJP2 and
Phytanic acid Dietary restriction of Below 10 mg/day
BAAT. Nat Genet 34:91–96
accumulation phytanic acid intake
Cartier N, Hacein-Bey-Abina S, Bartholomae C et al (2009)
Plasmapheresis, Hematopoietic stem cell gene therapy with a lentiviral vector in
lipapheresis X-linked adrenoleukodystrophy. Science 326:818–823
Avoidance of sudden Appropriate caloric De Vet EC, Ijlst L, Oostheim W et al (1998)
weight loss intake Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome
biogenesis disorders and identification of the point mutation under-
lying a single enzyme deficiency. J Biol Chem 273:10296–10301
Table 24.20 Therapeutic options for Peroxisomal bile acid/amino Dick D, Horvath R, Chinnery PF et al (2011) AMACR mutations cause
acid transferase deficiency late-onset autosomal recessive cerebellar ataxia. Neurology
Defect/deficiency Medication Dosage 76:1768–1770
Ebberink MS, Mooijer PAW, Gootjes J et al (2010) Genetic classifica-
Vitamin D Vitamin D 2,500 U/day
tion and mutational spectrum of more than 600 patients with a
Vitamin E Alpha-tocopherol 50 mg/day Zellweger syndrome spectrum disorder. Hum Mutat 32:59–69
acetate Ferdinandusse S, Denis S, Clayton PT et al (2000) Mutations in the
Vitamin K Vitamin K Up to 1 mg/day gene encoding peroxisomal alpha-methylacyl-CoA racemase cause
(phytomenadione) or more adult-onset sensory motor neuropathy. Nat Genet 24:188–191
Accumulation of Glycocholic acid 15 mg/day Ferdinandusse S, Denis S, Mooyer PAW et al (2006a) Clinical and bio-
unconjugated cholic acid chemical spectrum of D-bifunctional protein deficiency. Ann Neurol
59:92–104
Ferdinandusse S, Kostopoules P, Denis S et al (2006b) Mutations in the
Monitoring of weight and plasma phytanic acid levels gene encoding peroxisomal sterol carrier protein X (SCPx) cause
with appropriate modifications of the diet should be done leukoencephalopathy with dystonia and motor neuropathy. Am J
regularly. Hum Genet 78:1046–1052
Ferdinandusse S, Denis S, Hogenhout EM et al (2007) Clinical, bio-
chemical, and mutational spectrum of peroxisomal acyl-coenzyme
24.11 Peroxisomal Bile Acid-CoA, Amino Acid N- A oxidase deficiency. Hum Mutat 28:904–912
Acyltransferase (BAAT) Deficiency Gilbert EF, Opitz JM, Spranger JW et al (1976) Chondrodysplasia
The therapeutic options for this disorder are depicted in punctata-rhizomelic form. Pathologic and radiologic studies of
Table 24.20. three infants. Eur J Pediatr 123:89–109
Gould SJ, Valle D (2000) Peroxisome biogenesis disorders: genetics
and cell biology. Trends Genet 16:340–345
Acknowledgements The authors gratefully acknowledge Mrs. Maddy Gould SJ, Raymond GV, Valk D (2001) The peroxisome biogenesis
Festen-Sidler and Mrs. J.A. van Leeuwen-Wesselingh for their expert disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The
preparation of the manuscript and Mr. J.P.N. Ruiter for the artwork. metabolic and molecular bases of inherited disease, 8th edn.
Supported by grants from the Dutch Organisation for Scientific Research McGraw-Hill, New York, pp 3181–3217
(NWO), Medical Sciences, the European Leukodystrophy Association Grønberg S, Krätzner R, Spiegler J et al (2010) Typical cMRI pattern as
(Project no FP7-Health-2009-single-stage-241622), the European Union diagnostic clue for D-bifunctional protein deficiency without appar-
Peroxisome Project (Project no LSHG-CT-2004-512018) and the ent biochemical abnormalities in plasma. Am J Med Genet A
Prinses Beatrix Fonds (Project no MAR-03-216), Den Haag, the 152A:2845–2849
Netherlands. Jenkinson EM, Clayton-Smith J, Mehta S et al (2011) Perrault syn-
drome: further evidence for genetic heterogeneity. J Neurol.
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Orphanet J Rare Dis 6:8
Lysosomal Storage Disorders
Including Neuronal Ceroid 25
Lipofuscinoses
Carla Hollak, Matthias Kettwig, Lars Schlotawa,

and Robert Steinfeld
Contents Summary
25.1 Introduction ..................................................................... 400 Metabolic disorders that affect the lysosomal degradation of
complex carbohydrates, lipids, and proteins are associated
25.2 Nomenclature................................................................... 401
with a wide variety of clinical symptoms. At the severe end
25.3 Metabolic Pathway .......................................................... 403 of the clinical spectrum, neurological symptoms are com-
25.4 Gaucher Disease, Saposin C Deficiency mon and go along with significant brain pathology. The high
and Limp2 Deficiency ..................................................... 406 abundance of sphingolipids in brain partially explains the
25.5 Fabry Disease................................................................... 407 major neurodegenerative effect of disturbed sphingolipid
breakdown. In fact, sphingolipidoses frequently present with
25.6 Sign and Symptoms......................................................... 408
an involvement of the cerebral white matter. Other lysosomal
25.7 Diagnosis and Reference Values..................................... 423 disorders, in particular the neuronal ceroid lipofuscinoses
25.8 Diagnostic Flow Charts .................................................. 424 (NCLs), primarily affect the cerebral grey matter. The NCLs
25.9 Diagnostics ....................................................................... 426 share the lysosomal accumulation of autofluorescent storage
material (lipofuscin) and often result from the disturbed deg-
25.10 Treatment ......................................................................... 427
radation of hydrophobic proteins. Table 25.1 lists lysosomal
25.11 Follow-Up and Monitoring of Gaucher Disease ........... 428 storage diseases that are associated with neurodegeneration.
25.12 Follow-Up and Monitoring of Krabbe Disease ............. 430 At the milder end of the clinical spectrum, variable degrees
of organ involvement exist, such as renal and cardiac disease
References ...................................................................................... 431
in Fabry disease and hepatosplenomegaly with bone sym-
toms in Gaucher disease. Neurological involvement is less
prominent in these disorders.
C. Hollak Table 25.1 Lysosomal storage diseases associated with

Division of Endocrinology and Metabolism, neurodegeneration
Department of Internal Medicine, F5-170, Lysosomal storage
Academic Medical Center, University of Amsterdam, Clinical symptoms Storage material disease
PO box 22700, 1100 DE Amsterdam, The Netherlands Facial dysmorphism, Mucopolysaccharides,
Mucopolysaccharidoses,
e-mail: c.e.hollak@amc.uva.nl dysostosis multiplex, oligosaccharides, oligosaccharidoses,
M. Kettwig • L. Schlotawa hepatosplenomegaly, sphingolipids mucolipidoses, multiple
Department of Pediatrics, variable sulfatase deficiency,
University Medical Center Göttingen, neurodegeneration GM1-gangliosidosis,
Robert-Koch-Str. 40, 37075 Göttingen, Germany galactosialidosis
e-mail: matthias.kettwig@med.uni-goettingen.de; Hepatosplenomegaly, Sphingolipids, lipids Sphingolipidoses,
lars.schlotawa@med.uni-goettingen.de significant M. Wolman
neurodegeneration
R. Steinfeld (*)
Department of Pediatrics, Major Sphingolipids, GM2-gangliosidoses,
University of Göttingen, neurodegeneration proteins MLD, M. Krabbe,
Robert-Koch-Str. 40, 37075 Göttingen, Germany neuronal ceroid
e-mail: rsteinfeld@med.uni-goettingen.de lipofuscinoses
400 C. Hollak et al.
25.1 Introduction The neuronal ceroid lipofuscinosis (NCL) are a group of

neurodegenerative diseases of childhood and adulthood that
This chapter deals with lysosomal storage disorders associ- are characterized by the lysosomal accumulation of autofluo-
ated with CNS involvement and neurodegeneration and atten- rescent material, most commonly subunit c of mitochondrial
uated forms of disease. The majority of disorders are caused ATP synthase and sphingolipid activator proteins A and C. The
by the inherited defect of a lysosomal hydrolase that is incidence of the NCL is estimated to be 1:20.000. The major
involved in the degradation of sphingolipids and lipids. clinical symptoms include psychomotor regression, movement
Mucolipidoses and multiple sulfatase deficiency result from disorder, myoclonic epilepsy, progressive loss of vision,
the disturbed targeting or processing of multiple lysosomal behavioral disturbance, and cognitive decline leading to
enzymes. Therefore, the multisystemic symptoms that are dementia. The early-onset forms of NCL are characterized by
found with mucolipidoses, multiple sulfatase deficiency, severe brain atrophy, decerebration, and premature death. Five
GM1-gangliosidosis, and galactosialidosis resemble the clini- clinical groups – congenital (CNCL), infantile (INCL), late
cal picture of mucopolysaccharidoses and oligosaccharido- infantile (LINCL), juvenile (JNCL), and adult (ANCL) – refer
ses. However, GM1-gangliosidosis and galactosialidosis share to the age of disease onset and are caused by mutations in at
clinical features and metabolic pathology with the sphingo- least 13 different genes (see Table 25.2 below). The majority
lipidoses as well. Two sphingolipidoses, M. Gaucher and M. of NCL cases are associated with autosomal recessive muta-
Fabry, have been well studied and were the first lysosomal tions in the PPT1 (CLN1), TPP1 (CLN2), and CLN3 genes. In
storage diseases that could be treated by enzyme replacement addition, congenital NCL (CLN10), variant late-infantile
therapy (ERT). Lysosomal diseases that primarily manifest forms (CLN5, CLN6, CLN7, and CLN8), progressive myo-
with neurological symptoms (GM2-gangliosidoses, MLD, clonic epilepsies (CLN8 and CLN14), variant juvenile forms
M. Krabbe, neuronal ceroid lipofuscinoses) are often diag- (“CLN9” and CLN12), and various adult forms (CLN4A,
nosed with significant delay and require brain imaging to CLN4B, CLN11, CLN13) are considered NCL disorders.
assess the brain pathology. Nearly all of these diseases can be Specific mutations in early-onset NCL genes are associated
subdivided in different clinical forms by age of onset and with slower disease progression mimicking a late-infantile,
severity due to residual activity of the corresponding enzyme. juvenile, and adult NCL phenotype (see table below).
Table 25.2 Genetic heterogeneity of NCL disorders

Disease abbreviation
(inheritance) Gene symbol Protein (type) Typical NCL disease Atypical NCL disease
CLN10 CTSD Cathepsin D (lysosomal enzyme) Congenital Late infantile, juvenile,
(autosomal recessive) adult
CLN1 PPT1 Palmitoyl protein thioesterase 1 Infantile Late infantile, juvenile,
(autosomal recessive) (lysosomal enzyme) adult
CLN14/EPM3 KCTD7 KCTD7 (potassium channel) Infantile
(autosomal recessive)
CLN2 TPP1 Tripeptidyl peptidase 1 (lysosomal Late infantile Juvenile
(autosomal recessive) enzyme)
CLN5 CLN5 CLN5 (soluble lysosomal protein) Late infantile Juvenile, adult
CLN6 CLN6 CLN6 (ER transmembrane protein) Late infantile Adult
CLN7 MFSD8 MFSD8 (lysosomal transporter) Late infantile Juvenile
CLN8 CLN8 CLN8 (ER-Golgi transmembrane Late infantile Juvenile (EPMR)
(autosomal recessive) protein)
CLN3 CLN3 CLN3 (Golgi-lysosome transmembrane Juvenile
(autosomal recessive) protein)
CLN12/KFS ATP13A2 ATP13A2 (P-type ATPase, lysosomal Juvenile
(autosomal recessive) cation transporter)
CLN4A CLN6 CLN6 (ER transmembrane protein) Adult
CLN4B DNAJC5 Soluble cysteine string protein alpha Adult
(autosomal dominant) (synaptic vesicle protein)
CLN11 GRN Progranulin (autocrine growth factor) Adult
CLN13 CTSF Cathepsin F (lysosomal enzyme) Adult
25 Lysosomal Storage Disorders Including Neuronal Ceroid Lipofuscinoses 401
Most NCL diseases are inherited in an autosomal recessive and visual failure. After the onset of symptoms, the disease
manner. Only the CLN4B subtype of adult NCL follows an progresses in a program-like manner over 2–3 years leading
autosomal dominant inheritance. There is a rough genotype- to chair boundness, spasticity, blindness, and dementia
phenotype correlation in the sense that several missense (Steinfeld et al. 2002).
mutations with minor residual enzymatic activity in the pal- The leading symptom of juvenile NCL (CLN3) is progres-
mitoyl protein thioesterase 1 (CLN1), the tripeptidyl pepti- sive visual failure beginning at the age of 4–9 years. Cognitive
dase 1 (CLN2), and cathepsin D are correlated with protracted decline and motor deterioration follow and finally lead to
clinical courses that are recognized as atypical NCL disease death in the third or fourth decades. Seizures are a variable
forms (Das et al. 2001; Steinfeld et al. 2004, 2006). feature. Several other juvenile NCL forms (CLN1, CLN7,
Congenital NCL (CLN10) begins in utero and is associ- CLN10) may also initiate with visual disturbance followed
ated with intrauterine growth retardation and fetal micro- by movement disorder and cognitive decline. Other atypical
cephaly. At birth, affected infants show intractable seizures, juvenile forms (CLN2 and CLN5) initially present with
spasticity, and central apnea. These newborns usually sur- motor symptoms and behavioral problems before they
vive only days, seldom weeks. develop visual disturbance.
The infantile NCL is the most rapidly progressive form Several variant forms of late-infantile NCL (CLN1,
and presents between 6 and 18 months with hyperexcitabil- CLN5, CLN6, CLN7, CLN8) have clinical presentations
ity, muscular hypotonia, psychomotor retardation, myo- intermediate between the classical late-infantile (CLN2) and
clonic seizures, visual failure, and microcephaly. Later, by juvenile forms. Mutations in the CLN8 gene may cause
the age of 3 years, loss of motor abilities, increasing spastic- either a subtype of Turkish variant late-infantile NCL or a
ity, and lack of environmental contact comprise the clinical mild progressive myoclonic epilepsy with normal life span
picture. occurring in Finland (Ranta et al. 1999). Adult NCL (Kufs
The classical late-infantile NCL (CLN2) is characterized disease, CLN4A, CLN4B, CLN11, CLN13) is distinguished
by normal development to the age of 2 years, followed by from the other NCL types by the absence of visual failure
developmental delay, motor regression, myoclonic seizures, and an onset at 20–30 years of age.
25.2 Nomenclature

No. Disorder Alternative name Abbreviation symbol localisation Affected protein No. Sub type
25.3 GM1- Beta-galactosidase-1 GLB1 3p22.3 Beta-galactosidase 230500 All forms
gangliosidosis deficiency
25.4 Galactosialidosis Protective protein/ GSL CTSA 20q13.12 Protective protein/ 256540 All forms
cathepsin A cathepsin A
deficiency
25.5.1 Sandhoff disease GM2-gangliosidosis HEXB 5q13.3 Beta-subunit of 268800 All forms
O-variant hexosaminidase
25.5.2 Tay-Sachs GM2-gangliosidosis TSD HEXA 15q23 Alpha-subunit of 272800 All forms
disease B-variant hexosaminidase
25.5.3 GM2- GM2 activator GM2A 5q33.1 GM2 activator 272750 All forms
gangliosidosis deficiency
AB variant
25.6.1 Krabbe disease Globoid cell GLD GALC 14q31.3 Galactocerebrosidase 245200 All forms
leukodystrophy
25.6.2 Krabbe disease- Saposin A deficiency PSAP 10q22.1 Saposin A 611722 All forms
like disorder due
to saposin A
deficiency
25.7.1 Metachromatic Arylsulphatase A MLD ARSA 22q13.33 Arylsulphatase A 250100 All forms
leukodystrophy deficiency
25.7.2 Metachromatic Saposin B deficiency PSAP 10q22.1 Saposin B, 249900 All forms
leukodystrophy- cerebroside sulfatase
like disorder due activator
to saposin B
deficiency
25.8.1 Gaucher disease Glucocerebrosidase GBA 1q22 Glucocerebrosidase 230800 All forms
deficiency

25.8.2 Gaucher Saposin C deficiency PSAP 10q22.1 Saposin C 610539 All forms
disease-like
disorder due to
saposin C
deficiency
25.9 Action Myoclonus- AMRF, SCARB2/ 4q21.1 Scavenger receptor 254900 All forms
myoclonus-renal neuropathy syndrome EPM4 LIMP-2 class B, member 2/
failure syndrome progressive lysosomal integral
myoclonic epilepsy membrane protein 2
type 4
25.10 Combined Prosaposin PSAPD PSAP 10q22.1 Saposin A-D 611721 All forms
saposin deficiency
deficiency
25.11 Fabry disease Alpha-galactosidase GLA Xq22.1 Alpha-galactosidase 301500 All forms
A deficiency
25.12 Farber disease Farber ASAH1 8p22 Acid ceramidase 228000 All forms
lipogranulomatosis
25.13 Niemann-Pick Sphingomyelinase NPD SMPD1 11p15.4 Acid 257200, All forms
disease type A or deficiency sphingomyelinase 607616
B (ASM)
25.14.1 Mucolipidosis II I-cell disease GNPTAB 12q23.2 Alpha/beta-subunit of 252500 All forms
alpha/beta N-acetylglucosamine-
1-phosphotransferase
25.14.2 Mucolipidosis III Pseudo-Hurler GNPTAB 12q23.2 Alpha/beta-subunit of 252600 All forms
alpha/beta polydystrophy N-acetylglucosamine-
25.14.3 Mucolipidosis III Pseudo-Hurler GNPTAG 16p13.3 Gamma-subunit of 252605 All forms
gamma polydystrophy N-acetylglucosamine-
25.15 Multiple Sulphatase- MSD SUMF1 3p26.1 Formylglycine 272200 All forms
sulphatase modifying factor 1 Generating Enzyme
deficiency (SUMF1) deficiency
25.16 Lysosomal acid Wolman disease, CESD LIPA 10q23.31 Acid lipase 278000 All forms
lipase deficiency cholesteryl ester
storage disease
25.17.1 Niemann-Pick NPC1 NPC1 18q11.2 NPC1 protein 257220 All forms
disease type C1
25.17.2 Niemann-Pick NPC2 NPC2 14q24.3 NPC2 protein 607625 All forms
disease type C2
25.18.1 Ceroid Santavuori-Haltia CLN1 PPT1 1p34.2 Lysosomal palmitoyl 256730 All forms
lipofuscinosis, disease protein thioesterase-1
neuronal, 1
25.18.2 Ceroid Jansky-Bielschowsky CLN2 TPP1 11p15.4 Lysosomal 204500 All forms
lipofuscinosis, disease tripeptidyl-
neuronal, 2 peptidase-1
25.18.3 Ceroid Batten Spielmeyer- CLN3 CLN3 16p11.2 Lysosomal 204200 All forms
lipofuscinosis, Vogt disease transmembrane
neuronal, 3 CLN3 protein
25.18.4 Ceroid Kufs disease CLN4A CLN6 15q23 CLN6 204300 Adult
lipofuscinosis, recessive type form
neuronal, 4A
25.18.5 Ceroid Kufs disease CLN4B DNAJC5 20q13.33 DNAJC5 162350 All forms
lipofuscinosis, dominant type
neuronal, 4B
25.18.6 Ceroid Lysosomal CLN5 CLN5 CLN5 13q22.3 Lysosomal CLN5 256731 All forms
lipofuscinosis, protein deficiency protein
neuronal, 5
25.18.7 Ceroid CLN6 late infantile CLN6 CLN6 15q23 CLN6 601780 All forms
lipofuscinosis, variant
neuronal, 6

25.18.8 Ceroid CLN7 Turkish CLN7 MFSD8 4q28.2 Major facilitator 610951 All forms
lipofuscinosis, variant superfamily
neuronal, 7 domain-containing
protein-8 (MFSD8)
25.18.9 Ceroid CLN8 disease, late CLN8 CLN8 8p23.3 CLN8 600143 Late
lipofuscinosis, infantile variant infantile
neuronal, 8 form
25.18.10 Ceroid CLN8 disease, CLN8 CLN8 8p23.3 CLN8 610003 Juvenile
lipofuscinosis, progressive epilepsy form
neuronal, 8, with mental
Northern retardation
Epilepsy variant
25.18.11 Ceroid Ceroid CLN10 CTSD 11p15.5 Cathepsin D 610127 All forms
lipofuscinosis, lipofuscinosis,
neuronal, 10 neuronal, cathepsin
D-deficient
25.18.12 Ceroid Progranulin LN11 GRN 17q21.31 Progranulin 614706 Adult
lipofuscinosis, deficiency form
neuronal, 11
25.18.13 Ceroid Kufor-Rakeb CLN12/ ATP13A2 1p36.13 Lysosomal type 5 606693 Juvenile
lipofuscinosis, syndrome, Parkinson PARK9 P-type ATPase form
neuronal, 12 disease 9
25.18.14 Ceroid Kufs disease CLN13 CTSF 11q13 Cathepsin F 615362 Adult
lipofuscinosis, recessive type form
neuronal, 13
25.18.15 Ceroid Progressive CLN14/ KCTD7 7q11.22 Potassium channel 611726 Infantile
lipofuscinosis, myoclonic epilepsy EPM3 tetramerization form
neuronal, 14 type 3 domain-containing
protein 7
CLN9 is not genetically defined yet. SGSH gene muta- thereby leading to cell pathology. Membrane-rich tissues or
tions usually cause mucopolysaccharidosis type IIIA. SGSH tissues with a high membrane turnover, e.g., myelin in the
mutations that are associated with high residual sulfamidase central and peripheral nervous system or cells of the reticu-
activity may also cause adult onset of neuronal ceroid loendothelial system like macrophages with a high amount
lipofuscinosis. of glycosphingolipids and sphingolipids, are prone to an
early cellular dysfunction in case of a hydrolase defect.
This primarily affects CNS, liver, and spleen to a variable
25.3 Metabolic Pathway extend in every disease due to the different content of
defined glycosphingolipids and sphingolipids in cell mem-
Glycosphingolipids and sphingolipids are components of branes. A detailed pathway of the lysosomal degradation of
cell membranes. They are formed of ceramide, a molecule sphingolipids is given in Fig. 25.1 (Schulze et al. 2009;
formed of sphingosine and long-chain fatty acids. After Kolter and Sandhoff 2006; Kolter et al. 2002).
synthesis in the ER, glucose and carbohydrates are added to Single lysosomal hydrolase deficiencies comprise disor-
ceramide in a series of modification steps in the Golgi ders like gangliosidosis, galactosialidosis, Krabbe disease,
apparatus before the newly synthesized glycosphingolipids metachromatic leukodystrophy, M. Gaucher, M. Fabry,
reach membrane compartments by vesicular membrane Farber disease, Wolman disease, and Niemann-Pick disease
transport. Glycosphingolipids comprise gangliosides, and types A and B.
cerebrosides, named according to the composition of their In gangliosidosis the deficiency of β-galactosidase leads
sugar chain, and sulfatides in case of sulfation of cerebro- to the disease pattern of GM1-gangliosidosis (Wenger et al.
sides. Sphingomyelin is a sphingolipid composed of 1978; D’Azzo et al. 1982; Galjart et al. 1988), and the defi-
ceramide and phosphocholine or phosphoethanolamine ciency of hexosaminidase subunit alpha and beta as well as
instead. The degradation of glycosphingolipids and sphin- the deficiency of GM2-activator leads to the phenotype of
golipids is driven by a stepwise degradation through a Tay-Sachs disease (B variant), Sandhoff disease (0 variant),
series of specific hydrolases in the lysosome after their and AB variant of GM2-gangliosidosis (Adams and Green
transport via the endosomal pathway. Hydrolase defects 1986; Beck et al. 1998; Hendriksz et al. 2004).
result in accumulation and lysosomal storage of substrates Galactosialidosis is characterized by combined deficiency of
NeuAc
I Gal-ß(1-3)-GalNAc-Gal-Glc-Cer
Gal-ß(1-3)-GalNAc-Gal-Glc-Cer
GA1
GM1
GM1- GM1-ß-galactosidase GM1-

Gangliosidosis GM2-Activator, Sap-B Gangliosidosis GM1-ß-galactosidase
NeuAc
I GalNAc-ß-(1-4)-Gal-Glc-Cer GalNAc-ß(1-3)Gal-Glc-Cer
GalNAc-ß(1-4)-Gal-Glc-Cer GA2 Globoside
GM2
Tay-Sachs,
ß-Hexosaminidase A Sandhoff ß-HexosaminidaseA,B Sandhoff ß-HexosaminidaseA,B
Sandhoff,
GM2-Activator GM2-Activator
AB variant
A(2,3)NeuAc-Gal-ß(1-4)-Glc-Cer Sialidosis Gal-ß(1-4)-Glc-Cer Fabry Gal-α(1-4)-Gal-Glc-Cer

GM3 Salidase
Lactosylceramide α-Galactosidase A
Globotriaosylceramide
Sap-B Sap-B
GalCer-ß--galactosidase
GM1-ß-galactosiidase
Sap-B andSap-C
Glc-ß(1-1)-Cer Gal-α(1-4)-Gal-Cer
Glucosylceramide Digalactosylceramide
Gaucher Glucosylceramid-ß-glucosidase α-GalactosidaseA

Sap-C Fabry Sap-B
Glucosylceramid-ß-glucosidase
Sphingomyelinase Sap-A Gal-ß(1-1)-Cer
Sphingomyelin Ceramide Galactosylceramide
Krabbe
Niemann-Pick A and B
Acid ceramidase Metachromatic Acrylsulfatase A

Farber Sap-C Sap-B
leukodystrophy
°O3S-3-Gal-Cer
Sphingosine
Sulfatide
Fig. 25.1 Degradation pathway of sphingolipids. The major metabolites are framed, the enzymes and activator proteins that catalyze the degrada-
tion step are colored blue, and the disorders resulting from the corresponding enzyme defect are highlighted in red
β-galactosidase and neuraminidase (sialidase) activity due to to impaired GM2-ganglioside hydrolysis: HEXA which
a defect in the protective protein/cathepsin A (PPCA). PPCA encodes β-hexosaminidase A subunit-α, HEXB which
stabilized and routed the β-galactosidase/neuraminidase encodes β-hexosaminidase A/B subunit-β, and GM2A which
complex on their way from endoplasmic reticulum to encodes the GM2-activator. Mutations of HEXA lead to
lysosome and protected them against rapid proteolysis impaired β-hexosaminidase A activity with normal
(Abaroa et al. 2011). While β-galactosidase cleaves β-hexosaminidase B activity in Tay-Sachs disease (B vari-
β-galactosyl residue from GM1-gangliosides, glycoproteins, ant). Mutations of HEXB lead to impaired β-hexosaminidase
and glycosaminoglycans, neuraminidase (sialidase) cleaves A and B activity in Sandhoff disease (0 variant). And at last
sialic acid mainly N-acetylneuraminic acid from glycopro- mutations in GM2A lead to failure of GM2-activator in
teins. PPCA itself has a catalytic deaminase/esterase activity GM2-gangliosidosis AB variant.
on neuropeptides, but this activity is presumably irrelevant In Krabbe disease cerebroside-beta-galactosidase catabo-
for the pathophysiology of galactosialidosis disease. lizes galactosylceramide to ceramide and galactosylsphingo-
In mammalian neuronal tissue, there are two isoenzymes sine to sphingosine. The degradation step further requires the
of β-hexosaminidase, and each isoenzyme consists of two saposin A protein that presents the substrate to the enzyme.
polypeptide chains. β-Hexosaminidase A is a heterodimer Mutations in the GALC gene encoding cerebroside-beta-
(α/β), whereas β-hexosaminidase B is a homodimer (β/β). galactosidase lead to impaired enzymatic function with stor-
Only the β-hexosaminidase A is able to cleave N-acetyl-d- age of galactosylceramide in the pathognomonic “globoid
glucosamine and N-acetyl-d-galactosamine from GM2- cells,” macrophages with storage material, in the CNS as
gangliosides in presence of GM2-activator, a substrate-specific well as toxic galactosylsphingosine in oligodendrocytes and
cofactor. Due to this fact, there are three genes that can lead Schwann cells (Wenger et al. 2000).
Arylsulfatase A (ASA) conducts the desulfation of 3-O- clature: MLII, I-cell disease; MLIII pseudo-Hurler polydys-
sulfogalactosyl-containing glycolipids. 3-O-sulfogalactosyl- trophy) (Cathey et al. 2008). The term mucolipidosis
containing glycolipids are mainly present in the myelin describes the clinical combination of symptoms of muco-
sheaths of central and peripheral nervous system. The desul- polysaccharidosis and sphingolipidosis. The nomenclature
fation requires the combined action of ASA and saposin B, a reflects the genetic defect. ML II/III is caused by a defect
nonenzymatic protein, supposed to present the substrate to of the UDP-N-acetylglucosamine/lysosomal enzyme
ASA, similar to the action of saposin A and cerebroside-beta- N-acetylglucosamine 1-phosphotransferase (GlcNAc phos-
galactosidase (see above). Deficiency of either ASA activity, photransferase). GlcNAc phosphotransferase is a protein
which is much more common, or saposin B causes meta- complex with hexameric α2β2γ2 subunit structure localized in
chromatic leukodystrophy (Gieselmann and Krageloh-Mann the Golgi. GlcNAc phosphotransferase is involved in the
2010; Grossi et al. 2008) enzymatic reaction transferring mannose-6-phosphate to
Farber disease is a rare disorder due to the deficiency newly synthesized lysosomal hydrolases allowing their
of the lysosomal hydrolase acid ceramidase with accumula- entrance into the mannose-6-phosphate-receptor pathway,
tion of undegraded ceramide. An accumulation of macro- the major intracellular route for the delivery of synthesized
phages and histiocytes in different tissues as well as hydrolases to the lysosome (Storch and Braulke 2005). Two
ceramide accumulation in brain cells defines the genes encode the GlcNAc phosphotransferase subunits.
histopathology. GNTBAB encodes the α-/β-subunits (Tiede et al. 2005);
Wolman disease and the milder phenotype cholesteryl GNTBG encodes the γ-subunit (Raas-Rothschild et al.
ester storage disease are due to the deficiency of lysosomal 2004). Mutations result in defective GlcNAc phosphotrans-
acid lipase, which is needed to cleave cholesteryl esters in ferase activity leading to missorting of lysosomal hydrolases
various tissues. The deficiency results in a massive accumu- into the secretory pathway and hypersecretion into the extra-
lation of cholesteryl esters and triglycerides in lysosomes of cellular space. ML II α/β is the most severe form with nearly
most body tissues (Scriver 2002). absent GlcNAc phosphotransferase activity. Patients with
Deficiency of lysosomal acid sphingomyelinase in ML III α/β or ML III γ display reduced GlcNAc phos-
Niemann-Pick type A and B that catalyzes the degradation of photransferase activity. The phenotype is attenuated (Cathey
sphingomyelin to ceramide and phosphocholine results in et al. 2008).
the accumulation of sphingomyelin and other lipids in the Multiple sulfatase deficiency (MSD) is caused by muta-
monocyte-macrophage system. Residual enzymatic activity tions in the SUMF1 gene encoding the Cα-formylglycine-
of sphingomyelinase is much more reduced in patients pre- generating enzyme (FGE). FGE posttranslationally activates
senting with the severe form type A compared to milder newly synthesized sulfatases in the ER by oxidizing a cyste-
activities in patients with type B, displaying a more attenu- ine residue in a conserved amino acid sequence, the so-called
ated course of disease (Schuchman 2007). sulfatase signature, forming the active site of sulfatases and
Beside single lysosomal hydrolase deficiencies leading necessary for catalytic activity. Mutations in SUMF1 lead to
to glycosphingolipid and sphingolipid storage in lyso- misfolding and rapid degradation of FGE variants as well as
somes, the group of sphingolipids comprises diseases due compromised FGE activity resulting in catalytic inactivity of
to deficiencies of activator proteins, defects of posttransla- all cellular sulfatases, among them at least 7 lysosomal
tional modification of lysosomal hydrolases, and lipid traf- sulfatases supposed to function on sulfatides (Dierks et al.
ficking, primarily displaying the clinical picture of 2003; Cosma et al. 2003).
sphingolipidosis or in combination with clinical signs of The majority of cases of Niemann-Pick type C (NPC1)
mucopolysaccharidosis (Klima et al. 1993; Vielhaber et al. are caused by a defective protein of so far undiscovered
1996; Dierks et al. 2009). For the function of different mode of action in the late endosome involved in the LDL
lysosomal hydrolases in the degradation of cerebrosides cholesterol distribution in the cell (Lloyd-Evans et al.
and gangliosides, the so-called saposins, a group of intra- 2008).
lysosomal activator proteins, are a prerequisite. Defective Combined saposin deficiency is characterized by the
saposin proteins lead to lysosomal hydrolase dysfunction absence of all four types of saposin (saposin A–D) and
and substrate accumulation resulting in a nearly identical already manifests in the newborn period with hypotonia,
clinical phenotype (Spiegel et al. 2005; Christomanou myoclonus, and hepatosplenomegaly. Patients usually
et al. 1986). develop generalized brain atrophy and epileptic seizures and
In case of mucolipidosis, a defect in the posttranslational die during infancy. As in the case of the isolated saposin defi-
glycosylation of lysosomal hydrolases results in a missorting ciencies, the enzymatic activities of lysosomal enzymes
of lysosomal hydrolases to the extracellular space (Storch (e.g., glycosylceramidase, galactosylceramidase, and ceram-
and Braulke 2005). Mucolipidosis are subdivided into the idase) may be normal in leukocytes but are reduced in cul-
forms ML II α/β, ML III α/β, and ML III γ (previous nomen- tured fibroblasts. In addition, increased urinary excretion of
glycosphingolipids, such as globotriaosylceramide, is found. painful bone crises or avascular necrosis. Although the
The diagnosis is confirmed by detection of (often truncating) majority is diagnosed during adolescence/adulthood, severe
mutations in the PSAP gene. manifestations may already be present during early child-
hood. Spleens can become massively enlarged, which neces-
sitated splenectomy before enzyme replacement therapy was
25.4 Gaucher Disease, Saposin C available (Beutler 1988). After splenectomy, these patients
Deficiency and Limp2 Deficiency are vulnerable to septicaemia and progressive skeletal, liver
and pulmonary disease. Once extreme bone marrow infiltra-
The lysosomal enzyme β-glucocerebrosidase (β-GCase) is tion has occurred, the risk for bone complications increases
involved in the degradation of the natural glycosphingolipid (Hollak et al. 2001). This can be shown by a reduction in fat
glucocerebroside (or glucosylceramide; GC) into glucose signal in the bone marrow on MRI. Skeletal complications
and ceramide (Brady et al. 1966). Deficient activity of this include avascular necrosis, specifically of the femoral head,
enzyme causes Gaucher disease. The enzyme β-GCase is but also in other joints, pathological fractures and bone cri-
activated by saposin C, which is derived from proteolytic ses. These crises resemble the acute bone pain seen in sickle
cleavage of prosaposin. Deficiency of saposin C results in a cell disease and can only be managed by supportive measures
decrease in β-GCase and gives rise to a clinical variant of such as morphinomimetics. Imaging of the skeleton shows
Gaucher disease with neuronopathic features (Christomanou characteristic, although not pathognomonic, features includ-
1986). More recently, another disease subtype associated ing osteopenia, sclerosis, erlenmeyer flask deformities of the
with deficiency of β-GCase in skin fibroblasts, but not in leu- femurs, lytic lesions and fractures (Maas et al. 2002). On
cocytes, has been described. In this disorder, lysosomal inte- MRI, decrease in fat signal and sometimes areas with high
gral membrane protein type 2 (LIMP-2) is deficient. This water signal can be seen. This last feature does not always
protein has been shown to be the mannose-6-phosphate inde- indicate acute disease. Early AVN and infarcts can also be
pendent trafficking receptor for β-GCase (Reczek et al. recognized on MRI. While severe, progressive disease in
2007), which may result in tissue dependent decreases in children and young adults has been described, on the other
intralysosomal β-GCase activities (Berkovic et al. 2008; end of the clinical spectrum, a diagnosis of Gaucher disease
Balreira et al. 2008). Clinically this disorder had previously can be made as a coincidental finding in an otherwise healthy
been described as action myoclonus renal failure syndrome. person. The very mild patients have usually little disease pro-
gression (Beutler et al. 1995). Gaucher disease type 1 is also
Demographics and Clinical Presentations associated with other conditions, including a higher risk for
Gaucher Disease malignancies, specifically multiple myeloma. As a large pro-
Gaucher disease has first been described by Philippe Gaucher portion (up to 20 %) of adult Gaucher disease patients have a
in 1882 and is now recognized as one of the most prevalent monoclonal protein, it is clear that there is an increased risk
lysosomal storage disorders. Its inheritance is autosomal and patients should be monitored for this (De Fost et al.
recessive and the disease is panethnic, but there are some 2008). In addition, several cases of hepatocellular carcinoma
ethnic predilections. In Western countries, type I Gaucher have been reported, presumably associated with advanced
disease or the non-neuronopathic variant is most common liver involvement. Pulmonary hypertension and renal and
and has a high prevalence in the Ashkenazi Jewish popula- cardiac involvement have all been reported. The increased
tion of 1 in 850 (Zimran and Elstein 2010). In general, the risk for these conditions should be kept in mind during clini-
prevalence is believed to be around 1 in 75,000 (Meikle et al. cal follow-up. Individual factors that put patients at risk for
1999; Poorthuis et al. 1999), but it is likely that this is an these complications are currently under investigation.
underestimation since milder variants remain undiagnosed. Type 2 and 3 Gaucher disease are the neuronopathic
The neuronopathic variants are extremely rare, with variable Gaucher disease forms which are traditionally distinguished
ethnic background, although clusters of these patients have based upon the onset of neurological disease and the rate of
been described in Norbotten (Sweden), Poland and in the deterioration. Type 2 patients always present with very early
Jenin Arab population (Vellodi et al. 2009). psychomotor developmental delay and a rapidly fatal course.
Type 1 Gaucher disease has a wide phenotypic variability. However, an early diagnosis can also be made in a type 3
In general, the build up of undegraded GC in macrophages patient and considerable overlap of disease manifestations
results in hepatosplenomegaly, bone marrow involvement, between type 2 and 3 disease exist. Therefore, neuronopathic
cytopenia and skeletal disease (Grabowski 2008). Most forms are increasingly regarded as a phenotypic continuum
patients with this disorder present with unexplained thrombo- (Hruska et al. 2008). Since management decisions will be
cytopenia and splenomegaly. More exceptionally, the first made primarily based upon the course of progression, Vellodi
symptoms can be related to skeletal involvement, such as and co-workers have suggested to use the terms “acute” and
“chronic” (Vellodi et al. 2009) instead of type 2 and type 3. 25.5 Fabry Disease
Acute neuronopathic disease then refers to onset at <1 year of
age with progressive neurological features including bulbar Demographics and Clinical Presentations
and pyramidal signs and cognitive impairment. Chronic neu- The enzyme α-galactosidase A (aGalA) is deficient in Fabry
ronopathic disease encompasses all patients with neurological disease, which results in storage of glycosphingolipids,
disease manifestations in the context of Gaucher disease, who mainly globotriaosylceramide (ceramide trihexoside, CTH,
do not have the acute form (Vellodi et al. 2009). The chronic Gb3) in multiple cell types (Desnick et al. 2003). Most
neuronopathic disease group harbours several variants: the involved are cells of the vascular system, including endothe-
subtype living in Northern Sweden (Norbotten) with relatively lial and smooth muscle cells. Storage can also be found in
mild neurological features, but extensive visceral disease is podocytes in the kidney, cardiomyocytes, sweat glands and
sometimes referred to as type 3a, to distinguish these from peripheral nerves. Fabry disease is X-linked, with severe
patients with more prominent neurological features (type 3b). manifestations in male patients without any residual aGalA
Another atypical variant of chronic neuronopathic disease is activity. In carrier females and in non-classical male patients
referred to as type 3c and has first been reported in a group of the disease is much more variable. Variants in males have
Jenin Arab patients (Abrahamov et al. 1995). Homozygosity been described in which only the heart or kidney is involved.
for the D409H mutation in these patients is associated with The disease is panethnic, with an estimated birth prevalence
valvular heart disease with cardiovascular calcifications in of classically affected patients of ~1:40,000 (Desnick 2003).
addition to oculomotor apraxia and mild visceromegaly. However, awareness for the disease after the introduction of
Saposin C Deficiency or Prosaposin Deficiency enzyme replacement therapy has resulted in the identifica-
Patients with saposin C deficiency or prosaposin deficiency tion of large groups of patients. For example, neonatal
have been described with clinical features of neuronopathic screening studies have shown a much higher prevalence of
Gaucher disease, including the characteristic horizontal oph- around 1:3000 or higher (Spada et al. 2006; Hwu et al. 2009).
thalmoplegia, epilepsy and pyramidal and cerebellar signs A large proportion of these cases were presumably individu-
(Pampols et al. 1999) but also non-neuronopathic variants als presenting with late onset manifestations. Indeed, in con-
with extensive visceral involvement have been described trast to the majority of classically affected males, all of these
(Tylki-Szymanska et al. 2007). Diagnosis can be difficult as patients have residual enzyme activity. Whether such indi-
β-GCase activity is normal. The clinical presentation, pres- viduals will develop Fabry related complications has not
ence of Gaucher cells, high levels of chitotriosidase and stor- been sufficiently investigated. There is increasing evidence
age of glucosylceramide will point to the direction of saposin that many of the, often private, mutations are not disease
C deficiency. Mutation analysis of the PSAP gene can con- causing (Froissart et al. 2003; Terryn et al. 2013) and thus a
firm the diagnosis. Treatment with substrate reduction ther- definite diagnosis of Fabry disease needs to be made with
apy (miglustat, Zavesca tm) has been attempted but did not utmost care.
have any effect (Tylki Szymanska 2007). In classically affected males, severe pain in hands and feet
LIMP2 Deficiency caused by acroparesthesia is one of the earliest symptoms
Patients diagnosed with action myoclonus-renal failure syn- starting in the first or second decade of life. Some children
drome have been identified as carrying a mutation in the may have severe abdominal pain as well or unexplained peri-
SCARB2/LIMP2 gene. Several non-sense mutations in this ods of fever. Angiokeratoma may start to appear at the same
gene have been shown to impair trafficking of β-GCase to age, located primarily in the genital area and around the
the lysosome. Deficiency of active β-GCase that differs umbilicus (Germain 2010). Gradually, further storage in kid-
between tissues can hamper a diagnosis, as only fibroblasts ney heart and brain results in end-organ damage: proteinuria
and not leukocytes have low activity. Mutation analysis is one of the first signs of kidney involvement and is associ-
needs to be performed for a final diagnosis. The diversity in ated with further deterioration in both kidney and heart
β-GCase deficiency throughout the body gives a totally dif- (Wanner et al. 2010). The heart may become extremely
ferent clinical picture, although some of the neurological hypertrophic, with areas of fibrosis. Already at an early
features resemble those found in neuronopathic Gaucher dis- stage, most patients have bradycardia or other rhythm distur-
ease. However, no visceromegaly is present and no evidence bances and many need to receive a pacemaker or ICD later in
of systemic storage of glucosylceramide is established in life (Germain 2010). White matter lesions in the brain are
bone marrow and kidney biopsies, which is in agreement believed to represent small vessel involvement, and there is
with the presence of normal chitotriosidase activities (Chaves an increased risk of stroke/TIA. Hearing loss, both acute and
et al. 2011). Interestingly, in one case benefit from substrate chronic, are probably also related to ischemia in the brain
reduction therapy on neurological features was described. and has frequently been reported in Fabry disease. Classically
This needs to be further explored. affected males usually have more advanced kidney disease
than non-classical males and females and they may progress often. They also have a reduced life expectation of around 10
to end-stage renal failure in their forties. In general, their life years (MacDermot et al. 2001), although there is probably
expectation is reduced with around 20 years (Schiffmann some bias in studies reporting on this, since many asymp-
et al. 2009). In females renal disease can occur, but cardiac tomatic females may remain undiagnosed or are not regu-
manifestations and brain white matter lesions are seen more larly followed in the clinic.
25.6 Sign and Symptoms
Table 25.3 GM1-gangliosidosis

Cardiovascular Cardiomyopathy ± ± ± – –
CNS Dystonia ± ± ++ ++ ++
Gait disturbance – – ± ++ ++
Hypotonia +++ +++ + – –
Mental deterioration +++ +++ ++ ± ±
Seizures ++ ++ ++
Spasticity +++ +++ ++ ± ±
Speech disturbances – – ± ++ ++
Startle response, exaggerated ++ ++ + – –
Digestive Ascites, edema ± – – – –
Hepatosplenomegaly ++ ++ ± – –
Eye Cherry-red spot ++ ++ ± – –
Corneal clouding + + ± ± ±
Optic atrophy ++ ++ ± ± ±
Hematological Vacuolated lymphocytes + + + + +
Musculoskeletal Coarse facial features ++ ++ + – –
Dysostosis multiplex ++ ++ ± – –
Gingival hypertrophy, ++ + ± – –
macroglossia
Routine laboratory cMRI – atrophy ++ ++ + – –
cMRI – leukodystrophy ++ ++ + + +
Special laboratory DNA mutation analysis + + + + +
Mucopolysaccharides ↑ ↑ ↑ – –
(unsaturated keratan sulfate)
Oligosaccharides (U) ↑↑↑ ↑↑↑ ↑↑ ↑ ↑
There is no involvement of peripheral nerve system
Table 25.4 Galactosialidosis

Cardiovascular Cardiomyopathy +++ ± + + +
Valvular thickening ± +++ – – –
CNS Ataxia – – + ++ ++
Mental deterioration +++ ± + + +
Myoclonus – – ++ ++ ++
Seizures ± ± + + +
Spasticity + + +
Dermatological Angiokeratoma ± ± + ++ ++
Telangiectasia +++ ± ± ± ±
Digestive Hepatosplenomegaly +++ +++ ++ – –
Eye Cherry-red spot + + +++ ++ ++
Corneal clouding + + + + +
Vision, impaired ± ± +++ +++ +++

Hematological Foam cells +++ +++ +++ +++ +++
Vacuolated lymphocytes +++ +++ +++ +++ +++
Musculoskeletal Coarse facial features + + + + +
Dysostosis multiplex ++ +++ +++ + +
Edema ++
Growth retardation + +++ ++ ++ ++
Hernias + – – – –
Renal Renal failure, proteinuria +++ ++
Other Fetal hydrops ++
Special laboratory Alpha-neuraminidase activity ↓↓↓ ↓↓↓ ↓↓ ↓ ↓
Beta-galactosidase activity ↓↓↓ ↓↓↓ ↓↓ ↓ ↓
Cathepsin A – activity2 ↓-n ↓-n ↓-n ↓-n ↓-n
DNA mutation analysis + + + + +
Sialic acid-rich ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
oligosaccharides (U)1
1
Atypical forms with absence of sialyloligosacchariduria are described (Darin et al. 2009)
2
Catalytic activity of PPCA is an indication for galactosialidosis but is not needed for diagnosis of galactosialidosis, because not all mutations that
avoid the interaction of the multienzyme complex lead to poor or absent catalytic activity of PPCA
Table 25.5.1 Sandhoff disease

CNS Ataxia – ± ± ++ ++
Choreoathetosis – – – ++ ++
Dementia – +++ ++ + +
Dystonia – – – ++ ++
Hypotonia – +++ ++ + +
Mental deterioration – +++ ++ ± ±
Seizures – ± ± + +
Spasticity – +++ +++ + +
Speech disturbances – +++ ++ ++ ++
Startle response, exaggerated – +++ ++ + +
Digestive Hepatosplenomegaly – ± ± ± ±
Eye Cherry-red spot – ++ ++ ± ±
Vision, impaired – ++ ++ + +
Genitourinary Urinary incontinence – ++ ++ + +
Hematological Vacuolated lymphocytes – + + + +
Musculoskeletal Doll-like face – ++ ++ – –
Macrocephaly – – ++ + +
Muscle weakness – +++ +++ ++ ++
Routine laboratory cMRI – atrophy – ++ ++ + +
cMRI – leukodystrophy – ++ ++ ± ±
Visual evoked potential – ↓↓↓ ↓↓↓
Special laboratory Beta-hexosaminidase activity ↓↓↓ ↓↓↓ ↓↓↓ ↓↓ ↓
Aa
Beta-hexosaminidase activity ↓↓↓ ↓↓↓ ↓↓↓ ↓↓ ↓
A + Ba
Oligosaccharides (U) – ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
a
Enzyme activity is measured with synthetic substrates without need of GM2-activator
Table 25.5.2 Tay-Sachs disease

CNS Ataxia – ± ± ++ ++
Dementia – +++ ++ ± ±
Dystonia – – – ++ ++
Hypotonia – +++ ++ ++ ++
Megalencephaly – ++ + – –
Mental deterioration – +++ ++ ± ±
Psychiatric symptoms – – +++ ± ±
Psychosis – – – ++ ++
Seizuresa – ++ ++ ± ±
Spasticity – +++ +++ + +
Speech disturbances – +++ ++ ± ±
Startle response, exaggerated – +++ ++ ± ±
Digestive Hepatosplenomegaly – ± ± ± ±
Eye Cherry-red spot – ++ ++ ± ±
Vision, impaired – ++ ++ ± ±
Genitourinary Urinary incontinence – ++ ++ – –
Musculoskeletal Macrocephaly – – ++ – –
Muscle weakness – +++ +++ ++ ++
Special laboratory Beta-hexosaminidase activity ↓↓↓ ↓↓↓ ↓↓↓ ↓↓ ↓↓
Ab
Beta-hexosaminidase activity n-↑ n-↑ n-↑ n-↑ n-↑
A + Bb
Oligosaccharides (U) ↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
a
Seizures develop in infancy with disease duration time
There is no neonatal form of Tay-Sachs disease
Chronic/adult form is clinical related to an atypical Friedreich’s ataxia (spinocerebellar ataxia); juvenile form is clinical related to juvenile spinal
muscular atrophy (Kugelberg-Welander syndrome)
b
Enzyme activity is measured with synthetic substrates without need of GM2-activator
Table 25.5.3 GM2-gangliosidosis AB variant

CNS Megalencephaly – ++ + – –
Mental deterioration – +++ ++ – –
Muscular hypotonia – ++ ++ – –
Psychiatric symptoms – – +++ – –
Spasticity – ++ ++ – –
Speech disturbances – +++ ++ – –
Startle response, – +++ + – –
exaggerated
Eye Cherry-red spot – ++ ++ – –
Vision, impaired – ++ ++ – –
Genitourinary Urinary incontinence – ++ ++ – –
Special laboratory Beta-hexosaminidase n-↑ n-↑ n-↑ – –
activity A
DNA mutation analysis + + + – –
Oligosaccharides (U) n-↑ n-↑ n-↑ – –
Oligosaccharides (U) ↑↑ ↑↑↑ ↑↑↑ – –
All forms of GM2-gangliosidosis AB variant are infantile; no chronic forms described
There is no possibility to measure the functionality of GM2-activator, so in clinical suspicion of GM2-gangliosidosis with normal activity of
β-hexosaminidases, DNA mutation analysis of GM2A should be done
Tables 25.6.1 and 25.6.2 Krabbe disease and Krabbe disease-like disorder due to saposin A deficiency
CNS Ataxia – – – ++ ++
Irritability – +++ ++
Leukodystrophy – +++ +++ ++ ++
Nerve conductive velocity – ↓↓↓ ↓↓↓ ↓ ↓-n
Neurological deterioration – +++ +++
Neuropathy – +++ +++
Seizures – +++ +++
Spasticity – +++ +++
Digestive Feeding difficulties – +++ +++
Ear Deafness – ++
Eye Blindness – +++ +++
Other Fever – +++ +++
Routine laboratory Protein (CSF) – ↑↑↑ (↑) (↑) (↑)
Tables 25.7.1 and 25.7.2 Metachromatic leukodystrophy and Metachromatic leukodystrophy-like disorder due to saposin B deficiency
CNS Ataxia – – +++ ++ ++
Dysarthria – – +++ ++ ++
Emotion ability – – + ++ +++
Gait disturbance – – – ++ ++
Irritability – +++ ++
Leukodystrophy – +++ +++ ++ ++
Nerve conductive velocity – ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Neurological deterioration – +++ +++
Neuropathy – +++ +++
Psychosis – – – + +++
Seizures – +++ +++ ± ±
Spasticity – +++ +++ ± ±
Musculoskeletal Muscle weakness – +++ +++
Routine laboratory Protein (CSF) – ↑↑↑ ↑↑↑ ↑↑↑ n
Special laboratory Sulfatide (U) – ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 25.8.1 Gaucher disease

CNS Retardation, psychomotor – +++ ++ ± –
Seizures – +++ ++ ± –
Dermatological Collodion skin, collodion baby +++ – – – –
Digestive Hepatosplenomegaly ++ +++ +++ +++ +++
Liver cirrhosis – – – – ±
Eye Eye movements, abnormal – +++ ++ ± –
Hematological Anemia – + +++ ++ +
Foam cells ++ +++ +++ +++ +++
Pancytopenia – + + + +
Thrombocytopenia – + +++ +++ ++
Musculoskeletal Bone pain – – + +++ ++
Kyphosis – – ± ++ ±
Osteoporosis – – ± ++ +
Pathological fractures – – ± + ++
Respiratory Restrictive lung disease – ++ ++ + ±
Other Early death +++ +++ ± – –
Special laboratory Beta-d-glucosidase ↓↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Chitotriosidase ↑↑ ↑↑ ↑↑↑ ↑↑↑ ↑↑
Tables 25.8.2 and 25.9 Gaucher disease-like disorder due to saposin C deficiency and Action myoclonus-renal failure syndrome
CNS Retardation, psychomotor – ++ ++ ± –
Seizures, myoclonic – ++ ++ ± –
Digestive Hepatosplenomegaly ++ +++ +++
Eye Eye movements, abnormal – +++ ++ ± –
Hematological Anemia – + ++ ++ +++
Foam cells ++ +++ +++ +++ +++
Thrombocytopenia – + ++ +++ +++
Musculoskeletal Bone pain ± + ++
Pathological fractures – – ± + ++
Special laboratory Beta-d-glucosidase n n n n n
Chitotriosidase ↑↑ ↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 25.10 Combined saposin deficiency

CNS Cerebral and cerebellar atrophy + +++ – – –
Hyperkinesia ++ +++ – – –
Hypotonia ± ++ – – –
Myoclonus ++ +++ – – –
Retardation, psychomotor ++ +++ – – –
Seizures, myoclonic + ++ – – –
Digestive Hepatosplenomegaly ++ +++ – – –
Hematological Foam cells ++ +++ – – –
Special laboratory Ceramidase (fibroblasts) ↓↓ ↓↓↓ – – –
Chitotriosidase ↑↑ ↑↑ – – –
Galactosylceramidase ↓↓ ↓↓ – – –
(fibroblasts)
Glycosylceramidase ↓↓ ↓↓↓ – – –
(fibroblasts)
Table 25.11 Fabry disease

Cardiovascular Cardiomyopathy, hypertrophic – – – – ++
CNS Cerebral infarction/stroke-like – – – ± ++
encephalopathy
Neuropathic pain – – ± ++ +
Dermatological Angiokeratoma – – ± ++ ++
Digestive Abdominal pain + + +
Ear Hearing loss, sensorineural – – – ± +
Eye Cornea verticillata + + +
Renal Proteinuria – – ± ± ++
Renal failure, chronic – – – – ++
Special laboratory Globotriaosylceramide (↑) ↑ ↑ ↑ ↑
Globotriaosylsphingosine ↑ ↑ ↑↑ ↑↑ ↑↑
Table 25.12 Farber disease

CNS Deep tendon reflexes ↓↓ ↓ – –
Hypotonia ± ++ – –
Retardation, psychomotor – ++ ± – –
Seizures – ++ + – –
Dermatological Subcutaneous nodules +++ +++ +++ – –
Digestive Hepatosplenomegaly ++ + + – –
Eye Cherry-red spot – + – – –
Hematological Foam cells ± ± ± – –
Lymphadenopathy – + – – –
Musculoskeletal Arthritis +++ +++ +++ – –
Respiratory Hoarseness +++ +++ +++ – –
Lung infiltrates – ++ + – –
Other Failure to thrive – ++ – –
Routine laboratory Protein (CSF) n-↑ n-↑ – –
Special laboratory Acid ceramidase activity (LC) ↓↓↓ ↓↓↓ ↓↓↓ – –
Table 25.13 Niemann-Pick disease type A or B

CNS Hypotonia +++ – – – –
Retardation, psychomotor +++ – – – –
Seizures ± – – – –
Digestive Feeding difficulties +++ – – – –
Hepatosplenomegaly +++ +++ +++ +++ +++
Jaundice ↑↑ – – – –
Liver cirrhosis – +++ +++ +++ +++
Ear Deafness +++ – – – –
Eye Cherry-red spot +++ ± ± ± ±
Vision loss +++ – – – –
Hematological Foam cells +++ +++ +++ +++ +++
Lymphadenopathy +++ – – – –
Pancytopenia – + + + +
Thrombocytopenia +++ – – – –
Respiratory Hypoxia – +++ +++ +++ +++
Pulmonary interstitial changes – +++ +++ +++ +++
Other Failure to thrive +++ – – – –
Special laboratory Sphingomyelinase ↓↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 25.14.1 Mucolipidosis II alpha/beta

Cardiovascular Cardiomyopathy ± ± + – –
Valvular thickening +++ +++ +++ – –
CNS Cortical atrophy ++ +++ +++ – –
Mental retardation +++ +++ +++ – –
Motor retardation +++ +++ +++ – –
Muscular hypotonia +++ +++ +++ – –
Speech disturbances +++ +++ +++ – –
Digestive Hepatosplenomegaly +++ ++ ++ – –
Ear Recurrent otitis media + +++ +++ – –
Eye Corneal clouding + ++ ++ – –
Musculoskeletal Coarse facial features +++ +++ +++ – –
Gingival hypertrophy + +++ +++ – –
Hernias +++ +++ +++ – –
Hip dislocation +++ +++ +++ – –
Joint contractures ++ +++ +++ – –
Special laboratory CNS, hypomyelination +++ +++ +++ – –
Enzyme activity (cells) ↓↓↓ ↓↓↓ ↓↓↓ – –
Enzyme activity (serum) ↑↑↑ ↑↑↑ ↑↑↑ – –
Glycosaminoglycans n-↑ n-↑ n-↑ – –
MRI: cortical and subcortial +++ +++ +++ – –
atrophy
MRI: thin corpus callosum +++ +++ +++ – –
Oligosaccharides (U) ↑↑↑ ↑↑↑ ↑↑↑ – –
Table 25.14.2 Mucolipidosis III alpha/beta

Cardiovascular Aortic insufficiency – ± ± ± –
CNS Mental retardation – + + + +
Motor retardation – + + + +
Digestive Hepatomegaly – – – – –
Hernias – ± +++ ++ –
Musculoskeletal Coarse facial features – ± ++ +++ –
Corneal clouding – ± ++ + –
Gingival hypertrophy, – ± ± ± ±
macroglossia
Hip dislocation – ± + + –
Joint contractures – – +++ +++ –
Osteodystrophy – ± ++ +++ +++
Special laboratory Enzyme activity (cells) – ↓↓ ↓↓ ↓↓ ↓↓
Enzyme activity (serum) – ↑↑ ↑↑ ↑↑ ↑↑
Glycosaminoglycans – n-↑ n-↑ n-↑ n-↑
Oligosaccharides (U) – n-↑ n-↑ n-↑ n-↑
Table 25.14.3 Mucolipidosis III gamma

CNS Mental retardation – – + + –
Ear Recurrent otitis media – – ++ ++ ++
Musculoskeletal Coarse facial features – – ± ± ±
Joint contractures – – ++ ++ ++
Special laboratory Enzyme activity (cells) – – ↓ ↓ ↓
Enzyme activity (serum) – – ↑ ↑ ↑
Glycosaminoglycans – – n-↑ n-↑ n-↑
Oligosaccharides (U) – – n-↑ n-↑ n-↑
Table 25.15 Multiple sulfatase deficiency

CNS Gait disturbance – +++ +++
Hydrocephalus ± ++ ++
Leukodystrophy – +++ +++
Mental retardation +++ +++ ++
Motor retardation +++ +++ + – –
Muscular hypotonia +++ ± ±
Nerve conductive velocity – ↓↓↓ ↓↓↓
Neurological deterioration +++ +++ +++
Seizures ± ± ±
Spasticity ± +++ +++
Speech disturbances – +++ +++
Dermatological Ichthyosis +++ ++ ± +
Digestive Hepatosplenomegaly +++ ± ± ++
Musculoskeletal Coarse facial features +++ + + +
Dysostosis multiplex +++ ± ±
Routine laboratory Protein (CSF) – ↑↑↑ ↑↑↑
Special laboratory CNS, hypomyelination +++ – – – –
Glycosaminoglycans ↑↑↑ ↑↑↑ ↑↑↑
Sulfatide (U) ↑↑↑ ↑↑↑ ↑↑↑
Table 25.16 Lysosomal acid lipase deficiency

Cardiovascular Atherosclerosis, severe – – + ++ +++
CNS Retardation, psychomotor + + – – –
Digestive Abdominal distension + + – – –
Failure to thrive ++ ++ – – –
Hepatosplenomegaly +++ +++ + + +
Steatorrhea + + – – –
Vomiting + + – – –
Endocrine Adrenal calcification + + – – –
Routine laboratory Cholesterol (S) n-↑ n-↑ n-↑ n-↑ n-↑
Triglycerides (S) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Acid lipase activity ↓↓↓ ↓↓↓ ↓ ↓ ↓
Tables 25.17.1 and 25.17.2 Niemann-Pick disease type C1 and Niemann-Pick disease type C2
CNS Action dystonia – + + +
Ataxia – – +++ +++ ±
Behavioral disorder – – – + +
Clumsiness – +++ +++ +++
Cognitive dysfunction – +++ +++
Dysarthria – + + + ±
Dystonia – + + + ±
Gait disturbance – +++ +++
Gelastic cataplexy – + + +
Language difficulties – +++ +++
Psychosis – – – + +
School problems (difficulties in – +++ +++
writing, attention)
Seizures – ± ± ±
Vertical gaze palsy – + + + –
Digestive Hepatosplenomegaly +++ +++ ± ± –
Jaundice, cholestatic +++ – – – –
Hematological Foam cells + + + + +
Routine laboratory Blue histiocytes + + + + +
Special laboratory Chitotriosidase ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Filipin test ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 25.18.1 Ceroid lipofuscinosis, neuronal, 1

CNS Ataxia – + +++ +++ +++
Cerebral and cerebellar atrophy ± ++ +++ +++ +++
Developmental regression – ++ +++ +++ +++
Developmental regression – ++ +++ +++ +++
Dystonia – + +++ +++ +++
Epilepsy – ± +++ +++ +++
Language difficulties – + +++ +++ +++
Movement disorder – + +++ +++ +++
Myoclonic epilepsy – ± +++ +++ +++
Myoclonus – + +++ +++ +++
Neurodegenerative disease – ++ +++ +++ +++
Seizures, myoclonic – ± +++ +++ +++
Seizures, tonic-clonic – ± +++ +++ +++
Spasticity – ± +++ +++ +++
Speech delay – + +++ +++ +++
Eye Maculopathy/retinopathy – ± +++ +++ +++
Optic atrophy – + +++ +++ +++
Optic atrophy – + +++ +++ +++
Vision loss ± + +++ +++ +++
Musculoskeletal Microcephaly ± ++ +++ +++ +++
Microcephaly ± ++ +++ +++ +++
Routine laboratory cMRI – atrophy ± ++ +++ +++ +++
EEG, abnormal – + +++ +++ +++
ERG, abnormal – + +++ +++ +++
SSEP, abnormal – ± +++ +++ +++
VEP, abnormal – ± +++ +++ +++
Special laboratory DNA mutation analysis +++ +++ +++ +++ +++
Enzymatic activity (DBS) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Enzymatic activity (FB) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Enzymatic activity (LC) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Storage material in electron microscopy ++ +++ +++ +++ +++
White matter abnormalities – ++ ++ ± ±

CNS Ataxia n ± +++ +++ +++
Cerebral and cerebellar atrophy n ± +++ +++ +++
Developmental regression n ± +++ +++ +++
Developmental regression n n +++ +++ +++
Dystonia n ± +++ +++ +++
Movement disorder n n +++ +++ +++
Myoclonic epilepsy n ± +++ +++ +++
Myoclonus n ± +++ +++ +++
Neurodegenerative disease n n +++ +++ +++
Seizures n n +++ +++ +++
Seizures, myoclonic n n +++ +++ +++
Seizures, tonic-clonic n n +++ +++ +++
Spasticity n n +++ +++ +++
Speech delay n + +++ +++ +++
Eye Optic atrophy n n +++ +++ +++
Pigmentary retinopathy n n +++ +++ +++
Retinal dystrophy n n +++ +++ +++
Vision loss n n +++ +++ +++
Routine laboratory cMRI – atrophy n + +++ +++ +++
EEG, abnormal n ± +++ +++ +++
ERG, abnormal n n +++ +++ +++
SSEP, abnormal n ± +++ +++ +++
VEP, abnormal n n +++ +++ +++
Storage material in electron ++ +++ +++ +++ +++
microscopy
White matter abnormalities n ± + ± ±

Cardiovascular Cardiac arrhythmia n n n + ++
Cardiomyopathy n n n + ++
CNS Anxiety n n n ++ ++
Behavioral disorder n n ++ +++ +++
Behavior, aggressive n n n ++ ++
Cerebral and cerebellar atrophy n n n + ++
Cognitive dysfunction n n + ++ +++
Depression n n n ++ ++
Developmental regression n n ++ +++ +++
Developmental regression n n + ++ +++
Dysarthria n n + ++ +++
Extrapyramidal movement disorder n n n ++ +++
Gait disturbance n n n ++ +++
Hallucinations n n n ++ ++
Hypokinesia n n n ++ +++
Movement disorder n n + ++ +++
Neurodegenerative disease n n + ++ +++
Psychiatric symptoms n n n ++ +++
Seizures n n n + ++
Seizures, complex partial n n n + ++
Seizures, tonic-clonic n n n + ++
(continued)
Table 25.18.3 (continued)

Eye Optic atrophy n n + ++ +++
Pigmentary retinopathy n n ++ +++ +++
Retinal dystrophy n n ++ +++ +++
Hematological Vacuolated lymphocytes ++ ++ ++ ++
Musculoskeletal Rigidity n n n ++ +++
Routine laboratory cMRI – atrophy n n ± + ++
EEG, abnormal n n + ++ +++
ERG, abnormal n n ++ +++ +++
SSEP, abnormal n n + ++ +++
VEP, abnormal n n ++ +++ +++
Storage material in electron microscopy ++ +++ +++ +++
White matter abnormalities n n + + +
Table 25.18.4 Ceroid lipofuscinosis, neuronal, 4A

CNS Ataxia – – – – ++
Behavioral disorder – – – – +
Behavioral disorder – – – – +
Cerebral and cerebellar atrophy – – – – ++
Cognitive decline – – – – ++
Extrapyramidal movement disorder – – – – +++
Movement disorder – – – – ++
Myoclonic epilepsy – – – – +++
Myoclonus – – – – ++
Neurodegenerative disease – – – – +++
Seizures – – – – ++
Seizures, tonic-clonic – – – – ++
Spasticity – – – – ++
Routine laboratory EEG, abnormal – – – – ++
Storage material in electron microscopy +
Table 25.18.5 Ceroid lipofuscinosis, neuronal, 4B

CNS Ataxia – – – – ++
Behavioral disorder – – – – ++
Behavioral disorder – – – – ++
Cerebral and cerebellar atrophy – – – – +
Cognitive decline – – – – ++
Extrapyramidal movement disorder – – – – ++
Movement disorder – – – – ++
Myoclonus – – – – ++
Neurodegenerative disease – – – – +++
Psychiatric symptoms – – – – ++
Seizures – – – – ++
Seizures, tonic-clonic – – – – +++
Spasticity – – – – ++
Routine laboratory EEG, abnormal – – – – ++
Storage material in electron microscopy +

CNS Ataxia n ± +++ +++ +++
Dystonia n ± +++ +++ +++
Spasticity n n + ++ +++
Speech delay n n + ++ +++
Eye Macular dystrophy n n +++ +++ +++
Optic atrophy n n ++ +++ +++
Storage material in electron microscopy ++ +++ +++ ++ ++

CNS Ataxia n ± +++ +++ +++
Dystonia n ± +++ +++ +++
Speech delay n n ++ ++ +++
Eye Optic atrophy n n ++ +++ +++

CNS Ataxia n ± +++ +++ +++
Behavioral disorder n n + ++ +++
Dystonia n ± +++ +++ +++

CNS Ataxia n ± +++ +++ +++
Behavioral disorder n n + ++ +++
Dystonia n ± +++ +++ +++
Seizures, complex partial n n +++ +++ +++
Vision loss n n ++ +++ +++
Table 25.18.10 Ceroid lipofuscinosis, neuronal, 8, northern epilepsy variant

CNS Ataxia – – + ++ ++
Behavioral disorder – – + ++ +++
Behavioral disorder – – + ++ ++
Cerebral and cerebellar – – – ± +
atrophy
Cognitive decline – – + ++ ++
Movement disorder – – + ++ ++
Myoclonus – ± ++ ++ ++
Neurodegenerative disease – – ++ + +
Seizures – – +++ +++ +++
Seizures, complex partial – – +++ +++ +++
Seizures, tonic-clonic – – +++ +++ +++
Speech delay – – ++ ++ +++
Routine laboratory EEG, abnormal – – – +++ +++
Storage material in electron ++ ++ ++ ++ ++
microscopy

CNS Ataxia n + +++ +++ +++
Cerebral and cerebellar atrophy +++ +++ + ++ +
Congenital encephalopathy +++ +++ – – –
Developmental regression – – + ++ ++
Epilepsy +++ +++ + + +
Language difficulties – – ++ +++ +++
Movement disorder +++ +++ + ++ ++
Myoclonus – – + + +
Neurodegenerative disease +++ +++ ++ ++ ++
Seizures +++ +++ + + +
Spasticity +++ +++ + + +
Eye Optic atrophy + ++ ++
Optic atrophy + ++ ++
Pigmentary retinopathy +++ +++ +++
Retinal dystrophy ++ +++ ++
Vision loss ++ +++ +++
Musculoskeletal Microcephaly +++ +++ – – –
Microcephaly +++ +++ – – –
Routine laboratory cMRI – atrophy +++ +++ + ++ +
EEG, abnormal +++ +++ + + +
ERG, abnormal +++ +++ +++
SSEP, abnormal ++ ++ +
VEP, abnormal +++ +++ +++
Storage material in electron +++ +++ +++ +++ +++
microscopy

CNS Ataxia − − − − +
Cerebellar atrophy − − − − +
Language difficulties − − − − +
Movement disorder − − − − +
Seizures − − − − +
Eye Retinal dystrophy − − − − ++
Vision loss − − − − ++
Routine laboratory cMRI − atrophy − − − − +
EEG: abnormal − − − − +
ERG: abnormal − − − − ++
Storage material in electron +++
microscopy

CNS Akinesia n n n ++ +++
Behavioral abnormalities n n n + ++
Cognitive dysfunction n n + ++ +++
Dysarthria n n n + ++
Extrapyramidal movement n n n + ++
disorder
Gait disturbance n n n ++ +++
Movement disorder n n n + ++
Myoclonus n n n + +++
Neurodegenerative disease n n n + ++
Musculoskeletal Rigiditiy n n n ++ +++
Storage material in electron +++ +++
microscopy

CNS Ataxia − − − − +
Behavioral disorder − − − − +
Cerebral and cerebellar atrophy − − − − +
Cognitive decline − − − − ++
Dysarthria − − − − +
Extrapyramidal movement − − − − ++
disorder
Movement disorder − − − − +
Neurodegenerative disease − − − − ++
Seizures − − − − +
Seizures, tonic clonic − − − − +
Routine cMRI − atrophy − − − − +
laboratory
Special DNA mutation analysis +++ +++ +++ +++ +++
laboratory Storage material in electron ±
microscopy

CNS Ataxia − − ++ +++ +++
Cerebral and cerebellar + ++ +++ +++
atrophy
Developmental regression − − +++ +++ +++
Epilepsy − + +++ +++ +++
Hypokinesia − − ++ +++ +++
Language difficulties − − +++ +++ +++
Movement disorder − − +++ +++ +++
Myoclonic epilepsy − + +++ +++ +++
Neurodegenerative disease − − +++ +++ +++
Seizures, myoclonic − + +++ +++ +++
Speech delay − − +++ +++ +++
Eye Optic atrophy − − +++ +++ +++
Vision loss − − +++ +++ +++
Musculoskeletal Microcephaly − − +++ +++ +++
Routine Laboratory cMRI − atrophy ± ++ +++ +++ +++
EEG: abnormal − + +++ +++ +++
Special Laboratory DNA mutation analysis +++ +++ +++ +++ +++
Storage material in electron +++ +++ +++
microscopy
25.7 Diagnosis and Reference Values Specific tests for sphingolipidoses and neuronal ceroid
lipofuscinoses should be performed in specialized laborato-
The diagnostic work-up of the sphingolipidoses and neuro- ries. Diagnostic procedures include quantitative analysis of
nal ceroid lipofuscinoses should begin with the clinical metabolites in body fluids (urine, serum, CSF) and fibroblasts
assessment of the patients and should consider the general as well as specific enzyme assays of lysosomal hydrolases. In
classification as given in Table 25.19. The characteristic case the disorder is not associated with an enzyme deficiency
combination of symptoms determines the further diagnostic or in order to confirm the diagnosis, DNA mutational analy-
investigations as outlined in the diagnostic flow charts in sis in the affected gene can be performed. DNA testing is
Sect. 25.8 (see Figs. 25.2–25.4). Commonly these comprise particularly important for prenatal diagnosis. Further details
full blood count and smear, fundoscopy, cardiac assessment on the specific investigations are listed in Sect. 25.9 (see
(ECG/ECHO), skeletal status (X-ray), cranial MRI, and Table 25.21). Reference values for selected lysosomal
electrophysiology (VEP, SEP, ERG). enzyme assays are listed in Tables 25.19 and 25.20.
Table 25.19 Reference values for fluorometric enzyme assays given as ratio enzyme/beta-galactosidase activity (normal ranges depend on assays
and detection devices)
Enzyme/beta-
No. Disorder Enzyme galactosidase (%) Material
25.3 GM1-gangliosidosis Beta-galactosidase 54–146 Leukocytes
25.5.1 Sandhoff disease Beta-hexosaminidase A + B 284–1,345 Leukocytes
25.5.2 Tay-Sachs disease Beta-hexosaminidase A 46–171 Leukocytes
25.6.1 Krabbe disease Galactocerebrosidase 0.2–3.2 Leukocytes
25.8.1 Gaucher disease Beta-d-glucosidase 2,2–22 Leukocytes
25.11 Fabry disease Alpha-galactosidase 5,9–59 Leukocytes
25.13 Niemann-Pick disease type A or B Sphingomyelinase 0.2–0.8 Leukocytes
25.18.1 Ceroid lipofuscinosis, neuronal, 1 Palmitoyl protein thioesterase 1 28–105 Leukocytes
25.18.2 Ceroid lipofuscinosis, neuronal, 2 Tripeptidyl peptidase 1 18–95 Leukocytes
25.18.11 Ceroid lipofuscinosis, neuronal, 10 Cathepsin D 3,129–4,758 Leukocytes
Table 25.20 Reference values for spectrophotometric enzyme assays given as ratio enzyme/beta-hexosaminidase activity (normal ranges depend
on assays and detection devices)
Enzyme/beta-
No. Disorder Enzyme hexosaminidase (%) Material
25.3 GM1-gangliosidosis Beta-galactosidase 6.6–33 Leukocytes
25.5.1 Sandhoff disease Beta-hexosaminidase A + B 53–147 Leukocytes
25.7.1 Metachromatic leukodystrophy Arylsulfatase A 1.7–8.5 Leukocytes
Facial dysmorphism, dysostosis multiplex, organiomegaly,

variable neurodegeneration
Urine analysis
GAGs
MRI:
Oligosaccharides
Leukodystrophy
Fundoscopy: Urine:
Cherry red spot Sulfatides
Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme

assay assay assay assay assay assay
Oligo- Galacto- GM1 Mucopoly- Multiple Sulfatase

Mucolipidosis
saccharidosis sialidosis Gangiosidosis saccharidosis Deficiency
Genetic analysis
Fig. 25.2
Fig. 25.3
Clinical signs of organomegaly with variable neurodegeneration
Cardiac
Vacuolated lymphocytes/Foam cells
involvment
Cherry red Vertical gaze

Subcutanous
spot Adrenal palsy
nodules Angioceratomas
Intersitiell calcification Ataxia
Hoarseness
lung disease Dystonia
Massive elevated Ceramides

Chitotriosidase (urine)
Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme

assay assay assay assay assay assay
NP Type A/B Farber Wolman NP Typ C Gaucher Fabry
neg.
Sap C
AMRF
Genetic analysis
Clinical signs of major neurodegeneration without organomegaly

(psychomotor regression, ataxia, blindness, spasticity, seizures)
cMRI and fundoscopy
cMRI: Cherry red spot Retinopathy + cMRI: brain

leukodystrophy vacuolated lymphos atrophy
Central and peripheral ERG ↓ and VEP ↓ Abnormal ERG,

demyelination VEP, SEP, EEG
Macrocephaly, cMRI: T2-
Sulfatides hypodense thalami, atrophy PCR for frequent 1kb Enzyme assay for:
in urine ↓ No deletion in CLN3 gene CLN1,CLN2,CLN10
Yes
Enzyme Enzyme Enzyme Genetic analysis

assay assay assay pos. neg.
MLD Krabbe GM2-Gangliosidosis CLN3 CLN3,5,6,7,8

neg. in Fibrobl. M. sandhoff, Tay-Sachs
neg. neg.
neg.
sap B sap A GM2-Activator other NCL genes
Fig. 25.4 Genetic analysis

25.9 Diagnostics
Table 25.21 Specific laboratory investigations for the diagnostic work-up of sphingolipidoses and neuronal ceroid lipofuscinoses
DNA mutation Prenatal
No. Disorder Metabolite analysis diagnostic feature Enzyme assay analysis diagnosis
25.3 GM1-gangliosidosis GAGs (U); oligosaccharides (U) DBS, L, FB L, FB A, CV
25.4 Galactosialidosis Oligosaccharides (U), vacuolated FB L, FB A, CV,
lymphos oligoa
25.5.1 Sandhoff disease Oligosaccharides (U) L, S, FB L, FB A, CV
25.5.2 Tay-Sachs disease Oligosaccharides (U) L, S, FB L, FB A, CV
25.5.3 GM2-gangliosidosis AB variant Oligosaccharides (U) – L, FB A, CV
25.6.1 Krabbe disease Protein (CSF) L, FB L, FB A, CV
25.6.2 Krabbe disease-like disorder due to Low galactocerebrosidase activity in L L L, FB A, CV
saposin A deficiency
25.7.1 Metachromatic leukodystrophy Sulfatides (U) L, FB L, FB A, CV
25.7.2 Metachromatic leukodystrophy-like Sulfatides (U) – L, FB A, CV
disorder due to saposin B deficiency
25.8.1 Gaucher disease Chitotriosidase (S) DBS, L, FB L, FB A, CV
25.8.2 Gaucher disease-like disorder due to Chitotriosidase (S) – L, FB A, CV
saposin C deficiency
25.9 Action myoclonus-renal failure – L, FB L, FB A, CV
syndrome
25.10 Combined saposin deficiency – L, FB L, FB A, CV
25.11 Fabry disease Ceramides (U) DBS, L, S FB L, FB A, CV
25.12 Farber disease – FB L, FB A, CV
25.13 Niemann-Pick disease types A or B Vacuolated lymphos L, FB L, FB A, CV
25.14.1 Mucolipidosis II alpha/beta Oligosaccharides (U), vacuolated S, FB L, FB A, CV
lymphos
25.14.2 Mucolipidosis III gamma Oligosaccharides (U), vacuolated S, FB L, FB A, CV
lymphos
25.14.3 Mucolipidosis III alpha/beta Oligosaccharides (U), vacuolated S, FB L, FB A, CV
lymphos
25.15 Multiple sulfatase deficiency GAGs (U), Sulfatides (U), vacuolated L, FB L, FB A, CV
lymphos
25.16 Lysosomal acid lipase deficiency Cholesterol, vacuolated lymphos L L, FB A, CV
25.17.1 Niemann-Pick disease type C1 Cholesterol analysis, Chitotriosidase (S), – L, FB A, CV
vacuolated lymphos
25.17.2 Niemann-Pick disease type C2 Cholesterol analysis, Chitotriosidase (S), – L, FB A, CV
vacuolated lymphos
25.18.1 Ceroid lipofuscinosis, neuronal, 1 EM storage material DBS, L, FB L, FB A, CV
25.18.3 Ceroid lipofuscinosis, neuronal, 3 EM storage material, vacuolated lymphos – L, FB A, CV
25.18.4 Ceroid lipofuscinosis, neuronal, 4A EM storage material – L, FB A, CV
25.18.5 Ceroid lipofuscinosis, neuronal, 4B EM storage material – L, FB A, CV
25.18.6 Ceroid lipofuscinosis, neuronal, 5 EM storage material – L, FB A, CV
25.18.10 Ceroid lipofuscinosis, neuronal, 8, EM storage material – L, FB A, CV
northern epilepsy variant
a
Sialyloligosaccharides in amniotic fluid supernatant
GAGs glycosaminoglycans, DBS dried blood spot, L leukocytes, S serum/plasma, FB fibroblasts, A amniotic fluid cells, CV chorionic villi, EM
electron microscopy, lymphos lymphocytes
25.10 Treatment et al. 1991). In patients with advanced liver, lung, or bone
disease, the effectiveness is more limited. But in those initi-
Gaucher Disease ating therapy before irreversible damage has occurred, the
Before enzyme replacement therapy became available, the responses are very good. Many dosing regimens have been
treatment of a Gaucher disease patient has been symptomatic explored, with higher doses generally leading to more robust
(Beutler 1988). Splenectomy was performed usually in case responses (Grabowski et al. 2009). However, individualiza-
of grossly enlarged spleens, leading to infarcts, abdominal tion of dosing has become standard practice, since doses
discomfort, or severe cytopenia secondary to hypersplenism. between 15 and 60 U/kg eow may all produce sustained
After splenectomy, cytopenia is usually immediately resolved. responses. It needs to be emphasized that many patients do
However, the patient is at risk for further liver involvement, not need to be treated as they have very mild, nonprogressive
hepatopulmonary syndrome, and pulmonary hypertension. In disease manifestations (Beutler et al. 1995). The precise cri-
addition, splenectomized patients more frequently develop teria to start ERT in type 1 Gaucher disease are not unequivo-
bone crises and pathological fractures (Mikosch and Hughes cal. Some believe that most patients need ERT, while others
2010). Also, absence of the spleen puts them at a higher risk tend to be more conservative. In general, children with
of septicemia from pneumococci and other encapsulated bac- symptoms are always treated, while adults with stable dis-
teria. Thus, splenectomy should be avoided whenever possi- ease and mild splenomegaly without severe platelet count
ble. Bone crises usually present as acute, circumscript, severe, (e.g., below 60 x 109/l) and without severe bone marrow
persistent pain in a long bone, pelvis, or spine, often with infiltration may remain untreated. Currently, the risk factors
signs of inflammation. No other treatment than supportive for late complications, such as multiple myeloma, osteopo-
care with bed rest and nonsteroidal anti-inflammatory drugs rosis, or liver/lung disease, are unknown. Whether early ini-
or morphine is available. Orthopedic procedures such as joint tiation of ERT can prevent this is unclear and needs to be
replacement in case of avascular necrosis are sometimes nec- investigated. Patients with the acute neuronopathic form of
essary. Bleeding has been a major problem related to throm- Gaucher disease do not benefit from ERT, and it is now gen-
bocytopenia, thrombocytopathy and decreased clotting erally accepted that these should not be treated (Vellodi et al.
factors (Hollak et al. 1997). Coagulation studies, including 2009). In a very young patient with neurological symptoms,
PT and aPTT, need to be performed around every surgical it may be difficult to decide whether this represents an acute
procedure or pregnancy. Supplementation with coagulation or a chronic form. In such a case, it may be an option to dis-
factors or plasma may be indicated. For patients with severe cuss temporary treatment with the option to stop when the
skeletal disease, especially those with fractures and osteopo- course of disease is rapidly progressive (Vellodi et al. 2009).
rosis, bisphosphonates have been used. While they lead to an In patients with the chronic neuronopathic form, ERT may
increase in bone density, the clinical relevance of this treat- certainly alleviate the symptoms related to hepatospleno-
ment is unclear. Also, new evidence points towards decreased megaly and partially also skeletal disease. However, there is
osteoblast activity rather than increased bone loss, and pro- no evidence that ERT has reversed, stabilized, or slowed the
longed bisphosphonate therapy may further impair bone progression of neurological involvement. Detailed recom-
remodelling (Van Dussen et al. 2011). mendations for treatment are provided in the consensus
paper by Vellodi et al. (2009). In type 3c, ERT is usually not
Hematopoietic Stem Cell Transplantation (HSCT) indicated, as these patients do not have extensive visceral
Allogeneic bone marrow transplantation or HSCT has been disease. Over the last years, new ERTs have been developed.
used specifically in the past to treat neuronopathic Gaucher Currently, velaglucerase (VPRIV, Shire HGT) has also
disease (Ringdén et al. 1995). The aim of this approach is to received marketing authorization in the EU and the USA,
replace hematopoietic stem cells with healthy donor cells whereas taliglucerase (Uplyso, Protalix/Pfizer) has been
secreting normal enzyme. Since enzyme replacement ther- approved by the FDA only (Zimran et al. 2010; 2011). All
apy does not reach the central nervous system, HSCT may these enzymes have shown effectiveness, and choices for
still be an option for patients with chronic neuronopathic dis- treatment will probably depend on whether there are any
ease. However, this procedure is rarely performed nowadays potential differences in effectiveness or safety, as well as
considering the high risk and the beneficial effects of ERT. reimbursement strategies and costs.
Enzyme Replacement Therapy (ERT) Substrate Reduction Therapy (SRT)

The prospects of patients with type 1 Gaucher disease have An alternative approach is based on small molecules that
improved dramatically with the advent of ERT. Intravenous inhibit substrate synthesis. The first and only authorized
administration, usually once every two weeks, of recombi- treatment is miglustat (N-butyl deoxynojirimycin, Zavesca,
nant β-GCase (Cerezyme, Genzyme Corp. MA) has proven Actelion). This compound is an iminosugar, which inhibits
to result in reversal of most disease manifestations (Barton the glucosyltransferase involved in the first step in the
formation of complex glycosphingolipids. The rationale is importance is that good clinical responses are always
that the reduction in substrate is balanced against the residual accompanied by substantial decreases in chitotriosidase
β-GCase activity, which ultimately results in degradation of (Aerts et al. 2005) or in deficient patients, in CCL18/
stored glycolipids. Indeed, miglustat results in reductions in PARC, ACE, or ferritin levels. Absence of a response in
liver and spleen size, and gradual improvements in hemoglo- these parameters indicates failure of treatment. Table 25.22
bin and platelet counts (Cox et al. 2000). The official use of shows the minimal recommended follow-up procedures.
miglustat is for patients with mild to moderate type 1 Gaucher Because of the increased risk for multiple myeloma, pul-
disease who are unsuitable to receive ERT. Patients may pre- monary hypertension, Parkinson’s disease, and perhaps
fer the convenience of oral treatment above lifelong intrave- other conditions such as diabetes, it may be wise to
nous infusions. However, ERT remains the first choice of include relevant physical, laboratory, or imaging parame-
therapy (Cox et al. 2003). Although direct head-to-head ters in the follow-up (Zimran 2011). Skeletal imaging
comparative trials have not been conducted, ERT is believed should preferably be performed with MRI, as this allows
to be superior to SRT (Zimran 2011). In addition, many the assessment of the degree of bone marrow involve-
patients experience gastrointestinal side effects, which limits ment. Serial follow-up with skeletal X-rays is unneces-
the use of miglustat. Newer SRTs are currently under devel- sary and exposes patients at high levels of radiation (Poll
opment with a potentially better effectiveness/toxicity profile et al. 2002). DEXA scanning has difficulties in interpret-
(Lukina et al. 2010). SRT has been suggested to be used in ing results in pediatric patients and in adults with sclerotic
neuronopathic Gaucher disease. However, one small trial lesions may also give false outcomes. However, in those
failed to demonstrate effectiveness (Schiffmann et al. 2008), with milder disease, regular follow-up to detect early
and miglustat is not authorized for use in neuronopathic dis- osteopenia or osteoporosis is of importance.
ease. Some anecdotal reports suggest that miglustat may
have some effect, and therefore combination therapy with Fabry Disease
ERT is sometimes tried (Cox et al. 2003). It is expected that Multidisciplinary care for a Fabry disease patient is usually
the use of small molecules may further be explored as some required because of the complexity of the manifestations
act as chaperones by stabilizing the mutated β-GCase and (Desnick et al. 2003). Adequate pain management and early
hence increase its activity (Desnick and Schuchman 2002). treatment with ACE inhibitors or angiotensin receptor block-
ers may be needed in case of proteinuria or cardiac symptoms
(Jain and Warnock 2011). The use of aspirin is mostly recom-
25.11 Follow-Up and Monitoring mended, certainly after TIA or stroke, but whether use of anti-
of Gaucher Disease platelet therapy prevents new white matter lesions is unknown.
Cardiovascular risk factors independent of Fabry disease are
Follow-up should in principle be individualized, as the likely to influence the disease course and deserve adequate
heterogeneity of the disease and a number of associated identification and strict treatment, including hypertension, dia-
conditions precludes strict protocolized follow-up. betes, and hypercholesterolemia. Those with rhythm distur-
However, recommendations for follow-up of patients with bances, frequently bradycardia and AV blocks with ventricular
Gaucher disease have been previously published (Charrow escape tachycardias, may experience syncope. Those patients
et al. 2004; Baldellou et al. 2004; Grabowski 2004; carry a risk for acute cardiac death and need cardiological
Zimran 2011). For neuronopathic disease, a helpful monitoring (Pierre-Louis et al. 2009). Patients with end-stage
scheme for follow-up of neurological symptoms has been renal failure may benefit greatly from kidney transplantation.
published (Vellodi et al. 2009). Some of the recommenda- The graft survival is not different compared with other patients
tions for follow-up in non-neuronopathic Gaucher disease with renal failure (Ojo et al. 2000).
refer to earlier-defined therapeutic goals (Pastores et al.
2004). It should be kept in mind that these goals are in Enzyme Replacement Therapy
fact mean responses in blood counts, liver and spleen vol- In 2001, two recombinant enzymes were approved for use in
umes, and skeletal parameters, rather than clinically Fabry disease: agalsidase alpha (Replagal®) and agalsidase
meaningful endpoints. Dose adjustment should be made beta (Fabrazyme®), referred to as enzyme replacement ther-
on an individual basis rather than on achievement of these apy (ERT). While these enzymes are produced in different
goals. For example, in patients with very large spleens, cell systems, their biochemical properties are very similar.
platelet responses will initially be very slow, and in the They have been investigated in different doses: agalsidase
absence of clinically relevant bleeding episodes, there is beta at 1.0 mg/kg eow and agalsidase alpha at 0.2 mg/kg eow.
no justification for a dose increase (Hollak et al. 2012). In The first placebo-controlled trials showed some beneficial
general, bleeding, severe anemia, and symptomatic effect on reduction of storage in endothelial cells and improve-
organomegaly are alleviated within 12–24 months. Of ment in pain, with stable parameters of renal and cardiac
Table 25.22 Follow-up and monitoring of non-neuronopathic (type 1) Gaucher disease

Assessment Untreated Treated
Schedule Every 12–24 months Every 3 months during the first Every 12 months during the first
year, every 6 months thereafter 2–4 years, on an individual basis
thereafter
Physical examination X X X
Laborator CBC X X
Chitotriosidase or X X
CCL18/PARCa
ACE X X
Ferritin X X
Liver enzymes X X
M-protein X X
Ab testing Xc
Imaging Liver and spleen
volume (MRI, US)
Skeletal MRI X X
Skeletal X-raysb X X
DEXA scanb
QoL scores X X
a
CCL18/PARC is recommended in case of chitotriosidase deficiency
b
Skeletal X-rays and DEXA scans are recommended to be made only once, to serve as baseline; follow-up X-rays should only be made upon clini-
cal indication (Maas et al. 2002)
c
Testing for antibodies should be done at baseline and after 12 months. If negative, this should only be repeated in case of infusion-associated reac-
tions or lack of response
disease (Eng et al. 2001; Schiffmann et al. 2001; Hughes for early treatment, optimal dosing, and strategies to over-
2008). In a phase IV placebo-controlled trial, treatment with come the effects of antibodies.
agalsidase beta resulted in a reduction of Fabry-related com-
plications in patients with advanced disease at baseline Alternative or Investigational Treatments
(Banikazemi et al. 2007). However, complications can still Small molecules that act either as substrate inhibitors (see
occur during treatment, and more recent studies have shown Gaucher disease) or as chaperones, to enhance aGalA activity
that patients with advanced disease, specifically with renal in case of a missense mutation, are under investigation.
disease or cardiac fibrosis, will show further deterioration Miglustat (N-butyl deoxynojirimycin, Zavesca, Actelion),
(Schiffmann et al. 2006; Germain et al. 2007; Weidemann which is authorized as an oral treatment in mild to moderate
et al. 2009). The occurrence of antibodies may also influence type 1 Gaucher disease (Cox et al. 2003), has been investi-
the clinical outcome, although so far it has been difficult to gated in Fabry disease, but no results have been published.
establish clear effects on rate of progression of disease com- Since studies in mice have shown that these molecules may
plications. This may have to do with the chronic progressive reduce the storage of Gb3, there is a potential for substrate
nature of the disease with little effect of treatment in advanced inhibitors to be used as oral treatment in Fabry disease (Platt
cases. Biochemically, there is no doubt that antibodies have a et al. 2001). A similar iminosugar, the small molecule pharma-
deleterious effect, since several studies have shown that initial cological 1-deoxygalactonojirimycin (migalastat hydrochlo-
beneficial responses, i.e., reductions in Gb3 and lysoGb3 in ride, marketed as Amigal™ by Amicus Therapeutics, Inc.), is
plasma or urine, are reversed when antibodies emerge currently being studied in a phase 3 clinical trial as a therapeu-
(Linthorst et al. 2004; Rombach et al. 2012). Antibodies tic agent for Fabry disease. This compound acts as a pharma-
almost exclusively emerge in male patients, in particular cological chaperone to improve its stability and trafficking to
those without any residual enzyme activity. Their presence is the lysosome after binding to its active site (Asano et al. 2000).
frequently associated with infusion-associated reactions such Studies in mice expressing human mutant α-galactosidase A
as chills and fever. This can usually be managed by premedi- resulted in significant increases in α-galactosidase A activity
cation with corticosteroids and or slowing down of the infu- in various organs, with concomitant reductions in Gb3 (Ishii
sion rate. Benefits of ERT have also been reported in children, 2012). As this approach is only useful in patients with mis-
but whether early treatment can prevent the occurrence of late sense mutations with some preserved enzyme activity, patients
complications is still unknown. Many questions are thus without any aGalA activity cannot benefit from this approach.
unanswered, and future studies need to focus on the rationale Another application of chaperone therapy may be the
enhancement of the recombinant aGalA. In vitro studies Niemann-Pick Disease Type C

showed that coadministration with ERT may improve the Substrate reduction therapy with miglustat is the only possi-
pharmacological properties of recombinant aGalA (Benjamin ble therapeutic approach in NPC so far, leading to an arrest
et al. 2012). Gene therapy has not been developed as yet for of disease progression (Patterson et al. 2012). Cataplexy
Fabry disease, but studies are currently being conducted could be treated with clomipramine (Vanier 2010). Bone
(Yoshimitsu et al. 2007). marrow transplantation in a child with NPC did not improve
neurological deterioration (Hsu et al. 1999), whereas trials
with hematopoietic stem cell transplantation are in progress
25.12 Follow-Up and Monitoring in an animal model of NPC (Seo et al. 2011). Allogeneic
of Krabbe Disease bone marrow transplantation has been successful in one
patient with NPC2 (Bonney et al. 2010).
Earlier guidelines for follow-up have been published in 2003
(Desnick et al. 2003). Fabry disease is extremely variable. Gangliosidosis
Hence strict guidelines for follow-up cannot be provided. An Allogeneic bone marrow transplantation has been attempted
individualized approach to the patient with Fabry disease is in a child with infantile GM1-gangliosidosis. However,
the cornerstone of management and follow-up. ERT is usu- despite complete normalization of white blood cell
ally initiated in symptomatic patients, but those who are β-galactosidase levels, the patient continued to deteriorate
asymptomatic should receive follow-up as well. Early recog- neurologically (Shield et al. 2005).
nition of organ failure and adequate and timely initiation of Iminosugars inhibiting ganglioside biosynthesis have
appropriate therapy is important. ERT is probably of limited been investigated in rodents and have resulted in reduction of
effectiveness in advanced cases, and benefits should be out- ganglioside accumulation in the CNS, suggesting that sub-
weighed against the burden of frequent intravenous adminis- strate deprivation therapy may be a potentially effective early
tration and costs. In these advanced cases, follow-up intervention therapy for GM1-gangliosidosis (Kasperzyk
schedules will depend on the most severe end-organ damage, et al. 2005).
and general practice guidelines for patients with renal or car- Currently the inhibitor of ganglioside synthesis (miglus-
diac failure need to be followed. A proposal for follow-up tat) is tested to treat slowly progressive forms (Elliot-Smith
measures is shown in Table 25.23. et al. 2008).
Table 25.23 Follow-up and monitoring of Fabry disease

At first
Assessment visit Untreated Treated
Every 12–24 Every 3 months during the first Every 12 months during the
months year, every 6 months thereafter first 2–4 years, on an individual
basis thereafter
Physical examination General Neurologicala X X X X
Laboratory Creatinine/eGFR X X X
24 h urinary protein X X X
Electrolytes X X
Cholesterol profileb X
Gb3/lysoGb3 X
Ab testing Xd
Imaging Cardiac MRI or X X
USc X X
Brain MRIc X X
Other EKG X X X
Qol scores X X X
Audiogram X X X
Ocular examination X
a
At first visit, the presence of angiokeratoma and small fiber neuropathy should be investigated
b
Cholesterol profile and other cardiovascular risk factors should be recorded carefully at first visit and followed on an individual basis
c
MRI or US of the heart and MRI of the brain in adults only or upon clinical indication in children
d
Testing for antibodies should be done at baseline and after 12 months. If negative, this should only be repeated in case of infusion-associated
reactions or lack of response
Also for galactosialidosis bone marrow transplantation in Lysosomal Acid Lipase Deficiency
mice can fully correct the systemic organ pathology but not ERT with recombinant human acid lipase is under evalua-
the neurological phenotype (Zhou et al. 1995). tion. HMG-CoA reductase inhibitors are given in cholesteryl
For Sandhoff disease substrate reduction therapy with ester storage disease to prevent early complications of ath-
miglustat is successfully tested in mouse model (Baek et al. erosclerosis. Wolman disease treatment is presently only
2008), and in a few cases miglustat is tested for chronic form symptomatic.
of Sandhoff disease in human.
Furthermore intrathecal stem cell therapy and intrathecal Neuronal Ceroid Lipofuscinoses (NCLs)
enzyme replacement therapy are tested in mouse model At present only symptomatic treatment is available. The anti-
(Matsuoka et al. 2011), and pyrimethamine, a protein stabi- epileptic drugs of choice are lamotrigine and valproate.
lizer, is tested in mice (Clarke et al. 2011). Carbamazepine, phenytoin, and vigabatrin can exacerbate
In addition there are new appendages with highly phos- myoclonia and should therefore be avoided. To control myo-
phomannosylated enzyme replacement therapy for chronic clonia and spasticity, baclofen and tizanidine have been used.
forms of GM2-gangliosidosis (Tsuji et al. 2011). Other supportive means such as chest physiotherapy, nasogas-
tric or gastric tube feeding, orthopedic treatment, and psycho-
Krabbe Disease logical help are important. Gene therapy has been evaluated
Hematopoietic stem cell transplantation appeared to be with several NCL animal models as well as in few clinical
successful in asymptomatic infantile patients or slowly trials. One clinical trial with stem cell therapy has not showed
progressive later-onset forms of disease. Demyelination significant effect. Intrathecal enzyme replacement therapy is
and neurological deterioration was prevented or arrested, currently the most promising treatment option for the CLN1
respectively (Krivit et al. 1998; Escolar et al. 2005). and CLN2 disease and has been proven to preserve neurologi-
Therapeutic approaches using virus-mediated gene trans- cal functions and extend the life span of treated animals.
fer and a combination of gene transfer and stem cell trans-
plantation were performed in a mouse model of Krabbe
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2623–2634 R, Rosenbaum H, Giraldo P, Mehta A, Park G, Phillips M, Elstein
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118(6):1463–1471 trial with plant cell-expressed recombinant glucocerebrosidase,
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Oligosaccharidoses and Sialic Acid
Disorders 26
Zoltan Lukacs and Michael Beck
Contents Summary
26.1 Introduction ..................................................................... 437 Oligosaccharidoses comprises a group of disorders which
26.2 Nomenclature................................................................... 439 show glycoprotein excretion in urine and diminished activity
of lysosomal enzymes that are involved in the degradation
26.3 Metabolic Pathways ........................................................ 440
of sugar side chains. Among those are glycosylasparagi-
26.4 Signs and Symptoms ....................................................... 440 nase deficiency (aspartylglucosaminuria), α-l-fucosidase
26.5 Reference Values ............................................................. 443 deficiency (fucosidosis), α- and β-mannosidase deficiency
26.6 Pathological Values ......................................................... 444
(α- and β-mannosidosis), α-N-acetylgalactosaminidase defi-
ciency (Schindler and Kanzaki disease), and neuraminidase
26.7 Specimen Collection ........................................................ 445 deficiency (sialidosis). In contrast to the latter, where deg-
26.8 Prenatal Diagnosis........................................................... 445 radation of sialic acid containing glycosides is impaired,
26.9 DNA Testing ..................................................................... 445 Salla disease results from a defect in sialin, a membrane
transporter protein. Consequently, sialic acid is stored in the
26.10 Treatment ......................................................................... 446
lysosomes. The diseases are rare and present a wide pheno-
References ...................................................................................... 447 typic spectrum. Detection of oligosaccharides in urine and
enzyme activity measurements in leukocytes or fibroblasts
is usually diagnostic. Free sialic acid has to be assessed
by HPLC. For final confirmation, a molecular genetic test
should be carried out. To date mainly palliative therapies
can be provided.
26.1 Introduction
The term oligosaccharidoses comprises a group of disorders

that are marked by defective degradation of protein-bound
sugar side chains as a result of deficiency in certain lyso-
somal enzymes. Among those, aspartylglucosaminuria
(AGU) is the most common lysosomal storage disorder,
which directly involves glycoprotein catabolism. Jenner and
Pollit published the first report on AGU in two patients in the
Z. Lukacs (*) UK (Pollitt and Jenner 1968). Based on this study Palo real-
Metabolic Laboratory, Department of Pediatrics University ized that a founder effect in Finland resulted in a high fre-
Medical Center, Institute of Clinical Chemistry, quency of AGU in the Finnish population (Palo and Mattsson
Martinistr 52, 20246 Hamburg, Germany
1970). In contrast, α-mannosidosis presumably just shows a
e-mail: lukacs@uke.uni-hamburg.de
worldwide incidence of 1:500,000 live births (Malm and
M. Beck
Nilssen 2008).
Villa Metabolica, Universitäts-Kinderklinik,
Mainz Langenbeckstr. 2, 55131 Mainz, Germany Hurler-like symptoms, skeletal abnormalities, hear-
e-mail: beck@kinder.klinik.uni-mainz.de ing impairment, and neurological symptoms are common
438 Z. Lukacs and M. Beck
features of α-mannosidosis, sialidosis, and fucosidosis. mutations in the PLA2G6 gene that is in close proximity to
Until 1999, for the latter, less than 100 patients have been the NAGA gene may have caused neuroaxonal dystrophy in
reported (Willems et al. 1999). Even less, only 21 cases of the original patients with Schindler disease (Westaway et al.
β-mannosidosis have been described until 2009, which may 2007). Aula et al. first described Salla disease that is named
also be attributed to the relative mildness of the disease in after a geographic region in Finland where the patients’
humans (Sabourdy et al. 2009). In sialidosis, the enzyme families have lived (Aula et al. 1979). In contrast, to the
neuraminidase is deficient. In vivo, this enzyme is found other diseases described above, Salla disease is caused by
within a multienzyme complex consisting of cathepsin a mutation in a transporter protein (sialin) of the lysosomal
A, β-galactosidase, and N-acetylgalactosamine-6-sulfate membrane. This, results in hampered efflux of free sialic
sulfatase. It seems that cathepsin A stabilizes this struc- acid from the lysosome and progressive accumulation in the
ture and thus deficiency of that protein leads to galacto- cell.
sialidosis which clinically resembles sialidosis. However, For the diagnosis of these disorders, usually, thin-layer
β-galactosidase deficiency is also observed in those patients, chromatography combined with enzyme measurement and/
probably due to degradation of this enzyme in the cell. The or mutation analysis is employed. Free sialic acid will not
determination of neuraminidase activity poses an additional prominently show on thin-layer chromatography, and there-
problem in the diagnosis of the disease, because the enzyme fore, a dedicated high-performance-liquid-chromatography
is rather unstable in vitro and will lose activity either by method is necessary to assess the concentration of sialic acid
sonication or freezing of the sample. The diseases named in urine and fibroblasts. More recently, tandem-mass spec-
after Schindler and Kanzaki represent different phenotypic trometry has been applied for the determination of oligosac-
expressions of α-N-acetylgalactosaminidase (α-NAGA) charides and sialic acid.
deficiency. However, it seems that α-NAGA deficiency alone Unfortunately, despite progress in therapies for many
cannot explain the phenotypic variation among patients with lysosomal storage diseases, the oligosaccharidoses still lack
the same mutation. In addition, no clear correlation between treatment, e.g., enzyme replacement therapy. However, fur-
residual enzyme activity and the severity of the mutation ther understanding of disease mechanism and phenotypic
exists. Thus, Keulemans et al. concluded that other genetic differences between patients with the same mutation may
factors may contribute to the clinical picture (Keulemans help to pave the way to novel therapies and better care for the
et al. 1996). Recently, Westaway et al. suggested that existing patients.
26
26.2 Nomenclature
Chromosomal
No. Disorder Alternative name Abbreviation Gene symbol localization Affected protein OMIM No. Subtype
26.1 Aspartylglucosaminuria Glycosylasparaginase deficiency AGU AGA 4q34.3 Glycosylasparaginase 208400 All forms
26.2 Fucosidosis Alpha-l-fucosidase deficiency FUCO FUCA1 1p34 Alpha-l-fucosidase 230000 All forms
26.3 Mannosidosis Alpha-mannosidase B deficiency LAMAN MAN2B1 19p13.2–q12 Alpha-mannosidase B 248500 All forms
26.4 Beta-mannosidosis Lysosomal β-mannosidase LBMAN MANBA 4q22-q25 Beta-mannosidase 248510 All forms
deficiency
26.5 Schindler disease type I Alpha-N-acetylgalactosaminidase NAGA NAGA 22q13–qter/22q11 Alpha-N-acetylgalactosaminidase 609241 Infantile form
deficiency type I
26.6 Kanzaki disease Alpha-N-acetylgalactosaminidase NAGA NAGA 22q13–qter/22q11 Alpha-N-acetylgalactosaminidase 609242 Adult form
deficiency type II
26.7 Schindler disease type III Alpha-N-acetylgalactosaminidase NAGA NAGA 22q13–qter/22q11 Alpha-N-acetylgalactosaminidase 609241 Mild form
deficiency type III
Oligosaccharidoses and Sialic Acid Disorders
26.8 Sialidosis Mucolipidosis type I NEU NEU1 6p21.3 Alpha-neuraminidase 256550 All forms
26.9 Salla disease Sialuria, Finnish type SLC17A5 SLC17A5 6q14–q15 Sialin 604369 All forms
439
26.3 Metabolic Pathways Protein
Lysosomal enzymes that cleave sugar residues from glyco- 3

proteins are deficient in oligosaccharidoses (Fig. 26.1). In 4α 1
4β
aspartylglucosaminuria, glycosylasparaginase shows dimin- Asn
3 2
ished activity (Fig. 26.1; step 2). This enzyme requires prior 4α
hydrolysis of both peptide bonds joined to the asparagine
(Asn). This is achieved by lysosomal cathepsins. In addition,
the enzyme is inhibited by fucose that is bound to the core
reducing end of N-acetylglucosamine (Fig. 26.1; step 1). Sialic acid N-acetylglucosamine
This special property explains the reason in fucosidosis for
fucose
the many oligosaccharide fragments that retain the Asn resi-
galactose α–/β-mannose
due (Aronson 1999).
In α-mannosidosis oligosaccharides containing α-1-3 and Fig. 26.1 Degradation of complex oligosaccharides. Liberation of the
α-1-6 linkages accumulate, while in β-mannosidosis the glycoside from the protein by lysosomal cathepsins is followed by a
disaccharide Man-β1-4GlcNAc is the major storage material sequential degradation of the sugar moieties (steps 1 to 4). Galactose
(Fig. 26.1, step 4α/β)(Cooper et al. 1990). For α-NAGA defi- and N-acetylglucosamine are cleaved by b-galactosidase and hexosa-
minidase, respectively, between step 3 and 4
ciency the major compounds are sialylglycopeptides of the
O-linked type. However, they seem not to be the primary
lysosomal storage product (Keulemans et al. 1996). Sialidosis HO
OH
is characterized by increased amounts of sialylated oligosac- COO−
charides and glycoproteins (Fig. 26.1, step 3).
In Salla disease, a defect in the membrane transporter pro- O
NH OH
tein sialin impairs efflux of sialic acid (Fig. 26.2) from the
lysosomes, so that this compound accumulates in the cell. O HO
HO
Fig. 26.2
Table 26.1 Aspartylglucosaminuria

Cardiovascular Cardiomyopathy, hypertrophic ± ± ±
CNS Epileptic seizures ± ± ±
Mental retardation + ++ ++
Spasticity ± + +
Digestive Hepatosplenomegaly ± ± ± ±
Hematological Vacuolated lymphocytes ± ±
Musculoskeletal Coarse facial features ± + +
Dysostosis multiplex + + +
Hernias ± ± ±
Short stature ± ± ±
26 Oligosaccharidoses and Sialic Acid Disorders 441
Table 26.2 Fucosidosis

CNS Epileptic seizures ± + +
Mental retardation ± ++ ++
Spasticity ± ++ ++
Dermatological Angiokeratoma ± +++ +++
Digestive Hepatosplenomegaly ± ± ±
Ear Hearing loss ± ±
Eye Corneal clouding ± ± ±
Hematological Vacuolar lymphocytes ± ± ±
Musculoskeletal Coarse facial features ± + +
Dysostosis multiplex ± + +
Hernias ± ± ± ±
Short stature + +
Table 26.3 Mannosidosis

CNS Ataxia + ++ +++
Mental retardation ± + ++ +++
Psychosis + +
Spasticity ± ± ±
Ear Deafness, sensorineural ++ +++ +++ +++
Eye Corneal clouding, deposits ± ± ±
Hematological Immunodeficiency ++ ++ ++ +
Vacuolar lymphocytes + + + +
Musculoskeletal Coarse facial features ++ ++ + +
Dysostosis multiplex + + ± ±
Hernias ± + + + ±
Macrocephaly + + + ±
Table 26.4 Beta-mannosidosis

Spasticity ± ± ±
Ear Hearing loss + ++ +++
Musculoskeletal Coarse facial + +
features
Short stature ± ± ±
Table 26.5 Schindler disease type I

CNS Ataxia ±
Mental retardation + ++
Myoclonic epilepsy + +
Neuroaxonal dystrophy ++ ++
Seizures + ++
Spastic tetraplegia ± +++
Startle response, exaggerated + +
Ear Hearing loss + ++
Eye Optic atrophy + ++
Table 26.6 Kanzaki disease

Cardiovascular Cardiomyopathy, hypertrophic +
Dermatological Angiokeratoma ++
Lymphedema ±
Musculoskeletal Coarse facial features ±
Table 26.7 Schindler disease type III

CNS Developmental delay + ++
Mental retardation + ++
Seizures ++ ++
Table 26.8 Sialidosis

Digestive Hepatosplenomegaly ++ ++ ±
Ear Hearing loss +
Eye Cherry red spot ++ ++ ++ ++
Corneal clouding + ± ± ±
Hematological Vacuolated lymphocytes + ++
Musculoskeletal Coarse facial features ++ ++
Dysostosis multiplex ++ ++
Hernias + + +
Macrocephaly ± ±
Short stature ± ++
Renal Renal failure, chronic ± ±
Table 26.9 Salla disease

CNS Ataxia ± + +
Developmental delay + + ++
Hypotonia ± + ±
Mental retardation ± ++ ++ +++
Spasticity + + ++ +++
Digestive Hepatosplenomegaly + ++ ++ ±
Eye Nystagmus + +
Musculoskeletal Coarse facial features ± ± ± ± ±
Hernias ± ±
Reference values for analysis of the specific enzymes that are deficient in the respective disorders and free sialic acid. Activities are usually inde-
pendent of age and gender. Ranges are listed according to methods and materials used
Entry Protein Method Material Range (unit) Reference
1 AGU 2-AADGa Fibroblasts 45–333 (nmol/min*g) Aula et al. (1974)
2 AGU 2-AADGa Lymphocytes 91–243 (nmol/min*g) Aula et al. (1976)
3 AGU 2-AADGa Leukocytes 22–132 (nmol/min*g) Aula et al. (1976)
4 AGU 2-AADGa Plasma 45–170 (nmol/min*mL) Aula et al. (1976)
5 AGU AspAMC Lymphocytes 87–493 (μU/mg) Mononen et al. (1994)
6 AGU AspAMC Serum 10.6–28.2 (mU/L) Mononen et al. (1994)
7 AGU AspAMC Plasma 10.2–26.0 (mU/L) Mononen et al. (1994)
8 FUCO NitroFb Fibroblasts 2.3–41.9 (nmol/mg*h) Zielke et al. (1972)
9 FUCO MUFc DBS 0.2–2.1 (nmol/spot*21 h) HHd
10 FUCO MUFc Leukocytes 0.05–0.4 (nmol/mg*min) HHd
11 FUCO MUFc Fibroblasts 0.05–0.33 (nmol/mg*min) HHd
12 FUCO MUFc Plasma 3–8 (nmol/min*mL) HHd
13 LAMAN MUAMe DBS 0.3–1.3 (nmol/spot*21 h) HHd
14 LAMAN MUAMe Leukocytes 0.09–0.7 (nmol/min*mg) HHd
15 LBMAN MUBMf Plasma 240–800 (μM/h*L) Cooper et al. (1987)
16 LBMAN MUBMf Leukocytes 245–467 (μM/h*g) Cooper et al. (1988)
17 LBMAN MUBMf Fibroblasts 58–389 (μM/h*g) Cooper et al. (1988)
18 NAGA MUNACg Fibroblasts 40–130 (nmol/h*mg) Keulemans et al. (1996)
19 NAGA MUNACg Plasma 4–8.33 (nmol/s*L) Kanzaki et al. (1991)
20 NAGA MUNACg Lymphoblasts 10.5–27.2 (nmol/s*g) Kanzaki et al. (1991)
21 NEU MUNEUh Fibroblasts 17.6–189 (nmol/h*mg) Mueller et al. (1986)
22 Sialini HPLC Fibroblasts 2.6–20.4 (nmol/mg protein) Erikson et al. (2002)
23 Sialini HPLC Urine <1 a: 0–58 Erikson et al. (2002)
1–4 a: 0–40
4–15 a: 0–30
> 15 a: 0–21
(mmol/mol Crea)
a
2-Acetamido-N-(l-aspart-4′-oyl)-2-deoxy-β-glucopyranosylamine
b
p-Nitrophenyl-α-l-fucopyranoside
c
4-methylumbelliferyl-α-l-fucoside
d
Reference values of the Hamburg Metabolic Laboratory
e
4-methylumbelliferyl-α-d-mannopyranoside
f
4-Methylumbelliferyl-β-d-mannopyranoside
g
4-methylumbelliferyl-α-N-acetylgalactosaminide
h
4-methylumbelliferyl-α-neuraminide
i
Free sialic acid has been determined by HPLC
Pathological values for the analysis of the specific enzymes deficient in ods and analytical materials that either have been used at the Hamburg
the listed disorders and free sialic acid. Activities are usually indepen- University Medical Center or have been published in peer-reviewed
dent of age and gender. Pathological values are restricted to those meth- journals
Entry Protein Method Material Range (unit) Reference
1 AGU 2-AADGa Fibroblasts 0–9 (nmol/min*g) Aula et al. (1974))
2 AGU 2-AADGa Lymphocytes 0–5 (nmol/min*g) Aula et al. (1976)
3 AGU 2-AADGa Leukocytes 0 (nmol/min*g) Aula et al. (1976)
4 AGU 2-AADGa Plasma 0–2 (nmol/min*mL) Aula et al. (1976)
5 AGU AspAMC Lymphocytes 1.2–11.4 (μU/mg) Mononen et al. (1994)
6 AGU AspAMC Serum 0–1.2 (mU/L) Mononen et al. (1994)
7 AGU AspAMC Plasma 0–0.9 (mU/L) Mononen et al. (1994)
8 FUCO NitroPb Fibroblasts 0–0.08 (nmol/mg*h) Zielke et al. (1972)c
9 FUCO MUFd Plasmae 0 (nmol/mL*min) HHf
10 LAMAN MUAMg Leukocytes < 0.05h (nmol/mg*min) HHf
11 LBMAN MUBMi Plasma 0–10 (μM/h*L) Cooper et al. (1987)
12 LBMAN MUBMi Leukocytes < 1 (μM/h*g) Cooper et al. (1988)
13 LBMAN MUBMi Fibroblasts 4 (n = 1) (μM/h*g) Cooper et al. (1988)
14 NAGA MUNACj Fibroblasts 0.2–3.2 (nmol/h*mg) Keulemans et al. (1996)
15 NAGA MUNACj Plasma 0.05 (n = 1) (nmol/s*L) Kanzaki et al. (1991)
16 NAGA MUNACj Lymphoblasts 0.05 (n = 1) (nmol/s*g) Kanzaki et al. (1991)
17 NEU MUNEUk Fibroblastsl 0–5.3 (Sialidosis) Mueller et al. (1986)
0.8–17.0 (Galactosialidosis)
(nmol/h*mg)
18 Sialinm HPLC Fibroblasts < 1.3 (nmol/mg protein) Erikson et al. (2002)
19 Sialinm HPLC Urine 48–402 (nmol/mol Crea) Erikson et al. (2002)
a
2-Acetamido-N-(l-aspart-4′-oyl)-2-deoxy-β-glucopyranosylamine
b
p-Nitrophenyl-α-l-fucopyranoside
c
Patients with fucosidosis showed enzyme activities below 10 % of normal in fibroblasts
d
4-Methylumbelliferyl-α-l-fucoside
e
A polymorphism is responsible for enzyme activities of approx. 25 % of the control mean in ca. 10 % of the population. This affects only measure-
ments in fibroblasts and plasma. Activity in leukocytes remains unaffected (Willems et al. 1999)
f
Pathological values obtained in the Hamburg Metabolic Laboratory
g
4-Methylumbelliferyl-α-d-mannopyranoside
h
In affected individuals the residual activity is found to be between 5 and 15 % of normal activity. This may be due to the fact that other mannosi-
dases are present and can contribute some activity. Following immunoprecipitation, only 0.1–1.3 % of the normal activity has been detected (Malm
and Nilssen 2008)
i
4-Methylumbelliferyl-β-d-mannopyranoside
j
4-methylumbelliferyl-α-N-acetylgalactosaminide
k
4-methylumbelliferyl-α-neuraminide
l
Enzyme is labile to sonication and freezing. Activity in leukocytes is approx. 10-fold lower than in fibroblasts. For this reason, leukocyte activity
measurement is not recommended for diagnosis in sialidosis
m
Free sialic acid has been determined by HPLC
Preferably, prenatal diagnosis should be performed by

molecular genetic testing especially, if mutations from an
index patient in the family are already known. Enzyme test-
ing of amniotic cells is also possible. However, it has to be
checked whether maternal cells are present and thus may
contribute enzyme activity. In addition, interpretation of low
enzyme activity becomes problematic in cases of heterozy-
Lane: A B C D E F G gote carriers, especially, as few laboratories have a significant
number of normal controls to determine a statistically reli-
Fig. 26.3 Oligosaccharides in urine analyzed by thin-layer chroma- able reference range. In any case, requirements and the pos-
tography provide a simple tool for a primary biochemical diagnosis.
sibility of prenatal enzyme testing should be checked with
This figure shows examples of certain oligosaccharidoses (urines kindly
provided by Prof. Beck, Mainz, Germany). Urines from patients with the specialized laboratory before shipment of samples.
the following disorders have been analyzed: GM2-gangliosidosis; Recently, a method has been described to determine sialic
Sandhoff type (lane A), fucosidosis (lane B), GM1-gangliosidosis, acid in amniotic fluid and fibroblasts for the diagnosis of
patient 1 (lane C), aspartylglucosaminuria (lane D), mannosidosis (lane
Salla disease (van den Bosch et al. 2011).
E), sialidosis (lane F), GM1-gangliosidoses, patient 2 (lane G)
26.7 Specimen Collection 26.9 DNA Testing
For enzyme analysis, either fibroblasts, leukocytes/lympho- In aspartylglucosaminuria the AGA gene is comprised of 9
cytes, plasma, or dried blood spots can be used, depending exons. The disease is most frequent in Finland (1:17 000)
on the enzyme that should be determined. Fibroblasts should while rare in other countries. One founder mutation (C163S)
be taken by specialized staff in a hospital environment. They is responsible for 98 % of AGU alleles in Finland. Another
can be shipped in sterile growth medium or, if shipping times Finnish mutation, a 2-bp deletion in exon 2 of the gene con-
do not exceed 1–2 days, in physiological NaCl solution (also tributes another 1.5 % of Finnish cases. Furthermore, a range
sterile). For leukocytes and lymphocytes, whole blood is of mutations from deletions to a rearrangement has been
required (usually approx. 5 mL). Shipping times should be described in few patients worldwide. Aronson (Aronson
kept to a minimum (less than 3 days). Nevertheless, the like- 1999), Saarela (Saarela et al. 2001), and Saito (Saito et al.
lihood of hemolysis increases with prolonged shipping 2008) review the currently known mutations.
times. Plasma samples should be shipped after centrifugation Fucosidosis is caused by mutations in the FUCA1 gene
and removal of any cell pellet to avoid contamination with that is composed of 8 exons spanning approx. 23kbp.
hemolyzed cells during shipment. Dried blood specimens Willems et al. (1999) review currently known mutations.
can be prepared either from tubes of whole blood or directly There are no particular hot spots in the gene and no fre-
from venous/capillary blood. In the case of newborns, the quently occurring mutations exist.
usual procedures for taking newborn screening samples can For α-mannosidosis, which is caused by mutations in the
also be followed. It is especially important that samples are MAN2B1-gene spanning 21.5 kB over 24 exons, mostly pri-
prepared swiftly after the blood has been taken. In addition, vate mutations that are distributed throughout the gene have
they should be dried at room temperature (ca. 20 °C) over been described. However, Berg et al. (1999) found that the
night before shipment. The stability of enzymes varies mutation R750W appears to be more frequent and accounted
widely in dried condition, so that timely shipping and analy- for 30 % of all disease alleles in that study.
sis is always preferable. Until 2009 only 21 cases of β-mannosidosis in 17 families
Oligosaccharides are analyzed from urine. Spontaneous have been described worldwide. The gene consists of 17
urine samples (ca. 3–5 mL) can be shipped without additives exons. In the past years, mutations have been published by
at room temperature, provided no bacterial contamination is Alkhayat et al. (1998), Bedilu et al. (2002), Stensland et al.
present. Otherwise, samples should be shipped on dry ice. (2008), and Sabourdy et al. (2009).
The α-NAGA gene consists of nine exons coding for 411 transplantation (HSCT) or exogenously supplied enzyme.
amino acids. Up to 2001 only 11 patients from seven families If stable engraftment is achieved, the donor cells should be
are known with Schindler/Kanzaki disease. An overview of able to build up sufficient amounts of enzyme to correct the
mutations is given by Bakker et al. (2001). deficient activity and to be clinically efficient for the whole
Sialidosis is caused by mutations in the NEU1 gene that life of the patient (Krivit 2004). Clinical experience on the
consists of 6 exons and codes for a protein of 368 amino acid efficacy of HSCT in lysosomal storage disorders has been
moieties. Seyrantepe et al. (2003) review the currently known obtained in patients with mucopolysaccharidosis, Gaucher
mutations. disease, Krabbe disease, Niemann-Pick disease types A, B,
Salla disease is caused by mutations in the SLC17A5 gene and C, fucosidosis, α-mannosidosis, aspartylglucosaminuria,
that codes for a lysosomal membrane transporter (sialin). mucolipidosis II, and GM2-Gangliosidoses (Krivit 2004;
The disease is mainly found in Finland where the missense Malatack et al. 2003). However, whereas allogeneic HSCT
mutation R39C is responsible for 95 % of cases. An over- has been shown to be very successful in patients affected
view of mutations is given in the paper by Verheijen et al. by the severe form of mucopolysaccharidosis type I (Hurler
(1999). disease), this procedure has a limited effect in other lyso-
somal storage disorders such as oligosaccharidoses, if not
performed in early infancy. Arvio et al. (2001), for example,
26.10 Treatment have reported 5 patients with aspartylglucosaminuria who
underwent bone marrow transplantation. All of them had
Until now, no specific treatment is available for patients post-transplant complications, and two of them after 7 and 5
affected by an oligosaccharidoses; therefore management years of follow-up were more severely mentally retarded in
mainly consists of supportive care and treatment of comparison with non-transplanted patients of a similar age.
complications. Thus, the authors came to the conclusion that bone marrow
Emergency Treatment transplantation is not suitable for the treatment of patients
Not applicable with aspartylglucosaminuria after infancy. Malm et al.
Standard Treatment (2004) have performed allogeneic stem cell transplantation
Arvio et al. reported on 121 Finnish patients, whereby 22 of in two siblings affected by aspartylglucosaminuria who were
78 adults (28 %) and 1 of 43 children (2 %) suffered from carefully followed for a period of 5 years with regular clini-
epileptic seizures. These patients responded rather well to cal and biochemical investigations. During this time no clini-
treatment with carbamazepine (Arvio et al. 1993). In fucosi- cal deterioration was observed.
dosis, the occurrence of dystonic symptoms has been Enzyme replacement therapy has been studied in several
observed to not respond to drugs that are commonly used for animal models of oligosaccharide storage disorders:
the treatment of dystonia such as carbamazepine, l-dopa, Dunder and coworkers have given mice affected by aspar-
anticholinergics, or baclofen (Gordon et al. 1995). tylglucosaminuria different dosages of recombinant glyco-
The anesthetic management of patients with oligosac- sylasparaginase at the age of 1 week. In the mice who have
charidoses is often complicated by airway problems such as received the highest amount of enzyme, a reduction of the
difficult laryngoscopy secondary to dysmorphic facial fea- storage material aspartylglucosamine was observed not
tures, thoracic deformities, and impaired visualization of the only in the visceral organs but also in the brain tissue
vocal cords (Soltani et al. 2007). Preoperative examination (Dunder et al. 2010).
should be carefully performed to rule out the presence of car- Gene knockout mice of α-mannosidosis show cerebellar
diomyopathy and hepatosplenomegaly. In those cases, intu- abnormalities such as macrophage activation, astrogliosis,
bation by fiber-optic laryngoscopy is strongly recommended and Lamp1 upregulation that is similar to the alteration
in order to prevent the necessity of performing a observed in human patients. Using short-term enzyme
tracheostomy. replacement therapy, the pathological changes could par-
Experimental Treatment tially be reversed, leading to less activated macrophages and
Oral zinc therapy was attempted on a 4-year-old boy with astrocytes (Damme et al. 2011). Based on this animal model,
α-mannosidosis because it had been demonstrated before a clinical trial of enzyme replacement therapy in
that zinc sulfate is able to stimulate α-mannosidase activity α-mannosidosis patients has been initiated (website: Clinical
in vitro. This treatment, however, did not have any clinical Trials http://clinicaltrials.gov/ct2/show/NCT01285700?term
effect (Wong et al. 1993). =Mannosidosis&rank=3, last access at October 3, 2011).
The fact that a fraction of lysosomal enzymes that does not As lysosomal storage disorders in general represent
enter the lysosome is secreted from the cell and can be recap- well-characterized single gene disorders and in addition are
tured from other cells via specific receptors is the prereq- not subject to complex regulation mechanisms, they are
uisite for successful treatment with hematopoietic stem cell excellent candidates for therapy by gene transfer. In addi-
tion, an enzyme activity of only 15–20 % of the normal Cooper A, Hatton C, Thornley M, Sardharwalla IB (1988) Human beta-
level is sufficient for clinical efficacy (Sands and Davidson mannosidase deficiency: biochemical findings in plasma, fibro-
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23:462–465
The Mucopolysaccharidoses
27
Giancarlo Parenti and Edward J. Wraith
Contents Summary
27.1 Introduction ....................................................................... 450 The mucopolysaccharidoses are a group of inborn errors of
metabolism caused by the deficiency of lysosomal hydrolases
27.2 Nomenclature..................................................................... 450
that degrade glycosaminoglycans (GAGs). These disorders
27.3 Metabolic Pathway ............................................................ 450 are associated with a progressive accumulation of different
27.4 Signs and Symptoms ......................................................... 453 types of GAGs within the cells of various organs and are char-
27.5 Diagnosis ............................................................................ 458
acterized by somatic manifestations (facial dysmorphisms,
hepatosplenomegaly, cardiac, respiratory, and skeletal involve-
27.6 Treatment ........................................................................... 460
ment) and neurological, hematologic, and ocular symptoms.
27.7 Follow-Up and Monitoring ............................................... 463 These manifestations are variably associated in each disorder.
References ..................................................................................... 464 In most cases the disease phenotypes encompass a spectrum
ranging from severe to attenuated clinical forms.
Because of the multisystem involvement in these patients,
initial clinical assessment should include the evaluation of
different organs and systems. Biochemical and genetic inves-
tigations are required to confirm the diagnosis. These include
the analysis of urinary GAGs, the demonstration of a specific
enzyme defect, and the identification of mutations in the rel-
evant gene. For many of these disorders, prenatal diagnosis
is possible either by direct assay of the deficient enzyme or
by molecular analysis.
Management of all mucopolysaccharidoses requires sup-
portive care and multidisciplinary treatment of a variety of
systemic complications. Specific treatment is based on dif-
ferent approaches. Hematopoietic stem cell transplantation
should be considered for MPS IH patients under the age of 2
years who have normal or near-normal developmental scores
and for MPS VI patients.
Enzyme replacement therapy (ERT) with recombinant
human enzymes is presently available for MPS IH/S and
MPS IS, MPS II, and MPS VI and is under development for
MPS IV. ERT results in improvement of somatic manifesta-
G. Parenti (*) tions and of motor performance and in reduced GAG urinary
Department of Pediatrics, Federico II University, excretion. However, other clinical features respond to a
Via S, Pansini, 5, 80131 Naples, Italy
lesser extent to therapy; there is no evidence that any of the
e-mail: parenti@unina.it
recombinant proteins cross the blood–brain barrier.
E.J. Wraith
Other therapeutic approaches for the treatment of the
Manchester Academic Health Sciences Centre (Genetic Medicine),
St. Mary’s Hospital, mucopolysaccharidoses, such as substrate reduction therapy
Manchester, UK and gene therapy, are still experimental.
450 G. Parenti and E.J. Wraith
27.1 Introduction accumulation of different types of GAGs within the cells of

various organs, ultimately compromising their function. The
The disorders described in this chapter are a broad group of major sites of disease differ depending on the specific
lysosomal storage diseases caused by the deficiency of lyso- enzyme deficiency, and therefore the clinical presentation
somal hydrolases that degrade the glycosaminoglycans and approach to therapy is different for the various disease
(GAGs). These disorders are associated with a progressive subtypes.
27.2 Nomenclature

No. Disorder Alternative name Abbreviation symbol localization Affected protein No. Sub type
27.1 Hurler, Alpha-iduronidase MPS I IDUA 4p16.3 Alpha-iduronidase 607014, All forms
Scheie disease deficiency 607015,
607016
27.2 Hunter Iduronate 2-sulfatase MPS II IDS Xq28 Iduronate 2-sulfatase 309900 All forms
disease deficiency
27.3 Sanfilippo Heparan-N-sulfatase MPS IIIA SGSH 17q25.3 Heparan-N-sulfatase 252900 All forms
A disease deficiency
27.4 Sanfilippo N-Acetyl-alpha-d- MPS IIIB NAGU 17q21 N-Acetyl-alpha-d- 252920 All forms
B disease glucosaminidase glucosaminidase
deficiency
27.5 Sanfilippo Acetyl-CoA alpha- MPS IIIC MPS3C 8p11.1 Acetyl-CoA alpha- 252930 All forms
C disease glucosaminide glucosaminide
acetyltransferase acetyltransferase
deficiency
27.6 Sanfilippo N-Acetylglucosamine MPS IIID GNS 12q14 N-Acetylglucosamine 252940 All forms
D disease -6-sulfatase deficiency -6-sulfatase
27.7 Morquio N-Acetylgalactosamine MPS IVA GALNS 16q24.3 N-Acetylgalactosamine 253000 All forms
A disease -6-sulfatase deficiency -6-sulfatase
27.8 Morquio Beta-galactosidase MPS IVB GLB1 3p21.33 Beta-galactosidase 253010 All forms
B disease deficiency
27.9 Maroteaux- N-Acetylgalactosamine MPS VI ARSB 5q11-q13 N-Acetylgalactosamine 253200 All forms
Lamy disease -4-sulfatase deficiency -4 – sulfatase
27.10 Sly disease Beta-glucuronidase MPS VII GUSB 7q21.11 Beta-glucuronidase 253220 All forms
deficiency
27.11 Hyaluronidase MPS IX HYAL1 3p21.31 Hyaluronidase 601492 All forms
deficiency
27.3 Metabolic Pathway end of the oligosaccharide chains) or sulfatases, a deficiency

of one of these activities results in a block of further degrada-
The degradation of GAGs is performed through the sequen- tion of GAGs. The degradation pathways of HS, KS, and DS
tial action of lysosomal hydrolases. As these enzymes are are shown in Fig. 27.1.
either exoglycosidases (they cleave the sugar moiety at the
27 The Mucopolysaccharidoses 451
Fig. 27.1 Metabolic pathways of (a) a Heparan sulfate degradation

OSO2H
heparan sulfate, (b) keratan sulfate, and (c)
CH2OH COOH CH2
dermatan sulfate degradation. The diseases O O O O
OH O O O
associated with each specific enzyme COOH
OH OH OH OH
O
(n)
deficiency are indicated OSO3H NHOSO2H OSO3H NAc
MPS II Iduronate sulfatase OSO2H

CH2OH COOH CH2
O O O O
OH COOH O O O O
OH OH OH OH
(n)
OH NHOSO2H OSO3H NAc
MPS I Alpha-iduronidase OSO2H

CH2OH COOH CH2
O O O
O O O
OH
OH OH OH
(n)
NHOSO2H OSO3H NAc
MPS IIIA Heparan-N-sulfatase OSO2H

CH2OH COOH CH2
O O O
O O O
OH
OH OH OH
(n)
NH2 OSO3H NAc
MPS IIIC AcetylCoA:acetyltransferase

OSO3H
CH2OH COOH CH2
O O O
O O O
OH
OH OH OH
(n)
NAc OSO3H NAc
MPS IIIB N-acetylglucosaminidase OSO3H

COOH CH2
O O
O O
OH
OH OH
(n)
OSO3H NAc
Glucuronate sulfatase OSO3H

COOH CH2
O O
O O
OH
OH OH
(n)
OH NAc
MPS VII β-glucuronidase

OSO3H
CH2
O
OH O
OH
(n)
N-acetylglucosamine- NAc
MPS IIID
6-sulfatase
OH
CH2
O
OH O
OH
(n)
NAc
Fig. 27.1 (continued) b Keratan sulfate degradation
OSO3H OSO3H OSO3H

CH2 CH2 CH2OH CH2
OH OH
O O O O
O O O O
OH OH OH
(n)
OH NAc OH NAc
MPS IVA galactose 6-sulfatase

OH OSO3H OSO3H
CH2 CH2 CH2OH CH2
OH OH
O O O O
O O O O
OH OH OH
(n)
OH NAc OH NAc
MPS IVB β-galactosidase

OSO3H OSO3H
CH2 CH2OH CH2
OH
O O O
OH O O O
OH OH
(n)
NAc OH NAc
MPS IIID N-acetylglucosamine 6-sulfatase

OH OSO3H
CH2 CH2OH CH2
OH
O O O
OH O O O
OH OH
(n)
NAc OH NAc
GM2 gangliosidoses β-hexosaminidases A and B
c Dermatan sulfate degradation

OSO3H OH
CH2OH COOH CH2OH
O O O O
OH COOH O O O O
OH OH
(n)
OSO3H NAc OH NAc
MPS II Iduronate sulfatase

OSO3H OH
CH2OH COOH CH2OH
O O O O
OH COOH O O O
O
OH OH
(n)
OH NAc OH NAc
MPS I Alpha-iduronidase
OSO3H OH
CH2OH COOH CH2OH
O O O
O O O
OH OH
(n)
NAc OH NAc
N-acetyl galactosamine
MPSVI 4-sulfatase
OH OH
CH2OH COOH CH2OH
O O O
O O O
OH OH
(n)
NAc OH NAc
GM2 gangliosidoses β-hexosaminidases A and B

OH
COOH CH2OH
O O
OH O O
OH
(n)
OH NAc
MPS VII β-glucuronidase

OH
CH2OH
O
O
OH
(n)
NAc
27.4 Signs and Symptoms disturbance. The behavior disorder is characteristic and often
leads to the diagnosis. Somatic features are mild in these
The major (and evocative) clinical features of mucopolysac- patients. Children with MPS IVA (Morquio disease, type A)
charidoses include somatic manifestations (facial dysmor- have normal cognitive functions, but are affected by severe
phism, hepatosplenomegaly, cardiac, respiratory, and skeletal spondyloepiphyseal dysplasia, which in most patients leads to
involvement) and neurological, hematologic, and ocular extreme short stature, deformity of the chest, marked shorten-
symptoms. These disease manifestations are variably associ- ing and instability of the neck, and joint laxity. MPS IVB
ated in each disorder. The combination of the clinical fea- (Morquio disease, type B) has some features of the skeletal
tures may be an important clue for the differential diagnosis dysplasia of MPS IVA, but is much more variable in its effects.
in a patient in whom a mucopolysaccharidosis is suspected. It has some features of the skeletal dysplasia of MPS IVA;
In most cases the disease phenotypes encompass a spec- MPS VII (Sly disease) often presents as nonimmune hydrops
trum ranging from early-onset (severe) to late-onset (attenu- fetalis. Those patients who survive or who present later
ated) clinical forms. The clinical presentation of each disease resemble patients with MPS IH with respect to clinical pheno-
is reported in Tables 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, type and supportive management. Patients with the rare MPS
27.8, 27.9, 27.10, and 27.11 (signs and symptoms). IX (Natowicz disease) show a clinical picture limited to joint
Patients with the severe form of mucopolysaccharidosis involvement and proliferative synovitis (Imundo et al. 2011).
(MPS I; Hurler disease, MPS IH), MPS II (Hunter disease), The phenotype of patients with more attenuated forms of
and MPS VI (Maroteaux-Lamy disease) generally present MPS, e. g., MPS IH/S or MPS IS (Hurler-Scheie or Scheie
with facial dysmorphism and persistent respiratory disease in disease, respectively) is much more difficult to predict, and
the early years of life. Many patients will have undergone treatment needs in this group of patients may be very vari-
surgical procedures for recurrent otitis media and hernia able. The MPS disorders in general present as a continuum of
repair before the diagnosis is established. Infants with MPS clinical involvement, and even patients with the most attenu-
III (Sanfilippo A, B, C, or D disease) present with learning ated forms of Scheie syndrome may have severe disabilities,
difficulties and then develop a profound behavioral requiring major medical and surgical interventions.
Table 27.1 Hurler, Scheie disease (MPS I)a

Cardiovascular Cardiomyopathy – + + + +
Coronary artery disease – + + + +
Valvular thickening – ++ +++ +++ +++
CNS Behavioral disorder – – ± – –
Cervical myelopathy – – ++ ++ ±
Hydrocephalus – ++ ++ ++ –
Retardation and regression n ++ +++ ± –
Seizures – ± ± – –
Swallowing difficulties – + + + –
Digestive Diarrhea – + + + –
Hepatosplenomegaly – ++ +++ +++ +
Ear Hearing loss – ± + + +
Recurrent otitis media – ++ ++ ++ –
Eye Corneal clouding – + +++ +++ +++
Glaucoma – ± + – –
Retinal dystrophy – ± ± ++ ++
Musculoskeletal Atlantoaxial instability – ± + + –
Carpal tunnel syndrome – ± ++ ++ ++
Coarse facial features – ++ +++ ++ ++
Degenerative hip dysplasia – + ++ ++ ++
Dysostosis multiplex – +++ +++ ++ ++
Genu valgum – ± ± ± ±
Hernias – ++ ++ ++ +
Joint contractures – ++ +++ +++ ++
Kyphosis – + ++ + –
Macrocephaly – +++ +++ ++ +
(continued)

Respiratory Obstructive sleep apnea – ++ +++ +++ ++
Restrictive lung disease – ± + + ±
Upper airway obstruction – ++ +++ +++ ++
Special Laboratory Alpha-iduronidase activity ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Dermatan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Heparan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
a
MPS I presents with wide variability. Traditionally, the severe forms and the attenuated phenotypes are referred to as Hurler disease (MPS IH) and
Scheie disease (MPS IS), respectively. Intermediate phenotypes are known as Hurler/Scheie (MPS IH/S). In the table infancy and childhood phe-
notypes describe the MPS IH, whereas the adolescence and adult phenotypes refer to MPS IH/S and MPS IS (Scheie disease), respectively
Table 27.2 Hunter disease (MPS II)a

Cardiovascular Cardiomyopathy – + + + +
Valvular thickening – ++ +++ +++ +++
CNS Behavioral disorder – – ++ ++ –
Cervical myelopathy – – ++ ++ –
Hydrocephalus – ++ ++ ++ –
Retardation and regression – ++ +++ ++ –
Seizures – + + ++ –
Swallowing difficulties – ± + ± –
Digestive Diarrhea – ± + ± –
Hepatosplenomegaly – ++ +++ +++ ++
Ear Hearing loss – ++ ++ ++ ++
Recurrent otitis media – ++ ++ ++ ++
Eye Retinal dystrophy – – ± ± ±
Musculoskeletal Atlantoaxial instability – ± + + ±
Carpal tunnel syndrome – ± ++ ++ ++
Coarse facial features – ++ +++ ++ ++
Degenerative hip dysplasia – + ++ ++ +
Dysostosis multiplex – + + + +
Hernias – +++ +++ ++ +
Joint contractures – +++ +++ +++ ++
Kyphosis – + ++ + ±
Macrocephaly – +++ +++ ++ +
Respiratory Obstructive sleep apnea – ++ +++ +++ ++
Restrictive lung disease – ± + + ±
Upper airway obstruction – ++ +++ +++ ++
Special laboratory Dermatan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Iduronate-2-sulfatase (LC) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
a
Hunter disease phenotype is variable. The adolescence and adult phenotypes refer to the milder forms of the disease
Table 27.3 Sanfilippo A disease (MPS IIIA)

CNS Behavior, aggressive – ± +++ ++ +
Hyperactivity – ± +++ ++ +
Retardation and regression – ± +++ +++ +++
Seizures – – ± ++ ++
Sleep disturbances – ++ +++ ++ +
Swallowing difficulties – ± + +++ +++
Digestive Diarrhea – ++ ++ + –
Liver dysfunction – ± ± + +
Ear Hearing loss – ± ± ± ±
Musculoskeletal Coarse facial features – ± + + +
Dysostosis multiplex – ± ± ± ±
Special Laboratory Heparan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Heparan-N-sulfatase (LC) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 27.4 Sanfilippo B disease (MPS IIIB)

Sleep disturbances – ++ +++ ++ +
Digestive Diarrhea – ++ ++ + ±
Special laboratory Alpha-N-acetylglucosaminidase ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 27.5 Sanfilippo C disease (MPS IIIC)

Sleep disturbances – ± ++ ++ +
Special laboratory Acetyl-CoA:alpha-N-glucosaminide-N-acetyl ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
transferase (LC)
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 27.6 Sanfilippo D disease (MPS IIID)

CNS Behavior, aggressive – – +++ ++ +
Hyperactivity – – +++ ++ +
Retardation and regression – – +++ +++ +++
Seizures – – – ++ ++
Liver dysfunction – – ± + +
Ear Hearing loss – – ± ± ±
Musculoskeletal Coarse facial features – – + + +
Dysostosis multiplex – – ± ± ±
Special Laboratory Heparan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
N-Acetylglucosamine-6-sulfatase (LC) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 27.7 Morquio A disease (MPS IVA)

Cardiovascular Valvular thickening – – + + +
CNS Cervical myelopathy – – + ++ ++
Digestive Liver dysfunction – – + + +
Ear Hearing loss – – + + +
Eye Corneal clouding – – + ++ ++
Musculoskeletal Atlantoaxial instability – +++ +++ +++ +++
Coarse facial features – – ± ± ±
Degenerative hip dysplasia – – ++ ++ ++
Dysostosis multiplex – – + + +
Genu Valgum – – +++ +++ +++
Joint laxity – – + + +
Kyphosis – ++ ++ ++ ++
Odontoid hypoplasia – – ++ ++ ++
Short stature. – + +++ +++ +++
Sternal bulging – + ++ ++ ++
Respiratory Restrictive lung disease – – + + +
Special laboratory Chondroitin sulfate (U) n-↑ ↑ ↑ n-↑ n-↑
Keratan sulfate (U) n-↑ n-↑ n-↑ n n
N-Acetylgalactosamine-6-sulfatase (LC) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) – n-↑ n-↑ n n
Table 27.8 Morquio B disease (MPS IVB)

Cardiovascular Valvular thickening – – + + +
CNS Cervical myelopathy – – + ++ ++
Digestive Liver dysfunction – – + + +
Ear Hearing loss – – ± ± ±
Eye Corneal clouding – – + ++ ++
Musculoskeletal Atlantoaxial instability – – ++ ++ ++
Coarse facial features – – ± ± ±
Dysostosis multiplex – – + + +
Genu Valgum – – ++ ++ ++
Joint laxity – – + + +
Kyphosis – – ++ ++ ++
Odontoid hypoplasia – – + + +
Short stature. – – +++ +++ +++
Sternal bulging – – + + +
Respiratory Restrictive lung disease – – + + +
Special laboratory Beta-galactosidase ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Keratan sulfate (U) n-↑ n-↑ n-↑ n-↑ n-↑
Total GAGs (U) n-↑ n-↑ n-↑ n n
Table 27.9 Maroteaux-Lamy disease (MPS VI)

Cardiovascular Cardiomyopathy – ± + + +
Valvular thickening – + +++ +++ +++
CNS Spinal cord, compression – – ++ ++ ++
Digestive Hepatosplenomegaly – ++ +++ +++ ++
Ear Hearing loss – – + + +
Recurrent otitis media – + ++ ++ +
Eye Corneal clouding – + +++ +++ +++
Glaucoma – – + + +
Musculoskeletal Carpal tunnel syndrome – ± ++ ++ ++
Coarse facial features – – ++ ++ ++
Dysostosis multiplex – + +++ +++ +++
Hernias – + + + +
Joint contractures – + +++ +++ +++
Kyphosis – + ++ ++ ++
Short stature. – + ++ ++ ++
Sternal bulging – – + + +
Respiratory Obstructive sleep apnea – + ++ ++ ++
Upper airway obstruction – + ++ ++ ++
Special laboratory Dermatan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
N-Acetylgalactosamine-4-sulfatase ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 27.10 Sly disease (MPS VII)

Cardiovascular Valvular thickening – ++ ++ – –
CNS Retardation and regression – +++ +++ ± ±
Digestive Hepatosplenomegaly + ++ ++ ++ ++
Eye Corneal clouding – ± ± ± ±
Musculoskeletal Coarse facial features – ++ ++ ++ ++
Dysostosis multiplex – + + + +
Hernias – + + – –
Joint contractures – ++ ++ ++ ++
Macrocephaly + + – – –
Short stature. + + + + +
Others Fetal hydrops ++ – – – –
Special laboratory Beta-glucuronidase ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Chondroitin sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Dermatan sulfate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Total GAGs (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 27.11 Hyaluronidase deficiency (MPS IX)

Dermatological Cutaneous nodules and swelling – – ++ ++ ++
Musculoskeletal Degenerative hip dysplasia – – + + +
Joint contractures – – ++ ++ +
Scoliosis – – + + +
Special laboratory Hyaluronan (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Hyaluronidase (P) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Total GAGs (U) n n n n n
27.5 Diagnosis procedures include the search for metabolites (quantitative

and qualitative analysis of urinary GAGs, Fig. 27.3),
The first step in the diagnostic workup of mucopolysacchari- enzyme assay of specific hydrolases in plasma, leucocytes
doses is the clinical evaluation of patients and first-level or fibroblasts, and the molecular analysis of the relevant
diagnostic procedures (x-ray, blood smear, eye examination, gene (usually in DNA samples from peripheral blood, but
ECG, cardiac ECHO, etc.; see Fig. 27.2). DNA can be obtained also from other sources, such as
Laboratory tests to complete the diagnosis should be fibroblasts).
performed in laboratories with specific expertise. These
MPS investigation sheet
History and examination Physiotherapy ENT Dental Cardiology Psychology Eyes NCVs Radiology Lab tests
height, wight, OFC (see
pulse Fig. 27.3)
respiratory rate
oxygen saturation 6MWT Audiology ECG DQ Acuity CTS
QoL questionnaire PFTs (>5 years) EUA ECHO IOP
registry consent severity score sleep study fundo-
clinical photographs JROM scopy
video (selected ERG
patients) VEPs
Plain radiology CT MRI Scan DEXA

(from age
5 years)
Skull C spine AP and Hips Whole Trachea Brain Whole Hips

flexion/ext lat whole and length chest and spine
spine pelvis lower CCJ lateral
standing limbs
standing
toes
forward
Fig. 27.2 The mucopolysaccharidoses investigation sheet. History and clinical examination are associated with evaluation of multisystem
involvement and laboratory tests to confirm the diagnosis
Urinary GAG analysis
Elevated values Normal values
Electrophoretic
separation
Consider Phenotype
oligosaccharide TLC suggestive of
and other lysosomal MPS IV
storage diseases
DS & HS HS DS
Electrophoretic
separation
MPS I MPS II MPS VII MPS III MPS VI
KS & CS
Enzyme assay to confirm Enzyme assay Enzyme assay to confirm

diagnosis diagnosis
Enzyme assay
MPS IIIA MPS IIB MPS IIC MPS IID MPS IVA and B
Fig. 27.3 The assay of urinary GAGs and the identification of specific excretion patterns by electrophoretic separation allows a temptative diag-
nosis of the mucopolysaccharidosis type. The suspect must be confirmed by the enzymatic assay in the appropriate sample
Samples for the biochemical diagnosis Prenatal diagnosis

of mucopolysaccharidoses
No. Disorder Enzyme assay Source
Metabolite 27.1 MPS I α-Iduronidase CV, A
analysis Enzyme Molecular 27.2 MPS II Iduronate-2-sulfatase CV, A, AF
No. Disorder (GAGs) assay characterizatione
27.3 MPS IIIA Heparan-N-sulfatase CV, A
27.1 MPS I Ua Lb, FBc B (sulfamidase)
27.2 MPS II U Pd, L, FB B 27.4 MPS IIIB α-N-acetylglucosaminidase CV, A
27.3 MPS IIIA U L, FB B 27.5 MPS IIIC Acetyl-CoA:α-glucosaminide CV, A
27.4 MPS IIIB U Pd, L, FB B N-acetyltransferase
27.5 MPS IIIC U L, FB B 27.6 MPS IIID N-Acetyl CV, A
27.6 MPS IIID U L, FB B glucosamine-6-sulfatase
27.7 MPS IVA U L, FB B 27.7 MPS IVA N-Acetyl CV, A
27.8 MPS VIB U L, FB B galattosamine-6-sulfatase
27.9 MPS VI U L, FB B 27.8 MPS VIB β-Galactosidase CV, A
27.10 MPS VII U L, FB B 27.9 MPS VI N-Acetyl CV, A
27.11 MPS IX – P B galactosamine-4-sulfatase
27.10 MPS VII β-Glucuronidase CV, A
Abbreviations: U urine, P plasma, S serum, B blood, L leucocytes, FB
fibroblast, CV chorionic villi, A amniocytes 27.11 MPS IX Hyaluronidase –a
a a
Urine should be kept frozen (−20°). Possible interference with GAGs Not reported in the literature, but in principle can be performed by
assay may be due to low creatinine, urine concentration, proteinuria, DNA analysis
and medications. Heparan sulfate may be increased in other diseases
b
Lymphocytes and leucocytes should be kept at room temperature
c
Fibroblasts are obtained by skin biopsy and kept at room temperature 27.6 Treatment
d
Plasma should be kept frozen (−20°)
e
For all diseases DNA can be obtained from other sources
(e.g., fibroblasts) Because of the multisystem involvement in these patients,
treatment is multidisciplinary and encompasses both the
Urinary GAG reference valuesa “curative” and palliative elements.
Uronic acid N-sulfated
Carbazole Orcinol hexosamine MBTH Supportive Treatment
Age method method method Irrespective of the type, management of all of them requires
1–11 months 25.5 ± 18.3 15.2 ± 14.9 19.8 ± 22.0 supportive care and multidisciplinary treatment of a variety
12–23 38.9 ± 30.1 20.9 ± 16.1 31.3 ± 22.2 of systemic complications.
months Regular evaluation at a major center with special interest
2–3 years 16.9 ± 9.9 10.7 ± 8.1 15.8 ± 12.3 and expertise in the management of the diseases is important
4–6 years 16.8 ± 14.9 13.7 ± 11.4 15.8 ± 12.3 in the coordination of interdisciplinary input and to coordi-
7–8 years 7.4 ± 5.2 4.6 ± 3.2 20.9 ± 16.1
nate multispecialty treatment strategies. In addition to the
9–11 years 8.3 ± 4.4 5.0 ± 3.2 8.8 ± 5.1
neurological complications experienced by many, distortion
12–18 years 10.0 ± 2.7 5.0 ± 1.9 4.0 ± 3.0
a
and narrowing of the upper airway and deformities of the
From Thompson (2003)
chest present potential fatal anesthetic risks for most patients
Urinary GAG pathologic values with MPS. Even the most trivial procedures requiring gen-
eral anesthesia should be done at centers with anesthetists
Disorder Range of valuesa who are experienced with MPS disorders.
Carbazole Orcinol MBTH Because of the progressive nature of the diseases, indi-
No method method method Qualitative viduals with MPS need to be evaluated regularly in order to
27.1 MPS I 157–537 153–783 20–75 DS, HS
identify potential problems early at a time when intervention
27.2 MPS II 311–537 200–302 44–99 DS, HS
would decrease morbidity, prevent premature mortality, and
27.3 MPS IIIA 116–297 29–89 30–88 HS
enhance the quality of life of affected patients. Every patient
27.4 MPS IIIB 63–272 25–108 37–59 HS
with MPS is unique; therefore, treatment options need to be
27.5 MPS IIIC 180–242 48–135 43–57 HS
27.6 MPS IIID 14–27 7–14 2–6 HS
individually based.
27.7 MPS IVA KS, CS The table summarizes the types of problems experienced
27.8 MPS VIB KS by patients with MPS disorders and strategies for their
27.9 MPS VI 23–108 40–161 2–7 DS management.
27.10 MPS VII 148 103 14 DS, CS,
HS Specific Treatment
a
From Thompson (2003) Hematopoietic Stem Cell Transplantation (HSCT)
Supportive treatment of mucopolysaccharidoses In patients with MPS IH under the age of 2 years who
System Problem Intervention have normal or near-normal developmental scores (DQ >
Cardiovascular Cardiomyopathy Antifailure medication 70), HSCT should be considered, using either HLA-matched
Valve lesions Antifailure medication; bone marrow or umbilical cord blood cells as the donor
valve replacement cells. The best results are achieved with HLA-matched sib-
Coronary artery None ling donors. Successful engraftment is associated with reso-
disease
lution of hepatosplenomegaly and upper airway obstruction.
CNS Hydrocephalus Ventriculoperitoneal
shunt surgery Corneal clouding usually resolves slowly, but never com-
Atlantoaxial Surgical decompression pletely. Intraocular pressures may decrease. Cardiac
instability and fusion of cervical manifestations attributable to muscle involvement are cor-
spine rected, but valvular abnormalities are resistant to HSCT and
Cervical myelopathy Surgical decompression often progress. Improvements in joint mobility are routinely
and fusion
experienced, and growth may approach normal rates for
Seizures Anticonvulsant
medication children the same age. However, some skeletal abnormali-
Behavior problems Behavior management, ties, especially abnormalities of the spine, do not respond to
medication HSCT, and most severely affected children still require
Sleep disturbance Medication major orthopedic interventions (Prasad and Kurtzberg
Mental retardation Appropriate educational 2010).
support and interventions Enzyme Replacement Therapy
Digestive Hepatosplenomegaly None ERT has been demonstrated in randomized, double-blind,
Umbilical and Surgical repair
placebo-controlled studies to produce improvements in joint
inguinal hernias
Swallowing Pureed diet, small,
mobility, pulmonary function, and exercise tolerance in
problems frequent meals; patients with MPS IH/S and MPS IS, MPS II, and MPS VI
gastrostomy (Laronidase, MPS I, Elaprase, MPS II, and Naglazyme, MPS
Drooling Hyoscine glycopyrrolate, VI). However, the extent and sustainability of improvement,
botox injections whether other clinical features of the disease will also
Diarrhea Antimotility medication
respond to therapy, and the optimum dosage of the enzymes
Ear Recurrent otitis Antibiotic therapy; ENT
media surgerya
used are still unknown (Wraith 2006). Laronidase
Sensorineural Hearing aids
(Aldurazyme) is licensed in the European Union and the
deafness United States to treat the nonneurological aspects of MPS I
Eye Corneal clouding Avoid direct sunlight; disease; there is no evidence that any of the recombinant pro-
corneal transplantation teins cross the blood–brain barrier. An approach based on
Glaucoma Topical beta-blockers; repeated intrathecal injections of recombinant enzymes is
trabecular surgery
under evaluation (Dickson and Chen 2011) but is not rou-
Retinal dystrophy None
tinely used. Other approaches based on high doses of recom-
Musculoskeletal Degenerative hip Analgesics; orthopedic
dysplasia surgical correction binant enzymes or modified enzyme preparations are in a
Kyphosis or Bracing or orthopedic research/preclinical phase of development (Polito et al. 2010;
kyphoscoliosis surgical correction Grubb et al. 2010). In MPS II and VI, intravenous ERT has
Joint contractures Physiotherapy and produced similar benefits.
orthoses A role as an adjunct to HSCT in patients with MPS IH is
Genu valgum Osteotomies currently under investigation. ERT may have the least impact
deformities
in patients with the most attenuated forms of the disorders.
Respiratory Upper airway ENT surgerya
obstruction Treatment costs are greater in these patients than in patients
Obstructive sleep Oxygen therapy CPAP or with more severe forms of the disease because the dosage of
apnea BiPAP enzyme is based on body weight, and patients with attenu-
Restrictive lung Oxygen therapy CPAP or ated disease are relatively heavy, compared to patients with
disease BiPAP more severe phenotypes. ERT for MPS IV is currently under-
Dental Caries, dental Oral hygiene; dental going clinical trial.
abscess extraction
Different therapeutic options may be available for the
Peripheral nerve Carpal tunnel Surgical decompression
syndrome same disorder and the appropriate treatment should be con-
ENT ears, nose, throat, CPAP continuous positive airway pressure sidered in individual patients. Figure 27.4 shows the approach
a
Including various combinations of tonsillectomy, adenoidectomy, to the treatment of MPS I.
myringotomy, the insertion of ventilation tubes, and tracheostomy
Suggestive clinical features
Increased urine HS/DS
Decreased/absent IDUA
MPS IH (Hurler) MPS IH (Hurler/Scheie) MPS IH (/Scheie)
Start Aldurazyme 100 mg/kg/week i.v. Careful assessment &

follow-up
MPS IH (Hurler) MPS IH (Hurler/Scheie)

Potentially treatable complications e.g.cardiac
muscle disease or deteriorating respiratory
Under 2 yrs Over 2 yrs function
Consider trial with Aldurazyme for 2 months

HSCT Aldurazyme Aldurazyme
Continue Patient improves No change or

Aldurazyme deterioration
until stable
engraftment
Continue treatment Stop treatment
Fig. 27.4 Flow chart for the management of a-l-iduronidase deficiency (MPS IH, -IH/S, -IS). HSCT hematopoietic stem cell transplant by bone
marrow or umbilical blood cells. HS heparan sulfate, DS dermatan sulfate
Patients with severe central nervous system involvement Other Therapeutic Approaches
(MPS III, Sanfilippo disease) or severe bone dysplasia Other therapeutic approaches for the treatment of muco-
(MPS IVA, Morquio disease) present particular challenges polysaccharidoses are currently under investigation.
to management, as current therapies are poor in correcting Substrate reduction therapy (SRT) is based on the use of
the effects of the genetic lesion in brain and bone, small-molecule drugs that interfere with substrate synthesis,
respectively. reducing the flux of substrates to lysosomes. SRT is cur-
rently approved for other lysosomal storage diseases
Treatment of mucopolysaccharidoses by ERT (Gaucher disease and Niemann-Pick disease type C) and has
Route and been proposed also for some mucopolysaccharidoses.
No. Disorder Age Medication Dosage frequency Specifically, the isoflavone genistein and the chemical dye
27.1 MPS I All Laronidase 100 U/kg IV weekly rhodamine B have been proposed as SRT agents (Malinowska
27.2 MPS II All Elaprase 0.5 mg/kg IV weekly et al. 2010; Roberts et al. 2006). Genistein has some effect in
27.9 MPS VI All Naglazyme 1 mg/kg IV weekly MPS III mice at high dose but the results in humans are
difficult to interpret and formal clinical trials have not been 27.7 Follow-Up and Monitoring
conducted.
Gene therapy also represents a potential treatment for The objectives of monitoring patients with MPS
mucopolysaccharidoses (Ellinwood et al. 2011) and clinical disorders are:
trials are being planned to treat some of these disorders (e.g., 1. To provide ongoing support for the patient and family
MPS IIIA and B). 2. To anticipate complications, identify them early when
they occur, and treat them in order to decrease morbidity
3. To monitor specific therapies, such as HSCT and ERT, to
Therapeutic options for mucopolysaccharidoses
assess their effectiveness, and, in the case of ERT, to
Gene
adjust enzyme dosage
No. Disorder HSCT ERT SRT therapy
18.1 MPS I + + Preclinical Preclinical
studies studies
18.2 MPS II + Preclinical Preclinical
studies studies
18.3 MPS IIIA Under + Preclinical
evaluation studies
18.4 MPS IIIB + Preclinical
studies
18.5 MPS IIIC ? Preclinical
studies
18.6 MPS IIID ? Preclinical
studies
18.7 MPS IVA Under Preclinical
evaluation studies
18.8 MPS VIB Preclinical
studies
18.9 MPS VI + + Preclinical
studies
18.10 MPS VII Preclinical
studies
18.11 MPS IX
Recommended follow-up and monitoring of MPS disorders (Dickson and Chen 2011)
Initial Every 6 months Every 12 months Every 2 years
General Medical history and physical examinationa * *
Neurological Neurological examination * *
Developmental assessment * *
MRI of brain * *
MRI of spine * *
Ophthalmologicb Visual acuity * *
Retinal examination * *
Corneal examination * *
Auditory ENT consultation * *
Audiometry * *
Cardiac Chest radiograph (for heart size) * *
ECG * *
Echocardiogram * *
Respiratory Pulmonary function testsc * *
Sleep study * *
Gastrointestinal Spleen and liver volumesd * *
Musculoskeletal Skeletal radiographse * *
Laboratory studiesf Enzyme assayg *
Urinary GAG level * *
Urine analysis * *
Antibody testing *h *
a
Including measurement of height, weight, head circumference, and blood pressure
b
Including measurement of intraocular pressures
c
Forced vital capacity (FVC) and 1-s forced expiratory volume (FEV1)
d
Best measured by MRI or CT scan
e
AP and lateral views of the skull, PA view of the chest, lateral views of the spine, (including the cervical spine), AP view of the hips and pelvis,
single AP view of both hands together. In the case of MPS IV, include lateral views of the neck in flexion and extension to assess stability of the
atlanto-axial joint, and a single AP view of the upper cervical spine through the open mouth to assess the integrity of the odontoid process. These
studies are primarily for the assessment of disease in children; the menu and schedule for radiographic studies in adults would be more limited,
emphasizing the assessment of osteoarthritis
f
In patients who have undergone hematopoietic stem cells transplantation (HSCT), leucocytes alpha-l-iduronidase assays and VNTR analyses on
DNA extracted from peripheral blood should be done monthly from the time of transplantation, then every 6 months to assess engraftment
g
For assessment of response to enzyme replacement therapy or HSCT
h
To be performed initially 3 months after the start of ERT
Malinowska M, Wilkinson FL, Langford-Smith KJ, Langford-Smith A,

References Brown JR, Crawford BE, Vanier MT, Grynkiewicz G, Wynn RF,
Wraith JE, Wegrzyn G, Bigger BW (2010) Genistein improves neu-
Dickson PI, Chen AH (2011) Intrathecal enzyme replacement therapy ropathology and corrects behaviour in a mouse model of neurode-
for mucopolysaccharidosis I: translating success in animal models generative metabolic disease. PLoS One 5:e14192
to patients. Curr Pharm Biotechnol 12:946–955 Polito VA, Abbondante S, Polishchuk RS, Nusco E, Salvia R, Cosma
Ellinwood NM, Ausseil J, Desmaris N, Bigou S, Liu S, Jens JK, MP (2010) Correction of CNS defects in the MPSII mouse model
Snella EM, Mohammed EE, Thomson CB, Raoul S, Joussemet B, via systemic enzyme replacement therapy. Hum Mol Genet
Roux F, Chérel Y, Lajat Y, Piraud M, Benchaouir R, Hermening S, 19:4871–4885
Petry H, Froissart R, Tardieu M, Ciron C, Moullier P, Parkes J, Prasad VK, Kurtzberg J (2010) Transplant outcomes in mucopolysac-
Kline KL, Maire I, Vanier MT, Heard JM, Colle MA (2011) Safe, charidoses. Semin Hematol 47:59–69
efficient, and reproducible gene therapy of the brain in the dog Roberts AL, Thomas BJ, Wilkinson AS, Fletcher JM, Byers S (2006)
models of Sanfilippo and Hurler syndromes. Mol Ther 19: Inhibition of glycosaminoglycan synthesis using rhodamine B in a
251–259 mouse model of mucopolysaccharidosis type IIIA. Pediatr Res
Grubb JH, Vogler C, Sly WS (2010) New strategies for enzyme replace- 60:309–314
ment therapy for lysosomal storage diseases. Rejuvenation Res Thompson JN (2003) The mucopolysaccharidoses. In: Blau N, Duran
13:229–236 M, Blaskovics M, Gibson KM (eds) Physicians guide to the labora-
Imundo L, LeDuc CA, Guha S, Brown M, Perino G, Gushulak L, tory diagnosis of metabolic diseases, 2nd edn. Springer, Berlin, pp
Triggs-Raine B, Chung WK (2011) A complete deficiency of 377–388
Hyaluronoglucosaminidase 1 (HYAL1) presenting as familial juve- Wraith JE (2006) Limitations of enzyme replacement therapy: current
nile idiopathic arthritis. J Inherit Metab Dis 34(5):1013–1022 and future. J Inherit Metab Dis 29:442–447
Hyperoxalurias
28
Bernd Hoppe and Nenad Blau
Contents Summary
28.1 Introduction ..................................................................... 465 Hyperoxaluria was often shown to be an important promoter
28.2 Nomenclature................................................................... 467 of crystallization (Hoppe et al. 2008; Coe et al. 1992).
Urinary oxalate is mostly of endogenous origin, only ~10 %
28.3 Metabolic Pathway .......................................................... 467
derive from the daily nutritional intake (Williams and
28.4 Signs and Symptoms ....................................................... 468 Wandzilak 1989; Monico and Milliner 1999). Primary causes
28.5 Reference Values ............................................................. 469 are distinguished from secondary ones: The autosomal-
28.6 Pathological Values ......................................................... 470
recessive inherited primary hyperoxaluria (PH) types 1, 2,
and 3 are defects of the glyoxylate metabolism leading to
28.7 Diagnostic Flow Chart .................................................... 470 endogenous (primary) overproduction of oxalate (Hoppe
28.8 Specimen Collection ........................................................ 471 et al. 2009; Belostotsky et al. 2010). Although type III pri-
28.9 Prenatal Diagnosis and DNA Testing ............................ 471 mary hyperoxaluria was just recently described, further types
of PH are likely to exist (unclassified PH). Urinary excretion
28.10 Treatment ......................................................................... 471
of oxalate is strongly elevated (>1 mmol/1.73 m2 BSA/day,
28.11 Follow-Up/Monitoring .................................................... 473 normal <0.5) in all forms of PH, resulting in recurrent stone
28.12 Standard Protocol for Intercurrent Illness ................... 473 formation and/or nephrocalcinosis and in progressive kidney
References ...................................................................................... 473
damage with systemic calcium oxalate deposition (systemic
oxalosis), primarily in PH type 1. Systemic oxalosis in PH I
is a catastrophic situation that must be prevented by all
means. Yet, diagnosis is all too often missed or delayed until
end-stage renal failure occurs (in > a third of adult patients).
This is particularly unfortunate because progressive renal
damage can be delayed or even prevented by early interven-
tion (Hoppe et al. 2009).
Secondary hyperoxaluria is due either to excessive dietary
oxalate intake (dietary hyperoxaluria) or to increased intesti-
nal oxalate absorption (enteric, mostly based on chronic
inflammatory bowel syndromes, Hoppe et al. 2003).
Although the urinary oxalate excretion is usually <1
B. Hoppe (*) mmol/1.73m2 BSA/24 h, it may nevertheless lead to signifi-
Division of Pediatric Nephrology, Department of Pediatrics, cant morbidity, i.e., to recurrent urolithiasis or progressive
University Hospital, Adenauerallee 119, Bonn 53113, Germany nephrocalcinosis with renal failure.
e-mail: bernd.hoppe@ukb.uni-bonn.de
N. Blau
Division of Inborn Metabolic Diseases, Department of General
Pediatrics, University Children’s Hospital,
28.1 Introduction
Im Neuenheimer Feld 430, Heidelberg 69120, Germany
All currently known types of primary hyperoxaluria (PH I–
Division of Metabolism, University Children’s Hospital,
Steinwiesstrasse 75, Zürich 8032, Switzerland III) are rare, autosomal-recessive disorders of the glyoxylate
e-mail: nenad.blau@med.uni-heidelberg.de metabolism (Hoppe et al. 2009; Belostotsky et al. 2010). In
466 B. Hoppe and N. Blau
PH I low or absent activity of liver-specific peroxisomal 2-oxoglutarate aldolase. Little is known about the pathogenetic
alanine-glyoxylate aminotransferase (AGT) causes massive basis of PH III, but increased glyoxylate generation by acti-
hyperoxaluria (Danpure 1989). PH I is the most frequent and vating mutations was the mechanism proposed (Belostotsky
most problematic subtype. The underlying AGXT-gene com- et al. 2010). However, the finding of nonsense mutations in
prising 11 exons and is located on chromosome 2q36-37 the HOGA1 gene suggested that a loss of function leads to
(Purdue et al. 1991). Diagnosis is mostly based on complete glyoxylate accumulation and hence the production of oxalate
AGXT sequencing, and >150 mutations have been identified (Beck et al. 2013). What may be extrapolated from the lim-
throughout the gene. Liver biopsy, previously regarded as the ited data available is the fact that this subtype is about to
gold standard, may still be necessary in patients with the become the second most frequent form and shows the most
obvious clinical presentation of PH I, however, an inconclu- favorable outcome. Although PH III so far has no documented
sive genetic testing. The disease prevalence is approximately case of ESRF, initial presentation in infancy with uni- or
two patients per million populations (van Woerden et al. bilateral nephrolithiasis eventually complicated by urinary
2003). The highly elevated urinary excretion of oxalate and tract infections can be quite severe (Monico et al. 2011).
glycolate (>1 mmol/1.73 m2 body surface area/day, normal The management greatly depends on the degree of renal
<0.5) causes renal calculi (medullary) NC, or both (Hoppe function, especially in PH type 1. One third of patients with PH
et al. 2009). With disease progression and declining renal I respond to pharmacological doses of pyridoxine (Milliner
calculi, (medullary) NC, or both function, calcium oxalate et al. 1994). In case of renal failure, the waiting time until trans-
crystals are systemically deposited (Akhan et al. 1995; plantation has to be kept as short as possible, as no form of
Hoppe et al. 2009), as plasma oxalate (Pox) and plasma cal- dialysis is able to keep pace with the extreme amounts of endog-
cium oxalate saturation (ßPCaOx) correlate inversely with the enous oxalate production (Leumann and Hoppe 2001). Thus,
glomerular filtration rate (GFR, Hoppe et al. 1999). CaOx systemic oxalosis develops, which greatly reduces the success
supersaturation then leads to systemic CaOx crystal deposi- of transplantation (Jamieson 2005). Isolated kidney transplanta-
tion and hence a multisystemic disorder with extreme mor- tion in PH I is only a reasonable option in fully pyridoxine
bidity. There is a substantial genetic, biochemical, and responsive patients or, perhaps, in older patients. The transplan-
phenotypic heterogeneity ranging from infantile end-stage tation treatment of choice in all other patients with PH I is com-
renal failure (ESRF) already in infancy (infantile oxalosis) to bined liver-kidney transplantation. In infantile oxalosis
a late onset or oligosymptomatic course in advanced adult- hepatocyte transplantation as bridging procedure until com-
hood. Unfortunately, most patients will develop ESRF over bined liver-kidney transplantation is possible may be an option
time. Systemic oxalosis in PH I is a catastrophic situation (Beck et al. 2012b). Preemptive liver transplantation as enzyme
that must be prevented by all means. Yet, diagnosis is all too replacement therapy may be considered in patients with remain-
often missed or delayed until end-stage renal failure occurs ing stable kidney function (GFR >50 ml/min, Nolkemper et al.
(in > a third of adult patients) (Hoppe and Langman 2003; 2003). In PH type 2 isolated kidney transplantation is the treat-
van Woerden et al. 2003). This is particularly unfortunate ment of choice, as the underlying enzyme defect is not only
because progressive renal damage can be delayed or even located within the liver. Currently, no patient with PH type 3 has
prevented by early intervention. reached end-stage renal failure (Beck et al. 2013; Belostotsky
Primary hyperoxaluria type II (PH II) seems to be even et al. 2010; Monico et al. 2011).
more rare (about a tenth of the PH I fraction) or remains Distinction between PH and secondary hyperoxaluria
markedly underdiagnosed. It is characterized by increased may be difficult (Hoppe et al. 2003, 2009; Robijn et al.
urinary excretion of oxalate and l-glyceric acid due to a 2011). The latter is due either to excessive dietary oxalate
defect of d-glycerate dehydrogenase and hydroxypyruvate intake (dietary hyperoxaluria) or to increased intestinal oxa-
reductase (GRHPR) (Cregeen et al. 2003). The GRHPR gene late absorption (enteric). Patients with intestinal disease have
located on chromosome 9p11 is composed of 9 exons with an increased risk of hyperoxaluria, particularly after bowel
~17 currently known causative mutations (Cramer et al. resection (short bowel syndrome), after bypass surgery, in
1999). The clinical course of PH II is generally more benign, chronic inflammatory bowel disease or cystic fibrosis, and in
but symptoms may be clinically indistinguishable from PH I. other malabsorption syndromes. Although the urinary oxa-
ESRF occurs less frequently, has not been reported in child- late excretion is usually <1 mmol/1.73 m2 BSA/24 h, it may
hood, but still affects about 20 % of adults (Cregeen and nevertheless lead to significant morbidity, i.e., to recurrent
Rumsby 1999; Marangella et al. 1999). urolithiasis or progressive nephrocalcinosis with renal fail-
Recently, mutations in a third, 7 exons spanning gene ure and also to systemic oxalosis subsuming multisystemic
(HOGA1, formerly called DHDPSL) on chromosome 10 disorder like in PH type 1 (Beck et al. 2008). Therapy is pri-
were found to cause PH III (Blostosky et al. 2010; Monico marily directed towards the underlying disease, but addi-
et al. 2011). HOGA1 encodes for a mitochondrial 4-hydroxy- tional measures are very important.
28 Hyperoxalurias 467
28.2 Nomenclature
Chromosomal
28.1 Primary Alanine-glyoxylate PH I AGXT 2q36-37 Alanine-glyoxylate 259900 All forms
hyperoxaluria aminotransferase aminotransferase
type I deficiency
(peroxisomal)
28.2 Primary Glyoxylate PH II and GRHPR 9p13.2 d-Glycerate 260000 All forms
hyperoxaluria reductase PH III dehydrogenase
type II deficiency
28.3 Primary 4-Hydroxy-2- PH HOGA1 10q24.2 4-Hydroxy-2- 613616 All forms
hyperoxaluria oxoglutarate oxoglutarate aldolase
type III aldolase deficiency
(mitochondrial)
Fig. 28.1 Proposed pathways of

oxalate, hydroxypyruvate, and
Serine Pyruvate Glycine
4-hydroxy-2-oxoglutarate
metabolism in human liver AGT 28.1 AGT Oxalate
Hydroxypyruvate Alanine Glyoxylate GO

Glycolate
Peroxisome
Cytososl
Hydroxypyruvate L-glycerate Glycolate
LDH
NAD(P)H NAD(P)
GR
HPR D-GDH 28.2 Mitochondrium 28.3 NAD(P)H+
NAD(P)+ 4-Hydroxy-2-oxoglutarate Pyruvate + Glyoxylate
D-glycerate LDH
HOGA
Oxalate
Gluconeogenesis
Table 28.1 Primary hyperoxaluria type I

Dermatological Calcinosis cutis ± ± ± ± ±
Livedo reticularis ± ± ± ± ±
Pigmentary retinopathy ± ± ± ± ±
Hematological Pancytopenia ± ± ± ± ±
Musculoskeletal Bone pain ± ± ± ± ±
Growth retardation ++ ++ ++ ++
Pathological fractures ± ± ± ± ±
Radiolucent metaphyseal bands + + + + +
Renal Nephrocalcinosis ++ ++ ++ + +
Nephrolithiasis ± ± ± + ++
Renal colic ± ± + ++ ++
Renal failure, chronic +++ ++ ++ + +
Urinary infections ± ± ± ± ±
Other Failure to thrive +++ +++ +++ +++
Routine laboratory Creatinine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Hematuria ± ± ± ± ±
Urea (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Glycolic acid (P) n-↑ n-↑ n-↑ n-↑ n-↑
Glycolic acid (U) n-↑ n-↑ n-↑ n-↑ n-↑
Oxalic acid (P) ↑↑ ↑↑ n-↑ n-↑ n-↑
Oxalic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 28.2 Primary hyperoxaluria type II

Growth retardation ± ± ± ±
Radiolucent metaphyseal bands + + + + +
Renal Nephrocalcinosis + + + + +
Nephrolithiasis + + + ++ ++
Renal colic + + + ++ ++
Renal failure, chronic ± ± ± ± ±
Urinary Infections ± ± ± ± ±
Other Failure to thrive ± ± ± ± ±
Routine laboratory Creatinine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Urea (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Glyceric acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Oxalic acid (P) n-↑ n-↑ n-↑ n-↑ n-↑
Oxalic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 28.3 Primary hyperoxaluria type III

Radiolucent metaphyseal bands ± ± ± ± ±
Renal Nephrocalcinosis + + + + +
Nephrolithiasis ++ ++ ++ ++ ±
Renal colic ++ ++ ++ + +
Urinary infections ± ± ± ± ±
Routine laboratory Calcium (U) ± ± ± ± ±
Creatinine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Urea (P) n-↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Oxalic acid (P) ± ± ± ± ±
Oxalic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Metabolite Age Normal value

Oxalate (U) All ages <0.5 mmol (45 mg)/1.73 m2/day
Glycolate (U) <0.5 mmol (45 mg)/1.73 m2/day
l-Glycerate (U) <5 μmol/l
Oxalate (U) mmol/mol creatinine <6 months <325–360
7 months–2 years <132–174
2–5 years <98–101
5–15 years <70–82
>16 years <40
Oxalate (P) μmol/l 6.3 ± 1.4
Glycolate (U) mmol/mol creatinine <6 months <363–425
7 months–2 years <245–293
2–5 years <191–229
5–15 years <166–186
>16 years <99–125
Glycolate (P) μmol/l 7.9 ± 2.4
l-Glycerate (U) mmol/mol creatinine <6 months 14–205
7 months–2 years 14–205
2–5 years 14–205
5–15 years 23–138
>16 years <138
Calcium (U) mmol/kg body weight/24 h All age groups <0.1
Data from Leumann et al. (1990), Hoppe et al. (1999, 2008), and Marangella et al. (1992)
Disorder Oxalate (U) Oxalate (P) Glycolate (U) Glycolate (P) l-Glycerate (U) Calcium (U)
28.1 PH I ↑ ↑ ↑ ↑ n n
28.2 PH II ↑ ↑ n n ↑ n
28.3 PH III ↑ ↑ n n n ↑
Hydroxy-oxo-glutarate (HOG), which is elevated in urine and plasma in PH III patients
Suspected stone disease Renal failure of unknown

origin (any age)
Child/adult recurrent/ Child first/single Ultrasonography

multiple stones stone
Nephrocalcinosis and/or
Extended work-up Basic work-up multiple stones
Oxalate (U) Oxalate (U) Oxalate in spot urine or

elevated normal plasma oxalate
Signs of Search for other

malabsorption reasons Oxalate (U) Oxalate (U) normal
Oxalate (P) >30 umol/L Oxalate (P) <30 umol/L

(chronic RF) (chronic RF)
Oxalate (P) >70 umol/L Oxalate (P) <70 umol/L

YES NO
(PD, pre-HD) (PD, pre-HD)
Evaluate for Search for other

Glycolate (U/P) ↑ PH Ι likely reasons
secondary
hyperoxaluria
Glycerate (U/P) ↑ PH ΙΙ likely
(e.g. cystic fibrosis,
Crohn's disease, short
HOG (U) ↑ PH ΙΙΙ likely
bowel syndrome)
Consider genetic analysis

No mutation
AGXT GRHPR HOGA1
Unclassified PH
PH Ι PH ΙΙ PH ΙΙΙ
Fig. 28.2

Oxalate Urine Immediately acidify with 0.02 ml, 6 mol/l HCl/ml to pH <3. 1. Excess alimentary oxalate or precursors
Dilute 20-fold with 0.3 mol/l boric acid (increased enteral uptake)
2. Vitamin B6 deficiency
3. Chronic renal failure
4. Nutrition in prematures
5. Alkaline pH aids spontaneous
formation of oxalate from ascorbate
6. Precipitation of oxalate from urine
sample before analysis
Blood, Keep cold after collection
Lithium- Separate red cells and ultrafilter (cutoff, 10,000 Da) 2 ml
heparinized plasma on to 0.06 ml, 1 mol/l hydrochloric acid/ml. Dilute
ultrafiltrate 1:1 with 0.3 mol/l boric acid. All steps at 4 °C.
Store at –20 °C until analysis
Glycolate Urine Immediately acidify with 0.02 ml, 6 mol/l HCl/ml to pH <3.
Dilute 20-fold with 0.3 mol/l boric acid
Blood, Keep cold after collection
Lithium- Separate red cells and ultrafilter (cutoff, 10,000 Da) 2 ml
heparinized plasma on to 0.06 ml, 1 mol/l hydrochloric acid/ml. Dilute
ultrafiltrate 1:1 with 0.3 mol/l boric acid All steps at 4 °C. Store
at –20 °C until analysis
Glycerate Urine Same handling, store and ship at –20 °C
28.9 Prenatal Diagnosis and DNA Testing

Amniotic fluid metabolites Fetal liver biopsy (enzyme activity) CV (enzyme activity) DNA testinga (from CV or biopsy)
PH I Nondiagnostic 2 trimester 1 and 2 trimester +
PH II Nondiagnostic ? ? +
PH III Nondiagnostic ? ? +
a
In PH patients genomic DNA sequencing or linkage analysis (if an index patient exists)
28.10 Treatment necessary in all patients (Milliner 2005). Reliable and

repeated baseline values for Uox (in normal renal function) or
AGT Deficiency (28.1; PH I) Pox in chronic renal failure are essential. If reduction of Uox
Systematic evaluation of pyridoxine (vitamin B6) treat- or Pox is >30 %, continue with dose of best response in B6
ment, starting with 5 mg/kg body weight/day, increasing trial. If no effect, discontinue pyridoxine. Be aware of side
every six weeks by 5 mg until 20 mg/kg body weight/day is effect (e.g., neuropathy).
Additional measures depending on clinical subgroup

Clinical category GFR (% of normal) Pox (μmol/l) Measures
A. Stone former/nephrocalcinosis >80 % <10 Large fluid intake (>2.5 l/m2 BSA/day) plus
Alkali (Na/K) citrate 0.3–0.5 meq (0.1–0.15 mg)/kg body
weight/day or orthophosphate 20–60 mg/kg body weight
per day
B. Asymptomatic (no stones, no >80 % <10 Large fluid intake (>2.5 l/m2 BSA/day)
nephrocalcinosis); Uox <0.7 mmol/day/1.73 m2
on B6
C. Reduced renal function: adults 30–80 % >30 Same as A
Medication: reduced dosage may be required
(continued)
Clinical category GFR (% of normal) Pox (μmol/l) Measures

D. Reduced renal function: age 3–18 years 30–80 % >30 Same as C
(Preemptive liver transplantation may be considered
[personalized approach, centers decision])
E. Renal failure: age 3–50 years <30 %, ESRF >80 Combined liver-kidney transplantation
F. Renal failure: adult, age >50 years <30 %, ESRF >80 (Isolated kidney transplantation may be an option in
B6-responsive patient)
G. Renal failure: adult, fully pyridoxine <30 %, ESRF <80 (Isolated kidney transplantation may be considered)
responsive
H. Infantile oxalosis <30 %, ESRF >80 Combined liver-kidney transplantation or sequential liver
and kidney transplantation
28.2. Glyoxylate Reductase Deficiency (PH II) Beware: Pitfalls in Hyperoxaluria

• No effect of pyridoxine to be expected 1. Urinary oxalate excretion and oxalate/creatinine ratios
• Benefit of liver transplantation in renal failure not are falsely low in renal insufficiency because of oxalate
established retention.
• Other measures as for PH I 2. Ascorbic acid is a precursor of oxalate and may interfere
28.3. HOGA1 Deficiency (PH III) with oxalate determination.
• No benefit of pyridoxine to be expected 3. Poor compliance is a serious problem in patients with
• No patient with ESRD reported, hence transplant option hyperoxaluria who require long-term therapy.
not yet determined 4. Calcium restriction is contraindicated because it leads to
• Other measures as for PH I enhanced intestinal oxalate absorption.
• Dietary (low-hydroxyproline, low-oxalate) diet 5. Renal replacement therapy by dialysis (hemo, peritoneal)
recommendable should be avoided by all means or, at least, not be extended
28.4. Unclassified PH (Enzyme Defect Not Yet Defined) beyond 6 months in patients with primary hyperoxaluria.
• Trial by pyridoxine may be made. No form of dialysis is able to eliminate all oxalate gener-
• Other measures as for PH I. ated, thus ongoing systemic deposition of calcium oxalate
is inevitable.
Secondary hyperoxaluria
No. Symbol Medication/diet Dosage
No. Symbol Medication/diet Dosage
28.5 SDHyOx Diet Low-oxalate,
28.1 PH I Hepatocyte transplantation To be determined,
Fluid intake high-calcium dieta
in infantile oxalosis performed once Beck
Alkali citrate >2 l/m2 BSA/day
et al. (2012a)
0.3–0.5 meq
(0.1–0.15 mg)/kg 28.1 PH I Oxalate-degrading bacteria Hoppe et al. (2006,
body weight per day like Oxalobacter 2011a, b) last phase III
formigenes study with negative
28.6 SEHyOx Fluid intake >2 l/m2 BSA/day
Diet Low-oxalate, 28.2 PH II Oxalate degrading bacteria results!
Alkali citrate high-calcium dieta 28.3 PH III applies to all forms of
0.3–0.5 meq 28.4 PH ? PH, Oxalate degrading
(0.1–0.15 mg)/kg enzyme medication to the
28.5 SDHyOx
body weight per day secondary forms
28.6 SEHyOx
a
Diet: avoid oxalate-rich food, e.g., spinach, rhubarb, and beetroot
28.11 Follow-Up/Monitoring
28.1–28.4 Primary Hyperoxalurias

Biochemical monitoringa
a
Age Clinical monitoring Basicb Uox, Ucitrate Pox Renal ultrasonography
<1 year Monthly Monthly Monthly 3 monthly 3 monthly
1–10 years 3–6 monthly 3–6 monthly 6 monthly 6 monthly 6–12 months
11–18 years 4–6 monthly 4–6 monthly 6 monthly 6 monthly 6–12 months
>18 years 6 monthly 6 monthly 6 monthly 6 monthly 6–12 months
a
More frequently with reduced renal function
b
Renal function (serum creatinine), electrolytes, and blood gases. Urine: oxalate, calcium, citrate, uric acid, and creatinine; relative density; and
sediment
Coe FL, Parks JH, Asplin JR (1992) The pathogenesis and treatment of
28.12 Standard Protocol for Intercurrent kidney stones. N Engl J Med 327(16):1141–1152
Illness Cramer SD, Ferree PM, Lin K, Milliner DS, Holmes RP (1999) The
gene encoding hydroxypyruvate reductase (GRHPR) is mutated in
patients with primary hyperoxaluria type II. Hum Mol Genet
Make sure patient gets a high fluid intake at all times. Early
8:2063–2069
intravenous fluid administration is indicated in case of severe Cregeen DP, Rumsby G (1999) Recent developments in our under-
diarrhea, vomiting, infection, and high fever. A medical standing of primary hyperoxaluria type 2. J Am Soc Nephrol
emergency card with appropriate instructions is recom- 10:S348–S350
Cregeen DP, Williams EL, Hulton S, Rumsby G (2003) Molecular anal-
mended for patients going abroad.
ysis of the glyoxylate reductase (GRHPR) gene and description of
mutations underlying primary hyperoxaluria type 2. Hum Mutat
22(6):497
Secondary hyperoxalurias – monitoring depends primarily on the Danpure CJ (1989) Recent advances in the understanding, diagnosis
underlying pathology and treatment of primary hyperoxaluria type 1. J Inherit Metab Dis
12(2):210–224
Clinical Biochemical Renal
Hoppe B, Langman CB (2003) A United States survey on diagnosis,
Age monitoringa monitoringb ultrasonography
treatment, and outcome of primary hyperoxaluria. Pediatr Nephrol
0–12 months 3 monthly 2–3 monthly 6 monthly 18(10):986–991
1–16 years 6 monthly 6 monthly 6–12 monthly Hoppe B, Kemper MJ, Bökenkamp A et al (1999) Plasma calcium-
>16 years 6–12 monthly 6–12 monthly yearly oxalate supersaturation in children with primary hyperoxaluria and
a
Fluid intake, stone passage, and general health end stage renal disease. Kidney Int 56:268–274
b
Renal function (serum creatinine). Urine: oxalate, calcium, citrate, and Hoppe B, Leumann E, von Unruh G, Laube N, Hesse A (2003)
creatinine; relative density; and sediment. Plasma oxalate optional Diagnostic and therapeutic approaches in patients with secondary
hyperoxaluria. Front Biosci 8:e437–e443
Hoppe B, Beck B, Gatter N et al (2006) Oxalobacter formigenes: a
potential tool for the treatment of primary hyperoxaluria type 1.
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Hoppe B, Leumann E, Milliner D (2008) Urolithiasis in childhood. In:
Akhan O, Ozmen MN, Coşkun M et al (1995) Systemic oxalosis: Geary D, Schäfer F (eds) Comprehensive pediatric nephrology.
pathognomonic renal and specific extrarenal findings on US and CT. Elsevier/WB Saunders, New York, pp 499–525
Pediatr Radiol 25(1):15–16 Hoppe B, Beck BB, Milliner DS (2009) The primary hyperoxalurias.
Beck B, Habbig S, Feldkötter M et al (2008) How to handle the dilemma Kidney Int 75(12):1264–1271
of ESRF and systemic oxalosis in short bowel syndrome from Hoppe B, Groothoff JW, Hulton SA et al (2011a) Efficacy and safety of
Crohn’s disease – a potential application for Oxalobacter formi- oxalobacter formigenes to reduce urinary oxalate in primary hyper-
genes. Pediatr Nephrol 23:P065 oxaluria. Nephrol Dial Transplant 26:3609–3615
Beck BB, Habbig S, Dittlich K et al (2012a) First liver cell transplanta- Hoppe B, Dittlich K, Fehrenbach H et al (2011b) Reduction of plasma
tion in infantile oxalosis. Cell Transplant (submitted) oxalate levels by oral application of oxalobacter formigenes in 2
Beck BB, Habbig S, Dittrich K, Stippel D et al (2012b) Liver cell trans- patients with infantile oxalosis. Am J Kidney Dis 58(3):453–455
plantation in severe infantile oxalosis. Nephrol Dial Transplant Jamieson NV, European PHI Transplantation Study Group (2005) A
27(7):2984–9. doi:10.1093/ndt/gfr776. [Epub 2012 Jan 28] 20-year experience of combined liver/kidney transplantation for pri-
Beck BB, Baasner A, Buescher A, Habbig S et al (2013) Novel findings in mary hyperoxaluria (PH1): the European PH1 transplant registry
patients with primary hyperoxaluria type III and implications for experience 1984-2004. Am J Nephrol 25:282–289
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Plasma and urine glycolate assays for differentiating the hyperoxal- Nephrol 6(9):2289–2295
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Cystinosis
29
Elena Levtchenko and Francesco Emma
Contents Summary
29.1 Introduction..................................................................... 476 Infantile nephropathic cystinosis is an autosomal reces-
sive disorder caused by mutations in the CTNS gene that
29.2 Nomenclature .................................................................. 477
encodes for cystinosin, a lysosomal cystine carrier. With
29.3 Metabolic Pathways ........................................................ 478 time, cystine accumulation leads to cell dysfunction in
29.4 Signs and Symptoms ....................................................... 478 various tissues; the kidneys are initially more involved.
Most patients are asymptomatic at birth but present in the
first or second year of life with failure to thrive, polyuria,
29.6 Pathological Values ......................................................... 480 dehydration, and/or rickets, which are secondary to the
29.7 Diagnostic Flow Chart .................................................... 480 renal Fanconi syndrome. With very few exceptions, all
29.8 Specimen Collection........................................................ 481 patients have corneal cystine crystals by 18 months of age
that can help in recognising the disease. The diagnosis of
29.9 Prenatal Diagnosis .......................................................... 481
cystinosis is based on the demonstration of increased
29.10 Treatment Summary....................................................... 481 intra‐leucocyte cystine levels and of mutations in the
References ..................................................................................... 481 CTNS gene (detection rate >95 %). In Northern Europe,
75 % of mutated alleles carry a 57‐kb deletion; all other
types of mutations, generally resulting in a complete or
severe loss of function of the cystinosin protein, have
been described.
In the past decades, the introduction of cysteamine treat-
ment and improvements in dialysis techniques and renal
transplantation have considerably improved the prognosis
of cystinosis. Cysteamine significantly delays progression
to end‐stage renal failure, but cannot prevent it in most
cases. The majority of patients live into adulthood, but,
when not appropriately treated with cysteamine, develop
other symptoms related to cystine accumulation in various
tissues. These include retinal degeneration, hypothyroid-
ism, diabetes mellitus, exocrine pancreatic insufficiency,
E. Levtchenko
Department of Pediatric Nephrology, pubertal retardation and gonadal dysfunction, restrictive
University Hospitals Leuven, Catholic University Leuven, pulmonary disease, myopathy, neurological deterioration,
Herestraat 49, Leuven 3000, Belgium and liver involvement. Two milder forms of the disease
e-mail: elena.levtchenko@uzleuven.be
(juvenile cystinosis and ocular cystinosis), caused by hypo-
F. Emma (*) morphic mutations of the CTNS gene, have also been
Division of Nephrology and Dialysis,
reported; these patients present with milder symptoms later
Ospedale Pediatrico Bambino Gesù, IRCCS,
Piazza S. Onofrio 4, Rome, 00165, Italy in childhood or with isolated corneal cystine depositions
e-mail: francesco.emma@opbg.net during adulthood.
476 E. Levtchenko and F. Emma
29.1 Introduction The incidence of cystinosis varies among countries

between 0.5 and 1.0:100,000 live births; several founder
Cystinosis is an inherited autosomal recessive disease caused mutations have been observed in different regions, such as
by mutations in the CTNS gene, which encodes for cystino- the 57 kb deletion that originated in Germany around A.D.
sin, a cystine carrier that is primarily expressed at the lyso- 500 (Gahl et al. 2002).
somal level (Kalatzis et al. 2001). The CTNS gene was cloned In its most frequent form, termed infantile nephro-
in 1998 and is located on chromosome 17p13 (Town et al. pathic cystinosis (MIM 219800), children are usually asymp-
1998). To date, more than 100 different mutations have been tomatic at birth and develop normally during the first 2–3
identified with a detection rate that exceeds 95 %. months of life; they generally present around the age of
The CTNS gene is composed of 12 exons and spans 23 kb; 6 months with failure to thrive, vomiting, constipation, poly-
cystinosin is composed of 367 amino acids that are believed uria, excessive thirst, dehydration, and sometimes rickets
to form a seven transmembrane domain cotransporter with (Gahl et al. 2002; Wilmer et al. 2010). These symptoms result
two lysosomal targeting motifs (Cherqui et al. 2001). The from a renal Fanconi syndrome, which is characterised by
most common mutation, accounting for 75 % of the affected excessive urinary losses of water, amino acids, phosphate,
alleles in Northern Europe, is a 57‐kb deletion that removes uric acid, bicarbonate, glucose, sodium, potassium, low‐
the first 9 exons and part of exon 10 of the CTNS gene, the molecular‐weight proteins, and other solutes, as a conse-
upstream 5′ region of the CARKL gene, and the first two non‐ quence of a global defect in renal proximal tubular
coding exons of the TRPV1 gene (Gahl et al. 2002; Wilmer reabsorption mechanisms. Untreated children develop
et al. 2010). The function of the CARKL gene has been iden- chronic renal failure during early childhood that progresses to
tified in the phosphorylation of sedoheptulose, an intermedi- end‐stage renal disease in the first decade of life. Patients with
ate metabolite of the pentose phosphate pathway. The cystinosis have a characteristic facial appearance; typically
transient receptor potential channel vanilloid subfamily Caucasian children have blond hair, although children from
member 1 (TRPV1), encodes for an ion channel that is pri- other ethnic groups may retain normal or near‐normal pig-
marily expressed in sensory nerves and is activated by a wide mentation. With time, cystine crystals deposit in the cornea
range of chemical stimuli. Patients with a homozygous 57‐ and are nearly always observed by 18 months of age (Tsilou
kb deletion have elevated blood and urinary levels of sedo- et al. 2002). They may cause photophobia and chronic inflam-
heptulose. The contribution of the CARKL and TRPV1 genes matory changes of the anterior chamber, if not treated.
to the pathogenesis of the disease in patients with homozy- The diagnosis of cystinosis is based on the measurement
gous 57‐kb deletion has not been fully studied; no major dif- of intra‐leucocyte cystine levels by HPLC or tandem mass
ferences in the clinical evolution, however, have been spectrometry. Each abnormal result needs to be confirmed
reported when comparing these patients to patients carrying by molecular analysis of the CTNS gene. Intra‐leucocyte cys-
other CTNS gene mutations. These include smaller deletions, tine levels are increased up to 100‐fold in affected individu-
insertions, nonsense mutations, missense mutations, muta- als compared with control subjects; levels in heterozygous
tions within the promoter region, and splice site mutations. carriers are only slightly increased.
Intronic mutations affecting normal splicing have also been In the past three decades, improvements in renal trans-
reported in families whose standard genetic testing failed to plantation and introduction of treatment with the cystine‐
identify mutations in one or both alleles, which strongly sug- depleting agent cysteamine have considerably prolonged the
gests that cystinosis is a monogenic disorder and that cDNA life expectancy of patients with cystinosis (Markello et al.
sequence analysis should be performed when gene sequenc- 1993; Van Stralen et al. 2011); however, they have also
ing does not allow identification of mutations (Taranta et al. revealed hitherto unknown long‐term complications result-
2010). ing from cystine accumulation in other tissues (Gahl et al.
Recently, a transcript variant originating from an alterna- 2007). These include retinal degeneration, hypothyroidism,
tive splicing of exon 12 has been reported and was shown to diabetes mellitus, exocrine pancreatic insufficiency, pubertal
be expressed also in other cell compartments, including the retardation and gonadal dysfunction, restrictive pulmonary
plasma membrane (Taranta et al. 2008). The contribution of disease, myopathy, neurological deterioration, and liver
this isoform to the physiopathology of the disease is not involvement. A few female patients have given birth to nor-
known yet (Wilmer et al. 2010). mal healthy children, but all tested male patients are infertile
29 Cystinosis 477
(Gahl et al. 2002). Most late‐onset complications improve or “juvenile” or “intermediate” form (MIM 219900), which is
are prevented by cysteamine (Gahl 2003; Gahl et al. 2007; usually diagnosed during childhood or adolescence and is
Kleta et al. 2004); corneal cystine crystals do not respond to characterised by less severe renal symptoms, and a third
oral cysteamine and require topical administration. form that has been reported primarily in adults and is charac-
Cysteamine treatment should be optimised by regular mea- terised only by ocular symptoms and has therefore been
surements of intra‐leucocyte cystine levels that allow adapt- termed “ocular” or “non‐nephropathic” cystinosis (MIM
ing the dose to target levels (Levtchenko et al. 2005). 219750).
Renal transplantation is a well‐established treatment for The severity of the disease co‐segregates within family
patients reaching end‐stage renal disease; cystinotic patients members with very few exceptions.
are probably at lower risk of rejection. Although cyste- In vitro studies of residual cystine transport activity have
amine has considerably improved the outcome, progression shown that infantile cystinosis generally results from severe
to end‐stage renal diseases cannot always be prevented mutations leading to complete loss of function of cystinosin
beyond the second or third decade of life (Greco et al. 2010). (Attard et al. 1999). To date, the physiopathology of cell
Early recognition of complications, optimalisation of nutri- damage in cystinosis has not been fully elucidated. In vitro
tional intake, early treatment of the Fanconi syndrome and and in vivo studies have shown that cystinotic cells are more
rickets, and growth hormone therapy have also considerably prone to apoptosis and autophagy; cysteinylation of pro-
improved the clinical outcome. apoptotic kinases may be involved in this process; abnormal-
In addition to the infantile form, two other milder variants ities in glutathione and ATP metabolism have also been
have been identified (Gahl et al. 2002). These include the reported (Wilmer et al. 2010).
29.2 Nomenclature
Gene Chromosomal Affected

No. Disorder Alternative name Abbreviation symbol localisation protein OMIM no. Subtype
29.1 Cystinosis Infantile form – CTNS 17p13 Cystinosin 219800; 219900a ; All forms
nephropathic 219750b
a
Juvenile form
b
Ocular form
a b
NH2 NH2
CYSTINOSIN
CH CH2 S S CH2 CH
COOH Cystine COOH

NH2
SH CH2 CH2
ATP Cysteamine
Lysosome
Cystine H+
Lysosome NH2 NH2

ADP+Pi
CH CH2 S S CH2 CH2
Cytosol
COOH
Mixed disulfide of
cysteine and cysteamine
PQLC2 TRANSPORTER
Fig. 29.1 Panel a. Cystinosin is a lysosomal cystine carrier; cystine resembling lysine, which can exit the lysosome through a PQLC2 trans-
efflux from lysosomes is dependent on the proton gradient generated by porter (Jézègou et al. PNAS 2012). It should be noted that the mixed
the vacuolar H+ ATPase. Panel b. Mechanism of action of cysteamine: disulfide is far more soluble than cystine and thus does not form
in the presence of cystine, cysteamine forms a mixed disulfide molecule crystals
Table 29.1 Nephropathic cystinosis (infantile)

CNS Cognitive dysfunction ± ± ±
Peripheral neuropathy ±
Pyramidal signs ±
Stroke-like episodes ± ±
Swallowing difficulties ±
Digestive Hepatomegaly ± ±
Liver fibrosis ±
Splenomegaly ±
Endocrine Diabetes mellitus ± ±
Hypogonadism ± +
Hypothyroidism ± ± ±
Infertility (men) + +
Pancreatic dysfunction, ± ±
endocrine
Puberty, delayed +
Eye Photophobia ± + +
Retinopathy ± ± ± + +
Vision, impaired ± ±
29 Cystinosis 479

Musculoskeletal Dental defects + + +
Diaphragm dysfunction ±
Genu valgum ± ± ± ±
Muscle weakness ±
Myopathy, peripheral ±
Osteopenia ± ± ±
Osteoporosis ± ± ±
Pes planus ± ± ±
Renal osteodystrophy ± ± ±
Rickets ± ± ± ±
Scoliosis ± ± ±
Renal Nephrocalcinosis ± ± ± ±
Nephrolithiasis ± ± ±
Polyuria + + + ±
Renal Fanconi syndrome + + ± ±
Renal failure, chronic ± ± +
Other Corneal cystine crystals ± + + +
Failure to thrive + +
Routine laboratory Albumin (U) ↑ ↑ ↑
Bicarbonate (P) ↓ ↓ ↓-nb
Calcium (U) ↑ ↑ ↑b
Creatinine (P) n n n-↑ n-↑
Glucose (U) ↑ ↑ ↑b
Phosphate (P) n ↓ ↓-n ↓-nb
Phosphate (U) ↑ ↑ ↑ ↑b
Potassium (P) n ↓ ↓ ↓-nb
Potassium (U) ↑ ↑ n-↑b
Sodium (U) ↑ ↑ ↑b
Uric acid (P) n ↓ ↓-n n-↑b
Uric acid (U) ↑ ↑ ↑b
Special laboratory Amino acids (P) n n n n
Amino acids (U) ↑ ↑ ↑ n-↑b
Carnitine, total and free (P) n ↓ ↓-n n
Cystine (P) n n n n
Cystine (WBC, FB)c ↑ ↑ ↑ ↑
T4 n n ↓-n ↓-n
TSH (S) n-↑ n-↑ n-↑
Patients with juvenile cystinosis present with symptoms similar to the infantile form, but usually at older age. Renal Fanconi syndrome is usually
less pronounced
Patients with ocular cystinosis present only with ocular complaints related to corneal cystine crystals, but not with systemic symptoms
a
Patients have usually a kidney transplant at that age; symptoms and routine biochemical parameters depend on the graft function
b
Biochemical features of renal Fanconi syndrome decrease during the decline of the GFR. Urinary features of renal Fanconi syndrome mostly
persist even at advanced stages of renal failure and after renal transplantation if the diuresis of the native kidneys is still present
c
Cystine measurements in cultured fibroblasts (FB) are not recommended routinely. The diagnosis of cystinosis should be confirmed by molecular
diagnosis of the CTNS gene
Clinical and biochemical characteristics of patients with dysfunction, but some may have more pronounced protein-
juvenile or intermediate form of cystinosis resemble those of uria, even in the nephrotic range.
patients with the infantile form; however, renal symptoms Clinical characteristics of patients with the ocular form are
usually manifest later in life, often during adolescence. restricted to photophobia due to corneal cystine accumula-
Most of these patients have less severe proximal tubular tion; some degree of renal dysfunction can appear later in life.
29.5 Reference Values used; these can vary considerably according to differences in
the pre‐analytic and analytic protocols, as well as in the type
Reference values for intracellular cystine
of cells that are assayed.
Intracellular cystine can be performed for the initial diag-
Cystine (WBC) <0.1–0.2 nmol cystine/mg proteina
nosis and for monitoring treatment using either mixed leuco-
Cystine (PMN) <0.1–0.2 nmol cystine/mg proteina
a
cyte preparations or PMN leucocytes isolated from whole
Cystine is sometimes expressed as nmol ½ cystine/mg protein.
Conversion factor ½ cystine/mg protein = 2× cystine/mg protein
blood. PMN leucocytes are lysosome‐rich cells and accumu-
late more cystine compared to the lymphocytes. Therefore,
cystine determination in PMN cells is a method of choice
because it increases the sensitivity of measurements and it is
29.6 Pathological Values not influenced by changes in the leucocyte formula that is
depending on age or undercurrent infections (Levtchenko
Pathological values for intracellular cystine et al. 2004).
Cystine (PMN) Heterozygotes 0.09–0.65 nmol
cystine/mg protein
Patients at diagnosis >2 nmol cystine/mg 29.7 Diagnostic Flow Chart
protein
Patients under cysteamine <0.6 nmol cystine/ Cystinosis should be excluded in all children presenting with
therapy mg protein
renal Fanconi syndrome. In general, slit lamp examination
Cystine (WBC) Heterozygotes 0.05–0.5 nmol
cystine/mg protein
and measurement of intracellular cystine concentrations are
Patients at diagnosis >2 nmol cystine/mg performed simultaneously. The absence of corneal crystals
protein does not allow the exclusion of the diagnosis of cystinosis in
Patients under cysteamine <0.5 nmol cystine/ small children, as these are generally not present during the
therapy mg protein first months of life or may be too small to be detected by
non‐experienced examiners. Mutations have been found in
Few laboratories worldwide offer intracellular cystine mea- the majority of patients. cDNA sequencing as well as
surements. The reported pathological values are only indica- sequencing of the promoter region allows the detection of
tive; reference values of individual laboratories should be mutations in nearly all patients.
Renal Fanconi syndrome
Corneal crystals by Intra-leucocyte

slit lamp eye examination cystine levels
No Yes Normal High
Other primary or DNA testing

secondary causes of
renal Fanconi syndrome?
No mutation CTNS mutation
Yes No
Test cDNA Cystinosis
Idiopathic renal Fanconi syndrome
Fig. 29.2 Differential diagnosis of renal Fanconi syndrome in young children without syndromic futures
29 Cystinosis 481
29.8 Specimen Collection (1–3 mg/kg/day in 2–3 separate doses), but this therapy can
be nephrotoxic; it can be, however, particularly useful in the
Intracellular cystine measurement is a particularly sensitive first years of life. The doses of potassium, sodium, bicarbon-
test that has several methodological pitfalls and should be ate, and phosphate supplements should be regularly adapted;
performed in certified laboratories. Each laboratory should 1,25‐dihydroxycholecalciferol should be prescribed very
produce its own reference values for healthy subjects, het- early to prevent rickets. Carnitine supplementation is recom-
erozygotes carriers, patients at diagnosis, and patients under mended in some centres.
cysteamine treatment. Treatment with recombinant growth hormone improves
Blood is usually collected in heparin or citrate tubes growth of children that are growth retarded despite cyste-
according to the laboratory recommendation; results are sig- amine therapy. Other complications such as hypothyroidism,
nificantly influenced by the type of anticoagulant. After col- diabetes, or hypogonadism are treated with l‐thyroxin, insu-
lecting blood samples, specimens should be proceeded as lin, or testosterone, respectively.
soon possible. The pre‐analytic phase is the most delicate ACE inhibitors can decrease albuminuria and may delay
part of the analysis. Cystine is currently measured by high- progression of renal failure; however, they may also cause
performance liquid chromatography (HPLC) or tandem hypotension and impair renal function in patients that are
mass spectrometry (MS‐MS). Results are normalised per mg fluid and salt depleted (Greco et al. 2010; Levtchenko et al.
of proteins; sample contamination with other non‐leucocyte 2003). They should be used therefore with caution, prefera-
proteins (e.g. erythrocyte ghosts) may result in falsely low bly not in conjunction with indomethacin; the dose should be
values. Other methods for normalising cystine data (per reduced or stopped during the hot weather season.
number of cells, per mcg of DNA, relative to other cell
metabolites) are being tested.
References
29.9 Prenatal Diagnosis Attard M, Jean G, Forestier L et al (1999) Severity of phenotype in
cystinosis varies with mutations in the CTNS gene: predicted effect
Prenatal DNA analysis is feasible and has become the unique on the model of cystinosin. Hum Mol Genet 8:2507–2514
method for prenatal screening. In the past, cystine measure- Cherqui S, Kalatzis V, Trugnan G et al (2001) The targeting of cystino-
sin to the lysosomal membrane requires a tyrosine‐based signal and
ments have been performed on cultured amniotic cells, but a novel sorting motif. J Biol Chem 276:13314–13321
this approach has now been abandoned. Dohil R, Gangoiti JA, Cabrera BL et al (2010) Long-term treatment of
cystinosis in children with twice-daily cysteamine. J Pediatr
156:823–827
Gahl WA (2003) Early oral cysteamine therapy for nephropathic cysti-
29.10 Treatment Summary nosis. Eur J Pediatr 162:S38–S41
Gahl WA, Thoene JG, Schneider JA (2002) Cystinosis. N Engl J Med
Oral cysteamine efficiently depletes lysosomes from cystine 347:111–121
(recommended dose: 1.3–1.9 g/m2/day administered every Gahl WA, Balog JZ, Kleta R (2007) Nephropathic cystinosis in adults:
natural history and effects of oral cysteamine therapy. Ann Intern
6 h) (Gahl et al. 2002; Markello et al. 1993). Treatment Med 147:242–250
should be started as soon as the diagnosis is made and con- Greco M, Brugnara M, Zaffanello M et al (2010) Long-term outcome of
tinued lifelong (Gahl 2003; Kleta et al. 2004). Blood sam- nephropathic cystinosis: a 20-year single-center experience. Pediatr
ples for monitoring therapy should be drawn 6 h after the Nephrol 25:2459–2467
Jézégou A, Llinares E, Anne C et al (2012) Heptahelical protein PQLC2
last dose (Levtchenko et al. 2005). Side effects of cyste- is a lysosomal cationic amino acid exporter underlying the action of
amine are mostly restricted to gastrointestinal discomfort, cysteamine in cystinosis therapy. Proc Natl Acad Sci U S A 109:
bad breath, and sweat odour. Gastric tolerance can be E3434–43
improved by proton pump inhibitors. Non‐compliance due Kalatzis V, Cherqui S, Antignac C et al (2001) Cystinosin, the protein
defective in cystinosis, is a H(+)-driven lysosomal cystine trans-
to foul odour and breath and strict administration schedules porter. EMBO J 20:5940–5949
occurs frequently particularly in adolescents. A delayed‐ Kleta R, Bernardini I, Ueda M et al (2004) Long-term follow-up of
release cysteamine is being currently tested (Dohil well-treated nephropathic cystinosis patients. J Pediatr
et al. 2010; Langman et al. 2012). 145:555–560
Langman CB, Greenbaum LA, Sarwal M et al (2012) A randomized
Symptomatic therapy includes prescription of appropriate controlled crossover trial with delayed-release cysteamine bitar-
fluid and electrolyte intake, adequate nutrition, and preven- trate in nephropathic cystinosis: effectiveness on white blood cell
tion of rickets. Patients with cystinosis should always have cystine levels and comparison of safety. Clin J Am Soc Nephrol
free access to water; prolonged heat exposure should be 7:1112–1120
Levtchenko E, Blom H, Wilmer M et al (2003) ACE inhibitor enalapril
avoided. Young children frequently require tube feeding. diminishes albuminuria in patients with cystinosis. Clin Nephrol
Renal tubular waste is significantly reduced by indomethacin 60:386–389
Levtchenko E, de Graaf-Hess A, Wilmer M et al (2004) Comparison of Town M, Jean G, Cherqui S et al (1998) A novel gene encoding an
cystine determination in mixed leukocytes vs polymorphonuclear integral membrane protein is mutated in nephropathic cystinosis.
leukocytes for diagnosis of cystinosis and monitoring of cysteamine Nat Genet 18:319–324
therapy. Clin Chem 50:1686–1688 Tsilou ET, Rubin BI, Reed GF et al (2002) Age-related prevalence of
Levtchenko EN, van Dael CM, de Graaf-Hess AC et al (2005) Strict anterior segment complications in patients with infantile nephro-
cysteamine dose regimen is required to prevent nocturnal cystine pathic cystinosis. Cornea 21:173–176
accumulation in cystinosis. Pediatr Nephrol 21:110–113 Van Stralen KJ, Emma F, Jager KJ et al (2011) Improvement in the
Markello TC, Bernardini IM, Gahl WA (1993) Improved renal function renal prognosis in nephropathic cystinosis. Clin J Am Soc Nephrol
in children with cystinosis treated with cysteamine. N Engl J Med 6:2485–2491
328:1157–1162 Wilmer MJ, Emma F, Levtchenko EN (2010) The pathogenesis of cys-
Taranta A, Petrini S, Palma A et al (2008) Identification and sub-cellu- tinosis: mechanisms beyond cystine accumulation. Am J Physiol
lar localization of a new cystinosin isoform. Am J Physiol Renal Renal Physiol 299:F905–F916
Physiol 294:F1101–F1108
Taranta A, Wilmer MJ, van den Heuvel LP et al (2010) Analysis of
CTNS gene transcripts in nephropathic cystinosis. Pediatr Nephrol
25(7):1263–1267
Congenital Disorders of Glycosylation
30
Jaak Jaeken and Lambert van den Heuvel
Contents Summary
30.1 Introduction ................................................................... 484 Congenital defects of glycosylation (CDG) are genetic
30.2 Nomenclature................................................................. 486 diseases due to deficient glycosylation of glycoconjugates
(glycoproteins, glycolipids, and glycosylphosphatidylinositol
30.3 Metabolic Pathway ........................................................ 489
anchors). Since the first clinical description of patients with
30.4 Signs and Symptoms Tables ......................................... 490 CDG, this disease family has shown an exponential expan-
30.5 Reference Values ........................................................... 509 sion. We know actually nearly 50 CDG that can be divided
30.6 Pathological Values ....................................................... 509
in four groups: (1) defects in protein N-glycosylation
(n: 16), (2) defects in protein O-glycosylation (n: 11), (3)
30.7 Diagnostic Flow Chart .................................................. 510 defects in glycolipid and glycosylphosphatidylinositol
30.8 Specimen Collection ...................................................... 511 anchor glycosylation (n: 3), and (4) defects in multiple
30.9 Prenatal Diagnosis Table and Sample glycosylation pathways and in other glycosylation pathways
Requirements ................................................................. 511 (n: 18). In 2008–2009 a novel, transparent CDG nomencla-
30.10 DNA Testing Table and Sample Requirements .......... 511 ture was introduced that covers all (known and still to be
discovered) CDG. It consists of the official gene symbol
30.11 Treatment ....................................................................... 511
(unitalicized) followed by “-CDG.” The majority of CDG
References .................................................................................... 511 patients show neurological disease associated with variable
involvement of nearly all other organs. Only a few CDG are
pauci-organ/mono-organ diseases (e.g., TUSC3-CDG
(brain), EXT1/EXT2-CDG (cartilage), GNE-CDG (skeletal
muscles), SEC23B-CDG (erythrocytes)). All known CDG
present autosomal recessive inheritance except for MAGT1-
CDG (X-linked) and EXT1/EXT2-CDG (autosomal domi-
nant). Some CDG can be diagnosed clinically such as EXT1/
EXT2-CDG. Two screening techniques are available: serum
transferrin isofocusing (or other techniques such as capillary
zone electrophoresis) for the diagnosis of protein
J. Jaeken (*)
N-glycosylation disorders associated with sialic acid defi-
Division of Metabolic Diseases, Department of Pediatrics,
University Hospital Gasthuisberg, Leuven 3000, Belgium ciency and serum apolipoprotein C-III isofocusing for the
e-mail: jaak.jaeken@uz.kuleuven.ac.be diagnosis of core 1 mucin type O-glycans. Treatment possi-
L. van den Heuvel bilities are still frustratingly limited. An efficient therapy is
Department of Pediatrics, Center for Metabolic Disease, University available for only one CDG, namely, oral mannose for MPI-
Hospital Gasthuisberg, Herestraat 49, Leuven, B-3000, Belgium CDG. Since about 1 % of the human genome is involved in
Department of Laboratory Medicine, Institute for Genetic glycosylation, it appears that most CDG have still to be dis-
and Metabolic Disease, Nijmegen Medical Centre, covered. Elucidation of the basic defects of these CDG will
Radboud University, Nijmegen, The Netherlands
increasingly require next-generation sequencing techniques
e-mail: bert.vandenheuvel@med.kuleuven.be,
l.p.w.j.vandenheuvel@live.nl such as whole genome sequencing.
484 J. Jaeken and L. van den Heuvel
30.1 Introduction of its pure hepatic-intestinal presentation but particularly

because it is still the only efficiently treatable CDG (with
Congenital disorders of glycosylation (CDG) are genetic dis- oral mannose). The 13 remaining N-glycosylation disorders
eases caused by defective glycosylation of glycoproteins, have been reported in less than 10 patients each and show
glycolipids, and glycosylphosphatidylinositol anchors variable combinations and degrees of neurological disease,
(reviews in Jaeken 2011; Grünewald 2007; Jaeken and other organ involvement and dysmorphy: mannosyltransfer-
Matthijs 2007; reviews on specific aspects of CDG in Coman ase 6 deficiency (ALG3-CDG), mannosyltransferase 8 defi-
et al. 2008; Footitt et al. 2009; Morava et al. 2009). It should ciency (ALG12-CDG), glucosyltransferase 2 deficiency
be noted that recently a novel CDG nomenclature was intro- (ALG8-CDG), mannosyltransferase 2 deficiency (ALG2-
duced that covers all (known and still to be discovered) CDG. CDG), UDP-GlcNAc:Dol-P-GlcNAc-P transferase defi-
It consists of the official gene symbol (unitalicized) followed ciency (DPAGT1-CDG), mannosyltransferase 1 deficiency
by “-CDG” (Jaeken et al. 2008, 2009). Among the 48 known (ALG1-CDG), mannosyltransferase 7–9 deficiency (ALG9-
CDG, 45 are disorders of glycoprotein glycosylation. This CDG), mannosyltransferase 4–5 deficiency (ALG11-CDG),
CDG group comprises disorders of N-glycosylation, disor- flippase of Man5GlcNAc2-PP-Dol deficiency (RFT1-CDG),
ders of O-glycosylation, and disorders with a combined N- N-acetylglucosaminyltransferase 2 deficiency (MGAT2-
and O-glycosylation defect. C-glycosylation defects have CDG), glucosidase 1 deficiency (GCS1-CDG), oligosac-
not yet been reported. The N-glycosylation pathway encom- charyltransferase subunit TUSC3 deficiency (TUSC3-CDG),
passes three cellular compartments: the cytosol, the endo- and magnesium transporter 1 deficiency (MAGT1-CDG).
plasmic reticulum (ER), and the Golgi. It starts in the cytosol Eighteen N-glycosylation disorders are associated with
with the formation of the mannose donor GDP-mannose an O-glycosylation defect. These comprise defects in doli-
from fructose 6-phosphate, an intermediate of the glycolytic chol phosphomannose synthesis (DPM1-CDG, DPM3-
pathway. In the ER, the dolichol-linked oligosaccharide CDG), in dolichol phosphomannose utilization
GlcNAc2Man9Glc3 is assembled and subsequently trans- (MPDU1-CDG), in galactosylation (B4GALT1-CDG), in
ferred from dolichol to selected asparagines of nascent pro- sialylation (SLC35A1-CDG), in fucosylation (SLC35C1-
teins. Still in the ER this glycan starts to be processed by CDG), in dolicholphosphate synthesis (DK1-CDG,
trimming off the three glucoses and one of the mannoses. SRD5A3-CDG), in ER-Golgi intermediate compartment
This processing is continued in the Golgi by trimming off proteins (SEC23B-CDG), and in other Golgi-associated pro-
five mannoses and replacing them with two residues each of teins (COG7-CDG, COG1-CDG, COG8-CDG, COG4-
N-acetylglucosamine, galactose and finally sialic acid. CDG, COG5-CDG, COG6-CDG, GMAP210-CDG, and
O-glycosylation is usually limited to the Golgi and has no ATP6V0A2-CDG). The latter two groups comprise defects
processing pathway. The O-glycans are linked to the in proteins that are not only involved in glycosylation but
hydroxyl group of selected threonines and serines. also in other functions (such as Golgi pH regulation). It is
The first report on CDG was on patients who were subse- proposed to name them “CDG-plus.” The symptomatology
quently shown to have an N-glycosylation disorder, phos- of patients with a combined protein glycosylation defect is
phomannomutase 2 deficiency (PMM2-CDG) (Jaeken et al. very heterogeneous: neurological, muscular, ophthalmologi-
1980; Van Schaftingen and Jaeken 1995; Matthijs et al. cal, cardiological, hepatic, skeletal, hematological, dermato-
1997). It is a cytosolic assembly defect and turned out to be, logical, etc.
by far, the most frequent N-glycosylation disorder with The known protein O-glycosylation disorders can be
around 600 patients known worldwide. They show mild to divided on the basis of the monosaccharide which links
severe neurological disease and variable involvement of the glycan to the protein. Five groups have been described:
many other organs. Dysmorphy ranges from mild and aspe- (1) defects in O-xylosylglycan synthesis (EXT1/EXT2-
cific to a characteristic abnormal subcutaneous adipose tis- CDG, CHSY1-CDG, B4GALT7-CDG), (2) defect in O-N-
sue distribution with fat pads and nipple retraction. Mortality acetylgalactosaminylglycan synthesis (GALNT3-CDG),
is about 20 % in the first years of life (review in Grünewald (3) defect in O-xylosyl-/N-acetylgalactosaminylglycan syn-
2009). The second most frequent N-glycosylation disorder is thesis (SLC35D1-CDG), (4) defects in O-mannosylglycan
an ER assembly defect, glucosyltransferase 1 deficiency synthesis (POMT1/POMT2-CDG, POMGNT1-CDG), and
(ALG6-CDG). It is mainly a neurological disorder but milder (5) defects in O-fucosylglycan synthesis (LFNG-CDG,
than that of PMM2-CDG (some 37 patients known) B3GALTL-CDG). Nearly all these diseases have also a
(Sun et al. 2005). The third most reported N-glycosylation descriptive name, based on their typical clinical presentation:
disorder is again a cytosolic assembly defect, phosphoman- multiple cartilaginous exostoses (EXT1/EXT2-CDG), famil-
nose isomerase deficiency (MPI-CDG) (de Koning et al. ial tumoral calcinosis (GALNT3-CDG), Schneckenbecken
1998; Jaeken et al. 1998; Niehues et al. 1998; de Lonlay and dysplasia (SLC35D1-CDG), muscle-eye-brain disease
Seta 2009). It is a very remarkable disorder not only because (POMGNT1-CDG), and spondylocostal dysostosis type 3
30 Congenital Disorders of Glycosylation 485
(LFNG-CDG). Waardenburg syndrome (POMT1/POMT2- asialoprotein (CDG-II). A type 1 pattern points to an

CDG) is also a muscle-eye-brain disease but with a more assembly or transfer defect of the dolichol-linked glycan
severe expression than POMGNT1-CDG, while Peters plus (in the cytosol or the ER). Measurement of the phospho-
syndrome (B3GALTL-CDG) shows peculiar anterior eye mannomutase activity in fibroblasts or leukocytes is the
chamber abnormalities besides many other symptoms. next step because PMM2-CDG is by far the most frequent
Defects in glycosphingolipid glycosylation are repre- N-glycosylation defect. In case of a purely hepato-intestinal
sented by only one disorder: GM3 synthase (Amish infantile presentation, the activity should be measured of phospho-
epilepsy or ST3GAL5-CDG). Finally, two defects are known mannose isomerase, deficient in MPI-CDG, a treatable
in phosphatidylinositolglycan synthesis: PIGM-CDG (with disease. Finding a normal activity of these enzymes neces-
absence epilepsy and hepatic venous thrombosis) and PIGV- sitates analysis of the lipid-linked oligosaccharides (LLO)
CDG, a mental retardation-hyperphosphatasemia syndrome. in fibroblasts. A type 2 pattern indicates a glycan processing
All CDG are inherited in an autosomal recessive way defect (in the ER or the Golgi). Processing defects that are
except MAGT1-CDG (X-linked) and EXT1/EXT2-CDG not associated with sialic acid deficiency (GCS1-CDG and
(autosomal dominant). SLC35C1-CDG) show, of course, a normal transferrin IEF
Essentially two techniques are available for CDG screen- profile. CDG-II patients are further investigated by means
ing: serum transferrin isofocusing (IEF) detects protein of mass spectrometry of isolated serum N-glycans. In a
N-glycosylation disorders associated with sialic acid defi- small minority of patients, this will lead to the identification
ciency (Jaeken et al. 1984), and serum apolipoprotein C-III of specific defects such as MGAT2-CDG and B4GALT1-
(IEF) detects core 1 mucin type O-glycosylation defects CDG, but in the majority of patients, an aspecific glycan
(Wopereis et al. 2003). Serum transferrin IEF can be profile will be found. In the latter situation, the possibility
replaced by other techniques such as capillary zone electro- of an associated mucin type O-glycosylation defect should
phoresis (Carchon et al. 2004). In case of an abnormal be investigated by IEF of serum apolipoprotein C-III. In
transferrin IEF profile, first an artifact, a protein polymor- patients with an abnormal IEF of transferrin as well as of
phism and a secondary CDG (fructosemia, galactosemia, apolipoprotein C-III, it is recommended to look for a defect
alcohol abuse, bacterial sialidase, etc.) should be excluded. in one of the COG (conserved oligomeric Golgi complex)
Two types of abnormal transferrin IEF profiles can be subunits by mutation analysis of the eight COG subunit
distinguished: a type 1 pattern, characterized by an increase genes (Foulquier 2009). However, if the patient presents a
of di- and/or asialoprotein (CDG-I), and a type 2 pattern, cutis laxa syndrome, mutation analysis of the ATP6V0A2
characterized by an increase of tri-, di-, mono-, and/or gene is indicated.
30.2 Nomenclature
486
Alternative Chromosomal
No. Disorder name Abbreviation Gene symbol localization Affected protein OMIM No. Subtype
30.1 Phosphomannomutase 2 deficiency PMM2-CDG PMM2-CDG PMM2 16p13.2 Phosphomannomutase 2 601785 All forms
– CDG-Ia
30.2 Phosphomannose isomerase MPI-CDG MPI-CDG MPI 15q24.1 Phosphomannose isomerase 602579 All forms
deficiency – CDG-Ib
30.3 Glucosyltransferase 1 deficiency ALG6-CDG ALG6-CDG ALG6 1p22.3 Glucosyltransferase 1 603147 All forms
– CDG-Ic
30.4 Mannosyltransferase 6 deficiency ALG3-CDG CDG-Id ALG3 3q27.1 Mannosyltransferase 6 601110 All forms
– CDG-Id
30.5 Mannosyltransferase 8 deficiency ALG 12-CDG ALG12-CDG ALG 12 22q13.33 Mannosyltransferase 8 607143 All forms
– CDG-Ig
30.6 Glucosyltransferase 2 deficiency ALG 8-CDG CDG-Ih ALG 8 11q14.1 Glucosyltransferase 2 608104 All forms
– CDG-Ih
30.7 Mannosyltransferase 2 deficiency ALG 2-CDG CDG-Ii ALG 2 9q22.33 Mannosyltransferase 2 607906 All forms
– CDG-Ii
30.8 UDP-GlcNAc:Dol-P-GlcNac-P DPAGT1-CDG CDG-Ij DPAGT1 11q23.3 UDP-GlcNAc:Dol-P-GlcNac-P 608093 All forms
transferase deficiency – CDG-Ij transferase
30.9 Mannosyltransferase 1 deficiency ALG1-CDG ALG1-CDG ALG1 16p13.3 Mannosyltransferase 1 608540 All forms
– CDG-Ik
30.10 Mannosyltransferase 7–9 deficiency ALG9-CDG CDG-Il ALG1 11q23.1 Mannosyltransferase 7–9 608776 All forms
– CDG-Il
30.11 Mannosyltransferase 4–5 deficiency ALG11-CDG ALG11-CDG ALG11 13q14.3 Mannosyltransferase 4–5 613661 All forms
(ALG11-CDG-Ip)
30.12 Flippase of Man5GlcNAc2-PP-Dol RFT1-CDG RFT1-CDG RFT1 3p21.1 Flippase of Man5GlcNAc2-PP-Dol 612015 All forms
deficiency – CDG-In
30.13 N-acetylglucosaminyltransferase 2 MGAT2-CDG MGAT2-CDG MGAT2 14q21.3 N-acetylglucosaminyltransferase 2 212066 All forms
deficiency – CDG-IIa
30.14 Glucosidase 1 deficiency – CDG-IIb GCS1-CDG GCS1-CDG GCS1 2p13.1 Glucosidase 1 606056 All forms
30.15 Oligosaccharyltransferase subunit tusc TUSC3-CDG TUSC3-CDG TUSC3 8p22 Oligosaccharyltransferase subunit tusc 611093 All forms
3 deficiency 3
30.16 Magnesium transporter 1 deficiency MAGT1-CDG MAGT1-CDG MAGT1 Xq21.1 Magnesium transporter 1 300716, All forms
(MAGT1-CDG) 300853
30.17 Exostosin 1 deficiency EXT1-CDG EXT1-CDG EXT1 8q24.11 Exostosin 1 133700 All forms
30.18 Exostosin 2 deficiency EXT2-CDG EXT2-CDG EXT2 11p11.2 Exostosin 2 133701 All forms
30.19 Chondroitin sulfate synthase 1 CHSY1-CDG CHSY1-CDG CHSY1 15q26.3 Chondroitin sulfate synthase 1 605282 All forms
deficiency
30.20 Beta-1,4-galactosyltransferase 7 B4GALT7-CDG B4GALT7-CDG B4GALT7 5q35.3 Beta-1,4-galactosyltransferase 7 130070 All forms
deficiency
30.21 Polypeptide GALTNT3-CDG GALTNT3-CDG GALNT3 2q24.3 Polypeptide 211900 All forms
N-acetylgalactosaminyltransferase 3 N-acetylgalactosaminyltransferase 3
deficiency
J. Jaeken and L. van den Heuvel
30
30.22 UDP-glucuronic acid/UDP-N- SLC35D1-CDG SLC35D1-CDG SLC35D1 1p31.3 UDP-glucuronic acid/UDP-N- 269250 All forms
acetylgalactosamine dual transporter acetylgalactosamine dual transporter
deficiency
30.23 O-Mannosyltransferase 1 deficiency POMT1-CDG POMT1-CDG POMT1 9q34.13 O-mannosyltransferase 1 236670, All forms
613555,
609308
30.24 O-Mannosyltransferase 2 deficiency POMT2-CDG POMT2-CDG POMT2 14q24.3 O-mannosyltransferase 2 613150, All forms
613156,
613158
30.25 O-Mannose beta-1,2-N- POMGNT1- POMGNT1-CDG POMGNT1 1p34.1 O-Mannose 253280, All forms
acetyglucosaminyltransferase CDG beta-1,2-N- 613151,
deficiency acetyglucosaminyltransferase 613157
30.26 O-Fucose-specific beta-1,3-N- LFNG-CDG LFNG-CDG LFNG 7p22.3 O-Fucose-specific 609813 All forms
acetylglucosaminyltransferase beta-1,3-N-
Congenital Disorders of Glycosylation
deficiency acetylglucosaminyltransferase
30.27 O-Fucose-specific beta-1,3-N- B3GALTL-CDG B3GALTL-CDG B3GALTL 13q12.3 O-Fucose-specific 261540 All forms
glucosyltransferase deficiency beta-1,3-N-glucosyltransferase
30.28 Lactosylceramide alpha-2,3- ST3GAL5-CDG ST3GAL5-CDG ST3GAL5 2p11.2 Lactosylceramide 609056 All forms
sialyltransferase deficiency alpha-2,3-sialyltransferase
30.29 Phosphatidylinositolglycan, class M, PIGM-CDG PIGM-CDG PIGM 1q23.2 Phosphatidylinositolglycan, class M 610293 All forms
deficiency
30.30 Phosphatidylinositolglycan, class V, PIGV-CDG PIGV-CDG PIGV 1p36.11 Phosphatidylinositolglycan, class V 239300 All forms
deficiency (PIGV-CDG)
30.31 GDP-Man:Dol-P mannosyltransferase DPM1-CDG CDG Ie DPM1 20q13.13 GDP-Man:Dol-P mannosyltransferase 608799 All forms
subunit 1 deficiency - CDG Ie subunit 1
30.32 GDP-Man:Dol-P mannosyltransferase DPM3-CDG DPM3-CDG DPM3 1q22 GDP-Man:Dol-P mannosyltransferase 3 612937 All forms
3 deficiency
30.33 Dol-P-Man utilization 1 deficiency MPDU1-CDG CDG-If MPDU1 17p13.1 Dol-P-Man utilization 1 609180 All forms
30.34 Beta-1,4-galactosyltransferase 1 B4GALT1-CDG B4GALT1-CDG B4GALT1 9p21.1 Beta-1,4-galactosyltransferase 1 607091 All forms
deficiency - CDG-IId
30.35 UDP-GlcNAc epimerase/kinase GNE-CDG GNE-CDG GNE 9p13.3 UDP-GlcNAc epimerase/kinase 600737, All forms
deficiency 605820
30.36 CMP-sialic acid transporter SLC35A1-CDG SLC35A1-CDG SLC35A1 6q15 CMP-sialic acid transporter 603585 All forms
deficiency - CDG-IIf
30.37 GDP-fucose transporter deficiency SLC35C1-CDG LAD2 SLC35C1 11p11.2 GDP-fucose transporter 266265 All forms
30.38 Dolichol kinase deficiency - CDG-Im DK1-CDG DK1-CDG DK1 9q34.11 Dolichol kinase 610768 All forms
30.39 Steroid 5 alpha-reductase 3 deficiency SRD5A3-CDG SRD5A3-CDG SRD5A3 4q12 Steroid 5 alpha-reductase 3 612379 All forms
30.40 Component of COG complex 7 COG7-CDG COG7-CDG COG7 16p12.2 COG complex 7 608779 All forms
deficiency - CDG-IIe
30.41 Component of COG complex 1 COG1-CDG COG1-CDG COG1 17q25.1 COG complex 1 611209 All forms
deficiency - CDG-IIg
deficiency - CDG-IIh
deficiency
487
488
deficiency
30.46 Golgi-microtubule-associated protein, GMAP210-CDG GMAP210-CDG GMAP210 14q32.12 Golgi-microtubule-associated protein 200600 All forms
210 kD, deficiency
30.47 V0 subunit a2 of vesicular H(+)- ATP6VOA2- ATP6V0A2-CDG ATP6V0A2 12q24.31 Vesicular H(+)-ATPase subunit a2 219200, All forms
ATPase deficiency CDG 278250
30.48 COPII component SEC23B deficiency SEC23B-CDG SEC23B-CDG, SEC23B 20p11.23 COPII component SEC23B 224100 All forms
CDA II
J. Jaeken and L. van den Heuvel
Sia2GaI2Man3GlcNAc4-Prot
G
O GaI2Man3GlcNAc4-Prot
L 30.34
G Man3GlcNAc4-Prot
Mevalonate
I 30.13
Man3GlcNAc3-Prot
SiaGalMan3GlcNAc3-Prot
Polyprenol
30.39
E Glc2Man9GlcNAc2-Prot
Dolichol 30.14
N
30.38
D Glc3Man9GlcNAc2-Prot
Dolichol phosphate O
30.15
P 30.16
Glc3Man9GlcNAc2-PP-Dol
Dol-PP L
A 30.6
S Glc2Man9GlcNAc2-PP-Dol
GlcNAc-PP-Dol
M 30.6
30.8
I GlcMan9GlcNAc2-PP-Dol
GlcNAc2-PP-Dol C
30.3
30.9
Man9GlcNAc2-PP-Dol Fructose 6-phosphate
ManGlcNAc2-PP-Dol R
30.10 30.2
30.7 E
T Man8GlcNAc2-PP-Dol Mannose 6-phosphate
Man2GlcNAc2PP-Dol
I 30.5 30.1
C Man7GlcNAc2-PP-Dol Mannose 1-phosphate
Man3GlcNAc2-PP-Dol
U 30.10
30.11 L Man6GlcNAc2-PP-Dol GDP-mannose
Man4GlcNAc2-PP-Dol U
30.33 30.31
30.11 M 30.4 30.32
Man5GlcNAc2-PP-Dol Man5GlcNAc2-PP-Dol DoI-P-Man
Fig. 30.1 Abbreviated scheme of the synthesis of a biantennary GCS1-CDG; 30.15, TUSC3-CDG; 30.16, MAGT1-CDG; 30.31,
N-glycan linked to dolichol pyrophosphate (Dol-PP) and subsequently, DPM1-CDG; 30.32, DPM3-CDG; 30.33, MPDU1-CDG; 30.34,
in the Golgi apparatus, to proteins (Prot). Fucose modification has been B4GALT1-CDG; 30.38, DK1-CDG; 30.39, SRD5A3-CDG. GDP gua-
omitted. 30.1, PMM2-CDG; 30.2, MPI-CDG; 30.3, ALG6-CDG; 30.4, nosine diphosphate, Man mannose, GlcNAc N-acetylglucosamine, Dol
ALG3-CDG; 30.5, ALG12-CDG; 30.6, ALG8-CDG; 30.7, ALG2- dolichol, P phosphate, Glc glucose, Gal galactose, Sia sialic acid.
CDG; 30.8, DPAGT1-CDG; 30.9, ALG1-CDG; 30.10, ALG9-CDG; Dotted arrow indicates multiple steps
30.11, ALG11-CDG; 30.12, RFT1-CDG; 30.13, MGAT2-CDG; 30.14,
Table 30.1 Phosphomannomutase 2 deficiency: CDG-Ia

Pericardial effusion ± ± ± ± ±
CNS Cerebellar hypoplasia + ++ ++ ++ ++
Demyelination + + + + +
Epilepsy ± ± + ± ±
Hypotonia ++ ++ + + +
Retardation, psychomotor ++ ++ ++ ++ ++
Tendon reflexes, decreased ± + + + +
Dermatological Subcutaneous fat distribution, abnormal ± ± ± ± ±
Digestive Anorexia ± ± ± ± ±
Ascites ± ± ± ± ±
Diarrhea ± ± ± ± ±
Failure to thrive ± ± ± ± ±
Liver dysfunction + + + ± ±
Vomiting ± ± ± − −
Endocrine Hypogonadism (in females), hypergonadotropic − − − + +
Eye Pigmentary retinopathy ± + + + +
Strabismus ± ± ± ± ±
Musculoskeletal Cervical compressive myelopathy − − ± ± ±
Dysmorphic features ± ± ± ± ±
Dysostosis multiplex ± ± ± ± ±
Joint contractures − − ± ± ±
Osteopenia − ± + + +
Vertebral anomalies ± ± ± ± ±
Renal Proteinuria ± ± ± ± ±
Renal enlargement ± ± ± ± ±
Other Fetal hydrops ± − − − −
Routine laboratory Albumin (S) + + + + +
Cholesterol (S) ↓ ↓ ↓ ↓ ↓
Factor XI (B) ↓ ↓ ↓ ↓ ↓
Thyroxin-binding globulin (S) ↓ ↓ ↓ ↓-n ↓-n
Transaminases (P) ↑ ↑ ↑ n-↑ n-↑
Special laboratory Arylsulfatase A (S) ↑ ↑ ↑ n-↑ n-↑
Sialotransferrins (S), type 1 pattern ++ ++ ++ + −/+
Table 30.2 Phosphomannose isomerase deficiency: CDG-Ib

Cardiovascular Thromboses − ± ± ± ±
CNS Hypotonia ± ± ± ± ±
Digestive Diarrhea − +/++ +/++ ± ±
Liver fibrosis − + + + +
Protein-losing enteropathy − +/++ +/++ ± ±
Routine laboratory Albumin (S) + + + + +
Cholesterol (S) ↓ ↓ ↓ ↓ ↓
Factor XI (B) ↓ ↓ ↓ ↓ ↓
Special laboratory Arylsulfatase A (S) ↑ ↑ ↑ n-↑ n-↑
Sialotransferrins (S), type 1 pattern ++ ++ ++ + −/+
Table 30.3 Glucosyltransferase 1 deficiency: CDG-Ic

Axial hypotonia ++ ++ + ± −
Epileptic seizures + ++ + ± ±
Retardation, psychomotor +/++ +/++ +/++ +/++ +/++
Eye Strabismus ++ ++ ++ ++ ++
Routine laboratory Cholesterol (S) ↓ ↓ ↓ ↓ ↓
Factor XI (B) ↓ ↓ ↓ ↓ ↓
Special laboratory Arylsulphatase A (S) ↑ ↑ ↑ n-↑ n-↑
Lipid-linked Man9GlcNAc2 (FB) ↑ ↑ ↑ ↑ ↑
Sialotransferrins (S), type 1 pattern + + + + −/+
Table 30.4 Mannosyltransferase 6 deficiency: CDG-Id

CNS Axial hypotonia + ++ ++
Epilepsy ++ ++ ++
Hypertonia, extremities + + +
Microcephaly ± + ++
Digestive Diarrhea ± ± ±
Vomiting ± ± ±
Strabismus ± ± ±
Musculoskeletal Arachnodactyly ± ± ±
Club foot ± ± ±
Facial dysmorphism ± + +
Micrognathia ± ± ±
Special laboratory Lipid-linked Man5GlcNAc2 (FB) ↑↑ ↑↑ ↑↑ ↑↑
Sialotransferrins (S), type 1 pattern + ++ ++
Table 30.5 Mannosyltransferase 8 deficiency: CDG-Ig

Cardiovascular Cardiomyopathy, hypertrophic ± ± ±
Ventricular septal defect ± ± ±
CNS Agenesis, corpus callosum ± ± ±
Cerebellar hypoplasia ± ± ±
Hearing, impaired ± ± ±
Hypotonia ± ± ±
Retardation, psychomotor ++ ++ ++
Dermatological Hypoplastic nails ± ± ±
Inverted nipples ± ± ±
Feeding difficulties ± ± ±
Gastric tube feeding ± ± ±
Gastrointestinal dysmotility ± ± ±
Malrotation ± ± ±
Eye Retinal detachment ± ± ±
Strabismus ± ± ±
Genitourinary Hypospadias ± ± ±
Male genital hypoplasia ± ± ±
Micropenis ± ± ±
Hematological Thrombocytopenia ± ± ±
Musculoskeletal Club foot ± ± ±
Dwarfism ± ± ±
Edema ± − −
Facial dysmorphism ± ± ±
Skeletal dysplasia ± ± ±
Other Early death ± ± ±
Maternal HELLP syndrome ± − − − −
Prematurity ± − − − −
Routine laboratory Cholesterol (P) ↓-n ↓-n ↓-n
Hypoglycemia ± ± −
IGF BP3 ↓-n ↓-n ↓-n
IGF1 ↓-n ↓-n ↓-n
Immunoglobulins ↓-n ↓-n ↓-n
Transaminases (P) n-↑ n-↑ n-↑
Special laboratory Lipid-linked Man7GlcNA2 (FB) ↑ ↑ ↑
MRI: temporal hypoplasia, dilated external CSF spaces ± ± ±
Sialotransferrins (S), type 1 pattern + + +
Table 30.6 Glucosyltransferase 2 deficiency: CDG-Ih

Cardiovascular Ventricular septal defect ± ± ±
CNS Cerebellar hypoplasia ± ± ±
Cerebral atrophy ± ± ±
Hypotonia + + +
Retardation, psychomotor −/++ −/++ −/++
Seizures ± ± ±
Dermatological Edema, generalized ± ± ±
Excess skin ± ± ±
Inverted nipples ± ± ± ± ±
Digestive Cholestasis ± ± ±
Feeding difficulties ± ± ±
Hepatomegaly ± ± ±
Protein-losing enteropathy ± ± ±
Endocrine Hypothyroidism ± ± ±
Eye Cataract ± ± ±
Optic atrophy ± ± ±
Genitourinary Cryptorchidism ± ± ±
Hematological Anemia ± ± ±
Coagulopathy ± ± ±
Thrombocytopenia ± ± ±
Musculoskeletal Camptodactyly ± ± ±
Closure of fontanels, delayed ± ± -
Osteopenia ± ± ±
Renal Renal cysts ± ± ±
Renal tubulopathy ± ± ±
Other Facial dysmorphism ± ± ±
Routine laboratory Albumin (S) ↓ ↓ ↓
Special laboratory Lipid-linked Glc1Man9GlcNAc2 + + +
Lipid-linked Glc1Man9GlcNAc2 (FB) ↑ ↑ ↑
Table 30.7 Mannosyltransferase 2 deficiency: CDG-Ii

CNS Abnormal jitter − − − + +
Bulbar dysfunction − − − ± ±
Congenital myasthenic syndrome ± ± ±
Demyelination + + +
Epilepsy + + + − −
Facial weakness (mild) ± ± ± ± ±
Hyperreflexia + + +
Hypotonia + + + − −
Motor developmental delay + + + + +
Eye Cataract + + +
Coloboma + + + ± ±
Factor XI (B) ↓ ↓ ↓
Special laboratory Lipid-linked Man1GlcNAc2 and Man2GlcNAc2 (FB) ↑ ↑ ↑
Table 30.8 UDP-GlcNAc:Dol-P-GlcNac-P transferase deficiency: CDG-Ij

CNS Congenital myasthenic syndrome − − ± ± ±
Fetal hypokinesia phenotype ± ± − − −
Mental retardation ± ± ± ± ±
Microcephaly − ± ± ± ±
Retardation, motor + + + + +
Digestive Feeding difficulties ± ± ± ±
Eye Cataract ± ± − − −
Nystagmus ± ± ±
Musculoskeletal Contractures ± ± ±
Other Dysmorphism ± ± ± ± ±
Special laboratory Lipid-linked Man9GlcNAc2 (FB) ↓-n ↓-n ↓-n ↓-n
Muscle EM: type 2 fiber tubular aggregates ± ±
Sialotransferrins (S), type 1 pattern + + + +
Table 30.9 Mannosyltransferase 1 deficiency: CDG-Ik

CNS Brain MRI: cerebellar hypoplasia/cortical atrophy ± ± ±
Epilepsy + + +
Hypotonia, axial ± ± ±
Digestive Feeding difficulties ± ± ±
Eye Poor visual fixation ± ± ±
Strabismus ± ± ±
Musculoskeletal Facial dysmorphism ± ± ±
Other Fatal outcome ± ± ±
Special laboratory Hypoglycosylated glycoproteins (S, B) + + +
Lipid-linked GlcNAc2 (FB) ↑ ↑ ↑
Table 30.10 Mannosyltransferase 7–9 deficiency: CDG-IL

Cardiovascular Pericardial effusion ± ± ± ±
CNS Brain atrophy ± ± ±
Epilepsy + + +
Hypotonia + + +
Dermatological Inverted nipples ± ± ±
Splenomegaly ± ± ±
Eye Strabismus ± ± ±
Renal Renal cysts ± ± ±
Other Facial dysmorphism ± ± ±
Routine laboratory Albumin (S) ↓-n ↓-n ↓-n ↓-n
Cholesterol (S) ↓-n ↓-n ↓-n ↓-n
Factor XI (B) ↓-n ↓-n ↓-n
Special laboratory Lipid-linked Man6GlcNA2 and Man8GlcNA2 + + +
Table 30.11 Mannosyltransferase 4–5 deficiency (ALG11-CDG)

CNS Axial hypotonia + + +
Cerebral atrophy ± ± ±
Epilepsy ± + +
Hearing, impaired ± ± ±
Hyperreflexia ± ± ±
Hypertonia, extremities ± ± ±
Dermatological Fat pads ± ± ±
Inverted nipples ± ± ±
Vomiting, episodic ± ± ±
Retinal dystrophy ± ± ±
Respiratory Stridor, inspiratory ± ± ±
Other Early death ± ± ± − −
Routine laboratory Factor XI (B) ↓-n ↓-n ↓-n
Lactate (P) n-↑ n-↑ n-↑
Prolactin (S) n-↑ n-↑ n-↑
Special laboratory Lipid-linked Man3GlcNAc2 and Man4GlcNAc2 (FB) ↑ ↑ ↑
Table 30.12 Flippase of Man5GlcNAc2-PP-Dol deficiency: CDG-In

CNS Ataxia ± ± ± +
Cerebral atrophy ± ± ± −
Epilepsy + + + + +
Hearing, impaired + + + ±
Hypotonia + + + + +
Microcephaly ± ± ± − −
Retardation, psychomotor ++ ++ ++ + +
Dermatological Inverted nipples ± ± ± −
Digestive Failure to thrive + + + −
Feeding difficulties + + + −
Hepatomegaly ± ± ± +
Eye Vision, impaired + + + −
Hematological Thrombosis ± ± ± −
Other Dysmorphism ± ± ± +
Routine laboratory Creatine kinase (P) n n n n n
Factor XI (B) ↓ ↓ ↓ ↓ ↓
Transaminases (P) n n n n n
Special laboratory Lipid-linked Man5GlcNAc2 (FB) ↑ ↑ ↑ ↑ ↑
Sialotransferrins (S), type 1 pattern + + + + +
Table 30.13 N-acetylglucosaminyltransferase 2 deficiency: CDG-IIa

Cardiovascular Ventricular septal defect ± ± ± ± ±
CNS Cortical atrophy ± ± ± ± ±
Epilepsy, intractable ± ± ± ± ±
Hypotonia + + + + +
Movement, abnormal ± ± ± ± ±
Retardation, psychomotor ++ ++ ++ ++ ++
Digestive Big open mouth + + + + +
Diarrhea, chronic ± ± ±
Everted lower lip ± ± ± ± ±
Feeding difficulties ± ± ± ± ±
Gastroesophageal reflux ± ± ±
Gastrointestinal bleeding ± ± ±
Volvulus of the stomach ± ± ±
Ear Dysplastic ears + + + + +
Hearing loss ± ± ± ± ±
Endocrine Absent puberty − − − ± ±
Decreased body height ± + + + +
Eye Delayed visual maturation − ± ± − −
Myopia ± ± ± ± ±
Genitourinary Male genital hypoplasia ± ± ± ± ±
Hematological Bleeding tendency ± ± ± ± ±
Problematic lymphocyte growth ± ± ± ± ±
Recurrent infections ± ± ± ± ±

Musculoskeletal Foot deformity ± ± ± ± ±
Kyphosis ± ± ± ± ±
Muscular dystrophy − ± ± ± ±
Osteoporosis − ± ± ± ±
Pectus excavatum ± ± ± ± ±
Radius dislocation ± ± ± ± ±
Scoliosis ± ± ± ± ±
Respiratory Respiratory insufficiency − − − ± ±
Other Beaked nose ± ± ± ± ±
Drug reactions ± ± ± ± ±
Facial dysmorphism + + + + +
Fatal evolution before 1 year ± ± − − −
Gum hypertrophy − ± ± ± ±
Teeth malposition − ± ± ± ±
Routine laboratory Arylsulfatase A (S) n n n n n
ASAT ↑ ↑ ↑ ↑ ↑
Factor XI (B) ↓ ↓ ↓ ↓ ↓
Haptoglobin (S) ↓ ↓ ↓ ↓ ↓
Immunoglobulin G (S) ± ± ± ± ±
Thyroxin-binding globulin (S) ↓ ↓ ↓ ↓ ↓
Special laboratory Sialotransferrins (S), type 2 pattern + + + + +
Table 30.14 Glucosidase 1 deficiency: CDG-IIb

CNS Apnea ++ ++
Developmental delay + +
EEG, VEP, BAER abnormalities ++ ++
Epilepsy ++ ++
Hypokinesia + +
Hypotonia ++ ++
Neuropathy, myelinating + +
Digestive Gastric tube feeding + ++
Hepatomegaly + ++
Eye Long eye lashes + +
Short palpebral fissures + +
Genitourinary Hypoplastic labia majora + +
Hair Alopecia areata + +
Musculoskeletal Arched palate, high + +
Facial dysmorphism + +
Fingers, overlapping + +
Respiratory Respiratory insufficiency + +++
Routine laboratory ASAT ↑ ↑
EEG: burst-suppression, abnormal + +
VEP, abnormal +++ +++
Special laboratory Sialotransferrins (S) n n
Tetrasaccharide in urine + +
Table 30.15 Oligosaccharyltransferase subunit tusc 3 deficiency

CNS Retardation, psychomotor +/++ +/++ +/++ +/++ +/++
Special laboratory Sialotransferrins (S) − − − − −
Table 30.16 Magnesium transporter 1 deficiency (MAGT1-CDG)

CNS Psychomotor retardation (X-linked; non-syndromic) + + + + +
Hematological Epstein-Barr virus infection ± ± ± ± ±
Immunodeficiency, T cell ± ± ± ± ±
Other Magnesium transport defect ± ± ± ± ±
Neoplasm ± ± ± ± ±
Special laboratory Sialotransferrins (S) n n n n n
Table 30.17 Exostosin 1 deficiency

Musculoskeletal Chondrosarcoma ± ± ± ± ±
Functional joint impairment ± ± ± ± ±
Osteochondroma + +/++ +/++ +/++ +/++
Skeletal deformations ± ± ± ± ±
Table 30.18 Exostosin 2 deficiency

Musculoskeletal Chondrosarcoma ± ± ± ± ±
Functional joint impairment ± ± ± ± ±
Osteochondroma + +/++ +/++ +/++ +/++
Skeletal deformations ± ± ± ± ±
Table 30.19 Chondroitin sulfate synthase 1 deficiency

CNS Cerebellar vermis agenesis, hypoplasia ± ± ± ± ±
Ear Deafness, conductive ± ± ± ± ±
Deafness, sensorineural ± ± ± ± ±
Musculoskeletal Abducted thumbs + + + + +
Clinodactyly + + + + +
Hyperphalangism + + + + +
Kyphoscoliosis ± ± ± ± ±
Lateral/medial deviations of fingers/toes + + + + +
Pectus excavatum ± ± ± ± ±
Phalangeal duplications + + + + +
Preaxial brachydactyly + + + + +
Radiohumeral and/or radioulnar synostosis ± ± ± ± ±
Short fingers or toes I, II, III + + + + +
Short metacarpals/metatarsals + + + + +
Syndactyly + + + + +
Symphalangism + + + + +
Table 30.20 Beta-1,4-galactosyltransferase 7 deficiency

CNS Retardation, psychomotor +/++ +/++ +/++ +/++ +/++
Dermatological Atrophic scars + + + +
Hyperelastic, loose skin + + + + +
Eye Exophthalmia + + + + +
Musculoskeletal Arachnodactyly + + + + +
Dental defects − + + + +
Dwarfism + + + + +
Equinovarus + + + + +
Fingers, overlapping + + + + +
Frontal bossing + + + + +
Joint laxity + + + + +
Midface hypoplasia + + + + +
Progeroid face ± ± ± ± ±
Radioulnar synostosis + + + + +
Short clavicle + + + + +
Tiny face + + + + +
Table 30.21 Polypeptide N-acetylgalactosaminyltransferase 3 deficiency

Dermatological Cutaneous and subcutaneous calcifications +/++ +/++ +/++ +/++ +/++
Digestive Visceral calcifications ± ± ± ± ±
Ectopic calcifications + + + + +
Hyperostosis ± ± ± ± ±
Routine laboratory Phosphate (S) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 30.22 UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter deficiency

Digestive Abdominal distension ++ − − − −
Musculoskeletal Advanced ossification + − − − −
Club foot + − − − −
Dwarfism ++ − − − −
Nasal hypoplasia + − − − −
Platyspondyly ++ − − − −
Shortening of long bones +++ − − − −
Small ilia with snail-like appearance ++ − − − −
Thoracic hypoplasia +++ − − − −
Other Hydrops ++ − − − −
Perinatal lethality + − − − −
Table 30.23 O-Mannosyltransferase 1 deficiency

CNS Agenesis, corpus callosum ± ± ± ± ±
Cerebral cortical malformations, cobblestone lissencephaly ± ± ± ± ±
Encephalocele ± ± ± ± ±
Hydrocephalus ± ± ± ± ±
Retardation, psychomotor +/+++ +/+++ +/+++ +/+++ +/+++
Eye Buphthalmos ± ± ± ± ±
Cataract ± ± ± ± ±
Exophthalmia ± ± ± ± ±
Glaucoma ± ± ± ± ±
Megalocornea ± ± ± ± ±
Microphthalmia ± ± ± ± ±
Muscular dystrophy + + + + +
Other Fatal evolution before 1 year ± ± − − −
Walker-Warburg syndrome ± ± ± ± ±
Routine laboratory Creatine kinase (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 30.24 O-Mannosyltransferase 2 deficiency

Other Fatal evolution before 1 year ± ± − − −
Walker-Warburg syndrome ± ± ± ± ±
Table 30.25 O-Mannose beta-1,2-N-acetyglucosaminyltransferase deficiency

Myopia ± ± ± ± ±
Muscle-eye-brain disease ± ± ± ± ±
Table 30.26 O-Fucose-specific beta-1,3-N-acetylglucosaminyltransferase deficiency

Endocrine Decreased body height + + + + +
Musculoskeletal Long, slender fingers + + + + +
Scoliosis + + + + +
Vertebral anomalies of the whole spine + + + + +
Table 30.27 O-Fucose-specific beta-1,3-N-glucosyltransferase deficiency

CNS Hydrocephalus ± ± ± ± ±
Digestive Anteriorly placed anus ± ± ± ± ±
Gastroesophageal reflux ± ± ± ± ±
Malrotation ± ± ± ± ±
Ear Hearing loss ± ± ± ± ±
Eye Anterior eye chamber anomalies + + + + +
Hydronephrosis or hydroureter ± ± ± ± ±
Musculoskeletal Brachydactyly ± ± ± ± ±
Growth retardation ± ± ± ± ±
Table 30.28 Lactosylceramide alpha-2,3-sialyltransferase deficiency

Cortical atrophy − − + ++ ++
Developmental arrest − − + + +
Developmental regression − ± + + +
Epilepsy, intractable − ± + + +
Hyporeflexia ± + + + +
Hypotonia + + + + +
Irritability − + + + +
Digestive Anorexia − + + + +
Failure to thrive − + + + +
Gastric tube feeding − + + + +
Vomiting − + + + +
Special laboratory GM3 activity, plasma ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
GM3 ganglioside, plasma ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Lactosylceramide, plasma ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Sialotransferrins (S) n n n n n
Table 30.29 Phosphatidylinositolglycan, class M, deficiency

CNS Absence seizures − − + +
Hematological Portal and hepatic vein thrombosis + + +
Table 30.30 Phosphatidylinositolglycan, class V, deficiency (PIGV-CDG)

CNS Epilepsy ± ± ± ± ±
Hearing, impaired ± ± ± ± ±
Retardation, psychomotor − + + + +
Dermatological Hypoplastic nails ± ± ± ± ±
Digestive Anorectal anomalies ± ± ± ± ±
Musculoskeletal Cleft palate ± ± ± ± ±
Phalangeal malformations ± ± ± ± ±
Routine laboratory Alkaline phosphatase (P) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Table 30.31 GDP-Man:Dol-P mannosyltransferase subunit 1 deficiency – CDG Ie

CNS Acquired microcephaly − + + + +
Ataxia ± ± ± ±
Cerebral atrophy ± ± ± ±
Epilepsy ± ± ± ±
Hypotonia + + + +
Neuropathy, peripheral ± ± ± ±
Retardation, psychomotor ++ ++ ++ ++
Tremor ± ± ± ±
Hepatosplenomegaly ± ± ±
Eye Nystagmus ± ± ±
Retinal dystrophy ± ± ±
Strabismus + + +
Routine laboratory Creatine kinase (P) ± ±
Transaminases (P) ± ±
Special laboratory MRI: dentate nucleus lesions ± ±
Table 30.32 GDP-Man:Dol-P mannosyltransferase 3 deficiency

Cardiovascular Cardiomyopathy, dilated +
CNS Gait disturbance − − − + +
Retardation, psychomotor − − − − −
Stroke-like episodes − − − − +
Musculoskeletal Dysmorphic features − − − − −
Muscle weakness, proximal − − + + +
Pes planus − − − + +
Routine laboratory Creatine kinase (P) ↑↑
Factor XI (B) n n n n n
Transaminases (P) ↑
Special laboratory Sialotransferrins (S), type 1 pattern +
Table 30.33 Dol-P-Man utilization 1 deficiency

CNS Cerebral atrophy ± ± ± ± ±
Retardation, psychomotor − ++ ++ ++ ++
Tendon reflexes n n n n n
Dermatological Erythroderma ± ± ± ± ±
Hyperkeratosis ± ± ± ± ±
Ichthyosis ± ± ± ± ±
Digestive Feeding difficulties ± ± ± ± ±
Endocrine Growth hormone deficiency − − ± ± ±
Vision, impaired − ± ± ± ±
Transaminases (P) n n n n n
Special laboratory Lipid-linked Man5GlcNAc2 (FB) + + + + +
Lipid-linked Man9GlcNAc2 (FB) + + + + +
Table 30.34 Beta-1,4-galactosyltransferase 1 deficiency

CNS Hypotonia, axial + + − −
Retardation, psychomotor − − −
Digestive Diarrhea, recurrent episodes + + − −
Eye Myopia + + + +
Hematological Perinatal bleeding diathesis + − − − −
Routine laboratory Antithrombin (B) ↓ ↓ ↓ ↓
APTT ↑ ↑ ↑ ↑
ASAT (S) ++ ++ ++ ++
Creatine kinase (P) ↑ ↑ ↑ ↑
Special laboratory Hypogalactosylation Tf glycans + + + + +
Table 30.35 UDP-GlcNAc epimerase/kinase deficiency

Cardiovascular Cardiomyopathy − − − − ±
Musculoskeletal Muscle dystrophy, progressive − − − ± ++
Muscle wasting of limbs with sparing of quadriceps − − − ± +/+++
muscles
Respiratory Respiratory failure − − − − +
Routine Creatine kinase (P) n n n n-↑ n-↑
laboratory
Special laboratory Muscle histopathology: rimmed vacuoles and tubulofilaments − − − ± +
Table 30.36 CMP-sialic acid transporter deficiency

Cardiovascular Aortic insufficiency +
CNS Epilepsy
Retardation, psychomotor +
Hematological Macrothrombocytopenia +
Renal Proteinuria +
Special laboratory Sialotransferrins (S), type 2 pattern +
Table 30.37 GDP-fucose transporter deficiency

CNS Retardation, psychomotor − +++ +++ +++ +++
Dermatological Recurrent skin infections ± ± ± ± ±
Digestive Periodontitis − ++ ++ ++ ++
Endocrine Decreased body height + + + + +
Hematological Delayed separation umbilical cord − − − − −
Neutrophilia ++ ++ ++ ++ ++
Recurrent infections + + + + +
Musculoskeletal Facial dysmorphism ++ ++ ++ ++ ++
Routine laboratory Bombay blood phenotype + + + + +
T-and B-cell function n n n n n
Special laboratory Neutrophil motility ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Neutrophil rolling ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 30.38 Dolichol kinase deficiency

Cardiovascular Cardiomyopathy, dilated ± ± ± ± ±
Microcephaly − ± ± ± ±
Dermatological Hair abnormality ± ± ± ±
Ichthyosiform erythroderma ± ± ± ± ±
Digestive Failure to thrive ± ± ± ±
Endocrine Puberty, delayed − − − ± ±
Eye Nystagmus ± ± ± ±
Musculoskeletal Facial dysmorphism − − − − −
Other Early death − ± − − −
Routine laboratory Transaminases (P) n-↑ n-↑ n-↑ n-↑
Special laboratory Lipid-linked oligosaccharides (F) ↓ ↓ ↓ ↓
Table 30.39 Steroid 5 alpha-reductase 3 deficiency

CNS Cerebellar abnormalities ± ± ± ± ±
Midline brain malformations ± ± ± ± ±
Muscular hypotonia + + + + +
Dermatological Dry skin ± ± ± ± ±
Erythroderma ± ± ± ± ±
Ichthyosis ± ± ± ± ±
Endocrine Growth hormone deficiency ± ± ± ± ±
Coloboma ± ± ± ± ±
Nystagmus ± ± ± ± ±
Optic atrophy ± ± ± ± ±
Kyphosis − − − ± ±
Special laboratory Sialotransferrins (S), type 1 pattern ± ± ± ± ±
Table 30.40 Component of COG complex 7 deficiency

CNS Brain atrophy + + +
Epilepsy ± ± ±
Hyporeflexia + + +
Hypotonia + + +
Dermatological Wrinkly skin ± ± ±
Digestive Failure to thrive + + +
Feeding difficulties + + ±
Endocrine Decreased body height ± ± ±
Hematological Recurrent infections ± ± ±
Musculoskeletal Facial dysmorphism ± ± ±
Finger anomalies ± ± ±
Renal Neurogenic bladder ± ± ±
Obstructive uropathy ± ± ±
Renal tubulopathy ± ± ±
Other Fever of unknown origin + + +
Routine laboratory Creatine kinase (P) n-↑ n-↑ n-↑
Special laboratory Apolipoprotein C-III (S) isoelectrofocusing, abnormal + + +
Transferrin glycan hyposialylation and hypogalactosylation + + +

Cardiovascular Aortic insufficiency ± ± ±
Cardiomyopathy, hypertrophic ± ± ± ±
CNS Hypotonia ± ± ± ±
Microcephaly ++ ++ ++ ++ ++
Digestive Feeding difficulties ± ± − −
Hepatosplenomegaly − ± ± ±
Ear Hearing loss, conductive ± ± ± ±
Musculoskeletal Cerebrocostomandibular-like syndrome ± ± ± ± ±
Dwarfism ± + + + +
Special laboratory Apolipoprotein C-III (S) isoelectrofocusing, abnormal + + + + +
MRI: cerebral and cerebellar atrophy ± ± ± ± ±
Transferrin hyposialylation and hypogalactosylation (S) + + + + +

CNS Acquired microcephaly ± ± ±
Ataxia, cerebellar ± ± ±
Encephalopathic crisis, acute ± ± ±
Epilepsy + + +
Hypotonia + + +
Neuropathy, axonal motor + + +
Retardation, psychomotor − + +
Eye Strabismus + + +
Musculoskeletal Dwarfism + + +
Finger anomalies ± ± ±
Other Recurrent infections ± ± ±
Routine laboratory Creatine kinase (P) ↑ ↑ ↑
Factor XI (B) ↓-n ↓-n ↓-n
Transaminases (P) ↑ ↑ ↑
Special laboratory Apolipoprotein C-III (S) isoelectrofocusing, abnormal + + +
Hyposialylation of N- and O-glycans + + +
MRI/CT: cerebral and cerebellar atrophy, cystic white matter ± ± ±
changes, ventriculomegalia

CNS Cerebral and cerebellar atrophy ± ± ± ± ±
Hypotonia + + + + +
Retardation, psychomotor − +/++ +/++ +/++ +/++
Digestive Hepatomegaly ± ± ± ± ±
Ear Deafness ± ± ± ± ±
Eye Blindness ± ± ± ± ±
Genitourinary Neurogenic bladder ± ± ± ± ±
Musculoskeletal Dwarfism ± ± ± ± ±
Flexion contractures of fingers ± ± ± ± ±
Transferrin glycan hyposialylation + + + + +

Microcephaly ± ± ± ±
Dermatological Dry skin ± ± ± ±
Palmoplantar hyperkeratosis ± ± ± ±
Digestive Failure to thrive ± ± ± ±
Liver dysfunction + + + +
Hematological Recurrent infections + + + −
Musculoskeletal Facial dysmorphism ± ± ± ±
Other Enamel hypoplasia − + + +
Routine laboratory Transaminases (P) ↑ ↑ ↑ ↑
Special laboratory Sialotransferrins (S), type 2 pattern ± ± ± ±
Table 30.46 Golgi-microtubule-associated protein, 210 kD, deficiency

Musculoskeletal Achondrogenesis type IA + − − − −
Deficient ossification +++ − − − −
Macrocephaly (due to edema of soft tissues) + − − − −
Micromelia +++ − − − −
Other Early death or stillbirth + − − − −
Special laboratory Sialotransferrins (S)
Table 30.47 V0 subunit a2 of vesicular H(+)-ATPase deficiency

CNS Retardation, psychomotor ± ± ± ± ±
Dermatological Cutis laxa + + ± ± ±
Eye Amblyopia ± ± ± ± ±
Myopia ± ± ± ± ±
Musculoskeletal Closure of fontanels, delayed − − ± − −
Growth retardation ± ± ± ± ±
Joint laxity ± ± ± ± ±
Osteoporosis ± ± ± ± ±
Other Malformations (kidney, brain) ± ± ± ± ±
Routine laboratory Transaminases (P) ↑ ↑ ↑ ↑ ↑
MRI: cerebral cortical malformations ± ± ± ± ±
Sialotransferrins (S), type 2 pattern ± + + + +
Skin histology, abnormal elastin fibers + + ± ± ±
Table 30.48 COPII component SEC23B deficiency

Cardiovascular Cardiomyopathy − − − − +
Digestive Jaundice ± ± ± ± ±
Liver cirrhosis − − − − +
Endocrine Diabetes mellitus − − − − +
Hematological Anemia, dyserythropoietic +/+++ +/+++ +/+++ +/+++ +/+++
Splenomegaly ± ± ± ± ±
Routine laboratory Bilirubin (P) ↑/↑ ↑ ↑/↑ ↑ ↑/↑ ↑ ↑/↑ ↑ ↑/↑ ↑
Special laboratory Acidified serum test (HEMPAS test) + + + + +
Bi- and multinucleated erythroblasts (BM) + + + + +

Serum sialotransferrins (isoelectrofocusing) (centile values: P3–P97, Disease Sialotransferrin IEF pattern (S)
n = 96; all ages) 30.1–30.12 Type 1 patterna
Monosialotransferrins 0.0–3.7 30.31–30.34
Disialotransferrins 2.0–6.1 30.38–30.39
Trisialotransferrins 5.5–15.1 30.13 Type 2 patternb
Tetrasialotransferrins 48.5–65.3 30.40–30.45
Pentasialotransferrins 14.9–28.7 30.47
Hexasialotransferrins 2.3–8.1 All other CDG Normal pattern
a
Type 1 pattern: increase of di- and asialotransferrin and decrease of
tetra-, penta-, and hexasialotransferrin
*Enzyme analyses b
Type 2 pattern – increase of tri-, di-, mono-, and/or asialotransferrin
Phosphomannomutase (mU/mg protein) and decrease of tetra-, penta-, and hexasialotransferrin
Leukocytes 1.8–3.2 (range); 2.2 (median)
Fibroblasts 3.8 ± 0.9 (mean ± 1 SD) Disease Apolipoprotein C-III IEF pattern (S)
Phosphomannose isomerase (mU/mg protein) 30.40–30.45 Cathodal shift
Leukocytes 860–1,800 (nmol/h/mg protein; range) 30.47 Cathodal shift
Fibroblasts 6.8 ± 1.0 (mean ± 1 SD) All other CDG Normal pattern
Disease Lipid-linked oligosaccharides (F)

*Lipid-linked oligosaccharides in fibroblasts (HPLC)
30.3–30.12 Increase of the glycan intermediate(s) that is (are)
Man5GlcNAc2 Not detectable substrate(s) of the defective enzyme
Man9GlcNAc2 Not detectable 30.38–30.39 Normal pattern or generalized decrease
Glc3Man9GlcNAc2-PP-Dol Only significant compound All other CDG Normal pattern
Isoelectrofocusing of serum transferrin
Abnormal Normal
Protein variant
Artefact Isoelectrofocusing of
serum apolipoprotein
C -III
CDG with sialic acid-deficient glycoproteins
Abnormal
Normal
Mutation
analysis by
WES
Type 1 pattern Type 2 pattern
Secondary Primary Glycan structure

(galactosemia, analysis
fructose intolerance,
alcohol abuse,…)
Enzymatic tests
(PMM, PMI) Enzymatic tests (if available)
Deficient Normal
Mutation analysis by
WES
Mutation analysis Lipid- linked

of PMM2 or MPI oligosaccharides
Enzymatic tests (if available)
Mutation analysis
by WES
WES: whole exome sequencing (checking first established genes for CDG defects and afterwards
candidate genes for CDG defects).
Fig. 30.2
30.8 Specimen Collection Standard Treatment Table and Medication Requirements

30.2 Phosphomannose isomerase deficiency
Test Precondition Material Handling Pitfalls Mannose circumvents the defective step because it can be
Sialotransferrins S Frozen No directly converted to mannose 6-phosphate by hexokinases.
(−20 °C) EDTA Oral mannose, 0.2 g/kg of body weight per 4 h, is recom-
plasma mended. The clinical symptoms usually disappear rapidly
Apolipoprotein S Frozen but it takes several months for the serum transferrin isoform
C-III (−20 °C)
pattern to improve or normalize.
Lipid-linked FB Frozen
oligosaccharides (−20 °C) 30.37 GDP-fucose transporter deficiency
Phosphoman- WBC, Frozen Oral fucose, 150 mg/kg of body weight, five times a day,
nosemutase FB (−20 °C) abolishes or prevents infections and normalizes neutrophil
Phosphomannose WBC, Frozen counts in some patients (depending on genotype).
isomerase FB (−20 °C)
Dangers/Pitfalls
30.2 Higher mannose doses can induce osmotic diarrhea.
30.37 Higher fucose doses can induce autoimmune neutro-
30.9 Prenatal Diagnosis Table and Sample penia.
Requirements
Experimental Treatment and Medication Requirements
Disorder Material Timing, trimester 30.29 Phosphatidylinositol glycan, class M, deficiency
All (30.1–30.48) CV sampling cultured AFC I, II In this disorder there is a histone hypoacetylation at the pro-
moter of PIGM. The histone deacetylase inhibitor butyrate
Prenatal diagnostics is performed by DNA diagnostics on increases PIGM transcription, and treatment with butyrate
genomic DNA level in case the genetic defect has been (20 mg/kg of body weight three times a day) resulted in
established in a particular family. Maternal contamination excellent control of intractable epilepsy in an affected child.
has to be excluded by haplotype (CA repeat) testing of the All antiepileptic medication could be stopped. No side
AFC and maternal blood effects were noted.
30.10 DNA Testing Table and Sample References

Requirements
Almeida AM, Murakami Y, Baker A et al (2007) Targeted therapy for
inherited GPI deficiency. N Engl J Med 356:1641–1647
Disorder Material Methodology Carchon H, Chevigné R, Falmagne JB, Jaeken J (2004) Diagnosis of
All (30.1–30.48) F, WBC Direct sequencing of genomic DNA congenital disorders of glycosylation by capillary zone electropho-
resis of serum transferring. Clin Chem 50:101–111
Coman D, Irving M, Kannu P, Jaeken J, Savarirayan R (2008) The skel-
etal manifestations of the congenital disorders of glycosylation.
Clin Genet 73:507–515
30.11 Treatment de Koning TJ, Dorland L, van Diggelen OP et al (1998) A novel disor-
der of N-glycosylation due to phosphomannose isomerase defi-
ciency. Biochim Biophys Acta 245:38–42
Summary de Lonlay P, Seta N (2009) The clinical spectrum of phosphomannose
Besides the well-established symptomatic and supportive isomerase deficiency, with an evaluation of mannose treatment for
therapies for all CDG, there are very few specific/curative CDG-Ib. Biochim Biophys Acta 1792:841–843
treatments: mannose for MPI-CDG, fucose for some patients Footitt EJ, Karimova A, Burch M et al (2009) Cardiomyopathy in the
congenital disorders of glycosylation (CDG): a case of late presen-
with SLC35C1 (Wild et al. 2002), and benzoate for the neu- tation and literature review. J Inherit Metab Dis 32:S313–S319
rological symptoms of PIGM-CDG (Almeida et al. 2007). A Foulquier F (2009) COG defects, birth and rise! Biochim Biophys Acta
minority of patients with PMM2-CDG present recurrent 1792:896–902
stroke-like episodes (at least in part due to hyperaggregabil- Grünewald S (2007) Congenital disorders of glycosylation (CDG): rap-
idly enlarging group of (neuro)metabolic disorders. Early Hum Dev
ity of blood platelets). These episodes can be prevented more 83:825–830
or less efficiently by small doses of acetylsalicylic acid (~1 Grünewald S (2009) The clinical spectrum of phosphomannomutase 2
mg/kg of body weight per day). deficiency (CDG-Ia). Biochim Biophys Acta 1792:827–834
Jaeken J (2011) Congenital disorders of glycosylation (CDG): it’s
(nearly) all in it! J Inherit Metab Dis 34:853–858
Emergency Treatment Table and Medication Require- Jaeken J, Matthijs G (2007) Congenital disorders of glycosylation: a
ments rapidly expanding disease family. Annu Rev Genomics Hum Genet
Not applicable 8:261–278
Jaeken J, Vanderschueren-Lodeweyckx M, Casaer P et al (1980) Morava E, Wosik HN, Sykut-Cegielska J et al (2009) Ophthalmological
Familial psychomotor retardation with markedly fluctuating serum abnormalities in children with congenital disorders of glycosylation
proteins, FSH and GH levels, partial TBG-deficiency, increased type I. Br J Ophthalmol 93:350–354
serum arylsulphatase A and increased CSF protein: a new syn- Niehues R, Hasilik M, Alton G et al (1998) Carbohydrate-deficient gly-
drome? Pediatr Res 14:179 coprotein syndrome type Ib. Phosphomannose isomerase deficiency
Jaeken J, van Eijk HG, van der Heul C et al (1984) Sialic acid-deficient and mannose therapy. J Clin Invest 101:1414–1420
serum and cerebrospinal fluid transferrin in a newly recognized syn- Sun L, Eklund EA, Van Hove JLK, Freeze HH, Thomas JA (2005)
drome. Clin Chim Acta 144:245–247 Clinical and molecular characterization of the first adult congenital
Jaeken J, Matthijs G, Saudubray JM et al (1998) Phosphomannose isom- disorder of glycosylation (CDG) type Ic patient. Am J Med Genet
erase deficiency: a carbohydrate-deficient glycoprotein syndrome 137A:22–26
with hepatic-intestinal presentation. Am J Hum Genet 62:1535–1539 Van Schaftingen E, Jaeken J (1995) Phosphomannomutase deficiency is
Jaeken J, Hennet T, Freeze HH, Matthijs G (2008) On the nomenclature a cause of carbohydrate-deficient glycoprotein syndrome type I.
of congenital disorders of glycosylation (CDG). J Inherit Metab Dis FEBS Lett 370:318–320
31:669–672 Wild MK, Lühne K, Marquardt T, Vestweber D (2002) Leukocyte adhe-
Jaeken J, Hennet T, Matthijs G, Freeze HH (2009) CDG nomenclature: sion deficiency II: therapy and genetic defect. Cells Tissues Organs
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Genet 16:88–92
Part VI
Selected Disorder
Neurotransmitter Disorders
31
Thomas Opladen and Georg F. Hoffmann
Contents Summary
31.1 Introduction .................................................................... 516 Neurotransmitters include the catecholamines (dopamine,
norepinephrine, and epinephrine) and the indoleamines
31.2 Nomenclature .................................................................. 517
(serotonin and melatonin). They are chemical messengers,
31.3 Metabolic Pathways ....................................................... 518 which mediate, amplify, or modulate synaptic transmission
31.4 Signs and Symptoms ...................................................... 518 between neurons in the brain. Consequently, neurotransmit-
31.5 Reference Values............................................................. 522
ters are involved in central brain functions including control
of movements and behavior, neuronal excitation and inhibi-
31.6 Pathological Values......................................................... 523 tion, the regulation of body temperature, pain threshold,
31.7 Diagnostic Flow Chart ................................................... 524 memory, and a host of other processes. Inherited deficiencies
31.8 Specimen Collection of neurotransmitters encompass defects of neurotransmitter
(Incl. Pitfalls and Special Handling/Storage)............... 525 biosynthesis and catabolism, as well as defects of neurotrans-
31.9 Prenatal Diagnosis .......................................................... 525 mitter transporters. They result in a wide variety of clinical
signs and symptoms. This chapter will focus on primary dis-
31.10 DNA Testing .................................................................... 525
orders of serotonin and dopamine metabolism. Described
31.11 Treatment ........................................................................ 526 defects are deficiencies of tyrosine hydroxylase (TH), aro-
References .................................................................................... 527 matic l-amino acid decarboxylase (AADC), dopamine
ß-hydroxylase (DßH), monoamine oxidase A, as well as
the hereditary dopamine transporter syndrome and the brain
dopamin-serotonin vesicular transport (VMAT2) disease.
Neurotransmitter disorders are important to recognize
because early diagnosis and prompt therapeutic intervention
seem to improve motor and cognitive outcome. The disease
predominantly starts during infancy and early childhood.
The specific clinical presentation of individual neurotrans-
mitter diseases is determined by the type and severity of the
underlying disorder. The clinical phenotype is not character-
istic but can mimic that of other neurological disorders.
Although a detailed clinical history and physical examina-
tion are essential, the diagnosis is almost exclusively based
on the quantitative determination of neurotransmitters or
T. Opladen
Division of Inborn Metabolic Diseases, Department of Pediatrics, their metabolites in cerebrospinal fluid (CSF). The additional
University Children’s Hospital, Im Neuenheimer Feld 430, determination of pterin metabolites is needed for the differ-
Heidelberg D-69120, Germany entiation from deficiencies of BH4 metabolism. Every diag-
e-mail: thomas.opladen@med.uni-heidelberg.de
nosis must be confirmed by molecular testing. The aim of
G.F. Hoffmann (*) treatment is to restore neurotransmitter homeostasis.
Department of Pediatrics, University Children’s Hospital,
Bypassing the metabolic block using levodopa/carbidopa
Im Neuenheimer Feld 430, Heidelberg
D-69120, Germany together with dopamine agonists has led to remarkable clini-
e-mail: georg.hoffmann@med.uni-heidelberg.de cal improvement in TH deficiency. In patients with AADC
516 T. Opladen and G.F. Hoffmann
deficiency and with dopamine receptor deficiency, syndrome individual pterin species (Hoffmann et al. 1998). Apart from
treatment options are limited and in many cases not a strictly standardized sampling protocol, neurotransmitter
satisfactory. Patients with DβH deficiency benefit from analysis requires special logistics and transport as well as
dihydroxyphenylserine (DOPS) administration. While pati- demanding laboratory techniques, adequately established
entes with VMAT2 defects benefit from treatment with a age-related reference values, and finally experienced
dopamine-receptor agonist, no specific treatment with sus- interpretation in the clinical context (Bräutigam et al. 2002,
tained effect for MAO-A deficiency or dopamine transporter 1998). It is therefore performed in a limited number of
deficiency has been described. specialized laboratories worldwide. As a consequence only
small numbers of patients have been diagnosed, and a
substantial number is suspected to have remained
31.1 Introduction underdiagnosed. A generalized deficiency of dopaminergic
transmission can be recognized by raised levels of prolactin
Neurotransmitters are chemical messengers, which mediate, in blood. However, this is not a diagnostic test because of
amplify, or modulate synaptic transmission between neurons insufficient sensitivity as well as specificity. In addition, pro-
in the brain. Apart from neurotransmission they have impor- lactin levels can change in response to stress. To obtain reli-
tant functions during embryogenesis such as differentiation able values, three determinations should be done hourly. If
and neuronal growth (Herlenius and Lagercrantz 2001). these are in agreement, the value is to be trusted.
Inherited defects of neurotransmitter synthesis, breakdown, Tyrosine hydroxylase (TH) catalyzes the conversion of
and transport have been recognized as a cause of early onset, l-tyrosine to l-dopa. It is the rate-limiting step in the biosyn-
severe, and progressive encephalopathies. This chapter will thesis of dopamine, norepinephrine, and epinephrine. The
focus on primary disorders of serotonin and dopamine (bio- first committed defect, recessively inherited TH deficiency,
genic amine) metabolism. Established defects in biogenic is therefore characterized by decreased concentrations of
amine metabolism include deficiencies of tyrosine hydroxy- homovanillic acid in CSF in combination with normal
lase (TH) (EC 1.14.16.2) (Bartholome and Ludecke 1998; 5-hydroxyindoleacetic acid. According to severity of the
Ludecke and Bartholome 1995), aromatic l-amino acid neurological symptoms and the concentrations of the
decarboxylase (AADC) (EC 4.1.1.28) (Hyland et al. 1992), biogenic amines in CSF, patients were delineated into two
dopamine ß-hydroxylase (DßH) (EC 1.14.17.1) (Man in ’t clinical phenotypes: an infantile onset, progressive, hypoki-
Veld et al. 1987; Robertson et al. 1986), monoamine oxidase netic-rigid syndrome with dystonia (type A, less severe) and
A (MAO-A) (EC 1.4.3.4) (Brunner et al. 1993a), as well as a complex encephalopathy with neonatal onset (type B).
the recently characterized hereditary dopamine transporter However, both phenotypes overlap substantially to a contin-
deficiency (Kurian et al. 2010) and the brain dopamine–sero- uum of symptomatology and clinical severity (Willemsen
tonin vesicular transport disease (Rilstone et al. 2013). et al. 2010).
The diseases predominantly start during infancy and early Aromatic l-amino acid decarboxylase (AADC) is
childhood. The clinical presentation is determined by the required for the synthesis of both serotonin and the catechol-
type and severity of the underlying disorder. The phenotype amines. Therefore, the clinical phenotype of autosomal
is not characteristic and can be mistaken for other neurologi- recessively inherited AADC deficiency is due to a severe
cal disease entities (Kurian et al. 2011a). Important neuro- combined deficiency of serotonin and catecholamines and
logical symptoms are developmental delay, central and characterized by vegetative symptoms in addition to oculo-
peripheral hypotonia, hypo- or hyperkinesia, mixed pyrami- gyric crises, dystonia, and severe neurological dysfunction
dal and extrapyramidal motor disorders, epilepsy, and oculo- (Brun et al. 2010; Pons et al. 2004; Swoboda et al. 2003).
gyric crises. Cognitive function is generally less severely Biochemically, the deficiency leads to low levels of all
affected (Assmann et al. 2003; Brun et al. 2010; Willemsen biogenic amines and accumulation of 3-O-methyldopa,
et al. 2010). 5-hydroxytryptophan, and l-dopa in CSF, plasma, and urine.
Identical clinical pictures may result from abnormalities The aim of treatment in TH and AADC deficiencies is to
of tetrahydrobiopterin (BH4) metabolism (see Chap. 1). But restore neurotransmitter homeostasis. Bypassing the metabolic
in contrast to the majority of patients with BH4 deficiencies block using levodopa/carbidopa together with dopamine ago-
where hyperphenylalaninemia in newborn screening can nists leads to a good to excellent clinical response in most TH
guide an early way to diagnosis, hyperphenylalaninemia is deficient patients. AADC deficient patients have received dopa-
always missing in neurotransmitter disorders. Hence, the mine receptor agonists, monoamine oxidase inhibitors, anti-
diagnosis is depending on a diagnostic lumbar puncture, cholinergics, and selective serotonin reuptake inhibitors often
more explicitly the quantitative determination of neurotrans- in conjunction with vitamin B6 (cofactor for AADC). Not sur-
mitter metabolites in cerebrospinal fluid (CSF), i.e., prisingly, significant clinical improvement was only observed
the acidic metabolites of the biogenic monoamines and in attenuated clinical manifestations (Brun et al. 2010).
31 Neurotransmitter Disorders 517
Dopamine-ß-hydroxylase (DßH) catalyzes the conversion been reported (Cheung and Earl 2001). Up to now no effec-
of dopamine to norepinephrine. Autosomal recessive DßH tive treatment for MAO-A deficiency is available. Dietary
deficiency is characterized by primary autonomic failure intervention by avoiding foods rich in amines and cyprohep-
with complete absence of norepinephrine and epinephrine in tadine hydrochloride and sertraline hydrochloride have been
plasma in combination with increased levels of dopamine. used with uncertain or with no effect.
Serum DßH activity is low (Man in ’t Veld et al. 1987; In addition to enzyme deficiencies two transporter defects
Robertson et al. 1986). The initial symptoms can be seen are known to cause disorders of biogenic amines. The auto-
during the perinatal period with hypotension, muscle hypo- somal recessive dopamine transporter (DAT) deficiency syn-
tonia, hypothermia and hypoglycemia, delayed eye opening, drome is characterized by impaired dopamine transporter
and ptosis. Children exhibit a reduced ability to exercise function and was described in a group of so far 11 patients
because blood pressure cannot adapt adequately which with a characteristic mixed hyper- and hypokinetic move-
results in syncopes. Symptoms usually progressively worsen ment disorder with hyperkinesia as well as parkinsonian fea-
during early adulthood with severe orthostatic hypotension tures starting in early infancy (Assmann et al. 2004; Kurian
and also eyelid ptosis, nasal stuffiness, and retrograde ejacu- et al. 2009, 2011b). The disease progresses during childhood
lation (Senard and Rouet 2006). Oral administration of dihy- to severe parkinsonism-dystonia. Characteristic biochemical
droxyphenylserine (DOPS) increases blood pressure findings are elevated homovanillic acid including raised
resulting in clinical improvement. Very few pathogenic ratios of homovanillic acid to 5-hydroxyindoleacetic acid in
mutations have been identified. Variations in the DßH gene CSF. All known patients have a homozygous or compound
were further reported in patients with Gilles de la Tourette heterozygous mutation in the SLC6A3 gene encoding DAT.
syndrome, ADHD, and schizophrenia (Haavik et al. 2008). As yet various treatment attempts did not have sustained
X-linked monoamine oxidase (MAO) catalyzes the oxi- effect. The second transporter defect was identified in eight
dative deamination of a wide variety of monoamines includ- children from one consanguineous family showing clinical
ing dopamine, serotonin, and the catecholamines. Two forms symptoms of deficiencies of dopamine, serotonin, epineph-
of the enzyme are classified as MAO-A and MAO-B depend- rine and norepinephrine without detectable deficit of neu-
ing on their sensitivity to inhibitors and affinity for sub- rotransmitters in CSF. A mutation in SLC18A2 gene encoding
strates. MAO-A prefers serotonin, norepinephrine, and the VMAT2 protein was identified, leading to a defective
dopamine and MAO-B phenethylamine. MAO-A deficiency monoamine loading into synaptic vesicles (Rilstone et al.
leads conceptually to accumulation of the monoamine neu- 2013). The biochemical hallmarks of the condition are
rotransmitters in CSF, while levels of their metabolites in abnormalities of monoamine metabolites in urine but not
body fluids and compartments are decreased (Brunner et al. CSF. Homovanillic acid and 5-hydroxyindoleacetic acid
1993a). Male patients presented with mild mental retardation concentration in urine are elevated, while norepinephrine
and a tendency toward stereotyped hand movements, as well and dopamine concentrations are decreased. Treatment with
as behavioral problems with repeated occurrences of aggres- a direct dopamine-receptor agonist resulted in a dramatic and
sion (Brunner et al. 1993b). In addition, chronic episodes of sustained improvement of movement disorders. Treatment
flushing, diarrhea, headaches, and psychiatric problems have results were better in the younger affected child.
31.2 Nomenclature
No. Disorder name Abbreviation symbol localization Affected protein OMIM no. Subtype
31.1 Tyrosine hydroxylase l-dopa TH TH 11p15.5 Tyrosine-3- 191290 All forms
deficiency responsive hydroxylase
dystonia
31.2 Aromatic l-amino acid AADC DDC 7p12.1-12.3 Aromatic l-amino 608643 All forms
decarboxylase deficiency acid decarboxylase
31.3 Dopamine beta- hydroxylase DßH DßH 9q34 Dopamine 223360 All forms
deficiency beta-hydroxylase
31.4 Monoamine oxidase A MAO-A MAO-A Xp11-21 Monoamine oxidase 307850 All forms
deficiency A
31.5 Dopamine transporter SLC6A3 DAT SLC6A3 5p15.3 SLC6A3 transporter 126455 All forms
31.6 Dopamine-serotonin VMAT2 SLC18A2 10q25.3 Vesicular 193001 All forms
vesicular transport defect monoamine
transporter 2
Epinephrine Norepinephrine Normetanephrine
31.4 31.4
MHPG
MET 31.4
31.4
VMA
31.3
VLA
31.4
3MT HVA
3OMD
31.1 31.4
Tyrosine L-DOPA Dopamine DOPAC
31.2
31.4
Tryptophan 5HTP Serotonin 5HIAA
Fig. 31.1 Metabolism of serotonin and the catecholamines. 31.1 tyro- homovanillic acid, VMA vanillylmandelic acid, MHPG 3-methoxy-
sine hydroxylase, 31.2 aromatic l-amino acid decarboxylase, 31.3 4-hydroxyphenylglycol, MET metanephrine, 3MT 3-methoxytyramine
dopamine ß-hydroxylase, 31.4 monoamine oxidase. VLA vanillactic (− − −> represents several steps involved. Pathological metabolites
acid, 3OMD 3-O-methyldopa, 5HTP 5-hydroxytryptophan, 5HIAA used as markers in the differential diagnosis are shown within boxes)
5-hydroxyindoleacetic acid, DOPAC dihydroxyphenylacetic acid, HVA
Table 31.1 Tyrosine hydroxylase deficiency

Autonomic system Sweatinga ± + + + +
Temperature instability + + + + +
CNS Bradykinesia + + + + +
Dystonia – + + + +
Dystonic crisisa – + + + +
Epileptic seizuresb – + + + +
Hypokinesia + + + + +
Hypotoniaa + + + + +
Lethargy and irritability crises ± + + + +
Mental retardationa – + + + +
Sleep disturbances – + + + +
Tremora – + + + +
Digestive Droolinga ± + + + +
Eye Oculogyric crisis ± + + + +
Ptosis of eyelida ± + + + +
Musculoskeletal Growth retardation – + + + +
Rigidityc – + + + +

Other Complicated perinatal courseb ++ – – – –
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (CSF) n n n n n
Dopamine (U) – ↓-n ↓-n ↓-n ↓-n
Homovanillic acid, HVA (CSF) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Homovanillic acid, HVA (U) – ↓-n ↓-n ↓-n ↓-n
HVA/5HIAA (CSF) ↓ ↓ ↓ ↓ ↓
MHPG (CSF) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Norepinephrine (U) – ↓-n ↓-n ↓-n ↓-n
Prolactin (P) – ↑ ↑ ↑ ↑
VMA (U) – ↓-n ↓-n ↓-n ↓-n
Based on the presenting neurological features, TH deficiency can be conceptually divided in two phenotypes: an infantile onset, progressive,
hypokinetic-rigid syndrome with dystonia (type A, less severe), and a complex encephalopathy with neonatal onset (type B, more severe)
a
Predominant in type A
b
Only in type B
c
Predominant in type B
Table 31.2 Aromatic l-amino acid decarboxylase deficiency

Autonomic system Sweating – + + + +
Temperature instability – ± ± ± ±
CNS Athetosis – ± ± – ±
Chorea – ± ± ± –
Developmental delay – + + + +
Dysarthria – + + + +
Dystonia – + + + +
Head control, absent or poor – ± ± ± ±
Hyperkinesia – + + + +
Hypertonia – + + + +
Hypokinesia – + + + +
Hypotonia – ++ ++ ++ ++
Insomnia – + + + –
Irritability – + + + +
Digestive Drooling – + + + +
Feeding/swallowing difficulties – + + – –
Eye Eye movements, abnormal, oculogyric crisis – ++ ++ ++ ++
Poor visual fixation – ± ± – –
Ptosis of eyelid – + + + –
Respiratory Nasal congestion – + + + –
Special laboratory 3-O-Methyldopa (CSF) – ↑↑ ↑↑ ↑↑ ↑↑
5-Hydroxyindoleacetic acid, 5HIAA (CSF) – ↓ ↓ ↓ ↓
Homovanillic acid, HVA (CSF) – ↓ ↓ ↓ ↓
5-Hydroxytryptophan (CSF) – ↑ ↑ ↑ ↑
l-Dopa (CSF) – n-↑ n-↑ n-↑ n-↑
Vanillactic acid (U) – n-↑ n-↑ n-↑ n-↑
Table 31.3 Dopamine beta-hydroxylase deficiency

Autonomic system Hypothermia ± ± – – –
Cardiovascular Hypotension, orthostatic – – ± ++ +++
Syncope – – ± ++ +++
CNS Behavioral disorder, mild – – – ± ±
Eye Eyes opening, delayed ± – – – –
Ptosis of eyelid ± ± ± ± ±
Genitourinary Impaired ejaculation – – – – ++
Musculoskeletal Exercise Intolerance – – ± + +
Routine laboratory Glucose (P) n-↓ n-↓ – – –
Special laboratory 3-O-Methyldopa [P] – – – n-↑ n-↑
Dopamine (P) – – – ↑↑ ↑↑
Dopamine beta-hydroxylase (P) – – – ↓↓↓ ↓↓↓
Epinephrine (P) – – – ↓↓ ↓↓
Homovanillic acid, HVA (CSF) – – – ↑ ↑
Homovanillic acid, HVA (U) – – – ↑ ↑
l-Dopa (P, CSF) – – – ↑ ↑
MHPG (CSF) – – – ↓ ↓
Norepinephrine (P, U) – – – ↓↓↓ ↓↓↓
Vanillylmandelic acid, VMA (U) – – – ↓ ↓
Please note: Metabolite values have not been reported in children, but a similar pattern is predicted
Plasma DßH activity can be low to undetectable in normal individuals; therefore a low value is not diagnostic by itself
Table 31.4 Monoamine oxidase A deficiency

CNS Behavior, aggressive – – – + +
Mental retardation, mild – – + + +
Stereotyped hand movements – – – ± ±
Dermatological Flushing – – – ± ±
Special laboratory 3-Methoxytryramine (U) – – – ↑↑ ↑↑
5-Hydroxyindoleacetic acid, 5HIAA (CSF) – – – ↓ ↓
5-Hydroxyindoleacetic acid, 5HIAA (U) – – – ↓-n ↓-n
Homovanillic acid, HVA (U, CSF) – – – ↓ ↓
MAO-A (FB) – – – ↓↓ ↓↓
MAO-B (PLT) – – – n +
MHPG (U, CSF) – – – ↓-n ↓-n
Normetanephrine (U) – – – ↑↑ ↑↑
VMA (U) – – – ↓ ↓
Please note: Metabolite values have not been reported in children, but a similar pattern is predicted
Table 31.5 Dopamine transporter deficiency

Autonomic system Hyperthermia – ± ± ± –
Sweating – ± ± ± –
CNS Axial hypotonia – ± + + –
Bradykinesia – ± + + –
Bulbar dysfunction – – ± + –
Chorea – ± ± ± –
Dyskinesia – ± + + –
Dystonia – ± + + –
Dystonic crisis – – ± ± –
Hyperkinesia – ± ± ± –
Hypokinesia – ++ ++ ++ –
Hypomimia – ± + + –
Hypotonia, axial – ± + + –
Movement disorder – ++ ++ ++ –
Neonatal irritability ± – – – –
Parkinsonism, hypokinetic features – ± + + –
Pyramidal signs – – ± ± –
Tremor – ± ± ± –
Digestive Drooling – ± ± ± –
Feeding difficulties + – – – –
Eye Ocular flutter – – ± ± –
Musculoskeletal Rigidity – ± + + –
Special laboratory Homovanillic acid, HVA (U) ↑ ↑ ↑ ↑ –
HVA/5HIAA (CSF) ↑↑ ↑↑ ↑↑ ↑↑ –
Prolactin (P) – n-↑ n-↑ n-↑ –
Table 31.6 Dopamine-serotonin vesicular transport defect

Autonomic system Sweating – + + + +
CNS Ataxia – + + + +
Cognitive dysfunction n n + ++ ++
Dysarthria – – – ++ ++
Dysdiadochokinesis – – – ++ ++
Dystonia n + ++ ++ ++
Head control, absent or poor n + – – –
Hypertonia, extremities – – + + +
Hypomimia – + – – –
Hypotonia n + ++ ++ ++
Oculogyric crisis – ++ ++ ++ ++
Parkinsonism, hypokinetic features – + + ++ ++
Eye Ptosis of eyelid – – ++ ++ ++
Respiratory Nasal congestion – ++ ++ ++ ++
Other Hypernasal speech – – + + +
Profuse nasal secretion – + + + +
Special laboratory 5-Hydroxyindoleacetic acid, 5HIAA (U) – ↑↑ ↑↑ ↑↑ ↑↑
Dopamine (U) – ↓ ↓ ↓ ↓
Homovanillic acid, HVA (U) – ↑↑ ↑↑ ↑↑ ↑↑
Norepinephrine (U), Homovanillic acid, – ↓ ↓ ↓ ↓
HVA (CSF) and 5-Hydroxyindolacetic acid
(CSF) normal (in all age classes)
Enzyme analyses
Aromatic amino acid Fibroblast MAO-A
decarboxylase Plasma l-dopa decarboxylase Liver l-dopa decarboxylase (pmol/min/mg Plasma DβH
Age (pmol/min/ml plasma) (pmol/min/ml) (pmol/min/mg protein) protein) (nmol/min/ml)
Fetal – – 720–2,590 – –
<3 years 36–129 36–129 125–695 – –
Adult 24–43 24–43 – 10–350a 0–100b
a
After stimulation with dexamethasone Edelstein et al. (1986)
b
Plasma dopamine-β-hydroxylase: Approximately 5 % of the normal population have undetectable plasma DβH. The diagnosis of DβH deficiency,
therefore, cannot be made solely on the basis of undetectable plasma DβH. On the other hand its presence rules out the diagnosis
Biogenic amines in CSF

Age HVA [nmol/l] 5HIAA [nmol/l] 5HTP [nmol/l] l-Dopa [nmol/l] 3OMD [nmol/l]
0–90 days 484 1,146 302 952 0 20 0 15 0 310
91–210 days 427 989 159 528 0 17 0 15 0 128
211–365 days 403 919 170 412 0 17 0 15 0 50
1–2 years 364 870 155 359 0 15 0 15 0 50
3–5 years 313 824 130 362 0 15 0 15 0 50
6–8 years 260 713 110 247 0 15 0 15 0 50
9–12 years 220 560 90 237 0 10 0 15 0 50
13–15 years 190 507 75 203 0 10 0 15 0 50
>16 years 115 455 51 204 0 10 0 15 0 50
Adapted from Bräutigam et al. (1998)
These reference ranges were established in the author’s laboratory using the second milliliter of CSF (0.5 ml in children <1 year of life). Tubes
were placed immediately on ice and kept frozen at −20 °C until shipping
Blood and plasma neurotransmitters and metabolities (nmol/l) (HPLC, electrochemical (EC) or fluorescene (F) detection)
Age 3OMD (F) l-Dopa (F) 5HTP (F) NEa (EC) DAa (EC) Whole blood serotonin (F)
<3 <80 <25 <20 – – 550–1,780
Adult <80 <25 <20 0.5–3.1 0–0.7 450–980
3OMD 3-O-methyldopa, 5HTP 5-hydroxytryptophan, Ne norepinephrine, DA dopamine
a
Plasma catecholamines: Age-specific lower limits of plasma norepinephrine and dopamine have not been determined. Plasma norepinephrine
varies with posture, activity, volume status and dietary salt, but should be present in plasma even during resting conditions at concentrations of at
least 0.3 nmol/l
Urine neurotransmitters and metabolites (nmol/mmol creatinine) (HPLC, electrochemical (EC) or fluorescence (F) detection)
Age 3OMDa l-dopaa 5HTPa 5HIAAa HVAa NEa Ea DAa NMNa 3MTa VMAa 5HTa VLAa
(years) (F) (F) (F) (F) (F) (EC) (EC) (EC) (EC) (EC) (EC) (F) (EC)
<3 152–378 – – 5,500–7,300 7,000–8,500 22–140 2.5–26 76–1,350 5–143 45–480 500–2,500 130–170 <150
Adult 90–225 12–42 <5 300–5,100 1,000–2,800 10–53 2–11 60–225 55–200 60–145 800–2,200 11–68 <80
3OMD 3-O-methyldopa, 5HTP 5-hydroxytryptophan, Ne norepinephrine, E epinephrine, DA dopamine, NMN nor-metanephrine, 3MT
3-methoxytyramine, VMA vanilylmandelic acid, 5HT serotonin, HVA homovanillic acid, 5HIAA 5-hydroxyindoleacetic acid, VLA vanillactic acid
a
Unconjugagted
31
5HIAA HVA 3OMD l-Dopa 5HTP MHPG DOPAC Pro NE DA 5HT VLA
(CSF) (CSF) HVA/5HIAA (P, U, CSF) (P, U, CSF) (P, U, CSF) (U CSF) (P, U) (P) (P, U) E (P, U) (P, U) NMN (U) 3MT (U) VMA (U) (B) (U)
TH n ↓↓ ↓↓ n ↓ ↑ ↓-n ↓-n
AADC ↓↓ ↓↓ ↑↑↑ ↑↑ ↑↑ ↑ (U) ↑ ↓↓↓ ↓ ↑-n ↑ (U) ↓↓ ↑
(U)
DßH n ↑ (U) ↑-n ↑ (P,CSF) ↓↓ ↑↑ (P) ↓↓↓ ↓ ↑↑↑ ↓
MAO-A ↓ ↓ n ↓ ↑↑ ↑↑ ↑↑ ↓ ↑
Neurotransmitter Disorders
↓-n (U) ↓-n (U)

DAT n ↑ ↑↑ n-↑
↑-n (U)
VMAT2 n n ↑↑ ↓ (U) ↓ (U)
Abbreviations: P plasma, U urine, 3OMD 3-O-methyldopa, HVA homovanillic acid, MHPG 3-methoxy-4-hydroxyphenylglycol, VMA vanillylmandelic acid, 5HTP 5-hydroxytryptophan, 5HIAA
5-hydroxyindoleacetic acid, NE norepinephrine, E epinephrine, DA dopamine, NMN normetanephrine, 3MT 3-methoxytyramine, VMA vanillylmandelic acid, VLA vanillactic acid, Pro prolactin
523
Unknown neurological disorder presenting with

one or more of the following symptoms:
Parkinsonism, truncal hypotonia, limb
hypertonia, dystonia, dyskinesia, chorea,
oculogyric crises, ptosis, temperature
instability, excessive sweating, feeding
difficulties, hypersalivation or behavioral
abnormalities or orthostatic hypotension
Yes
Hyper PHE (NBS) BH4 Deficiency?
No
Perform analysis of HVA, 5HIAA, L-

Pterins /
Dopa, 5-HT, MHPG, Pterins (CSF)
HVA n (CSF) HVA HVA HVA HVA

5HIAA n (CSF) 5HIAA n 5HIAA 5HIAA n 5HIAA
MHPG 3OMD n 3OMD
3OMD n L-dopa
5-HT
HVA (U) HVA/ DA (U) DA (U) VLA (U)

5HIAA (U) 5HIAA NE (U) NME (U)
DO (U) E (U) 3MT (U)
NE (U) VMA (U) VMA (U)
SLC18A2 SLC6A3 DβB MAO-A TH AADC

mutation mutation activity (P) activity mutation activity (B)
DβB mutation (FB)
VMAT2 DAT DβH MAO-A TH AADC

deficiency deficiency deficiency deficiency deficiency deficiency
Fig. 31.2
31.8 Specimen Collection (Incl. Pitfalls

and Special Handling/Storage)

5HIAA CSF Before medication Collect 6 CSF fractions (0.5 ml each) Unstable in blood-
3OMD contaminated samples
l-Dopa Place tubes immediately on ice and keep frozen In the case of blood
HVA at −20° C (better −70° C) contamination,
MHPG Use the 1st fraction for other investigations centrifuge CSF first
BH4 (CSF cell count, lactate, glucose etc.)
BH2 Collect the 4th fraction for pterins (BH4) in tubes
Neo containing 1 mg DTE and 1 mg DTPA
(diethylenetriaminepentaacetic acid)
AADC activity Plasma 1 ml, heparin tube, centrifuge and store
immediately at −70 °C
5HT, 3MT, 3OMD, 24-h urine Diet free of biogenic 24-h urine collected into 6 M HCl, store at −20 °C Many foods, e.g.,
NMN, 5HIAA, VMA, amine-containing bananas, dates, contain
HVA, DA, E, MHPG, NE foodstuffs biogenic amines and
metabolites
5HT (serotonin) Whole blood 2 ml, EDTA tubes containing 6 mg ascorbic acid
store at −20 °C
DßH activity Plasma 1 ml, heparin tube, store at −70 °C
MAO-A activity Fibroblast Room temp.
Catecholamines and Plasma 1 ml, in EDTA tube, store at −70 °C
metabolites
VLA Urine Urine 10 ml, store at −20 °C
Abbreviations: MHPG 3-methoxy-4-hydroxyphenylglycol, HVA homovanillic acid, 5HIAA 5-hydroxyindoleacetic acid, 3OMD 3-O-methyldopa,
VMA vanillylmandelic acid, 5HTP 5-hydroxytryptophan, NE norepinephrine, 5HTP 5-hydroxytryptophan, E epinephrine, DA dopamine, NMN
normetanephrine, 3MT 3-methoxytyramine, VLA vanillactic acid, Pro prolactin, BH4 tetrahydrobiopterin, BH2 dihydrobiopterin, Neo neopterin,
DA dopamine, NMN normetanephrine, DßH dopamine-ß-hydroxylase
Material Timing
TH Chorionic villi I. trimester
Amniotic fluid cells (if mutations are known)
AADC Chorionic villi I. trimester
Liver biopsy for enzyme analysis (2. trimester)
DßH – –
MAO-A – –
DAT Chorionic villi I. trimester
31.10 DNA Testing
Tissue Methodology
TH Genomic DNA (and from cultures of skin fibroblasts, lymphoblasts, lymphocytes) PCR/SSCP sequencing
AADC Genomic DNA (and from cultures of skin fibroblasts, lymphoblasts, lymphocytes) PCR sequencing
DßH Genomic DNA PCR sequencing
MAO-A Cultured fibroblasts PCR sequencing
DAT Genomic DNA (and from cultures of skin fibroblasts, lymphoblasts, lymphocytes) PCR sequencing
VMAT2 Genomic DNA (and from cultures of skin fibroblasts, lymphoblasts, lymphocytes) PCR sequencing
31.11 Treatment including hypoglycemia due to catecholamine deficiency

(Manegold et al. 2009).
Summary 2. Lack of catecholamines may also render patients suscep-
Treatment in TH and AADC deficiencies is aimed at correction tible to sudden cardiac arrest, especially when they are
of the shortage of neurotransmitters. Bypassing the metabolic exposed to stressful situations (Anselm and Darras 2006).
block using levodopa/carbidopa only or together with dopamine 3. Therefore, clinical admission and close cardiac monitor-
agonists usually leads to a good to excellent clinical response in ing during acute illness and/or for any medical interven-
most TH deficient patients. In addition to l-dopa, inhibitors of tion is inevitable.
dopamine degradation such as selegiline can be used. Despite 4. Use glucose infusion during acute illness and/or during
appropriate treatment long-term cognitive development can any medical intervention.
be subnormal in these patients (Willemsen et al. 2010).
AADC deficient patients have received dopamine receptor Standard Treatment and Medication Requirements
agonists, monoamine oxidase inhibitors, anticholinergics, and Medication Dosage (mg/kg/day) Dose/day (n)
selective serotonin reuptake inhibitors in conjunction with TH Levodopa (l-dopa) (0.5) 1–10 2–6
vitamin B6 (cofactor for AADC). Therapy is usually initiated Carbidopa 10–25 % of dopa 2–6
with one of the dopamine agonists (bromocriptine or per- AADC Bromocriptine 0.25–0.5 3
golide), sometimes in combination with low-dose pyridoxine. Pergolide 0.006–0.05 2–3
In a second step, a monoamine oxidase inhibitor is added Selegiline 0.1–0.3 2–3
(e.g., selegiline). All other medications are added only if the Pyridoxine (B6) 50–200 3
initial treatment protocol fails to improve neurological symp- Tranylcypromide 0.1–0.5 2
Trihexyphenidyl 0.1–0.5 3
toms. Treatment should be carefully evaluated for at least
DßH dl-Dihydroxy- 250–500 2–3
three months and be discontinued in case of no or question- phenylserine
able positive effects. l-dopa treatment has been successful in (DOPS)
3 siblings with mutations affecting the l-dopa binding (Brun DAT L-dopa/carbidopa 2–6 2–6
et al. 2010; Chang et al. 2004). In general response to treat- VMAT2 Pramipexole Not known Not known
ment in AADC patients is variable and outcome appears to be Levodopa (l-dopa) 2–6 2–6
better in less severely affected patients and males than females.
Since orthostatic hypotension is the main symptom in
patients with dopamine-ß-hydroxylase (DßH), treatment
Dangers/Pitfalls
focuses on this aspect. Oral administration of dihydroxyphe-
1. l-Dopa/carbidopa and 5-hydroxytryptophan ther-
nylserine (DOPS), a synthetic precursor of noradrenalin,
apy should be introduced slowly and increased in
dramatically increases blood pressure resulting in clinical
steps of not more than 1 mg/kg over days or weeks.
improvement of postural symptoms.
2. Changes in dopamine receptor density can cause
Up to now no effective treatment for MAO-A deficiency
difficulties with treatment. Receptor hypersensitiv-
is available. Dietary intervention by avoiding foods rich in
ity in early diagnosed, severe cases means that
amines as well as cyproheptadine hydrochloride and sertra-
treatment with L-dopa/carbidopa should start at
line hydrochloride supplementation have been used with
very low doses (0.25–0.5 mg levodopa/kg/day)
uncertain or with no effect.
given frequently up to six times a day. Receptor
Various drug treatment strategies and surgical interven-
downregulation in late-diagnosed severe forms
tions like deep brain stimulators have only little, temporary,
means that treatment with L-dopa/carbidopa in the
or even no effect on the clinical symptoms of DAT deficient
maximally tolerated dose up to 10 mg levodopa/kg/
patients (Kurian et al. 2011b). In brain dopamine–serotonin
day should be maintained for as much as 6 months
vesicular transport disease treatment was initially started
before deciding it is unhelpful.
with levodopa leading to a worsening of the movement dis-
3. l-Dopa/carbidopa and 5-hydroxytryptophan ther-
order. In contrast, subsequent treatment with direct dopa-
apy may reduce CSF folate (5′-methyltetrahydrofo-
mine agonists was followed by immediate ambulation,
late in CSF is the major transport species for the
near-complete correction of the movement disorder, and
brain folate pool and is utilized by the single carbon
resumption of development (Rilstone et al. 2013).
transfer pathway to methylate l-dopa to 3-O-methyl-
dopa). Determine 5-methyltetrahydrofolate in CSF.
Emergency Treatment
Consider folinic acid (5-formyltetrahydrofolate)
Potential hazards and pitfalls:
substitution (10–20 mg/day). This may occur “nat-
1. Besides the neurological picture, patients with TH and
urally” in AADC deficiency, again requiring folate
AADC deficiency may develop additional non-
supplementation (Surtees and Hyland 1990).
neurological symptoms, especially autonomic symptoms
diagnosis of childhood-onset dystonia. Ann Neurol 54(Suppl

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troublesome side-effects occur (dry eyes or mouth, 1897–1904
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Creatine Disorders
32
Sylvia Stöckler, Olivier Braissant, and Andreas Schulze
Contents Summary
32.1 Introduction..................................................................... 529 Reduced creatine levels in the brain and in body fluids/tis-
sues are the common denominator of primary (AGAT,
32.2 Nomenclature .................................................................. 530
GAMT, X-linked creatine transporter (SLC6A8) deficiency)
32.3 Metabolic Pathway ......................................................... 531 and secondary (OAT deficiency) creatine disorders.
32.4 Signs and Symptoms ....................................................... 531 Developmental delay/intellectual disability, speech delay,
and behavioral problems, combined with epilepsy, and
movement disorders are characteristic clinical features of
32.6 Pathological Values ......................................................... 534 primary creatine deficiency syndromes. Chorioretinal degen-
32.7 Differential Diagnosis ..................................................... 534 eration is the clinical hallmark of OAT deficiency. Diagnostic
32.8 Diagnostic Flowchart ...................................................... 535 markers include high (GAMT) or low (AGAT) levels of gua-
nidinoacetate, a high urinary creatine excretion (SLC6A8
32.9 Prenatal Diagnosis .......................................................... 536
deficiency), and high plasma ornithine levels (OAT defi-
32.10 DNA Testing..................................................................... 536 ciency). Treatments comprise substitution of creatine (AGAT
32.11 Specimen Collection........................................................ 536 deficiency) combined with l-ornithine (GAMT deficiency)
32.12 Treatment......................................................................... 537
and arginine-restricted diet (GAMT and OAT deficiency).
Administration of creatine and of substrates for intracerebral
References ..................................................................................... 539 creatine synthesis (l-arginine and l-glycine) have limited
therapeutic effects in CrT deficiency. Despite availability of
causal treatments and improved outcomes after early recog-
nition, creatine disorders are still under-recognized. All
patients with unexplained developmental delay/intellectual
disability should be screened for these disorders.
32.1 Introduction
S. Stöckler (*)
Division of Biochemical Diseases, Creatine is synthesized by two enzymatic reactions: (1) the
BC Children’s Hospital, University of British Columbia,
formation of guanidinoacetate (GAA) from arginine and gly-
Room K3-205, 4480 Oak Street,
Vancouver, BC V6H 3V4, Canada cine by l-arginine/glycine amidinotransferase (AGAT) and
e-mail: sstockler@cw.bc.ca (2) the methylation of GAA by S-adenosyl-l-methionine/N-
O. Braissant guanidinoacetate methyltransferase (GAMT). Liver, pancreas,
Service of Biomedicine, Department of Laboratories, and kidney are the main sites of creatine synthesis. While the
University Hospital of Lausanne, Lausanne, Switzerland brain can synthesize creatine, the major proportion of brain
e-mail: Olivier.Braissant@chuv.ch
creatine is taken up from the blood via a Na+/Cl−-dependent
A. Schulze creatine transporter (CrT, SLC6A8) expressed at the blood–
Division of Clinical and Metabolic Genetics, Department of
brain barrier (Braissant 2012). Creatine plays a major role in
Pediatrics, The Hospital for Sick Children, University of Toronto,
555 University Avenue, Toronto, ON M5G 1X8, Canada storage and transmission of high-energy phosphates (ATP),
e-mail: andreas.schulze@sickkids.ca via its reversible conversion into phosphocreatine, catalyzed
530 S. Stöckler et al.
by creatine kinase(s). Creatine may also play a role in neuro- ranging from occasional to intractable seizures. Additional
transmission. Intracellular creatine and phosphocreatine are manifestations include failure to thrive, low muscular
non-enzymatically converted into creatinine, which is excreted mass, muscular hypotonia, and movement disorders
in urine. The daily creatinine excretion is directly proportional (mainly extrapyramidal). Pathological signal intensities of
to total body creatine content. Creatine synthesis is regulated the basal ganglia have been described in patients with
via AGAT activity. Because AGAT is inhibited by high con- GAMT deficiency.
centrations of l-ornithine, the hyperornithinemia in ornithine OAT Deficiency: A Secondary Creatine Deficiency
aminotransferase (OAT) deficiency is associated with second- Hyperornithinemia-associated gyrate atrophy of the choroid
ary creatine deficiency. and retina is an inherited metabolic eye disease, caused by
Primary Creatine Deficiencies: AGAT, GAMT, and CrT autosomal recessive deficiency of the l-ornithine: 2-oxoacid
Deficiencies Primary creatine deficiencies comprise two aminotransferase (OAT) (32.4) (Valle and Simell 2001).
autosomal recessive inborn errors of creatine synthesis, Clinical features include progressive chorioretinal degenera-
AGAT (32.2) and GAMT (32.1) deficiencies, and one tion with myopia, night blindness, and loss of peripheral
X-linked defect of creatine transport, CrT (32.3) deficiency vision starting late in the first decade, proceeding to tunnel
(Schulze 2003; Stockler-Ipsiroglu et al. 2012). The main vision and eventually leading to blindness in the third and
affected organ in primary creatine deficiencies is the brain, fourth decade. In addition to the ocular findings, some
which can be visualized as a strongly decreased or absent patients present systemic abnormalities. Most patients have
peak of creatine (phosphate) in the proton magnetic reso- normal intelligence. MRI findings include degenerative
nance spectrum (1H-MRS). Developmental delay and lesions in the white matter and premature atrophic changes.
intellectual disability are their common clinical presenta- Tubular aggregates and selected atrophy of the type II fibers
tion. Speech delay is prominent even in patients with mild/ of skeletal muscle do not cause muscle weakness, although
moderate intellectual disability. Many patients have an muscle performance of affected patients may be impaired
autistic behavior and/or various degrees of epilepsy when speed or acute strength is required.
32.2 Nomenclature
No. Disorder name Abbreviation Gene symbol localization Affected protein OMIM no. Subtype
32.1 Guanidinoacetate GAMT GAMT 19p13.3 Guanidinoacetate 612736 All forms
methyltransferase methyltransferase
deficiency
32.2 Arginine/glycine AGAT GATM 15q15.3 Arginine/glycine 612718 All forms
amidinotransferase amidinotransferase
deficiency
32.3 Creatine transporter X-linked CrT, SLC6A8 SLC6A8 Xq28 SLC6A8 transporter 300352 All forms
deficiency creatine (CrT, creatine
deficiency transporter)
syndrome
32.4 Ornithine Gyrate OAT, GACR OAT 10q26.13 Ornithine 258870 All forms
aminotransferase atrophy of aminotransferase
deficiency the choroid
and retina
32 Creatine Disorders 531
1: Creatine synthesis Arginine Glycine

kidney, liver, pancreas
brain
(32.2) AGAT 2-Oxoglutarate
ADP ATP
Phospho- CK
Guanidinoacetate Ornithine
guanidinoacetate
(32.4) OAT VitB6
S-adenosylmethionine
Glutamate
(32.1) GAMT ( Glutamate -
S-adenosylhomocysteine 5-semialdehyde )
Creatine Proline Pyrroline -

5-carboxylate
2: Creatine uptake
SLC6A8 (32.3)
mainly brain, muscle
ATP ADP
3: Creatine / Creatine Phosphocreatine
phosphocreatine / CK
CK system
mainly brain, muscle Non-enzymatic
conversion
neurotransmission ? Creatinine
4: Urinary creatinine
excretion Creatinine
Fig. 32.1 The pathways of creatine synthesis and transport
Table 32.1 Guanidinoacetate methyltransferase deficiency

CNS Autism n +++ +++ +++ +++
Basal ganglia abnormalities n + + + +
Developmental delay n +++ +++ +++ +++
Epilepsy n ++ ++ ++ ++
Movement disorder n + + + +
Speech delay n +++ +++ +++ +++
Routine laboratory Creatinine (U,P) ↓-n ↓ ↓ ↓ ↓
Special laboratory Cerebral creatine deficiency +++ +++ +++ +++ +++
Creatine (U,P,CSF) ↓-n ↓↓ ↓↓ ↓↓ ↓↓
Guanidinoacetate (U,P,CSF) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 32.2 Arginine/glycine amidinotransferase deficiency

CNS Developmental delay n +++ +++ +++ +++
Epilepsy n + + + +
Movement disorder n ± ± ± ±
Digestive Failure to thrive n + + + +
Routine laboratory Creatinine (U,P) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory Cerebral creatine deficiency +++ +++ +++ +++ +++
Guanidinoacetate (U,P,CSF) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 32.3 Creatine transporter deficiency

CNS Autism n +++ +++ +++ +++
Developmental delay n +++ +++ +++ +++
Epilepsy n + + + +
Digestive Constipation ± ± ± ±
Musculoskeletal Muscle mass, low ± + + +
Special laboratory Cerebral creatine deficiency +++ +++ +++ +++
Creatine/creatinine ratio (U) ↑↑ ↑↑ ↑↑ ↑↑
Table 32.4 Ornithine aminotransferase deficiency

CNS Cortical atrophy n ± ±
Intellectual disability n n ± ± ±
Neuropathy, sensory ± ± ±
Seizures n n n ± ±
Eye Adaptation, dark impaired n ± ++ +++ +++
Atrophy, gyrate of choroid and retina n n + ++ +++
Blindness n n n n +++
Cataract, posterior subcapsular n n n ± +
Chorioretinal degeneration n n + ++ +++
Myopia n ± + ++ +++
Retinal detachment n n ± ± ±
Vision, tunnel n n ± + +++
Musculoskeletal Muscle weakness n n ± ± ±
Routine laboratory Ammonia (B) n-↑ n n n n
Creatinine (P) ↓-n ↓-n ↓-n ↓-n
EEG: abnormal ± ±
Special laboratory 3-Amino-2-piperidone (U) ± ↑ ↑ ↑ ↑
Arginine + lysine (U) ± ↑ ↑ ↑ ↑
Creatine (U,P,CSF) ↓↓ ↓↓ ↓↓↓ ↓↓↓
Guanidinoacetate (U,P,CSF) ↓↓ ↓↓ ↓↓↓ ↓↓↓
Liver EM: abnormal mitochondria ± ±
MRS: Creatine/phosphocreatine (brain) ↓ ↓ ↓
MRS: Creatine/phosphocreatine (muscle) ↓ ↓ ↓
Muscle EM: abnormal mitochondria ± ± ±
Muscle EM: type 2 fiber atrophy and ± ± ±
tubular aggregates
Ornithine (P,U) ± ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
White matter abnormalities n ± ±
Urine [mmol/mol Amniotic fluid

Age range creatinine] Plasma [μmol/L] CSF [μmol/L] [μmol/L]
Guanidinoacetatea <10 years 20–208 0.6–2.4
10–15 years 15–152 0.9–3.0
>15 years 5–78 1.2–3.6
<4 years 9–128
4–15 years
Male >15 years
Female >15 years
Guanidinoacetateb 0–15 years 4–220 0.35–1.8 0.02–0.56
>15 years 3–78 1.0–3.5
No age range
established
Guanidinoacetatec No age range 0.036–0.22
established
Guanidinoacetated 1.6–4.4
Creatinea <10 years 29–1,551 34–124
10–15 years 19–1,046 28–104
>15 years 7–492 12–99
<4 years 12–682
4–15 years
Male >15 years
Female >15 years
Creatineb 0–4 years 6–1,208 17–109 17–87
4–12 years
>12 years 17–721
0–10 years 6–50
>10 years 11–244
No age range
established
Creatinec 0–10 years 24–53
>10 years 38–66
Creatined 28–88
Ornithinee 0–1 month 0–19 41–129 0.7–15.7
1–6 months 0–13 20–136 2.0–5.9
6–12 months 0–8 47–195 3–9
1–2 years 0–8 55–135 1.7–8.1
2–4 years 0–7 36–96
4–7 years 0–7
7–13 years 0–6
>13 years 0–5
<3 months
<12 months
Children
Adolescents
Men
Women
a
Method: LC-MS/MS. Reference values modified from Joncquel et al. (2011)
b
Method: GC-MS. Reference: Almeida et al. (2004)
c
Method: LC-MS/MS. Reference: Young et al. (2007)
d
Method: LC-/MS/MS. Reference: Cheillan et al. (2006)
e
Method: HPLC (amino acid analyzer). Reference: Shih (2003)
Plasma magnitude
of change CSF magnitude of Amniotic fluid
Urine [mmol/ compared to change compared magnitude of change
Guanidinoacetate Disorder mol creatinine] normal to normal Brain 1H-MRS compared to normal
32.1 >500a >10× >100× Elevated >3×b
GAMT
32.2 <0.3a <0.1× No data available/ No data available/ No data available/
AGAT presumably low presumably not detectable presumably low
32.3 Normal Normal No data available/ No data available/may be No data available/
CrT presumably normal elevated presumably normal
32.4 Low Low No data available/ No data available/ Mo data available/
OAT presumably normal presumably not detectable presumably normal
Creatine Disorder Urine [mmol/ Plasma [μmol/L] CSF [μmol/L] Brain 1H-MRS Amniotic fluid
mol creatinine] [μmol/L]
32.1 Low <7 1.4–19c Absent/very low Normalb
GAMT
32.2 Low Low No data available/ Absent/very low No data available/
AGAT presumably low presumably normal
32.3 High (>1400a) Normal May be normal Very low No data available/
CrT presumably normal
32.4 Low Low No data available/ Low No data available/
OAT presumably low presumably normal
Ornithine Disorder Plasma [μmol/L]
32.4 600–1400d
OAT
a
Method: GC-MS. Reference: Struys et al. (2008)
b
Method: LC-MS/MS. Reference: Cheillan et al. (2006)
c
Method: HPLC, fluorescence. Reference: Schulze A. Own observation in 3 patients
d
Please note: Plasma ornithine may not be elevated in neonates and in the first months of life
32.7 Differential Diagnosis methods used for determination of urinary GAA excretion
may not be sensitive enough in the below normal range. CrT
32.1–32.3: GAMT, AGAT, and CrT Deficiencies Cerebral cre- (SLC6A8) deficiency may represent a major cause of
atine deficiency as detected by 1H-MRS is the common bio- X-linked mental retardation accounting for 1–2 % percent in
chemical hallmark of AGAT, GAMT, and CrT deficiencies. males with intellectual disability (Rosenberg et al. 2004).
Increased levels of GAA in body fluids are pathognomonic Females may be moderately affected clinically, but it appears
for GAMT deficiency, whereas GAA levels are reduced in that urinary creatine/creatinine ratio and cerebral creatine
AGAT deficiency. Increased urinary creatine and decreased levels do not allow a reliable diagnosis in this group of
creatinine excretion, resulting in an increased urinary cre- patients. Primary creatine disorders may account for a high
atine-to-creatinine ratio, are associated with CrT deficiency. proportion of undiagnosed and potentially treatable genetic
Less than 100 patients are known with GAMT deficiency, mental retardation syndromes in children and adults.
and less than 15 patients are known with AGAT deficiency. Therefore, selective screening for these disorders (by urinary/
One reason for the low detection rate is the fact that determi- plasma metabolites, brain 1H-MRS, and/or DNA approach)
nation of urinary GAA requires a specific test, which often is should be included in the investigation of this population.
not requested by physicians assessing children with develop- 32.4: OAT Deficiency Marked hyperornithinemia outside
mental delay or intellectual disability. Diagnosis of AGAT the neonatal period is the biochemical hallmark of OAT defi-
deficiency is particularly challenging as existing analytical ciency. The most important laboratory test in this disease is
therefore the measurement of amino acids in plasma. In that requires pyridoxal phosphate (vitamin B6) for the revers-
affected individuals, plasma ornithine values range from 400 ible conversion of ornithine and 2-oxoglutarate to
to 1,400 μmol/L (controls: see table). The combination of Δ1-pyrroline-5-carboxylate and glutamate. In its deficiency
elevated plasma ornithine and characteristic ocular findings is the accumulation of ornithine causes secondary creatine defi-
highly specific for the disease. Ornithine is also increased in ciency through inhibition of AGAT activity, the rate limiting
the CSF and urine. Pathological urinary excretion of ornithine step in endogenous creatine synthesis (Näntö-Salonen et al.
and the other dibasic amino acids (arginine, lysine, cystine) 1999). Enzyme analysis is feasible in stimulated lymphocytes
typically occurs at plasma ornithine concentrations greater or fibroblasts. Prenatal diagnosis is potentially possible.
than 600 μmol/L. In plasma, lysine and creatine may be
decreased. In urine, creatine, creatinine, and GAA are usually
decreased. Investigation by in vivo 1H-MRS reveals a 32.8 Diagnostic Flowchart
decreased concentration of creatine and phosphocreatine in
brain and muscle. Mutation analysis can be performed to con- The differential diagnosis of primary and secondary creatine
firm the diagnosis. OAT is a mitochondrial matrix enzyme deficiencies is shown in Fig. 32.2.
Patient
Neurological MRS of brain Chorio-retinal

symptoms creatine degeneration
Cr: decreased ↓
GAA, creatine, creatinine (U,P,CSF) / ornithine (P) ornithine (P)
GAA: increased GAA: decreased GAA,Cr: normal GAA,Cr: decreased Orn: increased
↑ U,P,CSF ↓U,P U,P,CSF ↓ U,P ↑P
and and
Cr/Crn ratio: Orn: increased
increased ↑ U ↑ U,P,CSF
GAMT GATM SLC6A8 OAT

sequencing sequencing sequencing sequencing
(enzyme activity) (enzyme activity) (Cr uptake study)
32.1 GAMT 32.2 AGAT 32.3 CrT 32.4 OAT

deficiency deficiency deficiency deficiency
Fig. 32.2 Differential diagnosis in creatine disorders

Disorder Method/analytes Material Timing semester

32.1 GAMT activity Cultured amniotic cells II
Mutation analysis Chorionic villi, cultured amniotic cells I, II
GAA Amniotic fluid II
32.2 AGAT activity Cultured amniotic cells II
32.3 SLC6A8 activity Cultured amniotic cells II
32.4 OAT activity Cultured amniotic cells II
So far, prenatal diagnosis has been documented for

disorders 32.1, 32.2, and 32.4, but not for 32.3.
32.10 DNA Testing
Disorder Tissue Methodology Mutations

32.1 Genomic DNA (cultured FB, LYM, Direct sequencing, high-throughput http://www.LOVD.nl/GAMT
EBV-transformed lymphoblasts) sequencing
32.2 Genomic DNA (cultured FB, LYM, Direct sequencing, high-throughput http://www.LOVD.nl/GATM
32.3 Genomic DNA (cultured FB, LYM, Direct sequencing, high-throughput http://www.LOVD.nl/SLC6A8
32.4 Genomic DNA (cultured FB, LYM, Direct sequencing, high-throughput http://www.LOVD.nl/OAT
FB fibroblasts, LYM lymphocytes

Creatine Before creatine U 24 h urine or spot, store at −20 °C U: false positives in high-protein diet
supplementation CSF Store at −20 °C
P EDTA or heparinate, store at −20 °C P: probably not diagnostic in neonates
Creatinine Before creatine U 24 h urine or spot, store at −20 °C U: low in patients with reduced muscle
supplementation mass
CSF Store at −20 °C U: false positives in high-protein diet
P EDTA or heparinate, store at −20 °C P: probably not diagnostic in neonates
Creatine/creatinine ratio U Not sensitive in heterozygous females
to test SLC6A8 deficiency Can be normal in symptomatic patients
Guanidinoacetate U 24 h urine or spot, store at −20 °C
CSF Store at −20 °C
P EDTA or heparinate, store at −20 °C
DBS Room temperature
Ornithine U 24 h urine or spot, store at −20 °C
CSF Store at −20 °C
P EDTA or heparinate, store at −20 °C
GAMT, AGAT, SLC6A8, liver
OAT activities Store at −80 °C
FB
Cultured skin cells
LYM EBV-transformed lymphoblasts
DBS dry blood spot, FB fibroblasts, LYM lymphoblasts
32.12 Treatment females may benefit from creatine supplementation. Various

combinations of l-arginine and l-glycine (serving as precur-
Summary sors of intracerebral creatine synthesis) have also been
32.1–32.3: GAMT, AGAT, and CrT deficiencies Correction attempted. While some neurological and behavioral improve-
of cerebral creatine deficiency in GAMT and AGAT defi- ments associated with these treatments were observed in a
ciency is achieved via long-term supplementation of high few cases, in many others, the clinical benefit was inconclu-
dosages of creatine (given as creatine monohydrate). sive (Mercimek-Mahmutoglu et al. 2010; Valayannopoulos
Clinically this is associated with significant improvement et al. 2012; van de Kamp et al. 2012). Analogs of creatine,
and even normalization of abnormal developmental scores in like cyclocreatine, may represent interesting alternatives to
AGAT deficiency (Ndika et al. 2012) and with improvement treat CrT deficiency in the future (Kurosawa et al. 2012).
of seizures and movement disorder in GAMT deficiency 32.4: OAT deficiency A permanent reduction of plasma
(Mercimek-Mahmutoglu et al. 2006). In order to reduce the ornithine below 200 μmol/L slows or stops the chorioretinal
formation of GAA in GAMT deficiency, creatine supple- degeneration. Some patients respond to pharmacological
mentation is combined with pharmacological doses of doses of vitamin B6. Mainstay of treatment is substrate depri-
l-ornithine and/or dietary arginine restriction (Schulze et al. vation with a diet that consists of arginine restriction and
2001). Sodium benzoate has been proposed as an additional arginine-free amino acid supplements. Additional experi-
approach to reduce the production of GAA via conjugation mental approaches include augmentation of renal ornithine
with glycine to form hippuric acid; however, no clinical stud- excretion by administration of pharmacological doses of
ies have been performed to prove its effectivity. The pres- l-lysine (Elpeleg and Korman 2001) or the non-metabolizable
ymptomatic treatment of AGAT and GAMT deficiencies amino acid α-aminoisobutyric acid, and proline supplemen-
appears to completely prevent the phenotypic expression of tation. Since no form of therapy is unequivocally effective,
both diseases (Schulze and Battini 2007). combined treatment approaches seem necessary. Creatine
Treatment of male patients with high dosages of creatine administration improves the histologic abnormalities in mus-
is not effective due to the absence of a functional creatine cle, but does not halt the progress of chorioretinal
transporter at the blood–brain barrier (Braissant 2012), while degeneration.
Standard Treatment
No./symbol Age Medication/diet Dosage [mg/kg/day] Doses per day

32.1 GAMT All ages Creatine monohydrate 400 3–6
l-Ornithine aspartate 3–6
All ages Low dose 100a
Children High dose 800a
Adults 400a
All ages Sodium benzoate 100 3
l-Arginine intake 15–25b 3–5 daily meals
Essential amino acid mixture 0.2–0.7 g essential amino 3–5 daily meals
(arginine-free) acids/kg
32.2 AGAT All ages Creatine monohydrate 200–400c 3–6
32.4 OAT <14 years Pyridoxine hydrochloride diet (see below) 40–200e 2
Vitamin B6 responsive >14 years Pyridoxine hydrochloride diet (see below) 40–500e 2
formd
32.4 OAT All ages Diet (see below)
Vitamin B6 nonresponsive
formd
a
The aim of low-dose substitution is to provide sufficient amounts of ornithine to the urea cycle (target plasma ornithine concentration 100–
200 μmol/l). The aim of high-dose substitution is to potentially inhibit competitively AGAT activity by high intracellular ornithine concentrations
(Km = 300 μmol/l)
b
Corresponds to 0.4–0.7 g/kg natural protein. Essential amino acid mixture supplement is necessary in order to meet age-dependent physiological
amino acid/protein requirements
c
Lower dosages of creatine monohydrate might be sufficient for the restoration of the cerebral creatine pool in AGAT deficiency due to the absence
of GAA accumulation and no subsequent competitive inhibition of creatine uptake
d
Target plasma ornithine concentration <200 μmol/l
e
15–20 mg/day might be as effective in some patients as higher dosage (Weleber and Kennaway 1981)
Beware/Pitfalls
In individuals supplemented with creatine, intake of high doses (20 g/day in adults) has been associated with weight
gain (due to intracellular edema), muscle cramps, and impairment of renal function
Creatine monohydrate is freely available in drug stores, and attention has to be paid to the purity of the various prod-
ucts. In particular, after chemical synthesis from sarcosine and cyanamide, contamination with dicyanamide, which
liberates HCN in the acidic conditions of the stomach, may occur as a result of incomplete purification
Higher doses of ornithine should be given in a formulation of ornithine aspartate, but not as ornithine HCl. The latter
treatment can cause metabolic acidosis
Acute respiratory failure after institution of vitamin B6 reported in a few neonates with severe seizure disorder
Peripheral neuropathy associated with long-term ingestion of high-dosage vitamin B6 (>1,000 mg/day)
An al gorithm on how to test for vitamin B6

responsiveness is shown in Fig. 32.3.
Vitamin B6
< 6 y 200 mg/day
> 6 y 500 mg/day
for 1-2 months
Significant reduction YES plasma Orn YES Continue

of plasma Orn < 200 µM B6 treatment
NO NO
Dietary B6 treatment
Arg restriction + dietary Arg restriction
Continue dietary YES plasma Orn plasma Orn YES Continue B6 treatment
Arg restriction < 200-400 µM < 200 µM + dietary Arg restriction
NO NO
dietary Arg restriction

+ experimental trial with Continue B6 treatment
L-lysine or + dietary Arg restriction
α-aminoisobutyric acid
Keep plasma Orn Keep plasma Orn Keep plasma Orn

< 200-400 µM as low as possible < 200 µM
Fig. 32.3 Treatment in gyrate atrophy

Dietary Treatment (32.1–32.4)
Protein requirement [g/kg/ Arginine-free essential AAM

Age day] Natural protein [g/kg/day]a Typeb Protein equivalentc [g/day]
Infants 1.1–2.7 0.4 1 2–5
Children 1.0–1.7 0.3–0.5 2 10–25
Adults 0.8 0.25 2 30–75
a
According to Dewey et al. (1996)
b
Type 1, infantile formula; type 2, childhood formula
c
Spread as evenly as possible through the 24 h
Beware/Pitfalls
Overtreatment through protein restriction

No./symbol Age Medication/diet Dosage mg/kg per day Dosages per day Literature
32.3 All ages Creatine monohydrate 100–400 3–6 Valayannopoulos et al. (2012)
CrT All ages l-Arginine 400 3 van de Kamp et al. (2012)
All ages l-Glycine 150 3 Mercimek-Mahmutoglu et al.
(2010)
32.4 Adults Creatine monohydrate 1.5–2 g/day (1–1.5 g/m2/day) 2–3 Heinänen et al. (1999)
Adults l-Lysine 10–15 g/day (5 g/m2/day) 5a Peltola et al. (2000)
Elpeleg and Korman (2001)
OAT Adults α-Aminoisobutyric acid 0.1 5a Valle et al. (1981)
All ages l-Proline 65–488 3 Hayasaka et al. (1985)
a
Spread within the diet as evenly as possible through the 24 h
Dewey KG, Beaton G, Fjeld C, Lönnerdal B, Reeds P (1996) Protein

Beware/Pitfalls requirements of infants and children. Eur J Clin Nutr
50(Suppl1):S119–S147
In CRT, creatine administration might be efficient in
Elpeleg N, Korman SH (2001) Sustained oral lysine supplementation in
females but not in males ornithine delta-aminotransferase deficiency. J Inherit Metab Dis
In OAT, creatine administration corrects skeletal mus- 24:423–424
cle abnormalities, but not the progress of ophthal- Hayasaka S, Saito T, Nakajima H, Takahashi O, Mizuno K, Tada K
(1985) Clinical trials of vitamin B6 and proline supplementation for
mologic abnormalities
gyrate atrophy of the choroid and retina. Br J Ophthalmol
Studies of the long-term efficacy of these approaches 69:283–290
have not been reported, or the results are Heinänen K, Näntö-Salonen K, Komu M et al (1999) Creatine corrects
inconclusive muscle 31P spectrum in gyrate atrophy with hyperornithinaemia.
Eur J Clin Invest 29:1060–1065
Joncquel M, Briand G, Valayannopoulos V et al (2011) Determination
of new reference values for GAA and creatine from a large cohort of
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Almeida LS, Verhoeven NM, Roos B et al (2004) Creatine and guanidi-
Kurosawa Y, deGrauw TJ, Lindquist DM et al (2012) Cyclocreatine
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Heme Synthesis Defects
and Porphyrias 33
Ulrich Stölzel, Thomas Stauch, and Manfred O. Doss
Contents Summary
33.1 Introduction ...................................................................... 541 Porphyrias are metabolic disorders of the heme biosynthesis.
Clinically, they can be differentiated into acute and non-acute
33.2 Nomenclature.................................................................... 542
porphyrias. The symptomatic phase of acute hepatic porphyrias
33.3 Metabolic Pathways ......................................................... 543 is characterized by overproduction of neurotoxic porphyrin pre-
33.4 Signs and Symptoms ........................................................ 544 cursors and porphyrins. Acute intermittent porphyria (AIP),
variegate porphyria (VP), hereditary coproporphyria (HCP),
and Doss porphyria (ALADP) belong to this group of metabolic
33.6 Pathological Deviations and Values ................................ 549 disorders. The clinical presentation of the acute hepatic por-
33.7 Diagnostic Flowcharts...................................................... 550 phyria syndrome includes abdominal, psychiatric, neurological,
33.8 Specimen Collection ......................................................... 550 and cardiovascular symptoms. The diagnosis is based on an at
least tenfold increased urinary excretion of porphobilinogen
33.9 Treatment .......................................................................... 550
(apart from Doss porphyria and lead intoxication). Besides
References .................................................................................... 553 symptomatic therapy with non-porphyrinogenic drugs, electro-
lyte correction, and intensive monitoring, intravenous adminis-
tration of glucose and heme arginate is established for treatment.
Among the non-acute types like porphyria cutanea tarda, eryth-
ropoietic protoporphyria, and congenital erythropoietic por-
phyria, the accumulated porphyrins cause photosensitivity of
the skin and in some cases severe liver damage. X-linked proto-
porphyria (XLPP) represents a new type of protoporphyria, with
5-aminolevulinic acid synthase 2 gain of function leading to
high concentrations of free protoporphyrin IX. The location of
the deficient enzyme within the heme biosynthetic pathway
determines the pattern of the accumulated porphyrins. The
U. Stölzel (*) cDNA of all enzymes of heme biosynthesis have been charac-
Department of Internal Medicine II, terized, and mutations responsible for any of the porphyrias
Gastroenterology, Hepatology, Endocrinology, have been described. Besides light protection, there are different
Metabolic Disorders, Oncology, Saxony Porphyria Center,
therapies depending on the type of non-acute porphyria.
Klinikum Chemnitz gGmbH, Chemnitz 09116, Germany
e-mail: u.stoelzel@skc.de Ultimately, liver transplantation may be considered in therapy-
resistant cases of acute hepatic porphyrias and bone marrow
T. Stauch
Department of Clinical Chemistry and Toxicology, transplantation in severe cases of erythropoietic porphyrias.
German Competence Center for Porphyria Diagnosis
and Consultation, MVZ Labor PD Dr. Volkmann
und Kollegen GbR, Kriegsstr. 99, Karlsruhe 76133, Germany
e-mail: t.stauch@laborvolkmann.de
33.1 Introduction
M.O. Doss
Porphyrias are a heterogeneous group of metabolic disorders,
German Competence Center for Porphyria Diagnosis
and Consultation, Postfach 1220, which are based on genetic deficiencies along the heme syn-
Marburg an der Lahn 35002, Germany thesis (Bonkovsky et al. 2013; Puy et al. 2010) (Fig. 33.1).
542 U. Stölzel et al.
Clinically it can be differentiated between acute and non- In contrast to the acute type, chronic porphyrias such as
acute porphyrias, pathogenetically between hepatic and porphyria cutanea tarda (PCT) as a chronic hepatic porphyria
erythropoietic porphyrias. Apart from the recessive congeni- (CHP) and the erythropoietic porphyria (EPP) display char-
tal erythropoietic porphyria (Morbus Günther) and the two acteristics of chronic diseases (Stölzel and Doss 2009).
types of protoporphyria (EPP,XLPP) all other porphyrias are The diagnosis of clinical symptomatic porphyrias is based
classified as hepatic. on the biochemical analysis, in which each porphyria
The clinical presentation of acute hepatic porphyrias expresses its own specific diagnostic (excretory) pattern.
includes an abdominal – neuropsychiatric – cardiovascular Porphyrias are characterized by a huge molecular genetic
syndrome, whereas chronic hepatic and erythropoietic heterogeneity. They can be caused by a multitude of differ-
porphyrias present with dermatological symptoms due to ent mutations. Genetic analysis of clinically “homozygous”
photodermatosis (Stölzel et al. 1987; Stölzel and Doss porphyrias show that there is quite often a compound het-
2009). erozygosity. Rare homozygous forms can also be found
The symptomatic phase of acute hepatic porphyria is among dominant autosomal hereditary porphyrias. DNA
characterized by excessive accumulation and excretion of analysis can be helpful to screen asymptomatic carriers.
the porphyrin precursors 5-aminolevulinic acid (ALA) and However, since asymptomatic gene carriers of porphyric
porphobilinogen (PBG) as well as porphyrins (Bonkovsky disorder are common in the normal population, genetic test-
et al. 2013; Puy et al. 2010). ing is not recommended for diagnosing acute porphyrias.
Acute porphyrias are biochemically distinguished Recently, modifying genes have been identified that contrib-
according to the localization of the affected enzyme. Among ute to clinically overt disease and moreover disease severity.
acute porphyrias, the acute intermittent porphyria (AIP) is In addition there is a considerable interaction between gene
the most common, followed by the variegate porphyria defect and environmental factors.
(VP), the hereditary coproporphyria (HCP), and a rare Dual porphyrias were diagnosed in rare cases with clini-
recessive acute hepatic porphyria with 5-aminolevulinic cal and biochemical findings of two porphyrias. For exam-
acid-dehydratase deficiency (ALADP, synonym: porphobi- ple, coexistent AIP and PCT in the same individual was
linogen synthase defect porphyria, Doss porphyria), which described.
is biochemically very similar to lead poisoning (Doss et al. Apart from the genetic predisposed erythropoietic and
1979). Therefore, lead poisoning as toxic and toxo-genetic hepatic porphyrias, clinical asymptomatic secondary por-
acute porphyria can be included into the acute types of phyrinurias and porphyrinemias can be detected among a
porphyrias. number of different diseases and dysfunctions.
33.2 Nomenclature
Gene Chromosomal
33.1 X-linked Erythroid XLSA ALAS2 Xp11.21 5-aminolevulinate 300751 All forms
sideroblastic anemia 5-aminolevulinate synthase 2
synthase deficiency
33.2 X-linked Erythroid XLPP ALAS2 Xp11.21 5-aminolevulinate 300752 All forms
protoporphyria 5-aminolevulinate synthase 2
synthase gain of
function
33.3 5-aminolevulinate Doss porphyria ALADP ALAD 9q34 Delta-aminolevulinate 125270 Autosomal
dehydratase dehydratase recessive
porphyria
33.4 Acute intermittent Porphobilinogen AIP HMBS 11q23-11qter Porphobilinogen 176000 Autosomal
porphyria deaminase deficiency deaminase dominant
33.5 Congenital Uroporphyrinogen III CEP UROS 10q25.2-26.3 Uroporphyrinogen III 263700 Classic
erythropoietic synthase deficiency synthase form
porphyria Morbus Günther
33 Heme Synthesis Defects and Porphyrias 543
Gene Chromosomal
33.6 Porphyria cutanea Hepatic PCT/HEP UROD 1p34 Uroporphyrinogen 176100 All forms
tarda types I, II, and uroporphyrinogen decarboxylase
III and decarboxylase
hepatoerythropoietic deficiency chronic
porphyria hepatic porphyria
33.7 Hereditary Coproporphyrinogen HC CPOX 3q12 Coproporphyrinogen 121300 Autosomal
coproporphyria oxidase deficiency oxidase dominant
33.8 Porphyria variegata Protoporphyrinogen PV PPOX 1q22-q23 Protoporphyrinogen 176200 Autosomal
oxidase deficiency oxidase dominant
33.9 Erythropoietic Ferrochelatase EPP FECH 18q21.3 Ferrochelatase 177000 Autosomal
protoporphyria deficiency dominant
Heme Biosynthesis and Localisation of Enzyme Deficiency in Porphyrlas and Lead Poisoning
according to M.O. Doss and U. Stölzel
Hepatocyte Fe2+ Erythropoietic

Heme protoporphyria
k
ac
Glycine + edb Ferrochelatase lead poisoning
succinyl-CoA fe
e
tiv
Protoprophyrin Erythrocytes, Stool

ga
ne
Proto-Oxidase Variegate porphyria

X-linked ALA-Synthase
protoporphyria Protoprophyrinogen Stool
Mitochondrion
Urine δ-Aminolevulinic acid
Copro-Oxidase Hereditary
Doss-porphyria coproporphyria
lead poisoning lead poisoning
ALA-Dehydratase Coproporphyrinogen
Hypertyrosinemia Type I
Cytosol III I Urine, Stool
Urine Porphobilinogen
Pentacarboxyporphyrinogen Urine
Acute
PBG-Deaminase III I
intermittent Urine
Hexacarboxyporphyrinogen
porphyria UR0-111-Synthase III I Urine
Congenital Heptacarboxyporphyrinogen
erythropoietic Uroporphyrinogen
III I
porphyria I III Porphyria cutanea
URO-Decarboxylase tarda (PTC)
Urine Hepatoerythropoietic
porphyria
Fig. 33.1 The enzymes of the heme biosynthesis, their subcellular ALAS-1 is a ubiquitous enzyme. ALAS-2 is an erythroid-specific
localizations, and associated diseases. There are two differently regu- enzyme. X-linked protoporphyria is caused by ALAS-2 gain of func-
lated isoforms of 5-aminolevulinic acid synthase (ALA synthase). tion mutations
Overview on symptomatology Disorder Leading clinical symptoms Onset of symptoms Frequency

33.1 X-linked Anemia
sideroblastic anemia
33.2 X-linked Photosensitivity/liver Childhood Rare
protoporphyria disease/anemia
33.3 ALA dehydratase Abdominal and Childhood Very rare
deficiency neurological symptoms
Lead intoxication Abdominal and Childhood/ Rare
neurological symptoms/ adolescence
anemia/lead blue line
(Burton´s line)
2.1 Hypertyrosinemia Abdominal and Early childhood Rare
type 1a neurological symptoms
33.4 Acute intermittent Abdominal and After puberty 5–10: 100,000
porphyria neurological symptoms/ (female > male)
hyponatremia
33.5 Congenital Photosensitivity: blisters, Neonatal/childhood Very rare
erythropoietic porphyria erosions, mutilations/
anemia/hemolysis/
hepatosplenomegaly/
erythrodontia
33.6 Porphyria cutanea Liver disease/skin fragility/ Late adolescence and 20–50: 100,000
tarda and photosensitivity/ childhood,
hepatoerythropoietic hypertrichosis respectively
porphyria
33.7 Hereditary Abdominal and After puberty 0.5: 100,000
coproporphyria neurological symptoms, (female > male)
photosensitivity
33.8 Porphyria variegata Abdominal and After puberty 1: 100,000
neurological symptoms, (female > male)
photosensitivity
33.9 Erythropoietic Photosensitivity, some Infancy/childhood 0.5: 100,000
protoporphyria patients develop liver
disease
a
The hypertyrosinemia type 1 is not described in detail in this chapter, but the reader is referred to Chap. 2
Enzyme defects along the heme biosynthesis in porphyrias, regulated induction of ALAS1, and major clinical manifestation
Induction of Major Clinical Manifestation

Disorder in heme synthesis Enzyme ALAS1 Neurovisceral Cutaneous Anemia Liver
X-linked sideroblastic anemia (33.1) ALAS2 − ++
XLPP (33.2) ALAS2 − − ++ + −/+
ALADP (Doss) (33.3) ALAD + ++ − −/+ −
Lead intoxication ALAD + + − +a +b
Hypertyrosinemia type 1c (2.1) ALAD + +
AIP (33.4) PBGD + ++ − − −
CEP (Günther) (33.5) UROS − − ++ ++ −/+
PCT/HEP (33.6) UROD − − + −
HCP (33.7) CPO + + −/+ − +
VP (33.8) PPO + + −/+ − −
EPP (33.9) FECH − − + −/+ −/+
a
Lead inhibits also the enzymes CPO and FECH
b
“Lead hepatitis”
c
Hypertyrosinemia is not described in detail in this chapter, but the reader is referred to Chap. 2
Table 33.1 X-linked sideroblastic anemia

Hematological Anemia, microcytic, hypochromic + + +
Dimorphism (RBC) + +
Sideroblasts (BM) ↑ ↑ ↑
Routine laboratory Ferritin (S) ↑ ↑ ↑
Transferrin saturation ↑ ↑
Special laboratory 5-ALA synthase 2 (RBC) ↓ ↓ ↓
Protoporphyrin-Zn (RBC) ↓ ↓ ↓
Table 33.2 X-linked protoporphyria (XLPP)

Dermatological Burning sensation after sun exposure + + +
Photosensitivity ++ ++ ++
Special laboratory 5-ALA synthase 2 (RBC) ↑ ↑ ↑
5-aminolevulinic acid (U) n n n
Porphyrins, all (U) n n na na na
Protoporphyrin feces (or stool) ± ±
Protoporphyrin IX (RBC) ↑↑↑ ↑↑↑ ↑↑↑
Protoporphyrin IX-Zn (RBC) ↑↑ ↑↑ ↑↑
a
In case of extensive and prolonged hepatic exposure to protoporphyrin, there might be increases of coproporphyrin and also the higher carboxyl-
ated porphyrin fractions, such as uro- and heptacarboxyporphyrin in urine
Table 33.3 5-aminolevulinate dehydratase deficiency (Doss porphyria)

System Symptom Neonatala Infancya Childhood Adolescence Adulthood
Cardiovascular Tachycardia + + +
CNS Coma + + +
Hyperesthesia + + +
Motor neuropathy + + +
Seizures + + +
Digestive Abdominal pain + + +
Constipation + + +
Nausea + + +
Vomiting + + +
Musculoskeletal Muscle pain + + +
Renal Hypertension + + +
Routine laboratory Color red-brown w. pink fluorescence (U) + + +
Sodium (P) ↓-n ↓-n ↓-n
Special laboratory Coproporphyrin III (U) ↑ ↑↑↑ ↑↑↑
5-ALA (U) ↑↑ ↑↑↑ ↑↑↑
5-ALA dehydratase (RBC) ↓↓↓ ↓↓↓ ↓↓↓
PBG (U) n-↑ n-↑ n-↑
ALADP deficiency, see also Chap. 2 tyrosinemia type 1 (2.1) and lead poisoning
a
No data available
Table 33.4 Acute intermittent porphyria (AIP)

Cardiovascular Tachycardia + +
CNS Coma ± ±
Hyperesthesia ± ±
Motor neuropathy ± ±
Seizures ± ±
Digestive Abdominal pain + +
Constipation ± ±
Nausea ± ±
Vomiting ± ±
Musculoskeletal Muscle pain + +
Renal Hypertension + +
Routine laboratory Color red-brown w. red fluorescence (U) + +
Sodium (P) ↓-n ↓-n
Special laboratory 5-ALA (U) ↑↑↑ ↑↑↑
PBG (U) ↑↑↑ ↑↑↑
Porphyrins, all (U) ↑↑↑ ↑↑↑
Table 33.5 Congenital erythropoietic porphyria (Guenther disease, CEP)

Dermatological Skin blisters ± + + + +
Skin fragility ± + + + +
Skin scarring and mutilation n + + + +
Hematological Anemia, microcytic, hypochromic, hemolytic ± + + + +
Routine laboratory Color red-brown w. red fluorescence (U) + + + + +
Special laboratory Porphyrins type I isomers (P,U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Porphyrins, all (P,U) ↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 33.6 Porphyria cutanea tarda types I, II, and III and hepatoerythropoietic porphyria (PCT, HEP)
Childhood
System Symptom Neonatal Infancy (HEP) Adolescence Adulthood
Dermatological Skin blisters, Skin fragility + ± +
Hypertrichosis + ± +
Digestive Elevated liver enzymes + ± ±
(ALT > AST, γ-GT), Ferritin (S)
Routine laboratory Color red-brown w. red fluorescence (U) + ± +
Special laboratory 5-ALA/PBG (U) n n n
Porphyrins, all (P,U) ↑↑↑ ↑ ↑↑↑
Porphyrins, uro- and heptacarboxy > copro ↑↑↑ ↑ ↑↑↑
(P, U)
Isocoproporphyrin (F) ↑ n-↑ n-↑
Table 33.7 Hereditary coproporphyria (HCP)

System Symptom Neonatal Infancy Childhood Adolescense Adulthood
CNS Coma ± ±
Hyperesthesia ± ±
Seizures ± ±
Dermatological Blisters ± ±
Constipation ± ±
Nausea ± ±
Vomiting ± ±
Routine laboratory Color red-brown w. red fluorescence (U) + +
Magnesium (P), Sodium (P) n-↓ n-↓
Special laboratory 5-ALA (U) ↑↑ ↑↑
PBG (U) ↑↑ ↑↑
Coproporphyrin III (F) ↑↑↑ ↑↑↑
Table 33.8 Porphyria variegata (VP)

CNS Coma ± ±
Hyperesthesia ± ±
Seizures ± ±
Dermatological Blisters ± ±
Constipation ± ±
Nausea ± ±
Vomiting ± ±
Routine laboratory Color red-brown w. pink fluorescence (U) + +
Sodium (P) ↓ ↓
Special laboratory 5-ALA (U) ↑↑ ↑↑
PBG (U) ↑↑ ↑↑
Coproporphyrin III and ↑↑↑ ↑↑↑
Protoporphyrin IX (F)
Table 33.9 Erythropoietic protoporphyria (EPP)

Dermatological Edema in light-exposed ± + + +
areas
Photosensitivity, acute ± + + +
painful
Digestive Liver dysfunction n n-++ n-++ n-++
Pigment Gallstones + + +
Hematological Anemia ± ± ± ±
Microcytosis ± ± ± ±
Routine laboratory Ferritin (S) ↓-n ↓-n ↓-n ↓-n
Iron (S) ↓-n ↓-n ↓-n ↓-n
Special laboratory Free protoporphyrin (RBC) ↑↑ ↑↑ ↑↑-↑↑↑ ↑↑-↑↑↑
Coproporphyrin (U) n n-↑ n-↑↑ n-↑↑
Urinary excretion of porphyrin precursors and porphyrins

Metabolite 24-h value Related to creatinine
ALA <49 μmol <9.1 μmol/mmol
PBG <7.5 μmol <1.4 μmol/mmol
Uroporphyrin <33 nmol <6 nmol/mmol
Coproporphyrin I <51 nmol <10 nmol/mmol
Coproporphyrin III <102 nmol <20 nmol/mmol
Fecal porphyrin excretion

Metabolite Upper limit of reference
Coproporphyrin I <26 nmol/g dry weight
Coproporphyrin III <11 nmol/g dry weight
Protoporphyrin <80 nmol/g dry weight
Erythrocytic porphyrins
Metabolite
Zinc protoporphyrin IX <385 nmol/l
Protoporphyrin IX free <89 nmol/l
33.6 Pathological Deviations and Values
Biochemical markers in primary and secondary disorders of heme biosynthesis

33.2 33.3 2.1 33.4 33.5 33.6 33.7 33.8 33.9
Sample/metabolite XLPP ALADP Lead-intox HTT1 AIP CEP PCT/HEP HCP VP EPP
Urine
5-ALA n ↑↑ ↑↑ ↑-↑↑ ↑ n n ↑ ↑ n
PBG n n n-↑ n ↑ n n ↑ ↑ n
5-ALA/PBG n ↑ ↑ ↑ ↑ n n ↑ ↑ n
Porphyrin I isomers ↑
Uroporphyrins, total n-↑ n-↑ n-↑ n ↑ ↑↑ ↑↑ ↓-n ↓-n n-↑
Uroporphyrins I – – – ↑↑ ↑↑ – –
Uroporphyrins III ↑ – ↑↑
Heptacarboxyporphyrin n-↑ n-↑ n n ↑ ↑ n-↑ n-↑ n-↑
Hexacarboxyporphyrin n n-↑ n n ↑ ↑ n-↑ n-↑ n
Pentacarboxyporphyrin n n ↑ n-↑ ↑ ↑ n-↑ n-↑ n
Coproporphyrins, total n-↑ ↑↑ ↑↑ ↑↑ ↑↑ ↑↑ n-↑ ↑↑ ↑↑ n-↑
Coproporphyrin I > III >1 +
Coproporphyrin III > I + + + + +
Coproporphyrin III ↑↑ ↑↑ ↑ ↑
Feces
Porphyrin I isomers ↑
Uroporphyrins n n n n n-↑ n
Heptacarboxyporphyrin n n n n-↑ ↑ n
Hexacarboxyporphyrin n n n ↑ ↑ n
Pentacarboxyporphyrin n n n ↑ ↑ n
Isocoproporphyrin – ↑ –
Coproporphyrins n n n-↑ n-↑ ↑↑ ↑ ↑
Coproporphyrin III > I + +
Coproporphyrin I/III >1 <1 <1 >1
Protoporphyrin n-↑ ↑ n n ↑ n-↑↑
Red blood cells
Uroporphyrin ↑
Coproporphyrin ↑
Zinc protoporphyrin ↑↑ ↑ ↑↑ ↑ ↑
Protoporphyrin IX, free ↑ ↑ ↑ ↑ n-↑ ↑↑
Various
PBG deaminase ↓-na n n n n-↓ n
5-ALS-Dehydratase ↓↓ ↓-↓↓ ↓ n n n n n n
Uroporphyrinogen n n n-↓↓ n n n
decarboxylase
Emission maximum of 624–635 615–619 615–618 618–620 615–618 618–620 618–620 624–627 624–635
plasma fluorescence
spectrum on excitation with
405 nm (nm)
a
Normal activity only in case of non-erythroid splice site mutation variant
33.7 Diagnostic Flowcharts
Characteristic clinical presentation of porphyrias

Characteristic clinical presentation (scenario 1–3)*
1 2 3
Patient after puberty and mostly <40 years old Patient often >40 years old Patients in childhood
Abdominal pain, intermittent and colic-like, Easy vulnerability of the skin and blisters on Burning, itching, pain, edemas, and erythema
nausea, vomiting, constipation, and ileus sunlight-exposed skin areas on sunlight-exposed areas
symptoms
Tachycardia, hypertension
Dark-reddish urine Hypertrichosis close to the temples and to the Mild anemia with hypochromia and
Hyponatremia cheekbone as well as periorbital microcytosis
Peripheral motor neuropathy, paresthesias, Associated with
seizures 1. Iron overload
Psychiatric symptoms, behavioral changes, 2. Hepatitis C
hallucinations 3. Alcohol consumption
4. Estrogens (contraceptives,
postmenopausal)
Acute hepatic porphyrias (AIP, HCP,VP, Chronic hepatic porphyria porphyria cutanea Erythropoietic protoporphyria and XLPP****
ALADP) and lead intoxication and tarda and HEP***
hypertyrosinemia type 1**
*Please note: in order to allocate porphyrias, the schema was simplified, and a number of exceptions exists in clinical practice
**Diagnostic tools help to subclassify acute hepatic porphyrias using criteria listed in the Table 33.6
***The diagnosis of a PCT and HEP is based on an excessive increase of uro- and heptacarboxyporphyrin up to a proportion of 80 % of total
urinary porphyrin excretion increase. The excretion of PBG remains normal, while urinary ALS can be slightly elevated. The corresponding con-
centration of plasma uro- and heptacarboxyporphyrin and of stool isocoproporphyrin is increased. Characteristic red porphyrin fluorescence of the
liver biopsy, if exposed to long-wave UV light (Woods light, 366 nm)
****In XLPP up to 30 % of total elevated protoporphyrin is zinc bounded
Test Material Handling

Molecular genetic assays
Genomic DNA EDTA blood or –
isolated DNA
Test Material Handling
cDNA EDTA blood Rapid sample
First-line diagnostic tests: transport (<12 h)
Fluorescence scan 1 ml serum, plasma Light protected and
(heparin, EDTA) cool
5-Aminolevulinic 5 ml urine (spot Light protected and
acid sample or 24 h cool
Porphobilinogen collection)
(Porphyrin 33.9 Treatment
precursors)
Porphyrins 5 ml urine (spot Light protected and
sample or 24 h cool Acute Porphyrias (ALADP; 33.3, AIP; 33.4, HCP; 33.7,
collection, 5 g feces VP; 33.8)
Free protoporphyrin 3 ml EDTA blood or Light protected and General treatment strategies include admitting the patient in
Zinc protoporphyrin heparinized blood cool to an intensive care unit, analgesic therapy, glucose infusion,
Second-line diagnostic tests: correction of electrolyte abnormalities and stopping porphy-
Enzyme activity tests 5 ml heparinized Cool, not frozen rinogenic medication (Anderson et al. 2005). The most impor-
blood or ACD blood
tant therapeutic measures are summarized in the table below.
Therapy of Acute Porphyrias After infusion, preferably into a large vein to reduce local
toxicity, normal saline should be infused for 15 minutes to
1. Withdraw unsafe medication and intensive care unit observation reduce local toxicity. We suggest to dilute the human heme
2. Treatment with glucose or together with heme
arginate in 100 ml of human albumin (4–20 % depending on
Intravenous glucose (300–500 g/day) should be given for mild
symptoms (moderate pain, no paresis or hyponatremia). Glucose
country specific availability) instead of normal saline solu-
combined with insulin is considered to be more effective than tion in order to avoid damage of the veins. Heme as a lyophi-
glucose alone lized powder (Panhematin®; RECORDATI Rare Disease) is
CAVE: Large volumes of 10 % glucose may increase risk for available in the United States where human hemin
hyponatremia (Normosang®) lacks FDA approval. Prophylactic heme
Severe symptoms and neurological complications should be treated application within periods ranging from weekly to monthly
as soon as possible with intravenously given human heme arginate
(Normosang®, Orphan Europe) (3 mg/kg body weight for four is used to prevent repeated clinical manifestations. Frequent
consecutive days). After infusion, preferably into large vein normal treatment with heme leads to iron overload since 100 mg of
saline should be infused for 15 minutes to reduce local toxicity heme contains 8 mg iron. Moreover, heme induces the
We suggested to dilute the human heme arginate in 100 ml of human hemoxygenase 1. Recently heme arginate-associated inflam-
albumin (4–20 % depending on country availability) instead of
matory reactions such as elevation of interleukin 6 and meta-
normal saline solution to avoid damage of the veins. Heme as a
lyophilized powder (Panhematin®; RECORDATI Rare Disease) is bolic stress were observed. Taken together these effects may
available in the United States explain tachyphylaxis with subsequent need to shorten the
3. Monitoring and correction of electrolytes including sodium and interval between therapeutic applications (Bonkovsky et al.
magnesium 2013). Long-term application of heme arginate should be
Analgesic therapy: aspirin, gabapentin, morphine derivatives scrutinized critically.
In case of tachycardia and hypertension: metoprolol, propranolol, Heme therapy was found to be helpful to relieve symp-
valsartan
toms in lead intoxication and ALADP.
In case of unrest and vomiting: lorazepam, phenothiazines,
ondansetron Intravenously applied recombinant PBD reduced PBG
In case of ileus: neostigmine levels but did not show any benefit on clinical endpoints.
In case of respiratory failure: mechanical ventilation Ultimately, in severe and complicated disease, liver trans-
In case of infection: penicillin and derivatives, cephalosporins, plantation can cure the disease (Seth et al. 2007). Complete
imipenem, aminoglycosides normalization of porphyrin metabolism after liver transplan-
In case of paresis: physiotherapy tation proves the fact that acute porphyrias are diseases of the
Monitoring: urinary excretion of ALA, PBG, and porphyrins in AIP liver. Using adenoviral vectors the hepatic enzymes defects
and additionally fecal excreted porphyrins in HCP and VP
were reversed. So far seven patients have been included, and
patients as well as physicians wait for hopefully encouraging
short- and long-term results. Transplantation of hepatocytes
Glucose and more importantly intravenously applied may be an option for the future. Using small interfering RNA
heme downregulate the metabolic dysregulation (Doss and in order to silence hepatic ALAS 1 may be helpful to inhibit
Verspohl 1981). Mild cases can be treated with glucose alone ALA und PBG overproduction.
(Anderson et al. 2005). Neurological symptoms should be In women with acute porphyria, pregnancy is in general
immediately treated with heme in order to reverse and avoid not at risk, although progesterone potently induces liver
progress or persistence. The so-called glucose effect was heme production (Kühnel et al. 2002). However, pregnancy-
recently confirmed by elucidating regulatory links between associated vomiting and subsequent caloric deficiency
hepatic heme biosynthesis – and glucose metabolism via should be normalized promptly by glucose infusion.
nuclear receptors including PGC-1α (Handschin et al. 2005). For those women suffering from frequent attacks related
Glucose combined with insulin is considered to be more to menstrual cycle, gonadotropin-releasing hormone ana-
effective than glucose alone. On one hand, fasting worsens; logues, combined with low-dose estrogen patch to suppress
on the other hand, glucose can help to treat mild clinical menopausal symptoms, can be helpful. Before treatment
manifestations of acute porphyria. If tolerated, diet rich in with LH-RH- agonist is considered, low-dose hormonal
carbohydrates or oral glucose can be added to or replace oral contraceptives should be used under strict control of
intravenous application. Large volumes of 10 % glucose may the urinary excretion of porphyrin precursors and
increase risk for hyponatremia. Intravenously given human porphyrins.
heme (3 mg/kg body weight for four consecutive days, The H2 receptor antagonist cimetidine was successfully
Normosang®, Orphan Europe) increases the hepatic heme used in a few patients. The reports on LH-RH agonists and
storage, improves the function of hepatic hemoproteins, even on cimetidine are non consistent, and more data from
represses ALA synthase, and decreases the overproduction placebo controlled double blinded studies are not available
and consequently urinary excretion of ALA and PBG within so far. The H2 blocker cimetidine can obviously be used
days (Bonkowsky et al. 1971; Bonkovsky 1993). safely on patients with acute porphyria. Parenteral injected
magnesium was reported to be helpful in case of epileptic monitored in order to detect and to effectively treat a renewed
seizures. porphyria manifestation as soon as possible.
Avoiding porphyrinogenic medication, alcohol and physi- Based on experimental work, pharmacological data, and
cal stress is of major importance, as well as a balanced diet clinical reports, safe und unsafe drugs are listed (Table 33.10)
with a high percentage of carbohydrates. The patients should (Stölzel et al. 2009). International guidelines are useful to
be advised not to smoke. Patients with acute porphyria individually handle problems (www.drugs-porphyria.org,
should take special care to avoid infections and other dis- www.porphyria-europe.com).
eases, and the porphyrin precursors ALA and PBG should be
Table 33.10 Safe and probably safe drugs in acute hepatic Table 33.10 (continued)
porphyrias
Gastrointestinal tract Hormones Infection (bacterial)
Allergy, immune Senna Liothyronine (Ceftriaxone)
system, and respiratory Ursodeoxycholic acid Oxytocin (Cefuroxime)
Anesthesia tract Cancer
Tetracosactide (Ciprofloxacin)
(Atropine) Acetylcysteine Anakinra
(Ertapenem)
(Bupivacaine) (Betamethasone) Asparaginase
Gentamicin
(Glycopyrronium) Cromoglicic acid Basiliximab
(Imipenem)
(Isoflurane) Epinephrine Bleomycin
(Meropenem)
(Neostigmine) Etanercept Cisplatin
Methenamine
(Pancuronium) Immunoglobulins Daclizumab
(Moxifloxacin)
(Rocuronium) Infliximab Filgrastim
Netilmicin
Suxamethonium (Loratadine) Lenograstim
(Ofloxacin)
Salbutamol Pegfilgrastim
Phenoxymethylpenicillin
(Triamcinolone) Trastuzumab
Piperacillin
Circulation and heart Coagulation Diuretics Teicoplanin
Adenosine Abciximab Amiloride Tobramycin
(Acetylsalicylic acid) Antithrombin III (Furosemide) Vancomycin
Atenolol Dalteparin (Hydrochlorothiazide)
Infection (fungal) Infection (viral) Metabolism
(Atropine) Dipyridamole
(Amphotericin) (Abacavir) Alendronic acid
Candesartan Enoxaparin
(Caspofungin) Aciclovir (Bezafibrate)
Digoxin Heparin
(Didanosine) Colestyramine
Dobutamine Streptokinase
(Famciclovir) Insulin
Dopamine Urokinase
(Foscarnet) Metformin
Enalapril (Warfarin)
Ganciclovir (Nicotinic acid)
Eprosartan
(Lamivudine) Risedronic acid
Glyceryl trinitrate
(Ribavirin)
Lisinopril
(Tenofovir)
Magnesium
Valaciclovir
Metoprolol
Valganciclovir
Propranolol
Sotalol Neurology Pain Psychiatry
Valsartan (Gabapentin) Acetylsalicylic acid Chloral hydrate
Magnesium Buprenorphine Chlorpromazine
Gastrointestinal tract Hormones Infection (bacterial)
(Vigabatrin) (Fentanyl) Fluoxetine
Cimetidine Epoetin alfa Amikacin
Morphine Fluphenazine
Lactulose Glucagon Amoxicillin-clavulanate
(Paracetamol) (Haloperidol)
(Loperamide) (Goserelin) (Azithromycin)
Pethidine Lithium
(Ondansetron) Insulin Benzylpenicillin
(Lorazepam)
Ranitidine Levothyroxine (Cefotaxime)
Chro Porphyria cutanea trada; Hepatoerythropoietic benefit can obviously only be achieved in the early phase of
Porphyria (PCT/HEP; 33.6) hepatobiliary damage. In order to protect the liver, otherwise
Phlebotomy and treatment with low-dose chloroquine are damaging agents should be completely avoided. Therefore,
therapeutically effective in PCT. The therapeutic aim is the vaccination against hepatitis A und B is generally recom-
elimination of accumulated porphyrins from the liver and mended for patients with EPP. Vitamin D substitution is
other organs. The patients should be advised to avoid known advisible since patients avoid sunlight. Although intrave-
precipitation factors. For patients with advanced liver cirrho- nous heme does not downregulate ALAS 2 in bone marrow,
sis and reduced albumin synthesis, phlebotomy is clinical improvement of liver function was described in
contraindicated. patients with EPP treated with heme. In some cases, red cell
In most cases, the dosage of 125 mg chloroquine twice a transfusions improved EPP. Plasmapheresis and extracorpo-
week leads to metabolic and clinical remission (Koestler and real albumin dialysis (molecular adsorbents recirculation
Wollina 2005). In case of excessive urinary porphyrin excre- system (MARS), Prometheus) can be used for the elimina-
tion of more than 10 mg/day, the treatment can be started tion of protoporphyrin. Liver transplantation is the therapy
with weekly phlebotomy and after 1 month continued with of choice in EPP patients with cholestatic liver cirrhosis.
chloroquine for several months up to 1 year. Recently for the first time, bone marrow transplantation has
The treatment can be abandoned when the urinary por- been reported in EPP (Wahlin et al. 2007).
phyrin excretion stabilizes in a subclinical level (<0.3 mg/ Iron supplementation improves protoporphyrin overload,
day). liver damage, and anemia in a new type of X-linked
A complete normalization of the porphyrinuria is rare, protoporphyria.
and in particular, it cannot be expected in case of a genetic Congenital Erythropoietic Porphyria (CEP; 33.5)
disposed form. Since a low to a moderate increased porphy- Therapeutic measures have been dissatisfactory up to now.
rinuria does not cause clinical symptoms, further treatment is Light protection, blood transfusion in case of a severe ane-
not recommended. Steatosis of liver cells and siderosis can mia, and splenectomy can be considered.
regress after phlebotomy as well as after a therapy with chlo- Bone marrow transplantation was performed in some
roquine (Stölzel et al. 2003). Chronic hepatitis C is associ- cases. Two children were reported to be disease-free for 3 and
ated with PCT (Stölzel et al. 1995). After iron depletion, 2 years posttransplantation. In a mouse model, it has recently
treatment of chronic hepatitis C should be initiated with been demonstrated to repair mutations of UROS in bone mar-
combination of protease inhibitor, pegylated, interferon- row cells with the help of viral vectors. Advance in gene
alpha-2a, and ribavirin (Bonkovsky et al. 2013). therapy could be a real hope for affected patients in the future.
Erythropoietic Protoporphyria (EPP; 33.9)
The light sensitivity is symptomatically treated with beta-
carotene with a dosage of 50 up to 200 (300) mg/day. References
Monitoring of serum carotene levels is recommended
(11–15 μmol/L). EPP is characterized by sensitivity to Anderson KE, Bloomer JR, Bonkovsky HL, Kushner JP, Pierach CA,
Pimstone NR, Desnick RJ (2005) Recommendations for the diagno-
visible light; conventional sunscreens that protect against
sis and treatment of the acute porphyrias. Ann Intern Med
ultraviolet radiation (particularly UVB) are usually not 142:439–450
effective. Reflectant sunscreens based on titanium dioxide Bonkovsky HL (1993) Advances in understanding and treating ‘the lit-
or zinc oxide that cover both UVA, UVB, and visible light tle imitator’, acute porphyria. Gastroenterology 105:590–594
Bonkovsky HL, Guo JT, Hou W, Li T, Narang T, Thapar M (2013)
to a degree will be more effective. Afamelanotide is a new
Porphyrin and heme metabolism and the porphyrias. Compr Physiol
synthetic analogue of naturally occurring alpha-melano- 3:365–401
cyte-stimulating hormone (α-MSH). After binding to the Bonkowsky HL, Tschudy DP, Collins A, Doherty J, Bossenmaier I,
melanocortin-1 receptor, it induces physiologic melanogen- Cardinal R, Watson CJ (1971) Repression of the overproduction of
porphyrin precursors in acute intermittent porphyria by intravenous
esis independent of the potentially harmful effects of UV
infusions of hematin. Proc Natl Acad Sci U S A 68:2725–2729
light. This leads to increased skin pigmentation and photo- Doss M, Verspohl F (1981) The “glucose effect” in acute hepatic por-
protection. In contrast to acute porphyrias, for patients with phyrias and in experimental porphyria. Klin Wochenschr
EPP, there is in general no restriction for porphyrinogenic 59:727–735
Doss M, von Tiepermann R, Schneider J, Schmid H (1979) New type of
substances or medication.
hepatic porphyria with porphobilinogen synthase defect and inter-
Colestyramine and ursodeoxycholic acid are used for the mittent acute clinical manifestation. Klin Wochenschr
treatment of EPP-induced liver disease. Colestyramine and 57:1123–1127
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Spiegelman BM (2005) Nutritional regulation of hepatic heme bio-
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synthesis and porphyria through PGC-1alpha. Cell 122:505–515
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enhance the biliary elimination of protoporphyrin. Clinical Opin Pharmacother 6:377–383
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Disorders of Bile Acid Synthesis
and Biliary Transport 34
Hugh A. Lemonde, Paul Gissen, and Peter T. Clayton

34.1 Introduction.................................................................... 555
The primary bile acids produced in man are the taurine and
34.2 Nomenclature ................................................................. 557
glycine conjugates of cholic and chenodeoxycholic acid.
34.3 Metabolic Pathways ....................................................... 558 They are synthesised in the liver from cholesterol via numer-
34.4 Signs and Symptoms ...................................................... 559 ous modifications of the sterol nucleus and side chain.
A number of physiological roles have traditionally been
34.5 Reference Values ............................................................ 565
assigned to bile acids. These include the end products of cho-
34.6 Pathological Values ........................................................ 567 lesterol catabolism that account for approximately 90 % of
34.7 Diagnostic Flow Charts ................................................. 569 cholesterol excretion, the facilitation of bile flow by activat-
34.8 Specimen Collection....................................................... 571 ing bile solute pumps (via nuclear receptors such as the
farnesoid X receptor) and driving the osmotic excretion of
34.9 Prenatal Diagnosis and DNA Testing ........................... 571
water into the bile canaliculi and as biological detergents
34.10 Treatment........................................................................ 571 within the gut lumen enabling the absorption of fat-soluble
References .................................................................................... 576 compounds. More recently a wider role of bile acids is
becoming apparent as hormone regulators of metabolism,
with postulated effects on diverse processes such as carbohy-
drate and fat metabolism and the regulation of energy expen-
diture by thyroid hormone (Hylemon et al. 2009).
The conversion of cholesterol to the primary bile acids can
occur via different pathways, which are summarised in
Fig. 34.1. The two commonly described pathways are the
‘neutral’ pathway, starting with 7α-hydroxylation and subse-
quent nuclear modification prior to side-chain modification,
and the ‘acidic’ pathway that starts with side-chain modifica-
tion. The majority of the enzymes involved in bile acid synthe-
sis are shared between these pathways, with the notable
exception being those involved in 7α-hydroxylation (choles-
terol 7α-hydroxylase [neutral pathway] and the oxysterol
7α-hydroxylase [‘acidic pathway’]) and 12α-hydroxylation
(Russell 2003). The neutral pathway is considered the most
significant in human adult life, whereas the acidic pathway
seems to play a more prominent role in early life. Alternative
pathways that involve initial 24/25 hydroxylation are described
H.A. Lemonde • P. Gissen • P.T. Clayton (*) and are postulated to play an important role in cholesterol
Institute of Child Health, University College London metabolism in brain and lung. The common pathway for side-
and Great Ormond Street Hospital,
chain modification is completed via peroxisomal β-oxidation.
30 Guilford Street, London WC1N 1EH, UK
e-mail: hlemonde@doctors.org.uk; p.gissen@ich.ucl.ac.uk; Inborn errors of bile acid metabolism can present in a
p.clayton@ich.ucl.ac.uk variety of ways. Most of the errors that effect transformation
556 H.A. Lemonde et al.
of the steroid nucleus (disorders 34.1–34.4) produce abnor- either enzyme leads to the production of unconjugated bile
mal metabolites that are not substrates for active transport acids, which are substrates for active transport into bile thus
into bile and generally present with failure of bile flow (cho- driving bile flow, but are inefficient biological detergents.
lestasis) and malabsorption of fat and fat-soluble vitamins. Thus, patients with BAAT deficiency can present with ste-
Patients may present acutely with the effects of vitamin defi- atorrhoea and fat-soluble vitamin deficiency with mild or
ciency such as haemorrhage or hypocalcaemic seizures or absent jaundice.
more insidiously with prolonged neonatal jaundice, steator- The clinical presentation of bile acid-CoA ligase defi-
rhoea and rickets. Transaminases are significantly raised ciency is unclear as the only symptomatic child described
with a conjugated hyperbilirubinaemia, but gamma-glutamyl had a possible concurrent diagnosis of TPN-related cholesta-
transpeptidase (γGT) is characteristically normal. Notable sis and mutations in the bile salt export pump gene
exceptions include cholesterol 7α-hydroxylase deficiency, (Chong et al. 2012).
which presents with statin-resistant hyperlipidaemia and The simplest way to screen a symptomatic individual for
gallstones in later life. This presumably reflects the ability of inborn errors of bile acid synthesis is to analyse urine by a
the ‘acidic’ pathway to compensate for the deficit in the soft ionisation (usually electrospray) mass spectrometry
‘neutral’ pathway. Also, oxysterol 7α-hydroxylase defi- technique (ESI-MS). Other methods are available for some
ciency has been described in patients with neonatal cholesta- of the individual disorders. Prompt and accurate diagnosis of
sis, but also in patients presenting with hereditary spastic inborn errors of bile acid metabolism is paramount, as many
paraplegia (HSP). The acidic pathway has been implicated in of these disorders are amenable to simple oral therapy if
cholesterol catabolism in the central nervous system, and instituted before the onset of significant hepatic damage –
significantly elevated levels of 27-hydroxycholesterol have treatment is further discussed in Sect. 34.10.
been found in the CSF of patients with HSP. Defects in bile transporters are commonly identified in
Inborn errors that effect the modification of the choles- patients with inherited forms of cholestasis (e.g. conjugated
terol side chain (disorders 34.5, 34.6 and the peroxisomal hyperbilirubinaemia). The more severe protein defects mani-
disorders) produce cholanoids (bile acids and alcohols) that, fest in early life, whilst milder abnormalities may become
to some extent, can drive bile flow. Symptoms appear to be apparent only when the transport processes are under stress
caused mainly by accumulation of intermediates proximal to such as in pregnancy or after specific drug ingestion. Defects
the site of the block and conversion of these intermediates to of at least six proteins that facilitate transport of different bile
a product which is deposited in various tissues of the body constituents are known (Fig. 34.2). Bile constituents that
(Verrips et al. 2000). The deposition of cholestanol and cho- include bile acids, bilirubin, cholesterol, phospholipids and
lesterol in CTX can lead to the formation of cataracts, mental other products of metabolism are secreted into biliary cana-
retardation in the first decade and neurological regression liculi in an energy-dependent manner. Transmembrane trans-
with dementia and motor dysfunction in later life. The lipid porter proteins mediate the secretory function of hepatocytes
deposition also produces tendon xanthomata and premature and biliary epithelial cells.
atherosclerosis. CTX can, however, also cause cholestasis in Progressive familial intrahepatic cholestasis (PFIC) is
infancy. In several of the peroxisomal disorders, there is characterised by persistent conjugated hyperbilirubinaemia
impaired bile acid synthesis and some impairment of liver and in PFIC1 and PFIC2, low or normal serum γGT values
function, although other pathways are often impaired and and progressive liver damage that requires liver transplan-
neurological disease usually predominates. These disorders tation in childhood. Patients with PFIC1/2 have reduced
are considered elsewhere. α-Methylacyl-CoA racemase defi- concentrations of primary bile acids in bile. Mutations in
ciency (34.6) can present with neonatal cholestasis (Setchell ATP8B1 (PFIC1) and ABCB11 (PFIC2) were found to be
et al. 2003) and is considered in this chapter, but is also men- the cause of disease in the majority of patients, although
tioned in Chap. 24 on peroxisomal diseases. there is still a proportion of patients without mutations in
In the defects of bile acid amidation (34.7, 34.8), the either of the genes. There are some clinical differences in
primary bile acids are synthesised but the final step of con- the presentation of ATP8B1 and ABCB11 disease; most
jugation with glycine or taurine is defective. Bile acids syn- notably patients with ATP8B1 have a range of extrahepatic
thesised de novo are produced as cholyl-CoA esters and manifestations such as diarrhoea and episodes of pancreati-
require only the peroxisomal enzyme bile acid-CoA/amino tis. Patients with ABCB11 mutations are at increased risk of
acid N-acyltransferase (BAAT) to produce conjugated prod- hepatobiliary malignancy. In addition to the classical PFIC,
ucts (Pellicoro et al. 2007). Bile acids that are deconjugated some mutations in both ATP8B1 and ABCB11 cause the
by intestinal flora and returned to the liver via enterohepatic so-called benign recurrent intrahepatic cholestasis (BRIC),
circulation require bile acid-CoA ligase to form the bile acid- when cholestasis can completely resolve between relapses.
CoA esters prior to reconjugation by BAAT. Deficiency of It is now clear that a spectrum of severity between PFIC and
34 Disorders of Bile Acid Synthesis and Biliary Transport 557
BRIC exists. ABCB4 (PFIC3) deficiency results in impaired and SLCO1B3 genes, encoding organic anion-transporting
excretion of phosphatidylcholine (PC) into bile and can polypeptides OATP1B1 and OATP1B3, have to be present
result in a spectrum of cholestatic disorders including neona- simultaneously to cause Rotor syndrome.
tal hepatitis and biliary cirrhosis with patients typically hav-
ing high serum γGT values.
Mutations in ABCC2 result in Dubin-Johnson syndrome, 34.2 Nomenclature
a condition characterised by recurrent episodes of choles-
tatic jaundice without other clinical/biochemical indications Disorders of peroxisome biogenesis and defects of peroxi-
of hepatobiliary injury. Liver biopsy shows intrahepato- somal β-oxidation (such as d-bifunctional protein deficiency)
cyte deposits of dark pigment but no other abnormalities. affect bile acid synthesis but are considered elsewhere.
Rotor syndrome is phenotypically very similar to Dubin- α-Methylacyl-CoA racemase is located both in peroxisomes
Johnson syndrome and manifests with mild cholestatic and mitochondria; it is considered here because in common
jaundice that can be detected in the neonatal period or in with other disorders of bile acid synthesis, it can present with
childhood. It differs from Dubin-Johnson syndrome in that neonatal cholestatic jaundice without neurological abnor-
no intrahepatocyte pigment deposits can be found in Rotor malities. An identical argument can be made for BAAT defi-
syndrome patients and there is a delayed plasma clearance ciency. Both disorders can also be found in Chap. 24 on
of unconjugated bromsulphthalein. Mutations in SLCO1B1 peroxisomal disease.

No. Disorder Alternative name Abbreviation symbollocalisation Affected protein no. Subtype
34.1 3β-Hydroxy-Δ5-C27- 3β-Dehydrogenase C27-3β-HSD HSD3B7 16p11.2 3β-Hydroxy-Δ5-C27- 607764 All forms
steroid dehydrogenase/ deficiency steroid dehydrogenase/
isomerase deficiency isomerase
34.2 Δ4-3-Oxosteroid-5β- 5β-Reductase SRD5B1 AKR1D1 7q32-q33 Delta(4)-3-oxosteroid- 604741 All forms
reductase deficiency deficiency 5β-reductase
34.3 Oxysterol 7α-hydroxylase Spastic Paraplegia CYP7B1 CYP7B1 8q12.3 Oxysterol 603711 All forms
deficiency 5A 7α-hydroxylase
34.4 Cholesterol CYP7A1 CYP7A1 8q12.1 Cholesterol 118455 All forms
7α-hydroxylase deficiency 7α-hydroxylase
34.5 Sterol 27-hydroxylase Cerebrotendinous CTX CYP27A1 2q35 Sterol 27-hydroxylase 213700 All forms
deficiency Xanthomatosis
34.6 α-Methylacyl-CoA AMACR deficiency AMACR AMACR 5q13.2 α-Methylacyl-CoA 604489 All forms
racemase deficiency racemase
34.7 Bile acid-CoA: amino acid Bile acid amidation BAAT BAAT 9q31.1 Bile acid-CoA: amino 602938 All forms
N-acyltransferase defect acid N-acyltransferase
deficiency
34.8 Bile acid-CoA ligase BA CoA LD SLC27A5 19q13.43 Bile acid-CoA ligase 603314 All forms
deficiency
34.9 ATP8B1 deficiency Progressive familial PFIC1 ATP8B1 18q21.31 ATP8B1 (type 4 211600 All forms
intrahepatic P-type ATPase)
cholestasis type 1
34.10 ABCB11 deficiency Progressive familial PFIC 2 ABCB11 2q31.1 ABCB11 (bile salt 603201 All forms
intrahepatic export pump [BSEP])
cholestasis type 2
34.11 ABCB4 deficiency Progressive familial ABCB4 ABCB4 7q21.12 ABCB4 (MDR3) 602347 All forms
intrahepatic
cholestasis type 3
34.12 OATP1B1 and OATP1B3 Rotor syndrome OATP1B1 SLCO1B1 12p12.2-p12.1 OATP1B1 and 237450, All forms
disease and and and 12p12.2 OATP1B3 604843,
OATP1B3 SLCO1B3 605495
34.13 ABCC2 deficiency Dubin-Johnson ABCC2 or ABCC2 10q24.2 ABCC2 (cMOAT) 237500, All forms
syndrome DJS 601107
Alternative pathways such as those

initiated by 24/25 hydroxylation
HO
Cholesterol
34.5 34.4
CH2OH
‘Neutral’ pathway
HO OH
HO
27-Hydroxycholesterol 7α-Hydroxycholesterol
2 34.1 OH
1
COOH
O OH
O OH
HO
7α-Hydroxy-4-cholesten-3-one 7α,12α-Dihydroxy-4-cholesten-3-one
3β-Hydroxy-5-cholestenoic acid
34.2 34.2
OH
34.3
COOH
O OH O OH
HO OH 7α-Hydroxy-5β-cholestan-3-one 7α,12α-Dihydroxy-5β-cholestan-3-one
3β,7α-Dihydroxy-5-cholestenoic acid
3 3 OH
HO OH HO OH
34.1
5β-Cholestan-3α,7α-diol 5β-Cholestan-3α,7α,12α-triol
34.2 34.5
34.5
34.6 5β-Cholestan-3α,7α,27-triol
4 34.6
25R-3α,7α-dihydroxy-5β-Cholestanoyl- CoA
‘Acidic’ 34.6
pathway
25S-3α,7α-dihydroxy-5β-Cholestanoyl- CoA
Peroxisomal β-oxidation
OH
O O
C C
OH OH
HO OH HO OH
34.8
Chenodeoxycholyl CoA 34.8 Cholyl CoA
34.7 34.7
Taurochenodeoxycholic acid Taurocholic acid

Chenodeoxycholate Cholate
Glycochenodeoxycholic acid 5 5 Glycocholic acid
Fig. 34.1 Simplified scheme of the known pathways for the synthesis deficiencies are numbered: (1) 12α-hydroxylase, (2) sterol 27-hydroxy-
of bile acids from cholesterol, including enterohepatic recycling. lase catalyses both 27-hdroxylation and further oxidation to a carbox-
The ‘neutral’ pathway starts with conversion of cholesterol to ylic acid, (3) 3α-hydroxysteroid dehydrogenase, (4) very long chain
7α-hydroxycholesterol and the ‘acidic’ pathway with formation of acyl-CoA synthase (VLCS)/di-/trihydroxycholestanoic acid-CoA
27-hydroxycholesterol. Defined inborn errors are highlighted with ligase and (5) intraluminal bacterial deconjugation
crossed arrows. Enzymatic steps thus far not associated with known
HEPATOCYTE
Cholesterol
OATP1B1/3 PS PC
ATP8B1
ABCB4
BLOOD
ABCB11 ABCC2
Bile acids
Na Bilirubin Glucuronide
NTCP CANALICULUS
Fig. 34.2 Diagram illustrating the transporters involved in the genera- results in impaired excretion of phosphatidylcholine (PC) into bile and
tion of bile. ATP8B1, a member of the type 4 subfamily of P-type can result in a spectrum of cholestatic disorders including neonatal hep-
ATPases, is present in the apical membrane of many epithelial cells atitis and biliary cirrhosis. As PC is a major component of the mixed
including hepatocytes and enterocytes. ATP8B1 appears to translocate micelles into which salts of bile acids are emulsified, deficiency of
aminophospholipids such as phosphatidylserine (PS) from the outer to ABCB4 leads to hepatocyte and cholangiocyte damage by bile acids.
the inner leaflet of the plasma membrane bilayer but also has other func- The protein encoded by ABCC2 (ABCC2 or MRP2) is a member of the
tions such as facilitating polarised expression of other apical membrane multidrug resistance protein subfamily. It exports anionic glutathione
proteins. ABCB11 encodes the bile salt export pump (BSEP), which is and glucuronate conjugates (including bilirubin) from hepatocytes into
responsible for the ATP-dependent transport of taurine and glycine- canaliculi. ABCC2 is expressed on apical membranes of many epithelial
conjugated primary BA across the canalicular membrane. BSEP is a cells including hepatocytes, proximal renal tubules, gallbladder, small
member of the P-glycoprotein/multidrug resistance (MDR/ABCB) sub- intestine, bronchi and placenta. OATP1B1 and OATP1B3 localise to the
family of transporters. ABCB4, or multidrug resistance protein 3 sinusoidal membrane of hepatocytes and mediate sodium-independent
(MDR3), is a P-glycoprotein that translocates phospholipids from intercellular uptake of highly diverse compounds that include bilirubin gluc-
nal to external leaflet of the canalicular membrane. ABCB4 deficiency uronide, bile acids, steroid and thyroid hormones, and numerous drugs
Table 34.1 3β-Hydroxy-Δ5-C27-steroid dehydrogenase/isomerase deficiency

Cardiovascular Vit K responsive bleeding ± ± ±
CNS Seizures/tetany, hypocalcaemic ± ±
Dermatological Itching N/A ± ±
Digestive Cholestasis + ± ± ±
Giant cell hepatitis + +
Jaundice + ± ± ±
Liver cirrhosis ± ± ±
Steatorrhoea + + +
Musculoskeletal Rickets + + +
Routine laboratory Albumin (P) n n n n-↓
Alkaline phosphatase (P) ↑ ↑ ↑ n-↑
ASAT/ALAT (P) ↑ ↑ ↑ ↑
Bilirubin conjugated (P) ↑ n-↑ n-↑
Calcium (P) ↓-n ↓-n ↓-n
Cholesterol (S) ↓-n ↓-n ↓-n
Gamma-glutamyl transpeptidase (GGT) (P) n n n n
Prothrombin ratio n-↑ n-↑ n-↑ n-↑
(continued)

Special laboratory 25-Hydroxy-vitamin D (P) ↓ ↓ ↓
7α-Hydroxycholesterol + + + +
dehydrogenase (FB)
Chenodeoxycholic acid (P) ↓ ↓ ↓
HSD3B7 gene sequencing + + + +
LSI-MS for sulphate/glycine-conjugated + + + +
di/trihydroxy-5-cholenoic acids (m/z
469,485,526,542)-negative ion mode
Periportal inflammation + + +
Bridging fibrosis + +
Sulphated 3β,7α-dihydroxy and 3β,7α, + + +
12α-trihydroxy-5-cholenoic acids (P,U)
Vitamin A (P) ↓-n ↓-n ↓-n
Vitamin E (P) ↓ ↓ ↓
a
Described modes of presentation include, in order of frequency, neonatal cholestasis, rickets and symptomatic hypocalcaemia (Clayton et al.
1987; Subramaniam et al. 2010). Two asymptomatic patients have been described that were identified on family screening of affected siblings. It
is not clear whether these children would have progressed to disease state as they started on prophylactic treatment (Subramaniam et al. 2010)
b
Some patients have been described with an ESI-MS pattern that is predominated by non-sulphated metabolites including glycine-conjugated/
unconjugated compounds (m/z 405,446,462). This may reflect different endogenous sulphation of compounds, although certain experimental
conditions may promote the formation of doubly charged sulphate conjugates (m/z 234, 263), and these may not be detected if the low mass/charge
end of the urine spectrum is not included (Clayton et al. 2011)
Table 34.2 Δ4-3-Oxosteroid-5β-reductase deficiency

Digestive Ascites, oedema ± ±
Cholestasis + +
Hepatosplenomegaly ± ±
Jaundice + ±
Others Pseudoacinar transformation + ±
Routine laboratory Albumin (P) ↓-n ↓-n
Alkaline phosphatase (P) n-↑ n-↑
ASAT/ALAT (P) ↑ ↑
Bilirubin conjugated (P) ↑ n-↑
Cholesterol (S) ↓-n ↓-n
Gamma-glutamyl transpeptidase (GGT) (P) n-↑ n-↑
Prothrombin ratio ↑ n-↑
Special laboratory 7α-Hydroxy-3-oxo and 7α, + + + +
12α-dihydroxy-3-oxo-4-cholenoic
acids (U)
AKR1D1 gene + +
Allocholic and allochenodeoxycholic + + + +
acid (P)
Cortisol metabolites increased 5αH/5βH + + +
ratio (U)
Periportal and lobular inflammation ± ±
Small caniculae, few microvilli + ±
There is no biochemical test currently available that can confidently distinguish between a patient that has reduced activity of 5β-reductase as a
result of mutations in AKR1D1 and a patient that has reduced activity of the enzyme (resulting in excretion of 3-oxo-Δ4 bile acids) as a non-specific
consequence of severe liver damage in infancy/childhood (Clayton 1994). Only patients with a proven genetic abnormality of 5β-reductase have
been included in this chapter (Lemonde et al. 2003; Clayton 2011)
Table 34.3 Oxysterol 7α-hydroxylase deficiency

Cardiovascular Vit K responsive bleeding ±
CNS Ataxia, cerebellar ±
Progressive spastic paraplegia ± ± ±
Digestive Bile duct proliferation +
Cholestasis +
Giant cell hepatitis +
Hepatosplenomegaly +
Jaundice ± +
Eye Cataract ±
Optic atrophy ±
Routine laboratory Alkaline phosphatase (P) ↑
ASAT/ALAT (P) ↑
Bridging fibrosis +
Bilirubin-total/direct (P) ↑
Cholesterol (S) n
Gamma-glutamyl transpeptidase (GGT) (P) n
Glucose (P) ↓
Special laboratory 27-Hydroxycholesterol and ↑ ↑ ↑ ↑
3β-hydroxy-5-cholestenoic acid (P)
3β-Hydroxy-5-cholenoic acids (U) + ↑ ↑ ↑
CYP7B1 gene + + + + +
Periportal inflammation +
Vitamin E (P) ↓
White matter abnormalities +
Oxysterol 7α-hydroxylase deficiency can present with neonatal cholestasis (Setchell et al. 1998), but, interestingly, mutations of CYP7B1 have
been found in a number of cases of hereditary spastic paraplegia (SPG5 [Spastic paraplegia group 5]), who present later in life with a neurological
presentation without clinical history of liver disease (Arnoldi et al. 2012)
Table 34.4 Cholesterol 7α-hydroxylase deficiency

Cardiovascular Statin-resistant hyperlipidaemia ±
Digestive Gallstones ±
Only three patients (siblings) have been described with a deficiency in this enzyme (Pullinger et al. 2002). They were identified in the sixth decade
of life, amongst patients in a hyperlipidaemia clinic, by screening the CYP7A1 gene in patients who had significant statin-resistant hyperlipidaemia
and gallstone disease. Total production of bile acids was shown to be reduced, but no hepatic or neurological pathology was identified. One sibling
was found to have significant atherosclerotic disease
Table 34.5 Sterol 27-hydroxylase deficiency

Cardiovascular Ischaemic heart disease, ± ±
angina/myocardial infarct
CNS Ataxia ± ±
Demyelination and lipid deposition on MRI ±
Expressive dysphasia ± ±
Neuropathy ±
Parkinsonism ±
Regression, dementia ± ±
Seizures ± ± ±
Spastic paresis/pyramidal signs ± ± ±
Spinal cord, myelopathy ±
Dermatological Xanthomas, tendon ± ±
(continued)

Digestive Diarrhoea ± ±
Gallstones ±
Jaundice ± ± ±
Pigment granules in liver biopsy ±
Endocrine Adrenal insufficiency ±
Hypo or hyperthyroidism ±
Pituitary/hypothalamic dysfunction ±
Musculoskeletal Osteoporosis ±
Pes cavus ±
Respiratory Failure ±
Routine laboratory Cholesterol (S) n-↑ n-↑
EEG: +/− evoked potentials, abnormal ± ± ±
Special laboratory 25-Hydroxy-Vitamin D (P) ↓-n ↓-n
27-Hydroxylase (FB) ↓ ↓ ↓ ↓ ↓
Cholestane pentol glucuronides (U) + + + + +
Cholestanol (P) ↑ ↑ ↑ ↑ ↑
CYP27A1 gene + + + + +
(Verrips et al. 2000; Clayton et al. 2002)
Table 34.6 α-Methylacyl-CoA racemase deficiency

Cardiovascular Vit K responsive bleeding ±
CNS Cognitive decline ±
Developmental delay ±
Epilepsy +/− encephalopathy ± ± ±
Headache ± ±
Neuropathy ±
Spastic paraparesis ±
Tremor ±
Digestive Cholestasis ↑
Gallstones ± ± ±
Giant cell hepatitis +
Jaundice ± ±
Endocrine Hypothyroidism ±
Eye Pigmentary retinopathy ± ±
Vision, progressive loss ± ±
Routine laboratory Gamma-glutamyl transpeptidase (GGT) (P) ↑
Transaminases (P) ↑
Special laboratory (25R)-3α,7α,12α-trihydroxy-5β-cholestan- ↑ ↑ ↑ ↑ ↑
26-oic acid (THCA) (P)
25-Hydroxy-vitamin D (P) ↓
AMACR gene + + + + +
Taurotetra-[tri/penta]-hydroxycholestanoic ↑ ↑ ↑ ↑ ↑
acids (U) [m/z 556,572,588 on LSI-MS]
Vitamin E (P) ↓
a
Five patients have been described with an AMACR deficiency. Four presented in later life with neurological symptoms (Ferdinandusse et al. 2000;
Thompson et al. 2008) and one in the neonatal period with cholestasis (Setchell et al. 2003)
Table 34.7 Bile acid-CoA/amino acid N-acyltransferase deficiency

Dermatological Itching ± ± ±
Digestive Failure to thrive ± ±
Steatorrhoea ± ±
Musculoskeletal Rickets ± ±
Transaminases (P) n-↑ n-↑
Special laboratory BAAT gene + + + + +
LSI-MS showing unamidated bile acids ↑ ↑
(m/z 407,471,487,567,583)-negative ion
mode
Vitamin A (P) ↓-n ↓-n ↓-n
Vitamin D (P) ↓-n ↓-n ↓-n
Vitamin E (P) ↓-n ↓-n ↓-n
Table 34.8 Bile acid-CoA ligase deficiency

Digestive Bile duct proliferation ±
Jaundice ±
Liver cirrhosis ±
Other Bridging fibrosis ±
Routine laboratory Bilirubin conjugated (P) n-↑
Gamma-glutamyl transpeptidase (GGT) (P) n
Transaminases (P) n-↑
Special laboratory LSI-MS showing unamidated bile acids ↑ ↑
(m/z 407,471,487,567,583)-negative ion
mode
SLC27A5 gene + + + + +
Only two siblings have been described with bile acid-CoA ligase deficiency. One has remained asymptomatic, whilst the other with cholestatic
liver disease was identified after a premature birth and prolonged period of parenteral feeding and also had BSEP mutations. It is unclear to what
extent the clinical findings were as a result of the bile acid-CoA ligase deficiency, TPN-related cholestasis, BSEP deficiency or indeed a combina-
tion of all three
Table 34.9 ATP8B1 deficiency

Digestive Bland centrilobular cholestasis ± ±
Diarrhoea ± ± ±
Liver fibrosis ± ±
Pancreatitis ± ± ±
Steatorrhoea ± ± ±
Ear Deafness, sensorineural ± ± ±
Genitourinary Delayed puberty ±
Respiratory Pneumonia ± ±
Routine laboratory Bile acids (enzyme assay) (P) ↑ ↑ ↑ ↑
Chloride (sweat) ↑ ↑
Fat-soluble vitamin deficiency ± ± ± ±
Special laboratory ATP8B1 gene + + + +
Bile acids, normal ↑ ↑ ↑ ↑
Coarse granular bile on electron ± ±
microscopy
PFIC1 may be suspected because of the extrahepatic manifestations (not often seen in PFIC2). The extrahepatic manifestations do not resolve after
liver transplantation. At presentation patients with PFIC1 have higher alkaline phosphatase, but lower plasma bile acids, transaminases and albu-
min than PFIC2 patients (Pawlikowska et al. 2010)
Table 34.10 ABCB11 deficiency

Dermatological Itching + + +
Gallstones + + +
Jaundice + + + +
Liver cirrhosis ± ± ±
Risk of malignant hepatoma + +
Routine laboratory Bile acids (enzyme assay) (P) ↑↑ ↑↑ ↑↑ ↑↑
Chloride (sweat) ↑ ↑
Special laboratory ABCB11 gene + + + +
Filamentous bile on electron microscopy ± ± ± ±
Patients with BSEP deficiency (PFIC2) are more likely to have gallstones and portal hypertension than PFIC1 patients (Pawlikowska et al. 2010).
Patients with the D482G mutation have less rapidly progressive PFIC than those with other mutations
Table 34.11 ABCB4 deficiency

Digestive Gallstones ± ± ±
Jaundice, cholestatic + + + +
Liver dysfunction ± ± ± ±
Liver fibrosis/biliary cirrhosis ± ± + +
Haematological Splenomegaly ± ±
Routine laboratory Bile acids (enzyme assay) (P) ↑ ↑ ↑ ↑
Gamma-glutamyl transpeptidase (GGT) (P) ↑ ↑ ↑ ↑
Special laboratory ABCB4 gene + + + +
Portal inflammation with ductular ± ± ± ±
proliferation
Table 34.12 OATP1B1 and OATP1B3 disease

Digestive Jaundice + + + +
Routine laboratory Bilirubin conjugated (P) ↑ ↑ ↑ ↑
LFTs not including conjugated bilirubin n n n n
Special laboratory Clearance of unconjugated ↓ ↓ ↓ ↓
bromsulphthalein (P)
Coproporphyrin I (U) ↑↑ ↑↑ ↑↑ ↑↑
Normal liver biopsy, no pigment + + + +
SLCO1B1 and SLCO1B3 genes + + + +
(Van de Steeg et al. 2012)
Table 34.13 ABCC2 deficiency

Digestive Jaundice, episodic + + + +
Routine laboratory Bilirubin conjugated (P) n-↑ n-↑ n-↑ n-↑
LFTs not including conjugated bilirubin n n n n
Special laboratory ABCC2 gene + + + +
Clearance of unconjugated n n n n
bromsulphthalein (P)
Coproporphyrin I (U) n n n n
Pigment granules in liver biopsy + + + +
It is thought that the mild phenotype of ABCC2 deficiency is explained by upregulation of other transporters such as ABCC3
Carriers for Mutations in Hepatocyte Transporter premature birth and stillbirth. Cholestasis associated with the
Proteins administration of oral contraceptives is also more frequent in
Carriers of ATP8B1, ABCB11 and ABCB4 mutations are carriers of ABCB11 mutations. Low-phospholipid-associated
predisposed to intrahepatic cholestasis of pregnancy (ICP), cholelithiasis (LPAC) is a form of gallstone disease that
which is a third-trimester disorder that is characterised by occurs in younger patients which is associated with ABCB4
pruritus and elevated serum concentrations of bile acids (van mutations, recurs after cholecystectomy and appears to
der Woerd et al. 2010). It seems that the subtype with low respond well to UDCA. Mutations in ABCB11 and ABCB4
serum γGT values occurs in ABCB11 and ATP8B1 mutation have been associated with drug-induced cholestasis (DIC)
carriers, whilst carriers of ABCB4 typically have high γGT following administration of amoxicillin, clavulanic acid and
values. ICP is associated with fetal disease, fetal distress, risperidone.
Table 34.14 Determination of urinary cholanoid (bile acid and bile alcohol) profile by ESI-MS (electrospray ionisation mass spectrometry)
Ion Identity Normal Cholestasis
234 [M-2H]2− 3β,7α-Dihydroxy-5-cholenoic acid (SO4) − −
263 [M-2H]2− 3β,7α-Dihydroxy-5-cholenoic acid (Gly,SO4) or isomer − ±
391 Dihydroxycholanoic acids (e.g. chenodeoxycholic acid) − ±
405 3β,7α,12α-Trihydroxy-5-cholenoic acid − −
407 Trihydroxy-cholanoic acids (e.g. cholic acid) ± ±
444 7α-Hydroxy-3-oxo-4-cholenoic acid (Gly) − ±
446 3β,7α-Dihydroxy-5-cholenoic acid (Gly) − −
448 Dihydroxycholanoic acids (e.g. chenodeoxycholic acid) (Gly) ± ±/↑
453 3β-Hydroxy-5-cholenoic acid (SO4) − ±
460 7α,12α-Dihydroxy-3-oxo-4-cholenoic acid (Gly) − ±
462 3β,7α,12α-Trihydroxy-5-cholenoic acid (Gly) − −
464 Trihydroxy-cholanoic acids (e.g. cholic acid) (Gly) ± ±/↑
469 3β,7α-Dihydroxy-5-cholenoic acid (SO4) − −
Also steroid sulphatea ± −
471 Dihydroxycholanoic acid(s) (SO4) − ±c
480 3β-Hydroxy-5-cholenoic acid (Tau) − ±
Tetrahydroxy-cholanoic acids (Gly) ± ±
485 3β,7α,12α-Trihydroxy-5-cholenoic acid (SO4) − −
494 7α-Hydroxy-3-oxo-4-cholenoic acid (Tau) − ±
498 Dihydroxycholanoic acids (e.g. chenodeoxycholic acid) (Tau) ± ±/↑
510 7α,12α-Dihydroxy-3-oxo-4-cholenoic acid (Tau), 3β-Hydroxy-5-cholenoic acid (Gly,SO4) ± ±
514 Trihydroxy-cholanoic acids (e.g. cholic acid) (Tau) ± ±/↑
526 3β,7α-Dihydroxy-5-cholenoic acid (Gly,SO4) − ±
528 Dihydroxycholanoic acids (e.g. chenodeoxycholic acid) (Gly,SO4) ± ±/↑
530 Tetrahydroxy-cholanoic acids (Tau) ± ±/↑
542 3β,7α,12α-Tihydroxy-5-cholenoic acid (Gly,SO4) − −
567 Dihydroxycholanoic acids (Gluc) − ±c
572 Tetrahydroxycholestanoic acids (Tau) − −
583 Trihydroxy-cholanoic acids (Gluc) – ±c
611 Cholestanetetrols (Gluc) ± ±
613 27-Nor-cholestanepentol(SO4) (Gluc) ±/↑b ±
627 Cholestanepentols (Gluc) ±/↑ ±
643 Cholestanehexols (Gluc) ±/↑ ±
651 Dihydroxycholanoic acids (Gly,GlcNAc) − ±
701 Dihydroxycholanoic acids (Tau, GlcNAc) − ±
Cholanoid conjugates are abbreviated; Gly glycine conjugate, Tau taurine conjugate, SO4 sulphate, Gluc glucuronide, GlcNAc N-acetylglucosamine
conjugate
a
An ion of mass/charge ratio 469 can occur in urine samples from patients who do not have 3β-HSDH deficiency; the other ions which are charac-
teristic of 3β-HSDH (485,526,542) are not present, and GC-MS fails to show increased excretion of 3β,7α-dihydroxy-5-cholenoic acid
b
In normal neonates and young infants m/z 613 or 627 can be the base peak. However, m/z 627 is much less intense than in patients with CTX
c
These peaks are prominent in patients with cholestasis who are receiving ursodeoxycholic acid; spectra from patients on ursodeoxycholic acid treatment
are very difficult to interpret
The mass spectrometer scans negative ions over the range peak identities, e.g. m/z 74 for glycine conjugates, m/z 80 for
m/z 350–700, or sometimes 200–800, and draws a spectrum taurine conjugates, m/z 97 for sulphates and m/z 85 for
with the largest peak as 100 % intensity. In Table 34.14, glucuronides.
indicates that the peak is not detectable above the back- The values below refer to total plasma concentration
ground, ± indicates undetectable to 20 % of the largest peak determined by GC-MS analysis following hydrolysis of
and ↑ indicates 20–100 % intensity of largest peak. Daughter cholestanol esters.
ions generated in a collision cell can be used to help confirm
Table 34.15 Urinary cholanoid excretions determined by GC-MS Table 34.16 Plasma cholanoid concentrations
μmol/mmol % Total bile Concentration (μmol/l)
Cholanoid creatinine acid excretion Plasma cholanoid Normal Cholestatic
3β,7α-diOH-5-cholenoic acida <0.1 <2 % Chenodeoxycholic acid 0.22–12.4 25–359
3β,7α,12α-triOH-5-cholenoic acida <0.1 <2 % Cholic acid 0.05–4.55 7–317
7αOH-3-oxo-4-cholenoic acidb Traced d
Other cholanoids of diagnostic <0.25 <0.25
7α,12α-triOH-3-oxo-4-cholenoic acidb Traced d
significancea
Cholestanepentolsc <1.0 The data below refers to results obtained by GC-MS following hydroly-
a
Following mild solvolysis and enzymatic hydrolysis of glycine sis of glycine and taurine conjugates with cholylglycine hydrolase.
conjugates Normal plasma bile acid concentrations are higher in the postprandial
b
Following enzymatic hydrolysis of glycine and taurine conjugates period (½–3 h following a fat-containing meal) than in the fasting state.
c
Following hydrolysis with glucuronidase They are also higher in the neonatal period than later in infancy. For the
d
Amount of urinary bile acids in healthy neonates, including 3-oxo-Δ4 purposes of diagnosis of inborn errors, these differences are not of great
bile acids, has been shown to be elevated in the first month of life – importance and have not been included in the reference data
a
7α,12α diOH-3-oxo-cholenoic acid <13.0 pmol/mmol creatinine 3β,7α-Dihydroxy-5-cholenoic, 3β,7α,12α-trihydroxy-5-cholenoic,
(<30 % total bile acid excretion) and 7α-OH-3-oxo-cholenoic acid 7α-hydroxy-3-oxo-4-cholenoic, 7α,12α-diOH-3-oxo-4-cholenoic,
<0.4 pmol/mmol creatinine (<1 % total bile acid excretion). See Kimura allocholic, allochenodeoxycholic and 3α,7α, 12α-trihydroxy-5β-
et al. (1999) cholestanoic acid (THCA)
Table 34.17 Plasma cholestanol concentrations

Age <15 years >15 years Cholestatic
Cholestanol (P) (μmol/l) 1–9 4–18 4–50
Urinary cholanoid (bile acid and alcohol) profile by ESI-MS

34.5 Sterol 34.6 AMACR 32.8 Bile
34.1 34.2 27-hydroxylase 34.4 Oxysterol (+other peroxisome 34.7 acid-CoA
Ion Identity 3β-HSDH 5β-reductasea (CTX) 7α-hydroxylase disorders) BAAT ligase
2−
234 [M-2H] 3β,7α-Dihydroxy-5- ±/↑
cholenoic acid (SO4)
263 [M-2H]2− 3β,7α-Dihydroxy-5- ±/↑
cholenoic acid (Gly,SO4)
405 3β,7α,12α-Trihydroxy-5- ±/↑
cholenoic acid
407 Unconjugated cholic acid ↑ ↑
444 7α-Hydroxy-3-oxo-4- ↑
cholenoic acid (Gly)
446 3β,7α-Dihydroxy-5-cholenoic ±/↑
acid (Gly)
448 Dihydroxycholanoic acids −/± ± – –
(e.g. chenodeoxycholic acid)
(Gly)
453 3β-Hydroxy-5-cholenoic acid ↑
(SO4)
460 7α,12α-Dihydroxy-3-oxo-4- ↑
462 3β,7α,12α-Trihydroxy-5- ±/↑
464 Trihydroxy-cholanoic acids −/± ± – –
(e.g. cholic acid) (Gly)
469 3β,7α-Dihydroxy-5-cholenoic ↑
acid (SO4)
?also steroid sulphate
471 Chenodeoxycholic acid (SO4) ±/↑ ↑
480 3β-Hydroxy-5-cholenoic acid ↑
(Tau)
485 3β,7α,12α-Trihydroxy-5- ↑
cholenoic acid (SO4)
487 Cholic acid (SO4) ±/↑ ↑
494 7α-Hydroxy-3-oxo-4- ↑
cholenoic acid (Tau)
498 Dihydroxycholanoic acids −/± ± – –
(Tau)
510 7α,12α-Dihydroxy-3-oxo-4- ↑ ↑
cholenoic acid (Tau) or
3β-Hydroxy-5-cholenoic acid
(Gly, SO4)
514 Trihydroxy-cholanoic acids −/± ± – –
(e.g. cholic acid) (Tau)
526 3β,7α-Dihydroxy-5-cholenoic ↑
acid (Gly, SO4)
528 Dihydroxycholanoic acids ±
(Gly, SO4)
530 Tetrahydroxy-cholanoic acids ±
(Tau)
542 3β,7α,12α-Trihydroxy-5- ↑
cholenoic acid (Gly, SO4)
567 Chenodeoxycholic acid ±/↑ ↑
(Gluc)
(continued)
34.5 Sterol 34.6 AMACR 32.8 Bile

34.1 34.2 27-hydroxylase 34.4 Oxysterol (+other peroxisome 34.7 acid-CoA
Ion Identity 3β-HSDH 5β-reductasea (CTX) 7α-hydroxylase disorders) BAAT ligase
572 Tetrahydroxycholestanoic ±/↑b
acids (Tau)
583 Cholic acid (Gluc) ± ±/↑ ↑
611 Cholestane terols (Gluc) ↑
613 27-Nor-cholestanepentol(s) ±/↑
(Gluc)
627 Cholestanepentols (Gluc) ↑
643 Cholestanehexols (Gluc) ±/↑
a
In patients considered to have a genetic deficiency of 5β-reductase, the ESI-MS spectrum shows peaks due to 3-oxo-Δ4 bile acids that are four to
five times larger than those due to the corresponding saturated bile acids (i.e. 444 > 448,460 > 464,494 > 498, 514 > 510). The saturated bile acids
may not be detectable above the background. By contrast an ESI-MS spectrum that shows 3-oxo-Δ4 peaks of similar size to the corresponding satu-
rated bile acid (i.e. 444 ≡ 448) indicates severe hepatocyte damage due to something other than genetic 5β-reductase deficiency. In these patients
the excretion of 3-oxo-Δ4 bile acids will disappear when the hepatocyte function improves
b
In patients with peroxisomal biogenesis, defects over the age of 18 months the ESI-MS analysis may give a negative result
Further analysis of plasma cholanoid profile by GC-MS

32.5 Sterol 32.6
34.1 34.2 27-hydroxylase 32.3 Oxysterol α-Methylacyl- Peroxisomal
Plasma cholanoids (μmol/l) 3β- HSDH 5β-reductase (CTX) 7α-hydroxylase CoA racemase disorders
Chenodeoxycholic acid ↓(<0.1) ↑/↓(0–25) ↓ n n n/↑
Cholic acid n/↓(0–4.5) n/↓ n/↓ n n n/↓
3β,7α-Dihydroxy-5-cholenoica ↑↑(1–80)
3β,7α,12α-Trihydroxy-5-cholenoica ↑(0.05–30)
3β,7α-Dihydroxy-5-cholestenoic ↑↑(3–40)
7α-Hydroxy-3-oxo-4-cholenoic ↑(0.8–10.0)b
7α,12α-Dihydroxy-3-oxo-4-cholenoic ↑(0.3–2.0)b
Allochenodeoxycholic acid ↑(0.5–10.0)c
Allocholic acid ↑(0.5–8.0)c
3β-Hydroxy-5-cholenoic acid ↑↑(1.4–87)
3β-Hydroxy-5-cholestenoic acid ↑↑(7–24)
3α,7α-Dihydroxy-5β-cholestan-26-oic acid ↑↑(0.1–0.8)g ↑↑(0.5–12)
[DHCA]
3α,7α,12α-Trihydroxy-5β-cholestan-26-oic ↑↑(0.7–23.8)g ↑↑(0.8–30)
acid [THCA]
5β-Cholestane-3α,7α,12α,25-tetrol ↑↑
d f e
Other compounds detected in cholanoid
profile
Cholestanol ↑(19–400)
a
These compounds are present almost entirely as sulphates in the plasma of patients with 3β-HSDH deficiency and will not be detected unless
plasma is subjected to a mild solvolysis procedure
b
3-Oxo-Δ4-bile acids constitute >10 % of total plasma bile acids
c
Allo-bile acids constitute >20 % of total
d
7α-Hydroxycholesterol, 7α-hydroxy-cholest-4-en-3-one, 7α,12α-dihydroxy-cholest-4-en-3-one, 5β-cholestane-3α,7α,12α-triol
e
C29-dicarboxylic acid and tetrahydroxycholestanoic acids in disorders of peroxisome biogenesis. Varanic acid in disorders of d-bifunctional
protein and thiolase
f
27-Hydroxycholesterol
g
These compounds are [25R]-DHCA/THCA acid stereoisomers
GC-MS analysis is used to confirm the identities of ions in 25 pentols. Tandem mass spectrometry (e.g. liquid secondary
the ESI-MS urine spectrum and to show that the excretion of ion tandem MS[LSI-MS/MS]) is an alternative method to
abnormal cholanoids is >20 times normal. In the case of GC-MS and can rapidly confirm the identity of a number of
5β-reductase deficiency GC-MS analysis should show that diagnostic ions that are found in the LSI-MS/ESI-MS spec-
3-oxo-Δ4 bile acids account for >70 % of the total urinary bile trum of urine. These include sulphated and taurine-conjugated
acid excretion. In the case of sterol 27-hydroxylase deficiency abnormal metabolites such as those observed in 3β-HSDH
(CTX), GC-MS analysis should indicate that the major cho- deficiency (34.1), 5β-reductase deficiency (34.2), oxysterol
lestane pentols in the urine are 3, 7, 12, 22, 25 and 3, 7, 12, 23, 7α-hydroxylase deficiency (34.3) and peroxisomal disorders.
34.7 Diagnostic Flow Charts Immunostaining can detect the absence of proteins
involved in bile acid metabolism or biliary secretion in a
There are also some syndromes which include low γGT liver biopsy.
cholestasis as well as extrahepatic features, e.g. the arthro-
gryposis, renal dysfunction and cholestasis (ARC) and
Åagenaes (cholestasis lymphoedema) syndromes.
Low γGT Cholestasis
Urine bile acid profile
High excretion of normal bile

Abnormal bile acids detected
acids (m/z 448, 464, 498, 514)
Progressive familial Inborn error of bile acid

intrahepatic cholestasis synthesis
Liver Bx plus DNA analyses

Refer to Figure 34.7.2
ATP8B1 ABCB11
Fig. 34.3 Diagnostic flowchart (PFIC 1) (PFIC 2)
for low γGT cholestasis
Urine -ve ion ESI- or

LSI- MS Analysis of urine by tandem MS
to confirm the presence of 3β,
7α-diOH- and 3β,7α,12α- Y
triOH-5-cholenoic acids
Major ions m/z 469, 3β-Hydroxy-Δ5-C27-steroid
485, 526 and 542 Y Analysis of urine/plasma by dehydrogenase deficiency
GC-MS following mild solvolysis
and deconjugation: ↑↑3β,7α-
Y
diOH- and 3β,7α,12α-triOH-5-
N cholenoic acid
Normal saturated bile acids (m/

Probably secondary to
Major ions m/z 444, z 448, 464, 498, 514) present in
Y severe liver damage
460, 494 and 510 Y large amounts in urine
N
GC-MS analysis following cholyl- N
glycine hydrolase deconjugation:
N 7α-OH-3-oxo- and 7α,12α- Possible primary
diOH-3-oxo-4-cholenoic acids deficiency of Δ4-3-
>70% total bile acid excretion Y
oxosteroid 5β-reductase
Analysis of urine by tandem MS

Oxysterol 7α -hydroxylase
Major ions m/z to confirm the presence of 3β-
Y deficiency
453,480 and 510 Y OH-5-cholenoic acids
Analysis of urine/plasma by GC-

MS following mild solvolysis and
deconjugation:↑↑3β-OH-5-
N cholenoic and cholestenoic Y
(plasma only) acids
GC-MS analysis following

glucuronidase deconjugation:
Sterol 27-hydroxylase
Major ion m/z 627 ↑↑ Cholestanepentol
deficiency (CTX)
Y glucuronides (esp. 3,7,12,23,25 Y
N and 3,7,12,22,25 pentols)
Analysis of urine by tandem MS

Major ion m/z 572 to confirm the presence of C27 Peroxisomal disorder
Y Y
bile acids
GC-MS analysis of plasma

following cholylglycine
hydrolase deconjugation:
↑C27 bile acids e.g. 3α,7α,12α- Y
triOH-cholestanoic acid (THCA)
N
HPLC analysis of plasma
following cholylglycine α-Methyl-acyl-CoA
hydrolase deconjugation racemase deficiency
showing [25R]-THCA Y (AMACR deficiency)
stereoisomer
GC-MS analysis of plasma +/-

Fig. 34.4 Diagnostic flow chart Major ion m/z 407 Amidation defect
deconjugation confirms
for disorders of bile acid Y Y
unconjugated bile acids
synthesis

Test Conditions Material Handling Pitfalls
Urine cholanoid profile No bile acid therapy Urine ≥0.5 ml Ambient temp. 12 h, Drugs and radiographic contrast
by LSI-MS or ESI-MS 4 °C for 48 h, −20 °C media may produce large peaks
for >6 months on the LSI-MS spectrum
Cholanoids from urine adsorbed Ambient temp. 48 h
on C18 cartridge (volume of urine
and creatinine recorded)
Further analysis of urinary No bile acid therapy Urine ≥2.0 ml (can be sent on C18 As above
cholanoids by GC-MS cartridge as above)
Plasma bile acids No bile acid therapy Plasma/serum 0.5–2.0 ml Ambient temp. 12 h,
4 °C for 48 h, −20 °C
for >6 months
Plasma cholestanol No bile acid therapy Plasma/serum 0.2–1.0 ml As above
34.9 Prenatal Diagnosis and DNA Testing cholestanol and the urinary excretion of bile alcohols and
can reverse the patient’s neurological disability, with clear-
Routine specific DNA testing for the inborn errors of bile ing of the dementia, improved orientation, a rise in intelli-
acid metabolism and biliary secretion is not generally avail- gence quotient and enhanced strength and independence
able and is conducted on a research basis only. The advent of (Berginer et al. 1984).
whole-exome sequencing will potentially increase the num- Patients with PFIC usually require treatment for fat-
ber of patients identified with these disorders. Prenatal diag- soluble vitamin deficiency. They often have severe pruri-
nosis can be undertaken using DNA analysis. tus which is difficult to treat but may respond to drugs
such as rifampicin, cholestyramine and ursodeoxycholic
acid. Rifampicin has been shown to inhibit the expression
34.10 Treatment of the enzyme autotaxin which is involved in the origin of
pruritus (Kremer et al. 2012). No treatments are yet avail-
Summary able that can correct the underlying transport defect.
Treatment for most of the synthesis disorders is simple and, Ursodeoxycholic acid promotes bile flow and can proba-
if instituted before the onset of significant hepatic damage, bly protect biliary epithelial cells and hepatocytes from
effective. Liver function tests and biopsy appearances can be damage during cholestasis. It is of proven benefit in
normalised by treatment with oral chenodeoxycholic acid ABCB4 deficiency, but, in ATP8B1 and ABCB11 defi-
and/or cholic acid. These bile acids enter the enterohepatic ciencies, there are conflicting reports of any benefit. Some
circulation and drive bile flow and also inhibit the endoge- patients with ATP8B1 and ABCB11 deficiencies have
nous production of abnormal bile acids. In some, however, benefitted from partial external biliary diversion or ileal
liver damage progresses requiring liver transplantation. exclusion surgery. However, in all three PFIC disorders,
Treatment of the consequences of acute vitamin K deficiency liver damage is progressive and most children ultimately
is important and can be immediately life saving, especially in require liver transplantation.
the case of hypocalcaemic seizures and haemorrhage sec- The Dubin-Johnson and Rotor syndromes generally pro-
ondary to vitamin K deficiency. Not only can the treatment duce mild (and often intermittent) conjugated hyperbilirubi-
of cholestasis be successful, but the treatment can improve naemia which has no important consequences and no
neurological sequelae in these conditions. In CTX, treatment progressive liver disease. So treatment is not required except
with chenodeoxycholic acid reduces the rate of synthesis of for severe neonatal cases.
Initial Management of the Cholestatic Infant with a Bile Acid Synthesis Defect
Cholestatic infant
3β-HSD / 5β-reductase / AMACR

deficiency / CTX / Oxysterol 7α-hydroxylase
deficiencies
Prolonged clotting times
i/v vitamin K daily,

check clotting after 48h
INR <1.5 or
INR 1.5– 4 INR > 4
Oxysterol 7α-hydroxyase
deficiency
Chenodeoxycholic acid Cholic acid

Fig. 34.5 Initial management of
15 mg/kg/d 15 mg/kg/da * Start transplant work-up
the cholestatic infant with a bile
Ketovite tabs iii daily Ketovite tabs iii daily
acid synthesis defect. aor cholic
Ketovite elixir 5ml dailyb Ketovite elixir 5ml dailyb
acid 7.5 mg/kg/day plus
chenodeoxycholic acid 7.5 mg/
kg/day. brapid healing of rickets
may require more vitamin D (as *
1,25-dihydroxycholecalciferol)
and a calcium supplement * No improvement in LFT’s and clotting
Treatment of the Consequences of Fat-Soluble Vitamin treatment with a vitamin supplement containing all four fat-
Malabsorption soluble vitamins, e.g. Ketovite tabs, iii daily (provides 15 mg
Once coagulopathy has been corrected and rickets healed, α-tocopheryl acetate and 1.5 mg acetomenaphthone), plus
bile acid replacement therapy should be adequate to prevent Ketovite elixir 5 ml daily (provides 2,500 units of vitamin A
any manifestations of fat-soluble vitamin malabsorption; and 400 units ergocalciferol).
however, it is wise to continue for ca.3 months after starting
Treatment for Medication Dose Route Target

Vitamin K deficient Vitamin K (phytomenadione) 1 mg daily i/v slowlya Normal clotting times
bleeding
Hypocalcaemia 10 % Calcium gluconate (plus 0.1–0.3 ml/kg/dose i/v slowly Normal ionised calcium
(fits, tetany) 1,25-dihydroxycholecacliferol, see below)
Rickets 1,25-Dihydroxycholecalciferol 0.25–1.00 μg/day Oral Normal calcium, healing
of rickets. Avoidance of
hypercalcaemia
Vitamin E Alpha-tocopherol acetate or water-soluble/ 50 mg Oral Normal plasma vitamin E
deficiency miscible vit E preparation
Vitamin A Vitamin A or water miscible analogue 2,500 Units e.g. Ketovite Oral Normal plasma vitamin A
deficiency elixir 5 ml daily
Basic defect Chenodeoxycholic acid and/or cholic acid See individual disorders Oral Normal liver function tests,
etc.
a
Immediate treatment of a coagulopathy caused by vitamin K deficiency may be life saving but intravenous phytomenadione can cause anaphylaxis
Specific Treatment Strategies
3β-Hydroxysteroid-Δ5-C27-steroid dehydrogenase deficiency

Basic defect Chenodeoxycholic acid Initial dose 12–18 mg/kg/day Oral Normalisation of
After 2 months 9–12 mg/kg/day Oral liver function tests,
or prevention of
Chenodeoxycholic acid 7 mg/kg/day Oral fat-soluble vitamin
plus malabsorption
Cholic acid 7 mg/kg/day Oral
Consequences of fat-soluble vitamin malabsorption See above See above See above See above
Δ4-3-Oxosteroid-5β-reductase deficiency
Basic defect Chenodeoxycholic acid 8 mg/kg/day Oral Normal liver function tests
plus
Cholic acid 8 mg/kg/day Oral
or
Cholic acid alone 15 mg/kg/day Oral
Failure to respond to bile acid replacement Liver transplant
(Clayton et al. 1996; Clayton 2011)
Patients with 5β-reductase deficiency usually present with patients in whom excretion of 3-oxo-Δ4 bile acids is second-
cholestatic liver disease in infancy. It is important to distin- ary to severe liver damage caused by another genetic disorder
guish patients with mutations in the 5β-reductase gene from (e.g. tyrosinaemia) or an acquired disorder (e.g. hepatitis B).
Oxysterol-7α-hydroxylase deficiency
Basic defect Chenodeoxycholic acid 11 mg/kg/day Oral Normal liver function tests
Failure to respond to bile acid replacement Liver transplant
The three patients described initially were treated with therapy (Chong et al. 2010). If the liver disease does not
ursodeoxycholic acid +/− liver transplantation, either as a respond to bile acid treatment, cholestasis will persist until
result of initial severe liver disease or lack of chenodeoxy- liver transplantation can be undertaken (Mizuochi et al.
cholic acid available for therapy. One recently described 2011). Therefore, these children require forms of the fat-
patient, postulated to have a deficiency of oxysterol soluble vitamins that are water soluble or can be given by
7α-hydroxylase, responded well to chenodeoxycholic acid injection.
Cholesterol 7α-hydroxylase Treatment for Medication Dose Route Targets

deficiency
Hyperlipidaemia Atorvastatin 40–80 mg/day (adult dose) Oral Normal cholesterol
plus 4–7 g/day (adult dose) Oral
Niacin
Sterol 27-hydroxlase deficiency (CTX)

Cholestasis in infancy Cholic acid 7–15 mg/kg/day Oral Normal liver function tests
Consequences of fat-soluble See above See above See above See above
vitamin malabsorption
Dementia, neurological Chenodeoxycholic acid 750 mg daily (adult dose) Oral Reduced bile alcohol excretion, plasma
disease, xanthomata cholestanol, improved neurology
α-Methylacyl-CoA racemase deficiency

Cholestasis in infancy Cholic acid 15 mg/kg/day Oral Normal liver function tests
Consequences of fat-soluble vitamin See above See above See above See above
malabsorption
Pristanic acid accumulation ?contributing Low phytanate diet Prevention/amelioration of neurological
to neurological disease disease
Bile acid-CoA: amino acid N-acyltransferase deficiency (BAAT deficiency)

Cholestasis in infancy Ursodeoxycholic acida 30 mg/kg/day (3 doses) Oral Normal liver function
Glycocholic acidb 15 mg/kg/day Oral tests and jaundice
a
One UK patient has been described who showed a good response to ursodeoxycholic acid
b
An initial report of 6 US patients suggests glycocholic therapy improves vitamin D absorption and helps normalise growth (Heubi et al. 2009)
Bile acid-Co ligase deficiency

Cholestasis in infancy Ursodeoxycholic acid 30 mg/kg/day (3 doses) Oral Normal liver function tests
The requirement for treatment in this condition is unclear. ursodeoxycholic acid for presumed TPN-related cholestasis,
Only two siblings have been described with a bile acid- and only after resolution of cholestasis and termination of
CoA ligase deficiency, one of whom was asymptomatic and therapy was a diagnosis made. The patient remains well and
did not require treatment. Another sibling was treated with has not required further therapy.
ATP8B1 disease (PFIC1)

Pruritus Cholestyramine 1 month–1 year: 1–9 g/day Orala Relief of pruritus
1–6 years: 2–18 g/day Monitor for folate deficiency;
6–12 years: 4–24 g/day supplement if necessary
12–18 years: 4–36 g/day
Rifampicin 5 mg/kg/day Relief of pruritus
Monitor LFT’s and stop if serious rise
in transaminase
Pruritus and progression Ursodeoxycholic acid 30 mg/kg/day (3 doses) Oral Relief of pruritus
of liver disease Partial external biliary diversion
Cirrhosis /liver failure Liver transplantationb
a
For lower doses, a single dose in the morning can be used; higher doses need three times daily regimen
b
Liver transplantation does not cure the extrahepatic problems
ABCB11 disease (PFIC2)

Pruritus Cholestyramine See above See above See above
Rifampicin
Pruritus and progression of liver disease Ursodeoxycholic acid
Partial external biliary diversion
Cirrhosis /liver failure Liver transplantation
ABCB4 disease (PFIC3)

Progressive damage to bile ducts, cirrhosis Ursodeoxycholic acid 30 mg/kg/day (3 doses) Oral
Cirrhosis, liver failure Liver transplantation
OATPB1 and OATPB3 disease (Rotor syndrome)

Jaundice Outside neonatal period not necessary. Severe neonatal jaundice; see below, Table for ABCC2
disease
Risk of drug toxicity Avoid drugs unless there is a strong indicationa
a
The OATPB1 and OATPB3 organic anion transporters are involved in the hepatic uptake and metabolism of many drugs. Defective metabolism
could lead to increased toxicity so all drugs should be used with caution and only when there is a strong indication
ABCC2 disease (Dubin-Johnson syndrome)

Jaundice Outside neonatal period not necessary
Severe neonatal jaundice Phenobarbitone 5 mg/kg/day in 2 doses Oral Reduction in plasma conjugated bilirubin
Ursodeoxycholic acid 30 mg/kg/day (3 doses) Oral Reduction in plasma conjugated bilirubin
(Kimura et al. 1991; Regev et al. 2002)
Alternative Therapies/Experiment Treatment

Cerebrotendinous xanthomatosis
Mode of treatment Dose Route Comment
Lovastatin (mevinolin) [HMG-CoA reductase inhibitor] 6.25 mg twice daily (adult dose) Oral Insufficient data to assess efficacy
Low density lipoprotein apheresis Insufficient data to assess efficacy
(Lewis et al. 1983)
Lemonde HA, Custard EJ, Bouquet J et al (2003) Mutations in SRD5B1,

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Berginer VM, Salen G, Shefer S (1984) Long-term treatment of cere- Mizuochi T, Kimura A, Suzuki M et al (2011) Successful heterozygous
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Chong CPK, Mills PB, McClean P et al (2012) Bile acid-CoA ligase Pellicoro A, Van den Heuvel FAJ, Geuken M et al (2007) Human and
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Clayton PT (1994) Delta 4-3-oxosteroid 5 beta-reductase deficiency port. Hepatology 45:340–348
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Disorders of Polyol Metabolism
35
Mirjam M.C. Wamelink, Vassili Valayannopoulos,
and Cornelis Jakobs
Contents Short Summary

35.1 Introduction ..................................................................... 577 Three inborn errors in the pentose phosphate pathway (PPP)
are known.
35.2 Nomenclature ................................................................... 578
Glucose-6-phosphate dehydrogenase deficiency is a defect
35.3 Metabolic Pathways ........................................................ 579 in the first, irreversible step of the pathway. As a consequence
35.4 Signs and Symptoms ....................................................... 580 NADPH production is decreased, making erythrocytes vul-
nerable to oxidative stress. Drug- and fava bean-induced hae-
molytic anaemia is the main presenting symptom of this
defect. G6PD deficiency is an X-linked disorder. As this is a
35.7 Diagnostic Flow Charts................................................... 582 haematological disorder, it is not discussed further.
35.8 Specimen Collection ........................................................ 582 Deficiency of ribose-5-phosphate isomerase has been
described in one patient who presented with developmental
35.9 Prenatal Diagnosis ........................................................... 583
delay and a slowly progressive leucoencephalopathy.
35.10 DNA Testing ..................................................................... 583 Transaldolase deficiency has been diagnosed in 19 unre-
35.11 Treatment Summary ....................................................... 583 lated families. All patients presented in the neonatal or ante-
References ..................................................................................... 583 natal period with hepatosplenomegaly, liver function
problems, hepatic fibrosis and haemolytic anaemia.
Moreover, the most common 57-kb deletion in nephro-
pathic cystinosis patients has been shown to also cause
deficiency of sedoheptulokinase, resulting in urinary accu-
mulation of sedoheptulose (Wamelink et al. 2008d).
In this chapter we focus on the two recently reported
defects, namely, deficiency of ribose-5-phosphate isomerase
(RPI) and transaldolase (TALDO) deficiency.
35.1 Introduction
The pentose phosphate pathway (PPP), also known as the

hexose monophosphate shunt, provides an alternative path-
way for glucose oxidation. It is present in the cytosol of all
M.M.C. Wamelink (*) C. Jakobs cells and has two major functions: (1) production of NADPH,
Metabolic Unit, Department of Clinical Chemistry, which is used as a reducing agent in many biosynthetic path-
VU University Medical Center, Amsterdam, The Netherlands ways and is also important for protection against oxidative
e-mail: m.wamelink@vumc.nl
damage, and (2) synthesis of ribose-5-phosphate, which is
V. Valayannopoulos required for nucleotide and nucleic acid synthesis. The
Reference Center for Inherited Metabolic Disorders,
pathway can be divided into two branches. The oxidative
Necker-Enfants Malades Hospital and
Paris Descartes University, Paris, France branch consists of three irreversible reactions, which results
e-mail: vassili.valaya@nck.aphp.fr in NADPH and pentose phosphate production. The
578 M.M.C. Wamelink et al.
non-oxidative branch of the pathway reconverts pentose pathology includes hepatocellular damage and degeneration
phosphates into glucose-6-phosphate (glucose-6P), and two of hepatocytes or hyperplastic and enlarged hepatocytes with
of the intermediates, fructose-6P and glyceraldehyde-3P, are fibrosis and cirrhosis.
also glycolytic intermediates. The reactions in the non- TALDO is located in the reversible part of the PPP and
oxidative branch are reversible. The flow of glucose-6P recycles pentose phosphates into glycolytic intermediates
through the PPP or glycolysis is dependent upon the cellular in concerted action with transketolase. Its deficiency
requirement for NADPH, ribose-5P and ATP. results in the accumulation of seven-carbon sugars
Transaldolase (TALDO) deficiency was first described in (sedoheptulose, mannoheptulose) from sedoheptulose-7-
2001 (Verhoeven et al. 2001) in a single patient presenting phosphate and polyols (erythritol, arabitol, ribitol, sedo-
with cirrhosis, with 30 additional patients diagnosed from 18 heptitol, perseitol) and erythronic acid derived from the
different families (Verhoeven et al. 2005; Valayannopoulos pathway intermediates (Verhoeven et al. 2001, 2005;
et al. 2006; Wamelink et al. 2008a, b; Tylki-Szymańska et al. Wamelink et al. 2005a, b, 2007, Wamelink et al. 2008c;
2009; Eyaid et al. 2013). Engelke et al. 2010). Elevations are striking in the neonatal
The patients were born to consanguineous Turkish, period and more subtle in older patients. In plasma and
United Arabic Emirates, Pakistani, Polish and Saudi Arabian CSF, there may be only minor polyol elevations. In a
couples, suggesting rare alleles in the human population. majority of TALDO-deficient patients, elevated urinary
A great phenotypic variability has been reported in concentration of the citric acid cycle intermediates
TALDO patients. Most patients displayed the first symptoms 2-oxoglutaric acid and fumaric acid were detected, indi-
of the disease in the neonatal or antenatal period where intra- cating a possible disturbed mitochondrial metabolism in
uterine growth retardation, oligohydramnios and hydrops TALDO deficiency (Engelke et al. 2010).
fetalis have been described leading to a medical termination RPI deficiency was described in a patient with a slowly
of pregnancy in a 28-week fetus presenting also with a poly- progressive leucoencephalopathy with accumulation of the
malformative syndrome (Valayannopoulos et al. 2006). pentitols ribitol and arabitol in brain and body fluids (van der
Neonates present with hepatosplenomegaly, bleeding, abnor- Knaap et al. 1999; Huck et al. 2004). Since then, no addi-
mal liver function tests, cholestatic jaundice and elevated tional patients with RPI deficiency have been described. The
liver enzymes. Hepatic fibrosis and cirrhosis (in older patient with RPI deficiency presented with impaired psycho-
patients) are the pathological liver hallmarks. Anaemia and motor development and epilepsy followed by neurological
thrombocytopenia are also a constant finding in patients. regression with cerebellar ataxia and peripheral neuropathy.
Most patients showed dysmorphic features, neonatal At diagnosis, he suffered from serious mental retardation,
oedema and congenital heart defects (septal defects, cardio- but somatic examination was normal.
myopathy, tetralogy of Fallot). MRI showed extensive abnormalities of the cerebral
Renal manifestations and endocrine disorders have been white matter. MRS revealed abnormal resonances, corre-
frequently reported. Mild transient hypotonia was described sponding to arabitol and ribitol.
in several patients, but mental and motor developments were The diagnosis of RPI deficiency can be made by the
normal in the majority, where assessment was possible (five analysis of sugars and polyols in urine, plasma or CSF.
patients died before the age of 6 months). Urinary ribitol and arabitol, as well as xylulose, are ele-
Unlike RPI deficiency, brain MRI and MRS did not reveal vated. High concentrations of the pentitols are also observed
abnormalities in patients with TALDO deficiency. Liver in CSF.
35.2 Nomenclature
No. Disorder name Abbreviation Gene symbol localisation Affected protein OMIM no. Subtype
35.1 Transaldolase TALDO TALDO TALDO1 11p15.5–p15.4 Transaldolase 606003 All forms
35.2 Ribose-5- RPI deficiency RPI RPIA 2p11.2 Ribose-5- 608611 All forms
phosphate phosphate
isomerase isomerase
deficiency
35 Disorders of Polyol Metabolism 579
D-Glucose-6-phosphate
NADH+
NADPH
D-6-phosphoglucono-d-lactone
D-6-phosphogluconate
D-Arabitol NADH+
NADPH
D-Ribulose-5-phosphate Ribitol
D-Xylulose
A
D-Xylulose-5-phosphate D-Ribose-5-phosphate D-Ribose
Sedoheptitol
D-Sedoheptulose D-Sedoheptulose-7-phosphate D-Glyceraldehyde-3-phosphate
B
D- mannoheptulose
D-Erythrose-4-phosphate D-Fructose-6-phosphate
Perseitol
Erythritol
D-Glucose-6-phosphate
Fig. 35.1 The pentose phosphate pathway. The conversion of the sugar phosphates into their corresponding sugars and polyols (dotted arrows)
has not been proven in humans. A ribose-5-phosphate isomerase, B transaldolase
Table 35.1 Transaldolase deficiency

Cardiovascular Cardiac anomalies/malformations ± ± ±
Cardiomyopathy ± ± ±
Patent ductus arteriosus ± ± ±
CNS Hypotonia ± ± n n n
Retardation, psychomotor ± ±
Dermatological Cutis laxa ± ± ±
Digestive Hepatosplenomegaly + + + ± +
Jaundice ± ±
Liver cirrhosis ± ± ± ± ±
Liver failure, acute ± ±
Liver failure, progressive + + + + +
Endocrine Adrenal insufficiency ± ± ±
Hyperinsulinism ± ±
Hypogonadotropic hypogonadism ± ±
Hypothyroidism ± ± ±
Genitourinary Clitoral hypertrophy ± ±
Cryptorchidism ± ± ±
Micropenis ± ± ±
Oligohydramnios, prenatally ± – – – –
Haematological Haemolytic anaemia + ± ± ± ±
Leucopenia ± ± ± ± ±
Musculoskeletal Dysmorphic features ++ + ±
Renal Hypertension – ± ±
Nephrocalcinosis ± ± ±
Renal dysgenesis/hypoplasia ± ± ± ±
Renal tubulopathy ± ± ± ± ±
Other Death ± ± ± ±
Fetal hydrops ± – – – –
Intrauterine growth retardation ± – – – –
Routine laboratory Albumin (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Alkaline phosphatase (P) n-↑ n-↑ n-↑ n
ASAT/ALAT (P) ↑ ↑ n-↑ n-↑ n-↑
Bilirubin (P) n-↑ n-↑ n-↑ n-↑ n-↑
Ferritin (S) n-↑ n-↑ n-↑ n-↑
Gamma-glutamyl transpeptidase n-↑ n-↑ n-↑ n-↑ n
(GGT) (P)
Haemoglobin (B) ↓↓ ↓-n ↓-n ↓-n ↓-n
Prothrombin time ↑ ↑ ↑ n-↑ n-↑
Special laboratory Arabitol (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Erythritol (U) ↑↑ ↑↑ ↑ ↑ ↑
Erythronic acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Mannoheptulose (U) ↑ ↑ ↑ ↑ ↑
MRI: brain n n n n
Perseitol (U) ↑ ↑ ↑ ↑ ↑
Respiratory chain activity (M) ↓-n ↓-n ↓-n
Ribitol (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Sedoheptulose (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Sedoheptitol (U) ↑ ↑ ↑ ↑ ↑
Sedoheptulose-7-P (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 35.2 Ribose-5-phosphate isomerase deficiency

CNS Ataxia + + +
Epilepsy – – ++ ++ ++
Leucoencephalopathy ++ ++ ++
Neuropathy + ++ ++
Retardation, psychomotor ++ ++ ++ ++
Spasticity + + +
Eye Optic atrophy + + +
Haematological MR spectroscopy brain ++ ++ ++
Special laboratory Arabitol (U, P, CSF) ↑↑↑ ↑↑↑ ↑↑↑
Ribitol (U, P, CSF) ↑↑↑ ↑↑↑ ↑↑↑
Xylulose (U) ↑↑ ↑↑ ↑↑

CSF (umol/l)a
Reference
Carbohydrate Age values Carbohydrate Reference values
Erythritola 0–4 months 89–209 Erythritol 12–33
4 months–5 years 38–148 Ribitol <5
5–80 years 14–88 Arabitol 9–39
Ribitola 0–4 months 0–25 a
Gas chromatography-flame ionisation detection (Jansen et al. 1986)
5–80 years 0–7
Arabitola 0–4 months 30–151
5–80 years 6–60
Sedoheptuloseb 0–1 month <40
1 month–80 years <9
Mannoheptuloseb 0–80 years <3
Sedoheptitola 0–80 years <1
Perseitola 0–80 years <1
Sedoheptulose-7-Pb 0–1 year <0.07
1–10 years <0.06
10–80 years n.d.
Erythronic acidc 0–3 months <120
3 months–80 years <50
a
Liquid chromatography tandem mass spectrometry (LC-MS/MS;
Wamelink et al. 2005a)
b
LC-MS/MS (Wamelink et al. 2007)
c1
H-NMR (Engelke et al. 2010)
Plasma (umol/l)a
Carbohydrate Reference values
Erythritol <5
Ribitol <5
Arabitol <5
a
Urine (mmol/mol creatinine) Plasma (umol/l)a

Carbohydrate Age 35.1 35.2 Carbohydrate 35.1 35.2
Erythritola 0–4 months 427–976 Erythritol 8–33 N
4 months–5 years 139–1,131 Ribitol 6–13 14–30
5–80 years 90–629 N Arabitol 15–32 90–163
Ribitola 0–4 months 188–722 N normal
4 months–5 years 63–432 a
5–80 years 43–313 123–186
Arabitola 0–4 months 216–588
4 months–5 years 163–588 CSF (umol/l)a
5–80 years 99–583 1,021–1,612 Carbohydrate 35.1 (n = 1) 35.2
Sedoheptuloseb 0–1 month 1,361 N Erythritol N <5
1 month – 80 years 70–5,300 Ribitol 19 891–1,249
Mannoheptuloseb 0–80 years 6–112 N Arabitol N 5,234–5,535
Sedoheptitola 0–80 years 2–27 N N normal
a
Perseitola 0–80 years 3–67 N Gas chromatography-flame ionisation detection (Jansen et al. 1986)
Sedoheptulose-7-Pb 0–1 year 2.5–42.1 N
1–10 years 0.5–23.8
10–80 years 1.8–8.5
Erythronic acidc 0–3 months 2,900 –
N normal
a
Liquid chromatography tandem mass spectrometry (LC-MS/MS;
Wamelink et al. 2005a)
b
LC-MS/MS Wamelink et al. (2007)
c1
H-NMR Engelke et al. (2010)
Fig. 35.2 Diagnostic

flow charts for TALDO Hepatosplenomegaly, liver function problems,
Leuko encephalopathy, neurodegeneration
and RPI deficiency hepatic fibrosis, haemolytic anemia
Urinary polyols Urinary polyols
Urinary C7-carbohydrates Plasma and CSF polyols
TALDO enzyme in fibroblasts or lymphoblasts RPI enzyme in fibroblasts or lymphoblasts

TALDO1 DNA analysis RPIA DNA analysis
Specimen Storage
Polyols U, P, CSF Frozen
C7-carbohydrates U, BSP Frozen
Transaldolase FIB, LYB, ERY Room temperature
Liver Frozen
Ribose-5-phosphate isomerase FIB, LYB Room temperature
Urine can be spotted on filter paper and sent on room temperature for analysis of polyols (sugars are not stable in spotted urine and therefore unreli-
able for diagnosis)
Disorder Material Testing Trimester

35.1 CV, AMN DNA I, II
Cell-free amniotic fluid Metabolite (II), III (performed only ones at 28 weeks gestation)
35.2 CV, AMN DNA I, II
35.10 DNA Testing
Disorder Material Gene locus

35.1 Transaldolase deficiency Any DNA source (WBC, FB, CV, AMN) 11p15
35.2 RPI deficiency Any DNA source (WBC, FB, CV, AMN) 2p11
Huck JHJ, Verhoeven NM, Struys EA et al (2004) Ribose-5-phosphate

35.11 Treatment Summary isomerase deficiency: new inborn error in the pentose phosphate
pathway associated with a slowly progressive leukoencephalopathy.
Am J Hum Genet 74:745–751
There is no specific treatment for TALDO and RPI defi- Jansen G, Muskiet FA, Schierbeek H et al (1986) Capillary gas chro-
ciency. For TALDO deficiency treatment is symptomatic and matographic profiling of urinary, plasma and erythrocyte sugars and
focused on liver disease, which is the main cause of morbid- polyols as their trimethylsilyl derivatives, preceded by a simple and
rapid prepurification method. Clin Chim Acta 157:277–293
ity and mortality of this condition. Liver transplantation has Tylki-Szymańska A, Stradomska TJ, Wamelink MM et al (2009)
never been performed so far in affected patients. Correction Transaldolase deficiency in two new patients with a relative mild
of anaemia and low clotting factors may require transfusions phenotype. Mol Genet Metab 97:15–17
of packed red cells and fresh frozen plasma. Valayannopoulos V, Verhoeven N, Mention K et al (2006) Transaldolase
deficiency: a new cause of hydrops fetalis and neonatal multi-organ
Surgical correction of heart septal defects and medical disease. J Pediatr 149:713–717
treatment of tubulopathy, arterial hypertension and endo- van der Knaap MS, Wevers RA, Struys EA et al (1999)
crine disorders may be required. In RPI deficiency support- Leukoencephalopathy associated with a disturbance in the metabo-
ive therapy and rehabilitation for the neurological lism of polyols. Ann Neurol 46:925–928
Verhoeven NM, Huck JH, Roos B et al (2001) Transaldolase deficiency:
complications are indicated. liver cirrhosis associated with a new inborn error in the pentose
phosphate pathway. Am J Hum Genet 68:1086–1092
Emergency or Standard Treatment Verhoeven NM, Wallot M, Huck JHJ et al (2005) A newborn with
No emergency or standard treatment exists for TALDO or severe liver failure, cardiomyopathy and transaldolase deficiency.
RPI deficiency. Wamelink MM, Smith DE, Jakobs C et al (2005a) Analysis of polyols
in urine by liquid chromatography-tandem mass spectrometry: a
Experimental Treatment useful tool for recognition of inborn errors affecting polyol metabo-
In the TALDO knockout mouse model, lifelong administra- lism. J Inherit Metab Dis 28:951–963
Wamelink MM, Struys EA, Huck JH et al (2005b) Quantification of
tion of the potent antioxidant N-acetylcysteine prevented sugar phosphate intermediates of the pentose phosphate pathway by
acetaminophen-induced liver failure and blocked hepatocar- LC-MS/MS: application to two new inherited defects of metabo-
cinogenesis in Taldo1−/− mice. This treatment has not been lism. J Chromatogr B Analyt Technol Biomed Life Sci 823:18–25
proposed in human subjects with TALDO deficiency so far Wamelink MM, Smith DEC, Janssen EEW et al (2007) Detection of
transaldolase deficiency by quantitation of novel seven-carbon
(Hanczko et al. 2009). chain carbohydrate biomarkers in urine. J Inherit Metab Dis
30:735–742
Wamelink MM, Struys EA, Salomons GS et al (2008a) Transaldolase
References deficiency in a two year-old boy with cirrhosis. Mol Genet Metab
29:532–536
Engelke UFH, Zijlstra FSM, Mochel F et al (2010) Mitochondrial Wamelink MMC, Struys EA, Jakobs C (2008b) The biochemistry,
involvement and erythronic acid as a novel biomarker in transaldol- metabolism and inherited defect of the pentose phosphate pathway:
ase deficiency. Biochim Biophys Acta 1802:1028–1035 a review. J Inherit Metab Dis 31:703–717
Eyaid W, Al Harbi T, Anazi S et al (2013) Transaldolase deficiency: Wamelink MM, Struys EA, Gonzales M et al (2008c) Retrospective
report of 12 new cases and further delineation of the phenotype. detection of transaldolase deficiency in amniotic fluid. Prenat Diagn
J Inherit Metab Dis 36:997–1004 28:460–462
Hanczko R, Fernandez DR, Doherty E et al (2009) Prevention of Wamelink MM, Struys EA, Jansen EE et al (2008d) Sedoheptulokinase
hepatocarcinogenesis and increased susceptibility to acetaminophen- deficiency due to a 57-kb deletion in cystinosis patients causes uri-
induced liver failure in transaldolase-deficient mice by nary accumulation of sedoheptulose: elucidation of the CARKL
N-acetylcysteine. J Clin Invest 119:1546–1557 gene. Hum Mutat 29:532–536
Cholesterol Synthesis Disorders
36
Richard I. Kelley and Lisa Kratz
Contents Summary
36.1 Introduction.................................................................... 585 Cholesterol has several essential functions in normal cell
physiology. In addition to being a major component of cel-
36.2 Nomenclature ................................................................. 588
lular membranes, cholesterol serves as the precursor of bile
36.3 Metabolic Pathway ........................................................ 589 acids and steroid hormones and plays an important role in
36.4 Signs and Symptoms ...................................................... 590 embryonic development. For many years, mevalonate kinase
36.5 Reference and Pathological Values............................... 595
deficiency (MKD) was the only known genetic disorder of
the cholesterol biosynthetic pathway (Hoffmann et al. 1993).
36.6 Diagnostic Flowchart ..................................................... 597
However, the discovery in 1993 of increased levels of
36.7 Specimen Collection....................................................... 598 7-dehydrocholesterol (7DHC) and hypocholesterolemia in
36.8 Prenatal Diagnosis and DNA Testing ........................... 599 patients with Smith-Lemli-Opitz syndrome (SLOS) (Irons
et al. 1993) heralded the emergence of a new group of meta-
36.9 DNA Analysis ................................................................. 599
bolic disorders – inborn errors of post-squalene cholesterol
36.10 Treatment........................................................................ 599 biosynthesis. To date, human disorders have been identified
References .................................................................................... 599 for all but 2 enzymes involved in cholesterol biosynthesis:
HSD17β7, a ketosteroid reductase that is part of a C4
demethylase complex, and TM7SF2, a sterol 14-reductase.
The discovery of the biochemical bases of these rare genetic
disorders has not only provided biochemical methods for
their diagnosis but also allowed the delineation of the spec-
trum of their clinical and biochemical phenotypes.
36.1 Introduction
Mevalonate Kinase Deficiency. Mevalonic kinase deficiency

(MKD) is a rare, autosomal recessive disorder with principal
characteristics of (1) periodic autoinflammatory crises asso-
ciated with arthralgia, edema, diarrhea, and morbilliform
rash; (2) developmental delays, often profound; (3) hepato-
splenomegaly secondary to extramedullary hematopoiesis;
R.I. Kelley (*)
Department of Metabolism and Pediatrics, and (4) marked mevalonic aciduria (Hoffmann et al. 1993).
Kennedy Krieger Institute, Johns Hopkins University, More severely affected patients have dysmorphic facial fea-
707 N. Broadway, Baltimore, MD 21205, USA tures, failure to thrive, rhizomelic dwarfism, cataracts, and
e-mail: rkelley3@jhmi.edu
anemia. Biochemically more mildly affected patients
L. Kratz included in the spectrum of MKD may have only minimal
Department of Neurogenetics,
psychomotor retardation, hypotonia, myopathy, and ataxia.
Kennedy Krieger Institute,
707 N. Broadway, Baltimore, MD 21205, USA A third group of patients with mevalonic kinase deficiency
e-mail: kratz@kennedykrieger.org have recurrent, every 3- to 4-week febrile crises associated
586 R.I. Kelley and L. Kratz
with hyperimmunoglobulinemia D (hyper-IgD syndrome), found, the mother should always be tested, because some
minimally increased urine levels of mevalonic acid, and none carrier mothers have had no clinical manifestations of the
of the developmental or physical abnormalities associated disorder or only mild short stature.
with classical MKD (Houten et al. 1999). The diagnosis of Although mutations in EBP that cause CDPX2 were once
all forms of MKD is based on finding increased urinary lev- assumed to be uniformly lethal in hemizygous males early in
els of mevalonic acid, deficient activity of mevalonate kinase gestation, we have found sterol-∆8-isomerase deficiency in
activity in cultured cells, or disabling mutations in MVK. more than 10 46,XY males based on a diagnostic CDPX2
Some success in reducing the severity of attacks in the milder sterol profile and hemizygosity or mosaic heterozygosity for
hyper-IgD form of MKD has been achieved by treatment a damaging mutation in EBP (Aughton et al. 2003; Milunsky
with corticosteroids or other immunomodulatory drugs, et al. 2003). Males who are hemizygous for mutations in EBP
including montelukast, anakinra, and etanercept (van der have a malformation syndrome quite distinct from that of het-
Hilst et al. 2008). erozygous CDPX2 females, including microcephaly, Dandy-
Smith-Lemli-Opitz Syndrome. The most common known Walker variants, and agenesis of the corpus callosum. Other
disorder of cholesterol biosynthesis, with an estimated inci- findings in hemizygous males include seizures, hypotonia,
dence of 1 in 80,000 births, is Smith-Lemli-Opitz syndrome developmental delays, 2- and 3-toe syndactyly, polydactyly,
(SLOS) (Kelley and Hennekam 2000). This well-known and heart defects, while both chondrodysplasia punctata and
autosomal recessive malformation syndrome is character- other skeletal abnormalities are sometimes absent.
ized clinically by distinctive facial and oral anomalies, limb CHILD Syndrome and CK Syndrome. Congenital hemi-
and genital malformations, and mild to profound develop- dysplasia, ichthyosis, and limb defects (CHILD syndrome)
mental disabilities (Cunniff et al. 1997). In the biochemically is a rare (fewer than 50 patients reported) X-linked dominant
more severely affected patients, malformations of the kidney, disorder occurring in females and defined by unilateral ich-
heart, and gastrointestinal system are also common. With the thyosiform skin lesions and ipsilateral reduction deformities
identification in 1993 of a biochemical marker for SLOS, of the limbs (Happle et al. 1980). Concomitant anomalies on
increased plasma or tissue levels of 7-dehydrocholesterol the affected side include punctate epiphyseal calcifications
(7DHC), and later identification of causative mutations in of vertebrae and long bones, abnormal calcification of the
DHCR7 encoding the last enzyme in the cholesterol biosyn- laryngeal and tracheal cartilages, and hypoplasia of inter-
thetic pathway, the clinical spectrum for this disorder has nal organs, especially the kidney. This clinical syndrome
broadened to include biochemically minimally affected is most often caused by a deficiency of 3ß-hydroxysteroid
patients with no discrete malformations and normal intelli- dehydrogenase, an enzyme encoded by NSDHL (NAD(P)
gence at one extreme and severely affected fetuses dying H steroid dehydrogenase-like), which is one of four pro-
early in gestation at the other. Although the detection of an teins constituting the 4α-methylsterol-4-demethylase com-
increased plasma level of 7DHC remains an accurate and plex (König et al. 2000). Patients with NSDHL-deficiency
efficient screening test for SLOS, rare patients, fewer than CHILD syndrome have abnormal levels of 4 α-methylsterols
0.1 %, will have normal plasma levels of both cholesterol in affected skin and cultured cells, but plasma sterol levels
and 7DHC, in which case mutation analysis or metabolite typically are normal (Liu et al. 1999). A few patients with
analysis in cultured cells must be undertaken. In many unilateral skin and skeletal lesions given an initial diagno-
patients, treating with supplements of cholesterol or a statin sis of “CHILD syndrome” have later been found to have
(to upregulate residual DHCR7 activity) leads to clinical and a deficiency of sterol-∆8-isomerase, the cause of X-linked
biochemical improvement. dominant Conradi-Hünermann syndrome (Grange et al.
3ß-Hydroxysteroid-∆8,∆7-Isomerase Deficiency. 2000). Conversely, a rare patient given a clinical diagno-
X-linked dominant Conradi-Hünermann syndrome (CDPX2) sis of CDPX2 at birth has been found later to have a defi-
is a rare disorder characterized in females by a variable com- ciency of NSDHL by biochemical or genetic analysis
bination of (1) bilateral and asymmetric shortening of long (R. Kelley unpublished 2001). Although males hemizygous
bones; (2) punctate calcifications of epiphyses, trachea, and for NSDHL deficiency were for many years assumed to die
larynx; (3) segmental cataracts; and (4) patches of ichthyosi- early in utero, missense mutations in NSDHL have recently
form erythroderma that mostly follow the lines of Blaschko been identified in several males from two unrelated families
(Silengo et al. 1980). The cause is a deficiency of with CK syndrome, an X-linked recessive intellectual dis-
3ß-hydroxysteroid-∆8,∆7-isomerase (sterol-∆8-isomerase, ability syndrome (McLarren et al. 2010).
emopamil-binding protein) which converts 8(9)-cholestenol Desmosterolosis. Desmosterolosis is an especially rare
into lathosterol (Kelley et al. 1999; Braverman et al. 1999). disorder of cholesterol synthesis caused by damaging muta-
The diagnosis of CDPX2 can be made by detection of tions in DHCR24, the gene encoding 3ß-hydroxysteroid-∆24-
increased plasma levels of 8(9)-cholestenol or by mutation reductase (desmosterol reductase) (Waterham et al. 2001),
analysis. If a mutation in sterol-∆8-isomerase (EBP) is and characterized biochemically by increased plasma and
36 Cholesterol Synthesis Disorders 587
tissue levels of desmosterol (cholesta-5,24-dien-3ß-ol) rarer homozygous form of PHA has clinically recognizable
(Fitzpatrick et al. 1998; Andersson et al. 2002; Zolotushko features, including psychomotor retardation, macrocephaly,
et al. 2011, L. Kratz unpublished 2004). While all 9 known ventricular septal defect, polydactyly, and short metacarpals
patients with desmosterolosis have had structural brain (Speeckaert et al. 2009). Such a mild biochemical abnormal-
abnormalities, especially agenesis of the corpus callosum ity in a lethal disorder like Greenberg dysplasia presumably
(9/9), other variably present abnormalities have been reflects the functional importance of a second microsomal
observed: microcephaly (5/9), macrocephaly (2/9), cleft pal- enzyme with sterol-14-reductase activity, TM7SF2, for
ate (3/9), renal agenesis/hypoplasia (2 patients), rhizomelic which an associated deficiency syndrome remains unknown.
shortness (2 patients), neonatal seizures (3/9), nystagmus P450 Oxidoreductase Deficiency. P450 oxidoreduc-
and strabismus (3/9), large joint contractures (6/9), and tase (POR) functions as an electron donor for numer-
severe psychomotor delays in the 6 surviving patents. ous enzymes of steroid and sterol metabolism, including
Lathosterolosis. Lathosterolosis, the rarest of known 14-alpha demethylase (CYP51), which converts lanosterol
inborn errors of cholesterol metabolism with only three to 4,4-dimethylcholesta-8(9),14,24-trien-3ß-ol early in
patients from two families, is caused by a deficiency of post-squalene cholesterol biosynthesis. Because POR also
lathosterol 5-desaturase (SC5D), which converts lathosterol serves as an electron donor for multiple enzymes involved
to 7-dehydrocholesterol (Brunetti-Pierri et al. 2002; in glucocorticoid and sex steroid synthesis, such as ste-
Krakowiak et al. 2003). The patients surviving birth and roid 17-hydroxylase (CYP17) and steroid 21-hydroxylase
described in the literature had phenotypic findings compati- (CYP21), the phenotype of POR deficiency also includes
ble with the diagnosis of SLOS but, in contrast to SLOS, also clinical and biochemical signs of cortisol and sex hormone
had progressive hepatosplenomegaly secondary to intracel- deficiency (Arlt 2007). The most severely affected individu-
lular accumulation of mucopolysaccharides and lipids. Sterol als, those with classical Antley-Bixler syndrome, present
analysis showed a marked increase in the level of lathosterol with craniosynostosis, multiple skeletal abnormalities, and
in plasma and/or cultured fibroblasts but, unlike most indi- ambiguous genitalia, whereas more mildly affected individ-
viduals with SLOS, a normal plasma cholesterol level. uals may have only mild facial dysmorphisms in addition to
Homozygosity for disabling mutations in SC5D was identi- endocrine steroid hormone deficiencies. A suspected diag-
fied in both families. The strong phenotypic resemblance of nosis of POR deficiency is most easily made by demonstrat-
both infants to SLOS, a disorder routinely screened in the ing increased urine levels of pregnanediol and pregnenetriol
evaluation of children with dysmorphic features and often relative to metabolites of cortisol followed by confirmation
just developmental delays, suggests that lathosterolosis is by sequencing of POR (Shackleton et al. 2004). Although
one of the rarest known autosomal recessive enzymopathies, impaired cholesterol synthesis and increased levels of
having an estimated incidence substantially less than 1 in a lanosterol and dihydrolanosterol can be shown in cultured
million births. lymphoblasts, the plasma sterol profile in POR deficiency
Hydrops-Ectopic Calcification-“Moth-Eaten” (HEM)/ is normal. Yet to be reported in the literature is a primary
Greenberg Skeletal Dysplasia. Hydrops-ectopic calcification- deficiency of the POR-requiring lanosterol demethylase
moth-eaten skeletal dysplasia (HEM) or “Greenberg dys- CYP51, a deficiency of which would be expected to cause
plasia” is a rare, autosomal recessive, prenatally lethal more severe sterol abnormalities than found in POR defi-
skeletal dysplasia characterized by fetal hydrops, severe ciency that would be detectable in plasma.
short-limbed dwarfism, disorganized chondro-osseous mes- Sterol Methyl C4 Oxidase Deficiency. Sterol methyl C4
enchymal proliferation, and abnormal bone mineralization oxidase, encoded by SC4MOL, is one of three enzymes and
(Konstantinidou et al. 2008). Radiographic findings include one structural protein comprising the heteromultimeric
marked limb shortening with a “moth-eaten” appearance of enzyme complex, 4α-methylsterol-4-demethylase. The first
the long bones, platyspondyly, ectopic epiphyseal calcifica- of two unrelated patients shown to have SC4MOL deficiency
tions, and abnormal laryngeal and tracheal calcifications. was an adolescent female with congenital cataracts, mild
Based on the finding of small amounts of 14-unsaturated developmental delays, microcephaly, short stature, persistent
sterols in cartilage and cultured fibroblasts or in chondro- hypocholesterolemia, and a severe ichthyosiform erythro-
cytes from affected fetuses, molecular studies confirmed derma that developed after age 2 years (He et al. 2011). The
pathogenic mutations in the lamin B receptor gene, LBR second as yet unpublished patient presented with congenital
(Waterham et al. 2003), which encodes a fusion protein carry- cataracts, microcephaly, hypotonia, and moderate develop-
ing both sterol-∆14-reductase enzymatic activity and nuclear mental delays but by age 3 years had normal skin and a nor-
lamin B receptor function. Interestingly, heterozygosity for mal plasma cholesterol level (R. Kelley and L. Kratz,
mutations in LBR causes the benign Pelger-Huët anomaly unpublished data 2009). Both patients had increased levels
(PHA), an autosomal dominant phenomenon of abnormal of 4α-methyl and 4,4α-dimethyl sterols in plasma, and both
lobation of neutrophils (Hoffmann et al. 2002). A much were homozygous for pathologic mutations in SC4MOL.
The combination of simvastatin treatment and cholesterol 4,4-dimethylcholesta-8(9),14,24-trien-3ß-ol. One patient

supplementation in the first patient led to resolution of the ich- has been identified with biochemical evidence and molecu-
thyosiform erythroderma and improved growth and develop- lar confirmation for a deficiency of this enzyme (L. Kratz
ment (M. He and J. Vockley, personal communication 2011). unpublished observations 2012). The clinical phenotype
Lanosterol Demethylase Deficiency. Sterol 14-α- of this patient includes microcephaly, congenital cataracts,
demethylase (lanosterol demethylase), encoded by the cyto- short stature, persistent cheek erythema, decreased pigmen-
chrome P450 gene CYP51, catalyzes an early, rate-limiting tation, developmental delays, and behavioral abnormalities.
step in post-squalene cholesterol biosynthesis (Rozman et al. Sterol analysis in plasma and cultured lymphoblasts revealed
1996). This enzyme, in conjunction with P450 oxidoreduc- increased levels of lanosterol and dihydrolanosterol, and two
tase, removes the 14-α methyl group in lanosterol to form inherited mutations in CYP51 were found.
36.2 Nomenclature

No. Disorder Alternative name Abbreviation symbol localization Affected protein no. Sub type
36.1.1 Mevalonate kinase Mevalonic aciduria MKD MVK 12q24.1 Mevalonate kinase 610377 Classic
deficiency form
36.1.2 Hyper-IgD Mevalonic aciduria HIDS MVK 12q24.1 Mevalonate kinase 260920 Mild form
syndrome
36.2 Smith-Lemli- 7-Dehydrocholesterol SLOS DHCR7 11q13.4 7-Dehydrocholesterol 270400 All forms
Opitz syndrome reductase deficiency reductase
36.3.1 Chondrodysplasia Conradi-Hünermann CDPX2 EBP Xp11.23 3β-Hydroxysteroid- 302960 Female
punctata 2: female syndrome delta8,
delta7-isomerase
36.3.2 Chondrodysplasia CDPX2 EBP Xp11.23 3β-Hydroxysteroid- 302960 Male
punctata 2: male delta8, delta7-
isomerase
36.4.1 CHILD syndrome Congenital CHILD NSDHL Xq28 3-Beta- 308050 Female
hemidysplasia with hydroxysteroid
ichthyosiform dehydrogenase
erythroderma and limb
defects
36.4.2 CK syndrome NSDHL deficiency CKS NSDHL Xq28 3-Beta- 300831 Male
hydroxysteroid
dehydrogenase
36.5 Desmosterolosis Desmosterol reductase DHCR24 DHCR24 1p32.3 3β-hydroxysteroid- 602398 All forms
deficiency deficiency delta24-reductase
36.6 Lathosterolosis Sterol C5-desaturase SC5D SC5D 11q23.3 Sterol C5-desaturase 607330 All forms
36.7 Greenberg skeletal Hydrops-ectopic HEM DHCR14 1q42.12 3β-Hydroxysteroid- 215140 All forms
dysplasia calcification-moth- Dysplasia delta14-reductase
eaten skeletal
dysplasia
36.8 Antley-Bixler POR ABS POR 7q11.2 Cytochrome P450 201750 All forms
syndrome deficiency oxidoreductase
36.9 SC4MOL Sterol SC4MOL SC4MOL 4q32.3 Sterol 607545 All forms
deficiency C4-methyloxidase C4-methyloxidase
deficiency
36.10 Lanosterol CYP51 deficiency CYP51A1 7q21.2 Lanosterol 14-alpha 601637 All forms
demethylase demethylase
deficiency
36.1
Squalene Mevalonate-PO4 Mevalonate 3-Hydroxy-3-methylglutaryl-CoA
4,4', 14-Trimethylcholesta-8(9),24-dien-3β-ol 36.5 4,4', 14-Trimethylcholest-8(9)-en-3β-ol

(Lanosterol) (Dihydrolanosterol)
36.8, 36.10 36.8, 36.10

36.5
4,4'-Dimethylcholesta-8(9), 14,24-trien-3β-ol 4,4'-Dimethylcholesta-8(9), 14-dien-3β-ol
36.7 36.7
4,4'-Dimethylcholesta-8(9), 24-dien-3β-ol 36.5 4,4'-Dimethylcholest-8(9)-en-3β-ol
36.4, 36.9
36.4, 36.9
4α-Methylcholesta-8(9),24-dien-3β-ol 36.5 4α-Methylcholest-8(9)-en-3β-ol
36.4, 36.9 36.4, 36.9
36.5 Cholest-8(9)-en-3b-ol ?
Cholesta-8(9),24-dien-3β-ol Cholesta-5,8(9)-dien-3b-ol
(Zymosterol) (8(9)-Cholestenol) (8-Dehydrocholesterol)
36.3
36.3
36.3
36.5 Cholest-7-en-3β-ol
Cholesta-7,24-dien-3β-ol
(Lathosterol)
36.6 36.6
Cholesta-5, 7 ,24-trien-3β-ol Cholesta-5, 7-dien-3β-ol

(7-Dehydrodesmosterol) 36.5
(7-Dehydrocholesterol)
36.2 36.2
Cholesta-5,24-dien-3β-ol 36.5 Cholest-5-en-3β-ol

(Desmosterol) (Cholesterol)
Fig. 36.1 The pathway of cholesterol biosynthesis. 36.1 3ß-hydroxysteroid-∆24-reductase (desmosterol reductase); 36.6
Mevalonate kinase; 36.2 3ß-hydroxysteroid-∆7-reductase 3ß-hydroxysteroid-∆5-desaturase(lathosteroldesaturase);36.73ß-hydroxysteroid-
(7-dehydrocholesterol reductase); 36.3 3ß-hydroxysteroid-∆8, ∆14-reductase; 36.8 cytochrome P450 oxidoreductase; 36.9 sterol
∆7-isomerase (sterol-∆8-isomerase); 36.4 3ß-hydroxysteroid dehy- C4-methyloxidase of the 4α-methylsterol-4-demethylase complex;
drogenase of the 4α-methylsterol-4-demethylase complex; 36.5 36.10 sterol 14-α-demethylase (lanosterol demethylase)
Table 36.1.1 Mevalonate kinase deficiency

CNS Ataxia + +
Cerebellar hypoplasia + + ++
Hypotonia ++ + +
Retardation, psychomotor + ++ ++
Hematological Anemia, leukocytosis, ± ± ±
thrombocytopenia
Hepatosplenomegaly ++ ++ ++
Musculoskeletal Dysmorphic features + + +
Hypotonia, muscular ++ ++ ++
Respiratory Respiratory failure + + +
Routine laboratory Cholesterol (S) ↓-n ↓-n ↓-n
Creatine kinase (P) n-↑ n-↑ n-↑
Immunoglobulin D n-↑ n-↑ n-↑
Special laboratory Leukotriene E4 (P) ↑ ↑ ↑
Mevalonic acid (U) ↑↑↑ ↑↑↑ ↑↑↑
Ubiquinone-50 (P) ↓-n ↓-n ↓-n
Table 36.1.2 Hyper Ig D syndrome

Digestive Diarrhea ± ± ± ±
Malabsorption ± ± ± ±
Hematological Anemia, leukocytosis, ± + + + ±
thrombocytopenia
Hepatosplenomegaly ± ± ± ± ±
Musculoskeletal Hypotonia, muscular n ± ± ± n
Routine laboratory Cholesterol (S) n n n n n
Erythrocyte sedimentation rate n-↑ n-↑ n-↑ n-↑
Immunoglobulin D n-↑ n-↑ n-↑ n-↑
Special laboratory Leukotriene E4 (P) ↑ ↑ ↑ ↑ ↑
Mevalonic acid (U) ↑ ↑ ↑ ↑ ↑
Ubiquinone-50 (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Table 36.2 Smith-Lemli-Opitz syndrome

CNS Agenesis/hypogenesis, corpus ± ± ± ± ±
callosum
Behavior, aggressive n n + + +
Cerebellar hypoplasia ± ± ± ± ±
Developmental delay + + + + +
Holoprosencephaly ± ± ± ± ±
Hypotonia, axial + + ± n n
Irritability + + + + +
Self-injury n n ± ± ±

Dermatological Excess digital whorls + + + + +
Digestive Feeding difficulties + + + ± ±
Hirschsprung disease ± ± ± ± ±
Intestinal dysmotility + + ± ± ±
Liver dysfunction ± ± n n n
Pyloric stenosis ± ± n n n
Ear Deafness, sensorineural ± ± ± ± ±
Ptosis of eyelid ± ± ± ± ±
Female to ambiguous genitalia in 46, ± ± ± ± ±
XY
Renal hypoplasia or agenesis ± ± ± ± ±
Musculoskeletal Club foot ± ± ± ± ±
Growth retardation + + + + +
Polydactyly, postaxial ± ± ± ± ±
Rhizomelia ± ± ± ± ±
Toe syndactyly, 2–3 or 2–5 + + + + +
Respiratory Pulmonary hypoplasia ± ± ± ± ±
Pulmonary lobation, abnormal ± ± ± ± ±
Other Anteverted nares + + + ± ±
Broad alveolar ridges + + + + +
Cleft palate ± ± ± ± ±
Epicanthal folds + + + + +
Micrognathia + + + + +
Routine laboratory Cholesterol (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Special laboratory 7-Dehydrocholesterol (P) ↑ ↑ ↑ ↑ ↑
8-Dehydrocholesterol (P) ↑ ↑ ↑ ↑ ↑
Table 36.3.1 Chondrodysplasia punctata 2: female

Dermatological Alopecia + + + + +
Dystrophic nails ± ± ± ± ±
Follicular atrophoderma ± + + + +
Ichthyosiform erythroderma + + n n n
Musculoskeletal Cervical compressive myelopathy ± ± ± ± ±
Midface hypoplasia ± ± ± ± ±
Polydactyly ± ± ± ± ±
Punctate calcifications of epiphyses, + + ± ± ±
trachea, and larynx
Rhizomelia ± ± ± ± ±
Renal Renal dysgenesis/hypoplasia ± ± ± ± ±
Other Micrognathia ± ± ± ± ±
Special laboratory 8(9)-Cholestenol (P) ↑ ↑ ↑ ↑ n-↑
8-Dehydrocholesterol (P) ↑ ↑ ↑ ↑ n-↑
Table 36.3.2 Chondrodysplasia punctata 2: male

CNS Agenesis/hypogenesis, corpus + + +
callosum
Dandy-Walker malformation + + +
Developmental delay + + +
Hydrocephalus ± ± ±
Hypotonia + + +
Microcephaly + + +
Seizures ± ± ±
Dermatological Hypoplastic nails ± ± ±
Ichthyosis ± ± ±
Ear Stenotic canals ± ± ±
Macular hypoplasia, bilateral ± ± ±
Ptosis of eyelid ± ± ±
Genitourinary Cryptorchidism ± ± ±
Hypospadias ± ± ±
Musculoskeletal Chondrodysplasia punctata ± ± ±
Midface hypoplasia ± ± ±
Nasal hypoplasia ± ± ±
Polydactyly ± ± ±
Toe syndactyly, 2–3 or 2–5 ± ± ±
Other Anteverted nares ± ± ±
Cleft palate ± ± ±
Special laboratory 8(9)-Cholestenol (P) ↑ ↑ ↑
8-Dehydrocholesterol (P) ↑ ↑ ↑
Table 36.4.1 CHILD syndrome

CNS Hypoplasia, unilateral ± ± ± ± ±
Dermatological Alopecia ± ± ± ± ±
Dystrophic nails ± ± ± ± ±
Erythema + + + + +
Ichthyosiform skin lesions + + + + +
demarcated at the midline
Genitourinary Renal hypoplasia or agenesis ± ± ± ± ±
Musculoskeletal Absent or hypoplastic long bones ± ± ± ± ±
and/or phalanges
Minor contralateral bone and skin ± ± ± ± ±
abnormalities
Punctate calcification of skeletal and ± ± n n n
nonskeletal cartilage
Unilateral limb defects + + + + +
Special laboratory 4-Carboxymethyl sterol (tissue, ↑ ↑ ↑ ↑ ↑
lymphoblasts)
4-Methyl and 4,4-dimethyl sterols ↑ ↑ ↑ ↑ ↑
(tissue, lymphoblasts)
Table 36.4.2 CK syndrome

CNS Behavioral abnormalities + + + +
Cerebral cortical malformations + + + + +
Cognitive dysfunction + + + +
Seizures + + + + +
Musculoskeletal Thin body habitus + + +
Other Dysmorphic features ± ± ± ± ±
Special laboratory 4-Methyl and 4,4-dimethyl sterols ↑ ↑ ↑ ↑ ↑
(lymphoblasts)
Table 36.5 Desmosterolosis

Cardiovascular Anomalous pulmonary venous ± ± ±
return
Patent ductus arteriosus ± ± ±
CNS Agenesis/hypogenesis, corpus + + +
callosum
Cerebral cortical malformations ± ± ±
Intellectual disability + +
Ventriculomegaly ± ± ±
Genitourinary Renal hypoplasia or agenesis ± ± ±
Rhizomesomelia ± ± ±
Respiratory Pulmonary hypoplasia ± ± ±
Other Cleft palate ± ± ±
Routine laboratory Osteosclerosis ±
Special laboratory Desmosterol (P) ↑ ↑ ↑
Table 36.6 Lathosterolosis

Cardiovascular Cardiac, anomalies/malformations ± ± ±
CNS Hypotonia + + ±
Intellectual disability ++ ++
Microcephaly + + +
Eye Cataract ++ ++ ++
Corneal clouding ± ± ±
Ptosis of eyelid + + +
Genitourinary Hypospadias ± ± ±
Growth retardation + + +
Polydactyly, postaxial + + +
Toe syndactyly, 2–3 or 2–4 + + +
Other Anteverted nares + + +
Bitemporal narrowing ± ± ±
Broad alveolar ridges + + +
Cleft palate ± ± ±
Micrognathia + + +
Special laboratory Lathosterol (P) ↑ ↑ ↑
Table 36.7 Greenberg skeletal dysplasia

Digestive Omphalocele ±
Musculoskeletal Chondrodysplasia punctata +
Disorganized chondro-osseous +
proliferation and mineralization
Hypoplasia, severe midface +
Platyspondyly +
Polydactyly, postaxial ±
Rhizomelic shortness, severe +
Respiratory Hypolobated, underdeveloped lungs ±
Other Cystic hygroma ±
Fetal hydrops +
Intrauterine death or stillbirth +
Laryngeal and tracheal calcification +
Special laboratory Cholesta-8,14-dien-3β-ol (tissue, FB) ↑
Table 36.8 Antley-Bixler syndrome

Ear Dysplastic ears ± ± ± ± ±
Endocrine Adrenal insufficiency ± ± ± ± ±
Genitourinary Clitoral hypertrophy ± ± ± ± ±
Hypoplastic labia majora ± ± ± ± ±
Male genital hypoplasia ± ± ± ± ±
Musculoskeletal Arachnodactyly ± ± ± ± ±
Femoral bowing and fractures ± ± ± ± ±
Midface hypoplasia + + + + +
Phalangeal malformations ± ± ± ± ±
Radio-humeral and/or radioulnar ± ± ± ± ±
synostosis
Renal Hydronephrosis ± ± ± ± ±
Renal dysgenesis/hypoplasia ± ± ± ± ±
Respiratory Choanal atresia or stenosis ± ± ± ± ±
Other Craniosynostosis ± ± ± ± ±
Special laboratory 17-OH-pregnenolone (P) n-↑ n-↑ n-↑ n-↑ n-↑
17-OH-progesterone (P) n-↑ n-↑ n-↑ n-↑ n-↑
ACTH (P) n-↑ n-↑ n-↑ n-↑ n-↑
Androstenedione (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Cortisol, ACTH stimulated (S) ↓ ↓ ↓ ↓ ↓
Cortisol, baseline (S) n n n n n
DHEA (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Pregnanediol (U) ↑ ↑ ↑ ↑ ↑
Pregnanediol (U) ↑ ↑ ↑ ↑ ↑
Table 36.9 SC4MOL deficiency

CNS Developmental delay + + + +
Hypotonia + + + +
Dermatological Dermatitis, psoriasiform ± ±
Eye Cataract + + + +
Musculoskeletal Arched palate, high ± ± ± ±
Frontal bossing ± ± ± ±
Osteoporosis ± ± ± ±
Short stature + + + +
Other Failure to thrive + + + +
Special laboratory 4-Methyl and 4,4-dimethyl sterols ↑ ↑ ↑ ↑
(P, lymphoblasts, FB)
Table 36.10 Lanosterol demethylase deficiency

CNS Developmental delay + + +
Microcephaly + + +
Self-injury +
Dermatological Erythema + + +
Hypopigmentation + + +
Eye Cataract + + +
Musculoskeletal Short stature + + +
Reference Values
Organic acids urine (SID GC-MS)

Age Mevalonic acid(mmol/mole creat.)
All ages 0.06–0.21a
a
Hoffmann (1993)
Sterols: plasma (GC-MS)

7-Dehydro- 8-Dehydro- Cholesta- 4α-Methyl
Cholesterol cholesterol cholesterol 8(9)-en-3ß-ol Desmosterol Lathosterol sterols 4,4′α-Dimethyl
Age (mmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) sterols (μmol/L)
Birth–1 week 1.86 0.10 <0.02 0.07 1.79 2.46 <0.05 <0.02
(0.96–3.00) (<0.02–0.31) (<0.03–0.77) (0.52–4.16) (0.73–4.35)
1–3 months 3.15 0.16 <0.02 <0.02 2.85 2.75 <0.05 <0.02
(2.38–4.12) (0.03–0.49) (1.04–6.50) (0.98–4.51)
3–18 months 3.81 0.19 <0.02 <0.02 2.57 2.23 <0.05 <0.02
(2.96–4.43) (<0.02–0.57) (0.26–5.98) (0.93–4.35)
18 months–3 years 3.86 0.16 <0.02 <0.02 2.04 2.56 <0.05 <0.02
(2.91–4.96) (<0.02–0.52) (0.52–5.46) (0.78–7.38)
3–16 years 3.83 0.19 <0.02 <0.02 1.60 2.05 <0.05 <0.02
(2.89–4.74) (0.03–0.52) (0.26–3.64) (0.44–6.86)
>16 years 4.36 0.28 <0.02 <0.02 1.95 3.11 <0.05 <0.02
(2.66–6.02) (0.10–0.52) (0.21–4.42) (1.45–4.33)
Sterols: plasma (GC-MS) cont

Age Lanosterol (μmol/L) Dihydrolanosterol (μmol/L)
All ages <0.35 <0.23
Sterols: cultured cells (GC-MS)

7-Dehydro- Cholesta-8(9)- 4α-Methyl 4,4′α-Dimethyl Dihydro- Cholesta-8,14-
cholesterol en-3ß-ol Desmosterol Lathosterol sterols sterols Lanosterol lanosterol dien-3ß-ol
LYM LYM LYM LYM LYM LYM LYM LYM FB
0.17 0.35 0.27 1.45 2.31 0.65 (<0.01–3.5) 0.19 0.06 0.27
(0.06–0.35) (<0.01–0.99) (0.07–0.69) (0.14–4.30) (<0.1–9.7) (<0.01–0.67) (<0.01–0.33) (<0.01–0.69)
Units: % ratio to total sterols
Note: Overall, most sterol levels in fibroblasts are comparable to levels in lymphoblasts
Pathological Values
36.1 Mevalonate kinase deficiency/hyper-IgD syndrome 36.6 3ß-Hydroxysteroid -∆5-desaturase deficiency (lathosterolosis)
Type Mevalonic acid (mmol/mol creat.) (U) Lathosterol
Classic MKDa 3,165–51,433 P, FB, tissue
Hyper-IgD syndromeb ↑
Acute 21–143
Well 4.4–10.3
a
Hoffmann (1993)
b
Kelley, unpublished data 36.7 3ß-Hydroxysteroid -∆14-reductase deficiency (Greenberg
dysplasia)
36.2 3ß-Hydroxysteroid-∆7-reductase deficiency (Smith-Lemli-Opitz Cholesta-8,14-dien-3ß-ol
syndrome) LYM, soft tissue
Cholesterol 7-Dehydrocholesterol 8-Dehydrocholesterol ↑
P P, LYM, FB, tissue, AF, P, LYM, FB, tissue, AF, CV
CV
n-↓ ↑ ↑ 36.8 Cytochrome P450 oxidoreductase deficiency (Antley-Bixler
syndrome)
36.3 3ß-Hydroxysteroid-∆8, ∆7-isomerase deficiency (CDPX2) Lanosterol Dihydrolanosterol
8(9)-Cholestenol 8-Dehydrocholesterol Cholesterol LYM LYM
P, LYM, FB, TIS P, LYM, FB, TIS P ↑ ↑
↑ ↑ n
36.9 Sterol C4-methyloxidase-like deficiency (SC4MOL deficiency)
36.4.1 3ß-Hydroxysteroid dehydrogenase deficiency: CHILD syndrome 4,4′α-Dimethyl 4α-Carboxymethylcholest-
4α-Methyl sterols sterols 8(9)-en-3-beta-ol
4α-Methyl sterols 4α -Carboxymethylcholest-8(9)-en-3-beta-ol
P, LYM, FB, skin P, LYM, FB, skin P, LYM, FB, skin flakes
LYM, Skin flakes Skin flakes flakes flakes
↑ ↑ ↑ ↑ n
36.4.2 3ß-Hydroxysteroid dehydrogenase deficiency: CK syndrome

36.10 Sterol 14-α-demethylase deficiency (lanosterol demethylase
4α-Methyl sterols 4α -Carboxymethylcholest-8(9)-en-3-beta-ol deficiency)
LYM LYM
Lanosterol Dihydrolanosterol
n-↑ n
P, LYM P, LYM
↑ ↑
36.5 3ß-Hydroxysteroid-∆24-reductase deficiency (desmosterolosis)
Desmosterol Cholesterol
P, LYM, FB, tissue P
↑ n
36.6 Diagnostic Flowchart
Recurrent/cyclical febrile crises,

hyper lgD ± dysmorphic facies
Urine organic acid

analysis
Undetectable
↑ Mevalonate
mevalonate
> 0.6 mmol/mol

Quantify mevalonate creatinine MVK mutation and/or
by isotope-dilution MVK enzymatic
GC/MS analysis
<0.6 mmol/mol
creatinine
↓ or nl cholesterol &
Quantify plasma precursor sterols Evaluate other
cholesterol and proximal steps in
precursor sterols cholesterol synthesis
Fig. 36.2 Diagnostic flow chart for mevalonic aciduria
SLOS-Iike
clinical syndrome
Plasma sterol
analysis
Yes
>10 0.5 –10
μmol/L 7-Dehydro- μmol/L Use of drug that Yes Able to
cholesterol level inhibits DHCR7? discontinue drug ?
> 0.5
No No
μmol/L
Normal Lathosterol Sterol analysis in DHCR7
level cultured cells mutation analysis
7DHC Single Normal

High
High mutation sequence
SC5D Smith-Lemli- Smith-Lemli Further clinical &

mutation analysis Opitz syndrome Opitz syndrome genetic evaluation
Fig. 36.3 Diagnostic flow chart heterozygote
for Smith-Lemli-Opitz syndrome
and related disorders
Fig. 36.4 Diagnostic flow chart Chondrodysplasia punctata

for chondrodysplasia punctata
CHILD syndrome,
Chondrodysplasia
ichthyosiform
punctata, ichthyosis
erythroderma
Erythrocyte Normal Plasma cholesterol

plasmalogen levels precursor analysis
Low
Rhizomelic CDP or
↑ 8(9)-cholestenol & Normal cholesterol ↑ 4-Methyl, dimethyl,
other peroxisomal
8-dehydrocholesterol precursor levels & carboxysterols
biogenesis disorder
Peroxisomal Sterol analysis in Probable sterol-4-

Probable Conradi-
complementation or cultured cells demethylase
Hünermann syndrome
mutation analysis or affected skin complex deficiency
EBP mutation Disabling NSDHL mutation

analysis mutation analysis
not found
Disabling Disabling
mutation mutation
Test for mutation in

Conradi-Hünermann NSDHL
other demethylase
syndrome (CDPX2) deficiency
complex gene
Specimen collection
Disorder Test Preconditions Material Handling Pitfalls
a
36.1 Organic acid analysis None U (random) Keep frozen (−20 °C)
b
36.2 Sterol analysis None P, LYM, FB, AF, CV, tissue Keep frozen (−20 °C)
c
36.3 Sterol analysis None P, FB, LYM, tissue Keep frozen (−20 °C)
c,d
36.4.1 Sterol analysis None Skin flakes, LYM, FB NA
e
36.4.2 Sterol analysis None LYM NA
f
36.5 Sterol analysis None P, LYM, FB, tissue Keep frozen (−20 °C)
36.6 Sterol analysis None P, LYM, FB, tissue Keep frozen (−20 °C)
36.7 Sterol analysis None FB, tissue Keep frozen (−20 °C)
d,g
36.8 Steroid analysis None U Keep frozen (−20 °C)
Sterol analysis None LYM
36.9 Sterol analysis None P, LYM, FB, skin flakes Keep frozen (−20 °C)
36.10 Sterol analysis None P, LYM Keep frozen (−20 °C)
a
Quantification of MVA by stable isotope GC-MS necessary for certain diagnosis of hyper-IgD syndrome in non-acute samples
b
Haloperidol and other “sigma” ligands may increase the level of 7DHC and 8DHC; 7DHC and 8DHC are subject to degradation over time in
plasma at room temperature
c
In females, there may be skewed X-inactivation in favor of normal allele
d
Sterol abnormality not detectable in plasma
e
CK syndrome is best diagnosed by molecular analysis
f
Amiodarone may increase the plasma level of desmosterol
g
Although the sterol abnormality may be detected in cultured lymphoblasts, urine steroid analysis is the preferred method for diagnosis
36.8 Prenatal Diagnosis and DNA Testing Aughton D, Kelley R, Metzenberg A, Pureza V, Pauli R (2003) X-linked
dominant chondrodysplasia punctata (CDPX2) caused by single
gene mosaicism in a male. Am J Med Genet 116A:255–260
36.9 Prenatal diagnosis Braverman N, Lin P, Moebius FF, Obie C, Moser A, Glossmann H,
Wilcox WR, Rimoin DL, Smith M, Kratz L, Kelley RI, Valle D
Disorder Material Timing, trimester (1999) Mutations in the gene encoding 3 beta-hydroxysteroid-delta
36.1 AF, AFC, CV, CCV I, II 8, delta 7- isomerase cause X-linked dominant Conradi-Hünermann
36.2 AF, CV, maternal I, II syndrome. Nat Genet 22:291–294
urine (steroid II Brunetti-Pierri N, Corso G, Rossi M, Ferrari P, Balli F, Rivasi F,
analysis) Annunziata I, Ballabio A, Dello Russo A, Andria G, Parenti G
36.3 AF, CV I,II (2002) Lathosterolosis, a novel multiple-malformation/mental
retardation syndrome due to deficiency of 3b-hydroxysteroid-D5-
36.8 Maternal urine II
desaturase. Am J Hum Genet 71:952–958
(steroid analysis)
Cunniff C, Kratz LE, Moser A, Natowicz MR, Kelley RI (1997) Clinical
Prenatal diagnosis is possible for all disorders by DNA sequencing and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz
36.3 – Experience by biochemical prenatal testing limited to female syndrome and abnormal cholesterol metabolism. Am J Med Genet
fetuses with skeletal abnormalities in utero 68:263–269
36.4, 36.5, 36.6, 36.7, 36.9, 36.10 – No experience by biochemical pre- FitzPatrick DR, Keeling JW, Evans MJ, Kan AE, Bell JE, Porteous ME,
natal testing thus far Mills K, Winter RM, Clayton PT (1998) Clinical phenotype of des-
mosterolosis. Am J Med Genet 75:145–152
Grange DK, Kratz LE, Braverman NE, Kelley RI (2000) CHILD syn-
drome caused by deficiency of 3beta-hydroxysteroid-delta8, delta7-
36.9 DNA Analysis isomerase. Am J Med Genet 90:328–335
Happle R, Koch H, Lenz W (1980) The CHILD syndrome: congenital
hemidysplasia with ichthyosiform erythroderma and limb defects.
Disorder Specimen Methodology Eur J Pediatr 134:27–33
All disorders Any DNA source Sequence analysis He M, Kratz LE, Michel JJ, Vallejo AN, Ferris L, Kelley RI, Hoover JJ,
Jukic D, Gibson KM, Wolfe LA, Ramachandran D, Zwick ME,
Vockley J (2011) Mutations in the human SC4MOL gene encoding
a methyl sterol oxidase cause psoriasiform dermatitis, microceph-
36.10 Treatment aly, and developmental delay. J Clin Invest 121:976–984
Hoffmann GF, Charpentier C, Mayatepek E, Mancini J, Leichsenring
M, Gibson KM, Divry P, Hrebicek M, Lehnert W, Sartor K, Trefz
There is no required emergent metabolic management for dis- FK, Rating D, Bremer HJ, Nyhan WL (1993) Clinical and biochem-
orders of cholesterol biosynthesis, but serious or life- ical phenotype in 11 patients with mevalonic aciduria. Pediatrics
threatening physical anomalies requiring acute medical or 91:915–921
Hoffmann K, Dreger CK, Olins AL, Olins DE, Shultz LD, Lucke B,
surgical intervention are common. For SLOS, cholesterol
Karl H, Kaps R, Müller D, Vayá A, Aznar J, Ware RE, Sotelo Cruz
supplementation has become standard of care. In addition, N, Lindner TH, Herrmann H, Reis A, Sperling K (2002) Mutations
when there is an acute life-threatening condition, such as in the gene encoding the lamin B receptor produce an altered
pneumonia or major surgery, and oral cholesterol replace- nuclear morphology in granulocytes (Pelger-Huët anomaly). Nat
Genet 31:410–414
ment therapy is not possible, intravenous banked plasma
Houten SM, Kuis W, Duran M, de Koning TJ, van Royen-Kerkhof A,
(“fresh-frozen” plasma) can be a valuable parenteral source Romeijn GJ, Frenkel J, Dorland L, de Barse MM, Huijbers WA,
of cholesterol. Statins may also be used to increase residual Rijkers GT, Waterham HR, Wanders RJ, Poll-The BT (1999)
activity of DHCR7. Steroid therapy may be required for Mutations in MVK, encoding mevalonate kinase, cause hyperim-
munoglobulinaemia D and periodic fever syndrome. Nat Genet
severely affected patients during periods of stress, usually
22:175–177
with a cholesterol level less than 0.5 mM, because of gluco- Irons M, Elias ER, Salen G, Tint GS, Batta AK (1993) Defective cho-
corticoid and/or mineralocorticoid deficiency. This is also lesterol biosynthesis in Smith-Lemli-Opitz syndrome. Lancet
required for patients with P450 oxidoreductase deficiency. 341:1414
Kelley RI, Hennekam RC (2000) The Smith-Lemli-Opitz syndrome.
Treatment schemes for non-SLOS sterol disorders are limited
J Med Genet 37:321–335
due, in part, to the rarity of these disorders. Cholesterol sup- Kelley RI, Wilcox WG, Smith M, Kratz LE, Moser A, Rimoin DS
plementation in conjunction with statin therapy has caused (1999) Abnormal sterol metabolism in patients with Conradi-
improved growth and development in one patient with Hunermann-Happle syndrome and sporadic lethal chondrodyspla-
sia punctata. Am J Med Genet 83:213–219
SC4MOL deficiency.
König A, Happle R, Bornholdt D, Engel H, Grzeschik KH (2000)
Mutations in the NSDHL gene, encoding a 3beta-hydroxysteroid
dehydrogenase, cause CHILD syndrome. Am J Med Genet
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Disorders of Adrenals and Gonads
37
Anna Lauber-Biason
Contents Summary
37.1 Introduction ................................................................... 602 Defects in steroid biosynthesis in adrenals and gonads
37.2 Nomenclature................................................................. 605 lead to complex and profound clinical consequences that
can be grouped in four categories: (1) defects of salt–water
37.3 Metabolic Pathways ...................................................... 606
homeostasis and sexual development (DSD), (2) defects of
37.4 Signs and Symptoms ..................................................... 607 salt–water homeostasis, (3) defects of sexual development,
37.5 Reference Values ........................................................... 612 and (4) end-organ steroid hormone resistance. Among
37.6 Pathologic Values/Differential Diagnosis .................... 614
the members of the first group, lipoid adrenal hyperpla-
sia is characterized by lack of all steroid hormones, with
37.7 Diagnostic Flowchart .................................................... 615 consequent 46, XY DSD and salt loss in the first weeks
37.8 Specimen Collection ...................................................... 615 of life. 17a-Hydroxylase deficiency leads also to 46, XY
37.9 Prenatal Diagnosis......................................................... 615 DSD associated with hypertension and hypokalemia.
3b-Hydroxysteroid dehydrogenase deficiency causes
37.10 Treatment Summary ..................................................... 615
incomplete virilization in male fetuses, together with salt
References .................................................................................... 616 loss. 21-Hydroxylase deficiency, whose nonclassic form
is one of the most common autosomal recessive diseases
in humans, is responsible for female ambiguous genitalia
at birth and salt loss. 11b-Hydroxylase deficiency differs
from 21-hydroxylase deficiency for the absence of salt
wasting and later presence of hypertension and hypokale-
mia. Defects of P450 oxidoreductase, a cofactor common
to 21-hydroxylase, 17a-hydroxylase, and aromatase, lead
to a complex combined defect of all three enzymes.
Enzymatic defects of the second group cause either salt-
wasting symptoms in the neonatal period, spontaneously
resolving in adulthood, as in the case of corticosterone methyl
oxidase II deficiency, or hypertension and hypokalemia as
in the cases of glucocorticoid-suppressible hyperaldosteron-
ism and apparent mineralocorticoid excess. The third group
of defects includes enzymatic blocks of the last steps of sex
hormones biosynthesis. 17,20-Lyase, 17b-hydroxysteroid
dehydrogenase, 5a-reductase, and aldo-keto reductase defi-
ciencies determine incomplete virilization of the male fetus.
In 17,20-lyase deficiency, there is no spontaneous puberty
in males and females; in the latter two male puberty occurs.
A. Lauber-Biason Aromatase deficiency is a cause of nonadrenal 46, XX DSD.
Faculty of Science, Department of Medicine,
The end-organ resistance syndromes, which are still an exclu-
University of Fribourg, Chemin du Musée 5,
Fribourg 1700, Switzerland sion diagnosis, represent a further challenge for future diag-
e-mail: anna.lauber@unifr.ch nostic and therapeutic applications. The treatment of these
602 A. Lauber-Biason
defects is based on exogenous administration of the deficient consequent compensatory rise of ACTH production causes
hormones and corrective surgery in intersexuality. Given the hyperplastic growth of the adrenal glands. Blocks of the ini-
rarity of most of these diseases, prenatal diagnosis is pos- tial steps of the steroidogenic pathway impair the production
sible only in a family at risk. In the case of 21-hydroxylase of all the three types of steroids, causing abnormalities in the
deficiency, however, advances in prenatal diagnosis allowed salt–water homeostasis and in sexual differentiation. That is
in utero treatment. Progress in molecular analysis of steroid the case in lipoid adrenal hyperplasia, where no conversion
biosynthesis and action defects will allow a better prenatal of cholesterol to any steroid takes place. This rare cause of
diagnosis and treatment of such diseases. CAH is characterized by salt loss and DSD in XY individu-
als. In XX subjects internal and external genitalia are female,
and the syndrome cannot clinically be separated from con-
37.1 Introduction genital adrenal hypoplasia (Prader and Gurtner 1955). The
molecular bases of such a defect have been recently clarified
The biosynthesis of steroid hormones is a fascinating process as mutations in the Steroidogenic Acute Response Protein
in which the neutral lipid cholesterol, a normal constituent of (StAR) (Lin et al. 1995). 17α-Hydroxylase deficiency leads
lipid bilayers, is transformed via a series of hydroxylation, to 46, XY DSD, due to the lack of precursors for testoster-
oxidation, and reduction steps into a vast array of biologi- one. In XX individuals, there is primary amenorrhea and
cally active compounds: mineralocorticoids, glucocorticoids, absent development of estrogenic secondary sexual charac-
and sex hormones. The majority of these transformations teristics. Both sexes display hypertension and hypokalemic
occur in the adrenal, testis, and ovary, although other tissues, alkalosis due to accumulation of mineralocorticoid precur-
such as liver, kidney, placenta, brain, and skin are also quite sors, which do not need 17α-hydroxylation for their synthe-
active. sis (Biglieri et al. 1966). Adrenal hyperplasia and
Steroid hormone action is a complex process that is only glucocorticoid deficiency are less marked than in the other
recently beginning to be understood. Steroid hormones bind types of CAH, because of the ability of corticosterone of
to specific intracellular receptors which upon dimerization suppressing ACTH. Male patients affected by CAH due to
interact with the DNA in the nucleus. As a result, gene activ- 3β-hydroxysteroid dehydrogenase deficiency display incom-
ity is modulated and a hormone-specific response occurs plete prenatal masculinization due to the impaired synthesis
(Evans 1988). It is logical that abnormalities in any step of of bioactive androgens and salt loss due to lack of mineralo-
this cascade will interfere with the action of a particular hor- corticoid (Bongiovanni 1962). XX subjects have normal
mone. Defects in the receptor or the post-receptor machinery female external genitalia or mild virilization due to the action
will lead for the most part to abnormalities of action of one of the weak androgen DHEA. 21-Hydroxylase deficiency
specific hormone. Abnormalities of steroid hormone produc- accounts for most cases of CAH (80–90 %, depending on the
tion, however, can result in more profound and complex ethnic group). Clinical consequences of 21-hydroxylase
effects. A block in the pathway of steroid biosynthesis leads deficiency arise from overproduction of androgens. Affected
to the lack of hormones downstream and accumulation of the females with the classical 21-hydroxylase deficiency are
upstream compounds that can activate other members of the born with ambiguous genitalia. Postnatally, untreated
steroid receptor family. The consequences of such defects patients of both sexes manifest rapid somatic growth with
can be schematically categorized in three groups: (1) defects accelerated skeletal maturation, early closure of the epiphy-
leading to defects of sexual development (DSD) and salt– ses, and short adult stature. Other symptoms include exces-
water balance, (2) defects leading to abnormalities of salt– sive pubic and body hair and decreased fertility. Seventy-five
water balance, and (3) defects leading to DSD. Abnormalities percent of patients with classic 21-hydroxylase deficiency
of end-organ action of steroid hormones, exemplified by the also have reduced synthesis of aldosterone with salt loss.
hormone insensitivity syndromes, can be grouped in a fourth Patients with nonclassical disease are born without symp-
category. toms of prenatal androgen exposure. Subsequently they may
The first group of steroid biosynthesis defects includes remain asymptomatic or may develop signs of androgen
the so-called congenital adrenal hyperplasia (CAH), a col- excess. Deficiency of 21-hydroxylase is inherited as an auto-
lective name given to a group of disorders characterized by somal recessive trait closely linked to the HLA major histo-
inherited inability of the adrenals to secrete cortisol. The compatibility complex on the short arm of chromosome 6.
37 Disorders of Adrenals and Gonads 603
While classic 21-hydroxylase deficiency is found in about 1 are generally normal. In 17,20-lyase (or 17,20-desmolase)
in 14.000 births, nonclassic deficiency is far more frequent, deficiency, there is no male or female pubertal development.
occurring in up to 3 % of persons among certain ethnic Cortisol is normal, and there is no hypertension. The defi-
groups (New et al. 1989). Steroid 11β-hydroxylase deficient enzyme is the same as in 17α-hydroxylase deficiency,
ciency, which is responsible for 10–20 % of cases of CAH, and it is unknown why the defect manifests itself as
produces symptoms of androgen excess similar to those in 17α-hydroxylase in some and as 17,20-lyase deficiency in
21-hydroxylase deficiency. The blocked enzymatic step also other families. Possibly, estrogen replacement induces a con-
results in accumulation of 11-deoxycorticosterone which has version to 17α-hydroxylase deficiency (Zachmann et al.
mineralocorticoid activity, leading in untreated patients to 1982). In 17β-HSD deficiency, there is some male pubertal
hypertension (New et al. 1989). development in XY individuals due to androstenedione and
The second group of diseases includes rare defects in the often gynecomastia due to estrone. In XX individuals, there
final step in the biosynthesis of mineralocorticoid and gluco- is mild virilization and insufficient development of estro-
corticoid that lead to water–sodium disequilibrium. Adrenal genic sexual characteristics. In 5α-reductase deficiency, male
corticosterone methyl oxidase II deficiency impairs the syn- puberty is present in XY subjects because testosterone is suf-
thesis of aldosterone with consequent salt loss in the neona- ficient and dihydrotestosterone (DHT) is not necessary for
tal period. Some patients, however, become completely expression of male sexual secondary characteristics. In XX
asymptomatic later in life. Glucocorticoid-suppressible individuals there are no symptoms. Interestingly, at the time
hyperaldosteronism is an autosomal dominant disease char- of expected puberty, the patients affected by these deficien-
acterized by mineralocorticoid hypertension due to an abnor- cies display some degree of virilization. Particularly high is
mal stimulatory action of ACTH on aldosterone synthesis. the incidence of 5α-reductase deficiency in the Dominican
This is due to unequal crossing over between the zona glo- Republic (Imperato-McGinley et al. 1986). Patients of both
merulosa 11β-hydroxylase (angiotensin II-regulated) and the sexes affected by aromatase deficiency show a delayed
zona fasciculata 11β-hydroxylase (ACTH-regulated) genes somatic development and slower skeletal maturation, with
(White and Pascoe 1992). Defects in the inactivation of cor- consequent tall adult stature. Female patients affected by
tisol, such as 11 β-hydroxysteroid dehydrogenase type II defi- aromatase deficiency display various degrees of genital
ciency, can lead to hypertension with hypokalemia in absence ambiguity, due to the lack of prenatal exposure to estrogens,
of elevated levels of mineralocorticoids. The mechanism and signs of hyperandrogenism, such as acne (Conte et al.
underlying such phenomenon is the prolonged half-life of 1994).
cortisol that binds to the relatively unselective mineralocorti- In the fourth class are grouped defects in the action of
coid receptor in the kidney and acts like a mineralocorticoid, steroid hormones due to receptor defect. Androgens exert
causing the so-called apparent mineralocorticoid excess syn- their effects in mediating the development of normal male
drome (New 1994). The disorder in which cortisone is over- phenotype via a single receptor protein, the androgen recep-
produced was tentatively named “apparent cortisone tor (AR), which is encoded on the X chromosome.
reductase deficiency” (AERD). This seems to be a rare con- Abnormalities that alter the function of this receptor result in
dition characterized by failure to convert cortisone to corti- a range of abnormalities of male phenotypic development,
sol, a reaction catalyzed by 11β-hydroxysteroid called complete and partial androgen insensitivity syndrome
dehydrogenase type I. This disease is caused by an increased (CAIS and PAIS, respectively). These phenotypes range
metabolic rate for cortisol at the expense of ACTH-mediated from normal female (female habitus, normal female breast
androgen excess, without any clinical symptoms of hyper- development, absent pubic and axillary hair, female external
cortisolism (Biason-Lauber et al. 2000). genitalia, no internal genital organs, and undescended testes)
The third group is characterized by deficiencies of to those that are characterized by only minor degrees of
enzymes responsible for the final steps of sex hormone syn- undervirilization and/or infertility (McPhaul and Griffin
thesis, such as 17,20-lyase, 17 β-hydroxysteroid dehydroge- 1999).
nase (17β-HSD), 5α-reductase, and aromatase. Deficient Estrogen resistance was described only in one case (Smith
activity of 17,20-lyase, 17β-HSD, and 5α-reductase enzymes et al. 1994) in a 28-year-old man. He was 204 cm tall and had
leads to male pseudohermaphroditism with varying genital incomplete epiphyseal closure, with a history of continued
ambiguity in XY individuals. In XX individual the genitalia linear growth into adulthood despite otherwise normal
pubertal development. He was normally masculinized and P450c21 , and CYP51A1 (Miller 1986). In 1985, a clinical
had bilateral axillary acanthosis nigricans. Glucose tolerance report described a patient with genital ambiguity and an
was impaired and hyperinsulinemia was present. Although abnormal urinary steroid profile suggesting partial combined
rare, the estrogen resistance was of crucial importance for deficiencies of what was then thought to be three distinct ste-
the understanding of skeletal physiology, since it demon- roidogenic enzymes: 17α-hydroxylase, 17,20 lyase, and
strated that estrogen is important for bone maturation and 21-hydroxylase (Peterson et al. 1985). Affected girls are
mineralization in men as well as in women. born with ambiguous genitalia, indicating intrauterine andro-
Progesterone prepares the endometrium for blastocyst gen excess. After birth, however, virilization does not prog-
implantation and allows maintenance of pregnancy. ress and amounts of circulating androgens are low or normal.
Complete end-organ resistance to progesterone would be Conversely, affected boys are sometimes born undermascu-
incompatible with reproductive competence in females. linized. The majority but not all of patients described to date
Males would not be expected to be affected since proges- with POR deficiency also had a pattern of skeletal malforma-
terone has no known function in men. Failure of the uterus tions termed Antley–Bixler syndrome (ABS). This disorder
to respond to progesterone would lead to the development is characterized by craniosynostosis (premature fusion of
of a “constantly proliferative” endometrium incompatible bones of the skull), radioulnar or radiohumeral synostosis,
with blastocyst implantation. Partial resistance to proges- bowed femora, and other more variable skeletal disorders
terone, on the other hand, would be expected to be associ- (Antley and Bixler 1975; Crisponi et al. 1997). Findings of
ated with various degrees of incomplete maturation of the biochemical investigations of urinary steroid excretion in
endometrium, expressed clinically as infertility or early affected patients have shown accumulation of steroid metab-
abortions. The syndrome presents with the clinical and his- olites, indicating impaired C17 and C21 hydroxylation, sug-
tologic picture of a luteal phase defect in which the life gesting concurrent partial deficiencies of the 2 steroidogenic
span of the corpus luteum and the plasma progesterone enzymes, P450C17 and P450C21. As these steroidogenic
concentrations are normal or slightly elevated (Chrousos enzyme activities were known to be catalyzed by type II
et al. 1986). cytochrome P450 enzymes, it was suggested that this patient
Glucocorticoid resistance is characterized by high levels had a disorder in the electron donor for these enzymes, P450
of cortisol (without stigmata of Cushing syndrome), resis- oxidoreductase (Miller 1986). This fascinating idea remained
tance of the hypothalamic–pituitary–adrenal axis to dexa- dormant until Flueck first reported four cases of POR defi-
methasone, and an affinity defect of the glucocorticoid ciency (Fluck et al. 2004). Since that time, about 50 addi-
receptor. Some of the affected patients presented with tional patients have been reported (for review see Fluck et al.
hypertension and hypokalemia due to illegal activation of 2008; Scott and Miller 2008).
the mineralocorticoid receptor by cortisol (Lipsett et al. The other defect concerns the so-called backdoor path-
1986). way of fetal androgen synthesis (Fig. 37.1b). Following
Pseudohypoaldosteronism (type I) is characterized by salt development of the fetal bipotential gonad into a testis, male
wasting in infancy that is responsive to supplementary genital differentiation requires testicular androgens. Fetal
sodium but not to mineralocorticoids. Marked aldosterone Leydig cells produce testosterone that is converted to dihy-
excess is present in all reported cases, and the renin level is drotestosterone in genital skin, resulting in labioscrotal
increased in most. Salt supplementation often can be discon- fusion. An alternative “backdoor” pathway of dihydrotestos-
tinued after infancy without adverse effects, even though terone synthesis that bypasses testosterone has been
aldosterone excess is persistent. Sweat and salivary glands described in marsupials, but its relevance to human biology
and colonic mucosa are unresponsive to mineralocorticoids has been uncertain. The classic and backdoor pathways share
as is the distal renal tubule. The basic defect in this disease many enzymes, but a 3α-reductase, AKR1C2, is unique to
resides in the mineralocorticoid receptor NR3C2 (Geller the backdoor pathway (Fig.37.1). Human AKR1C2 muta-
et al. 1998). tions cause disordered sexual differentiation, establishing
More recently, two new defects in steroid synthesis were that both pathways are required for normal human male gen-
identified. An intriguing case is that of P450 oxidoreductase ital development. These observations show that fetal dihy-
(POR) deficiency. Cytochrome P450 oxidoreductase is a fla- drotestosterone acts both as a hormonal and as a paracrine
voprotein that donates electrons to all microsomal P450 factor, substantially revising the classic paradigm for fetal
enzymes, including the steroidogenic enzymes P450c17 , male sexual development (Fluck et al. 2011).
37
37.2 Nomenclature
Chromosomal OMIM
37.1 Lipoid adrenal hyperplasia StAR deficiency StAR deficiency STAR 8p11.2 Steroidogenic acute 201710 All forms
regulatory protein
37.2 Cholesterol side-chain cleavage P450scc CYP11A1 15q23–q24 P450 side-chain cleavage 118485 All forms
deficiency
37.3 17-Alpha-hydroxylase Congenital adrenal hyperplasia P450c17 deficiency CYP17A1 10q24–25 Cytochromome P450 202110 All forms
17-alpha-hydroxylase
37.4 3-Beta-hydroxysteroid Congenital adrenal hyperplasia 3-Beta-HSD HSD3B2 1p11-q13 3-Beta-hydroxysteroid 201810 All forms
dehydrogenase type II deficiency dehydrogenase type II
37.5 21-Hydroxylase deficiency Congenital adrenal hyperplasia CAH CYP21A2 6p21.3 P450 21-hydroxylase 201910 All forms
37.6 11-Beta-hydroxylase type I Congenital adrenal hyperplasia CAH CYP11B1 8q21–22 P450 11-beta- 202010 All forms
deficiency hydroxylase type 1
37.7 Corticosterone methyl oxidase Aldosterone synthase deficiency CMO deficiency CYP11B2 8q21–22 Corticosterone methyl 124080 All forms
deficiency oxidase
37.8 Glucocorticoid-suppressible GRA CYP11B1/B2 8q21–22 11-Beta-hydroxylase I/II 103900 All forms
hyperaldosteronism
37.9 Apparent mineralocorticoid excess 11-Beta-hydroxysteroid AME HSD11B2 16q22 11-Beta-hydroxysteroid 218030 All forms
dehydrogenase 2 deficiency dehydrogenase type 2
37.10 Cortisone reductase deficiency 11-Beta-hydrosteroid CRD HSD11B1/H6PDH 1q36/1q32-q41 11-Beta-hydrosteroid 600713 All forms
dehydrogenase type 1 defect dehydrogenase type 1
37.11 17,20-Lyase deficiency P450c17 deficiency CYP17A1 10q24–25 Cytochromome P450 202110 All forms
17-alpha-hydroxylase
37.12 17-Beta-hydroxysteroid 17β-HSD HSD17B3 9 17-Beta-hydroxysteroid 264300 All forms
dehydrogenase deficiency deficiency dehydrogenase type 3
37.13 5-Alpha-reductase type II Pseudovaginal perineoscrotal 5-Alpha-reductase SRD5A2 2p23 5-Alpha-reductase 264600 All forms
deficiency hypospadia deficiency type II
37.14 Aromatase deficiency Aro deficiency CYP19A1 15q21.1 P450 aromatase 107910 All forms
37.15 P450 oxidoreductase deficiency POR deficiency POR 7q11.2 P450 oxidoreductase 613571 All forms
37.16 Androgen insensitivity syndrome Testicular feminization AIS AR Xq11-q12 Androgen receptor 300068 Classic form
37.17 Estrogen resistance Estrogen receptor defect ESR1 6q25.1 Estrogen receptor 133430 All forms
37.18 Progesterone resistance PGR defect PGR Progesterone receptor 264080 All forms
37.19 Glucocorticoid resistance Glucocorticoid receptor defect GCCR defect NR3C1 5q31 Glucocorticoid receptor 138040 All forms
37.20 Pseudohypoaldosteronism Mineralocorticoid resistance PHA1 NR3C2 4q31.1 Mineralocorticoid 264350 All forms
receptor
37.21 3a-Hydroxysteroid dehydrogenase Aldo-keto reductase 2 deficiency, AKR1C AKR1C 10p15.1 AKR1C2 600450 All forms
deficiency backdoor pathway defect
605
Fig. 37.1 (a) Classical a Cholesterol Classic pathway

pathway of steroidogenesis.
(b) The alternative pathway P450scc/StAR
of fetal androgen synthesis P450 c17 P450c17/Cytb5
Pregnenolone 17OH-Pregnenolone DHEA
3β HSD POR 3β HSD POR 3β HSD
Progesterone
P450 c17 17OH-Progesterone P450 c17 Androstenedione
POR
P450c21 P450c21
Deoxycorticosterone 11-Deoxycortisol
(DOC) 17β HSD3
P450c11
P450c11
Corticosterone Cortisol Testosterone

(B) Aldo synthase
(Corticosterone Methyl 11βHSD1
H6PDH 11 HSD2 5α-Reductase2 P450Aro
Oxidase I)
18OH Corticosterone Cortisone
Aldo synthase DIHYDROTESTOSTERONEEstradiol
(Corticosterone Methyl (DHT)
Aldosterone Oxidase II)
MINERALOCORTICOIDS GLUCOCORTICOIDS SEX HORMONES

(Salt) (Sugar/Stress) (Sex)
b
Backdoor pathway
Cholesterol
P450scc/StAR
P450 c17 P450c17/Cytb5
Pregnenolone 17OH-Pregnenolone DHEA
POR 3βHSD POR
3 HSD 3βHSD
P450 c17 P450 c17 Androstenedione
Progesterone 17OH-Progesterone
5α-Reductase1
17OH-DHP 17βHSD3
AKR1C2/4 (red)
17OH-Allopregnanolone Testosterone
P450 c17 5α-Reductase2

POR
Androsterone Androstanedione
17βHSD
Androstanediol
AKR1C4/RODH(Ox)
DIHYDROTESTOSTERONE
(DHT)
Table 37.1 Lipoid adrenal hyperplasia

Endocrine Adrenal hyperplasia +++ +++ +++ +++ +++
Adrenal insufficiency +++ +++ +++ +++ ++
Puberty, delayed n.a. n.a. n.a. +++ +++
Genitourinary Cryptorchidism +++ +++ +++ +++ +++
Female external genitalia regardless genetic sex +++ +++ +++ +++ +++
Female to ambiguous genitalia in 46, XY +++ +++ +++ +++ +++
Other Dehydration +++ ++ ++ + +
Routine laboratory Alkalosis ++ ++ + + +
Potassium (P) ↑↑↑ ↑↑↑ ↑↑ ↑↑ ↑↑
Sodium (P) ↓↓↓ ↓↓↓ ↓↓ ↓-n ↓-n
Special laboratory ACTH (P) ↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
Steroids (U, P) ↓↓↓ ↓↓↓ ↓↓ ↓-n ↓-n
Table 37.2 Cholesterol side-chain cleavage deficiency

Endocrine Adrenal hyperplasia − − − − −
Adrenal insufficiency +++ or +/− ++ or +/− ++ or +/− + or +/− + or +/−
Genitourinary Ambiguous genitalia in 46, XY ++ ++ ++ ++ ++
Cryptorchidism +++ +++ +++ if not +++ if not +++ if not
corrected corrected corrected
Routine laboratory Potassium (P) ↑↑ ↑↑ ↑ n-↑ n-↑
Sodium (P) ↓↓ ↓↓ ↓ ↓-n ↓-n
Table 37.3 17-Alpha-hydroxylase

CNS Headache ± ± + ++ +++
Digestive Vomiting, episodic ± ± + ++ +++
Endocrine Adrenal hyperplasia + + + + +
Adrenal insufficiency − − − − −
Puberty, delayed n.a. n.a. n.a. +++ +++
Genitourinary Cryptorchidism +++ +++ +++ +++ +++
Female external genitalia regardless genetic sex +++ +++ +++ +++ +++
Lack of pubertal sex development n.a. n.a. n.a. +++ +++
Ovarian cysts − − ± ++ +++
Renal Hypertension − ± + ++ +++
Routine laboratory Alkalosis + + + ++ ++
Potassium (P) ↓ ↓ ↓ ↓↓ ↓↓
Sodium (P) (↑) (↑) ↑ ↑↑ ↑↑
Special laboratory ACTH (P) (↑) (↑) (↑) n n
Cortisol, sex hormones, aldosterone (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Deoxycorticosterone, corticosterone (U,P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Progesterone (P) ↑↑ ↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 37.4 3-Beta-hydroxysteroid dehydrogenase type II

Endocrine Adrenal hyperplasia ++ ++ ++ + +
Adrenal insufficiency +++ +++ ++ ++ ++
Genitourinary Ambiguous genitalia +++ +++ +++ +++ +++
Clitoral hypertrophy ++ ++ ++ +++ +++
Cryptorchidism ++ ++ ++ ++ ++
Virilization + ++ ++ ++ ++
Other Dehydration +++ ++ ++ + +
Routine laboratory Potassium (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Sodium (P) ↓↓↓ ↓↓↓ ↓↓ ↓↓ ↓
Special laboratory 17-OH-pregnenolone (P) ↑↑ ↑↑ ↑↑ ↑↑↑ ↑↑↑
ACTH (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Cortisol, sex hormones, aldosterone (P) ↓↓ ↓↓ ↓↓ ↓↓↓ ↓↓↓
DHEA sulfate (P) ↑↑ ↑↑ ↑↑ ↑↑↑ ↑↑↑
Table 37.5 21-Hydroxylase deficiency

Endocrine Adrenal hyperplasia + ++ +++ +++ +++
Adrenal insufficiency +++ +++ +++ ++ ++
Genitourinary Fertility, decreased n.a. n.a. n.a. n.a. n.a.
Genital ambiguity in 46, XX +++ +++ ++ ++ ++
Virilization n.a. + ++ +++ +++
Musculoskeletal Short stature n.a. n.a. n.a. ++ +++
Other Accelerated growth ± ± ++ +++ n.a.
Dehydration +++ ++ + ± ±
Dehydration +++ +++ ++ ++ only in stress ++ only in stress
Routine laboratory Potassium (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
Sodium (P) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓ ↓↓
Special laboratory 17-OH-progesterone (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
ACTH (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Aldosterone (P) ↓ ↓↓ ↓↓ ↓ ↓
Androgens (DHEAS, ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
androstenedione, testosterone) (P)
Cortisol (P) ↓-n ↓-n ↓-n n n
Progesterone, 17-OH-progesterone ↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
and androgen metabolites (U)
Renin activity or renin (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 37.6 11-Beta-hydroxylase type I deficiency

Cardiovascular Hypertension ± ± + ++ +++
CNS Headache ± ± + ++ ++
Digestive Vomiting, episodic ± ± + ++ ++
Endocrine Adrenal hyperplasia ++ ++ ++ ++ ++
Adrenal insufficiency − − − − −
Genitourinary Fertility, decreased n.a. n.a. n.a. + ++
Genital ambiguity in 46, XX +++ +++ +++ +++ +++
Genital ambiguity in 46, XX ++ ++ ++ ++ ++
Musculoskeletal Short stature − − − ± +++
Other Accelerated growth ± ± ++ +++ n.a.
Routine laboratory Potassium (P) ↓↓ ↓↓ ↓ ↓↓ ↓↓
Sodium (P) ↑ ↑↑ ↑↑ ↑↑ ↑↑
Special laboratory 17-OH-progesterone (P) ↑ ↑ ↑ ↑ ↑
ACTH (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Deoxycorticosterone, 11-deoxycortisol ↑↑ ↑↑ ↑↑↑ ↑↑↑ ↑↑↑
(compound S), androgens (P)
Table 37.7 Corticosterone methyl oxidase deficiency

Digestive Failure to thrive ++ ++ ± − −
Other Dehydration ++ ++ ± − −
Routine laboratory Potassium (P) ↑↑ ↑ n-↑ n n
Sodium (P) ↓↓ ↓ ↓-n n n
Special Laboratory 18-Oxocortisol (U) ↑↑ ↑↑ n-↑ n n
Aldosterone (P) ↓↓ ↓↓ ↓-n n n
Aldosterone/renin ratio ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 37.8 Glucocorticoid-suppressible hyperaldosteronism

CNS Headache ± ++ ++ +++
Routine laboratory Potassium (P) ↓ ↓↓ ↓↓ ↓↓ ↓↓
Special laboratory 18-Oxocortisol (U) ↑ ↑↑ ↑↑ ↑↑
Aldosterone (P) ↑ ↑↑ ↑↑ ↑↑
Aldosterone after dexamethasone (P) n n n n
Table 37.9 Apparent mineralocorticoid excess

CNS Headache ± + ++ ++
Routine laboratory Potassium (P) ↓ ↓↓ ↓↓ ↓↓ ↓↓
Sodium (P) ↑ n-↑ n-↑ n-↑ n-↑
Special laboratory Tetrahydrocortisol/tetrahydrocortisone ratio (U) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 37.10 Cortisone reductase deficiency

Endocrine Adrenal hyperplasia ± ± ± + +
Precocious pseudopuberty in 46, XY n.a n.a. + ++ n.a.
Genitourinary Polycystic ovaries n.a. n.a. n.a. ++ +++
Virilization − − − +++ +++
Special laboratory ACTH (P) n-↑ ↑ ↑↑ ↑↑
Adrenal androgens (DHEAS, androstenedione) (P) n.a. n.a. ↑ ↑↑↑ ↑↑↑
Tetrahydrocortisol/tetrahydrocortisone ratio (U) ↓ ↓ ↓↓ ↓↓
Table 37.11 17,20-Lyase deficiency

Endocrine Adrenal hyperplasia − − − − −
Genitourinary Ambiguous genitalia in 46, XY +++ +++ +++ +++ +++
Cryptorchidism + + + + +
Special laboratory 17-OH-progesterone (P) ↑ ↑ ↑ n-↑ n-↑
Cortisol, aldosterone (P) n n n ↓-n under HRT ↓-n under HRT
Progesterone (P) n n n n n
Sex hormones n.a. n.a. ↓↓ ↓↓ ↓↓↓
Table 37.12 17-Beta-hydroxysteroid dehydrogenase type III deficiency

Endocrine Gynecomastia at puberty − − − ++ +
Genitourinary Cryptorchidism +++ ++ ++ ++ +
Special laboratory Androgens (DHEAS, androstenedione, testosterone) (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Androstenedione (P) n-↑ n-↑ n-↑ n-↑ n-↑
Gonadotropins (FSH, LH) (P) n-↑ n-↑ ↑ ↑↑ ↑↑↑
Testosterone (P) ↓↓ ↓↓ ↓↓ ↓↓↓ ↓↓↓
Table 37.13 5-Alpha-reductase type II deficiency

Genitourinary Cryptorchidism +++ +++ +++ ++ ++
Female to ambiguous genitalia +++ +++ +++ ++ ++
in 46, XY
Virilization − − − +++ +++
Virilization n.a. n.a. n.a. +++ ++
Special laboratory 5-α/5-β reduced metabolites ratio (U) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Dihydrotestosterone (DHT) (P) ↓↓ Usually not Usually not ↓↓↓ ↓↓↓
measurable without measurable without
hCG stimulation hCG stimulation
Testosterone (P) n n n n-↑ n-↑
Testosterone/dihydrotesterone ratio ↑↑ n.m. n.m. ↑↑ ↑↑
Table 37.14 Aromatase deficiency

Genitourinary Genital ambiguity in 46, XX +++ +++ +++ +++ +++
Ovarian cysts − − ± ++ ++
Virilization − − ± ++ ++
Musculoskeletal Somatic development, delayed − − + ++ +++
Stature, tall n.a. n.a. − ++ +++
Other Maternal virilization during pregnancy ++ ++ ++ ++ ++
Obesity (male) − − − ++ +++
Special laboratory Gonadotropins (FSH, LH) (P) ↑ n.a. n.a. ↑↑ ↑↑↑
Table 37.15 P450 oxidoreductase deficiency

Genitourinary Ambiguous genitalia +++ ++ ++ ++ ++
Genital ambiguity in 46, XX +++ +++ +++ +++ ++
Virilization ++ ± − − −
Musculoskeletal Antley–Bixler syndrome ++ ++ ++ ++ ++
Skeletal abnormalities + + + + +
Special laboratory 17-OH-progesterone (P) ↑↑ n n n n
Androgens (DHEAS, androstenedione, testosterone) (P) ↑↑ ↓-n ↓-n ↓-n ↓-n
Table 37.16 Androgen insensitivity syndrome

Dermatological Absent pubic and axillary hair n.a. n.a. +++ +++ +++
Genitourinary Absent uterus +++ +++ +++ +++ +++
Cryptorchidism +++ +++ +++ +++ +++
Special Laboratory Androgens (DHEAS, androstenedione, n-↑ n n n for 46, XY n for 46, XY
testosterone) (P)
Anti-Müllerian hormone (AMH) (P) n for 46, XY n for 46, XY n for 46, XY n for 46, XY n for 46, XY
Dihydrotestosterone (DHT) (P) n n n n n
Gonadotropins (FSH, LH) (P) n-↑ n n n-↑ n-↑
Table 37.17 Estrogen resistance

Endocrine Glucose intolerance n n n ↓ ↓↓
Genitourinary Genitalia n n n n n
Musculoskeletal Incomplete epiphyseal closure n.a. n.a. n.a. +++ +++
Somatic development, delayed n.a. n.a. + ++ +++
Stature, tall n.a. n.a. + ++ ++
Special laboratory Androgens (DHEAS, androstenedione, testosterone) (P) n n n n n
Estradiol (P) (↑) (↑) (↑) ↑↑ ↑↑
Gonadotropins (FSH, LH) (P) (↑) (↑) ↑ ↑↑ ↑↑↑
Table 37.18 Progesterone resistance

Genitourinary Endometrium n.a. n.a. n.a. Immature Immature
Infertility, female n.a. n.a. n.a. n.a. +++
Special laboratory Progesterone (P) n.a. n.a. n.a. Inadequately low in luteal phase Inadequately low in luteal phase
Table 37.19 Glucocorticoid resistance

Cardiovascular Hypertension ± ++ +++
Endocrine Cushing stigmata − − − − −
Routine laboratory Alkalosis ± ± + ++ ++
Hypokalemic alkalosis − − ± ++ +++
Potassium (P) n n ↓-n ↓↓ ↓↓
Special laboratory ACTH (P) n-↑ n-↑
Cortisol (P) n n n ↑ ↑
Cortisol (U) n n n ↑↑ ↑↑
Cortisol after dexamethasone Unresponsive (no Unresponsive
suppression (U) suppression) (no suppression)
Table 37.20 Pseudohypoaldosteronism

Renal Renal salt loss ± ± + ++ ++
Routine laboratory Potassium (P) ↑ ↑ ↑ ↑ ↑
Sodium (P) ↓↓ ↓↓ ↓ ↓ ↓
Special laboratory Aldosterone (P) ↑ ↑↑ ↑↑ ↑ ↑
Renin activity or renin (P) ↑ ↑ ↑ ↑ ↑
Table 37.21 3 Alpha hydroxysteroid dehydrogenase type III (AKR1C2)-deficiency

Characteristic clinical findings Genital ambiguity in 46, XY + + + + +
Special laboratory Testosterone n n n n n
DHT ↓ n n n n
37.5 Reference Values In the case of the semiquantitative urinary steroid profile using
gas chromatography–mass spectrometry (GC-MS), the inter-
Important note: Steroid hormone reference values vary enor- pretation of the results is even more complex and should be
mously depending on the assay and the clinical chemistry made by an expert in such assay. For these reasons, only the
laboratory. Therefore, the following values must be consid- diagnostic ratios are mentioned in this chapter
ered an example and not be used for everyday clinical practice. For more information see also Shackleton
37
Plasma
ACTH 17OHP D4A DOC

Age pmol/L FSH/LH mIU/mL nmol/L DHEA nmol/L nmol/L T nmol/L DHT nmol/L E2 pmol/L Aldo pmol/L pmol/L B pmol/L F nmol/L
<6 years 4.4–22.2 N/A 0.3–9.5 Male, 0.9–4.5; N/A Male, 0.28–0.49; Male, <0.1–0.4; Male, <37; N/A 1.5–7.5 0.6–2.16 AM
female, 0.7–4.5 female, 0.17–0.45 female, <0.1–0.3 female, <26 171–536
PM
64–340
8–10 years 4.4–22.2 N/A 0.3–9.5 Male, 1.8–4.7; N/A Male, 0.28–0.49; Male, <0.1–0.4; Male, <37; m, 83–250; 1.5–7.5 0.6–2.16 AM
female, 2.6–6.2 female, 0.17–0.45 female: <0.1–0.3 female, 30–65 j,11–832a 171–536
PM
64–340
10–12 years 4.4–22.2 N/A 0.3–9.5 Male, 6.3–12.3; N/A Male, 0.28–0.49; Male, <0.1–0.4; Male, <37; m. 83–250; 1.5–7.5 0.6–2.16 AM
female, 8.1–18.3 female, 0.17–0.45 female, <0.1–0.3 female, 30–65 j, 11–832 171–536
PM
64–340
14–16 years 4.4–22.2 FSH male, 2–17; 0.3–9.5 Male: 8.3–18; 1.4–7.9 Male, 2.91–6.24; Male, 1–10.4; Male, 62–85; m, 83–250; 1.5–7.5 0.6–2.16 AM
female, 4–20 female: 7.8–21.2 female, 0.31–0.83 female, 0.2–1.1 female, 73–250 j,11–832 171–536
LH male, 4–18; PM
female, 5–25 64–340
Adult ″ ″ 0.3–9.5 Male, 10.6–29; 1.4–7.9 Male, 10.4–34.7; Male, 1–10.4; Male, 62–184; m, 83–250, 1.5–7.5 0.6–2.16 AM
female, 9.8–27.7 female, 1.04–2.43 female, 0.2–1.1 female, follicular j, 11–832 171–536
73–367 and luteal PM
367–1836 64–340
ACTH adrenocorticotropin hormone, FSH follicle-stimulating hormone, LH luteinizing hormone, 17OHP 17-hydroxy progesterone, DHEA dehydroepiandrosterone, D4A androstenedione, T
testosterone, DHT dihydrotestosterone, E2 estradiol, Aldo aldosterone, DOC deoxycorticosterone, B corticosterone, F cortisol
Notes: asodium intake 100–200 meq/day recumbent (m), upright (j); sodium intake 10 meq/day recumbent, 333–999; upright, 472–3,800
613
37.6 Pathologic Values/Differential Diagnosis
Plasma
Disease ACTH FSH/LH 17OHP DHEA D4A T DHT E2 Aldo DOC B F

37.1 ↑↑↑ ↑ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
37.2 ↑ ↑-n ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓-n
37.3 ↑-n ↑ ↓ ↓ ↓ ↓ ↓ ↓ n ↑↑ ↑↑ n
37.4 ↑↑ ↑↑ ↑ ↑↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓-n
37.5 ↑↑ ↑ ↑↑↑ ↑↑ ↑↑↑ ↑↑ ↑↑ n n ↓-n ↓-n n-↓
37.6 ↑ ↑ ↑ ↑↑ ↑↑↑ ↑↑ ↑ n n ↑↑ ↓ n
37.7 n n n n n n n n ↓ ↑ ↑1 n
37.8 n n n n n n n n ↑2 n-↑ n-↑ n
37.93 n n n n n n n n n n n n
37.10 n-↑ n n-↑ ↑ ↑ ↑ ↑ n n n n ↑
37.11 n ↑↑ ↑ ↓ ↓ ↓ ↓ ↓ n n n n
37.12 n ↑↑ n n ↑ ↓ ↓ ↓ n n n n
37.13 n ↑ n n n n-↑ ↓↓ n n n n n
37.14 n ↑↑ n n n ↑ ↑ ↓↓ n n n n
37.15 n-↑ ↑ ↑-↓ ↑-↓ ↑-↓ ↓- ↓-↑ ↓ n n n n
37.16 n ↑ n (XY) n (XY) n (XY) n (XY) n (XY) ↑ (XY) n n n n
37.17 n n n n n n n ↑↑ n n n n
37.18 n ↑↑ n-↑ n n n n n n n n n
37.19 n-↑ n n n n n n n n n n ↑↑
37.20 n n n n n n n n ↑ ↑ n-↑ n
37.21 n n n n n n n n n n n n
ACTH adrenocorticotropin hormone, FSH follicle-stimulating hormone, LH luteinizing hormone, 17OHP 17-hydroxy progesterone, DHEA dehy-
droepiandrosterone, D4A androstenedione, T testosterone, DHT dihydrotestosterone, E2 estradiol, Aldo aldosterone, DOC deoxycorticosterone,
B corticosterone, F cortisol
Notes: 1. Low 18-oxocortisol (aldosterone urinary metabolite, not detectable via gas chromatographic urine profile). 2. Elevated 18-oxocortisol
(aldosterone urinary metabolite not detectable via gas chromatography/mass spectrometry urine profile). 3. No direct metabolite of testosterone
measurable not detectable via gas chromatographic urine profile
Special Tests
Disorder Test Procedure Results

37.9 Sodium (P) restriction 3–5 days of 10 mmol sodium intake. Increase two to threefold
Aldosterone morning plasma levels over basal levels
37.8 Dexamethasone suppression test 1 mg dexamethasone for 2 days p.o. Decrease of aldosterone
37.17 Aldosterone morning plasma levels Decrease of cortisol
Urine
Pathologic F/E >1

37.2 (17α-Hydroxylase deficiency) (THF + 5aTHF)/THE >3
(THA + THB + 5aTHB)/(THE + THF + 5aTHF) >10 (F + E)/(THE + THF + 5aTHF) >0.1
(THA + THB + 5aTHB)/(AN + ET) >10 37.10 (11ß-HSD1 deficiency)
100*THDOC/(THE + THF + 5aTHF) >10 (THF + 5aTHF)/THE <0.1
37.5 (21-Hydroxylase deficiency) 37.13 (5α-Reductase deficiency)
17HP/(THE + THF + 5aTHF) >0.25 ET/AN >5
PT/(THE + THF + 5aTHF) >0.25 THF/5aTHF >10
100*PT’ONE/(THE + THF + 5aTHF) >10 37.20 (Pseudohypoaldosteronismus)
37.6 (11β-Hydroxylase deficiency) THALDO (μg/24 h) >90
100*THS/(THE + THF + 5aTHF) >10 *100*THALDO/(THE + THF + 5aTHF) >5
100*THDOC/(THE + THF + 5aTHF) >10 THA tetrahydro compound A, THB tetrahydrocorticosterone, THE
37.7 (CMO deficiency) tetrahydrocortisone, THF tetrahydrocortisol, AN androsterone,
18OHTHA/THALDO >20 ET etiocholanolone, THDOC tetrahydrodeoxycortisone, 17HP
metabolites of 17-OH-progesterone (pregnanediol, pregnanetriol,
(THA + THB + 5aTHB)/(THE + THF + 5aTHF) >1
17-OH-pregnanolone), PTONE pregnanetriolone, THALDO tetrahy-
37.9 (11ß-HSD2 deficiency) droaldosterone, THS tetrahydrodeoxycortisol
Abnormal external genitalia
Gonads palpable Gonads not palpable
17OH-Progesterone Normal Normal Normal High Normal or low

or low
Karyotype XY poly X+ Y XX, XY or XX XX XY

XX/XY or X0/XY
46 XY, DSD Seminiferous Ovotesticular DSD 46, XX DSD Congenital Male Gonadal
(Male pseudohermaphroditism)* tubule dysgenesis (True hermaphroditism) Non-adrenal adrenal dysgenesis
(Klinefelter syndrome female pseudo- hyperplasia
and its variants) hermaphroditism (37.4, 37.5)
(37.13)
Ovotesticular DSD
(True Hermaphroditism) 46XY, DSD*
(37.1–6, 37.11–16, 37.21)
Fig. 37.2 Simplified diagnostic flowchart in case of abnormal external genitalia in a neonate
37.7 Diagnostic Flowchart Initial studies: plasma 17-OH-progesterone, DHEA,

androstenedione, testosterone, DHT, and estradiol
Diagnosis of Disorders of Sex Development (DSD) and Serum electrolytes
Intersexuality Sonogram of kidneys, ureters, and pelvic content
1. History: family history, pregnancy (hormones, virilization) 2. Provisional diagnosis: “abnormal external genita-
Palpation of inguinal region and labioscrotal folds: rectal lia” → proceed as in Fig. 37.2.
examination, karyotype

Plasma or serum quantitative None Frozen plasma or serum Keep frozen (−20 °C) None, except laboratory
steroids, ACTH and gonadotropins until analyzed error
Urine for gas chromatography– None Fresh or frozen 24 h urine (discard Keep frozen (−20 °C) None, except laboratory
mass spectrometry analysis of the morning urine of the first day but until analyzed error
urinary metabolites collect the morning urine of the
second and final day)

Disorder Material Timing, trimester
All disorders (except 37.9 and 37.15–18 that are adult-onset diseases) CV, AF I, II
37.10 Treatment Summary from case to case. It is therefore mandatory the creation of a
multidisciplinary team of experts especially when the
The therapeutic management in disorders of steroid synthe- patient has a DSD and not try to manage these complex
sis and action can be distinguished in two phases: cases alone.
1. Management of life-threatening situations (adrenal cri-
sis): emergency Phase 1: Initial Treatment
2. Management of lifelong consequences: no emergency Disorders 37.1, 37.2, 37.5, 37.7, 37.20 (salt-losing forms)
Whereas the therapeutic intervention in phase 1 is 1. Parenteral isotonic saline
straightforward, those for phase 2 are complex and different 2. Hydrocortisone (cortisol)
Note: salt loss symptoms do not appear before 6–10 days Chrousos GP, McLusky NJ, Brandon DD, Tomita M, Renquist DM,
after birth Loriaux DL, Lipsett MB (1986) Progesterone resistance. In:
Chrousos GP, Loriaux DL, Lipsett MB (eds) Steroid hormone resis-
Disorders 37.3, 37.6, 37.8, 37.9, (hypertensive, hypokale- tance: mechanisms and clinical aspects. Plenum Press, New York
mic forms) Conte FA, Grumbach MM, Ito Y, Fisher CR, Simpson ER (1994) A
1. Restoration of potassium concentrations syndrome of female pseudohermaphroditism, hypergonadotropic
2. Diuretic therapy to reduce blood pressure hypogonadism, and multicystic ovaries associated with missense
mutations in the gene encoding aromatase (P450arom). J Clin
Endocrinol Metab 78:1287–1292
Phase 2: Disorders 37.1–6, 37.11–16, 37.21 (DSD) Crisponi G, Porcu C, Piu ME (1997) Antley-Bixler syndrome: case
1. Sex assignment report and review of the literature. Clin Dysmorphol 6:61–68
2. Reconstructive genital surgery Evans RM (1988) The steroid and thyroid hormone receptor superfam-
ily. Science 240:889–895
After the adrenal emergency has been managed, type and Fluck CE, Tajima T, Pandey AV, Arlt W, Okuhara K, Verge CF, Jabs EW,
timing of genital surgery, the correspondent sex assignment, Mendonca BB, Fujieda K, Miller WL (2004) Mutant P450 oxidore-
and necessity and timing of gonadectomy should be discussed ductase causes disordered steroidogenesis with and without Antley-
case-by-case in a multidisciplinary team of experts in DSD. Bixler syndrome. Nat Genet 36:228–230
Fluck CE, Pandey A, Huang N, Agrawal V, Miller WL (2008) P560
oxidoreductase deficiency- a new form of congenital adrenal hyper-
Emergency Treatment plasia. Karger, Basel
All the salt-losing forms (37.1, 37.2, 37.5, 37.7, 37.20) are Fluck CE, Meyer-Boni M, Pandey AV, Kempna P, Miller WL, Schoenle
prone to adrenal crisis in stress situations. Therefore: EJ, Biason-Lauber A (2011) Why boys will be boys: two pathways
of fetal testicular androgen biosynthesis are needed for male sexual
differentiation. Am J Hum Genet 89:201–218. doi:10.1016/j.
Initial hydrocortisone i.v. Infants and preschool children: 25 mg
ajhg.2011.06.009
School-age children: 50 mg Geller DS, Rodriguez-Soriano J, Vallo Boado A, Schifter S, Bayer M,
Adults: 100 mg Chang SS, Lifton RP (1998) Mutations in the mineralocorticoid
receptor gene cause autosomal dominant pseudohypoaldosteronism
Successive i.v. hydrocortisone doses 3–4× the mainte- type I. Nat Genet 19:279–281. doi:10.1038/966
nance doses/day, divided in 4 doses (every 6 h) Imperato-McGinley J, Gautier T, Peterson RE, Shackleton C (1986) The
prevalence of 5 alpha-reductase deficiency in children with ambigu-
Standard Treatment ous genitalia in the Dominican Republic. J Urol 136:867–873
Lin D, Sugawara T, Strauss JF 3rd, Clark BJ, Stocco DM, Saenger P, Rogol
Disorders 37.1, 37.2, 37.5, 37.7, 37.20 A, Miller WL (1995) Role of steroidogenic acute regulatory protein in
adrenal and gonadal steroidogenesis. Science 267:1828–1831
Suggested Lipsett MB, Tomita M, Brandon DD, De Vroede MM (1986) Cortisol
Type of glucocorticoid dose (mg/day) Daily doses resistance in men. In: Chrousos GP, Loriaux MB (eds) Steroid hor-
Hydrocortisone (HC) 15–25 2–3 mone resistance: mechanism and clinical aspects. Plenum Press,
Prednisone 5–7.5 2 New York
Prednisolone 4–6 2 McPhaul MJ, Griffin JE (1999) Male pseudohermaphroditism caused
by mutations of the human androgen receptor. J Clin Endocrinol
Dexamethasone 0.25–0.5 1
Metab 84:3435–3441
Fludrocortisone (mineralocorticoid) 0.05–0.2 1 Miller WL (1986) Congenital adrenal hyperplasia. N Engl J Med
314:1321–1322
Patients affected by the salt-losing forms of steroid syn- New MI (1994) The prismatic case of apparent mineralocorticoid
thesis defects should be informed of the necessity to an excess. J Clin Endocrinol Metab 79:1–3
emergency treatment (37.15) New MI, White PC, Pang SA, Dupont B, Speiser PW (1989) The adre-
nal hyperplasias. In: Beaudet AR, Scriver CR, Sly W, Valle D (eds)
Disorders 37.3, 37.6, 37.8, 37.9 (hypertensive, hypokalemic) The metabolic basis of inherited disease. McGraw-Hill, New York
DSD (37.3, 37.6, 37.8, 37.9): Induction of puberty accord- Peterson RE, Imperato-McGinley J, Gautier T, Shackleton C (1985)
ing to sex assignment. Male pseudohermaphroditism due to multiple defects in steroid-
biosynthetic microsomal mixed-function oxidases. A new variant of
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Prader A, Gurtner HP (1955) The syndrome of male pseudohermaphro-
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Leukotrienes
38
Ertan Mayatepek
Contents Short Summary

38.1 Introduction ...................................................................... 617 In the biosynthesis of leukotrienes, hereditary primary
38.2 Nomenclature.................................................................... 618 defects have been detected in three of the enzymatic steps:
LTC4 synthase, γ-glutamyl transpeptidase and membrane-
38.3 Metabolic Pathway ........................................................... 619
bound dipeptidase. A deficiency of these enzymes results in
38.4 Signs and Symptoms ........................................................ 619 abnormal levels and profiles of cysteinyl leukotrienes in
38.5 Reference Values............................................................... 620 CSF, urine and/or plasma. Patients with LTC4 synthase defi-
38.6 Pathological Values .......................................................... 621
ciency seem to be most severely affected with symptoms
including muscular hypotonia, psychomotor retardation,
38.7 Diagnostic Flow Chart ..................................................... 621 microcephaly and failure to thrive. The other two defects, i.e.
38.8 Specimen Collection ......................................................... 622 gamma-glutamyl transpeptidase deficiency and membrane-
38.9 Prenatal Diagnosis ............................................................ 622 bound dipeptidase deficiency, have been less well studied.
A better understanding of the role of leukotrienes in the brain
38.10 DNA Testing ...................................................................... 622
and their pathophysiological role is a prerequisite for the
38.11 Treatment .......................................................................... 622 suggestion of possible therapeutic approaches. At present,
References .................................................................................... 622 there exist no treatment options. Although at present only a
limited number of patients have been identified with defects
in the synthesis of leukotrienes, it is possible that such disor-
ders are underdiagnosed since leukotrienes are usually not
included in the routine metabolic workup, although careful
interpretation of urine amino acid profiles may give some
clues. Analysis of leukotrienes, preferably in the CSF, is rec-
ommended in all patients with neurological symptoms who
have no apparently obvious metabolic cause.
38.1 Introduction
Leukotrienes comprise a group of biologically active lipid

mediators derived from C20 polyunsaturated fatty acids, pre-
dominantly arachidonic acid via the 5-lipoxygenase pathway
(Lewis et al. 1990; Mayatepek and Hoffmann 1995). They
include the cysteinyl leukotrienes (LTC4, LTD4 and LTE4) as
well as LTB4. Synthesis of the primary cysteinyl leukotriene,
LTC4, results from conjugation of the unstable LTA4 with
glutathione and is mediated by LTC4 synthase. Stepwise
E. Mayatepek
cleavage of glutamate and glycine from LTC4 by γ- glutamyl
Moorenstr. 5, Düsseldorf 40225, Germany transpeptidase and membrane-bound dipeptidase yields
e-mail: mayatepek@med.uni-duesseldorf.de LTD4 and LTE4, respectively. During the last decade
618 E. Mayatepek
leukotrienes have been mainly investigated because of their 1998; Mayatepek et al. 1999). Onset of symptoms started
role as inflammatory mediators, especially in asthma bron- already in the neonatal period or infancy. The clinical picture
chiale. Their role in the CNS is yet poorly understood, but was mainly characterised by severe muscular hypotonia,
there is increasing evidence that they are messengers or mod- psychomotor retardation, failure to thrive, microcephaly and
ulators of CNS activity. death in infancy. All general and specific biochemical inves-
A few disorders have been identified causing secondary tigations were negative. There was a complete absence of the
disturbances in leukotriene elimination and degradation, e.g. primary cysteinyl leukotriene, LTC4, and its metabolites in
defective hepatobiliary elimination of cysteinyl leukotrienes the CSF.
as seen in the Dubin-Johnson syndrome (Mayatepek and It is thought that the absence of LTC4, especially in the
Lehmann 1996), impaired ω- oxidation of LTB4 in the Sjögren- brain, is at least in part responsible for the observed neuro-
Larsson syndrome (Willemsen et al. 2000) or altered β- oxida- logical symptoms.
tion in disorders of peroxisome biogenesis such as the The second step in cysteinyl leukotriene synthesis is
Zellweger syndrome (Mayatepek et al. 1993). However, these mediated by γ-glutamyl transpeptidase. Besides elevated
conditions do not affect leukotriene synthesis itself. Patients glutathione in urine and plasma, these patients are character-
with the Sjögren-Larsson syndrome, an inborn error of lipid ised by the unique finding of LTC4 excretion in urine and
metabolism, are characterised clinically by congenital ichthy- absence of LTD4 in plasma (Mayatepek et al. 2004). The
osis, mental retardation and spasticity. However, they also suf- clinical picture is heterogeneous, varying from mental retar-
fer from severe pruritus. In this disorder degradation of LTB4 dation and psychosis to a nearly normal phenotype.
is defective, resulting in increased levels of LTB4 that might be To date, only one adolescent male patient has been identi-
involved in the pathogenesis of pruritus. With respect to the fied with membrane-bound dipeptidase deficiency present-
agonising pruritus, at least some patients with Sjögren-Larsson ing with mental retardation, motor impairment and peripheral
syndrome might benefit from treatment with zileuton (in a neuropathy (Bellet et al. 1999; Mayatepek et al. 2005). The
dosage of up to 600 mg four times a day), which is capable to enzyme is involved in the third step of the synthesis of cyste-
inhibit an increased synthesis of LTB4 (Willemsen et al. 2001). inyl leukotrienes, resulting in the synthesis of LTE4.
In the synthesis of the leukotrienes, hereditary primary Endogenous urinary LTE4 represents the index metabolite
defects have been detected in three of the enzymatic steps: for the generation of cysteinyl leukotrienes in vivo.
LTC4 synthase, γ-glutamyl transpeptidase and membrane- Defects in the pathway of the synthesis of LTB4 have not
bound dipeptidase. The latter two enzymes are part of the so- been reported yet.
called gamma-glutamyl cycle. Their role in this cycle is Because of the very limited number of patients identified
further illustrated in Chap. 42 of this book. Defects in the bio- so far and because of the lack of profound understanding of
synthesis of leukotrienes seem to represent a new group of the role of leukotrienes in the brain and their pathophysio-
neurometabolic disorders (Mayatepek 2000). Deficiency of logical significance in deficiency states, there exist at present
these enzymes results in abnormal levels and profiles of cys- no treatment or experimental therapeutic approaches. It is
teinyl leukotrienes in CSF, urine and/or plasma (Mayatepek possible that such disorders are still underdiagnosed, sug-
et al. 2000). In general, the yet known primary defects in the gesting that leukotriene analysis should be included in the
synthesis of cysteinyl leukotrienes seem to be rare. routine metabolic workup in patients with neurological
So far, LTC4 synthase deficiency has been identified symptoms who have no apparently obvious other metabolic
in two independent patients (Mayatepek and Flock cause.
38.2 Nomenclature
Chromosomal
38.1 Leukotriene C4 LTC4 synthase LTC4D LTC4S 5q35 Leukotriene C4 246530 All forms
synthase deficiency deficiency synthase
38.2 Gamma-glutamyl Glutathionuria GGTD GGT1 22q11.1-q11.2 Gamma-glutamyl 231950 All forms
transpeptidase transpeptidase
deficiency
38.3 Membrane-bound Cysteinylglycinase MBDD DPEP1, RDP, 16q24.3 Cysteinylglycinase 179780 All forms
dipeptidase deficiency MDP, MBD1
deficiency
38 Leukotrienes 619
Fig. 38.1 Metabolic pathway of leukotriene biosynthesis Arachidonic acid

and metabolism including known metabolic defects: 38.1 LTC4
synthase, 38.2 γ-glutamyl transpeptidase, 38.3 membrane-bound 5 – LOAP/ 5 - LO
dipeptidase. LT Leukotriene, 5-LOAP 5-lipoxygenase-activating
protein, 5-LO 5-lipoxygenase, LTA4H leukotriene A4 hydrolase 5-Hydroperoxyeicosatetraenoic acid
5 - LO
LTA4
38.1
LTA4H
Glutathione
LTB4 LTC4
38.2
LTD4
38.3
N-Acetyl-LTE4 LTE4
20 – OH – LTB4 20 – OH – LTE4
20 – COOH – LTB4 20 – COOH – LTE4
(C2) – COOH – LTB (C2) – COOH - LTE
Table 38.1 Leukotriene C4 synthase deficiency

CNS Absent head control + +
Encephalopathy, progressive + +
Hypotonia ± +
Lack of facial expression + +
Minimal spontaneous movements + +
No visual contact + +
Tendon reflexes, decreased + +
Musculoskeletal Dysmorphic features ± ±
Microcephaly ± +
Other Death +
Symmetric extension in the legs ± +
(continued)
620 E. Mayatepek

Routine laboratory EEG, abnormal + +
EMG, abnormal + +
Special laboratory Glutathione (RBC) n n
LTB4 (CSF, P) n-↑ n-↑
LTC4 (CSF, P) ↓ ↓
LTC4 synthesis in nucleated cells (WBC) ↓ ↓
LTD4 (CSF, P) ↓ ↓
LTE4 (CSF, P, U) ↓ ↓
Table 38.2 Gamma-glutamyl transpeptidase deficiency

CNS Psychosis ± ± ±
Retardation, psychomotor ± ± ±
Special Gamma-glutamyl transpeptidase ↓ ↓ ↓ ↓ ↓
laboratory (WBC,FB)
Glutathione (RBC) n n n n n
Glutathione (U) ↑ ↑ ↑ ↑ ↑
LTB4 (U, P) n n n n n
LTC4 (U, P) ↑ ↑ ↑ ↑ ↑
LTD4 (U, P) ↓ ↓ ↓ ↓ ↓
LTD4 synthesis in nucleated cells ↓ ↓ ↓ ↓ ↓
(WBC)
LTE4 (U, P) ↓ ↓ ↓ ↓ ↓
Table 38.3 Membrane-bound dipeptidase deficiency

CNS Motor impairment +
Neuropathy +
Retardation, +
psychomotor
Ear Deafness, partial +
Musculoskeletal Foot deformity +
Routine laboratory EEG, abnormal +
EMG, abnormal +
Special laboratory Cysteine, bound (U) ↑
Cystine (U) ↑
Cysteinylglycine (U, P) ↑
Glutathione (RBC) n
LTD4 (U) ↑
LTE4 (U) ↓
38.5 Reference Values LTB4 (CSF) 55.5–183.3 pmol/L

LTB4 (P) 27.3–35.4 nmol/L
LTB4 (U) <5 nmol/mol cr.
Metabolite Value Unit
20-Hydroxy-LTB4 (U) <5 nmol/mol cr.
LTC4 (CSF) 37.2–100.5 pmol/L 20-Carboxy-LTB4 (U) <5 nmol/mol cr.
LTC4 (P) 13.8–17.2 nmol/L Cysteinylglycine (U) <1 mmol/mol cr.
LTC4 (U) <5 nmol/mol cr. Cysteinylglycine (P) <16 μmol/L
LTD4 (CSF) 31.6–69.6 pmol/L
LTD4 (P) 23.2–28.4 nmol/L
LTD4 (U) <5 nmol/mol cr. Leukotrienes were measured by either specific immunoas-
LTE4 (CSF) 46.1–124.2 pmol/L say or GC-MS after purification with Sep-Pak C18 extraction
LTE4 (P) 27.0–32.9 nmol/L and reversed-phase HPLC. There exists no age dependency
LTE4 (U) 15.8–45.2 nmol/mol cr. of leukotriene concentrations in CSF, plasma and urine
38 Leukotrienes 621
38.2, γ-glutamyl transpeptidase 38.3, membrane-bound

38.1, LTC4 synthase deficiency deficiency dipeptidase deficiency
CSF (pmol/L)
LTC4 <1
LTD4 <1
LTE4 <1
LTB4 n-↑
Plasma (nmol/L)
LTC4 <3 ↑
LTD4 <3 <3
LTE4 <3 <3
LTB4 ↑ n
Cysteinylglycine n n 30
Urine (nmol/mol cr.)
LTC4 <5 ↑ <5
LTD4 <5 <5 ↑
LTE4 <5 <5 <5
LTB4 <5 <5 <5
Cysteinylglycine n n 49–72
(mmol/mol cr.)
WBC
LTC4 synthesis ↓ ↑
LTD4 synthesis ↓ ↓
CSF
Leukotrienes
Cysteinyi-Leukotrienes LTC4 normal or ↑

( LTC4, L TD4, LTE4) ↓
LTE4 (U) ↓ Urine Leukotrienes
Cysteinyi-Leukotrienes (P) ↓ LTC4 ↑ LTD4 ↑
LTC4-synthesis (WBC) ↓ LTC4 (P) ↑ Cystinylglycine (U) ↑
LTC4-synthase γ-Glutamyl Membrane-bound

activity ↓ transpeptidase dipeptidase
activity ↓ activity ↓
Fig. 38.2 Diagnostic flow chart

for defects in the synthesis of LTC4-synthase γ-Glutamyl Membrane-bound
cysteinyl leukotrienes. LT deficiency transpeptidase dipeptidase
Leukotriene
622 E. Mayatepek

Leukotrienes (CSF) Before medication CSF 0.5 ml, immediately kept in liquid Leukotrienes are very susceptible to
nitrogen, store at −70 °C oxidative degradation; collection
and storage in polypropylene tubes,
if possible under argon; long-term
storage may result in lower contents
Leukotrienes (U) Before medication Urine 10 ml, freeze immediately As above; leukocytes or bacterial
(preferably −70 °C) contamination may cause artificial
higher contents
Leukotrienes (P) Before medication Plasma from 1 ml, centrifuge, remove and As above; leukotrienes are easily
heparinised blood freeze immediately (preferably artificially generated and released
−70 °C) from leukocytes during blood
sampling
Leukotrienes (WBC) Before medication Monocytes, Stimulation with calcium As above
neutrophils ionophore A 23 187 (10 μmol/L
for 15 min. at 37 °C)
Cysteinylglycine, Before medication Urine/plasma 1 ml, frozen (−20 °C)
glutathione
tion to pathobiology in human diseases. N Engl J Med 323:

38.9 Prenatal Diagnosis 645–655
Mayatepek E (2000) Leukotriene C4 synthesis deficiency: a member of
Prenatal diagnosis has not been performed yet. a probably underdiagnosed new group of neurometabolic diseases.
Eur J Pediatr 159:811–818
Mayatepek E, Flock B (1998) Leukotriene C4-synthesis deficiency: a
new inborn error of metabolism linked to a fatal developmental syn-
38.10 DNA Testing drome. Lancet 352:1514–1517
Mayatepek E, Hoffmann GF (1995) Leukotrienes: biosynthesis, metab-
DNA analysis has not been performed yet. olism and pathophysiological significance. Pediatr Res 37:1–9
Mayatepek E, Lehmann WD (1996) Defective hepatobiliary leukotri-
ene elimination in patients with the Dubin-Johnson syndrome. Clin
Chim Acta 249:37–46
38.11 Treatment Mayatepek E, Lehmann WD, Fauler J et al (1993) Impaired degrada-
tion of leukotrienes in patients with peroxisome deficiency disor-
ders. J Clin Invest 91:881–888
There exist no treatment options for all three primary defects Mayatepek E, Lindner M, Zelezny R et al (1999) A severely affected
in the synthesis of cysteinyl leukotrienes (38.1–38.3). infant with absence of cysteinyl leukotrienes in cerebrospinal fluid:
further evidence that leukotriene C4-synthesis deficiency is a new
Emergency Treatment neurometabolic disorder. Neuropediatrics 30:5–7
Mayatepek E, Zelezny R, Hoffmann GF (2000) Analysis of leukotri-
No emergency treatment available. enes in cerebrospinal fluid of a reference population and patients
with inborn errors of metabolism: further evidence for a pathogno-
Standard Treatment monic profile in LTC4-synthesis deficiency. Clin Chim Acta
No standard treatment available. 292:155–162
Mayatepek E, Okun JG, Meissner T et al (2004) Synthesis and metabo-
lism in gamma- glutamyl transpeptidase deficiency. J Lipid Res
Experimental Treatment 45:900–904
There exist no experimental treatment options. Mayatepek E, Badiou S, Bellet H et al (2005) A patient with neurologi-
cal symptoms and leukotriene metabolism: a new defect in leukotri-
ene biosynthesis. Ann Neurol 58:968–970
Willemsen MAAP, De Jong JGN, Van Domburg PHMF et al (2000)
References Defective inactivation of leukotriene B4 in patients with Sjögren-
Larsson syndrome. J Pediatr 136:258–260
Bellet H, Rejou F, Vallat C et al (1999) Cystinylglycinuria: a new neu- Willemsen MAAP, Lutt MAJ, Steijlen PM et al (2001) Clinical and
rometabolic disorder? J Inherit Metab Dis 22:231–234 biochemical effects of zileuton in patients with the Sjögren-Larsson
Lewis RA, Austen KF, Soberman RJ (1990) Leukotrienes and other syndrome. Eur J Pediatr 160:711–717
products of the 5-lipoxygenase pathway: biochemistry and rela-
Disorders of Copper
and Zinc Metabolism 39
Peter M. van Hasselt and Roderick H.J. Houwen
Contents Summary
39.1 Introduction ...................................................................... 624 Copper and zinc are both essential trace elements and are
required for the proper function of many important metal-
39.2 Nomenclature ................................................................... 625
loenzymes. The uptake of both copper and zinc requires
39.3 Metabolic Pathways ......................................................... 626 specific, but different, carriers in the intestine. Copper
39.4 Signs and Symptoms ........................................................ 627 excretion takes place only in the liver, and its homeosta-
sis is tightly controlled, as copper can be highly toxic.
The balance between uptake and excretion of copper is
39.6 Pathological Values .......................................................... 630 disturbed in Wilson disease and Menkes disease, as well
39.7 Diagnostic Flow Chart ..................................................... 630 as in two milder variants of Menkes disease: the occipital
39.8 Specimen Collection ......................................................... 631 horn syndrome and X-linked distal hereditary neuropathy.
Both Wilson and Menkes diseases have specific patho-
39.9 Prenatal Diagnosis............................................................ 631
physiological manifestations, which are for Wilson disease
39.10 DNA Testing ...................................................................... 631 mainly related to copper accumulation in the liver and brain
39.11 Treatment Summary ........................................................ 631 and for Menkes disease to a systemic shortage of copper,
39.12 Follow-Up .......................................................................... 632
resulting in an insufficient function of copper-containing
enzymes, such as tyrosinase, lysyl oxidase and dopamine-
References ...................................................................................... 632
beta hydroxylase. The absence of another copper-contain-
ing enzyme, ceruloplasmin, which is due to mutations in
the corresponding gene, will result in iron overload in the
liver, brain and pancreas, pointing to the role of this serum
protein in iron metabolism. It is further discussed in the
chapter on iron metabolism.
Zinc is a cofactor for over 100 enzymes and, as such,
is involved in all major metabolic pathways. It is essen-
tial for nucleic acid metabolism and protein synthesis and
its regulation through the so-called zinc-finger proteins.
Zinc deficiency, either hereditary or acquired, therefore
has major detrimental effects. Conversely, high serum zinc
has few, probably because of its binding to albumin and
α2-macroglobulin. Acrodermatitis enteropathica, the main
inborn error of zinc metabolism, is due to mutations in
the intestinal carrier for zinc and will result in periorifi-
cial and acral dermatitis, diarrhoea, infections and growth
retardation.
P.M. van Hasselt • R.H.J. Houwen (*)
Department of Metabolic Diseases and Pediatric Gastroenterology,
University Medical Center Utrecht,
Lundlaan 6, 3584 EA Utrecht, The Netherlands
e-mail: p.vanHasselt@umcutrecht.nl; r.houwen@umcutrecht.nl
624 P.M. van Hasselt and R.H.J. Houwen
39.1 Introduction reasons. Therefore, it is generally used for confirmatory

purposes only.
Wilson disease is an autosomal recessive disorder of copper Menkes disease is an X-linked recessive disorder caused
metabolism due to mutations in ATP7B, encoding the by mutations in ATP7A, encoding ATP7A, a copper-
copper-transporting ATPase ATP7B (de Bie et al. 2007; Ala transporting ATPase, which is expressed in all tissues,
et al. 2007). With a defective ATP7B, copper excretion from except the liver. In patients afflicted by this disease, cellular
the liver is reduced, resulting in a gradual accumulation of uptake of copper is normal, but it cannot be exported (Kaler
copper in the liver and secondarily in the brain, the kidneys 1998). Consequently, copper efflux from intestinal cells is
and the eye. At a young age patients generally present with severely reduced, insufficient copper reaches the circulation,
hepatic symptoms, which can be quite fulminant, while at an and insufficient copper is available for incorporation into
adult age a presentation with neurological symptoms is more cuproenzymes. Amongst the copper-requiring enzymes in
prevalent. Nevertheless, even patients well into their fifth or the brain is dopamine-β-hydroxylase, essential for catechol-
sixth decade can present with hepatic symptoms (Ferenci amine biosynthesis and peptidylglycine monooxygenase,
et al. 2007), while in the paediatric age group, presentation which is involved in the processing of neuropeptide precur-
with neurological symptoms is not unusual. sors. Deficient activity of these and other copper-containing
ATP7B is also necessary for the incorporation of copper enzymes, such as cytochrome c oxidase, which is crucial for
into ceruloplasmin. When ATP7B is defective, ceruloplas- cellular respiration, is probably responsible for the progres-
min is excreted into the circulation without copper (apo- sive neurological deterioration in Menkes disease, which
ceruloplasmin), which has a shorter half-life, leading to the generally becomes manifest at the age of 2–3 months. Other
characteristic low serum ceruloplasmin seen in Wilson dis- enzymes influenced by copper deficiency are sulfhydryl oxi-
ease. As almost all serum copper is bound to ceruloplasmin, dase, necessary for keratin cross-linking, giving pili torti
a low level of this protein will result in a low serum copper. when deficient, and lysyl oxidase, essential for collagen
In severe Wilson disease, however, a huge amount of cop- cross-linking. Hence, patients generally also manifest abnor-
per can be released from the liver, giving a normal or almost mal skin laxity as well as vascular tortuosity and bladder
normal serum copper. Most of this copper is unbound, the diverticula.
so-called free serum copper, which is toxic and can induce At birth serum copper and ceruloplasmin may be below
haemolysis. A low serum ceruloplasmin can also be seen in the usual range, but this is not diagnostic, as these low levels
carriers of an ATP7B mutation and in aceruloplasminaemia, are normal in this age category. Abnormal levels of catechol-
so this parameter alone is insufficient to make a diagnosis of amines and their metabolites are quite specific for Menkes
Wilson disease. The Kayser-Fleischer ring, a greenish-brown disease, especially the ratio between dopamine to norepi-
deposit of copper molecules at the limbus of the cornea, is nephrine as well as the ratio of dihydroxyphenylacetic acid
almost always seen in patients presenting with neurological to dihydroxyphenylglycol in plasma (Kaler et al. 2008). The
disease, but is absent in up to 50 % of patients presenting cellular copper retention characteristic of Menkes disease
with hepatic symptoms. Most characteristically 24-h urinary can be measured by the increased accumulation of copper
copper excretion is elevated in Wilson disease. However, in cultured fibroblasts (Kim et al. 2003). Final diagnosis
this parameter too can be falsely elevated, for example, in requires identification of the mutation.
severe liver disease or when the collecting vessels are con- Occipital horn syndrome is also characterised by ATP7A
taminated. Conversely, 24-h urinary copper excretion can be mutations, but these are generally milder, permitting some
– almost – normal, such as in patients with a still low cop- ATP7A expression (Moeller et al. 2000). The effects are
per load, e.g. in younger siblings of a patient (Nicastro et al. especially seen in connective tissue, e.g. bone demineralisa-
2010). Finally, liver copper is elevated in Wilson disease, but tion and exostoses, predominantly at the occipital insertion
it is also abnormal in patients with cholestatic liver disease. of the paraspinal muscles, from which the name originates
As no single laboratory parameter is conclusive for making (Tsukahara et al. 1994). In addition these patients have uri-
the diagnosis of Wilson disease, generally a combination of nary tract diverticula and an abnormal skin and joint laxity.
several tests is used. Commonly a numerical value is given X-linked distal hereditary neuropathy is an even rarer,
to the absence or presence of the most distinguishing clinical and milder phenotype, with in fact a normal serum copper.
signs, i.e. Kayser-Fleischer rings and extrapyramidal neuro- Patients afflicted by this disease present with symptoms of
logical symptoms, as well as to specific laboratory param- distal muscular atrophy and weakness, generally in young
eters, i.e. serum ceruloplasmin, urinary copper excretion and adulthood (Kennerson et al. 2010).
liver copper concentration (Ferenci et al. 2003). A score of SLC33A1 deficiency with low serum copper and cerulo-
4 or more is considered to be highly suggestive for Wilson plasmin is a rare disorder in which serum ceruloplasmin and
disease (see also Sect. 39.7). Genetic testing of ATP7B serum copper are low, due to a defect in intracellular protein
mutations may still take up to 3 months, mostly for logistic acetylation. Clinical symptomatology, most notably a severe
39 Disorders of Copper and Zinc Metabolism 625
psychomotor retardation, is probably due to a combination initially localised at the acral and periorificial sites (Neldner
of a low serum copper and other, currently insufficiently and Hambidge 1975; Aggett 1983). Later on these lesions
understood, consequences of SLC33A1 deficiency (Huppke become more pustular and hyperkeratotic. In addition some
et al. 2012). patients have diarrhoea, which can be severe and result in
MEDNIK syndrome is caused by mutations in AP1S1, failure to thrive. Irritability, apathy and later on depression
which encodes the small subunit σ1A of the adaptor pro- are common. Infections are frequent, probably due to the
tein-1 (AP1) complex. When deficient a low serum copper disturbed cellular and humoral immunity associated with
and ceruloplasmin, in combination with mental retardation, zinc deficiency. If untreated, symptoms may worsen, and
variable intestinal pseudo-obstruction, ichthyosis and raised the disease can even be fatal. Nevertheless, before treatment
transaminases are seen (Martinelli et al. 2013). The adap- became available most patients survived into adulthood.
tor protein-1 complex is necessary for the intracellular traf- Serum zinc levels are usually low, although normal values
ficking of ATP7A, ATP7B and possibly other membrane are found in at least 15 % of the patients (Van Wouwe 1989).
proteins through its involvement in clathrin-coated vesicle Measurement of zinc in other tissues, such as hair or blood
assembly. A disturbance of this pathway will give specific cells, does not improve diagnostic accuracy. In addition sev-
abnormalities in copper metabolism, which resemble some eral other conditions, e.g. chronic diarrhoea, can cause low
aspects of both Menkes diseases and Wilson disease, as well serum zinc. Other tests contribute to a certain extent, such as
as other symptoms. low urinary zinc excretion or low alkaline phosphatase activ-
Acrodermatitis enteropathica is an autosomal recessive ity, for which zinc is a cofactor. The defect in intestinal zinc
disorder of zinc transport due to mutations in the SLC39A4 transport can be proven with radiolabeled zinc. However,
gene encoding ZIP4, a zinc transporter expressed at the api- this procedure is cumbersome and only available in a few
cal membrane of the enterocytes (Wang et al. 2002). When places. Genetic testing will take time, so a practical approach
this transporter is deficient, it is impossible to absorb suf- is to start zinc therapy whenever the diagnosis is suspected.
ficient zinc from the intestinal contents, at least at a normal In patients with zinc deficiency, a response should be dis-
luminal zinc concentration. cernible within a week. If genetic testing is equivocal, it can
Clinical symptoms develop after breast-feeding is stopped, be considered to stop oral zinc to discriminate between true
because a zinc transport ligand is allegedly present in breast acrodermatitis enteropathica, which should relapse quickly,
milk. Patients will develop severe erythematous dermatitis, and acquired zinc deficiency.
39.2 Nomenclature
Gene Chromosomal
No. Disorder Alternative name Abbreviation symbol localisation Affected protein OMIM no. Subtype
39.1 Wilson disease Hepatolenticular WND (WD) ATP7B 13q14.3 ATP7B 277900 All forms
degeneration
39.2 Menkes disease Kinky (steely) hair MNK (MK) ATP7A Xq21.1 ATP7A 309400 All forms
disease
39.3 Occipital horn syndrome X-linked cutis laxa OHS ATP7A Xq21.1 ATP7A 304150 All forms
39.4 X-linked distal spinal SMAX3 ATP7A Xq21.1 Copper- 300489 All forms
muscular atrophy transporting
P-type ATPase
39.5 Acrodermatitis enteropathica AEZ (AE) SLC39A4 8q24.3 ZIP4 201100 All forms
39.6 SLC33A1 deficiency with Congenital cataracts, CCHLND SLC33A1 3q25.31 SLC33A1 614482 All forms
low serum copper and hearing loss and low
ceruloplasmin serum copper and
ceruloplasmin
39.7 MEDNIK syndrome MEDNIK AP1S1 7q22.1 Adaptor-related 609313 All forms
complex
protein 1
Enterocyte Hepatocyte
Apical CTR1 Apical
Bile
canaliculus
ER
TGN TGN
Nucleus
ER
Basolateral Basolateral
CTR1
Fig. 39.1 Schematic representation of the key elements of copper leave the enterocyte, resulting in a systemic shortage of copper. Right
homeostasis (Adapted from de Bie et al. (2007)). Left panel: dietary panel: copper from the portal circulation is taken up through CTR1,
copper from the intestine is taken up by the enterocytes through CTR1 transported to ATP7B in the trans-Golgi network (TGN). This protein
and transported intracellularly to ATP7A, localised in the trans-Golgi will export copper to the bile canaliculus. When this pathway is defec-
network (TGN). This protein will export copper to the portal circula- tive, as in Wilson disease, the hepatocytes will gradually accumulate
tion. When ATP7A is defective, as in Menkes disease, no copper can copper, which above a threshold level will induce cellular damage
Metabolic Pathway for Zinc Zip2 and Zip4, only disease-causing mutations in the latter
Two groups of proteins involved in zinc transport are have been identified, giving a zinc absorption defect in the
involved in cellular zinc homeostasis: the Zip family that enterocytes and leading to acrodermatitis enteropathica.
mediates zinc transport from outside the cell into to the Similarly for the zinc export proteins identified in humans,
cytoplasm and the ZnT family that mediates export of zinc, ZnT1, ZnT2, ZnT4, mutations in the latter have found to
either into the extracellular space or into intracellular organ- be associated with a low breast milk concentration. For the
elles. The exact role of each of these zinc transport proteins others no disease-causing mutations have been identified in
in the various cell types is not known at present. For Zip1, humans.
Table 39.1 Wilson disease

CNS Ataxia n n ± + ++
Basal ganglia lesions n n ± + ++
Clumsiness n n ± + +
Dysarthria n n n + ++
Dystonia n n ± + ++
Handwriting n n ± + +
Irritability n n ± + +
Movement, abnormal n n ± + ++
Neurological symptoms n n ± + ++
Psychiatric symptoms n n n ± ±
Speech disturbances n n n + ++
Tremor n n ± + +
Digestive Abdominal pain n n ± ± ±
Ascites n n ++ ++ +
Drooling n n ± + +
Hepatosplenomegaly n n ++ ++ ++
Jaundice n n + ++ +
Liver dysfunction n n ++ ++ ++
Liver failure, acute n n ++ ++ +
Eye Cataract n n ± ± ±
Haematological Coagulopathy n n + ++ ++
Haemolysis n n + ++ +
Haemolytic anaemia n n + ++ +
Leukopenia n n + ++ ++
Thrombocytopenia n n + ++ ++
Renal Renal tubular acidosis n n + + +
Routine laboratory Albumin (S) n n ↓-n ↓-n ↓-n
ASAT/ALAT (P) n n + + +
Bilirubin (P) n n n-↑ n-↑ n-↑
Prothrombin ratio n n n-↑ ↑ ↑
Special Laboratory Ceruloplasmin (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Copper (liver) n n-↑ ↑ ↑ ↑
Copper (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Copper (U) n n-↑ n-↑ ↑ ↑↑
Table 39.2 Menkes disease

Autonomic system Hypothermia + + +
Cardiovascular Arterial ruptures n ± ±
Tortuous arteries n ± ±
CNS Convulsions n + +
Encephalopathy, progressive ± ± +
Mental retardation ± + +
Spasticity ± ± +
Dermatological Cutis laxa ± + +
Kinky hair ± + ++
Digestive Feeding difficulties ± + +
Genitourinary Bladder diverticula n ± ±
Haematological Anaemia ± ± ±
Neutropenia ± ± ±
Musculoskeletal Connective tissue abnormalities ± + +
Hernias n ± ±
Peculiar facies + + +
Special laboratory Ceruloplasmin (S) ↓ ↓ ↓
Copper (CSF) ↓ ↓ ↓
Copper (duodenal) ↑ ↑ ↑
Copper (liver) ↓ ↓ ↓
Copper (S) ↓ ↓ ↓
Table 39.3 Occipital horn syndrome

Cardiovascular Hypotension, orthostatic n n + + +
Dermatological Cutis laxa ± ± + + +
Digestive Diarrhoea ± ± + + +
Genitourinary Bladder diverticula n ± + + +
Musculoskeletal Exostosis (occipital horn) ± ± + + +
Renal Urinary infections n ± + + +
Special laboratory Ceruloplasmin (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Copper (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Table 39.4 X-linked distal spinal muscular atrophy

Musculoskeletal Absent/weak tendon reflexes n n n n ±
Muscle weakness, distal n n n n +
Special laboratory Copper (S) n n n n n
Table 39.5 Acrodermatitis enteropathica

CNS Apathy ± + + ± ±
Depression n n ± ± ±
Irritability ± ++ ++ + +
Dermatological Alopecia n + ++ ++ ++
Dermatitis ± ++ +++ ++ ++
Digestive Anorexia n ++ ++ + +
Diarrhoea ± ++ ++ + +
Failure to thrive n + ++ ++ ++
Other Frequent infections n + + ± ±
Routine laboratory Alkaline phosphatase (P) ↓-n ↓ ↓ ↓ ↓
Special laboratory Zinc (S) ↓-n ↓-n ↓-n ↓-n ↓-n
Zinc uptake (duodenal) ↓ ↓ ↓ ↓ ↓
Table 39.6 SLC33A1 deficiency with low serum copper and ceruloplasmin
CNS Cerebral and cerebellar atrophy + ++ ++
Hypomyelination (MRS) + ++ ++
Ear Hearing loss + ++ ++
Eye Cataract + + +
Special laboratory Ceruloplasmin (S) ↓ ↓ ↓
Copper (S) ↓ ↓ ↓
Table 39.7 MEDNIK syndrome

CNS Retardation, psychomotor + + + +
Dermatological Erythroderma + + +
Hyperkeratosis + + +
Ichthyosis + + + +
Digestive Intestinal pseudo-obstruction ± + + + +
Liver dysfunction + + + +
Ear Deafness + + +
Routine laboratory ASAT/ALAT (P) ↑ ↑ ↑ ↑ ↑
Bile acids (enzyme assay) (P) ↑ ↑ ↑
Special laboratory Ceruloplasmin (S) ↓ ↓ ↓ ↓
Copper (S) ↓ ↓ ↓ ↓
MRI: cerebral atrophy + + + +
VLCFA ↑ ↑ ↑ ↑
Copper, ceruloplasmin and zinc

Serum copper 11–22 μmol/la Serum zinc 11.9–19.4 μmol/la
Serum ceruloplasmin 0.2–0.5 g/l
Urinary copper <0.6 μmol/24 ha Urinary zinc 4.6 ± 2.6 μmol/24 ha
Liver copper <55 μg/g dry weighta
a
Atomic absorption. Ala et al. (2007) and Van Wouwe (1989)
Cu (S) Cu (U) Cpl (S) Liver copper Zn (S) Zn (urine)

μmol/l μmol/24 h g/l μg/g dry weight μmol/l μmol/24 h
Wilson disease
Presymptomatic >0.6 <0.2 >250
Symptomatic >1.6 <0.2 >250
Menkes disease <11 <0.2
Occipital horn syndrome n–↓ n–↓
X-linked distal spinal muscular atrophy n
Acrodermatitis enteropathica 7.1 ± 5.0 1.5 ± 0.9
Ala et al. (2007), Nicastro et al. (2010), Kaler (1998), Tsukahara et al. (1994), Kennerson et al. (2010), and Van Wouwe (1989)
Extrapyramidal symptoms present (2 points)

Kayser-Fleischer rings present (2 points)
Serum ceruloplasmin 0.1-0.2 g/L (1 point)
Serum ceruloplasmin <0.1 g/L (2 points)
Diagnosis
Score 0-1 Score 2-3 Score ≥ 4 established
urinary urinary
copper copper
>1.6 µmol/d* > 1.6 µmol/d*
Mutation
analysis
> 250µg/g
hepatic 2 mutations
copper
Score ≤ 3
1 mutation
Normal or< 250µg/g

no mutation
Fig. 39.2 Diagnostic flow chart for Wilson disease (Modified from Ferenci et al. (2003) and European Association for the study of the Liver
(2012)). *In asymptomatic children the cutoff can be lowered to 0.6 μmol/day (Ala et al. 2007)
Menkes Disease (N, 0.04 ± 0.03; Menkes, 0.83 ± 0.71), as well as the ratio of
Diagnosis should be suspected in any child with symptoms plasma dihydroxyphenylacetic acid to dihydroxyphenylglycol
suggestive of Menkes disease or a positive family history. A (N, 1.5 ± 0.4; Menkes, 13.0 ± 6.6) (Kaler et al. 2008). Serum
quick confirmation is essential, as an early diagnosis is a pre- copper and ceruloplasmin are generally low, but this may not
requisite for effective treatment. This can be obtained by deter- be discriminatory as levels tend to be low in infancy. Analysis
mining the ratio between plasma dopamine to norepinephrine of ATP7A for mutations confirms a biochemical diagnosis.
Occipital Horn Syndrome 39.11 Treatment Summary

In a patient with symptoms suggestive of occipital horn syn-
drome, the serum copper and serum ceruloplasmin are gen- Wilson disease treatment is directed towards reducing the
erally low but can be normal too. Therefore, genetic analysis amount of copper in the body. To this end several chelators
of ATP7A is mandatory for establishing a diagnosis. are available, i.e. penicillamine and trientine, as well as zinc.
Penicillamine seems to be preferred in patients with hepatic
Acrodermatitis Enteropathica problems, while in patients with neurological problems, zinc
In any patient who develops erythematous dermatitis at the can be considered (European Association for the study of the
acra and periorificial when breast-feeding is stopped, this Liver 2012; Wiggelinkhuizen et al. 2009). This intervention
diagnosis should be suspected. Serum zinc levels are usu- may also be the treatment of choice in patients still with-
ally low, although normal values are found in at least 15 % out clinical symptoms, e.g. those identified through family
of the patients (Aggett 1983). The diagnosis is proven when screening. Therapy might fail, however, both in patients with
mutations in SLC39A4 are found. However, this procedure neurological problems, leaving residual neurological prob-
will take time, so a practical approach is to start zinc therapy lems, and in patients with hepatic problems, especially in
whenever the diagnosis is suspected. A response should be end-stage cirrhosis or liver failure. For those patients liver
discernible within a week. transplantation is an option.
Menkes disease patients formerly mostly died before the
age of three, but with current supportive care, a longer sur-
39.8 Specimen Collection vival is common. In addition treatment with copper histidine,
the physiological copper complex in humans, may improve
Wilson disease can be suspected when obtaining a falsely outcome, especially when started in the first weeks of life.
increased urinary copper excretion due to contamination of When some residual activity of the ATP7A protein is pres-
the collection vessels. To this purpose it is essential to use ent, normal or near-normal intellectual development may
copper-free urine collection containers. Tubes for routine even be attained (Kaler et al. 2008).
blood collection are copper- and zinc-free. Acrodermatitis Enteropathica. Adding oral zinc at a dose
of 200–400 mg zinc sulphate per day (50–90 mg elemental
zinc/day) is sufficient to overcome the transport defect in the
39.9 Prenatal Diagnosis intestine, probably because other low-affinity transporters
can import this cation at higher luminal concentrations. With
Prenatal diagnosis in each condition discussed is possible by this treatment prognosis is excellent (Neldner and Hambidge
gene sequencing when the defect in the family is known. 1975; Van Wouwe 1989).
MEDNIK syndrome might respond to zinc acetate therapy.
In the only patient fully tested, in addition to the abnormali-
39.10 DNA Testing ties listed in the introduction, basal ganglia abnormalities,
cerebral atrophy, behavioural disturbances and severe itch-
For each of the disorders discussed, the gene is known, and ing were present, and urinary copper excretion as well as
by sending in EDTA blood, the gene that is suspected to be liver copper was raised. Oral zinc acetate induced a partial
mutated can be sequenced. response of both clinical and laboratory abnormalities.
Standard Treatment
Therapy Application Dose Duration

39.1 Wilson disease Chelating agents Orally Children, 25 mg/kg/day Lifelong
(d-penicillamine, trientine) Adults, 1–2 g in 4 doses
Zinc salts (zinc acetate) Orally 1–5 years, 2 × 25 mg/day Lifelong
6–16 years, 3 × 25 mg/day
>16 years, 3 × 50 mg/day
39.2 Menkes disease Cu-histidinate SC 250 μg twice daily Lifelong
250 μg daily after 1 year
39.3 Acrodermatitis Zinc sulphate Orally 50–90 mg Zn2+/day Lifelong
enteropathica Zinc acetate
39.12 Follow-Up with molecular defects and disease phenotypes. J Med Genet
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the effect of therapy. Initially, this is quite frequent and will Ferenci P, Caca K, Loudianos G et al (2003) Diagnosis and phenotypic
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tological, and laboratory investigations aimed at copper
disease. Gastroenterology 132:1294–1298
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liver (synthesis, bilirubin, transaminases). These parameters cause a lethal autosomal-recessive disorder with congenital
should all improve. On stable, long-term chelator therapy, cataracts, hearing loss, and low serum copper and ceruloplasmin.
Am J Hum Genet 90:61–68
urinary copper excretion should be 3–8 μmol/24 h, while a
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urinary copper excretion in excess of 1.6 μmol/24 h after 2 form of copper deficiency. Am J Clin Nutr 67S:1029S–1034S
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Kennerson ML, Nicholson GA, Kaler SG et al (2010) Missense muta-
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Menkes copper ATPase. J Med Genet 40:290–295
will be monitored by regular clinical evaluation, with spe-
Martinelli D, Travaglini L, Drouin CA et al (2013) MEDNIK syn-
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In addition serum copper will be followed regularly to mea- tate therapy. Brain 136:872–881
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tions of the ATP7A gene lead to different phenotypes: classical
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Menkes disease or occipital horn syndrome. Am J Hum Genet
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Nicastro E, Ranucci G, Vajro P, Vegnente A, Iorio R (2010)
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Wang K, Zhou B, Kuo YM et al (2002) A novel member of a zinc trans-
Aggett PJ (1983) Acrodermatitis enteropathica. J Inherit Metab Dis porter family is defective in acrodermatitis enteropathica. Am J
6:39S–43S Hum Genet 71:66–73
Ala A, Walker AP, Ashkan K, Dooley JS, Schilsky ML (2007) Wilson’s Wiggelinkhuizen M, Tilanus MEC, Bollen CW, Houwen RHJ (2009)
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de Bie P, Muller P, Wijmenga C, Klomp LWJ (2007) Molecular patho- the initial treatment of Wilson disease. Aliment Pharmacol Ther
genesis of Wilson and Menkes disease: correlation of mutations 29:947–958
Iron Metabolism Disorders
40
Vineta Fellman
Contents 40.1 Summary and Introduction

40.1 Summary and Introduction............................................. 633
Hereditary Hemochromatosis (40.1)
40.2 Nomenclature ................................................................... 635
Hereditary hemochromatosis (Pietrangelo 2010) is charac-
40.3 Metabolic Pathway........................................................... 636 terized by iron accumulation in several tissues, i.e., in the
40.4 Signs and Symptoms ........................................................ 637 parenchymal cells of the liver, heart, pancreas, and other
40.5 Reference and Pathological Values ................................. 639
endocrine organs. This toxic accumulation occurs as a result
of increased intestinal uptake of dietary iron.
40.6 Specimen Collection ......................................................... 639
Hereditary hemochromatosis is a common disorder in
40.7 Prenatal and DNA Testing............................................... 639 France, Britain, Ireland, and Northern Europe. In the majority
40.8 Treatment .......................................................................... 639 of cases, it is caused by a homozygous c.845G > A mutation
in the HFE gene, located on chromosome 6p21.3, resulting in
References .................................................................................... 639
the C282Y change in the protein. The mutation is considered
to have appeared about 1,500 years ago. The prevalence of
C282Y homozygosity in Caucasians is about 1:250. However,
the penetrance is low and depends on additional risk factors,
e.g., male gender, alcohol use, and unknown factors. The age
at disease onset is 40–50 years. Another mutation (H63D) in
HFE may, when present as combined heterozygosity together
with C282Y, cause steatosis or chronic hepatitis but with
a less severe iron accumulation in the liver than in C282Y
homozygosity (Cheng et al. 2009). HFE protein is expressed
in the crypt cells of duodenum and regulates iron transport by
decreasing the iron uptake by transferrin.
The clinical picture of HFE-related hemochromatosis ranges
from biochemical abnormalities in iron metabolism to severe
disease with organ damage, including hepatosplenomegaly,
liver cirrhosis, diabetes mellitus, hyperpigmentation, cardiomy-
opathy, and hypogonadism (Pietrangelo 2010). Initial diagnostic
findings are elevated serum iron levels with increased transfer-
rin saturation and increased serum ferritin levels. Increased liver
iron content verifies the diagnoses, assessed either with liver
biopsy, specific iron content imaging, or magnetic resonance
imaging. Diabetes mellitus is a risk factor for severe iron accu-
mulation in HFE C287Y homozygotes (Wood et al. 2012).
In homozygotes for C287Y or in compound heterozy-
gotes C287Y/H63D with increased serum ferritin level,
V. Fellman
Department of Pediatrics, Lund University, 22185 Lund, Sweden treatment should be initiated with repeated blood withdraw-
e-mail: vineta.fellman@med.lu.se ings to achieve a serum ferritin level of 30–100 ug/L.
634 V. Fellman
Hereditary Hemochromatosis (40.2) (Knisely et al. 2004). It usually presents after birth with
Hepcidin, a peptide hormone synthesized and secreted by the hypochromic microcytic anemia and iron overload. The
liver, regulates iron release from duodenal enterocytes and overload has been attributed to hepcidin deficiency, since
macrophages by interaction with ferroportin expressed on transferrin is a regulator of hepcidin (Bartnikas 2012).
their surfaces. Hepcidin decreases the release of iron into
blood stream. HFE, transferrin receptor 2 (Tfr2), and hemo- Pulmonary Hemosiderosis (40.5)
juvelin (HJV) are all needed to adjust an optimal hepcidin Idiopathic pulmonary hemosiderosis is a rare chronic disor-
secretion. If any of these regulators is failing, hepcidin is der, where iron accumulation is mainly found in the lungs.
decreased resulting in an unrestricted flow of iron into the The symptoms consist of a combination of iron-deficiency
plasma and hemosiderosis (Pietrangelo 2010). Mutations in anemia, recurrent hemoptysis, and diffuse infiltrates in chest
these iron regulators are much less common than those in x-rays (Poggi et al. 2011). It has been associated with celiac
HFE and affect both Caucasians and non-Caucasians without disease (Lane-Hamilton syndrome), in which case gluten-
a gender difference. free diet should be instituted (Sethi et al. 2011).
In Tfr2, the most common mutation causing hemochro-
matosis is Y250X leading to a truncated protein. Typically, GRACILE Syndrome (40.6)
the patient is 30–40 years old and presents with cardiomy- The neonatal mitochondrial disorder GRACILE syndrome
opathy, endocrinopathy, and liver disease. Transferrin satura- (Fellman disease, MIM 603358) belongs to the “Finnish
tion and serum ferritin are both elevated. disease heritage,” which is a group of recessively inherited
Juvenile hemochromatosis appears in 15–20-year-old diseases accumulated in Finland because of founder effect
individuals with endocrinologic disturbances (impotence or and genetic drift. The acronym is composed of the typical
amenorrhea) with or without cardiomyopathy. Transferrin findings: fetal growth restriction (−4SD), aminoaciduria
saturation is high, as also serum ferritin level. The causative (Fanconi-type tubulopathy), cholestasis (with steatosis and
mutation has been G320V in HJV in about half of the cases. cirrhosis), iron overload, lactic acidosis, and early infant
In some families, the disease has been associated with muta- death (Fellman et al. 1998; Visapaa et al. 2002). The char-
tions in the hepcidin gene (HAMP) (Pietrangelo 2010). acteristic histopathological findings include severe iron
Mutations in ferroportin cause a distinct iron overload accumulation in the hepatocytes in all cases and slight
disorder with increased serum ferritin level despite a normal accumulation in spleen and pancreas in some infants
or low transferrin saturation (Pietrangelo 2004). In addition, (Fellman 2002; Rapola et al. 2002). Serum transferrin level
two mutations in ferroportin (C326S and C326Y) have been is low and transferrin saturation increased. Serum ferritin
ascribed to cause hepcidin resistance, thus resulting in concentrations have been increased in all cases. The reason
increased absorption of dietary iron and accumulated iron in for the iron accumulation is unknown but most probably
the hepatocytes (Pietrangelo 2010). relates to disturbance of placental iron transport. The
underlying genetic cause is a homozygous c.232A > G
Neonatal Hemochromatosis (40.3) mutation in the BCS1L gene resulting in the substitution of
Severe liver failure is uncommon in the neonatal period. One glycine 78 for serine (S78G) in the protein (Visapaa et al.
causative pathology is the accumulation of iron in the liver 2002). The genotype-phenotype consistency in Finland
associated with severe liver fibrosis, the so-called neonatal (Fellman et al. 2008) enables mutation-based diagnostics,
hemochromatosis. None of the described mutations in any of including antenatal in families with a previous case
the iron regulators have been found in this disorder. In some (Fellman et al. 2002).
cases, intrauterine infection, e.g., cytomegalovirus, has been BCS1L is a nuclear gene that encodes a mitochondrial
associated with liver hemosiderosis. Recent studies suggest protein which is a member of the so-called AAA family, i.e.,
that neonatal hemochromatosis may be linked to maternal ATPases associated with various cellular activities (Hanson
immune system dysregulation (Collardeau-Frachon et al. and Whiteheart 2005). BCS1L acts as a chaperone for incor-
2012). poration of the Rieske iron-sulfur protein into complex III
(ubiquinol-cytochrome c reductase) of the respiratory chain.
Atransferrinemia (40.4) The mutation results in deficiency of complex III, but exact
Atransferrinemia or hypotransferrinemia is a rare disease, pathophysiological mechanisms are still unclear (Kotarsky
generally inherited in an autosomal recessive pattern et al. 2010).
40 Iron Metabolism Disorders 635
Since the first publications on the GRACILE syndrome mutations in BCS1L than the typical Finnish homozygous
(Fellman et al. 1998) and its causative mutation (Visapaa S78G mutation. No treatment has so far been effective in this
et al. 2002), international interest in the gene and its disor- mitochondrial disorder.
ders has increased. More than 70 patients, mainly infants,
have been reported with diseases caused by BCS1L muta- Neurodegeneration with Brain Iron Accumulations (40.7)
tions (Kotarsky et al. 2010; Tuppen et al. 2010). Other muta- There are many reasons for excess iron in the brain; both iron
tions in the BCS1L gene than c.232A > G have been associated deficiency and iron overload may be associated with the con-
with a wide spectrum of phenotypes, ranging from mild condition. The predilection areas for iron accumulation are in
genital neurosensory hearing loss and distorted hair, the hall- basal ganglia. Pantothenate kinase-associated neurodegen-
marks of Björnstad syndrome (Hinson et al. 2007), to eration (PKAN, NBIA1) and PLA2G6-associated neurode-
tubulopathy alone or combined with hepatopathy with some generation (PLAN, NBIA2) are the main etiologies, but
iron accumulation and/or encephalopathy (Gil-Borlado et al. several other genetic causes have been identified (including
2009). FA2H, C19orf12, ATP13A2, CP, and FTL) (Schneider and
In GRACILE syndrome, the functional deterioration of Bhatia 2013). The list of identified causative genes continues
the liver with cholestasis and steatosis leading to a starvation- to expand and includes mitochondrial membrane protein-
like condition is the most probable cause for the failure to associated neurodegeneration (e.g., C19orf12 mutations)
thrive in the infants, who survive only a few days or weeks. (Dogu et al. 2013). With increasing knowledge of mitochon-
GRACILE-like disorders have been found in several coun- drial disorders and their relation to iron metabolism, new
tries (Spain, Turkey, New Zealand) in infants with other etiologies will be revealed.
40.2 Nomenclature
Gene Chromosomal Affected OMIM

No. Disorder Alternative name Abbreviation symbol localization protein no. Subtype
40.1 Hereditary HLAH1 HFE HFE 6p21.3 Iron transport 235200 All forms
hemochromatosis (type 1) protein
40.2.1 Hereditary HLAH2a HFE2A HJV 1q21.1 Hemojuvelin 602390 Juvenile form
hemochromatosis (type 2a)
40.2.2 Hereditary HLAH2b HFE2B HAMP 19q13.1 Hepcidin 602390 Juvenile form
hemochromatosis (type 2b)
40.2.3 Hereditary TfR2-related HFE3 TFR2 7q22 Transferrin 604250 Juvenile form
hemochromatosis (type 3) hereditary receptor 2
hemochromatosis
40.3 Neonatal hemochromatosis Congenital NH Maternal 231100 Neonatal form
alloimmune immune system
hepatitis dysregulation
40.4 Atransferrinemia Familial TF TF 3q22.1 Transferrin 209300 All forms
hypotransferrinemia
40.5 Pulmonary hemosiderosis ? 178550 All forms
40.6 GRACILE syndrome Fellman disease GRACILE BCS1L 2q33-37 BCS1L 603358 Early infantile
form
40.7 Neurodegeneration with Pantothenate NBIA1 PANK2 20p13 Pantothenate 234200 All forms
brain iron accumulation 1 kinase-associated kinase
neurodegeneration
(PKAN)
636 V. Fellman
Enterocyte
Fe2+
Fe3+ precursor cell
Intestine
Fe3+
FR
Cu – CP
DMT1
Tf
FT Fe2+
FT
FT Fe2+ TfR
FT DMT1
Fe2 – Tf
IREG1 Heph
TfR
sla Fe3+
Fe2+
HFE
Blood Tf
Fe2 – Tf
Fig. 40.1 Schematic drawing of the key elements of iron homeosta- internal pH due to the action of a proton pump (not shown). This leads
sis and defects in aceruloplasminemia and hemochromatosis. Intestinal to the dissociation of the iron from transferrin. Iron leaves the endo-
iron is reduced by an unknown ferric reductase (FR) and transported somes via DMT1. Apo-transferrin and transferrin receptors recycle to
into intestinal cells by the divalent metal transporter DMT1 (formerly the plasma membrane for reuse. This iron uptake mechanism is found
called Nramp2 or DCT1) and also by other routes. Inside cells, iron is in most cell types, including enterocyte precursor cells. Excess iron
stored as ferritin (FT) and hemosiderin. In erythroid cells, most of the can leave at least some cell types by an unknown mechanism involv-
iron moves to mitochondria, where it is incorporated into protoporphy- ing ceruloplasmin (CP), a non-membrane multicopper ferroxidase.
rin to make a heme. On the basolateral side, iron leaves the epithelium Hereditary hemochromatosis results from mutations in HFE (originally
via a basolateral transporter, IREG1, followed by oxidation through the called HLA-H), a protein with sequence similarity to major histocom-
action of hephaestin (Heph), a membrane-bound ceruloplasmin-like patibility complex class I molecules. HFE forms a heterodimer with
multicopper ferroxidase. Iron-loaded transferrin (Fe2-Tf) binds to the β2-microglobulin, and some mutations that lead to hemochromatosis
transferrin receptor (TfR) on the surface of cells. The receptor-trans- interrupt this interaction and thus lead to excess iron accumulation.
ferrin complex, localized in clathrin-coated pits (TTTT), is invaginated Defects in a second transferrin receptor, TfR2, have recently been
and forms endosomes. These specialized endosomes acquire a low implicated in type 3 hemochromatosis
Table 40.1 Hereditary hemochromatosis (type 1)

Cardiovascular Cardiomyopathy ± +
Dermatological Hyperpigmentation ±
Digestive Abdominal pain ± ± ± ±
Liver fibrosis ± +
Musculoskeletal Arthralgia ± ± +
Routine laboratory ASAT/ALAT (P) ↑
Bilirubin (P) n-↑
Ferritin (S) ↑ ↑ ↑ ↑
Glucose (P) n-↑
Iron (liver) ↑ ↑ ↑
Iron (U) ↑
Transferrin saturation ↑ ↑ ↑ ↑
Table 40.2.1 Hereditary hemochromatosis (type 2a)

Cardiovascular Cardiomyopathy ++
CNS Fatigue +
Digestive Hepatopathy +
Liver cirrhosis +
Endocrine Hypogonadism ++
Musculoskeletal Arthralgia +
Routine laboratory Ferritin (S) ↑
Glucose (P) n-↑
Iron (liver) ↑
Transferrin saturation ↑
Table 40.2.2 Hereditary hemochromatosis (type 2b)

Cardiovascular Cardiomyopathy +
Endocrine Hypogonadism +
Glucose (P) ↑
Iron (liver) ↑
Table 40.2.3 Hereditary hemochromatosis (type 3)

Cardiovascular Cardiomyopathy ± +
Dermatological Hyperpigmentation +
Digestive Abdominal pain ± ± ± ±
Liver fibrosis ± +
Musculoskeletal Arthralgia ± ± +
Routine laboratory Ferritin (S) ↑ ↑ ↑ ↑
Glucose (P) n-↑
Iron (liver) ↑ ↑ ↑
Transferrin saturation ↑ ↑ ↑ ↑
638 V. Fellman
Table 40.3 Neonatal hemochromatosis

Digestive Liver fibrosis +
Iron (liver) ↑
Table 40.4 Atransferrinemia

Hematological Anemia, hypochromic + + + +
Hemosiderosis + +
Musculoskeletal Growth retardation + +
Other Recurrent infections + +
Routine laboratory Iron (liver) ↑
Transferrin (S) ↓ ↓ ↓ ↓
Table 40.5 Pulmonary hemosiderosis

Hematological Anemia + +
Musculoskeletal Growth retardation + +
Respiratory Lung bleeding + +
Routine laboratory Iron (S) ↓ ↓
Table 40.6 GRACILE syndrome

Digestive Cholestasis +++ (↑) ND ND ND
Ear Deafness, sensorineural +++ ++ ND ND ND
Renal Renal tubulopathy, proximal +++ (↑) ND ND ND
Others Early death +++ ++ ND ND ND
Intrauterine growth retardation +++ ND ND ND ND
Routine laboratory Iron (S) +++ ± ND ND ND
Lactic acidosis +++ (↑) ND ND ND
Table 40.7 Neurodegeneration with brain iron accumulation 1

CNS Dementia +
Depression +
Extrapyramidal signs + + +
Eye Optic atrophy +
Retinopathy ± ±
Vision, impaired ± + +
Routine laboratory Iron (brain) ↑ ↑ ↑

Serum Hb (g/L) ± 2SD Iron (μmol/L) Ferritin (μg/L) Transferrin (g/L; range)
Newborn 185 ± 30 6.4–33.0 110–503 1.8 (1.4–2.29)
3–6 months 115 ± 20 6.4–33.0 4–405 2.03 (1.58–2.57)
6–12 months 120 ± 15 6.4–33.0 4–405 –
2–6 years 125 ± 10 6.4–33.0 4–405 2.39 (1.86–3.03)
6–12 years 135 ± 20 6.4–33.0 4–405 2.17 (1.97–3.19)
12–18 years (w) 140 ± 20 6.4–33.0 9–79 2.17 (1.97–3.19)
12–18 years (m) 145 ± 15 6.4–33.0 9–59 2.17 (1.97–3.19)
>18 years (w) 140 ± 20 6.6–26.0 6–81 2.0–3.4
>18 years (m) 155 ± 20 10.6–28.0 30–233 –
CSF – 0.4 (0.2–0.6) – 14.4 mg/L
HFE >30 >300 (up to 5,000) >70
40.6 Specimen Collection In ferroportin deficiency, treatment by phlebotomy is dif-

ficult because of anemia.
Iron: Fasting in serum or hair (at RT) In perinatal hemochromatosis, supportive treatment in a
Ferritin and transferrin: Fasting in serum (at RT) neonatal intensive care unit is essential; liver transplanta-
tion is usually required. A multiple-drug regimen, called
the “antioxidant cocktail” (Roberts et al. 2006), has been
40.7 Prenatal and DNA Testing used extensively with some success. Not all infants respond
to this regimen, but early institution of treatment may favor
Possible for all disorders except for neonatal hemochromato- success. Monitoring subsequent pregnancies closely
sis (40.3) and pulmonary hemosiderosis (40.5) appears critically important. Surviving infants appear to
stabilize clinically; they may develop cirrhosis or have no
residual liver disease. Incidental hepatocellular carcinoma
40.8 Treatment has been reported in three infants. Recurrent iron accumu-
lation in the liver graft occurred in one infant after
Summary transplantation.
The term “hemochromatosis” refers to iron accumulation in
parenchymal cells of the liver and other tissues.
Approximately 90 % of primary hemochromatosis is due to
mutations in the HFE gene. Other types of primary hemo- References
chromatosis are rare. Secondary hemochromatosis is usually
Bartnikas TB (2012) Known and potential roles of transferrin in iron
related to congenital hemolytic anemia requiring chronic
biology. Biometals 25:677–686
transfusion or to dietary excess in a genetically susceptible Cheng R, Barton JC, Morrison ED, Phatak PD, Krawitt EL et al (2009)
individual (Bantu siderosis) (Roberts et al. 2006). Differences in hepatic phenotype between hemochromatosis
patients with HFE C282Y homozygosity and other HFE genotypes.
J Clin Gastroenterol 43:569–573
Standard and Emergency Treatment
Collardeau-Frachon S, Heissat S, Bouvier R, Fabre M, Baruteau J et al
In hereditary hemochromatosis, classic form treatment is (2012) French retrospective multicentric study of neonatal hemo-
indicated even if cirrhosis has developed, and symptoms chromatosis: importance of autopsy and autoimmune maternal
relating to extrahepatic disease may improve on treatment. manifestations. Pediatr Dev Pathol 15:450–470
Dogu O, Krebs C, Kaleagasi H, Demirtas Z, Oksuz N et al (2013) Rapid
Vitamin C supplements should be avoided.
disease progression in adult-onset mitochondrial membrane protein-
In juvenile hemochromatosis, treatment with phlebotomy associated neurodegeneration. Clin Genet 84:350–355. doi:10.1111/
is indicated. cge.12079
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Fellman V (2002) The GRACILE syndrome, a neonatal lethal meta- Pietrangelo A (2010) Hereditary hemochromatosis: pathogenesis, diag-
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Fellman V, Visapaa I, Vujic M, Wennerholm UB, Peltonen L (2002) Rapola J, Heikkila P, Fellman V (2002) Pathology of lethal fetal
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newborn infant. Acta Obstet Gynecol Scand 81:398–402 183–193
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Purine and Pyrimidine Disorders
41
Jörgen Bierau and Ivan Šebesta
Contents Summary
41.1 Introduction ...................................................................... 642 Genetic defects of purine and pyrimidine metabolism
represent a group of relatively new disorders. Xanthinuria,
41.2 Nomenclature.................................................................... 643
the first genetic metabolic purine disorder, was described in
41.3 Metabolic Pathways ......................................................... 645 children as the cause of renal stones in 1954, and a genetic
41.4 Signs and Symptoms Tables ............................................ 647 basis for the Lesch-Nyhan syndrome accompanied by gout
in childhood and adolescence with serious neurological
41.5 Reference and Pathological Values ................................. 654
impairment was recognised in 1967. The number of enzyme
41.6 Diagnostic Flow Chart ..................................................... 656 defects identified since then has increased rapidly and now
41.7 Sample Collection and Storage ....................................... 657 totals 27. Not surprisingly, knowledge at the clinical level
41.8 Prenatal Diagnosis Table and DNA Analysis ................. 657 has been unable to keep the pace, a problem compounded
by the broad spectrum of presentation and genetic heteroge-
41.9 Treatment Summary ........................................................ 657
neity. Clinical presentations may involve renal, musculo-
References .................................................................................... 659 skeletal, neurological, immunological and haematological
systems. Although these disorders are generally paediatric
problems, some disorders can manifest themselves in
patients at any age from birth to advanced adulthood.
Adding to this complexity is the rapidly expanding group of
defects in mitochondrial purine or pyrimidine metabolism.
Their recent description means that awareness is limited
and affected patients are frequently misdiagnosed or remain
undiagnosed, particularly in adults where such defects are
increasingly being recognised as the basis of serious illness.
Although some conditions are relatively benign, others can
have serious consequences. Currently, there appear to be
few laboratories worldwide providing the necessary com-
J. Bierau (*)
prehensive diagnostic services for detailed purine and
Laboratory Biochemical Genetics, Department of Clinical
Genetics, Maastricht University Medical Centre, pyrimidine investigations. Recent advances increasingly
5800, 6202 AZ Maastricht, The Netherlands implicate these defects in adults as well as children.
e-mail: jorgen.bierau@mumc.nl Consequently, effort is required to alert physicians to the
I. Šebesta broad spectrum of clinical presentation. The proper indica-
Institute of Medical, Biochemistry and Laboratory Medicine, tions and detection of new cases not only will help a num-
Institute of Inherited Metabolic Disorders,
ber of unfortunate patients but will almost certainly lead to
First Faculty of Medicine, Charles University,
Kateřinská 32, 12108 Prague 2, Czech Republic the illumination of the still unknown pathogenesis of these
e-mail: isebes@lf1.cuni.cz disorders.
642 J. Bierau and I. Šebesta
41.1 Introduction proliferative effects on vascular smooth muscle cells and

causes dysfunction of endothelial cells (Feig et al. 2008;
Purines and pyrimidines are omnipresent in all living Mazzali et al. 2010). Thus, uric acid has a role as a true car-
creatures. Not only are they the building blocks of our genetic diovascular risk factor, particularly for the development of
code in the form of DNA and RNA, but they also fulfil hypertension or renal disease, and so lowering uric acid in
numerous roles in biological processes, including energy subjects with asymptomatic hyperuricemia with allopurinol
transport, signal transduction and cell cycle progression of might improve endothelial dysfunction (Kanbay et al. 2011).
carriers of glycans and lipids and antioxidants, both in the Monitoring and controlling hyperuricemia may be more
intra- and extracellular matrices. It is therefore easy to com- important for physicians than previously thought. Another
prehend that the metabolism of these compounds is a com- focus of attention that has arisen in the last decade is the set
plex and tightly regulated interplay of a myriad of processes of mitochondrial DNA depletion syndromes, such as thymi-
and that any disturbance herein may have clinical dine kinase 2 and deoxyguanosine kinase deficiencies.
consequences. Despite the fact that these conditions are often considered to
Because of the diversity of biological functions of purines be mitochondrial diseases and lack obvious purine or pyrim-
and pyrimidines, the clinical presentation of the disorders idine marker metabolites, these are clearly defects of purine
covered in this chapter is extremely diverse. Even in one fam- and pyrimidine metabolism.
ily the clinical presentation may vary widely from patient to The laboratory diagnosis of purine and pyrimidine defects
patient. Some enzyme defects manifest themselves as clearly is made by any combination of the recognition of abnormal
distinct subtypes (Sebesta et al. 2006; Zanella et al. 2006). In concentrations of metabolites in urine, plasma, CSF or eryth-
general, there is not one presenting clinical sign that irrefut- rocytes; decreased/increased enzyme activities in red or
ably makes a clinical diagnosis (Simmonds and van Gennip white blood cells, in cultured fibroblasts and in very rare
2003; van den Berghe et al. 2006; van Gennip et al. 2006). cases in the liver of muscle biopsies; and finally molecular
Some conditions, however, have clear clinical signs, and, genetic anomalies. Analytical techniques have evolved
when present, the analysis of purines and pyrimidines is strongly in the past years and still are doing so. Especially
called for. Examples of these are kidney and bladder stones, the remarkable technological advances in tandem mass spec-
hyperuricemia (de Brouwer et al. 2010), hypouricemia trometry are of great value for the analysis of purines and
(Sebesta et al. 2011), gout, and self-mutilation (Micheli et al. pyrimidines. When using more traditional techniques such as
2011). In most other cases, the constellation of symptoms is thin-layer chromatography or classical HPLC with diode
not very conclusive (Assmann et al. 2006), and one should array detection, only a set of common, abundant metabolites
have a liberal policy concerning screening purine and pyrimi- could easily be detected and quantified in control subjects,
dine metabolites in body fluids. and patients were recognised by abnormal concentrations or
In the past, disorders in purine and pyrimidine metabo- abnormal metabolites. Next-generation liquid chromatogra-
lism were considered to be paediatric conditions, and in phy in the form of ultra performance liquid chromatography
their classical presentation many of these disorders still are (UPLC) has greatly increased the sensitivity of liquid chro-
(Camici et al. 2010). However, in the last decade, the focus matography. The greatest advantage, however, has been
is also shifting towards adult medicine. An example hereof made in tandem mass spectrometry. Virtually all nucleoside
is MNGIE syndrome caused by deficiency of thymidine intermediates of purine and pyrimidine metabolism can be
phosphorylase (Hirano et al. 2006). Another example is that detected in the urine in one comprehensive analytical run
the pharmacogenetic significance of the pyrimidine degra- (Hartmann et al. 2006; van Kuilenburg et al. 2008).
dation defects, in general, and dihydropyrimidine dehydro- Metabolites are detected at levels down to submicromolar
genase, in particular (van Kuilenburg 2004), and the concentrations.
purine-metabolising enzymes thiopurine methyltransferase Nowadays, molecular analysis is available, for all disor-
(Weinshilboum et al. 1999) and inosine triphosphatase ders (Database used 2011). Prenatal diagnosis is feasible and
(Bierau et al. 2007) are important topics of research and can be applied to chorionic villi, amniotic fluid or cells.
laboratory diagnosis nowadays. In addition, recent studies When the mutation(s) in the family is (are) known, molecu-
have found that soluble uric acid has proinflammatory and lar diagnostics is the option of choice.
41
41.2 Nomenclature
Gene Chromosomal
41.1 Phosphoribosyl pyrophosphate X-linked Charcot-Marie-Tooth PRPPS PRPS1 Xq21.32-q24 Phosphoribosyl pyrophosphate 311850 All forms
synthetase 1 defects disease-5 synthetase
41.2 Phosphoribosyl pyrophosphate PRPS1 PRPS1 Xq22-q24 Phosphoribosyl pyrophosphate 300661 All forms
synthetase 1 superactivity synthetase 1
41.3 Adenylosuccinate lyase ADSL deficiency ADSL ASL 22q13.1 Adenylosuccinate lyase 103050, 608222 All forms
deficiency
41.4 AICAR transformylase/IMP AICAribosiduria AICAR ATIC 2q35 AICAR transformylase/IMP 608688, 601731 All forms
Purine and Pyrimidine Disorders
cyclohydrolase deficiency cyclohydrolase

41.5 Adenosine monophosphate Myoadenylate deaminase AMPD AMPD1 1p21-p13 Adenosine monophosphate 102770 All forms
deaminase deficiency deficiency deaminase
41.6 Inosine monophosphate IMPDH1 IMPDH1 7q31.3-q32 Inosine monophosphate 146690 Juvenile and
dehydrogenase deficiency dehydrogenase adult form
41.7 Adenosine deaminase Severe combined ADA ADA 20q13.11 Adenosine deaminase 608958 All forms
deficiency immunodeficiency (SCID)
41.8 Purine nucleoside T-cell immunodeficiency PNP PNP 14q13.1 Purine nucleoside 613179, 164050 All forms
phosphorylase deficiency phosphorylase
41.9 Xanthine dehydrogenase Xanthinuria type I XDH XDH 2p23-p22 Xanthine dehydrogenase 278300, 607633 All forms
deficiency (oxidase)
41.9.1 Combined xanthine oxidase Xanthinuria type II XDH/AO Xanthine oxidase and aldehyde 603592 All forms
and aldehyde oxidase oxidase
deficiency
41.10 Adenine 2,8-Dihydroxyadeninuria APRT APRT 16q24.3 Adenine 102600 All forms
phosphoribosyltransferase phosphoribosyltransferase
deficiency
41.11 Hypoxanthine-guanine Lesch-Nyhan syndrome HGPRT HPRT1 Xq26-q27.2 Hypoxanthine-guanine 300322, 308000 All forms
phosphoribosyltransferase phosphoribosyltransferase
deficiency
41.12 Dihydroorotate dehydrogenase Miller syndrome POADS DHODH 16q22 Dihydroorotate dehydrogenase 263750, 126064 All forms
deficiency
41.13 Orotic aciduria type I Uridine monophosphate – UMPS 3q21.2 Uridine monophosphate 258900 All forms
synthase deficiency synthase
41.14 Orotic aciduria type II Orotidine-5-monophosphate – UMPS 3q21.2 Uridine monophosphate 258920 All forms
decarboxylase deficiency synthase
41.15 Pyrimidine-5′-nucleotidase I UMP hydrolase I deficiency – UMPH1 7p14.3 Pyrimidine-5′-nucleotidase I 266120, 606224, All forms
deficiency 191720
41.16 Pyrimidine 5′-nucleotidase Uridine-5′-monophosphate – UMPH1 7p14.3 Uridine-5′-monophosphate 266120, 197720 All forms
superactivity hydrolase superactivity hydrolase
(continued)
643
Gene Chromosomal
644
41.17 Thymidine phosphorylase Mitochondrial MNGIE TYMP 22q13.32-qter Thymidine phosphorylase 131222, 603041 All forms
deficiency neurogastrointestinal
encephalopathy syndrome
41.18 Dihydropyrimidine Thymine-uraciluria DPD DPYD 1p22 Dihydropyrimidine 274270, 612779 All forms
41.19 Dihydropyrimidinase Dihydropyrimidinuria DHP DPYS 8q22 Dihydropyrimidinase 222748, 613326 All forms
deficiency
41.20 Beta-ureidopropionase – UPB1 22q11.2 Beta-ureidopropionase 613161, 606673 All forms
deficiency
41.21 Beta-alanine alpha- Hyper-beta-alaninaemia – Beta-alanine-2-ketoglutarate 237400 All forms
ketoglutarate transaminase transaminase
deficiency
41.22 Beta-aminoisobutyrate- – Beta-aminoisobutyrate- 210100 All forms
pyruvate transaminase pyruvate transaminase
deficiency
41.23 Deoxyguanosine kinase Mitochondrial DNA depletion MTDPS3 GGUOK 2p13 Mitochondrial deoxyguanosine 251880, 601465 All forms
deficiency syndrome 3 (hepatocerebral kinase
type)
41.24 Thymidine kinase 2 deficiency Mitochondrial DNA depletion MTDPS2 TKS 16q22 Thymidine kinase 2 609560,188250 All forms
syndrome 2 (myopathic type)
41.25 Mitochondrial ribonucleotide Mitochondrial DNA depletion MTDP8A RRM2B 8q23.1 Mitochondrial ribonucleotide 604712 All forms
reductase subunit 2 deficiency syndrome 8A and 8B and MTDP8B reductase
41.26 Thiopurine S-methyltransferase Decreased thiopurine tolerance – TPMT 6p22.3 Thiopurine S-methyltransferase 610460,187680 All forms
deficiency
41.27 Inosine triphosphatase Inosine-5′-triphosphate – ITPA 20p13 Inosine-5′-triphosphate 147520 All forms
deficiency pyrophosphohydrolase pyrophosphohydrolase
deficiency
J. Bierau and I. Šebesta
41 Purine and Pyrimidine Disorders 645
PRPPS
Ribose-5-P PRPP
8 steps
SAICARP SAICAr
ADSL
dATP ATP GTP dGTP
AICARP AICAr
SAdo
ATIC
RR RR
dADP ADP FAICARP GDP dGDP
S-AMP ATIC
ADSL
dAMP AMP IMP XMP GMP dGMP
AMPDA IMPDH
HPRT
dGUOK
ADA
2'-Deoxy APRT Adenosine Inosine Xanthosine Guanosine
Adenosine PNP 2'-Deoxy
PNP PNP Guanosine
PNP
Adenine HPRT
Hypoxanthine Guanine
ADA PNP PNP
XO
XO Xanthine Urate
2'-Deoxy
Inosine XO
2,8-dihydroxy Adenine
PNP
Fig. 41.1 Overview of purine metabolism. Boxed metabolites indicate RR ribonucleotide reductase, HPRT hypoxanthine-guanine phosphori-
marker metabolites. Only enzymes with disorders are mentioned. bosyltransferase, dGUOK deoxyguanosine kinase, PNP purine nucleo-
Enzyme abbreviations: PRPPS phosphoribosyl pyrophosphate synthe- side phosphorylase, XO xanthine oxidase (dehydrogenase), AMPDA
tase, ADSL adenylosuccinate lyase, ATIC, AICAR transformylase/IMP adenosine-5′-monophosphate deaminase, APRT adenine phosphoribos-
cyclohydrolase, IMPDH inosine-5′-monophosphate dehydrogenase, yltransferase, ADA adenosine deaminase
Urea cycle Cytidine Uridine
HCO 3– + Glutamine HCO 3 – + Glutamine

CPS1 CPS2
Hyperammonaemia CTP
Carbamoyl-P Carbamoyl-P
Aspartate ACT CAD
OTC UTP
Ornithine Citrulline Carbamoylaspartate
DHO RR
Dihydroorotate Dihydroorotate UMPS UDP dUDP
DHODH
OPRT OMPDC TS
Orotate Orotate OMP UMP dUMP dTMP
PRPP +PRPP
Mitochondrion
Orotidine Uridine 2'-deoxy Thymidine
Uridine
TP
Uracil TP Thymine
DPD DPD
Dihydrouracil Dihydrothymine
DHP DHP
N-Carbamoyl- N-Carbamoyl-
propionate isobutyrate
UP UP
β-Alanine β-Aminoisobutyrate
Fig. 41.2 Overview of pyrimidine metabolism. Boxed metabolites synthetase 1, OTC ornithine transcarbamoylase, DHODH dihydrooro-
indicate marker metabolites. Only pyrimidine key enzymes and tate dehydrogenase, UMPS UMP synthase complex consisting of oro-
enzymes with known disorders are mentioned. For clarity, the link with tate phosphoribosyltransferase (OPRT) and OMP decarboxylase
the urea cycle is also depicted. Enzyme abbreviations: CAD complex of (OMPDC), RR ribonucleotide reductase, TS thymidylate synthase, TP
carbamoyl phosphate synthetase 2 (CPS2), aspartate carbamoyltrans- thymidine phosphorylase, DPD dihydropyrimidine dehydrogenase,
ferase (ACT) and dihydroorotase (DHO), CPS1 carbamoyl phosphate DHP dihydropyrimidinase, UP ß-ureidopropionase
41.1–41.6: Defects of Purine Nucleotide Biosynthesis
Table 41.1 Phosphoribosyl pyrophosphate synthetase 1 defects

CNS Ataxia +
Hypotonia + +
Mental retardation + +
Motor retardation + +
Tetraplegia +
Eye Optic atrophy + + +
Vision, progressive loss + + +
Haematological Recurrent infections + + +
Table 41.2 Phosphoribosyl pyrophosphate synthetase 1 superactivity

CNS Ataxia ± ± ±
Mental retardation ± ±
Ear Deafness, sensorineural + + + +
Musculoskeletal Dysmorphic features ± ± ±
Renal Urolithiasis ± ± ±
Routine laboratory Gout + +
Uric acid (P) n-↑ n-↑ n-↑ ↑↑ ↑↑
Uric acid (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Special laboratory Hypoxanthine (U) ↑ ↑ ↑ ↑ ↑
Phosphoribosyl pyrophosphate (RBC) ↑ ↑ ↑ ↑ ↑
PRPP synthetase activity (RBC, FB) ↑ ↑ ↑ ↑ ↑
Table 41.3 Adenylosuccinate lyase deficiency

CNS Autism ± ± ± ± ±
Cerebellar hypoplasia ± ± ± ± ±
Special laboratory Enzyme (erythrocyte) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
SAICAriboside (U, P, CSF) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Succinyladenosine (U,P, CSF) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 41.4 AICAR transformylase/IMP cyclohydrolase deficiency

CNS Mental retardation +++ +++ +++
Eye Blindness +++ ++ +++
Musculoskeletal Dysmorphic features ++ ++ ++
Special laboratory AICAriboside (U) ↑↑↑ ↑↑↑ ↑↑↑
Enzyme (fibroblasts) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 41.5 Adenosine monophosphate deaminase deficiency

Musculoskeletal Exercise intolerance ± ± ±
Muscle cramps ± ± ±
Others Acquired, associated with neuromuscular rheumatological +
disorders
Routine laboratory Creatine kinase (P) + + +
Special laboratory Enzyme (muscle biopsy) ↓ ↓ ↓
Ischaemic muscle exercise tolerance test (plasma NH3) ↓ ↓ ↓
Table 41.6 Inosine monophosphate dehydrogenase deficiency

Eye Leber amaurosis-like +
Pigmentary retinopathy + + +
41.7–41.9: Defects of Purine Nucleotide Breakdown
Table 41.7 Adenosine deaminase deficiency

Haematological CD19+ cells ↓↓↓ ↓↓ ↓ ↓ ↓
CD4+ cells ↓↓↓ ↓↓ ↓ ↓ ↓
CD8+ cells ↓↓↓ ↓↓ ↓ ↓ ↓
Immunodeficiency, severe combined (SCID) +++ ++ + + +
Immunoglobulins, low +++ ++ + + +
Lymphopaenia +++ ++ + + +
Special laboratory dATP (RBC) ↑↑↑ ↑↑ ↑↑ ↑↑ ↑
Deoxyadenosine (U) ↑↑↑ ↑↑ ↑↑ ↑↑ ↑
Enzyme (erythrocyte) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 41.8 Purine nucleoside phosphorylase deficiency

CNS Developmental delay + + +
Spastic diplegia ++ ++ ++
Tetraparesis ++ ++ ++
Haematological CD4+ cells ↓-n ↓-n ↓-n
Immunodeficiency, T-cell + + + + +
Routine laboratory Uric acid (U,P) ↓↓ ↓↓ ↓↓ ↓ ↓
Special laboratory dGuo (U,P) ↑↑ ↑↑ ↑↑ ↑ ↑
Dino (U,P) ↑↑ ↑↑ ↑↑ ↑ ↑
Enzyme (erythrocyte) ↓↓↓ ↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 41.9.1 Xanthine dehydrogenase deficiency

Musculoskeletal Myopathy + +
Renal Renal failure, acute ± ± ± ± ±
Urolithiasis ± ± ± ± ±
Urolithiasis, xanthine stones ± ± ± ± ±
Routine laboratory Uric acid (U,P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Special laboratory Allopurinol to oxipurinol conversion + + + + +
Hypoxanthine (P) ↑ ↑ ↑ ↑ ↑
Hypoxanthine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Xanthine (P) ↑ ↑ ↑ ↑ ↑
Xanthine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 41.9.2 Combined xanthine oxidase and aldehyde oxidase deficiency

Musculoskeletal Myopathy ± ±
Urolithiasis, xanthine stones ± ± ± ± ±
Routine laboratory Uric acid (U,P) ↓↓ ↓↓ ↓↓ ↓↓↓ ↓↓
Special laboratory Allopurinol to oxipurinol conversion ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Xanthine (P) ↑ ↑ ↑ ↑ ↑
Xanthine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
41.10, 41.11: Defects of Purine Salvage Pathway
Table 41.10 Adenine phosphoribosyltransferase deficiency

Renal Renal colic + + + + +
Renal failure, acute ± ± ± ± ±
Renal failure, chronic + + + +
Routine laboratory Haematuria + + + + +
Special laboratory 2,8-Dihydroxyadenine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Adenine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Enzyme (erythrocyte) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 41.11 Hypoxanthine-guanine phosphoribosyltransferase deficiency

CNS Cerebral palsy + + + + +
Choreoathetosis + + +
Pyramidal signs + + + + +
Self-mutilation + + + + +
Spasticity + + + + +
Urinary infections + + +
Urolithiasis + + + + +
Routine laboratory Haematuria + + +
Uric acid (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Uric acid (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑ ↑↑
Special laboratory AICAriboside (U) ↑ ↑ ↑ ↑
Enzyme (erythrocyte) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Xanthine (U) ↑ ↑ ↑ ↑ ↑
41.12–41.14: Defects of Pyrimidine Biosynthesis
Table 41.12 Dihydroorotate dehydrogenase deficiency

Ear Deafness, conductive ± ± ± ±
Eye Coloboma ± ± ± ± ±
Others Miller syndrome + + + + +
Table 41.13 Orotic aciduria type I

Haematological Anaemia, megaloblastic +++ +++ +++ +++ +++
Immunodeficiency, T-cell ± ± ± ± ±
Recurrent infections ± ± ± ± ±
Renal Urolithiasis + + + + +
Special laboratory Enzyme (erythrocyte) ↓ ↓ ↓ ↓ ↓
Orotic acid (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Orotic acid (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 41.14 Orotic aciduria type II

Special laboratory Enzyme (erythrocyte) ↓ ↓ ↓ ↓ ↓
Orotic acid (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Orotic acid (U) ↑↑↑ ↑↑↑ ↑↑ ↑↑↑ ↑↑↑
Orotidine (P) ↑ ↑ ↑ ↑ ↑
Orotidine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
41.15–41.22: Defects of Pyrimidine Nucleotide Breakdown
Table 41.15 Pyrimidine-5′-nucleotidase I deficiency

Haematological Anaemia, non-spherocytic haemolytic with basophilic stippling + + + + +
Splenomegaly + + + +
Musculoskeletal Myoglobinuria ± ± ± ± ±
Special laboratory Enzyme (erythrocyte) ↓↓↓ ↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Glutathione (RBC) ↓-n ↓-n ↓-n ↓-n ↓-n
Pyrimidine nucleotides (erythrocyte) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 41.16 Pyrimidine 5′-nucleotidase superactivity

CNS Developmental delay + +
Fits + +
Hyperactivity + +
Seizures + +
Haematological Recurrent infections + +
Routine laboratory Uric acid (U) ↓ ↓
Special laboratory Enzyme (fibroblasts) ↑↑ ↑↑
Phosphoribosyl pyrophosphate (FB) ↓ ↓
Table 41.17 Thymidine phosphorylase deficiency

CNS Areflexia + +
Hypodense white matter + +
Leucoencephalopathy + +
Neuropathy, myelinating + +
Anorexia + +
Diarrhoea + +
Gastrointestinal dysmotility + +
Gastroparesis + +
Intestinal pseudo-obstruction + +
Malabsorption + +
Malnutrition, chronic + +
Vomiting + +
Musculoskeletal Muscle weakness + +
Myopathy + +
Special laboratory Deoxyuridine (P) ↑ ↑
Deoxyuridine (U) ↑↑ ↑↑
Enzyme (wbc) ↓↓↓ ↓↓↓
Ragged red fibres + +
Thymidine (P) ↑ ↑
Thymidine (U) ↑↑ ↑↑
Table 41.18 Dihydropyrimidine dehydrogenase deficiency

CNS Autism ± ± ± ±
Cerebral atrophy ± ± ± ± ±
Hyperactivity ± ± ± ± ±
Motor retardation ± ± ± ±
Digestive Feeding difficulties ±
Eye Coloboma ± ± ± ±
Eye movements, abnormal ± ± ± ± ±
Nystagmus ± ± ± ±
Optic atrophy ± ± ± ±
Musculoskeletal Microcephaly ± ± ± ±
Others Failure to thrive ±
Severe 5-fluorouracil toxicity (hetero- and homozygotes) +++
Special laboratory 5-OH-Methyluracil ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Enzyme (fibroblasts) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Enzyme (wbc) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Thymine (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Thymine (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Uracil (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Uracil (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
White matter abnormalities ± ± ± ±
Table 41.19 Dihydropyrimidinase deficiency

CNS Mental retardation ± ± ± ±
Special laboratory Dihydrothymine (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Dihydrothymine (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Dihydrouracil (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Dihydrouracil (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 41.20 Beta-ureidopropionase deficiency

CNS Dystonia ± ± ± ± ±
Hypotonia + +
Speech disturbances ± ± ±
Special laboratory Dihydrothymine (P) ↑ ↑ ↑ ↑ ↑
Dihydrothymine (U) ↑ ↑ ↑ ↑ ↑
Dihydrouracil (P) ↑ ↑ ↑ ↑ ↑
Dihydrouracil (U) ↑ ↑ ↑ ↑ ↑
N-Carbamoyl-β-alanine (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
N-Carbamoyl-β-alanine (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
N-Carbamoyl-β-aminoisobutyric acid (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
N-Carbamoyl-β-aminoisobutyric acid (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Table 41.21 Beta-alanine alpha-ketoglutarate transaminase deficiency

CNS Seizures + +
Somnolence + +
Special laboratory Beta-alanine (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Table 41.22 Beta-aminoisobutyrate-pyruvate transaminase deficiency

Others No clinical significance
Special laboratory Beta-aminoisobutyrate (U) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
41.23–41.25: Defects of Mitochondrial Metabolism
Table 41.23 Deoxyguanosine kinase deficiency

Autonomic system Hypothermia + +
CNS Cerebral atrophy + +
Encephalopathy, progressive ++ ++
Hypotonia + +
Seizures + +
Tendon reflexes, increased + +
Digestive Failure to thrive ++ +
Hepatosplenomegaly + +
Jaundice +++ +++
Liver fibrosis + +
Liver steatosis + +
Eye Eye movements, abnormal + +
Nystagmus + +
Nystagmus + +
Musculoskeletal Microcephaly ± ±
Routine laboratory Albumin (P) ↓ ↓
Alpha-fetoprotein (P) (PGDH) ↑↑↑ ↑↑↑
ASAT/ALAT (P) ↑↑↑ ↑↑↑
Bilirubin (P) ↑↑↑ ↑↑↑
Glucose (P) ↓↓ ↓↓
Lactate (P) ↑↑↑ ↑↑↑
Special laboratory Amino acids, all + +
Sialotransferrins (S) + +
Table 41.24 Thymidine kinase 2 deficiency

CNS Hypotonia + +
Musculoskeletal Atrophy + +
Muscle weakness + +
Routine laboratory Creatine kinase (P) ↑ ↑
Lactate (P) ↑ ↑
Special laboratory Mitochondrial DNA depletion (M) + +
Ragged red fibres + +
Respiratory chain activity (M) ↓ ↓
Table 41.25 Mitochondrial ribonucleotide reductase subunit 2 deficiency

CNS Dysmyelination +
Seizures +
Feeding difficulties +
Feeding difficulties + + ±
Gastrointestinal dysmotility +
Ptosis of eyelid +
Musculoskeletal Muscle weakness +
Renal Renal tubulopathy + + ±
Routine laboratory Lactate (P) ↑↑ ↑↑ ↑
Special laboratory Amino acids, all + + ±
Mitochondrial DNA depletion (M) +++ +++
Respiratory chain activity (M) ↓↓↓ ↓-n
41.26, 41.27: Pharmacogenetic Defects
Table 41.26 Thiopurine S-methyltransferase deficiency

Others Thiopurines, decreased tolerance + + + +
Table 41.27 Inosine triphosphatase deficiency

Haematological Anaemia, ribavirin induced + +
Others Thiopurines, decreased tolerance ± ± ± ± ±
Special laboratory Erythrocyte ITP accumulation ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Urine purines (LC-MS/MS)

Compound rangea 0–1 year (n = 16) 1–5 years (n = 47) 5–16 years (n = 27) >16 years (n = 26) Pathological valuesb
2,8-Dihydroxy adenine 0–5.9 0–6.0 0–1.2 0–2.2 >10
SAICArc 0–2.0 0–0.9 n.d. 0–0.3 >10
Urated 820–1,026 527–790 326–436 222–287 <100 (all)
>3,500 (0–5 years)
>500 (>5 years)
Hypoxanthine 1–71.9 1–88.1 1–14.1 1–14.0 >100
<1
Xanthine 0–63.4 0–54.7 0–21.7 0.3–10.7 >70
<1
Adenine 0–4.8 0–2.8 0–0.9 0–0.6 >10
AICAR 0–4.5 0–3.0 0–1.7 0–1.6 >10
Inosine 0–6.1 0–4.5 0–1.2 0–0.6 >30
Guanosine 0–2.7 0–1.2 n.d. n.d. >30
Deoxyguanosine n.d. n.d. n.d. n.d. >10
Deoxyinosine 0–2.7 0–1.2 n.d. n.d. >10
SAdo 0.1–15.8 0–11.7 0–4.9 0–2.8 >20 (0–16)
>0 (>16)
Adenosine 0–4.4 0–4.7 0–3.9 0–2.8 >10
Deoxyadenosine 0–3.0 0–4.7 n.d. 0–0.6 >10
a
b
All categories, unless indicated
c
Not detectable (<0.1 μmol/l) with most HPLC-UV methods
d
95 % CI
Urine pyrimidines (LC-MS/MS)

Compound rangea 0–1 year (n = 16) 1–5 years (n = 47) 5–16 years (n = 27) >16 years (n = 26) Pathological valuesb
Orotidine 0–10.1 0–7.8 0.2–1.8 0–2.1 >100c
Orotate 0–1.4 0–3.0 0–2.5 0–2.0 >500
Dihydroorotate 0–18.6 0–16.0 0–8.6 0–3.9
N-carbamoyl-ß-alanine 0–35.9 0–15.6 0–4.7 0–4.3 >500
Dihydrouracil 0–29.6 0–8.1 0–3.7 0–2.6 >100
Uracil 0–100.9 0–66.6 0–16.1 0–9.7 > 110
5-OH-Me-uracil 0–4.9 0–10.1 0–2.0 0–3.6 > 10
Pseudouridine 26.5–216.5 17.7–134.6 16–56.9 10.2–43.5
N-carbamoyl-ß-aminoisobutyrate 0–17.6 0–12.0 0–1.4 0–1.8 >500
Uridine 0–6.8 0–2.1 0–1.6 0–1.1
Dihydrothymine 0–10.3 0–4.6 0–3.0 0–1.1 >100
Thymine 0–8.0 0–4.2 0–1.6 0–0.9
Deoxyuridine 0–3.0 0–1.7 0–0.6 n.d. >5
Thymidine 0–1.1 0–0.9 n.d. n.d. >5
n.d. not detectable
a
b
All categories, unless indicated
c
Beware of allopurinol use
Plasma purines (HPLC-UV and LCMS/MS) Plasma pyrimidines (HPLC-UV and LCMS/MS)
Compound rangea All ages Compound rangea All ages
2,8-Dihydroxy adenine Orotidine 0–0.1
SAICAr n.db Orotate 0–0.5
Urate 220–230 Dihydroorotate
Hypoxanthine 0–10.0b N-carbamoyl-ß-alanine
Xanthine 0–7.0b Dihydrouracil
Adenine 0.3 Uracil 0–0.35
AICAR 5-OH-Me-uracil
Inosine 0–2.5 Pseudouridine
Guanosine n.d.b N-carbamoyl-ß-aminoisobutyrate
Deoxyguanosine Uridine 0–8.5
Deoxyinosine Dihydrothymine
SAdo Thymine
Adenosine n.d.b Deoxyuridine n.d.
Deoxyadenosine Thymidine 0–0.2
n.d. not detectable (= <0.1 μmol/L) n.d. not detectable (= <0.1 μmol/l)
a
μmol/l a
μmol/l
b
n = 200 samples
Signs and symptoms Suspious case history Laboratory tests
Automutilation Hyperuricaemia
Gout Hypouricaemia
(especially in the young and Combined B- and T-cell
women) deficiency
Severe combined T-cell deficiency
immunodeficiency (SCID) Non-spherocytic anaemia
Unexplained psychomotor with basophilic stippling
retardation, seizures or hyptonia
Urate in serum and urine
Purine and pyrimidine metabolites

in urine
Enzyme activity assays

(Erythrocytes, white blood cells, fibroblasts)
Molecular genetic analysis
Fig. 41.3 The justification for detailed purine and pyrimidine purine and pyrimidine metabolites in urine. It is highly recommended
investigations includes three aspects: (a) clinical signs and symptoms to estimate both plasma and urine concentrations at once to be able to
(some are very characteristic such as self-mutilation or gout in young assess the excretion and confirm or exclude overproduction of uric acid.
people and women or SCID (severe combined immunodeficiency syn- The exclusion of secondary causes of hyperuricemia/hypouricemia
drome); others are shown on Tables 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, (such as nephropathy, tissue breakdown, Fanconi syndrome and
and 41.7), (b) a case history suspicious of an inborn error of metabo- uricosuric drugs) is very important. Confirmation of diagnosis consists
lism, and (c) characteristic laboratory tests. If a patient meets these of enzyme assay and finally molecular genetic analysis
criteria, the first steps are measurement of uric acid in body fluid and
41.7 Sample Collection and Storage material from which DNA or, in the case of exome sequenc-
ing, RNA can be extracted is suitable for molecular analysis.
This section is taken from the ERNDIM advisory document There are good and reliable databases to be found on the
for the analysis of purines and pyrimidines (Bierau 2010). Internet.
Urine
Traditionally 24-h urine collection or overnight collection is 41.9 Treatment Summary
preferred for diagnostic purposes. In practice, many
laboratories use urine portions for diagnostic purposes. No Enzyme deficiencies with hyperuricemia and gout represent
preservatives are added to the sample. the most frequent disorders of purine metabolism (Becker
The recommended procedure for 24-h urine collection is 2001). In addition, the incidence of gout is increasing. Novel
as follows: during the collection period, the urine aliquots therapeutic interventions for controlling hyperuricemia,
are kept refrigerated (4 °C), and after completion the urine is especially in patients with deleterious features of gout, have
sent to the laboratory in a well-isolated package and stored in raised renewed interest. One of the reasons was the new find-
the refrigerator for max. 1 week at 4 °C until analysis or ing that uric acid plays a role as a primary risk factor for the
stored frozen at −20 °C when analysis is carried out after development of hypertension and renal disease (Feig et al.
more than 1 week but within 2 months. For longer periods, 2008; Mazzali et al. 2010). Control of hyperuricemia may
storage at −80 °C is recommended. become more important to paediatricians than formerly
Dipstick tests for nitrite and pH should be carried out directly thought (Joosten et al. 2010; Kanbay et al. 2011).
after receipt of the urine in order to check for bacterial contami- Febuxostat (generic name) was introduced in 2009. This
nation. In addition, qualitative tests for glucose, reducing sub- non-purine compound inhibits xanthine oxidase more effi-
stances, sulphite and ketone bodies should be performed. ciently than allopurinol. Recent trials demonstrated that
Analysis should not be performed in severely bacterially con- febuxostat at single doses from 80 to 240 mg was superior
taminated samples (pH >7 and/or nitrite is positive). to allopurinol (which has long been the drug of choice) in its
urate-lowering efficacy. The biological agent pegloticase
Plasma (PEGylated mammalian recombinant uricase) is approved
The analysis of purines and pyrimidine can be performed in for the therapy of progressive gout refractory to regular oral
plasma obtained from blood anticoagulated with heparin as agents. The use of xanthine oxidase inhibitors is recom-
well as EDTA. This can be adjusted according to local proto- mended for the overproduction-type hyperuricemia, while
cols. In the case of capillary blood, clean and disinfect skin uricosuric agents are recommended for the underexcretion
thoroughly before taking the blood sample, to avoid contam- type. However, when uricosuric drugs are used, urinary out-
ination from the skin surface. put must be sustained, and, in addition, alkalinisation of the
Plasma samples should be stored at −20 °C or at −80 °C, urine to prevent urolithiasis must be considered.
if stored for a prolonged period. Plasma samples should be Benzbromarone, a main uricosuric agent, has been contro-
deproteinised before analysis. versial because of serious hepatotoxicity in some cases.
Newer uricosuric agents (MBX102, tranilast) are in clinical
CSF development (Becker 2011). In the treatment of purine and
Please refer to your own hospital protocol for the lumbar pyrimidine disorders, it is important to be aware of several
puncture procedure. precautions: the dosage of allopurinol in overproduction
CSF samples should be deproteinised before analysis. hyperuricemia (HPRT, PRPS) should be reduced in the
CSF samples should be stored at −80 °C. cases of renal insufficiency (risk of xanthine nephropathy)
(van Gennip et al. 2006); administration of fluorinated
pyrimidine analogues in dihydropyrimidine dehydrogenase
41.8 Prenatal Diagnosis Table and DNA deficiency can be catastrophic; in thiopurine methyltransfer-
Analysis ase deficiency, there may be enhanced toxicity of
mercaptopurines.
Providing a table listing all disorders and materials in which Specific treatment is available for a small number of other
molecular analysis can be performed is obsolete because of purine and pyrimidine disorders at present as, in many cases,
the modern sequencing techniques. All genes and their chro- our understanding of how a particular point in metabolism
mosomal localisations are listed above. In essence, any produces these relatively new disorders is still incomplete.
Standard Treatment
No. Disorder Age Medication Dosage Target

41.1 Phosphoribosyl pyrophosphate synthetase No established treatment
1 superactivity defects
41.2 Phosphoribosyl pyrophosphate synthetase Child Allopurinol 20 mg/kg/day Serum uric acid
1 superactivity Adult 200–600 mg/day <416 μmol/l (adult)
<360 μmol/l (child)
41.3 ADSL None established
41.4 AICAR transformylase deficiency No established treatment
41.5 Adenosine monophosphate deaminase Any D-ribose 200 mg/kg/day
deficiency Xylitol 15–20 g/day
41.6 Inosine monophosphate dehydrogenase
deficiency
41.7 Adenosine deaminase deficiency Any Bone marrow transplantation
Enzyme replacement therapy:
Carrier erythrocyte encapsulated native ADA
Polyethylene glycol-conjugated bovine ADA
Autologous haematopoietic stem cell gene therapy
PEG-ADA 60 U/kg/week— Maintenance dosage
initial dosage ADA activity in plasma
30 U/kg/week 25–150 μmol/h/ml
41.8 Purine nucleoside phosphorylase Bone marrow transplantation
deficiency Stem cell transplantation
41.9 Xanthine oxidase (dehydrogenase) Any Low-purine diet, high fluid intake
deficiency Vigorous exercise should be avoided
41.9.1 Combined xanthine oxidase and aldehyde Dextromethorphan (may be used as anticonvulsant)
oxidase deficiency
41.10 Adenine phosphoribosyltransferase Child Allopurinol 10 mg/kg/day Urinary −2,8-DHA
deficiency Adult 200–300 mg/day virtually 0
41.11 Hypoxanthine-guanine Child Allopurinol 20 mg/kg/day Serum uric acid
phosphoribosyltransferase deficiency Adult 200–600 mg/day <416 μmol/l (adult)
<360 μmol/l (child)
41.13 Orotic aciduria type I Any Uridine 50–300 mg/kg/day Normal Hb, MCV
megaloblastosis
41.14 Orotic aciduria type II Any Uridine 50–300 mg/kg/day Normal Hb, MCV
megaloblastosis
41.15 Pyrimidine −5′-nucleotidase deficiency Any Splenectomy
41.16 Pyrimidine −5′-nucleotidase superactivity Any Uridine 1,000 mg/kg/day No seizures
41.17 Thymidine phosphorylase deficiency Bone marrow transplantation
Stem cell transplantation
Carrier erythrocyte entrapped TP
PEG-ADA polyethylene glycol-modified bovine adenosine deaminase
Experimental Treatment
No. Disorder Age Medication

41.3 Adenylosuccinate lyase deficiency Any D-ribose (one patient)
41.23 Deoxyguanosine kinase deficiency Carnitine and bicarbonate (one
patient)
Treatment Pitfalls
No. Disorder Pitfalls

41.2 Phosphoribosyl pyrophosphate synthetase 1 In any overproduction hyperuricemia (incl. HPRT def., primary gout), uricosuric
superactivity agents are contraindicated (they may induce acute renal failure)
In case of chronic renal failure, the dosage of allopurinol needs to be lowered, and
monitoring metabolites of allopurinol and xanthine in plasma is useful (maintain
oxipurinol <100 μmol/l and xanthine <40 umol/l)
41.7 Adenosine deaminase deficiency Graft-versus-host disease in bone marrow transplant
Gene therapy: immunity to gene-transfer system
41.8 Purine nucleoside phosphorylase deficiency Graft-versus-host disease in bone marrow transplant
41.9 Xanthine oxidase (dehydrogenase) deficiency Urolithiasis after dehydration, infection or intense physical activity is common
41.10 Adenine phosphoribosyltransferase In acute or chronic renal failure, the dosage of allopurinol needs to be lowered (see
deficiency disorder 41.2)
2,8-DHA stones may be radiolucent
41.11 Hypoxanthine-guanine Uricosuric agents are contraindicated (see disorder 41.2)
phosphoribosyltransferase deficiency In acute or chronic renal failure, the dosage of allopurinol needs to be lowered (see
disorder 41.2)
Allopurinol can successfully control overproduction of UA (kidney stones, gouty
arthritis) but does not correct neurological and behavioural features
Physical restrain (or removal of teeth) is sometimes required to prevent self-mutilation
41.13 Orotic aciduria type I Uridine dosage may be limited by diarrhoea
41.14 Orotic aciduria type II Uridine dosage may be limited by diarrhoea
41.18 Dihydropyrimidine dehydrogenase deficiency The use of 5-halogenated pyrimidines (5-fluorouracil) should be avoided at any time
41.19 Dihydropyrimidinase deficiency The use of 5-halogenated pyrimidines (5-fluorouracil) should be avoided at any time
41.26 Thiopurine S-methyltransferase deficiency Dosage of mercaptopurines needs to be adjusted dependent on residual thiopurine
methyltransferase activity
Hirano M, Nishino I, Nishigaki Y et al (2006) Thymidine phosphory-

References lase gene mutations cause mitochondrial neurogastrointestinal
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Assmann B, Göhlich G, Baethmann M et al (2006) Clinical findings Joosten H, Strunk AL, Meijer S et al (2010) An aid to the diagnosis of
and a therapeutic trial in the first patient with beta-ureidopropionase genetic disorders underlying adult-onset renal failure: a literature
deficiency. Neuropediatrics 37:20–25 review. Clin Nephrol 73:454–472
Becker MA (2001) Hyperuricemia and gout. In: Scriver CR, Beaudet al, Kanbay M, Huddam B, Azak A et al (2011) A randomized study of
Sly WS, Valle D (eds) The metabolic and molecular bases of allopurinol on endothelial function and estimated glomerular filtra-
inherited disease, 8th edn. McGraw-Hill, New York tion rate in asymptomatic hyperuricemic subjects with normal renal
Becker MA (2011) Treatment of the hyperuricemia of gout in the function. Clin J Am Soc Nephrol 6:1887–1894
United States. In: Book of abstracts of 14th international sympo- Mazzali M, Kanbay M, Segal MS et al (2010) Uric acid and hyperten-
sium on purine and pyrimidine metabolism in man, p. 51, February sion: cause or effect? Curr Rheumatol Rep 12:108–117
12–21, 2011, Tokyo, Japan. Medical Toyou Co., Ltd, Tokyo Micheli V, Camici M, Tozzi MG et al (2011) Neurological disorders of
Bierau J (2010) ERNDIM Advisory Document of the quantitative anal- purine and pyrimidine metabolism. Curr Top Med Chem 11:
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of inosine triphosphatase. Pharmacogenomics 8:1221–1228 Acids 27:648–655
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dromes and inborn errors of purine metabolism. Neurochem Int renal hypouricemia. Nucleosides Nucleotides Nucleic Acids
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Hum Genet 86:506–518 pyrimidine metabolism. In: Fernandes J, van den Berghe G, Walter
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Disorders of Glutathione
and γ-Glutamyl Cycle 42
Nenad Blau and Carlo Dionisi-Vici
Contents Summary
42.1 Introduction ..................................................................... 661 The γ-glutamyl cycle, comprising six enzymes, harbors four
hereditary defects: γ-glutamylcysteine synthetase, glutathi-
42.2 Nomenclature ................................................................... 662
one synthetase, γ-glutamyl transpeptidase, and
42.3 Metabolic Pathway .......................................................... 663 5-oxoprolinase. Defects have also been identified in
42.4 Signs and Symptoms ....................................................... 663 γ-glutamyltranspeptidase and dipeptidase (cysteinylglyci-
nase); these conditions affect the biosynthesis of leukotri-
enes and will be discussed in Chap. 38.
Deficiency of either of the two synthetases results in
42.7 Diagnostic Flow Chart .................................................... 666 decreased levels of glutathione and thus increased sensitivity
42.8 Specimen Collection ........................................................ 666 to oxidative stress that results in hemolytic anemia.
Glutathione synthetase deficiency occurs with different
42.9 Prenatal Diagnosis ........................................................... 667
severity; the mild form is only associated with hemolytic
42.10 DNA Analysis ................................................................... 667 anemia, whereas moderate and severe glutathione synthetase
42.11 Treatment ......................................................................... 667 deficiency is associated also with metabolic acidosis, pro-
References ..................................................................................... 668 gressive neurological symptoms, and recurrent bacterial
infections. 5-Oxoproline (pyroglutamic acid) is overpro-
duced in glutathione synthetase deficiency due to lack of
feedback inhibition. Treatment involves acidosis correction;
administration of vitamin E, vitamin C, and N-acetylcysteine;
and avoidance of drugs inducing hemolysis. γ-Glutamyl
transpeptidase deficiency is associated with glutathionuria,
cysteinylglycinase deficiency with cystinylglycinuria, and
5-oxoprolinase deficiency with 5-oxoprolinuria.
42.1 Introduction
Glutathione, which is produced and broken down in the

N. Blau (*)
Division of Inborn Metabolic Diseases, γ-glutamyl cycle, participates in free radical scavenging,
Department of General Pediatrics, University Children’s Hospital, defense against oxidative stress, redox reactions, formation
Im Neuenheimer Feld 430, Heidelberg 69120, Germany of deoxyribonucleotides, xenobiotics metabolism, and amino
Division of Metabolism, acid transport. Patients with genetic defects in four of the six
University Children’s Hospital, Zürich, Switzerland γ-glutamyl cycle enzymes have been reported and they are
e-mail: nenad.blau@med.uni-heidelberg.de
all inherited as autosomal recessive traits (Larsson and
C. Dionisi-Vici Anderson 2001).
Division of Metabolism, Department of Pediatric Medicine,
The biosynthesis of the tripeptide glutathione (γ-glutamyl
Bambino Gesù Children’s Research Hospital, IRCCS,
Piazza Sant’Onofrio 4, Rome 00165, Italy cysteinylglycine) is catalyzed by γ-glutamylcysteine synthe-
e-mail: carlo.dionisivici@opbg.net tase and glutathione synthetase. The initial degradative step is
662 N. Blau and C. Dionisi-Vici
catalyzed by γ-glutamyl transpeptidase, which transfers the prevent accumulation of 5-oxoproline in body fluids.
γ-glutamyl group to an acceptor, for example, an amino acid, Treatment of patients with glutathione synthetase deficiency
to form γ-glutamyl amino acids. The latter are typically sub- includes acidosis correction and supplementation with the
strates of γ-glutamyl cyclotransferase which catalyzes release antioxidants vitamin E, vitamin C, and N-acetylcysteine, as
of the γ-glutamyl residue as 5-oxoproline (pyroglutamic acid) well as avoidance of drugs known to precipitate hemolytic
which is converted back to glutamate by 5-oxoprolinase. crises in patients with glucose-6-phosphate dehydrogenase
Glutathione acts as a feedback inhibitor of γ-glutamylcysteine deficiency.
synthetase. Deficiency of γ-glutamylcysteine synthetase or glutathi-
γ–Glutamylcysteine synthetase deficiency has been one synthetase results in low intracellular levels of glutathi-
described in more than ten patients in more than six fami- one. This can be demonstrated in erythrocytes, leukocytes,
lies. All had hemolytic anemia, and in addition, two siblings and cultured fibroblasts. Increased 5-oxoproline can only be
also had cerebellar involvement, neuropathy, myopathy, and determined via analysis of organic acids by gas chromatog-
aminoaciduria. Glutathione synthetase deficiency has been raphy–mass spectrometry (GC-MS). Analysis of the
reported in more than 50 patients in more than 40 families. γ-glutamyl cycle enzymes in erythrocytes or nucleated cells
According to clinical symptoms, glutathione synthetase is required for the diagnosis. The human genes for
deficiency can be classified as mild, moderate, or severe γ-glutamylcysteine synthetase and glutathione synthetase
(Beutler et al. 1999). Patients with mild glutathione synthe- have been mapped and cloned and mutations in the genes
tase deficiency show hemolytic anemia as their only clinical have been characterized (Larsson and Anderson 2001;
symptom. Patients with moderate glutathione synthetase Ristoff et al. 2000, 2001; Njalsson et al. 2000).
deficiency usually present in the neonatal period with meta- γ-Glutamyl transpeptidase deficiency has been identified
bolic acidosis, 5-oxoprolinuria, and hemolytic anemia. in five patients who excrete glutathione in their urine and
Patients with severe glutathione synthetase deficiency also have elevated plasma glutathione. Three of the five patients
develop progressive neurological symptoms (e.g., mental have CNS symptoms. Increased levels of urinary glutathione
retardation, seizures, spasticity) and may also develop recur- can be demonstrated by various chromatographic techniques.
rent bacterial infections, due to defective granulocyte func- The human γ-glutamyl transpeptidase gene is a multigenetic
tion. Several patients have died in early life due to acidosis family with several of its loci located on chromosome 22
and electrolyte imbalance. The acidosis is due to the over- (Larsson and Anderson 2001).
production of 5-oxoproline as a consequence of defective A tentative deficiency of cysteinylglycinase has been
feedback regulation of the early steps of the γ-glutamyl found in one patient with distinct neurological abnormalities.
cycle. As a consequence, accumulating γ-glutamylcysteine Its chromosomal location is 16q24.3. See also Chap. 38 for
will be cleaved by γ-glutamylcyclotransferase and the the latter two defects.
amount of its product 5-oxoproline then surpasses the 5-Oxoprolinase deficiency has been identified in eight
capacity of 5-oxoprolinase. Patients with moderate and patients who lack a consistent clinical syndrome. Urinary
severe glutathione synthetase deficiency usually excrete excretion of 5-oxoproline is elevated but less than in glutathi-
gram quantities of 5-oxoproline in urine. Patients with mild one synthetase deficiency. Erythrocytes contain an incom-
glutathione synthetase deficiency maintain cellular levels of plete γ-glutamyl cycle; they lack both γ-glutamyl
glutathione which usually, but not always, is sufficient to transpeptidase and 5-oxoprolinase (Almaghlouth et al. 2012).
42.2 Nomenclature
Gene Chromosomal
42.1 Glutathionuria Gamma-glutamyl GGT1 GGT1 22q11.1-q11.2 Gamma-glutamyl 231950 All forms
transpeptidase transpeptidase
deficiency
42.2 Oxoprolinuria 5-Oxoprolinase 5-Ooxoprolinase 260005 All forms
deficiency
42.3 Gamma- Hemolytic anemia GGCS GCLC 6p12 Gamma- 230450 All forms
glutamylcysteine due to GGCS glutamylcysteine
synthetase deficiency deficiency synthetase
42.4.1 Glutathione synthetase 5-Oxoprolinuria GSS 20q11.2 Glutathione 266130 Mild form
deficiency, mild synthetase
42.4.2 Glutathione synthetase 5-Oxoprolinuria GSS 20q11.2 Glutathione 266130 Severe
deficiency, severe synthetase
42 Disorders of Glutathione and γ-Glutamyl Cycle 663
Fig. 42.1 The γ-glutamyl cycle ADP

for the biosynthesis and Amino acid
degradation of glutathione
Glutathione
including known metabolic
defects: 42.1 γ-glutamyl
transpeptidase, 42.2 42.4
5-oxoprolinase, 42.3 γ
-glutamylcysteine synthetase, 42.4 42.1
glutathione synthetase. DP
dipeptidase (cysteinylglycinase), Cysteinylglycine
GGCT γ-glutamyl cyclotransferase.
Metabolites that show pathological ATP
levels in the various enzymatic DP
defects are marked in bold. Note Glycine
the role of excess 5-oxoproline g-Glutamylcysteine
(pyroglutamic acid) as a marker for
two of the four disorders g-Glutamylamino acid ADP
Cysteine
GGCT
42.3
Amino acid
5-oxoproline ATP
Glutamate
42.2
ATP ADP
Table 42.1 Glutathionuria

CNS Mental retardation ± ± ±
Psychosis ± ± ±
Other No consistent clinical picture
Special laboratory Gamma-glutamyltranspeptidase (WBC,FB) ↓ ↓ ↓ ↓ ↓
Glutathione (P) ↑ ↑ ↑ ↑ ↑
Glutathione (RBC) n n n n n
Glutathione (U) ↑ ↑ ↑ ↑ ↑
Table 42.2 Oxoprolinuria

CNS Retardation, psychomotor ± ± ± ± ±
Digestive Colitis ± ±
Diarrhea ± ±
Musculoskeletal Microcephaly ± ±
Renal Renal colic ± ± ±
Urolithiasis ± ± ±
Othert No consistent clinical picture
Special laboratory 5-Oxoprolien (U) ↑ ↑ ↑ ↑ ↑
5-Oxoprolinase (WBC, FB) ↓ ↓ ↓ ↓ ↓
Routine laboratory Acidosis (B) n n n n n
Table 42.3 Gamma-glutamylcysteine synthetase deficiency

Neuropathy ± ± ± ± ±
Psychosis ± ± ± ± ±
Hematological Anemia, hemolytic + + + + +
Hemolytic anemia + + + + +
Musculoskeletal Myopathy ± ± ± ± ±
Routine Hemoglobin (B) ↓ ↓ ↓ ↓ ↓
laboratory Reticulocytes (B) ↑ ↑ ↑ ↑ ↑
Special Amino acids, all ± ± ± ± ±
laboratory Gamma-glutamylcysteine synthetase (RBC,FB) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Glutathione (RBC) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 42.4.1 Glutathione synthetase deficiency, mild

Hematological Anemia, hemolytic + + + + +
Hemolytic anemia + + + + +
Routine laboratory Hemoglobin (B) ↓ ↓ ↓ ↓ ↓
Reticulocytes (B) ↑ ↑ ↑ ↑ ↑
Special laboratory 5-Oxoproline (U) n-↑ n-↑ n-↑ n-↑ n-↑
Gamma-glutathione synthetase (RBC,FB) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Glutathione (RBC) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 42.4.2 Glutathione synthetase deficiency, severe

Neurological symptoms ± ± ± ± ±
Eye Adaptation, dark impaired ± ± ±
Corneal clouding ± ± ±
Pigmentary retinopathy ± ± ±
Hematological Hemolytic anemia + + + + +
Musculoskeletal Myopathy ± ± ± ± ±
Other Recurrent bacterial infections ± ± ± ± ±
Recurrent bacterial infections ± ± ± ± ±
Routine laboratory Hemoglobin (B) ↓ ↓ ↓ ↓ ↓
Reticulocytes (B) ↑ ↑ ↑ ↑ ↑
Special laboratory 5-Oxoproline (U) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Gamma-glutathione synthetase (RBC,FB) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Glutathione (RBC) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓

Metabolite
5-Oxoproline (U) <10 mmol/mol creat
Glutathione (RBC) 4.6–10.9 nmol/mg Hb
Glutathione 5-Oxo-proline Acid–base balance Reticulocytes Hemolytic anemia

(RBC) (B) (U) (P) (U) (B) (B) (B)
42.1 Glutathionuria N ↑ ↑ N N N N
42.2 Oxoprolinuria N N N ↑ N N N
42.3 γ Glutamylcysteine ↓↓ N – N N ↑ ↑
synthetase deficiency
42.4.1 Glutathione synthetase ↓↓ N – N-↑ N ↑ ↑
deficiency, mild
42.4.2 Glutathione synthetase ↓↓ N – ↑↑↑ Acidosis ↑ ↑
deficiency, severe
Hemolytic anemia
Glutathione decreased N Not due to

(RBC, FB) γ-glutamyl cycle defect
Y
Go to Chapters 39 and 41
γ-Glutamylcysteine Y γ-Glutamylcysteine
synthetase decreased
synthetase decreased
(RBC)
5-Oxoproline N
increased
(U)
Y Mild
Glutathione synthetase
glutathione synthetase
decreased (RBC, FB)
Y deficiency
Glutathione synthetase N Y
5-Oxoprolinase decreased 5-oxoprolinase
decreased
(WBC, FB) deficiency
(RBC, LYM, FB)
Y N
Glutathione synthetase Secondary

deficiency 5-oxoprolinura
(mild, moderate, severe)
Fig. 42.2 Diagnostic flow chart for disorders of the γ-glutamyl cycle presenting with hemolytic anemia

Glutathione – RBC, B, FB Frozen (−20 °C) Assays that do not detect oxidized glutathione
tend to underestimate glutathione in stored
samples
γ-Glutamylcysteine synthetase – RBC, LYM, FB Frozen (−20 °C)
Glutathione synthetase – RBC, LYM, FB Frozen (−20 °C)
γ-Glutamyl transpeptidase – RBC, FB, P Frozen (−20 °C)
γ-Glutamyl cyclotransferase – RBC, LYM, FB Frozen (−20 °C)
5-Oxoprolinase – WBC, FB Frozen (−20 °C)

5-Oxoproline – U Frozen (−20 °C) Excretion of 5-oxoproline has been found in
patients with inborn errors of metabolism outside
the γ-glutamyl cycle (e.g., homocystinuria, OCT
deficiency, cystinosis) and in patients receiving
certain drugs (vigabatrin, paracetamol) and
specific diets (acid hydrolyzed protein formula).
The combination of paracetamol and
flucloxacillin may result in a fatal form of
5-oxoprolinuria. Urine glutamine may decompose
to form 5-oxoproline
Mutation analysis (DNA – FB, WBC, CV, Cells in culture Prenatal diagnosis is greatly facilitated if the
sequencing) AFC (room mutant allele in the specific family is known
temperature)
Prenatal diagnosis is greatly facilitated if the mutant allele(s) in Patients who are deficient in γ-glutamylcysteine synthe-
the specific family is known tase or glutathione synthetase should avoid drugs that can
Disorder Tissue Timing, trimester induce hemolytic crises in patients with glucose-6- phos-
42.4.2 CV I phate dehydrogenase deficiency, e.g., phenobarbital,
AF II acetylsalicylic acid, and sulfonamides.
AFC III
Treatment Summary
For γ-glutamylcysteine synthetase deficiency, the recom-
42.10 DNA Analysis mended treatment is to avoid drugs and foods known to
precipitate hemolytic crises in patients with glucose-
6-phosphate dehydrogenase deficiency. Early supplementa-
Disorder Tissue Methodology
42.3 B, WBC, LYM DNA sequencing
tion with the antioxidant vitamins C and E seems to prevent
42.4.1/2 FB, WBC, LYM, CV, AFC DNA sequencing damage to the CNS in patients with GSH synthetase defi-
ciency (Ristoff et al. 2001). In analogy, supplementation
with vitamins C and E might be worth testing also in
42.11 Treatment patients with γ-glutamylcysteine synthetase deficiency.
However, no studies of this treatment have yet been made.
Initial Treatment Treatment of glutathione synthetase deficiency in the neo-
Defects that lead to decreased levels of glutathione can be natal period involves the correction of acidosis and electro-
treated according to two complementary strategies: avoid- lyte imbalance and early treatment with the antioxidants
ance of drugs that lead to oxidative stress and supplementa- vitamins E and C to prevent damage to the CNS (Ristoff
tion with compounds that may act as free radical scavengers et al. 2001).
(e.g., vitamin C, vitamin E, and N-acetylcysteine). The lesions in the brain of patients with GSH synthetase
The only disorder of the γ-glutamyl cycle for which treat- deficiency resemble those seen after intoxication with the
ment principles have been developed is glutathione synthetase toxic compound mercury, i.e., Minamata disease, and it has
deficiency (42.4) (Larsson and Anderson 2001). The initial therefore been suggested that treatment with antioxidants
symptoms in the neonatal period may be metabolic acidosis may be beneficial (Skullerud et al. 1980). The goal of treat-
and jaundice. Acidosis usually needs to be corrected with ment in patients with GSH synthetase deficiency is to cor-
sodium bicarbonate, THAM, or sodium citrate. Patients may rect the acidosis and to compensate for the lack of antioxidant
benefit from oral administration of vitamin E (10 mg/kg/day) capacity in the cells. A long-term follow-up study of 28
and vitamin C (100 mg/kg/day). Trials have also been made patients showed that early supplementation with the antioxi-
with N-acetylcysteine and glutathione esters which increased dant vitamins C and E is useful for preventing damage to the
glutathione in leukocytes and plasma. Both these compounds CNS in patients with GSH synthetase deficiency (Ristoff
lead to increased intracellular levels of glutathione. However, et al. 2001). Recommended treatment does not normalize
no decrease in the excretion of 5-oxoproline has been reported. the elevated excretion of 5-oxoproline in urine.
No. Disorder Treatment/diet Dosage (mg/kg/day)

42.1 γ-Glutamyl transpeptidase (GT) No treatment has been recommended
deficiency
42.2 5-Oxoprolinase deficiency No treatment has been recommended
42.3 γ-Glutamylcysteine synthetase Avoid drugs and foods known to precipitate hemolytic crises in
deficiency patients with glucose-6-phosphate dehydrogenase deficiency
Vitamins C (ascorbic acid) 100
Vitamin E (α-tocopherol) 10
42.4 Glutathione (GSH) synthetase Avoid the drugs and foods known to precipitate hemolytic crises in
deficiency patients with glucose-6-phosphate dehydrogenase deficiency
Correction of acidosis (bicarbonate, citrate, or THAM)
Vitamin C (ascorbic acid)a 100
Vitamin E (α-tocopherol)b 10
a
A trial with short-term treatment of GSH synthetase-deficient patients with vitamin C has been reported to increase the levels of lymphocyte GSH
(Jain et al. 1994). Vitamin C and GSH can spare each other in a rodent model (Martensson and Meister 1991)
b
Vitamin E has been claimed to correct the defective granulocyte function (Boxer et al. 1979)
Alternative Therapies/Experimental Trials
No. Disorder Treatment/diet Dosage (mg/kg/day)

42.1 γ-Glutamyl transpeptidase (GT) deficiency No treatment has been recommended
42.2 5-Oxoprolinase deficiency No treatment has been recommended
42.3 γ-Glutamylcysteine synthetase deficiency No treatment has been recommended
42.4 Glutathione (GSH) synthetase deficiency N-Acetylcysteine (NAC)a 15
Glutathione estersb
a
Since N-acetylcysteine (NAC) protects cells in vitro from oxidative stress, it has been suggested that NAC supplements (15 mg/kg/day) should be
given to GSH-deficient patients. However, today we know that patients with GSH synthetase deficiency accumulate cysteine and, in our opinion,
NAC; therefore it should not be recommended (Ristoff et al. 2002)
b
Glutathione esters have been tried in animal models of GSH deficiency and in two patients with GSH synthetase deficiency (Anderson et al. 1994)
(W. Rhead 1995, personal communication). The GSH esters, which are more lipid soluble, are readily transported into cells and converted intracel-
lularly into GSH. The esters increase GSH levels in several tissues, but their use is limited because of associated toxic effects, i.e., when they are
hydrolyzed to release GSH; alcohols are produced as a by-product
Follow-Up/Monitoring
No. Disorder Clinical investigations Laboratory investigations

42.1 γ-Glutamyl transpeptidase (GT) deficiency Neurological investigations
42.2 5-Oxoprolinase deficiency Neurological investigations Acid–base balance
42.3 γ-Glutamylcysteine synthetase deficiency Neurological investigations Hb, reticulocytes
42.4 Glutathione (GSH) synthetase deficiency Neurological investigation Acid–base balance
Eye examination (retinal pigmentations, corneal opacities) Hb, reticulocytes
Boxer LA, Oliver JM, Spielberg SP, Allen JM, Schulman JD (1979)
References Protection of granulocytes by vitamin E in glutathione synthetase
deficiency. N Engl J Med 301:901–905
Almaghlouth IA, Mohamed JY, Al-Amoudi M, Al-Ahaidib L, Al-Odaib Jain A, Buist NR, Kennaway NG, Powell BR, Auld PA, Martensson J
A, Alkuraya FS (2012) 5-Oxoprolinase deficiency: report of the first (1994) Effect of ascorbate or N-acetylcysteine treatment in a patient
human OPLAH mutation. Clin Genet 82:193–196 with hereditary glutathione synthetase deficiency. J Pediatr
Anderson ME, Levy EJ, Meister A (1994) Preparation and use of glu- 124:229–233
tathione monoesters. Methods Enzymol 234:492–499 Larsson A, Anderson ME (2001) Glutathione synthetase deficiency and
Beutler E, Gelbart T, Kondo T, Matsunaga AT (1999) The molecular other disorders of the gamma-glutamyl cycle. In: Scriver CR,
basis of a case of gamma-glutamylcysteine synthetase deficiency. Beaudet AL, Sly WS, Valle D, Childs B, Vogelstein B (eds) The
Blood 94:2890 metabolic and molecular bases of inherited disease. McGraw-Hill,
New York, pp 2205–2216
Martensson J, Meister A (1991) Glutathione deficiency decreases tissue Ristoff E, Mayatepek E, Larsson A (2001) Long-term clinical outcome
ascorbate levels in newborn rats: ascorbate spares glutathione and in patients with glutathione synthetase deficiency. J Pediatr
protects. Proc Natl Acad Sci U S A 88:4656–4660 139:79–84
Njalsson R, Carlsson K, Olin B, Carlsson B, Whitbread L, Polekhina G, Ristoff E, Hebert C, Njalsson R, Norgren S, Rooyackers O, Larsson A
Parker MW, Norgren S, Mannervik B, Board PG, Larsson A (2000) (2002) Glutathione synthetase deficiency: is gamma-
Kinetic properties of missense mutations in patients with glutathi- glutamylcysteine accumulation a way to cope with oxidative stress
one synthetase deficiency. Biochem J 349:275–279 in cells with insufficient levels of glutathione? Inherit Metab Dis
Ristoff E, Augustson C, Geissler J, de Rijk T, Carlsson K, Luo JL, 25:577–584
Andersson K, Weening RS, van Zwieten R, Larsson A, Roos D Skullerud K, Marstein S, Schrader H, Brundelet PJ, Jellum E (1980)
(2000) A missense mutation in the heavy subunit of gamma- The cerebral lesions in a patient with generalized glutathione defi-
glutamylcysteine synthetase gene causes hemolytic anemia. Blood ciency and pyroglutamic aciduria (5-oxoprolinuria). Acta
95:2193–2196 Neuropathol 52:235–238
Disorders of Lipoprotein Metabolism
43
Robert A. Hegele and Serena Tonstad
Contents Summary
43.1 Introduction ...................................................................... 672 Disorders of lipoprotein metabolism – dyslipoproteinemias –
43.2 Nomenclature.................................................................... 674 can be classified based on the primary biochemical distur-
bance, such as high or low plasma levels of low-density
43.3 Metabolic Pathway ........................................................... 676
lipoprotein (LDL) cholesterol, or high-density lipoprotein
43.4 Signs and Symptoms ........................................................ 676 (HDL) cholesterol, or triglyceride (TG), or some combination
43.5 Reference Values ............................................................... 682 of these (Hegele 2009). Lipoproteins are physiological trans-
43.6 Pathological and Diagnostic Values ................................ 683
porters of hydrophobic lipids and fat-soluble vitamins through
plasma from their site of origin (intestine or liver) to their site
43.7 Diagnostic Flow Chart ..................................................... 684 of uptake and disposition. Abnormal levels of certain plasma
43.8 Specimen Collection ......................................................... 685 lipids and lipoproteins increase the risk of cardiovascular dis-
43.9 Prenatal Diagnosis ............................................................ 685 ease (CVD) end points, such as myocardial infarction and
stroke, and other complications such as pancreatitis.
43.10 DNA Testing ...................................................................... 686
Numerous genetic and environmental factors contribute to
43.11 Treatment Summary ........................................................ 686 interindividual variation in plasma concentrations of lipids
References .................................................................................... 689 and lipoproteins. Several monogenic dyslipidemias are now
defined at the molecular genetic level; those with a mono-
genic basis typically present earlier in life, while those that
present later in life also have genetic determinants, but their
expression further depends on interactions with nongenetic
environmental or lifestyle factors. Early diagnosis is central to
specific dietary, lifestyle, and pharmacologic interventions to
delay death, disability, and medical complications. For
R.A. Hegele (*) instance, to prevent premature cardiovascular disease in
Blackburn Cardiovascular Genetics Laboratory, monogenic dyslipoproteinemias – such as heterozygous
Robarts Research Institute, University of Western Ontario, familial hypercholesterolemia – it is important to screen sub-
406-100 Perth Drive, London ON N6A 5K8, Canada
jects at risk; make the appropriate diagnosis, which may
e-mail: hegele@robarts.ca
include DNA analysis; and initiate treatment, which includes
S. Tonstad
diet, exercise, and lipid-lowering medications. Here we dis-
Section of Preventive Cardiology, Department of Preventive
Medicine, Oslo University Hospital, Oslo, Norway cuss the current understanding of genetic determinants, clini-
cal manifestations, and treatment of disorders of lipoprotein
Department of Health Promotion and Education,
Loma Linda University, Loma Linda, CA, USA metabolism, focusing on defined monogenic disorders that
e-mail: stonstad@llu.edu are diagnosed throughout the life span.
672 R.A. Hegele and S. Tonstad
43.1 Introduction gene: apo B-48 mRNA is produced by unique editing of

the apo B-100 mRNA. Other apolipoproteins include apo
Disorders of lipoprotein metabolism are usually diagnosed C-II, a cofactor for the enzyme lipoprotein lipase (LPL);
biochemically upon documenting deviation in levels of cer- apo E, a ligand for receptor-mediated endocytosis; and apo
tain plasma lipids or lipoproteins. The most important A-I, an activator of lecithin-cholesterol acyl transferase
plasma lipids clinically are cholesterol and triglyceride (or (LCAT).
triacylglycerol, TG). Cholesterol is (1) a component of cell Plasma lipid and lipoprotein concentrations generally
membranes; (2) the precursor for steroid hormones and vita- follow a right-skewed Gaussian distribution in the general
min D and for oxysterols and bile acids, both activators of population. Median levels vary by age and sex: in general,
nuclear hormone receptors involved in sterol metabolism; older age and male sex are associated with a less favorable
and (3) required for the activation of neuronal signalling lipid profile. There are also differences in median levels
molecules. Approximately 80 % of circulating cholesterol is across ethnic groups. Epidemiologically, CVD risk is
derived from endogenous synthesis by the liver and extrahe- directly related to plasma levels of total and LDL choles-
patic tissues (Hegele 2009). Fat or carbohydrate not required terol and inversely related to plasma levels of HDL choles-
by the liver for energy or synthesis is converted to TG terol. While the inverse association between HDL and CVD
through several biosynthetic pathways. TG is a key energy is indisputable, a causal relationship remains uncertain, in
source that is comprised of three fatty acids (FA), each part because not all genetic disorders causing very low HDL
ester-linked to a carbon on a glycerol backbone. TG is syn- are associated with CVD, in contrast to the uniform pres-
thesized in intestinal epithelial and liver cells, transported ence of CVD in genetic disorders causing high LDL. Plasma
through plasma and after lipolysis at the endothelial surface, LDL cholesterol and apo B concentration are directly cor-
delivers free FA (FFA) to peripheral cells for beta-oxidation related, as are plasma HDL cholesterol and apo A-I concen-
or storage. tration. The ratio of total cholesterol to HDL cholesterol and
Lipids must be transported through plasma within sphe- also the ratio of apo B to A-I are even better predictors of
roidal macromolecular complexes called lipoproteins. CVD risk. Although the relationship between plasma TG
These contain phospholipid (PL), fat-soluble antioxidants and CHD has been confounded by the association of ele-
and vitamins, and (cholesteryl ester) CE in their hydropho- vated TG with depressed HDL cholesterol, elevated TG,
bic core and free cholesterol (C), PL, and apolipoprotein especially non-fasting TG and familial hypertriglyceride-
(apo) molecules in their hydrophilic coat. Lipoproteins are mia (HTG), appear to be independently associated with
distinguished from each other by size, density, electropho- CVD risk.
retic mobility, composition, and function. The main A chronic excess of circulating LDL alters physiological
TG-carrying lipoproteins are the chylomicron (CM) and arterial endothelial dilation, which is an early manifestation
very low-density lipoprotein (VLDL). The main choles- of vascular dysfunction. LDL that does not undergo regu-
terol-carrying lipoproteins are low-density lipoprotein lated receptor-mediated endocytosis is taken up in an unreg-
(LDL) and high-density lipoprotein (HDL). Intermediate- ulated manner by scavenger receptors on the arterial wall
density lipoprotein (IDL), physiologically a short-lived macrophages. Entrapped LDL lipids can become oxidized,
species, contains approximately equimolar amounts of cho- generating toxic intermediates that induce cytokine produc-
lesterol and TG. tion and inflammation. Arterial wall macrophages can
Apolipoproteins provide plasma lipoproteins with become engorged with cholesterol from LDL, creating
structural stability and solubility. They also serve as foam cells, which are a key component of atherogenic
ligands for receptors and activators of key enzymes. For plaques. Lipids engulfed by macrophages become oxidized,
instance, apolipoprotein (apo) B-100 (B100) of VLDL, generating toxic intermediates, which induce cytokine pro-
IDL, and LDL is synthesized in the liver, while apo B-48 duction and inflammatory cell chemotaxis. Occlusive
(B48) of CM and CM remnants (CMR) is synthesized in plaques, often the result of rupture compounded by throm-
the small intestine. Apo B-100 (4356 amino acids) and bosis, result in CVD end points, such as myocardial infarc-
B-48 (2152 amino acids) are both products of the APOB tion or stroke.
43 Disorders of Lipoprotein Metabolism 673
Several important genetic determinants of lipoprotein Common dyslipidemias are often associated with other
levels have been identified through studies of monogenic conditions, such as type 2 diabetes, obesity, alcoholism,
disorders (Hegele 2009). Monogenic disorders comprise a hypothyroidism, pregnancy, renal failure, or drug use, and
rare patient subgroup found at the extremes of population- hence are termed secondary. Secondary dyslipidemias often
specific lipoprotein distribution. The molecular basis of have a strong genetic component, since some exacerbating
many monogenic high and low lipoprotein syndromes was conditions are frequently but not universally associated, sug-
elucidated using biochemical approaches or classical linkage gesting that subjects who develop secondary dyslipidemia
analysis. Studies of these diseases have helped define key might have a subtle inherited metabolic defect that confers
pathways, such as receptor-mediated endocytosis through susceptibility. For example, abdominal obesity and meta-
the LDL receptor and sterol efflux from cells via ATP- bolic syndrome are becoming increasingly common not just
binding cassette proteins. Several causative genes with rare in adults but also in adolescents and even children. It is
mutations have resurfaced as loci with common small-effect important to determine whether there is a strong secondary
genetic variants that underlie lipoprotein variation in popula- factor underlying the dyslipidemia, since this may guide the
tion studies. Many of these disorders are autosomal reces- preferred means of intervention.
sive, due to homozygous mutations in causative genes. This chapter will focus primarily on genetic disorders that
Importantly, some affected patients are compound heterozy- affect the concentrations of two plasma lipoproteins, namely,
gotes, a category hereafter implicitly included whenever the LDL and HDL, and one plasma lipid, namely, TG, which
term “homozygous” is used. have been connected to CVD and related disorders. Each dis-
Some of the classical Fredrickson – or World Health order has specific clinical features, including physical mani-
Organization (WHO) – hyperlipoproteinemia (HLP) pheno- festations across a range of tissues and organ systems, a
types have a monogenic basis. For instance, HLP type 2A is distinctive biochemical profile, and often a discrete molecu-
defined by LDL cholesterol >95th percentile of the normal lar genetic basis, although genetic heterogeneity is increas-
population distribution. But ~10 % of these subjects have a ingly recognized for several clinical syndromes. Below, the
discrete monogenic syndrome, such as heterozygous famil- clinical features, molecular genetics, diagnosis, and treat-
ial hypercholesterolemia (HeFH), or the phenotypically ment specific to each disorder will be discussed within each
similar disorders familial defective apo B due to binding subsection. The increased ease of DNA sequencing means
defects in the APOB gene, and autosomal dominant hyper- that this technology is often the most direct and cost-effective
cholesterolemia due to gain-of-function mutations in the path to a diagnosis for monogenic dyslipoproteinemias
PCSK9 gene. (Hegele 2009).
43.2 Nomenclature
674
Chromosomal
43.1.1 Familial hypercholesterolemia Hyperlipoproteinemia HeFH LDLR 19p13.3 Low-density lipoprotein 143890, Autosomal
heterozygous (LDLR) type 2A receptor 606945 dominant
43.1.2 Familial hypercholesterolemia Hyperlipoproteinemia HoFH LDLR 19p13.3 Low-density lipoprotein 143890, Autosomal
homozygous (LDLR) type 2A receptor 606945 recessive
43.1.3 Familial defective Autosomal dominant HCHOLA2 APOB 2p24-p23 Apolipoprotein B 144010 All forms
apolipoprotein B (APOB) hypercholesterolemia type 2
(binding-defective apo B)
43.1.4 Autosomal dominant Autosomal dominant HCHOLA3 PCSK9 1p32.3 Proprotein convertase 603776, All forms
hypercholesterolemia hypercholesterolemia type 3 subtilisin/kexin type 9 607786
(PCSK9 gain of function)
43.1.5 Autosomal recessive ARH1 LDLRAP1 1p36-p35 Low-density lipoprotein 603813, All forms
hypercholesterolemia receptor associated protein 605747
(ARH) 1
43.2.1 Abetalipoproteinemia (MTP) Bassen-Kornzweig syndrome ABL MTP 4q24 Microsomal triglyceride 200100, All forms
transfer protein 157147
43.2.2 Hypobetalipoproteinemia HBL APOB 2p24-p23 Apolipoprotein B 144010, All forms
(APOB) 605019
43.2.3 PCSK9 deficiency with Hypobetalipoproteinemia PCSK9 PCSK9 1p32.3 Proprotein convertase 607786; All forms
low LDL (PCSK9 loss of function) deficiency subtilisin/kexin type 9 613589
43.2.4 Familial combined ANGPTL3 deficiency FCH ANGPTL3 1p31.1-p22.3 Angiopoietin-like 605019, All forms
hypolipidemia (ANGPTL3) protein 3 604774
43.3.1 Cholesteryl ester transfer Familial CETP deficiency CETP 16q21 Cholesteryl ester transfer 607322, All forms
protein deficiency (CETP) hyperalphalipoproteinemia protein 143470
type 1
43.3.2 Hepatic lipase deficiency HL deficiency LIPC 15q21-q23 Hepatic triglyceride 612797, All forms
(LIPC) lipase 614025
43.3.3 Scavenger receptor B1 SRB1 deficiency SRB1 deficiency SCARB1 12q23.31 Scavenger receptor B1 601040, All forms
deficiency (SCARB1) 610762
43.4.1 Tangier disease (ABCA1) Primary familial TD, FHA ABCA1 9q31 ATP-binding cassette, 600046, All forms
hypoalphalipoproteinemia subfamily member A1 205400
R.A. Hegele and S. Tonstad
43.4.2 Apolipoprotein A-I deficiency Apolipoprotein A-I structural N/A APOA1 11q23 Apolipoprotein A-I 107680, All forms
(APOA1) mutations 604091
43.4.3 Familial LCAT deficiency Lecithin-cholesterol acyl LCAT deficiency LCAT 16q22 Lecithin-cholesterol 606967, All forms
(complete) transferase deficiency (LCAT) acyl transferase 245900,
43.4.4 Familial LCAT Fish eye disease FED LCAT 16q22.1 Lecithin-cholesterol acyl 136120, All forms
deficiency (partial) transferase 606967
43.5.1 Lipoprotein lipase deficiency Hyperlipoproteinemia type 1 HLP type 1 LPL 8p22 Lipoprotein lipase 609708, All forms
(LPL) 238600
43.5.2 Apolipoprotein C-II deficiency APOC2 APOC2 19q13 Apolipoprotein C-II 608083, All forms
(APOC2) deficiency 207750
43.6.1 Familial combined Hyperlipoproteinemia type 2B FCHL; HLP type USF1a 1q22-q23 Upstream stimulatory factor 144250, All forms
hyperlipidemia 2 and several others 602491
43.6.2 Dysbetalipoproteinemia Hyperlipoproteinemia type 3 DBL; HLP type APOE 19q13 Apolipoprotein E 107741 All forms
43 Disorders of Lipoprotein Metabolism
(APOE) 3
43.7.1 Sitosterolemia Phytosterolemia N/A ABCG5/ABCG8 2p21 ATP-binding cassette, 210250, All forms
(ABCG5/ABCG8) subfamily G, members 5 605460,
and 8 (sterolin 1 and 2) 605459
43.7.2 Elevated Lipoprotein(a) (LPA) N/A LPA 6q25-q26 Apolipoprotein(a) moiety of 152200 All forms
Lp(a)
a
And several others (a complex trait)
675
43.5.2
43.5.1
C
LPL 43.2.1
-II
43.2.2
B B
E
43.6.2 Intestine
E
Remnant
Chylomicron
43.4.2
Liver
43.3.3
Remnant receptor SR-B1 A-I HL A-I ABCA1
Cholesterol pool
43.1.5 ARH PCSK9 43.1.4 43.4.1
A-
43.2.3
II
LDL receptor
43.2.1 HDL 2 LCAT HDL 3 LDL receptor
43.1.1
43.2.2 43.4.3
43.1.2 CETP 43.4.4
43.3.1 Cell
43.1.3
HL
-II
LPL
C
B B B
43.3.2
E
VLDL IDL LDL
Fig. 43.1 Lipoprotein metabolic pathways
Table 43.1.1 Familial hypercholesterolemia heterozygous (LDLR)

Cardiovascular Carotid or femoral bruits n n n n +
Myocardial ischemia n n n n ++
Dermatological Xanthelasma n n n n +
Xanthomas, tendon n n n n ++
Eye Arcus cornealis n n n n +
Routine laboratory HDL cholesterol (P) ↓-n ↓-n ↓-n ↓-n ↓-n
LDL cholesterol (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Triglycerides (S) n n n n n
Special laboratory Apo B (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
LDLR gene mutation, + + + + +
heterozygous, on DNA
sequencing
Table 43.1.2 Familial hypercholesterolemia homozygous (LDLR)

Cardiovascular Calcified aortic valve n n n ↑ ↑↑↑
Cardiac catheterization n n Coronary Coronary Coronary
atherosclerosis atherosclerosis atherosclerosis
Cardiac echo n n AV disease AV disease AV disease
Carotid Doppler n n Stenosis Stenosis Stenosis
Carotid or femoral bruits n n n + ++
Myocardial ischemia n n + + +++
Dermatological Xanthelasma n n + ++ +++
Xanthomas, tendon n n + ++ +++
Eye Arcus cornealis n n + ++ +++
Routine laboratory HDL cholesterol (P) ↓ ↓ ↓ ↓ ↓
LDL cholesterol (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Special laboratory Apo B (P) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
LDLR gene sequencing + + + + +
Table 43.1.3 Familial defective apolipoprotein B (APOB)

Xanthomas, tendon n n n n ++
LDL cholesterol (P) ↑ ↑ ↑ ↑ ↑
Special laboratory Apo B (P) ↑ ↑ ↑ ↑ ↑
APOB gene sequencing + + + + +
Table 43.1.4 Autosomal dominant hypercholesterolemia

Myocardial ischemia n n n n +
Xanthomas, tendon n n n n +
Special laboratory Apo B (P) ↑ ↑ ↑ ↑ ↑
PCSK9 gene sequencing + + + + +
Table 43.1.5 Autosomal recessive hypercholesterolemia (ARH)

Cardiovascular Carotid or femoral bruits n n n n ++
Myocardial ischemia n n n + +++
Dermatological Xanthelasma n n + ++ +++
Xanthomas, tendon n n + ++ +++
Eye Arcus cornealis n n n + +++
LDL cholesterol (P) ↑↑ ↑↑ ↑↑ ↑↑↑ ↑↑↑
Special laboratory Apo B (P) ↑↑ ↑↑ ↑↑ ↑↑↑ ↑↑↑
ARH gene sequencing + + + + +
Table 43.2.1 Abetalipoproteinemia (MTP)

CNS Ataxia n n + ++ ++
Deep tendon reflexes n n ↓ ↓ ↓
Digestive Malabsorption + + + + +
Eye Pigmentary retinopathy n n + ++ ++
Hematological Acanthocytosis + + ++ ++ ++
Bleeding tendency n n + + +
LDL cholesterol (P) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Prothrombin ratio ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Triglycerides (S) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Special laboratory Apo B (P) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
MTP gene sequencing + + + + +
Small intestinal biopsy Lipid-laden Lipid-laden Lipid-laden Lipid-laden Lipid-laden
Vitamin A (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Vitamin E (P) ↓↓ ↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 43.2.2 Hypobetalipoproteinemia (APOB)

CNS Ataxia n n + ++ ++
Deep tendon reflexes n n ↓ ↓ ↓
Digestive Malabsorption + + + + +
Eye Pigmentary retinopathy n n + ++ ++
Hematological Acanthocytosis + + ++ ++ ++
Bleeding tendency n n + + +
LDL cholesterol (P) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Prothrombin ratio ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Triglycerides (S) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Special laboratory Apo B (P) ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓
APOB gene sequencing + + + + +
Small intestinal biopsy Lipid-laden Lipid-laden Lipid-laden Lipid-laden Lipid-laden
Vitamin A (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Vitamin E (P) ↓↓ ↓↓ ↓↓↓ ↓↓↓ ↓↓↓
Table 43.2.3 PCSK9 deficiency with low LDL

LDL cholesterol (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Triglycerides (S) ↓ ↓ ↓ ↓ ↓
Special laboratory Apo B (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
PCSK9 gene + + + + +
sequencing
Table 43.2.4 Familial combined hypolipidemia (ANGPTL3)

LDL cholesterol (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Triglycerides (S) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Special laboratory ANGPTL3 gene + + + + +
sequencing
Apo B (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 43.3.1 Cholesteryl ester transfer protein deficiency (CETP)

Routine laboratory Cholesterol (S) n n n n ↑
HDL cholesterol (P) ↑ ↑ ↑ ↑↑ ↑↑↑
Triglycerides (S) n n n n-↑ n-↑
Special laboratory CETP gene sequencing + + + + +
Table 43.3.2 Hepatic lipase deficiency (LIPC)

Cardiovascular Coronary artery disease n n n n +
Routine laboratory Cholesterol (S) n n n ↑ ↑
HDL cholesterol (P) n n n n ↑
Triglycerides (S) n n n ↑ ↑
Special laboratory Hepatic ↓ ↓ ↓ ↓ ↓
lipase activity – post-heparin
LIPC gene sequencing + + + + +
Lipoprotein electrophoresis n n n n Broad beta
Table 43.3.3 Scavenger receptor B1 deficiency (SCARB1)

Hematological Platelet function, n n n + +
abnormal
HDL cholesterol (P) ↑ ↑ ↑ ↑↑ ↑↑↑
Special laboratory SCARB1 gene + + + + +
sequencing
Table 43.4.1 Tangier disease (ABCA1)

CNS Neuropathy, peripheral n n n + +
Digestive Hepatosplenomegaly n n n + +
Eye Arcus cornealis n n n n n
Hematological Tonsils, enlarged orange + + + + +
HDL cholesterol (P) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Triglycerides (S) n n ↑ ↑ ↑
Special laboratory ABCA1 gene sequencing + + + + +
Apolipoprotein A-I level ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 43.4.2 Apolipoprotein A-I deficiency (APOA1)

Special laboratory APOA1 gene sequencing + + + + +
Apolipoprotein A-I level ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Table 43.4.3 Familial LCAT deficiency (complete)

Eye Arcus cornealis n n n + +
Corneal clouding, deposits n n n n +
Proteins, total (U) n n n n ↑
Triglycerides (S) n n ↑ ↑ ↑
Urea and creatinine (P) n n n n ↑
Special laboratory Apolipoprotein A-I level ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Cholesterol esterification rate ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Cholesterol, unesterified (P) ↑ ↑ ↑ ↑ ↑
LCAT activity (FB) ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
LCAT gene sequencing + + + + +
Renal biopsy n n n n Abnormal
Table 43.4.4 Familial LCAT deficiency (partial)

Eye Arcus cornealis n n n + +
Corneal clouding, deposits n n n n +
Triglycerides (S) n-↑ n-↑ n-↑ n-↑ n-↑
Urea and creatinine (P) n n n n n
Urine protein n n n n n
Special laboratory Apolipoprotein A-I level ↓↓ ↓↓ ↓↓ ↓↓ ↓↓
Cholesterol esterification rate n n n n n
Cholesterol, unesterified (P) n n n n n
LCAT activity (FB) ↓ ↓ ↓ ↓ ↓
LCAT gene sequencing + + + + +
Renal biopsy n n n n n
Table 43.5.1 Lipoprotein lipase deficiency (LPL)

Dermatological Xanthomas, eruptive + + + + +
Digestive Abdominal pain + + + + +
Pancreatitis + + + ++ ++
Eye Lipemia retinalis + + + + +
Routine laboratory Cholesterol (S) ↑ ↑ ↑ ↑ ↑↑
Triglycerides (S) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Special laboratory Lipoprotein ↓ ↓ ↓ ↓ ↓
lipase activity – post-heparin
LPL gene sequencing + + + + +
Table 43.5.2 Apolipoprotein C-II deficiency (APOC2)

Dermatological Xanthomas, eruptive + + + + +
Digestive Abdominal pain + + + + +
Pancreatitis + + + ++ ++
Eye Lipemia retinalis + + + + +
Routine laboratory Cholesterol (S) ↑ ↑ ↑ ↑ ↑↑
Triglycerides (S) ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑
Special laboratory APOC2 gene sequencing + + + + +
Lipoprotein lipase ↓ ↓ ↓ ↓ ↓
activity – post-heparin
Table 43.6.1 Familial combined hyperlipidemia

Routine laboratory HDL cholesterol (P) ↓ ↓ ↓ ↓↓ ↓↓
Triglycerides (S) ↑ ↑ ↑ ↑↑ ↑↑
Special laboratory Apo B (P) ↑ ↑ ↑ ↑↑ ↑↑
Table 43.6.2 Dysbetalipoproteinemia (APOE)

Claudication n n n n +
Dermatological Xanthomas, palmar n n n n +
Xanthomas, n n + + ++
tuberoeruptive
HDL cholesterol (P) n n n n ↓-n
Triglycerides (S) n n n n ↑
Special laboratory APOE gene E2/2 E2/2 E2/2 E2/2 E2/2
Lipoprotein electrophoresis n n n n Broad beta
band
Table 43.7.1 Sitosterolemia (ABCG5/ABCG8)

Xanthomas n n + + ++
Special laboratory ABCG5 or ABCG8 + + + + +
gene sequencing
Sitosterols (P) + + + + +
Table 43.7.2 Elevated lipoprotein (a) (LPA)

HDL cholesterol (P) n n n n n
LDL cholesterol (P) n n n n n
Special laboratory Lp(a) (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
LPA genotype associated + + + + +
with higher levels
Infant Child Adolescent Adult Other comments

Total cholesterol 2.0–3.5 2.5–4.0 3.0–4.5 3.5–5.5 Values shown cover approximate
(mmol/L) normal ranges across many
demographics; there is further
variation by age, sex, and ethnic
group
Triglyceride (mmol/L) 0.3–1.2 0.3–1.5 0.3–1.7 0.5–1.7
LDL cholesterol 1.0–2.4 1.0–3.0 1.2–3.5 1.4–4.5 Treatment target LDL-C level for
(mmol/L) high CVD risk adult is 1.8; there is
no specific treatment target LDL-C
level for children
HDL cholesterol 0.8–1.6 1.0–1.6 0.9–1.6 0.9–1.8 (men)
(mmol/L) 1.0–2.4 (women)
Apo B (g/L) 0.6–1.0 0.6–1.0 0.6–1.2 0.7–1.4
Apo A-I (g/L) 1.0–1.5 1.0–1.5 1.0–1.5 1.0–1.6
Apo B to A-I ratio 0.4–0.8 0.4–1.0 0.5–1.1 0.6–1.2
Lp(a) (g/L) <0.30 <0.30 <0.30 <0.30 Values represent 90th percentile in
most populations
43.6 Pathological and Diagnostic Values
Disorder Triglyceride Cholesterol LDL-C HDL-C Apo B Other disorder-

number Disorder name (mmol/L) (mmol/L) (mmol/L) (mmol/L) (g/L) specific test
43.1.1 Familial hypercholesterolemia N >6.5 (child) >4.0 (child) Normal >1.8 Heterozygous LDLR
heterozygous >8.0 (adult) >6.0 (adult) or low gene mutation
43.1.2 Familial hypercholesterolemia N >10 (child) >7 (child) Normal >2.4 Homozygous LDLR
homozygous >13 (adult) >11 (adult) or low gene mutation
43.1.3 Familial defective N >6.5 (child) >4.0 (child) Normal >1.8 Heterozygous APOB
apolipoprotein B >8.0 (adult) >6.0 (adult) or low gene mutation
43.1.4 Autosomal dominant N >6.5 (child) >4.0 (child) Normal >1.8 Heterozygous PCSK9
hypercholesterolemia >8.0 (adult) >6.0 (adult) or low gene mutation
43.1.5 Autosomal recessive N >10 (child) >7 (child) Normal >2.4 Homozygous LDLR
hypercholesterolemia >13 (adult) >11 (adult) or low gene mutation
43.2.1 Abetalipoproteinemia <0.2 <1.5 <0.8 Normal <0.1 Homozygous MTP
or low gene mutation, normal
lipoproteins in parents
43.2.2 Hypobetalipoproteinemia <0.2 <1.5 <0.8 Normal <0.1 Homozygous APOB
or low gene mutation,
half-normal LDL
(range 0.5–
1.5 mmol/L) and apo
B in parents
43.2.3 PCSK9 deficiency with <0.2 <1.5 <0.8 Normal <0.1 Homozygous PCSK
low LDL or low (gene mutation,
half-normal LDL and
apo B in parents)
43.2.4 Familial combined <0.3 <1.8 <0.8 Normal <0.1 Homozygous
hypolipidemia or low ANGPTL3 gene
mutation, half-normal
LDL and apo B in
parents
43.3.1 Cholesteryl ester transfer N N N >2.5 N Homozygous or
protein deficiency heterozygous CETP
gene mutation
43.3.2 Hepatic lipase deficiency >3.0 >6.0 N or low >2.0 >1.4 Homozygous LIPC
gene mutation
43.3.3 Scavenger receptor B1 N N N >2.0 N Homozygous
deficiency SCARB1 gene
mutation
43.4.1 Tangier disease Increased N N or low <0.1 N Homozygous ABCA1
gene mutation
43.4.2 Apolipoprotein A-I deficiency N N N or low <0.2 N Homozygous APOA1
gene mutation
43.4.3 Familial LCAT deficiency N or increased N N or low <0.2 N Homozygous LCAT
(complete) gene mutation
43.4.4 Familial LCAT deficiency N or increased N N or low N N Homozygous LCAT
(partial) gene mutation
43.5.1 Lipoprotein lipase deficiency >10 >6.5 N or low <1.0 >1.5 Homozygous LPL
gene mutation
43.5.2 Apolipoprotein C-II deficiency >10 >6.5 N or low <1.0 >1.5 Homozygous APOC2
gene mutation
43.6.1 Familial combined >1.7 >6.0 >4.5 Low >1.5
hyperlipidemia
43.6.2 Dysbetalipoproteinemia >3.0 >6.0 Variable <1.0 >1.5 APOE E2/2 genotype
43.7.1 Sitosterolemia N >6.0 >4.0 N or low Increased plant sterols
measured by
chromatography
43.7.2 Elevated lipoprotein(a) N N N N N Lp(a) >0.300 g/L
a b
Rule out secondary causes: Rule out secondary causes:
Cholesterol >13 mmol/L nephrotic syndrome, primary 6.5 mmol/L < Cholesterol < 13 mmol/L nephrotic syndrome,
biliary cirrhosis, medications primary biliary cirrhosis,
Triglycerides hypothyroidism,
Triglycerides N medications
N
Rule out causes Gene sequencing:
Gene sequencing:
of LDLR
Rule out causes of LDLR increased TG ARH
increased TG ARH APOB
PSCK9
APOB
PSCK9
Heterozygous
mutation: HeFH or
related phenotype
Homozygous
mutation: HoFH or
d
related phenotype
HDL < 0.5 mmol/L Gene sequencing:
c
ABCA1
1.5 mmol/L < Cholesterol < 3.0 mmol/L
APOA1
Apo A - I level
LCAT
Apo B level n to 0
> 0.5 g/L < 0.5 g/L
LCAT activity Apo A-I deficiency
No further Gene sequencing:
evaluation APOB > 20 % < 20 %
PSCK9
ANGPTL3 Tangier disease LCAT deficiency
or uncertain
Heterozygous mutation:
hypobetalipoproteinemia Cholesterol esterification
or related phenotype rate
> 20 % < 20 %
Partial LCAT deficiency Complete LCAT

(fish-eye-disease) deficiency
Fig. 43.2 Diagnostic flow charts

Fig. 43.2 (continued) e f

Rule out secondary causes:
Cholesterol < 0.5 mmol/L TG > 10 mmol/L
uncontrolled diabetes, excessive
alcohol use, medications
Heparin test
60 units/kg IV Gene
Also check LDL-C sequencing:
and apo B level Post-heparin LPL activity LPL
< 10 % >10 % APOC2
LPL deficiency 2D gel N

electrophoresis Homozygous
or apo C-II assay mutation: LPL or
Absent apo C-II deficiency
Gene sequencing: N
Apo C-II deficiency
MTP
Other rare possibilities
APOB resolved with gene
PSCK9 sequencing: deficiencies
of LMF1, APOA5, GPIHBP1
ANGPTL3
g Rule out secondary causes:
uncontrolled diabetes,
excessive
alcohol use,
Increased TG and cholesterol
liver disease, renal
Homozygous mutation: APOE genotyping disease, medications
abetalipoproteinemia or
E2/2 not E2/2
related phenotype Gene sequencing:
Familial HDL LIPC (hepatic lipase)
dysbetalipoproteinemia cholesterol
n to
Heparin test FCHL
60 units/kg iv
Post-heparin hepatic lipase

activity
< 10 % >10 %
Hepatic lipase deficiency Uncertain
etiology
43.8 Specimen Collection 43.9 Prenatal Diagnosis
DNA for sequence analysis or genotyping: standard DNA Prenatal diagnosis for the purpose of pregnancy termination
collection procedures from blood, buccal swabs, or is not usually indicated for familial lipid disorders.
saliva. When the index of suspicion for a homozygous illness is
Skin fibroblasts for LDL-receptor activity assay: standard high, it is theoretically feasible to sequence genomic DNA
skin biopsy and storage. sequence of chorionic villus samples for prenatal diagnosis
Post-heparin plasma for lipase activity: baseline fasting of HoFH for two parents known to have HeFH: vast majority
plasma, followed by IV injection of 50–100 units of normal of mutations are found within the LDLR gene.
molecular weight heparin per kg body weight, followed in For other severe recessive disorders of lipoprotein metab-
30 min by a second plasma sample to assay total and frac- olism, e.g., LPL deficiency or abetalipoproteinemia, since
tionated lipase activities. carrier parents are asymptomatic, diagnosis of the condition
Other plasma biochemical activities: LCAT activity or within the family follows from diagnosis of an initial off-
cholesterol esterification rate can be measured from plasma, spring. Once the mutation(s) and their segregation within the
but several available assay kits are research based and are not family are identified, subsequent chorionic villus tissue can
standardized for clinical use. be tested with focused genomic DNA analysis.
43.10 DNA Testing
Clinical suspicion + biochemical disturbance DNA sequencing of gene(s) DNA genotyping

HeFH + LDL cholesterol >6.5 mmol/L LDLR, followed by APOB, PCSK9, and possibly
ARH
HoFH + LDL cholesterol >10 mmol/L LDLR, followed by ARH, APOB, and possibly
PCSK9
Hyperalphalipoproteinemia + HDL >2.2 mmol/L CETP, followed by LIPC and possibly SCARB1
Hypertriglyceridemia + TG >10 mmol/L LPL, followed by APOC2 and possibly APOA5,
LMF1, or GPIHBP1
ABL + LDL cholesterol >0.5 mmol/L MTP, followed by APOB and possibly PCSK9
Hypoalphalipoproteinemia + HDL >0.5 mmol/L ABCA1, followed by APOA1 and possibly LCAT
Familial dysbetalipoproteinemia (approximately equimolar total APOE E2/2 genotype
cholesterol and triglyceride elevations)
43.11 Treatment Summary carbohydrates, increase physical activity, and eliminate

alcohol in some cases. For most dyslipidemias, carbohy-
National agencies recommend LDL cholesterol as the pri- drate and protein intake should be 50–60 % and 15–20 %,
mary target of therapy to reduce CVD risk (Genest et al. respectively, whereas total fat should be <25–35 % of total
2009). LDL cholesterol goals are recommended for patients calories, with restrictions on dietary cholesterol (<200 mg/
stratified into particular risk categories with more stringent day), and saturated fat, <7 % of total calories/day. The
goals for patients at high CHD risk (Grundy et al. 2004). In intake of animal products should be reduced and fish, sea-
children, no strong evidence is available to show improve- food, and plant-based foods emphasized including whole
ments in end points in regard to achieved LDL cholesterol. grains, vegetables, berries, fruits, legumes, and nuts. Fish
A European consensus paper suggested that in children with oil supplements may be useful for hypertriglyceridemia. In
HeFH aged 10–14 years, a reduction of 30 % in LDL cho- general, the severity of the dyslipidemia dictates the inten-
lesterol could be targeted, while a goal of <2.5 mmol/L is sity of the intervention. For severe hyperchylomicronemia,
suggested after age 18. A US paper suggested a goal of a recommended fat intake is restricted further to 10–15 % of
50 % reduction or level <3.4 mmol/L (Kavey et al. 2006; total calories, with reductions in both saturated and unsatu-
McCrindle et al. 2007). It is important to consider potential rated fat. For patients with sitosterolemia, elimination of
adverse effects of medication, presence of other CHD risk fac- plant sterols is required. A specialized dietician can be very
tors, and the potential to reduce risk during the years required helpful in these circumstances.
for atherosclerosis to develop (Stein 2007). Intervention to Regular exercise and avoiding sedentariness (e.g., TV
reduce LDL cholesterol includes lifestyle management and watching) are important habits that may be established
if necessary, pharmacologic therapies (Tonstad 2010; Vuorio early. In addition to contributing to energy balance, exer-
et al. 2010). However, certain monogenic disorders are asso- cise lowers blood pressure and improves insulin resistance;
ciated with dramatically increased CVD risk, placing these more directly it increases the catabolism of TG-rich lipo-
individuals into the highest risk strata (Scientific Steering proteins and increases tissue lipolysis, leading to reduced
Committee on behalf of the Simon Broome Register Group TG and increased HDL cholesterol levels (Tonstad 2010).
1991). These individuals will require individualized thera- Recommendations relating to the intensity of regular exercise
pies for the individual conditions. depend on the health and fitness of the patient; children need
General dietary interventions depend on the pattern of to engage in activities equivalent to a daily total of 60 min
dyslipidemia and whether the child is overweight or obese. of moderately intense physical activity. Avoiding cigarettes
For normal weight children with elevated LDL cholesterol, and other tobacco products is imperative. Adolescents who
trans- and saturated fats should be replaced with monounsat- smoke need to be offered help to quit.
urated and polyunsaturated fats, and specific foods that may Finally, drug therapy may be started together with life-
have functional effects on the lipid profile (e.g., foods rich style interventions in high-risk patients. The patient’s level
in soluble fiber) should be added. In overweight or obese of risk guides the timing of treatment initiation and also the
children, treatment aims to additionally achieve a better intensity of the treatment. Moderate- or low-risk patients
balance between energy intake and expenditure. In children may be started on medication after a trial period of lifestyle
with high triglycerides, treatment goals are to replace sim- interventions. The main priority of drug therapy is to achieve
ple sugars and “high glycemic index” foods with complex the identified LDL cholesterol target level. Therefore, once
drug therapy has been decided upon for a general patient The response to drug therapy should be checked initially
with high CVD risk and dyslipidemia, an LDL-lowering after about 8 weeks. If the LDL cholesterol target is not
drug is almost always the first step. Among children and achieved, options include upward titration, switching to a
adolescents, some agencies have suggested guidelines for more potent member of the same drug class, or addition of a
pharmacologic treatment – particularly statins – although second-line agent. If the patient develops a drug-related
there is no consensus regarding the appropriate age to initiate adverse event, alternatives include a trial period off the medi-
therapy and appropriate target levels, as noted above (Kavey cation to confirm a temporal relationship, empirically switch-
et al. 2006; McCrindle et al. 2007; Stein 2007; Kwiterovich ing with the same class in the event that the side effect is
2008). Some groups have suggested initiation of statin treat- agent-specific rather than class-specific, or switching to a
ment at the age of 8 years or older in children with HeFH second-line drug, such as a bile acid sequestrant. In HeFH or
(McCrindle et al. 2007). Care should be given to choose FCHL (Kwiterovich 2008; Kuromori et al. 2002), combina-
drugs with documented reductions in cardiovascular disease tion regimens, such as statin plus either a bile acid seques-
in adults – no end point trials have been conducted in chil- trant or cholesterol absorption inhibitor, are often required to
dren (Genest et al. 2009). attain target levels.
Emergency Treatment Table
Medical emergency Treatment

Chylomicronemia syndrome (caused by any form Cessation of oral intake (NPO); intravenous saline to correct or prevent hypovolemia;
of severe hypertriglyceridemia), presenting with correction of secondary causes, such as uncontrolled hyperglycemia or alcohol use; with
sepsis, abdominal pain, pancreatitis hospitalization, NPO, and hydration, TG will fall with half-life of 2–3 days; there is
typically no need for plasmapheresis or heparinization
Acute coronary syndrome As per usual management in acute coronary situation due to atherosclerosis-related
myocardial ischemia, followed by long-term high-dose statin therapy (e.g., atorvastatin
80 mg/day) as maintenance therapy and possible combination therapy with ezetimibe,
bile acid sequestrant, niacin, or fibrate to ensure that lipid targets for secondary
prevention of coronary artery disease are being attained
Standard Treatment Table
Disorder
number Disorder name Diet Medications (adult regimens) Other
43.1.1 Familial Saturated fat Statin as foundation: simvastatin, In children and adolescents,
hypercholesterolemia <7–10 % of energy 10–80 mg/day; atorvastatin, 10–80 mg/ use half-maximal statin dose;
heterozygous (HeFH) intake day; pravastatin, 20–80 mg/day; half-maximal doses for other
lovastatin, 20–40 mg/day; fluvastatin, medications also
20–80 mg/day; rosuvastatin,
10–40 mg/day
Cholesterol <200 mg/ Additional agents for combination
day treatment: ezetimibe, 10 mg /day;
cholestyramine, 12 g/day or colestipol,
15 g/day in divided doses; niacin
preparations, 2–4 g/day
43.1.2 Familial As above As above As above, plus serial plasma
hypercholesterolemia exchange or plasmapheresis
homozygous (HoFH) or LDL apheresis
43.1.3 Familial defective As for HeFH As for HeFH As for HeFH
apolipoprotein B (FDB)
43.1.4 Autosomal dominant As for HeFH As for HeFH As for HeFH
hypercholesterolemia
(PCSK9)
43.1.5 Autosomal recessive As for HoFH As for HoFH As for HoFH
hypercholesterolemia
(ARH)
43.2.1 Abetalipoproteinemia Medium-chain TG, High doses of oral or parenteral
(ABL) essential FA preparations of vitamins A, D, E, and K
supplements
43.2.2 Hypobetalipoproteinemia as for ABL As for ABL Monitor blood levels of fat-
(HBL) soluble vitamins and titrate
therapy
43.2.3 PCSK9 deficiency with No specific diet No specific drug therapy Monitor blood levels of fat-
low LDL soluble vitamins and treat if
indicated
43.2.4 Familial combined No specific diet No specific drug therapy Monitor blood levels of fat-
hypolipidemia (ANGPTL3 soluble vitamins and treat if
deficiency) indicated
43.3.1 Cholesteryl ester transfer General prudent diet Consider statin therapy as in HeFH if
protein (CETP) deficiency and lifestyle for CHD indicated by global cardiovascular risk
prevention assessment
43.3.2 Hepatic lipase (LIPC) General prudent diet Consider statin therapy as in HeFH if
deficiency and lifestyle for CHD indicated by global cardiovascular risk
43.3.3 Scavenger receptor B1 General prudent diet Consider statin therapy as in HeFH if
(SCARB1) deficiency and lifestyle for CHD indicated by global cardiovascular risk
43.4.1 Tangier disease General prudent diet Consider statin therapy as in HeFH if
and lifestyle for CHD indicated by global cardiovascular risk
43.4.2 Apolipoprotein A-I General prudent diet Consider statin therapy as in HeFH if
(APOA1) deficiency and lifestyle for CHD indicated by global cardiovascular risk
43.4.3 Familial lecithin- General prudent diet Consider statin therapy as in HeFH if
cholesterol acyl transferase and lifestyle for CHD indicated by global cardiovascular risk
(LCAT) deficiency prevention assessment
(complete)
43.4.4 Familial LCAT deficiency General prudent diet Consider statin therapy as in HeFH if
(partial) and lifestyle for CHD indicated by global cardiovascular risk
43.5.1 Lipoprotein lipase (LPL) Fat <10–20 % of total Try fish oil capsules (EPA + DHA) Plasma exchange or apheresis is
deficiency energy. Medium- 1–2 g once daily; consider a fibrate almost never indicated or
chain triglyceride (e.g., fenofibrate 145–200 mg/day). definitively helpful in this
supplements condition
43.5.2 Apolipoprotein C-II Fat <10–20 % of total Try fish oil capsules (EPA + DHA) Plasma exchange or apheresis is
(APOC2) deficiency energy. Medium- 1–2 g once daily; consider a fibrate almost never indicated or
chain triglyceride (e.g., fenofibrate 145–200 mg/day). definitively helpful in this
supplements condition
43.6.1 Familial combined Balance diet and As for HeFH based on clinical As for HeFH
hyperlipidemia physical activity to assessment of cardiovascular
maintain body weight disease risk
in relation to growth.
Restrict sugars to
<10 % of energy,
saturated fat to <10 %
of energy
43.6.2 Dysbetalipoproteinemia Balance diet and As for HeFH based on clinical
physical activity to assessment of cardiovascular
maintain body weight disease risk
in relation to growth.
Restrict sugars to
<10 % of energy,
saturated fat to <10 %
of energy
43.7.1 Sitosterolemia Cessation of plant Ezetimibe, bile acid sequestrants, and
sterols, reduction of statins have been shown to have varying
dietary cholesterol effectiveness
43.7.2 Elevated lipoprotein(a) General prudent diet Consider statin therapy as in HeFH if
and lifestyle for CHD indicated by global cardiovascular risk
Experimental Treatment Table
Disorder
number Disorder name Experimental treatment
43.1.1 Familial hypercholesterolemia heterozygous (HeFH) APOB antisense RNA (mipomersen, Genzyme)
PCSK9 antibodies (Amgen, Pfizer, Sanofi-Regeneron, Lilly);
PCSK9 antisense RNA
CETP inhibitors (anacetrapib, Merck; evacetrapib, Lilly)
MTP inhibitor (lomitapide, Aegerion)
43.1.2 Familial hypercholesterolemia homozygous (HoFH) As for HeFH, also experimental LDLR liver-directed gene therapy
43.1.3 Familial defective apolipoprotein B (FDB) As for HeFH
43.1.4 Autosomal dominant hypercholesterolemia As for HeFH
43.1.5 Autosomal recessive hypercholesterolemia (ARH) As for HoFH
43.2.1 Abetalipoproteinemia (ABL) None
43.2.2 Hypobetalipoproteinemia (HBL) None
43.2.3 PCSK9 deficiency with low LDL None
43.2.4 Familial combined hypolipidemia (ANGPTL3) None
43.3.1 Cholesteryl ester transfer protein deficiency (CETP) None
43.3.2 Hepatic lipase deficiency (LIPC) None
43.3.3 Scavenger receptor B1 deficiency (SCARB1) None
43.4.1 Tangier disease (ABCA1) None
43.4.2 Apolipoprotein A-I deficiency (APOA1) None
43.4.3 Familial LCAT deficiency (complete) None
43.4.4 Familial LCAT deficiency (partial) None
43.5.1 Lipoprotein lipase deficiency (LPL) LPL gene therapy (alipogene tiparvovec, AMC, Amsterdam)
43.5.2 Apolipoprotein C-II deficiency (APOC2) None
43.6.1 Familial combined hyperlipidemia As for HeFH
43.6.2 Dysbetalipoproteinemia (APOE) As for HeFH
43.7.1 Sitosterolemia (ABCG5/ABCG8) As for HeFH
43.7.2 Elevated Lipoprotein(a) (LPA) As for HeFH
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Biochemical Phenotypes
of Questionable Clinical Significance 44
Stephen I. Goodman and Marinus Duran

44.1 Introduction ..................................................................... 691
It is noteworthy that one of the four inborn errors that com-
44.2 Nomenclature ................................................................... 693
prise Garrod’s tetrad is pentosuria, a biochemical abnormal-
44.3 Signs and Symptoms ....................................................... 694 ity whose primary clinical importance is its occasional
44.4 Disorders .......................................................................... 697 misdiagnosis as diabetes mellitus. Several other inborn errors
44.5 Concluding Remarks....................................................... 704
have little if any clinical relevance. In many of them, how-
ever, this realization has followed a lengthy period during
References ..................................................................................... 704
which they were thought to cause disease, and in most cases
the problem has been one of sampling bias.
The first subject to be described with a novel inborn error
is almost invariably screened because of clinical disease,
and any biochemical abnormality that is detected may ini-
tially be thought of as linked to the clinical phenotype. In
some cases the perceived association is strengthened or
weakened by studying other family members, particularly
siblings, but in most cases, the link between biochemical
and clinical phenotype remains an open issue until, with the
passage of time, more individuals with the defect are recog-
nized. And this can take a long time. Biochemical pheno-
types such as hydroxylysinemia (Goodman et al. 1972),
phosphohydroxylysinuria (Dorland et al. 1990), and beta-
mercaptocysteine-lactate disulfiduria (Crawhall et al. 1968),
despite their initial descriptions more than 20 years ago, are
too infrequently reported to allow firm conclusions about
pathogenicity.
Newborn screening programs have compressed the time
line considerably, and it is now possible to detect a biochem-
ical defect in infancy and to follow its effect, if any, on clini-
cal phenotype. Such programs have also identified many
enzyme-deficient subjects who would likely not have come
S.I. Goodman (*) to clinical attention, such as the clinically unaffected females
Department of Pediatrics, University of Colorado, with 3-methylcrotonylglycinuria and glutaric acidemia type
Denver School of Medicine, Mail Stop 8313, 1 whose infants tested positive for hydroxy-C5 carnitine
12800 East Nineteenth Avenue, Aurora, CO 80045, USA
ester and low carnitine, respectively.
e-mail: stephen.goodman@ucdenver.edu
This chapter will attempt to describe the biochemical phe-
M. Duran
notypes of some inborn errors whose clinical relevance is in
Laboratory Genetic Metabolic Diseases, Academic Medical Center,
F0-224 Meibergdreef 9, Amsterdam 1105AZ, The Netherlands question. Some, like hydroxykynureninuria, are primarily of
e-mail: m.duran@amc.uva.nl historical importance, although recognition of an abnormal
692 S.I. Goodman and M. Duran
compound in amino or organic acid analysis can cause An excellent example of the latter is hyperprolinemia type II,
consternation to patient and physician alike. Much of what in which an early impression that patients tended to have sei-
we write reflects the authors’ personal views of what is now zures was confirmed and shown to be due to sequestering of
known about these conditions, and it may well be that a con- vitamin B6 by accumulated Δ1-pyrroline-5-carboxylic acid.
dition that we now think is of questionable clinical A continuing discussion of the topics will almost certainly
significance will on more intense and detailed study revert. keep the field alive and fascinating.
44
44.2 Nomenclature
Chromosomal OMIM
No. Disorder Alternative name Abbreviation Gene symbol localization Affected protein No. Subtype
44.1 Iminogycinuria IG SLC36A2; 3p21.31; 5p15.33; Amino acid transporter 242600 Benign
SLC6A20; 5q33.1 form
SLC6A19
44.2 Hyperprolinemia type I Proline oxidase deficiency HP1 PRODH 22q11.21 Proline oxidase 239500 Benign
form
44.3 Hydroxyprolinemia Hydroxyproline oxidase deficiency HYPO POX 22q11.2 Hydroxyproline oxidase 237000 Benign
form
44.4 Histidinemia Histidase deficiency HAL HAL 12q23.1 Histidase 235800 Benign
form
44.5 Urocanase deficiency UROC UROC1 3q21.3 Urocanase 276880 Benign
form
44.6 Glutamate formiminotransferase Formiminoglutamic aciduria FIGLU FTCD 21q22.3 Formiminotransferase 229100 Benign
deficiency form
44.7 Carnosinemia and homocarnosinosis Carnosinase deficiency CARN CNDP1 18q22.3 Carnosinase 212200 Benign
form
44.8 Trimethylaminuria Fish odor syndrome TMA FMO3 1q24.3 Flavin-containing monooxygenase 602079 Benign
form
44.9 Dimethylglycinuria Dimethylglycine dehydrogenase DMGLY DMGDH 5q14.1 Dimethylglycine dehydrogenase 605849 Benign
Biochemical Phenotypes of Questionable Clinical Significance
deficiency form
44.10 Sarcosinemia Sarcosine dehydrogenase SDD SARDH 9q34.2 Sarcosine dehydrogenase 268900 Benign
deficiency form
44.11 Hyperlysinemia and saccharopinuria 2-Aminoadipic semialdehyde SDH AASS 7q31.32 2-Aminoadipic semialdehyde 268700 Benign
synthase deficiency synthase form
44.12 2-Ketoadipic and 2-aminoadipic 2-Ketoglutarate dehydrogenase KAA OGDH 7p13 2-Ketoglutarate dehydrogenase 204750 Benign
academia deficiency form
44.13 Glutaric aciduria type 3 Glutaryl-CoA oxidase deficiency, GA3 C7orf10 7p14.1 Glutaryl-CoA oxidase 231690 Benign
peroxisomal form
44.14 Hydroxykynureninuria Xanthurenic aciduria KYN KYNU 2q22.2 Kynureninase 236800 Benign
form
44.15 Cystathioninuria γ-Cystathionase deficiency CYSTA CTH 1p31.1 γ-Cystathionase 219500 Benign
form
44.16 3-Methylcrotonylglycinuria 3-Methylcrotonyl-CoA carboxylase 3MCC MCCC1 and 2 3q27.1; 5q13.2 3-Methylcrotonyl-CoA 210200 Benign
deficiency carboxylase form
44.17 2-Methylbutyrylglycinuria, benign 2-Methylbutyryl-CoA SBCAD ACADSB 10q26.13 2-Methylbutyryl-CoA 610006 Benign
dehydrogenase deficiency dehydrogenase form
44.18 Oxoprolinase deficiency OPLAHD OPLAH 8q24.3 Oxoprolinase 260005 Benign
form
44.19 Glycerol kinase deficiency, isolated Hyperglycerolemia GKD GK Xp21.2 Glycerol kinase 307030 Benign
form
44.20 Methionine adenosyltransferase MAT deficiency MAT MAT1A 10q23.1 Methionine adenosyltransferase 250850 Benign
deficiency form
44.21 Methylmalonyl-CoA epimerase Methylmalonic aciduria 3 MMAE MCEE 2p13.3 Methylmalonyl-CoA epimerase 251120 Benign
deficiency form
693
44.22 Short-chain acyl-CoA dehydrogenase SCAD deficiency SCAD ACADS 12q24.31 Short-chain acyl-CoA 606885 Benign
deficiency dehydrogenase form
Table 44.1 Iminogycinuria

Special laboratory Glycine (U) ↑ ↑ ↑ ↑ ↑
Hydroxyproline (U) ↑ ↑ ↑ ↑ ↑
Proline (U) ↑ ↑ ↑ ↑ ↑
Table 44.2 Hyperprolinemia type I

Special laboratory Glycine (U) n-↑ n-↑ n-↑ n-↑ n-↑
Hydroxyproline (U) n-↑ n-↑ n-↑ n-↑ n-↑
Proline (P) ↑↑ ↑↑ ↑↑ ↑↑ ↑↑
Proline (U) n-↑ n-↑ n-↑ n-↑ n-↑
Table 44.3 Hydroxyprolinemia

Special laboratory Hydroxyproline (P,U) ↑ ↑ ↑ ↑ ↑
Table 44.4 Histidinemia

Routine laboratory Ketones (U) + + + +
Special laboratory Histidase (FB) ↓ ↓ ↓ ↓ ↓
Histidine (P,U,CSF) ↑ ↑ ↑ ↑ ↑
Imidazole pyruvic acid (U) ↑ ↑ ↑ ↑ ↑
Table 44.5 Urocanase deficiency

Special laboratory Urocanic acid (U) ↑ ↑ ↑ ↑ ↑
Urocanoylglycine (U) ↑ ↑ ↑ ↑ ↑
Table 44.6 Glutamate formiminotransferase deficiency

Special FIGLU (U) ↑ ↑ ↑ ↑ ↑
laboratory Hydantoin-5-propionic acid (U) ↑ ↑ ↑ ↑ ↑
Table 44.7 Carnosinemia and homocarnosinosis

Special laboratory Anserine (U) ↑ ↑ ↑ ↑ ↑
Carnosine (P,U) ↑ ↑ ↑ ↑ ↑
Homocarnosine (CSF) ↑ ↑ ↑ ↑ ↑
44 Biochemical Phenotypes of Questionable Clinical Significance 695
Table 44.8 Trimethylaminuria

Routine laboratory Fish odor + + + + +
Special laboratory TMAO/TMA ratio (U) ↓ ↓ ↓ ↓ ↓
Trimethylamine (U) ↑ ↑ ↑ ↑ ↑
Table 44.9 Dimethylglycinuria

Routine laboratory Fish odor (U) +
Special laboratory Dimethylglycine (P) ↑
Dimethylglycine (U) ↑
Table 44.10 Sarcosinemia

Special laboratory Sarcosine (P,U) ↑ ↑ ↑ ↑ ↑
Table 44.11 Hyperlysinemia and saccharopinuria

Other No clinical significance +
Special laboratory Homocitrulline (U) n-↑ n-↑ n-↑ n-↑ n-↑
Lysine (P,U,CSF) ↑ ↑ ↑ ↑ ↑
N-Acetyl-lysine (U) n-↑ n-↑ n-↑ n-↑ n-↑
Saccharopine (P,U,CSF) ↑ ↑ ↑ ↑ ↑
Table 44.12 2-Ketoadipic and 2-aminoadipic academia

Special laboratory 2-Aminoadipate (P,U) ↑ ↑ ↑ ↑ ↑
2-Hydroxyadipate (U) ↑ ↑ ↑ ↑
2-Oxoadipate (U) ↑ ↑ ↑ ↑
Table 44.13 Glutaric aciduria type 3

Special laboratory 3-Hydroxyglutaric acid (U) n n n n n
Glutaric acid (P,U) ↑ ↑ ↑ ↑ ↑
Table 44.14 Hydroxykynureninuria

Special laboratory 3-Hydroxykynurenine (U) ↑ ↑ ↑
Kynurenine (U) ↑ ↑ ↑
Xanthurenic acid (U) ↑ ↑ ↑
Table 44.15 Cystathioninuria

Special laboratory Cystathionine (P,U) ↑ ↑ ↑
Table 44.16 3-Methylcrotonylglycinuria

CNS Lethargy ± ± ± ±
Somnolence ± ± ± ±
Digestive Diarrhea ± ± ± ±
Hepatosplenomegaly ± ± ± ±
Vomiting, episodic ± ± ± ±
Routine laboratory Ammonia (B) n-↑ n-↑ n-↑ n-↑
ASAT/ALAT (P) n-↑ n-↑ n-↑ n-↑
Glucose (P) ↓-n ↓-n ↓-n ↓-n
Uric acid (P) n-↑ n-↑ n-↑ n-↑
Special laboratory 3-Hydroxyisovaleric acid (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
3-Methylcrotonylglycine (U) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
C5-OH-acylcarnitine (P,B) ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑ ↑-↑↑↑
Table 44.17 2-Methylbutyrylglycinuria, benign

Special laboratory 2-Methylbutyrylglycine (U) ↑ ↑ ↑
C5-Acylcarnitine (P,B) ↑ ↑ ↑
Ethylhydracrylic acid (U) n-↑ n n-↑
Isobutyrylglycine (U) n-↑ n-↑ n-↑
Table 44.18 Oxoprolinase deficiency

Routine laboratory Acidosis n n n n n
Special laboratory 5-Oxoproline (U) ↑ ↑ ↑ ↑ ↑
Oxoprolinase (FB,LYM) ↓ ↓ ↓ ↓ ↓
Table 44.19 Glycerol kinase deficiency, isolated

Routine laboratory Glucose (P) ↓-n ↓-n ↓-n ↓-n ↓-n
Triglycerides, pseudo (P) ↑ ↑ ↑ ↑ ↑
Special laboratory Glycerol (P,U) ↑ ↑ ↑ ↑ ↑
Table 44.20 Methionine adenosyltransferase deficiency

Routine laboratory Cabbage-like odor + + +
Special laboratory Homocysteine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Methionine (P) ↑ ↑ ↑
Methionine sulfoxide (P,U) ↑ ↑ ↑
SAM/SAH ratio n n n n n
Table 44.21 Methylmalonyl-CoA epimerase deficiency

Special laboratory Methylmalonic acid (U) ↑ ↑ ↑ ↑
Table 44.22 Short-chain acyl-CoA dehydrogenase deficiency

Routine laboratory Glucose (P) ↓-n ↓-n
Special laboratory Butyrylcarnitine (P) n-↑ n-↑ n-↑ n-↑ n-↑
Butyrylglycine (U) n-↑ n-↑ n-↑ n-↑ n-↑
Ethylmalonic acid (U) ↑ ↑ ↑ ↑ n-↑
Methylsuccinic acid (U) ↑ ↑ ↑ ↑ n-↑
Short-chain acyl-CoA ↓ ↓ ↓ ↓ ↓
dehydrogenase (FB)
44.4 Disorders with iminoglycinuria is hyperprolinemia type II. This is usu-

ally quite simple, as plasma proline is very high in hyperpro-
44.1 Iminoglycinuria, i.e., increased amounts of proline, linemia type II, and Δ1-pyrroline-5-carboxylic acid (P5C)
hydroxyproline, and glycine in urine, is most commonly and Δ1-pyrroline-3-hydroxy-5-carboxylic acid (hydroxy-
seen in (premature) infants, when it is due to immaturity of a P5C) are increased in urine.
shared renal tubular transporter for these amino acids. P5C and hydroxy-P5C in the urine appear as discrete
Iminoglycinuria due to tubular immaturity usually disap- peaks on amino acid analysis and also cause the urine to
pears by 1 year of age. It can also be seen in hyperprolinemia react positively with o-aminobenzaldehyde (Efron 1965).
types I and II, when elevated proline in the glomerular filtrate When using sodium citrate buffers, hydroxy-P5C elutes just
saturates a transporter and competitively inhibits tubular after hydroxyproline, and P5C elutes just before proline
reabsorption of glycine and hydroxyproline, and when a (Goodman et al. 1974). Urine organic acid analysis of hyper-
transporter fails to operate properly due to mutation. The lat- prolinemia patients will reveal the presence of pyrrole-5-
ter is called familial iminoglycinuria and is inherited as an carboxylglycine, possibly the most accessible characteristic
autosomal recessive trait. Depending on which transporter is metabolite.
involved, the defect may also be expressed in the small intes- 44.2 Hyperprolinemia Type I (HPI) is due to a defect in
tine. Heterozygotes sometimes have hyperglycinuria without proline oxidase, an FAD-containing mitochondrial protein
hyperglycinemia. While occasionally found in symptomatic found mainly in liver, kidney, and brain, that converts proline
individuals, familial iminoglycinuria is probably without to Δ1-pyrroline-5-carboxylic acid (P5C). The condition was
clinical significance. initially described in a patient with Alport-like renal disease
The condition is easily recognized on urine amino acid (Schafer et al. 1962) and was subsequently reported in
analysis by the increases in proline, hydroxyproline, and gly- patients with a wide variety of renal and nonrenal manifesta-
cine, accompanied by normal concentration of proline in tions, including eye abnormalities, developmental delay, and
blood. Hyperprolinemia type I (see below) and iminoglycin- other neurologic manifestations. Evidence from subjects
uria of the newborn are clinically benign, so the only clini- identified by newborn screening shows that HPI is usually
cally relevant condition that must be excluded in an individual benign (e.g., Fontaine et al. 1970), and the early association
with clinical phenotypes is now thought to have been due to and glutamate via imidazolone propionic acid and formimi-
sampling bias although the reports about the association of noglutamic acid (FIGLU) appears to occur only in the liver.
this defect with various clinical problems such as epilepsia Histidinemia is caused by histidase deficiency. The enzyme
and schizophrenia are still appearing. The condition’s pri- defect is transmitted as an autosomal recessive trait.
mary importance is that it must be differentiated from hyper- Biochemical findings in histidinemia, which is the most
prolinemia type II, in which a defect in P5C dehydrogenase common inborn error in Japan, include increased histidine in
is often associated with mental retardation and/or seizures. plasma, urine, and cerebrospinal fluid, low concentrations of
The condition is easily recognized by finding increased urocanic acid in blood and sweat, and increased concentra-
proline (500–2,000 μM; normal <420) on amino acid analy- tions of histidine metabolites such as imidazole pyruvic acid
sis of plasma, usually accompanied by increased proline, in urine. Enzyme-deficient subjects differ widely in their
hydroxyproline, and glycine in urine. Iminoglycinuria is due plasma histidine levels and tolerance to oral histidine loads,
to saturation of the renal transporter for proline, hydroxypro- and this may well be due to differences in mutations and
line, and glycine by the increased proline in the glomerular residual enzyme activity.
filtrate. Histidine can be as high as 1,500 μM (normal <150). The
Plasma proline concentrations in hyperprolinemia type II increased imidazole pyruvic acid in urine can usually be
are often higher (>1,300 μM), and the urine contains P5C, detected by the ferric chloride test or the Phenistix reagent
hydroxy-P5C, and pyrrole-5-carboxylglycine (see above). strip. Diagnosis can be confirmed, if necessary, by demon-
44.3 Hydroxyprolinemia was described in 1962 (Efron strating histidase deficiency in skin and by mutation analysis
et al. 1962) in association with mental retardation and has (Kawai et al. 2005). Treatment with a histidine-restricted diet
since been described in several additional subjects with a normalizes the biochemical phenotype but is not indicated
variety of clinical manifestations and in several healthy for what is most likely a harmless condition.
hydroxyprolinemic siblings (Kim et al. 1997). In addition, 44.5 Urocanic Aciduria, which is caused by a deficiency
one hydroxyprolinemic baby has been detected by newborn of urocanase, is characterized by increased urocanic acid in
screening and continues to be clinically normal. It is now urine, with normal or slightly increased concentrations of
thought that, as with hyperprolinemia type I, deficiency of histidine in plasma and urine. Said to be very rare, the condi-
hydroxyproline oxidase does not cause clinical disease and tion is probably underdiagnosed because the increased uro-
that the early association with disease was due to sampling canic acid cannot be detected by the usual methods used to
bias. screen for amino and organic acids in blood and urine. Older
As in proline oxidation, hydroxyproline is normally oxi- methods used thin-layer chromatography and staining with
dized first to Δ1-pyrroline-3-hydroxy-5-carboxylic acid by the Pauly reagent, but more specific HPLC methods have
hydroxyproline oxidase and thence to hydroxyglutamic also been developed (e.g., Kuracka et al. 1996). Urocanic
semialdehyde by Δ1-pyrroline-5-carboxylic dehydrogenase, acid can appear in the HPLC analysis of urine purines and
the enzyme in the proline pathway that is deficient in hyper- pyrimidines using UV-detection. Alternatively, NMR analy-
prolinemia type II. sis of a urine sample will readily detect an increased uroca-
Hydroxyprolinemia, which is inherited as an autosomal nic acid (UF Engelke, SSIEM-symposium Istanbul 2010).
recessive trait, causes elevations of hydroxyproline in plasma While initially diagnosed in retarded individuals, several
and urine, without iminoglycinuria and with a normal excre- children identified by newborn screening have developed
tion of peptide-bound hydroxyproline. Its diagnosis is rela- normally without therapy, and the enzyme defect is now
tively simple and depends on demonstrating high thought to be benign in most instances.
hydroxyproline in plasma (200–500 μM; normal <50) and The condition is apt to be detected during investigation of
urine. increased histidine in blood and/or urine, when increased
44.4 Histidinemia, first described in two siblings by urocanic acid is found in urine instead of the decrease
Ghadimi et al. in 1961, was thought in the past to cause expected in histidinemia, which is much more common.
speech defects as an almost specific clinical phenotype. As Diagnosis can be confirmed by demonstrating reduced
more subjects with the condition have been found, however, enzyme activity in liver or by molecular analysis of the
many of them by newborn screening programs, it has been UROC1 gene (Espinós et al. 2009).
learned that most enzyme-deficient individuals have normal 44.6 Glutamate Formiminotransferase-Cyclodeaminase
intelligence and speech (Ishikawa 1987), and it is now Deficiency (FIGLU-Uria). Increased excretion of formimino-
thought that the condition is benign in most instances. l-glutamic acid (FIGLU), an intermediate in the oxidation of
Histidine is deaminated to trans-urocanic acid by histi- histidine, was first noted to result from inherited deficiency
dase (histidine ammonia-lyase), an enzyme found mainly in of formiminotransferase-cyclodeaminase (FTCD) when
the liver and in the stratum corneum of the skin, but further amino acid analysis became routine when evaluating chil-
metabolism of urocanic acid to 5-formyl-tetrahydrofolate dren with developmental disabilities. The compound does
not react with ninhydrin, but quickly breaks down to gluta- deficiency, which is easily confirmed by enzyme assay. CSF
mate during many analytic procedures. FIGLU excretion is homocarnosine in such individuals is normal, i.e., less than
also increased in folic acid and B12 deficiency and in cblC 15 μM. Because serum carnosinase also hydrolyzes anserine,
disease (see Chaps. 10 and 13). a dipeptide of β-alanine and 1-methyl-l-histidine found in
FTCD deficiency is a relatively common recessively skeletal muscle of birds and some mammals, normal subjects
inherited defect of histidine metabolism and has both severe excrete 1-methylhistidine in urine after eating fowl.
and mild phenotypes, with the former characterized by meg- Carnosinase-deficient subjects do not, however, and instead
aloblastic anemia, mental retardation, and elevated urine excrete intact anserine. Because enzyme activity is low in
FIGLU following a histidine load. The mild phenotype infancy and increases gradually to achieve adult levels in
includes high FIGLU in blood and urine without loading adolescence, it is important to compare enzyme activity with
and, if developmental delay is present, it is mild. appropriate age-matched control data.
Elevated FIGLU is now most frequently encountered dur- A diagnosis of homocarnosinosis will initially be sug-
ing analysis of plasma acylcarnitine esters (Malvagia et al. gested by increased carnosine in urine, even on a meat-free
2006). If folic acid deficiency and cblC disease are elimi- diet, accompanied by elevated homocarnosine in cerebrospi-
nated by appropriate studies, confirmation of the defect can nal fluid (50–75 μM; normal <15). As in serum carnosinase
be by enzyme analysis in the liver or by molecular analysis deficiency, anserine is excreted intact after eating anserine-
of the FTCD gene (Hilton et al. 2003). An additional diag- containing foods.
nostic marker is the urine hydantoin-5-propionic acid, a 44.8 Trimethylaminuria is a recessively inherited condi-
metabolite of the precursor imidazolone-propionic acid. It tion in which absorbed trimethylamine (TMA), formed by
can easily be detected by urine organic acid analysis. gut bacteria from dietary precursors such as lecithin and cho-
44.7 Carnosinase Deficiency and Homocarnosinosis. line, accumulates behind a defect in flavin-containing mono-
Formerly thought to be two conditions, carnosinase defi- oxygenase 3 (FMO3), the enzyme that oxidizes TMA to
ciency and homocarnosinosis are now thought to be variants TMA N-oxide (TMAO). TMA is not toxic to tissues, but is
of the same disorder. Carnosine is a dipeptide of β-alanine highly volatile, and FMO3-deficient subjects develop a pun-
and histidine and is present in several human tissues, including gent odor similar to that of rotting fish. Sometimes called
skeletal muscle and brain. Homocarnosine (γ-aminobutyryl- “fish odor syndrome,” the condition causes severe social
l-histidine) exists only in the brain, and in amounts much stigmatization and psychological distress to affected indi-
larger than those of carnosine. Both dipeptides can be hydro- viduals and to their families.
lyzed by carnosinase (EC 3.4.13.20), an enzyme that is pres- Usually suspected because of the odor, diagnosis is made by
ent in plasma and several tissues, including brain, and encoded demonstrating an abnormal ratio of TMA:TMAO in urine at
by the CN1 gene on human chromosome 18. A closely related rest or after a choline load; the latter is particularly useful when
CN2 gene is located near CN1 and encodes a much less spe- the odor is intermittent or mild and can consist most simply of
cific dipeptidase that hydrolyzes carnosine only under non- a marine fish meal. Analysis of TMA and TMAO in urine can
physiological conditions (Teufel et al. 2003). be quite cumbersome, but a recently described method using
Carnosinemia and carnosinuria due to recessively inher- tandem mass spectrometry (Johnson 2008) appears to be sim-
ited deficiency of carnosinase were initially described in ple, quick, and accurate. Alternatively, an in vitro NMR analy-
patients with neurological disease (Perry et al. 1967) but sis of the urine will unequivocally demonstrate the presence of
has since been observed repeatedly in normal subjects. It is TMA (see below). There is normally much more TMAO in
now thought that any relationship to clinical symptoms is urine than TMA, so the TMAO:TMA + TMAO ratio in normal
fortuitous. Homocarnosinosis, which may be caused by a subjects exceeds 0.92. The ratio in severely enzyme-deficient
more severe deficiency of the same enzyme, is much less subjects can be as low as 0.04.
common and is characterized by increased homocarnosine Several polymorphisms as well as severe and mild muta-
(the major form of bound GABA) in cerebrospinal fluid and tions have been described in the FMO3 gene, and the latter
increased carnosine in urine. It has been described only usually correlate with the severity of the metabolic block
once, in a healthy 72-year-old woman and in three of her (Hernandez et al. 2003). Treatment is by restricting the
four children, all of whom had severe progressive neuro- intake of high-choline- and lecithin-containing foods, in par-
logical disease and retinal pigmentation. The lack of clini- ticular fish, dairy products, eggs, and chocolate, and elimi-
cal disease in the mother in this family suggests that nating bacterial TMA production by gut sterilization.
neurological disease in her children was not related to ele- 44.9 Dimethylglycinuria. Dimethylglycine (DMG) is
vated CSF homocarnosine. formed by the transfer of a methyl- group from betaine, a
Persistent carnosinemia (0.005–0.03 μM; normal = not choline metabolite, to homocysteine, forming methionine.
detected) and carnosinuria on a meat- (and therefore carno- DMG is then converted to sarcosine by oxidative demethyl-
sine-) free diet strongly suggest a diagnosis of carnosinase ation catalyzed by DMG-dehydrogenase, a flavin-containing
enzyme that requires folate as a cofactor. Sarcosine is con- Not normally detectable in plasma or urine, plasma sarco-
verted to glycine by a similar and related enzyme. Electrons sine levels in sarcosinemia range from 50 to 700 μM, and urine
from the flavins of DMG and sarcosine dehydrogenase enter excretion ranges from 170 to 5,080 mmol/mol creatinine.
the respiratory chain via electron transfer flavoprotein (ETF) 44.11 Hyperlysinemia and Saccharopinuria. The initial
and ETF-ubiquinone oxidoreductase. Increased excretion of steps in the mitochondrial oxidation of lysine in the liver and
DMG has been observed in folate deficiency and in subjects kidney are catalyzed by a single bifunctional enzyme, i.e.,
receiving large doses of betaine as a therapeutic agent. 2-aminoadipic semialdehyde synthase. The first reaction is a
Theoretically one should expect DMG to accumulate in the condensation of lysine with 2-ketoglutarate, carried out by
multiple acyl-CoA dehydrogenation defect (MADD, glutaric lysine-2-ketoglutarate reductase, to form saccharopine, and
aciduria type II), but this has not been confirmed so far. the second, catalyzed by saccharopine dehydrogenase,
Dimethylglycinemia and -uria due to deficiency of dimeth- releases glutamate and 2-aminoadipic semialdehyde.
ylglycine dehydrogenase has been observed only once, in an Recessively inherited mutations in the bifunctional enzyme
adult (Moolenaar et al. 1999) with a fish-like odor, chronic cause familial hyperlysinemia when the first or both activi-
muscle fatigue, and increased serum creatine kinase. A search ties are deficient and saccharopinuria when appreciable
for trimethylamine in urine by 1H-NMR spectroscopy instead residual activity is retained in the first enzyme function.
detected an approximately 20-fold increase of DMG Although hyperlysinemia with and without saccharopin-
(450 mmol/mol creatinine; normal <26) and was confirmed by uria were initially recognized in mentally retarded children
GC-MS of the trimethylsilyl derivative. DMG was increased (Woody 1964; Carson et al. 1968), it soon became apparent
100-fold in plasma (221 μM; normal <5), and molecular anal- that many patients with hyperlysinemia who were diagnosed
ysis showed homozygosity for an inactivating mutation in the subsequently, some of whom were ascertained through new-
DMG dehydrogenase gene (Binzak et al. 2001). born screening programs, were clinically normal and that the
Except for the “fish odor,” which is characteristic of initial association of hyperlysinemia with neurodevelopmen-
dimethylglycine, the relationship between the enzyme defi- tal abnormalities may well have been due to ascertainment
ciency and the rest of the clinical phenotype is not clear and bias (Dancis et al. 1983). This may also be true of saccharo-
may well be fortuitous. The diagnosis should probably be pinuria, in which the added metabolite is rapidly cleared by
considered in the many patients with a fish-like odor who do the kidney, but we do not know of a large study that has dem-
not have trimethylamine oxidase deficiency. onstrated this conclusively.
44.10 Sarcosinemia. Sarcosine (monomethylglycine) is Plasma lysine in hyperlysinemia often exceeds 1,000 μM
converted to glycine, with oxidation of its methyl group to (normal <250) and is accompanied by impressive lysinuria
“active” formaldehyde by sarcosine dehydrogenase, a mito- together with increased levels of the two N-acetyllysines and
chondrial flavoprotein from which electrons enter the respira- homocitrulline, sometimes with minimal saccharopinuria.
tory chain through electron chain flavoprotein (ETF) and While only two patients have been described with saccharo-
ETF-ubiquione oxidoreductase (ETF:QO). Like dimethylgly- pinuria, plasma lysine is lower than in familial hyperlysin-
cine dehydrogenase, the enzyme requires folate as a cofactor. emia, and urine saccharopine is much higher. The relationship
Sarcosinemia, a biochemical phenotype characterized by of the enzyme defect to clinical manifestations is question-
increased sarcosine in blood and urine, was first described in able in both disorders.
a 10-month-old child (Gerritsen and Waisman 1966) with While saccharopinuria is difficult to miss – the compound
mental retardation and hypertonia and was subsequently runs as a 570 nm absorbing peak in the area of cystine on an
reported in association with a variety of clinical phenotypes amino acid analyzer – isolated hyperlysinemia and/or hyper-
and developmental issues. More recently it has been identi- lysinuria can represent a difficult diagnostic challenge. If
fied by newborn screening in subjects whose development elevated lysine is first detected in urine, plasma should be
appears to be normal (Levy et al. 1984). It is now thought examined to ensure that it is due to overflow and not to the
that the biochemical phenotype is without clinical effect in defects in renal transport that cause cystinuria and lysinuric
most instances. The enzyme defect has not been demon- protein intolerance. Hyperlysinemia must be distinguished
strated in affected individuals, but the SDH gene has been from the secondary hyperlysinemia that occurs in infantile
cloned (Eschenbrenner and Jorns 1999), opening the possi- pyruvate carboxylase deficiency, where it is accompanied by
bility of molecular analysis. hyperammonemia, lactic acidemia, and citrullinemia. In
Increased sarcosine is also occasionally found in the mul- addition, when finding isolated hyperlysinemia, one should
tiple acyl-CoA dehydrogenation defect (MADD, glutaric be aware of the possibility of dienoyl-CoA reductase defi-
aciduria type II), however, and it is important that the finding ciency. Two patients with this defect of mitochondrial fatty
of increased sarcosine in blood and/or urine be followed by a acid beta-oxidation also had hyperlysinemia.
careful search for the organic acids and/or acylcarnitine 44.12 2-Aminoadipic and 2-Ketoadipic Aciduria.
esters characteristic of GA2. 2-Aminoadipic acid, a lysine metabolite, is transaminated to
2-ketoadipic acid, which is probably oxidatively decarboxyl- strated for a missense mutation in C7orf10, which appears to
ated to glutaryl-CoA by 2-ketoglutarate dehydrogenase encode a mitochondrial enzyme with a coenzyme A transfer-
(Hirashima et al. 1967), or by another enzyme similar to the ase domain (Sherman et al. 2008). Mutations in this gene
dehydrogenases for pyruvate, branched-chain ketoacids, and were also present in several additional patients, and these
2-ketoglutarate. included homozygosity for a chain-terminating mutation in
Several subjects with 2-aminoadipic and/or 2-ketoadipic the child whose defect had been thought initially to be in a
acidemia and aciduria have been described, most of them ini- peroxisomal oxidase. Because the enzyme product may
tially ascertained because of developmental delay and a vari- esterify glutaric acid, it was suggested that the product of
ety of neurological manifestations. Because several siblings 2-ketoadipic dehydrogenase in the pathway of lysine oxida-
of these patients have been developmentally normal and tion is free glutaric acid, and not glutaryl-CoA. This seems
because a family is known in which all three biochemically unlikely in view of the relatively low glutaric acid excretion
affected individuals are clinically normal, it is now thought produced even by chain-terminating mutations in C7orf10,
that clinical manifestations in affected individuals are due to and it seems equally possible that the enzyme’s primary
sampling bias and that the condition is benign. function is to esterify glutarate from other sources, such as
A few individuals with 2-aminoadipic acidemia but with- bacteria in the gut.
out abnormal organic acids in urine have been thought to be Diagnosis, which is suspected by finding increased glu-
deficient in a 2-aminoadipic acid transaminase. Enzyme and/ taric acid in blood and/or urine without the abnormal organic
or molecular defects have not been demonstrated, however. acids and carnitine esters found in glutaric aciduria types 1
2-Aminoadipic acid concentrations in serum and urine and 2, can be confirmed by molecular analysis of C7orf10.
have been 34–120 μM (normal <5) and 66–350 mmol/mol 44.14 Hydroxykynureninuria. The oxidation of trypto-
creatinine (normal <17), respectively. The urine concentra- phan in the liver proceeds through kynurenine,
tion of 2-ketoadipic in one patient was 1 mmol/g creatinine. 3-hydroxykynurenine, and 3-hydroxyanthranilic acid to a
Because similar amino and organic acid profiles have been branch point; most of the carbon skeleton is then oxidized to
observed in Reye-like episodes associated with various acetyl-CoA via 2-ketoadipic acid and glutaryl-CoA, but
organic acidemias and other familial disorders, e.g., Elpeleg some is instead diverted to the biosynthesis of nicotinic acid
et al. (1990), diagnostic samples should be taken when the and nicotinamide.
patients are not acutely ill. Conversion of 3-hydroxykynurenine to 3-hydroxyanthranilic
The issue of disease vs non-disease in this instance is com- acid by kynureninase is B6 dependent, and renewed interest in
plicated by the recent observation that patients with severe vitamin deficiency and dependency states in the mid-1960s
and fatal disease due to mutations in NFU1, which encodes a prompted the examination of tryptophan metabolites in
protein involved in the biosynthesis of iron-sulfur clusters, patients with B6-sensitive disorders and/or symptoms reminis-
have large amounts of 2-ketoadipic and 2-ketoglutaric acids cent of pellagra. Hydroxykynureninuria was first described
in urine (Navarro-Sastre et al. 2011). The fact that enzyme (Komrower et al. 1964) in a mildly retarded 4-year-old girl
and/or gene defects have not been demonstrated in earlier with what had been thought to be nicotinic acid deficiency in
patients with 2-ketoadipic aciduria makes one wonder if infancy. Xanthurenic acid, kynurenine, and 3-hydroxykyn-
milder mutations in the same gene can cause biochemical urenine were increased in urine and were increased by oral
abnormalities with a less severe clinical phenotype. loading with l-tryptophan. A few children with similar metab-
44.13 Glutaric Aciduria Type 3. Clinically significant dis- olite excretion patterns have since been described without
orders in the pathway of lysine (and tryptophan) oxidation symptoms or with only mild pellagra-like findings, and it is
include glutaric aciduria types 1 and 2 and pyridoxine- now thought that the enzyme block is clinically relevant only
dependent seizures and are discussed in Chaps. 8 and 11. A if niacin intake is limited.
condition in which glutaric acid is increased in blood and The cumbersome paper and thin-layer chromatography
urine, but without accumulation of other organic acids and methods used to find and investigate the disorder in the past
carnitine esters typical of glutaric aciduria types 1 and 2, has are no longer necessary, and a recent patient was diagnosed
been called glutaric aciduria type 3. by finding a large peak of xanthurenic acid tri-TMS on urine
This condition was first described in a child with organic acid analysis. Subsequent measurements of xanth-
β-thalassemia and attributed to deficiency of peroxisomal urenic acid, kynurenine, and 3-hydroxykynurenine in urine
glutaryl-CoA oxidase (Bennett et al. 1991) but has since and plasma on that child, and on an affected brother, were
been described in several clinically diverse situations and in done by HPLC with UV-spectroscopic detection (Christensen
normal subjects, and the view has developed that whatever et al. 2007).
the primary defect, the condition is benign. Further, while it had not been possible to confirm kyn-
The defect was mapped in three subjects in the ureninase deficiency in this condition by enzyme assay, it
Pennsylvania Old Order Amish, and homozygosity demon- was demonstrated that both brothers were homozygous for
a threonine-to-alanine mutation in KYNU, the gene that most have easily detected peaks of 3-hydroxyisovaleric acid
encodes kynureninase. and 3-methylcrotonylglycine. The latter pattern is easily rec-
44.15 Cystathioninuria. Even when first described in a ognized, be it in an ill child or an asymptomatic female.
64-year-old mentally retarded woman (Harris et al. 1959) While it appears that the defect can sometimes cause clinical
and attributed to probable recessively inherited deficiency of disease, there is considerable disagreement about the man-
γ-cystathionase, the B6-requiring enzyme that cleaves cysta- agement of the asymptomatic mother and/or newborn
thionine to 2-ketobutyrate and cysteine, the suspicion was (Arnold et al. 2008).
that the metabolic abnormality was related only by chance to 44.17 2-Methylbutyrylglycinuria (2-Methylbutyryl-CoA
the clinical presentation. Large reviews suggest that the latter Dehydrogenase Deficiency). 2-Methylbutyryl-CoA is an inter-
is indeed the case; a review of patients ascertained in a bias- mediate in the oxidation of isoleucine. It is produced by oxida-
free manner found very few with symptoms, and even fewer tive decarboxylation of 2-keto-3-methylvaleric acid and is
whose symptoms could not be attributed to another cause normally oxidized to tiglyl-CoA by methylbutyryl-CoA dehy-
(Mudd et al. 2001). drogenase (SBCAD), one of several mitochondrial flavin-con-
Cystathionine is efficiently cleared by the kidney, and taining dehydrogenases that pass electrons into the respiratory
accumulation is much more easily detected in urine than in chain via ETF and ETF-ubiquinone oxidoreductase.
blood. Urine concentrations in enzyme-deficient individuals Deficiency of SBCAD was first observed in a 3-year-old
are up to 2,200 mmol/mol creatinine (normal <0.5). Urine boy with hypotonia and developmental delay and in his
cystathionine is often increased in newborns, probably asymptomatic mother (Andresen et al. 2000). The parents
because liver γ-cystathionase appears late in fetal life, in B6 were first cousins, and both mother and child were homozy-
deficiency, and in patients with functional neural tumors and gous for the same missense mutation in the SBCAD gene.
hepatoblastomas, presumably because of an imbalance Subjects that were subsequently described have had a variety
between production by cystathionine β-synthase and hydro- of clinical presentations, and many in fact have been com-
lysis and remethylation defects (Chap. 13). pletely normal. The enzyme deficiency is quite common in
Although rarely necessary, an inherited enzyme defi- the Hmong population in the midwest USA. A recent review
ciency may be confirmed by molecular analysis of the CTH of twelve non-Hmong enzyme-deficient subjects found that
gene (Wang and Hegele 2003). all eleven that had been detected by newborn screening were
44.16 3-Methylcrotonylglycinuria. 3-Methylcrotonyl- well; the oldest of them was 4 years of age (Alfardan et al.
CoA is formed by oxidation of isovaleryl-CoA, an interme- 2010). These findings suggest that the association between
diate in leucine metabolism, and is normally converted to the biochemical phenotype and clinical disease is
3-methylglutaconyl-CoA by a biotin requiring holocarboxyl- fortuitous.
ase. Deficiency of 3-methylcrotonyl-CoA carboxylase The diagnosis in newborns is usually suspected when an
(3MCC) can be isolated or, in biotin deficiency, biotinidase elevated C5 acylcarnitine is found on screening by tandem
deficiency and holocarboxylase synthase deficiency, com- mass spectrometry and is confirmed on urine organic acid
bined with deficiency of carboxylases specific for acetyl- analysis by finding a (usually) small peak of
CoA, propionyl-CoA, and pyruvate (see Chap. 19). 3MCC is 2-methylbutyrylglycine (MBG), often with small amounts of
probably an α6β6 dodecamer (Huang et al. 2010), and 3MCC isobutyrylglycine and ethylhydracrylic acid, and without
deficiency can be due to mutations in either subunit. isovalerylglycine. It is important to note that the amount of
Most early descriptions of isolated enzyme deficiency MBG in urine in this condition is often 10–50-fold less than
were in infants and children with acute disease in infancy the amount of isovalerylglycine excreted in isovaleric acide-
and in children with Reye-like symptoms, and there was lit- mia. Inherited defects of ETHE1 (ethylmalonic acid enceph-
tle doubt about the relevance of the biochemical defect to alopathy) may be accompanied by modestly elevated
clinical disease until newborn screening by tandem mass branched-chain acylglycines and should therefore be ruled
spectrometry detected increased hydroxy-C5 carnitine in out. While available, enzyme and molecular analysis is sel-
several infants whose mothers had asymptomatic enzyme dom indicated.
deficiency. It is not yet clear how many individuals with 44.18 Oxoprolinase Deficiency. Excretion of large
isolated carboxylase deficiency develop symptoms, but none amounts of pyroglutamic acid (5-oxoproline) can be due to
of the affected subjects picked up by newborn screening deficiency of glutathione synthetase, when it is invariably
developed any symptoms (Arnold et al. 2008). Kindreds associated with clinical disease (Chap. 42). Rarely, however,
with multiple “healthy” affected sibs had been reported it is due to inherited deficiency of oxoprolinase, a condition
before (Mourmans et al. 1995). with a much more benign clinical course. Oxoprolinase defi-
Though an occasional patient with 3MCC deficiency has ciency was first described in two siblings whose symptoms
isolated 3-hydroxyisovaleric aciduria (Wolfe et al. 2007), seemed unrelated to their enzyme block (Larsson et al. 1981)
and has since been reported in six additional patients, many adenosyltransferase. Over 60 patients with inherited defi-
of whom have been entirely normal. ciency of this enzyme are known, many of them detected by
As in glutathione synthetase deficiency, a large peak of high serum methionine on newborn screening. Most of these
pyroglutamic acid (>2 mol/mol creatinine; normal <0.05) is patients have remained free of symptoms, and the condition
seen on organic acid analysis. If not due to deficiency of glu- has long been thought to be benign (Gaull et al. 1981). The
tathione synthetase, the diagnosis of oxoprolinase deficiency latter may not be true in all instances, as a few patients with
can be made by demonstrating enzyme deficiency in white more severe enzyme deficiencies have developed demyelin-
blood cells or cultured fibroblasts or by molecular analysis of ating disorders.
the OPLAH gene (Almaghlouth et al. 2012). Plasma methionine in methionine adenosyltransferase
44.19 Glycerol Kinase Deficiency. Glycerol kinase phos- deficiency can be as high as 2,500 μM (normal <50), and
phorylates glycerol to glycerol-3-phosphate. X-linked defi- when detected, other causes of hypermethioninemia such as
ciency of this enzyme (GKD) was first described in two male metabolic liver disease, tyrosinemia type I, cystathionine
siblings with increased glycerol in blood and urine, psycho- β-synthase deficiency, glycine N-methyltransferase defi-
motor retardation, osteoporosis, myopathy, and adrenal hypo- ciency, and citrin deficiency must be excluded (Mudd 2011).
plasia (Guggenheim et al. 1980). Subsequent evaluation of Although the diagnosis is usually arrived at by exclusion,
many additional patients has shown that glycerol kinase defi- diagnosis can be confirmed by enzyme assay on liver or by
ciency by itself is important only in its ability to give a falsely molecular analysis of MAT1A, which encodes the catalytic
elevated estimation of serum triglyceride if the latter is mea- subunit of the two isoenzymes of methionine adenosyltrans-
sured by glycerol release after lipolysis, but not before. ferase in mammalian liver. Most of the mutations identified
Clinical phenotypes associated with GKD deficiency are to date are transmitted as autosomal recessives but one,
most often due to the fact that the GKD gene is closely linked R264H, causes reduced activity even when heterozygous and
to genes encoding ornithine transcarbamylase, congenital can cause the occasional situation in which hypermethionin-
adrenal hypoplasia, and Duchenne muscular dystrophy, and emia is transmitted as an autosomal dominant (Blom et al.
disease is often caused by deletions of several contiguous 1992).
genes (McCabe 2001). Isolated GKD may be associated with 44.21 Methylmalonyl-CoA Epimerase Deficiency.
mild fasting intolerance and hyperketonemia in childhood, Inherited methylmalonic acidemia is usually due to decreased
but causes no symptoms at all in adults (Sjarif et al. 2000). conversion of l-methylmalonyl-CoA to succinyl-CoA by
GKD deficiency is usually diagnosed when organic acid methylmalonyl-CoA mutase; in some cases, this is due to
analysis detects a large peak of glycerol in a patient being mutations of the MUT gene, which encodes the mutase, and
investigated for signs and symptoms due to deficiency of one in others it is due to defects in the biosynthesis of adenosyl-
of the adjacent genes, or during evaluation for pseudohyper- cobalamin, the mutase cofactor (Chap. 13). Very rarely, how-
triglycerolemia. Subsequent investigations will vary depend- ever, methylmalonic acidemia is due to deficiency of the
ing on how and why it was detected. The enzyme defect is epimerase (or racemase) responsible for the interconversion
easily demonstrated in many tissues, including leukocytes of d- and l-methylmalonyl-CoA.
and cultured fibroblasts, and molecular analysis of the GKD Three individuals in two families have been reported with
gene is also available. mild methylmalonic acidemia (uria) due to homozygosity
Plasma glycerol in GKD deficiency is 1.8–8.3 mmol/L for a nonsense mutation (R47X) in the MCEE gene (Bikker
(normal <0.27), and in urine is 90–193 mmol/mmol creati- et al. 2006; Dobson et al. 2006). The lone patient in one fam-
nine (normal = not detected); the latter is sufficiently high to ily was a girl who at age 2 years had retarded motor develop-
be seen on organic acid analysis despite its being poorly ment and signs of spasticity, possibly because she also had
extracted into organic solvents. Increased urine glycerol may sepiapterin reductase deficiency (Bikker et al. 2006). There
not be detected in the acidic fraction prepared by ion- were two siblings in the other family; a girl who presented at
exchange chromatography, but will be seen instead in the 1 year of age with a single episode of severe ketoacidosis and
neutral fraction. Further, glyceroluria due to GKD deficiency who then remained symptom-free on daily hydroxycobala-
must be distinguished from that due to glycerol in a variety min (1 mg po), and an older sister with congenital hydro-
of oral, rectal, and intravenously administered medications, cephalus, normal development, and no symptoms of
as well as direct glycerol contamination of blood and urine metabolic disease.
specimens. Urine methylmalonic acid in these patients has been as
44.20 Methionine Adenosyltransferase Deficiency. The high as 1,500 mmol/mol creatinine (normal <10), but
initial reaction in the transsulfuration pathway through which decreased considerably with age to approx. 50 mmol/mol
methionine is converted to homocysteine and cysteine is the creatinine. Suppression of gene expression in HeLa cells led
formation of S-adenosylmethionine (AdoMet) by methionine only to a small reduction in pathway activity, suggesting that
nonenzymatic conversion of d- to l- isomer contributes to 44.5 Concluding Remarks

flux through the pathway and perhaps explains the relatively
mild organic aciduria and symptoms even with a truncating The list of inborn errors of questionable clinical significance
mutation. provided is almost certainly incomplete, as future clinical
It now appears that epimerase deficiency is a relatively studies of patients detected by selective or newborn screen-
rare cause of methylmalonic aciduria and, since the diagnosis ing will show that some disorders do in fact cause disease in
might have prognostic significance, molecular analysis of the some patients, and that others do not. Further, newer diag-
MMCE gene may well become more widely used when eval- nostic methods like whole genome analysis or exome
uating patients detected in newborn screening programs. sequencing will add novel disorders to the mix, and these too
44.22 SCAD Deficiency. Short-chain acyl-CoA dehydro- will have to undergo intensive analysis to decide if they are
genase (SCAD) is one of the four chain length-specific acyl- indeed clinically relevant.
CoA dehydrogenases involved in mitochondrial β-oxidation
of fatty acids. Hexanoyl- and butyryl-CoA are its preferred
substrates, and like other members of the enzyme family,
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Part VII
General Subjects and Profiles
Emergency Diagnostic Procedures
and Emergency Treatment 45
Stephanie Grünewald, James Davison, Diego Martinelli,
Marinus Duran, and Carlo Dionisi-Vici

45.1 Introduction .................................................................... 709
45.1.1 The Acutely Unwell Neonate ........................................... 709 Metabolic emergencies need to be recognized early, and the
45.1.2 Late-Onset Patients .......................................................... 710 initiation of appropriate treatment without any delay deter-
45.2 Emergency Diagnostic Procedures ............................... 712 mines the overall outcome. A significant proportion of patients
45.2.1 Emergency Scenarios ....................................................... 712 with inborn errors of metabolism are at risk of developing a
45.3 Emergency Treatment.................................................... 714 metabolic emergency at some time in their life, particularly
45.3.1 Supportive Care ................................................................ 714 those children affected by an inborn error of metabolism that
45.3.2 Hyperammonemia ............................................................ 714 manifests as an acute intoxication. These patients usually
45.4 Practical Details.............................................................. 715 present as a neonate – typically after an initial symptom-free
45.4.1 Monitoring ....................................................................... 715 interval of some days – in an emergency situation. Treatment
45.4.2 Acidosis............................................................................ 716 needs to be initiated immediately even without having arrived
45.4.3 Hypoglycemia .................................................................. 716
45.4.4 Seizures ............................................................................ 716
at the exact diagnosis.
45.4.5 Postmortem Investigations ............................................... 717 Although there are numerous metabolic conditions that
can present with emergency situations, there is not much
References ..................................................................................... 717
variability of their manifestations, and there is a limited
number of therapeutic measures that are needed immediately
(Saudubray 2012; Dionisi-Vici and Ogier de Baulny 2012).
Furthermore, patients with an established diagnosis and on
an appropriate treatment may suffer from acute decompensa-
tions. Triggers for a metabolic crisis include both endoge-
nous and exogenous processes. Endogenous (“internal”)
processes, such as vomiting, fasting, or strenuous exercise,
all drive catabolism. Exogenous factors include exposure to
an inappropriate diet, such as high protein intake in amino-
acidopathies (AAs), organic acidurias (OAs) or urea cycle
S. Grünewald (*) • J. Davison disorders (UCDs); exposure to toxic substances such as dairy
Metabolic Unit, Great Ormond Street Hospital for Children, products in galactosemia or long-chain fat in fatty oxidation
NHS Foundation Trust and Institute for Child Health,
disorders (FAOs); or exposure to drugs such as valproate in
Great Ormond Street, London WC1N 3JH, UK
e-mail: stephanie.grunewald@gosh.nhs.uk mitochondrial disorders or cytochrome P450 inducers in
porphyrias.
D. Martinelli • C. Dionisi-Vici
Division of Metabolism, Department of Pediatric Medicine,
Bambino Gesù Children’s Research Hospital,
IRCCS Piazza Sant’Onofrio 4, 45.1.1 The Acutely Unwell Neonate
Rome 00165, Italy
e-mail: carlo.dionisivici@opbg.net
As part of the initial assessment of a neonate in an emer-
M. Duran
gency situation, information needs to be collected about:
F0-224 Meibergdreef 9, Amsterdam 1105AZ, The Netherlands • Pregnancy, delivery and family history
e-mail: m.duran@amc.uva.nl • Symptom-free period
710 S. Grünewald et al.
• First concerns Intractable convulsions are associated with amino acid dis-
• Progress of symptoms orders (glycine), vitamin B6 deficiency, or purine disorders
Typically, neonates will present with refusal of feeds, [adenylosuccinate lyase (ADSL) deficiency, molybdenum
weight loss and lethargy progressing to coma. cofactor deficiency].
An initial examination and baseline clinical chemistry 2. Liver failure: Hepatic manifestation of galactosemia,
will identify the key clinical and biochemical features: irrita- hereditary fructose intolerance, and tyrosinemia type I,
bility, seizures, lethargy, or coma as signs of encephalopathy; usually not manifest in the first few days of life, and are
hepatopathy with coagulopathy and increased transaminases/ amenable to emergency treatment.
hepatomegaly; cardiac failure; or others which help to nar- 3. Cardiac failure: In neonates, for fatty acid oxidation defects
row down the differential diagnosis and give guidance to the and Pompe disease there are specific treatments available.
most relevant diagnostic tests.
In neonates and young infants, the main clinical presenta-
tions are the following: 45.1.2 Late-Onset Patients
1. Neurological deterioration: Toxic encephalopathy is the
commonest presentation and most often associated with Older children, adolescents, or even adults may encounter any
urea cycle defects (UCDs); “classical” branched-chain of the above-mentioned clinical threats. Any type of coma or
organic acidemias (BCOAs) such as propionic, methylma- acute psychiatric symptom can be the presenting sign of a met-
lonic, and isovaleric acidemia; and maple syrup urine dis- abolic disorder. In addition, patients may suffer from recurrent
ease (MSUD). Toxicity in UCDs is caused by attacks of unexplained dehydration, abdominal pain, muscle
hyperammonemia and hyperglutaminemia, in BCOAs by pain and rhabdomyolysis or peripheral neuropathy.
metabolic acidosis with/without hyperammonemia, and in A summary of the most characteristic acute signs and dif-
MSUD by accumulating leucine and isocaproic acid. ferential diagnoses is listed in Table 45.1.
Table 45.1 Characteristic acute signs, symptoms and metabolic differential diagnoses
Sign Disease
Acute cardiorespiratory signs
Cardiac failure FAO, OXPHOS, Fabry, GSD III, GSD-II, PA, MMA, MPS-LYSO
Arrhythmia FAO, Kearns-Sayre, Danon, cardiac glycogenosis, triose phosphate isomerase, D-2-hydroxyglutaric aciduria,
thiamine deficiency/dependency states
Pulmonary hypertension MHTFR, OXPHOS, NFU1, LYSO, Cbl-C
Stridor Biotinidase, PDH, OXPHOS, Gaucher II, Krabbe
Acute neurological signs
Coma UCDs, BCOAs, MSUD, multiple carboxylase, GA-1, MADD, OXPHOS, FAO, ketolysis, hyperinsulinism,
gluconeogenesis
Seizures UCDs, BCOAs, hyperinsulinism, NKH, SO-XO def, PEROX, pyridoxine-dependent epilepsy, folinic acid-
responsive seizures, AGAT, biotinidase, cerebral folate receptor-alpha, creatine transporter, GAMT,
holocarboxylase synthase, Menkes disease/occipital horn syndrome, PGDH, phosphoserine phosphatase,
phosphoserine aminotransferase, PNPO def, OXPHOS, LYSO (Gaucher III, NP disease type C), NCL, GABA
transaminase, PDH, ADSL, GS, GLUT1
Oculogyric crisis Neurotransmitter disorders, PKU
Dystonia BCOAs and vitamin B12 metabolism disorders, GA-1, MEGDEL, Wilson disease, aceruloplasminemia,
biotin-responsive basal ganglia disease, neurotransmitter disorders, GAMT, homocystinuria, MHBD, HMG-
CoA lyase, HMG-CoA synthase, LSD, NKH, SCOT, beta-ketothiolase, Lesch-Nyhan
Stroke-like episode OXPHOS (MELAS), OTC, CDGs, homocystinuria, OAs, MSUD, Fabry, beta-ketothiolase
Ataxia MSUD, PDH, multiple carboxylase, UCDs, BCOAs and inborn errors of cobalamin metabolism, GLUT1,
Hartnup, CoQ10, CTX, LYSO (NP disease type C), NKH, SSADH, thiamine-responsive encephalopathy
Dementia Aceruloplasminemia, Wilson disease, alpha-mannosidosis, X-ALD, CTX, Gaucher disease type III, MPS I,
MPS II, MPS III, MPS IV; NP disease type C, MLD, MHBD, MELAS
Psychiatric signs UCDs, citrin, porphyria, MPS VII, MTHFR, Hartnup, creatine transporter, GAMT, homocystinuria, vitamin B12
metabolism errors, MSUD (variant), MELAS, MLD, BCOAs, NP disease type C, SSADH, Wilson disease
Sudden visual loss MELAS, LEBER, MMA, PA, homocystinurias, X-ALD, porphyria, biotinidase deficiency
Sudden hearing loss MELAS
Pain Fabry, Gaucher, Krabbe, prolidase def.
Acute peripheral neuropathy PDH, biotinidase, porphyria, MSUD (variant), MLD, tyrosinemia type I, LCHAD/MTP
45 Emergency Diagnostic Procedures and Emergency Treatment 711

Sign Disease
Basal ganglia lesions OXPHOS, PA, MMA, MSUD, beta-ketothiolase, GA I, MADD, HMG-CoA lyase, HMG-CoA synthase,
MHBD SCOT, biotin-responsive T1 transporter, Wilson disease, aceruloplasminemia, GAMT
Hydrocephalus MPS I, MPS II, MPS VI, Cbl-C, Cbl-D, MTHFR, Gaucher, NKH, mannosidosis
Myoglobinuria FAO, muscle GSD, OXPHOS, LIPIN1, porphyria, Wilson disease
Cramps FAO, muscle GSD
Acute hematological and vascular signs
Anemia hemolytic GSH synthase, gamma glutamylcysteine synthase, Wilson disease
Anemia nonhemolytic Orotic aciduria, inborn errors of cobalamin metabolism, methionine synthase, inborn errors of folate
metabolism, MK, Pearson syndrome, OXPHOS, thiamine-responsive megaloblastic anemia
Anemia non-macrocytic, Abetalipoproteinemia, adenylate kinase, adenosine triphosphatase carnitine uptake, porphyria, di-metal
hemolytic, or due to transporter 1, porphyria, galactosemia, glycolytic and pentose-phosphate enzyme, hemochromatosis, IRIDA,
combined mechanisms LCAT, MK, mitochondrial tyrosyl-tRNA synthetase, MLASA syndrome, pyroglutamic aciduria, pyrimidine
5-nucleotidase, red blood cell glycolysis defects, TALDO, Wilson disease, Wolman disease, X-linked
sideroblastic anemia, pyridoxine-responsive epilepsy
Neutropenia GSD1b, Barth syndrome, BCOAs
Pancytopenia Pearson syndrome, MMA, PA, Gaucher types I and III, NP types A and B, OXPHOS, adenylate kinase 2,
TALDO
Macrophage activation LPI, Cbl-C, Wolman disease, Gaucher disease, NP disease
syndrome, HLH
Coagulopathy CDGs, HHH
Subdural hematoma HHH, GA-I, Menkes disease
Thromboembolism CDGs, homocystinurias
Acute liver and digestive signs
Fetal hydrops LYSO (GM1 gangliosidosis, sialidosis type II, I-cell disease, Niemann-Pick type A/C, MPS VII,
galactosialidosis), TALDO, CDG, mevalonic aciduria
Acute liver failure Tyrosinemia type I, UCDs, citrin, OXPHOS, CDGs, HFI, FAO, galactosemia, TALDO, Wilson disease,
Wolman disease
Cholestatic jaundice Arginase, bile acid synthesis, CDG, CTX, cholesterol synthesis defects, citrin, galactosemia, LCHAD/MTP,
MK, NP-C, PEROX, TALDO, tyrosinemia type I
Pancreatitis FAO, OAs, GSD1, CBS, OXPHOS (MELAS), LPL
Intestinal pseudo-obstruction MNGIE, MK def
Recurrent vomiting UCDs, OAs, HFI
Acute endocrinological signs
Adrenal insufficiency SLO, ALD,OXPHOS
Hyperinsulinemic SCHAD, CDGs
hypoglycemia
Diabetes Abnormal proinsulin cleavage, diabetes, deafness and TRMA Kir 6.2, glucokinase, MMA, PA, IVA, ketolytic,
OXPHOS, Wolfram syndrome
Acute nephrological signs
Hemolytic uremic syndrome Cbl-C, MHTFR
Fanconi syndrome and renal Tyrosinemia type I, galactosemia, OXPHOS, cystinosis, HFI, Fanconi-Bickel, Wilson disease, LPI, CPT1
tubular acidosis
Acute renal failure Oxalosis, FAO
Nephrotic syndrome CoQ10, CDGs
Acute dermatological signs
Skin rash Biotinidase, MK, holocarboxylase synthetase, Hartnup, porphyria, steroid sulfatase
Orthostatic cyanosis, ETHE
petechiae
Abnormal urine and sweat odor
Sweaty feet odor IVA, 3-methylcrotonylglycinuria, MADD
Maple syrup odor MSUD
Musty odor PKU
Fish odor Trimethylaminuria, dimethylglycine dehydrogenase
Other
Febrile episodes MK
Maternal disease of LCHAD/MTP
pregnancy/HELLP
45.2 Emergency Diagnostic Procedures • Free fatty acids, 3-hydroxybutyrate, and insulin in the
hypoglycemic child
The initial routine laboratory investigations should give suf- • Purine analysis (suspected disorder of purine metabolism)
ficient information to allow immediate therapeutic decision
making. Most of the treatable disorders involve the pathways
of intermediary metabolism. Besides general clinical chem- 45.2.1 Emergency Scenarios
istry (complete blood count, C-reactive protein, electrolytes,
liver and kidney function tests), the baseline metabolic The most frequently observed metabolic emergencies pres-
screen (first-line investigations) should include the following ent biochemically as either hyperammonemia, metabolic
tests: acidosis, or hypoglycemia.
• Blood gas + anion gap [Na – (Cl + bicarbonate)]
• Lactate 45.2.1.1 Hyperammonemia
• Glucose Hyperammonemia should be considered in any unexplained
• Ammonia encephalopathy.
• Uric acid In defects affecting protein catabolism (urea cycle defects
• CK and organic acidurias), the overwhelming illness is mainly
• Urinary ketones due to hyperammonemia. Early initiation of appropriate
• Urinary reducing substances treatment is of utmost importance to provide the best possi-
These basic laboratory investigations should be available ble outcome. Table 45.2 lists the inborn errors of metabolism
in any hospital offering emergency treatment at any time. leading to hyperammonemia, guiding bedside differential
Appropriate blood and urine samples should be collected diagnosis.
for second-line investigations including:
• Blood/plasma acylcarnitines 45.2.1.2 Metabolic Acidosis
• Plasma amino acids Metabolic acidosis, defined as blood pH <7.2 and/or anion gap
• Urinary organic acids >16, is due to accumulation of acids such as keto acids, lactic
• Urinary orotic acid acid, or organic acids. Particularly in organic acidemias,
Results of the second-tier investigations should be increased keto acids (3-hydroxybutyrate and acetoacetate)
available in 24–48 h. will contribute to a high anion gap. Lactic acidemia is particu-
Depending on the differential diagnosis, the following larly seen in gluconeogenesis disorders or disorders of the oxi-
additional tests can be helpful: dative phosphorylation (OXPHOS) system. Table 45.3
• Galactose-1-phosphate (gal-1-p) and galactose-1-phosphate provides a list of inborn errors of metabolism leading to meta-
uridyltransferase (GALT) (suspected galactosemia) bolic acidosis.
Table 45.2 Bedside differential diagnosis of hyperammonemia

Disease
Urea cycle Organic acidurias Fatty acid oxidation Hyperammonemia Pyruvate carboxylase
Feature defects defects hyperinsulinism deficiency
Hyperammonemia ↑↑ ↑↑ ↑ ↑ ↑
Acidosis n-↑ ↑↑ n-↑ – ↑↑
Ketonuria – ↑↑ -(↑) – ↑
Glycemia n n-↑-↓ ↓ ↓↓ n-↓
Lactate – n-↑ n-↑ – ↑↑
ASAT-ALAT n-↑ – ↑↑ – n-↑
CK – – n-↑↑ – –
Uric acid – n-↑ n-↑ – n-↑
WBC/RBC/Plt – ↓ – – –
Weight lossa – ↑ – – n-↑
a
Only in neonates
↑ elevated, ↓ decreased, n-↑ normal to elevated, n-↓ normal to decreased, ↑↑ very elevated, (↓) inappropriately low, – no
45
Table 45.3 Metabolic acidosis

Fructose 1,6 Multiple
Organic GSDs (Ia–Ib) Ketolysis OXPHOS Ketotic bisphosphatase carboxylase
Disease acidurias MSUD GSD III defects defects PC def. PDH def. FAO defects hypoglycemia deficiency deficiencies
Lactate n-↑ n-↑ n-↑↑ n-↑ ↑↑ ↑↑ ↑↑ n-↑ n-↑ ↑
Ketone bodies ↑↑ ↑↑ n-↑ ↑↑ n-↑ ↑ (↓) ↑↑ ↑ ↑
Anion gap ↑↑ ↑ ↑ ↑ ↑↑ ↑↑ ↑↑ n-↑ ↑ ↑ ↑
Glucose n-↓-↑ n-↓ ↓↓ n-↓ n-↓ n-↓ n-↓ ↓ ↓ ↓ n-↓
Ammonia ↑↑ n-↑ n n-↑ n-↑ n-↑ n n-↑ n n-↑
Emergency Diagnostic Procedures and Emergency Treatment
ASAT-ALAT ↑ n-↑ n-↑ ↑↑ ↑↑

CK ↑ GSD III n-↑ ↑↑
WBC ↓ ↓ GSD Ib n-↓
Uric acid ↑ ↑ ↑ n-↑ n-↑ n-↑ n-↑ ↑ n-↑ n-↑ n-↑
Abnormal + + +/− + + + + + − +/− +
organic acids
Abnormal amino +/− + − − + + + − +/− − −
acids
Abnormal + − − +/− − − − + +/− − +
acylcarnitines
↑ elevated, ↓ decreased, n-↑ normal to elevated, n-↓ normal to decreased, ↑↑ very elevated, (↓) inappropriately low, + yes, – no
713
Table 45.4 Hypoglycemia

F 1,6 Adrenal FAO HMG
Disease GSD 1 GSD 3 HFI DP GALT HI-HA HI insuff. defects lyase KH OHPHOS HTy1 MSUD
Lactate ↑ n-↑ ↑ n-↑ n-↑ ↑↑ n-↑
ASAT-ALAT ↑ ↑ ↑ n-↑ ↑ n-↑ ↑ n-↑ n-↑ ↑
CK ↑ n-↑ n-↑
Uric acid ↑ n-↑ n-↑ ↑ n-↑ n-↑ n-↑ (↓) n-↑
FFA (↓) (↓) (↓) ↑ ↑ ↑ ↑
Triglycerides ↑
Ketone bodies ↑ n-↑ ↑ (↓) (↓) (↓) (↓) (↓) ↑ ↑ ↑
Ammonia ↑ n-↑ n-↑ n-↑ − n-↑
Anion gap ↑ n-↑ ↑ n-↑ n-↑ n-↑ ↑ n-↑ ↑ ↑ n-↑
Coagulation n-↓ n-↓ ↓ n-↓ n-↓ ↓
WBC ↓ (1b) n-↓
Abnormal ± ± + + + + + +
organic acids
Abnormal ± ± ± ± + +
amino acids
Abnormal n/+ + + (+)
acylcarnitines
Fasting + + + + + + + +
hypoglycemia
Post-meal + + + protein
hypoglycemia fructose galactose
↑ elevated, ↓ decreased, n-↑ normal to elevated, n-↓ normal to decreased, (↓) inappropriately low, + yes, – no
45.2.1.3 Hypoglycemia Many acutely sick patients, especially newborn infants,

Disorders of reduced fasting tolerance include disorders of will require circulatory and ventilatory support. Most of
glucose homeostasis (glycogen storage disorders, disorders them will need rehydration and correction of mineral and/or
of gluconeogenesis, galactosemia, and congenital hyper- electrolyte imbalances (Ca, P, Mg). Given their importance,
insulinism), disorders of inappropriate ketone synthesis such treatment should be started immediately and should not
(fatty acid) β-oxidation defects (FAOD), or inappropriate delay the initiation of more specific therapeutic measures.
ketone utilization (ketolysis defects). In disorders of FAOD Patients with a metabolic crisis might suffer from septice-
and hyperinsulinism, hypoglycemia will be combined mia, which can result in persistent catabolism and lead to
with hypoketosis. Affected patients become symptomatic therapeutic failure. Consequently, infections must be
with hypoglycemia, particularly after prolonged fasting. prevented, thoroughly investigated for and, if present, treated
Hypoglycemia can also be observed in disorders of amino aggressively.
acid metabolism (MSUD, tyrosinemia type 1). Glucose In disorders of the intoxication type, such as OAs,
administration needs to meet at least the rate of hepatic glu- AAs, UCDs, galactosemia, or fructose intolerance, the
cose production. oral intake of toxic precursors must be stopped
Table 45.4 provides a list of inborn errors of metabolism immediately.
leading to hypoglycemia.
45.3.2 Hyperammonemia
45.3 Emergency Treatment
While starting treatment of any child with (un)explained
45.3.1 Supportive Care hyperammonemia, the following blood and urine tests should
be done:
There is an extensive group of metabolic disorders with • Liver function tests
acute life-threatening presentation. Long-term outcome is • Plasma amino acids
often poorer in patients with early onset of symptoms and/or • Urinary organic acids (and orotate)
where initiation of treatment is delayed. • Blood/plasma acylcarnitine profile
Hyperammonemia is an acute toxic event. It is particu- these diseases hyperammonemia is usually associated with
larly harmful to the brain as it leads to astrocyte swelling and decreased levels of glutamine, with a risk of further depletion
brain edema, resulting in irreversible brain damage. The of the glutamine/glutamate pool (Filipowicz et al. 2006). In
prognosis of any hyperammonemic patient is strongly influ- the recently published guidelines of urea cycle defects
enced by the extent and duration of hyperammonemia, and (Häberle et al. 2012), the intravenous preparation containing
therefore treatment should not be delayed. The protein intake sodium benzoate/sodium phenylacetate has been recom-
needs to be stopped immediately and intravenous glucose mended in severe acute decompensation in undiagnosed
needs to be commenced to stop catabolism. patients with hyperammonemia as an “ultima ratio” drug.
To promote anabolism give: Arginine: It is normally synthesized in the urea cycle but in
urea cycle disorders may become a semi-essential amino acid,
Glucose to provide especially in ornithine transcarbamylase (OTC) and carbamyl
Age Weight (mg/kg/min) Glucose 10 % (ml/kg/day)
phosphate synthetase (CPS) deficiency. In citrullinemia and
0–2 10 150
argininosuccinic aciduria, the arginine additionally replaces
2–6 8 120
>6 <30 kg 6 90
ornithine that is not reformed in the cycle and thereby facili-
>6 30–50 4.5 67 tates the removal of ammonia in the form of citrulline or ASA.
>6 >50 3 45 In emergencies the dose is usually 250 mg/kg, up to 500 mg/
kg/day. Arginine is to be avoided in arginase deficiency.
If hyperglycemia occurs, consider insulin; do NOT reduce N-Carbamylglutamate

the glucose infusion. This is an alternative activator of carbamyl phosphate syn-
Hyperammonemia in a newborn patient should trigger thetase. N-Carbamylglutamate can be used to treat
investigations to exclude or confirm differential diagnosis N-acetylglutamate synthase deficiency or CPS deficiency. It
such as urea cycle defects, organic acidemias, and fatty acid has been shown to be helpful as an adjunctive medication in
oxidation disorders, as well as the more common neonatal the treatment of hyperammonemic crises secondary to
septicemias, liver failure, and congenital infections. In late- organic acidemias (Daniotti et al. 2001).
onset cases, hyperammonemia and metabolic decompensa- Extracorporeal detoxification may be required if ammo-
tion can be triggered by infection, catabolism, excessive nia levels exceed 400 umol/l and/or if ammonia levels do not
protein intake, medication, and other stressing events, both respond to conservative treatment. Of the available tech-
physiological and psychological (Broomfield and Grunewald niques, continuous venovenous hemodiafiltration (HF,
2012). Therapy must not be delayed and should proceed CVVHD) and hemodialysis (HD) are far more efficient than
simultaneously with the diagnostic workup. It is useful to peritoneal dialysis (PD) (Picca et al. 2001). Since the modal-
start with a combination of different drugs: ity of dialysis is dependent on the local facilities, a rapid
Benzoate: It is conjugated with glycine to form hippurate, referral of patients to centers with expert metabolic clini-
which is then rapidly excreted into the urine. Approximately cians and full dialysis facilities is the main determinant for
1 mol of nitrogen (=ammonia) is then lost for each mol of the success of treatment.
benzoate administered. The standard dose is 250 mg/kg/day
in 4–6 divided doses. This may be increased to 500 mg/kg/
day in an emergency. 45.4 Practical Details
Phenylbutyrate: It is first oxidized to phenylacetate and
then conjugated with glutamine to form phenylacetylgluta- Arginine, sodium benzoate, and sodium phenylbutyrate should
mine. This is excreted into the urine, and theoretically 2 mol be made up separately in 10 % glucose in a 50 ml syringe or
of nitrogen is removed for each mol of phenylbutyrate given. 500 ml bag (maximum concentration 2.5 g in 50 ml or 25 g in
The standard dose is 250 mg/kg/day in 4–6 divided doses. 500 ml bag) and given by a syringe pump or by an infusion
This may be increased to 500 mg/kg/day in an emergency. pump piggy-backed (Y- connector) into the main 10 % glu-
The use of these ammonia scavengers represents the main- cose infusion as close to the entry site as possible.
stay of therapy for detoxification of ammonia in UCDs. Their
use is still debated in BCOAs because of the potential risk of
increasing intramitochondrial accumulation of CoA esters 45.4.1 Monitoring
and of further depleting free CoA pools (Griffith et al. 1989).
However, sodium benzoate has been reported to be safe Monitor ammonia levels every 3 h. Add additives to i.v. fluids
(Walter et al. 1995), and most metabolic centers regularly use as needed and monitor electrolytes, taking into account
this drug in organic acidurias. The use of sodium phenylbu- sodium already given by sodium benzoate (7 mmol sodium
tyrate in organic acidurias raises further concern because in per 1 g) or sodium phenylbutyrate (5.4 mmol sodium per 1 g).
The aim is to restart protein by no later than 24–48 h and methylmalonyl-CoA mutase (and of methionine synthase),
monitoring of ammonia should be performed every 12 h. should be tried in all suspected cases; the recommended dose
Reintroducing feeds might necessitate a nasogastric tube. is 1 mg/day given parenterally (Deodato et al. 2006).
Cyanocobalamin is less efficient but may be used temporar-
ily until hydroxocobalamin is available. Biotin is the treat-
45.4.2 Acidosis ment of choice in both holocarboxylase synthetase and
biotinidase deficiency, while there are doubts whether biotin-
Start this treatment if the patient is obviously unwell, vomit- responsive forms of PA really exist; the recommended dose
ing, and drowsy and has a base deficit >8 mmol/l. Do not is 10 mg/day given either orally or i.v. (Grünewald et al.
delay if you are uncertain. 2004). Some patients with lactic acidosis due to primary or
If the child is dehydrated, give normal (0.9 %) saline secondary thiamine deficiency may benefit from thiamine
20 ml/kg immediately and repeat the saline bolus if poor cir- administration (Mayatepek and Schulze 1999; Pérez-Dueñas
culation persists as for a shocked non-metabolic patient. et al. 2013). Riboflavin is the vitamin of choice for treating
Give glucose 200 mg/kg at once (2 ml/kg of 10 % glucose new patients with MADD, as well as patients with the ribo-
or 1 ml/kg of 20 % glucose) over a few minutes. flavin transporter defect, who may have acute paralysis of
Continue with glucose 10 %/saline 0.45 % at 5 ml/kg/h, the diaphragm necessitating ventilation (Bosch et al. 2012).
but if this is not immediately available, continue with normal
saline until it is ready.
Exception: If there is a strong suspicion of an underlying 45.4.3 Hypoglycemia
mitochondriopathy resulting in disturbed energy supply, cor-
recting the metabolic acidosis is the major goal; glucose sup- Definition: Blood glucose <2.6 mmol/l (full-term neonates)
ply needs to be reduced to maintenance 5 % dextrose to • Obtain diagnostic blood/urine samples, when the child is
restrict the lactic acidosis. hypoglycemic, for:
If acidosis is still not corrected, start sodium bicarbonate. Lactate
Give sodium bicarbonate 8.4 % as half correction Free fatty acids
[0.15 × weight × base deficit (mmol/l)] over 20–30 min Insulin
diluted with i.v. fluids. One milliliter of sodium bicarbonate Cortisol
8.4 % contains 1 mmol of sodium bicarbonate and must be Blood spot acylcarnitine profile
diluted with at least 5 ml of 5 % glucose. Plasma amino acids
Repeat blood gases 30 min after the first dose. Consider Uric acid
continuous bicarbonate infusion, if the bicarbonate require- Urine ketones
ment remains high. Urine organic acids
To promote anabolism see above. • Give 2 ml/kg 10 % dextrose bolus and continue with infu-
Carnitine: In organic acidurias, such as PA and MMA, the sion 3–6 ml/kg/h.
intramitochondrial accumulation of CoA esters results in the • In a child with a suspected IEM, promote anabolism (see
inhibition of various pathways of intermediary metabolism, above).
as well as in depletion of free CoA and in increased levels of • Supplement sodium, potassium, and phosphate as
acylcarnitines (particularly propionylcarnitine) resulting in required.
carnitine insufficiency. Carnitine is therefore given to com-
pensate for secondary carnitine deficiency caused by urinary
excretion of carnitine-bound organic acids. As a rule, 45.4.4 Seizures
L-carnitine supplementation is never contraindicated in these
disorders. Only if a long-chain fatty acid oxidation defect is If intractable seizures dominate the clinical picture, folinic
suspected, the administration of carnitine should be avoided, acid-responsive, pyridoxine (B6)-responsive, and pyridoxine
at least as a bolus, because of the acute accumulation of toxic phosphate-responsive seizures should be considered (Mills
long-chain acylcarnitines and the risk of a fatal cardiac et al. 2005; Stockler et al. 2011; Rahman et al. 2013). In the
arrhythmia (Bonnet et al. 1999). case of a positive response, the therapeutic trials might give
Vitamin Therapy: Patients with isolated methylmalonic the diagnosis. Pyridoxine is administered i.v. The metabolic
academia might respond to vitamin B12 treatment. workup should always be started simultaneously. Higher
Pharmacological doses of specific vitamins should be sys- doses may be necessary to control seizures, at least initially.
tematically tested in each case. Vitamin responsiveness is Start with a single dose of 100 mg of pyridoxine and if the
more likely in late-onset forms than in patients presenting in patient is nonresponsive within 10 min, the dose should be
the newborn period. Hydroxocobalamin, the cofactor of increased and repeated up to 200 mg total before concluding
about pyridoxine nonresponsiveness. If there is uncertainty Bosch AM, Stroek K, Abeling NG et al (2012) The Brown-Vialetto-
about at least a partial response, pyridoxine should be contin- Van Laere and Fazio Londe syndrome revisited: natural history,
genetics, treatment and future perspectives. Orphanet J Rare Dis
ued with 5–15 mg/kg/day (up to 30 mg/kg/day) for 7 days 7:83
before final conclusions are made. If the response to pyridox- Broomfield A, Grunewald S (2012) How to use serum ammonia. Arch
ine is negative or doubtful, folinic acid should be adminis- Dis Child Educ Pract Ed 97(2):72–77
tered with 5 mg/kg/day in three doses intravenously or orally Daniotti M, la Marca G, Fiorini P et al (2001) New developments in the
treatment of hyperammonemia: emerging use of carglumic acid. Int
for 3 days. If negative, it may be followed by the administra- J Gen Med 4:21–28
tion of pyridoxal phosphate with 30 mg/kg/day in three doses Deodato F, Boenzi S, Santorelli FM et al (2006) Methylmalonic and
orally for 3 days. Severe infantile epileptic encephalopathy is propionic aciduria. Am J Med Genet C Semin Med Genet
one indication for CSF analyses testing neurotransmitter 142:104–112
Dionisi-Vici C, Ogier de Baulny H (2012) Emergency treatment. In:
substances, allowing the diagnosis of defects in the metabo- Saudubray JM, van den Berge G, Walter J (eds) Inborn metabolic
lism of biogenic amines and GABA transaminase deficiency diseases: diagnosis and treatment, 5th edn. Springer, Heidelberg, pp
(Hoffmann et al. 1998). 103–111
Filipowicz HR, Ernst SL, Ashurst CL et al (2006) Metabolic changes
associated with hyperammonemia in patients with propionic acide-
mia. Mol Genet Metab 88:123–130
45.4.5 Postmortem Investigations Griffith AD, Cyr DM, Egan SG et al (1989) Inhibition of pyruvate car-
boxylase by sequestration of coenzyme A with sodium benzoate.
In the event of death when metabolic disease is suspected, it Arch Biochem Biophys 269(1):201–207
Grünewald S, Champion MP, Leonard JV et al (2004) Biotinidase defi-
is important to store adequate amounts of biological fluids ciency: a treatable leukoencephalopathy. Neuropediatrics
and tissues for further diagnostic workup. In the case of sud- 35:211–216
den infant death syndrome (SIDS), it is important to recog- Häberle J, Boddaert N, Burlina A et al (2012) Suggested guidelines for
nize that defects of fatty acid β-oxidation may be responsible. the diagnosis and management of urea cycle disorders. Orphanet J
Rare Dis 7:32
In most cases, autopsy reveals an excess of fat droplets in the Hoffmann GF, Surtees RA, Wevers RA (1998) Cerebrospinal fluid
liver or heart, even in the absence of steatosis. investigations for neurometabolic disorders. Neuropediatrics
Should there be the suspicion of an underlying metabolic 29:59–71
disease in a deceased child, it is critical to collect the follow- Marín-Valencia I, Vilaseca MA, Thió M et al (2010) Assessment of the
perimortem protocol in neonates for the diagnosis of inborn errors
ing samples (Marín-Valencia et al. 2010): of metabolism. Eur J Paediatr Neurol 14:125–130
Mayatepek E, Schulze A (1999) Metabolic decompensation and lactic
acidosis in propionic acidaemia complicated by thiamine deficiency.
Sample/storage
Urine (−20°) Mills PB, Surtees RA, Champion MP et al (2005) Neonatal epileptic
Amino acids, organic acids, tubulopathy markers encephalopathy caused by mutations in the PNPO gene encoding
Blood (−20°) pyridox(am)ine 5′-phosphate oxidase. Hum Mol Genet
DNA, uric acid, amino acids 14:1077–1086
Dried blood spots (room temperature) Pérez-Dueñas B, Serrano M, Rebollo M et al (2013) Reversible lactic
acidosis in a newborn with thiamine transporter-2 deficiency.
Acylcarnitine profile
Pediatrics 131(5):1670–1675
CSF sample (−70°) Picca S, Dionisi-Vici C, Abeni D et al (2001) Extracorporeal dialysis in
Neurotransmitters, amino acids neonatal hyperammonemia: modalities and prognostic indicators.
Skin biopsy (fibroblasts, culture medium at room temperature) Pediatr Nephrol 16:862–867
Enzyme analysis, DNA Rahman S, Footitt E, Varadkar S et al (2013) Inborn errors of metabolism
causing encephalopathy. Dev Med Child Neurol 55:23–36
Saudubray JM (2012) Clinical approach to inborn errors of metabolism
Liver or muscle biopsies need to be considered in special in paediatrics. In: Saudubray JM, van den Berge G, Walter J (eds)
circumstances, for example, if there is the suspicion of an Inborn metabolic diseases: diagnosis and treatment, 5th edn.
Springer, Heidelberg, pp 3–52
underlying mitochondrial disorder. Those samples (and this Stockler S, Plecko B, Gospe SM Jr. et al (2011) Pyridoxine dependent
also applies for plasma amino acid samples) must be taken as epilepsy and antiquitin deficiency: clinical and molecular character-
soon as possible postmortem to ensure reliable results. The istics and recommendations for diagnosis, treatment and follow-up.
samples need to be stored at −70 °C immediately. Mol Genet Metab 104:48–60
Walter JH, Wraith JE, Cleary MA (1995) Absence of acidosis in the
initial presentation of propionic acidaemia. Arch Dis Child Fetal
Neonatal Ed 72:197–199
References
Bonnet D, Martin D, De Lonlay P et al (1999) Arrhythmias and conduc-
tion defects as presenting symptoms of fatty acid oxidation disor-
ders in children. Circulation 100:2248–2253
Newborn Screening for Inborn Errors
of Metabolism 46
Carol L. Greene and Dietrich Matern
Contents 46.1 What Is Newborn Screening?

46.1 What Is Newborn Screening? ...................................... 719
Newborn screening (NBS) is a system developed for early
46.2 History and Evolving Goals of NBS ............................ 719
identification and treatment of newborns who have condi-
46.3 Newborn Screening Is a System: tions for which treatment initiated in a timely fashion may
Stakeholders, Roles, and Responsibilities ................... 721
prevent death or disability. Since public health departments
46.4 Principles and Practice in the NBS Laboratory ......... 722 began screening for phenylketonuria using blood collected
46.4.1 Sensitivity and Specificity ................................................. 722 on filter paper in the early 1960s, NBS has expanded to
46.4.2 What Is a “False Positive” in NBS? .................................. 722
46.4.3 What Is a “False Negative” in NBS?................................. 723 include other conditions, using a variety of analytical meth-
46.4.4 Methodology ..................................................................... 725 ods. This chapter focuses on NBS as blood spot screening of
46.5 Before and After NBS: Roles of the
unselected newborns for evidence of inborn errors of metab-
Hospitals and Health Care Providers .......................... 730 olism. The principles considered here will also apply to NBS
46.5.1 Before Screening ............................................................... 730 using blood spot samples for conditions other than IEM
46.5.2 What to Do When a NBS Is Not Normal? ........................ 731 (including other genetic conditions such as cystic fibrosis or
46.5.3 Special Issues in Strategies for Testing
After Abnormal NBS ........................................................ 733
hemoglobinopathies and nongenetic conditions such as
46.5.4 What Does Screening Test For? ........................................ 733 hypothyroidism and infections including HIV and CMV)
46.5.5 Residual NBS Samples, QC, and Research ...................... 733 and to some aspects of point-of-care screening (including
46.5.6 Special Issues and New Challenges in NBS ..................... 734 hearing screening and pulse oximetry for critical congenital
Conclusion ................................................................................... 735 heart defects). In order for the practicing physician to be an
References .................................................................................... 735
effective partner in the NBS process, this chapter includes an
overview of the history, evolving goals, principles, method-
ology, short- and long-term follow-up issues, and future
challenges for NBS.
46.2 History and Evolving Goals of NBS
The Centers for Disease Control and Prevention of the USA

in 2011 included NBS as one of the last decade’s great public
health achievements (CDC 2011), and in recognition of the
C.L. Greene (*)
Department of Pediatrics, University of Maryland, complexity of the technical, scientific, logistic, and social
737 W Lombard St #199, Baltimore, MA 21201, USA elements of NBS, Hoffmann et al. (2010) call it “one of the
e-mail: cgreene@peds.umaryland.edu most complex scientific and medical endeavors.” Driscoll
D. Matern and McPherson (2010) provide a detailed history and analy-
Division of Laboratory Genetics, Departments of Laboratory sis of the principles and practice of NBS that will be invalu-
Medicine and Pathology, Medical Genetics, and Pediatric
able for those involved in NBS policy and systems
and Adolescent Medicine, Mayo Clinic College of Medicine,
200 First Street SW, Rochester, MN 55902, USA development. Here we provide a summary for the practicing
e-mail: matern@mayo.edu clinician.
720 C.L. Greene and D. Matern
NBS had its beginning 50 years ago when Robert Table 46.1 WHO 2008: Andermann et al. – review of classical and
Guthrie developed a simple and efficient test to measure emerging criteria for screening (Andermann et al. 2008)
phenylalanine in small blood samples collected by heel Wilson and Jungner classic screening criteria
stick from newborns on to filter paper (later called “Guthrie 1. The condition sought should be an important health problem
cards”). The reason for Guthrie’s interest in phenylalanine 2. There should be an accepted treatment for patients with
recognized disease
was that treatment for phenylketonuria had become avail-
3. Facilities for diagnosis and treatment should be available
able in the 1950, but proved to be of only limited value
4. There should be a recognizable latent or early symptomatic stage
when irreversible neurological damage had already
5. There should be a suitable test or examination
occurred (see Chap. 1). His goal then became to identify 6. The test should be acceptable to the population
PKU patients shortly after birth to allow for early initiation 7. The natural history of the condition, including development from
of dietary treatment to prevent development and progres- latent to declared disease, should be adequately understood
sion of neurodevelopmental damage. With the additional 8. There should be an agreed policy on whom to treat as patients
development of technology for testing small filter paper 9. The cost of case finding (including diagnosis and treatment of
samples for thyroid function, NBS programs focused ini- patients diagnosed) should be economically balanced in relation
to possible expenditure on medical care as a whole
tially on these two conditions, for both of which initiation
10. Case finding should be a continuing process and not a “once and
of effective treatment in the first weeks of life prevents for all” project
severe mental retardation. Additional conditions were Synthesis of emerging screening criteria proposed over the past
added over time, with the largest increase of screened con- 40 years
ditions occurring over the last 15 years driven by the appli- The screening program should respond to a recognized need
cation to blood spot testing of tandem mass spectrometry, The objectives of screening should be defined at the outset
which allows for the detection of more than 40 inborn There should be a defined target population
errors of metabolism by simultaneous measurement of There should be scientific evidence of screening program
effectiveness
amino acids and acylcarnitines.
The program should integrate education, testing, clinical services
Since the initial success of NBS in preventing mental and program management
retardation from PKU and hypothyroidism, there has been There should be quality assurance, with mechanisms to minimize
interest in screening babies for more conditions. The com- potential risks of screening
plex interaction of technical ability, medical knowledge, and The program should ensure informed choice, confidentiality, and
public health interest in the selection of conditions for which respect for autonomy
newborns should be screened was internationally recognized The program should promote equity and access to screening for the
entire target population
early in the history of NBS. In 1968, the World Health
Program evaluation should be planned from the outset
Organization (WHO) formally considered the rationale for
The overall benefits of screening should outweigh the harm
population screening and published principles summarized
by Wilson and Jungner (1968). While these nonbinding prin-
ciples have remained important in the ongoing development hyperphenylalaninemias. If it were a requirement that the
of NBS, significant advances in testing for and in treatment natural history of a condition was fully understood before
of inborn errors of metabolism have offered opportunities for NBS was initiated, we would never have achieved the
expansion of NBS that challenge these original principles. remarkable successes of NBS for PKU.
Around the world, discussions about NBS are evolving and Nevertheless, the international community has also had
WHO revisited the issues and in 2008 published a review by experiences that reinforce need for an adequate understand-
Andermann et al. (2008) of the established and emerging ing of the natural history of a condition before instituting
principles that currently govern decisions about population screening. Examples are screening for histidinemia and for
screening around the world (Table 46.1). neuroblastoma. Screening for histidinemia was stopped
One of the important elements of the discussion of because elevated histidine proved to not be of clinical signifi-
whether to screen for a condition is the understanding of the cance (Brosco et al. 2010). Perhaps more important is the
natural history of the disorder. In this context, it is critical to lesson from screening of babies’ urine in Quebec, Germany,
recognize that all medical knowledge expands iteratively. Austria, the UK, and Japan for neuroblastoma, an important
For example, before NBS for PKU, variant hyperphenylal- pediatric cancer. When diagnosed early, infants with neuro-
aninemia was not well recognized. Some of the babies blastoma respond well to a combination of surgery and che-
detected by NBS proved to have significant residual enzyme motherapy, but with late diagnosis of metastatic disease, the
activity, protecting them from the mental retardation caused condition is likely to be fatal. However, NBS for neuroblas-
by classic PKU due to phenylalanine hydroxylase (PAH) toma was halted after observation that the death rate from the
deficiency, while others proved to have primary disorders of tumor did not change despite early diagnosis and treatment
the synthesis or recycling of tetrahydrobiopterin, an impor- after NBS. It was concluded that the natural history of
tant cofactor of PAH (see Chap. 1). After more than 50 years neuroblastoma included previously unrecognized benign
of NBS, we still continue to learn more about the tumors that were picked up by the screen and that the tumors
46 Newborn Screening for Inborn Errors of Metabolism 721
detected by NB would have resolved without the invasive samples, laboratory analysis and reporting, and short-term
therapy (Riley et al. 2003; Maris and Woods 2008). follow-up (after any abnormal screen, until resolution with
Decisions about the conditions for which newborns determination of whether baby is affected) and long-term
should be screened continue to be based on a combination of follow-up of the babies who are proven to be affected. A
science, technical, financial, and practical considerations, 2008 statement of a federal Advisory Committee in the USA
with the general aim of improving the health of the popula- recognizes that ongoing treatment of those identified by
tion in general and of each baby. Considerations typically NBS as affected is an essential element of long-term follow-
include the frequency of the condition, the severity of the up, without which the benefits of NBS cannot be realized
condition if not diagnosed, and the availability (both in prin- (Kemper et al. 2008). The American Academy of Pediatrics
ciple and in practice) of strategies to improve outcome if the (2008) in 2008 provided detailed recommendations for the
diagnosis is known early. Technology that allows testing for primary care provider regarding NBS-related activities.
multiple metabolites at the same time improves the sensitiv- NBS is typically governed by a combination of national
ity and specificity of screening by permitting use of ratios and regional or local (e.g., state, province, canton, or other
between metabolites. However, this technology also chal- division) laws and regulations and is supported by either
lenges prior established principles of NBS by making it easy, public or private funding or by a combination of both. The
using a single test, to identify both conditions for which it hospital and health care provider must follow applicable
has been long agreed that screening is appropriate and also rules and regulations.
conditions that are either more rare or less responsive to ther- In addition to the basic and clinical research that is the
apy. Some of the stakeholders – families, health care provid- underpinning of NBS, the NBS system includes:
ers, and those responsible for development of public health • A process for developing, establishing, and maintaining
policy – are in favor of expanding NBS to include a broader policy around screening, including determination of what
range of conditions and some recommend holding to more conditions will be included on the NBS panel for which
limited screening. At one end of the spectrum are those who newborns are tested, what laboratories will perform the
point out the value of early diagnosis for any condition, no testing, and determination of how programs (including
matter how rare and no matter if it is not responsive to ther- the laboratory and the short-term and long-term follow-up
apy, because early diagnosis can avert a lengthy diagnostic activities) will be funded and managed.
odyssey and can provide opportunity to understand and avoid • Test development and validation leading to implementa-
recurrence risk and facilitate research that may lead eventu- tion of testing in a laboratory accessible to the population,
ally to therapy. On the other end of the spectrum are those determination of methodology to be used, criteria for
who emphasize the negative impact on families who are accepting samples, information required with sample,
given information predicting problems but for which they normal and abnormal values, and actions to be taken by
can take no action, as well as the burden on the public health laboratory in response to abnormal values.
system for no recognizable health benefit. The majority of • Collection of samples for testing by hospitals and/or indi-
stakeholders fall in between these extremes. vidual health care providers and transport of specimens to
Considering when information about a condition is suffi- the laboratory.
cient, when a screening test is sufficiently sensitive and spe- • Laboratory activities including determination of sample
cific, when therapy is sufficiently successful and available, quality, testing of sample, reporting of results to the sub-
and what is an appropriate balance between costs and bene- mitter of the sample, and making results available to the
fits leads to decisions about NBS. primary care provider and laboratory QC and QI.
The complex decision making is in some areas formal- • Short-term follow-up (may be carried out as part of the
ized. Currently in the USA, each state is responsible for laboratory activities or may be performed by a specially
making decisions about the conditions for which its new- designated team) that includes communication of any
borns are screened, and the federal government provides rec- abnormal result to the submitter of the sample, location of
ommendations for a “core panel” of conditions set by a the responsible health care provider, and, if no health care
formal advisory committee to the Secretary of Health and provider takes responsibility for follow-up, direct com-
Human Services (DHHS 2013). munication with the family, recommendations for appro-
priate diagnostic testing, and collection of results so that
the program is able to determine the outcome of each
46.3 Newborn Screening Is a System: abnormal NBS result.
Stakeholders, Roles, and • Confirmatory diagnostic testing typically involving
Responsibilities appropriate specialists and local and reference laborato-
ries working in collaboration with the primary care pro-
NBS is a system involving public programs, health care provider and the family; diagnostic testing is occasionally
viders, and professional organizations and babies and fami- carried out by the primary care provider under the direc-
lies. It includes the timely collection and shipment of tion of the short-term follow-up team.
• Long-term follow-up with treatment provided by Sensitivity/Specificity

appropriate specialists and with outcomes and other data Ideal situation
reported back to the public health system.
100% Sensitive and 100% Specific Test
• Clear and complete communication throughout the Frequency
process including discussion:
– Between the public health team and health care provid- Healthy Population Affected population
ers with respect to the screening test results, including
assurance of receipt of results and response to results
by hospitals and/or individual health care providers
– Between NBS providers and families, including dis-
cussion before collection of sample (in some states NI Abnl Concentration
including formal consent) and discussion of any abnor-
mal NBS results and results of diagnostic testing Sensitivity/Specificity
Where NBS is carried out, it is understood to be a profes- Typical situation
sional responsibility – and in some cases formal regulations
Frequency Sensitive Test
indicate a legal responsibility – that (1) the hospital assures
Specific Test
each newborn is tested and (2) the primary care provider
assures each newborn has been tested as determined to be
appropriate in the jurisdiction. Some jurisdictions specify Healthy Population
Affected population
limited reasons that a family may decline to have the baby
screened. Some jurisdictions provide only limited NBS test-
ing that does not satisfy professional guidelines for practic-
ing clinicians. In such cases, additional or supplemental
screening may be accessed by a hospital or by a health care A B Concentration
provider by sending samples to a laboratory that will accept
Fig. 46.1
special contracts or individual samples.
Each member of the health care team should be able to
provide the family with accurate information about NBS. All 46.4.1 Sensitivity and Specificity
members of the NBS team, whether clinical care providers,
laboratorians, or public health officials, should be knowl- Altered levels of key metabolites or of enzyme activity
edgeable advocates for NBS, including for quality improve- characterize the inborn errors of metabolism. For most con-
ment and policy development. ditions, it is expected that there will be some circumstances
in which there will be overlap between the results of quanti-
tative or semiquantitative analysis of biochemical test results
46.4 Principles and Practice in the NBS between the affected and the unaffected population.
Laboratory Figure 46.1 illustrates the principles of sensitivity and speci-
ficity of a screening test that examines the level of a metabo-
In order to identify those babies in need of further evaluation, lite that is expected to be elevated in a condition; the same
the laboratory must have a test that discriminates between principles will apply to a test that analyzes for low levels of
affected and unaffected individuals. Achieving this depends an analyte or for enzyme activity.
on a combination of science and technology both for test Sensitivity: the proportion of people with the disease
methodology and for definition of a normal or an abnormal who test positive. (A sensitive test has few false
screening test result. The goal is to maximize both sensitivity negatives.)
and specificity of the screening test, to minimize both false- Specificity: the proportion of people without the dis-
positive and false-negative screening test results. ease who test negative. (A specific test has few false
Test methodology and pathophysiology interact uniquely positives.)
for each condition for which newborns are screened. While
improvements in technology and information analysis have
markedly improved both sensitivity and specificity of 46.4.2 What Is a “False Positive” in NBS?
screening, it remains necessary for screening programs to
make decisions that recognize trade-offs between sensitivity The term “false positive” (FP) typically is used to describe
and specificity for each test and that consider the costs and the situation in which the NBS result is abnormal and
consequences of each decision for babies, families, health diagnostic testing finds no evidence that the baby has the
care providers, and the health care and public health conditions for which the screening test was abnormal. It is
systems. important for the clinician to recognize that FP is not
equivalent to lab error; lab error is usually the least likely For example, tyrosine is traditionally used as the biomarker
cause of a FP screening test. to detect tyrosinemia type I (TYR I, see Chap. 2); unfortu-
The possibility of a FP test result is an expected conse- nately, tyrosine concentrations in NBS samples from babies
quence of virtually all medical screening when attempting to with TYR I have significant overlap with the unaffected pop-
identify all affected individuals, whether the screening is a ulation. Therefore, even when accepting a high number of FP
simple physical examination (e.g., finding of a heart murmur results by lowering the cutoff for tyrosine, perfect sensitivity
in a baby who proves to have no heart defect) or involves any cannot reasonably be achieved and FN results cannot be
form of laboratory testing. In NBS, in some instances, an avoided. This has led some NBS programs to stop screening
abnormal screening result can be caused by the newborn’s for TYR I, while others have included succinylacetone, a
mother actually being affected with an inborn error of metab- specific marker for TYR I, into their MS/MS-based screen-
olism or a nutritional deficiency, such as vitamin B12 defi- ing (Table 46.3). Another important example is NBS for
ciency. Another possibility is that an abnormal analyte homocysteinurea, historically based on detection of eleva-
detected by the NBS could be the secondary result of a health tion of methionine that is found in cystathionine synthase
condition in the newborn that is not part of the screening deficiency (Chap. 3). As a result, NBS did not detect indi-
panel, as in the secondary elevation of tyrosine by other liver viduals with certain other forms of homocysteinurea due to
disease. This challenges the traditional definition of a FP cobalamin metabolism, who may have normal methionine.
result; some NBS programs consider these as FP results When more than one condition is detected by altered levels
whereas others consider such cases as true positive because of an analyte, the false-negative and false-positive rate should
clinically relevant information has been uncovered. be expected to vary for each condition since, although the
In NBS, abnormal screens may result from the basic method and the detection of the analyte will be the same, the
physiology of a condition, from other medical conditions physiologic expression of each disorder is unique.
that also affect relevant metabolites, and, less commonly, A normal NBS result should never lead a physician to
from sample mix-up. Causes of FP vary with the method of dismiss a screened condition from the differential diagnosis
screening as well as with the disorders for which babies are of a symptomatic patient. Causes of FN vary with the method
screened (Table 46.2). A common cause of FP testing in of screening as well as with the disorders for which babies
NBS is the result of using single analyte cutoffs that have are screened (Table 46.2).
been chosen to approach 100 % sensitivity at the cost of
specificity. Whether using single analyte data or incorporat-
ing ratios, means to improve the FP rate are now available at
no cost through the Region 4 MS/MS data project, a program False-Negative and False-Positive NBS: Example
that was funded by the Health Resources and Service While hypothyroidism detected by NBS is not typi-
Administration (HRSA) to support the implementation of cally an inherited condition, it is one of the first condi-
the uniform screening panel in the USA. The main goal of tions for which newborns were screened and continues
this project is to improve overall analytical performance to illustrate very well the complex physiologic causes
through a web-based data collection system that allows the of false positive and false negatives. Sick and very pre-
establishment of disease, carrier, and normal ranges for each mature babies have lower levels of thyroid hormone
analyte and analyte ratio (available at: www.clir-r4s.org). and are therefore on a physiologic basis more likely to
These data were the foundation of online interpretive tools trigger the NBS system even when they ultimately
that allow rapid review of the amino acid and acylcarnitine prove to be unaffected by congenital hypothyroidism.
profiles to identify newborns at high risk of being affected To minimize false positives due to physiology, many
with a screened condition. Application of these tools reduces screening programs use a system measuring both thy-
false-positive results due to dietary (e.g., total parenteral roid hormone and thyroid-stimulating hormone and
nutrition) or medication artifacts (e.g., cefotaxime). Of note, report as abnormal screen only those newborns with
this system is not only accessible to NBS laboratories but out-of-range result for both. However, when the level
also to follow-up personnel who may want to achieve a bet- of thyroid-stimulating hormone is used as either a pri-
ter assessment of the risk that the neonate is affected. mary or secondary marker in screening, a baby with
central hypothyroidism resulting from primary abnor-
mality of the pituitary will inevitably be missed since
46.4.3 What Is a “False Negative” in NBS? TSH is not elevated. These cases are missed not as a
result of an error in the laboratory but as a consequence
Again, it is important to recognize that screening is not diag- of the reasonable and necessary design of a screening
nostic testing. Screening is not expected to have perfect sen- system to identify the common cause of hypothyroid-
sitivity, and some analytes are particularly unreliable. It is ism while minimizing the false-positive rate from sick
important for the clinician to recognize that FN is not equiva- and premature newborns.
lent to lab error; lab error is typically the least likely cause.
Table 46.2 Commonlya used methods in blood spot NBS (historic or currently used)
724
Principle False positives False negatives Uses and comments

Bacterial Measures analyte by effect of analyte Physiologic variations of analyte levels, Physiologic variations of analyte The original method of screening for PKU and
inhibition assay level on growth of bacteria selected for effect of other medical conditions, effect levels, effects of antibiotics other IEM as designed by R. Guthrie and used until
dependence on analyte of intake (feeding, hyperalimentation) replaced by fluorescence and/or MS/MS
Fluorescent or Measures analyte by amount of Physiologic variations and effect of Physiologic variations of analyte PKU, maple syrup urine disease, homocystinuria,
other fluorescence or color compared with other medical conditions, effect of levels, effects of dietary intake of galactosemia
colorimetric standard intake (feeding, hyperalimentation) analytes
Immune assay Measures presence of protein based on Physiologic variation Physiologic variation; for CF it is Thyroid and thyroid-stimulating hormone (for
interaction with an antibody against the notable that immunoreactive hypothyroidism); immunoreactive trypsinogen
protein; various markers for levels, e.g., trypsinogen (IRT), the marker for (IRT) for cystic fibrosis; steroid hormone analytes
radioactivity for radioimmunoassay CF, is not elevated in babies with for congenital adrenal hyperplasia
(RIA) meconium ileus
Electrophoresis Measures presence or absence of Transfusion Transfusion Hemoglobinopathies; method in use for decades
protein with specific mass and charge also identifies carrier status
Enzyme assay Measures ability of enzyme in sample Heat inactivation of enzyme in Transfusion Galactosemia (by assay called “Beutler”),
to transform substrate to product; transport; deficiency of other pathway biotinidase deficiency, some lysosomal enzymes.
semiquantitative or quantitative required for generation of marker for Can identify carriers, identifies healthy individuals
determination product (Beutler assay for galactosemia with pseudodeficient states, and (especially for
depends of integrity of the enzyme lysosomal disorders) identifies affected individuals
G6PD); pseudodeficiency who may have adult-onset phenotype
MS/MS Tandem mass spectrometry, inferring Physiologic variations and effect of Physiologic variations of analyte PKU and other amino acidopathies, methylmalonic
levels of metabolites based on amounts other medical conditions, effect of levels, effects of dietary intake of acidemia and other organic acidemias, MCADD
of (and ratios of) molecular fragments intake (feeding, hyperalimentation), analytes and other disorders of fatty acid oxidation and
compared against isotopically labeled effect of some medications carnitine metabolism, some urea cycle disorders.
standards The accuracy of levels and the ability to examine
ratios dramatically improve sensitivity and
specificity compared with bacterial inhibition and
fluorescence
DNA mutation Analyzes for the presence or absence of Unless method identifies all mutations For virtually all conditions, there Occasionally used as primary screen but more often
analyses specific sequence changes or specific (see false-negative column), it may be will be false negatives if DNA is used as second-tier test for cystic fibrosis,
known deletions or duplications necessary to perform diagnostic testing the primary screen or required to medium-chain acyl-CoA dehydrogenase deficiency
on those identified by screening as be positive as a second-tier test in and galactosemia
having one mutation screening. The number of cases
Two mutations may be present in cis in missed depends on the number of Will identify carriers
an individual who is carrier but not mutations for which the sample is To screen for spinal muscular atrophy, DNA testing
affected screened and the frequency with must be used as primary method
which individuals in the screened
population have disease caused by
mutation(s) not on the panel
DNA repeat size Analyzes for the length of triplet repeat Hypothetically none Mosaicism for repeat size Fragile X; should also identify carriers
segments
DNA – TREC Quantifies fragments of DNA (cell Physiologic Some cases of adenosine Severe combined immune deficiency, including
receptor excision circles) generated in deaminase (ADA) deficiency several conditions which can present with immune
T-cell function maturation deficiency, such as T-cell deficiency due to deletion
of 22q
a
Includes methods used for IEM and for other (including nongenetic as well as non-metabolic) conditions. Less commonly used methodologies, e.g., HPLC, are not included here
C.L. Greene and D. Matern
46.4.4 Methodology Screening for CF illustrates the complexity around

second-tier testing strategies and around the use of DNA in
Most current laboratory techniques can be applied to the screening. Some laboratories use DNA as a second-tier test
quantitation or identification of disease markers once in NBS for cystic fibrosis (CF), where an abnormal biochem-
extracted from the dried blood spot sample (Table 46.2). Of ical determination of immunoreactive trypsinogen (IRT) will
note, the DBS sample must be considered less precise than prompt testing of the original sample for a set of common
plasma or serum given the collection method by heel stick CFTR mutations. However, because a second mutation can-
(vs. venous blood draw) and variability of the hematocrit. not be excluded with this approach, in these laboratories, any
Factors that need consideration in choosing a method elevated IRT is reported as presumptively abnormal when at
include the biomarker of interest itself (enzyme activity, least heterozygosity for one mutation is determined. This
analyte concentration, DNA), sensitivity and specificity, leads to a relatively high false-positive rate for CF screening
hardware and reagent cost, space requirements, and ability because of the high frequency of carrier status for CF. Use of
to measure more than one analyte at a time DNA as primary screen or use of DNA to determine that
(multiplexing). elevated IRT is a false positive can also lead to false-negative
Strengths and limitations of each method depend upon (a) screening for CF, when an affected baby has mutations not
the method, (b) cutoffs applied, (c) the pathophysiology of included on the panel of common CFTR mutations (Comeau
each IEM around the neonatal period, and (d) ongoing treat- et al. 2007); this is especially likely when a baby is not of
ment. For example, the reliable detection of PKU by either Northern European or Ashkenazi Jewish ancestry
Guthrie’s bacterial inhibition assay was compromised when (Ross 2008). In addition, the assumption in using DNA as a
an affected newborn happened to be treated with antibiotics second-tier screen is that there are two independent opportu-
prior to NBS sample collection (due to inhibition of bacterial nities to identify a CFTR mutation; in cultures that accept or
growth by the antibiotic despite the presence of high levels promote consanguineous marriage, many babies will be
of phenylalanine). Furthermore, when only measuring phe- expected to be homozygous based on descent, and the
nylalanine to screen for PKU, attention must be paid to the assumption behind the calculations for sensitivity of DNA as
feeding status of the newborn at sample collection. In par- a screen may not apply.
ticular with the introduction of early postnatal discharge, Additional second-tier tests have improved screening for
phenylalanine may be normal despite the baby being affected several conditions that were included in NBS programs with
with PKU because of insufficient protein intake prior to sam- the application of MS/MS-based testing (Table 46.3).
ple collection. Both of these problems were overcome by the Propionylcarnitine and methionine have particularly bene-
application of amino acid profile analysis, which measures fited from second-tier testing for methylcitric acid, methyl-
phenylalanine and other amino acids directly and allows for malonic acid, and total homocysteine. This assay not only
the calculation of the phenylalanine to tyrosine ratio – a help- improved the specificity and positive predictive value of
ful marker as it is independent of feeding status. Technologies NBS for inborn errors of propionic acid metabolism and
such as MS/MS are increasingly preferred as they allow for homocystinuria but also allowed for the detection of various
rapid and efficient measurement of multiple biomarkers defects of cobalamin metabolism that are associated with
simultaneously from the same DBS sample. Saving DBS low levels of methionine.
sample is of importance as more conditions are proposed for Improving specificity for the targeted condition will in
NBS, and second-tier testing is introduced to improve NBS some cases reduce sensitivity to alternative conditions that
performance. could have been recognized when screening used less spe-
“Second-tier testing” using a second method of testing on cific methods. For example, when screening for hepatorenal
the original sample will increase specificity. Examples of the tyrosinemia, reporting an out-of-range result for tyrosine
use of second-tier tests include NBS for congenital hypothy- only if the analyte succinylacetone is also present will reduce
roidism when a reduced concentration of T4 is followed by false-positive screens by excluding those babies who have an
measurement of TSH. While this improves NBS for hypo- environmental and typically benign (usually hyperalimenta-
thyroidism, this condition is commonly affected by both tion) cause of elevated tyrosine. However, those few babies
false-positive and false-negative results in preterm or acutely with some potentially useful incidental findings from “false
ill neonates. Accordingly, a two-tier testing approach is not positives” – for example, a baby with Wilson disease with
sufficient, and many NBS programs routinely screen prema- neonatal liver presentation – will no longer be identified. The
ture and NICU babies at least one more time between 2 and consequences of each methodologic choice will be weighed
6 weeks old. according to the principles of NBS.
Table 46.3 Measurable markers included in many NBS programs for inborn errors of metabolism and their differential diagnosis
Differential diagnosis
Method Informative marker Primary conditionsa Secondary conditionsa
FI-MS/MS Alanine (Ala) – –
Arginine (Arg) ARG CIT II
Argininosuccinic acid (ASA) ASL –
Citrulline (Cit) CIT I, ASL CIT II
Glutamic acid (Glu) – –
Glutamine + Pyroglutamic acid (Gln + Pyrog) – –
Glycine (Gly) – –
Leucine/isoleucine/OH proline (Xle) MSUD –
Lysine (Lys) – DE-RED
Methionine (Met) CBS MET
Ornithine + Asparagine (Orn + Asn) – –
Phenylalanine (Phe) PKU H-PHE, BIOPT (BS), BIOPT (REG)
Proline (Pro) – –
Succinylacetone (SUAC) TYR I –
Threonine (Thr) – CIT II
Tyrosine (Tyr) TYR I TYR II, TYR III
Valine (Val) MSUD –
Carnitine (C0) CUD CPT I
Propionylcarnitine (C3) PA, MCD, MUT, CBL A/B CBL C/D
Formiminoglutamic acid (FIGLU) – SCAD, IBG

Butyryl-/isobutyrylcarnitine (C4) – SCAD, IBG, GA II
Tiglylcarnitine (C5:1) BKT 2M3HBA
Isovaleryl-/2-methylbutyrylcarnitine (C5) IVA 2MBG, GA II
OH butyrylcarnitine (C4-OH) BKT 2M3HBA, S/MCHAD
Hexanoylcarnitine (C6) MCAD GA II
OH isovalerylcarnitine (C5-OH) 3MCC, HMG, BKT, MCD 2M3HBA, 3MGA
Octanoylcarnitine (C8) MCAD GA II, MCKAT

Malonyl-/OH octanoylcarnitine (C3DC) – MAL, MCKAT
Decadienoylcarnitine (C10:2) – DE-RED
Decenoylcarnitine (C10:1) MCAD GA II
Decanoylcarnitine (C10) MCAD GA II
Succinyl-/methylmalonylcarnitine (C4DC) MUT, Cbl A, B –
Glutaryl-/OH decanoylcarnitine (C5DC) GA I GA II
Dodecenoylcarnitine (C12:1) VLCAD –
Dodecanoylcarnitine (C12) VLCAD –
Methylglutarylcarnitine (C6DC) HMG –
Tetradecanedioylcarnitine (C14:2) VLCAD, LCHAD/TFP GA II
Tetradecenoylcarnitine (C14:1) VLCAD, LCHAD/TFP GA II
Tetradecanoylcarnitine (C14) VLCAD, LCHAD/TFP CACT, CPT II
Palmitoylcarnitine (C16) VLCAD CPT I (low), CACT, CPT II
OH hexadecenoylcarnitine (C16:1-OH) LCHAD/TFP –
OH palmitoylcarnitine (C16-OH) LCHAD/TFP –
Linoleylcarnitine (C18:2) LCHAD/TFP CPT II
Oleylcarnitine (C18:1) VLCAD, LCHAD/TFP CPT I (low), CACT, CPT II, GA II
Stearylcarnitine (C18) VLCAD CPT I (low), CACT, CPT II, GA II
OH oleylcarnitine (C18:1-OH) LCHAD/TFP –
OH stearylcarnitine (C18-OH) LCHAD/TFP –
Colorimetry Biotinidase BIOT –
Fluorometry Galactose-1-phosphate uridyltransferase GALT –
(GALT)
Total galactose GALT GALK, GALE
Glucose-6-phosphate dehydrogenase (G6PD) – –

Urgency of clinical
Other conditions Critical ratio Second-tier test Initial confirmatory testingb actionc
LA – – PAA, L&P Low
– – – PAA Moderate
– ASA/Arg – PAA, UOA High
PC, OTC, CPS – – PAA, UOA High
OTC, CPS Glu/Cit – PAA, UOA Uncertain
OXO-PRO, OTC, CPS Gln/Cit – PAA, UOA, OROT Uncertain
NKHG – – PAA, CSF-Gly Low
PDH (E3), OH-PRO – Allo-Ile PAA, UOA High
H-LYS – – PAA, PAC Low
MTHFR, Cbl E, Cbl G, Cbl D v1 Met/Phe tHcy PAA, tHcy High
H-ORN, HHH – – PAA, UAA Uncertain
Untreated maternal PKU Phe/Tyr – PAA, UPTR High
HPI, HPII, LA – – PAA Low
– – – PAA, UOA, AFP Moderate
– – – PAA Low
– – SUAC PAA, UOA Moderate
PDH (E3), VAL – Allo-Ile PAA, UOA High
Untreated maternal cases (CUD, GA C0/(C16 + C18) – PCarn, PAC Moderate
I, MCAD)d
SUCLA2, untreated maternal vit B12 C3/C2 MMA, MCA, tHcy PCarn, PAC, UOA High
defd
FIGLU aciduria – – PAC, UOA Low
EE, FIGLU aciduria – EMA UAG, UOA, PAC, UAC Low
– – – see C5-OH High
EE – – UAG, UOA, PAC High
– – – UOA, PAC Uncertain
– – – see C8 see C8
untreated cases of maternal 3MCC C5-OH/C0, C5-OH/C8 – UOA, PAC, UAC Low for 3MCC, High
for other conditions
– C8/C2 – UAG, UOA, PAC High
– C3DC/C10 – UOA, PAC High
– – – PAC, PAA Uncertain
– – – See C8 See C8
– – – See C8 See C8
SUCLA2 – MMA, MCA, tHcy UOA, PAC Low
– C5DC/C5OH – UOA, PAC, UAC High
– – – See C14:1
– – – See C14:2
– – – See C5-OH
– – – See C14:1
– C14:1/C2, C14:1/C16 – PAC, UOA, DNA High
– – – See C14:1
– – – PCarn, PAC, UOA High
– – – See C16-OH
– C16-OH/C16 – PCarn, PAC, UOA High
– – – See C16
– – – See C16
– – – See C16
– – – See C16-OH
– – – See C16-OH
– – – Biotinidase in serum Moderate
– – – GALT in RBC High
– – – GALT in RBC, Gal-1-P, High

GALK/GALE in RBC
G6PD – DNA G6PD in RBC High
(continued)

Differential diagnosis
Method Informative marker Primary conditionsa Secondary conditionsa
FI- or LC-MS/MS Galactocerebrosidase (GALC) – –
Acid alpha-glucosidase (GAA) – –
Alpha-galactosidase A ( GLA) – –
Acid sphingomyelinase (ASM) – –
Alpha-iduronidase (IDUA) – –
Lysophosphatidylcholines (LPCs) – –
FIA Immune reactive trypsinogen (IRT) Cystic fibrosis –
FIA or ELISA Thyroid-stimulating hormone (TSH) Congenital hypothyroidism –
FIA or ELISA Free T4 –
FIA, RIA or 17-Hydroxyprogesterone (17OHP) 21-Hydroxylase deficiency 11-Hydroxylase deficiency
ELISA
HPLC or Hemoglobin (Hb) Sickle cell disease –
electrophoresis Hemoglobin (Hb) Beta-thalassemia –
Hemoglobin (Hb) Hb S/C disease –
Hemoglobin (Hb) – –
Quantitative PCR T-cell receptor excision circle (TREC) SCID –
FIA CD3, CD45 –
RT-PCR 22q11.2 deletion – –
Post-PCR SMN1 and SMN2 – –
high-resolution
melt profiling
Bedside testing Otoacoustic emissions (OAEs) Hearing loss –
Pulse oximetry Cyanotic heart disease –

Transcutaneous bilirubin testing – –
Measurable markers included in many NBS programs for inborn errors of metabolism and their differential diagnosis. In addition, information
about the initial follow-up tests indicated for confirmation and assessment of the urgency of initiation of follow-up are provided. Also shown are
useful secondary markers and marker ratios as well as available second-tier tests
AC sum of selected species (C0 + C2 + C3 + C16 + C18 + C18:1), AFP alpha fetoprotein, Allo-Ile allo-isoleucine, ARG arginase deficiency, ASL
argininosuccinate lyase deficiency, BIOPT (BS) GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase deficiencies, BIOPT (REG) dihy-
dropteridine reductase and pterin-4α-carbinolamine dehydratase deficiencies, BIOT biotinidase deficiency, BKT beta-ketothiolase deficiency,
CACT carnitine-acylcarnitine translocase deficiency, Cbl cobalamin metabolism defect, complementation type A/B/C/D variant 1/E or F, CBS
cystathionine beta-synthase deficiency, CIT II citrullinemia type II (citrin deficiency), CPS carbamoyl phosphate synthetase type I deficiency, CPT
I carnitine palmitoyltransferase type I deficiency, CPT II carnitine palmitoyltransferase type II deficiency, CSF-Gly glycine in cerebrospinal fluid,
CUD carnitine uptake defect, DE-RED 2,4-Dienoyl-CoA reductase deficiency, DNA molecular genetic analysis, EE ethylmalonic encephalopathy,
EMA ethylmalonic acid, FIA fluorescence immunoassay, FI-MS/MS flow injection analysis tandem mass spectrometry, GA I glutaric aciduria type
I, GA II glutaric aciduria type II, Gal-1-P galactose-1-phosphate in red blood cells, GALE UDP-galactose-4-epimerase deficiency, GALK galacto-
kinase deficiency, GALT galactose-1-phosphate uridyltransferase deficiency, G6PD glucose-6-phosphate dehydrogenase deficiency, HHH hyper-
ornithinemia-hyperammonemia-homocitrullinuria syndrome, H-LYS alpha-aminoadipic semialdehyde synthase deficiency, HMG 3-hydroxy
methyl-glutaryl-CoA synthase deficiency, H-PHE mild phenylalanine hydroxylase deficiency, HPI hyperprolinemia type I (proline oxidase defi-
ciency), HPII hyperprolinemia type II (pyrroline-5-carboxylate dehydrogenase deficiency), IBG isobutyryl-CoA dehydrogenase deficiency, IVA
isovaleric acidemia, LA lactic acidemia (primary or secondary), LCHAD/TFP long-chain 3-hydroxy acyl-CoA dehydrogenase deficiency, trifunc-
tional protein deficiency, L&P plasma lactate and pyruvate, LC-MS/MS liquid chromatography-tandem mass spectrometry, MAL malonyl-CoA
decarboxylase deficiency, 2MBG 2-methylbutyrylglycinuria, MCAD medium-chain acyl-CoA dehydrogenase deficiency, 3MCC 3-methylcroto-
nyl-CoA carboxylase deficiency, MCD multiple carboxylase deficiency, MCKAT medium-chain 3-ketoacyl-CoA thiolase deficiency, MET methio-
nine adenosyltransferase I/III, S-adenosylhomocysteine hydrolase, and glycine N-methyltransferase deficiencies, 3MGA 3-methylglutaconic
aciduria type I, 2M3HBA 2-methyl 3-hydroxybutyric aciduria, MSUD maple syrup urine disease, MTHFR methylenetetrahydrofolate reductase
deficiency, MUT methylmalonyl-CoA mutase deficiency, NKHG non-ketotic hyperglycinemia, OAT ornithine aminotransferase deficiency, OH-
PRO hydroxyprolinemia (hydroxy-L-proline oxidase deficiency), OROT urine orotic acid, OTC ornithine transcarbamylase deficiency, OXO-PRO
5-oxoprolinemia (glutathione synthetase deficiency), PAA plasma amino acids, PAC plasma acylcarnitines, PC pyruvate carboxylase deficiency,
PCarn plasma carnitine, PDH (E3) pyruvate dehydrogenase component E3 deficiency (dihydrolipoamide dehydrogenase deficiency), PKU phe-
nylalanine hydroxylase deficiency, RBC red blood cells, RIA radioimmunoassay, SCAD short-chain acyl-CoA dehydrogenase deficiency, S/MCHAD
short-/medium-chain acyl-CoA dehydrogenase deficiency, SUCLA2 succinyl-CoA synthase deficiency, tHcy total homocysteine, TYR I tyrosin-
emia type I (fumarylacetoacetase deficiency), TYR II tyrosinemia type II (tyrosine aminotransferase deficiency), TYR III tyrosinemia type III
(4-hydroxyphenylpyruvate dioxygenase deficiency), UAA urine amino acids, UAC urine acylcarnitines, UOA urine organic acids, UPTR urine
pteridines, VAL valine transaminase deficiency, VLCAD very long-chain acyl-CoA dehydrogenase deficiency, WBC white blood cells
a
Primary and secondary conditions based on current recommendations by the US Secretary of Health and Human Services
b
Tests performed in other than dried blood spot samples; may include additional tests when newborn is clinically symptomaticc
c
Further information on follow-up requirements can be found at: http://www.ncbi.nlm.nih.gov/books/NBK55832/
d
Detection is possible because of secondary carnitine deficiency of the newborn
Urgency of clinical
Other conditions Critical ratio Second-tier test Initial confirmatory testingb actionc
Krabbe disease – DNA GALC in WBC, DNA High
Pompe disease – DNA GAA in WBC, DNA Moderate
Fabry disease – DNA GLA in WBC, DNA Moderate
Niemann-Pick A, B – DNA ASM in WBC, DNA Moderate
MPS I – DNA IDUA In WBC, DNA Moderate
X-ALD – VLCFA Moderate
– – DNA Sweat chloride, DNA Moderate
– – – TSH, free T4 High
– – TSH
– – Steroid profile serum 17OHP, electrolytes, High
glucose
other Hb-pathies – – Hgb electrophoresis, Hgb, CBC Moderate
Hgb H – –
– – – T- and B-cell quantitation High
– – –
22q11.2 DS – FISH, microarray High
SMA – Moderate
– – Auditory Comprehensive, High

brainstemresponse multidisciplinary hearing loss
(ABR) evaluation
– – Echocardiogram cardiology exam High
Hyper-bili/Kernicterus – – in-/direct plasma bilirubin High
46.5 Before and After NBS: Roles of the for families if and when the result is abnormal for another
Hospitals and Health Care Providers condition). Families need to understand that screening is
based on comparing blood levels in their baby to standard
Hospitals and health care providers should comply with all values and that any baby with a level outside the expected
applicable laws and regulations in their country, state, and range is called back for additional testing. Families should be
locality and be aware of and be prepared to be judged by any made aware that, although major improvements have reduced
applicable professional guidelines pertaining to NBS. the false-positive rate of NBS, statistically it remains more
Responsibilities of the hospitals and health care providers likely that a “positive” or abnormal result will ultimately
involve communication (with family, with each other, and prove to be a false positive, because of the need to design
with the screening laboratory and follow-up team), collec- screening programs to minimize the false-negative rate. The
tion and transport of samples, appropriate response to any family must also understand the importance of providing
abnormal results, documentation, and provision of informa- correct contact information so they can be reached if there is
tion as requested for public health follow-up and program an abnormal result.
QI/QC.
A useful analogy for families to help explain screening
for families who may have limited understanding of
46.5.1 Before Screening
statistics or biology is comparison to the eye chart
screening for vision done for school-age children and
In some places and programs, formal written consent is
easily recalled by most parents. Remind them that they
required before screening is performed, while most programs
have experienced screening when they were asked as a
consider NBS mandatory and part of routine standard of care
child to say which direction a letter on a chart (typi-
without requirement for written consent. Many programs
cally in English an “E”) is facing. Point out that if a
permit opting out of NBS for religious reasons, and some
child gives an incorrect answer on the vision screening
permit opt out for personal reasons, with documentation in
test, we don’t know if the child could not see the chart
such cases of family awareness of the risks to baby when
well or was simply not paying attention. When a child
NBS is not done. Regardless of whether consent is required,
“fails” the eye test, we do not know if a child really
families should be informed about NBS before it is done,
needs glasses until more testing is done. Explain that
including the purpose and value, what it can and cannot
the NBS is similar, in that when a baby “fails” the
detect, the time frame for testing and results, the possible
NBS, we do not yet know for certain whether the baby
outcomes of testing, and what the laboratory or program
really has a medical problem, but we do need to test the
does with any remaining sample. Ideally families will have
baby more specifically to find out if special treatment
been educated about NBS before time of delivery, however
is needed to keep the baby healthy.
studies have generally revealed in the USA that many fami-
lies are not aware of NBS even after the testing has been
performed (Tluczek et al. 2009). It is the responsibility of the Across the world, families are increasingly concerned
baby’s health care providers to assure that the family is aware with issues of confidentiality and the fate of any residual
of screening before the testing is performed, and most NBS blood spot sample. Families should be informed about the
programs provide educational materials for both health care policy of the program in which they are participating with
providers and families to assist in this process. Studies of respect to sample storage and any research use of the residual
health care providers (Davis et al. 2006) unfortunately show sample. For families interested in details, it will be important
significant deficits in knowledge and communication with to clearly distinguish between non-research uses that are
respect to NBS, and NBS programs should continue to make required as part of laboratory quality control and research.
efforts to educate providers so that accurate information is When the NBS program offers the opportunity to make
provided to families. If and when a local NBS program offers choices about any research use of residual sample, that infor-
a panel of tests that does not include all the testing recom- mation should be provided to the family.
mended by the health care provider’s professional guide- The NBS sample should be collected at the appropriate
lines, the health care provider should fulfill his or her time, according to the policy of each program. In general, for
professional responsibility by informing family about any programs using a single-screen system, the sample should be
supplemental testing that would complete the recommended collected when the baby is between 48 and 72 h old. This
testing. will (a) increase the likelihood of detection of conditions for
Some key elements of information to share with fami- which marker metabolite levels rise when they are no longer
lies are that NBS tests for a variety of conditions (in some cleared by the placenta and/or precursors are ingested and
places the NBS is still called “the PKU,” causing confusion (b) decrease the likelihood of some false positives that can
occur as a result of the normal physiologic changes around should be sent within 24 h of collection, and, in order to
birth. However, if a baby is ill and there is any possibility that reach the laboratory expeditiously, overnight delivery or
transfusion will be needed, a NBS sample should be col- courier delivery is preferred. Samples should be dry before
lected and sent immediately. Specific recommendations they are packed for transport, to minimize the likelihood of
about timing of sample and whether a repeat sample is degradation of enzyme function if exposed to excessive heat
required may vary, and the health care provider should famil- during transport.
iarize him or herself with local practice and standards for
ordinary and for special situations (AAP 2008). Professional
guidelines in the USA are that if a first sample was collected 46.5.2 What to Do When a NBS Is Not Normal?
before 24 h, a second sample should be collected.
Some programs require a second screen to be collected Each laboratory performing NBS testing has protocols for
between 10 and 14 days of age. In the USA, programs with informing the submitter of the sample and, if different, the
this requirement observe that a significant fraction of babies responsible health care provider if the NBS sample is not
with hypothyroidism are detected only on the second screen acceptable or if the results are nor normal.
(Maniatis et al. 2006). Methodology does not affect the per-
cent of babies with hypothyroidism detected only on second 46.5.2.1 Inadequate Samples
screen, since this results from the pathophysiology of The sample may be completely inadequate for any testing, as
hypothyroidism. when a sample arrives in the laboratory contaminated with
NBS samples should be collected in strict compliance alcohol or with separation of serum and red cells. Other sam-
with instructions of the local NBS program, typically by heel ples may be adequate for some testing and not adequate for
stick and dropping sample directly from heel as individual others, as when sample was collected before any enteral
drops on to appropriate filter paper provided by the labora- feeding for a baby who required early transfusion or the fol-
tory and which usually includes detailed instructions on low-up sample on the same baby after transfusion. If sample
sample collection. All alcohol should be wiped off heel to was not completely adequate for any reason, the health care
avoid dilution, and sample should not be overblotted or lay- provider should follow instructions of the laboratory regard-
ered. Collection into capillary glass or from venipuncture is ing when another sample is needed. A repeat sample may be
not in compliance with protocol in most jurisdictions. While needed immediately if sample was simply inadequate, or
there may be only small differences between the values of repeat sample may be needed in several months if baby was
the various metabolites in capillary or venous blood and transfused before any sample was collected, in order to
although there is very little change in levels from exposure to screen for conditions such as hemoglobinopathies or galac-
glass or plastic collection materials, small differences could tosemia for which transfusion confounds the screening.
be important in the screening setting where ranges are finely
tuned for small differences. Failure to comply with the stan- 46.5.2.2 Abnormal Results
dards of each program could expose the baby to failure of the If testing yields an abnormal result, the laboratory and short-
screening system and the health care practitioner to liability. term follow-up program will inform the submitter of the
Complete and legible information should be provided to sample or the primary care provider when known. If neces-
the laboratory, in compliance with the NBS program’s guide- sary, the NBS program will directly contact the family. In
lines. Laboratories need appropriate identifying information many programs certain abnormal results are automatically
for the baby, and at minimum information such as birth date, shared also with specialists in relevant disciplines (inborn
birth weight, gestational age, type and number of hours of errors of metabolism, endocrine, immunology, or hematol-
feeding, and hours of life at which sample was collected or ogy) to assist with next steps in evaluation. The health care
information (such as time of birth and time of sample collec- team should be provided with details of the laboratory results
tion) that will permit the laboratory to calculate that critical that will be combined with all relevant clinical information
information. Laboratories need to know whether the baby to determine next steps. In some cases, the laboratory infor-
has been transfused. Laboratories need identifying and con- mation alone is sufficient to select a course of action, espe-
tact information for the family, for the submitter of the sam- cially when a combination of laboratory results (e.g., first- and
ple, and for the health care provider who will be responsible second-tier results) is sufficiently informative that the only
for any follow-up. reasonable cause for a false-positive test would be sample
Samples should reach the laboratory in a timely fashion. mix-up. In some cases the laboratory may be able to catego-
Samples should never be batched at the originating hospital rize results with respect to the likelihood of an abnormal
or office before transport, as the conditions for which babies result being a true or a false positive, and clinical information
are screening include a number for which delay of screening may drive the pace and dictate some elements of the diagnos-
and diagnosis is deadly. Most programs agree the sample tic process.
Many NBS results are emergencies, requiring that baby

be immediately located, examined, and tested. A baby with shows one mutation for a mild variant (e.g., the Duarte
galactosemia, organic acidemia, MSUD, urea cycle disorder, mutation). The baby is immediately located, the pedia-
fatty acid oxidation disorder, or CAH could already be symp- trician had examined the baby yesterday, and the baby
tomatic and is at risk for imminent collapse and death. If the has already regained birth weight, and on the telephone
NBS result suggests a condition for which diagnostic evalu- the experienced and breast feeding mom reports that
ation can be safe after a wait of a day or two, a program’s baby is healthy and feeding well. Baby may have a
protocol may suggest that it could be appropriate for the mild variant or be a carrier, and diagnostic testing can
family to learn about the abnormal NBS result on the first be planned for the next working day, and diet does not
day that will allow the family to begin diagnostic testing need to be changed.
immediately to minimize their anxiety.
It will not be possible here to describe all possible results
of NBS and specify appropriate responses and diagnostic 46.5.2.3 Waiting for Results
evaluations, both because of the number of possibilities and While waiting for results of confirmatory evaluation for any
because of the rapid changes in the field. To facilitate appro- abnormal NBS, the baby should be observed for any signs
priate follow-up of abnormal NBS results, the American and symptoms of the condition. For any condition in which
College of Medical Genetics has developed ACT sheets and catabolism poses a threat, family should be advised to make
laboratory follow-up algorithms that are freely available on certain baby feeds well on a normal newborn schedule, at
the website of the US National Library of Medicine (http:// least every 3–4 h, and should call health care providers if
www.ncbi.nlm.nih.gov/books/NBK55832/) (ACMG 2013). baby is refusing to eat or not tolerating feeding. Except
In addition, a collaborative effort organized by the New when there is a high suspicion for classic galactosemia, diet
York-Mid-Atlantic Consortium explored in more detail should not be changed, and other specific treatment should
issues around diagnostic evaluation for conditions detected not be initiated without some confirmatory information.
by NBS (Kronn et al. 2010), and individual NBS programs Diagnostic confirmation indicating need to institute spe-
may have specific guidance for follow-up after an out-of- cific therapy could be a combination of diagnostic proof
range result. For information on the diagnosis of specific (such as amino acids in maple syrup urine disease) or
conditions, also see the appropriate chapters in this book. observation of clinical symptoms and supporting signs
Below, contrasting examples will serve here to illustrate (such as altered consciousness, acidosis, and positive urine
important principles and variables. ketones suggesting MSUD even while amino acid results
are pending). Caution should be exercised in overinterpre-
tation of minor symptoms such as ordinary small emesis
Contrasting Examples Illustrate Level of Risk for Baby due to reflux.
with Abnormal Screen for Galactosemia Families are under stress while waiting for results of
The NBS is reported on Saturday afternoon to the testing. Studies show that during this period families’ needs
health care provider for a now 5-day-old baby as for information and support vary; some would like infor-
abnormal for galactosemia; the laboratory screens for mation about the condition baby may have while others
both total galactose (TGAL) and Gal-1-PUT (GALT, prefer to wait to learn about the condition only if baby
using Beutler test) activity: proves to be affected (Schmidt et al. 2012). All families
Example 1: GALT activity is absent and TGAL is need to know what to expect during the diagnostic evalua-
elevated (with or without DNA confirmation on the tion, including what testing needs to be done (what kind of
screen). It is winter and there is no reason to expect the sample needs to be collected and when, where, and how),
sample was subjected to heat. Baby is presumed how long it will be until the results are back, and whether
affected and must be immediately located and exam- there is anything they need to watch for or to do. Families
ined; diagnostic testing (enzyme assay, may include need to be offered the opportunity to ask questions and be
also DNA) must be immediately sent. On the phone offered access to accurate information; websites that offer
mom says baby is fine, feeding well. Nevertheless, information about NBS and inborn errors of metabolism
baby should be called in and examined by a health care can be accessed through various federal and state portals
provider, and, in addition to sending diagnostic testing, around the world.
baby should have liver enzymes and bilirubin checked,
and diet must be immediately changed to exclude all 46.5.2.4 After the Results of Diagnostic Testing
galactose, even if mom is committed to breast feeding. Results of diagnostic testing may be completely normal, per-
Example 2: GALT activity is absent but TGAL is mitting baby to be dismissed from further follow-up.
normal, and DNA was done as a second-tier test and Evidence suggests that most families’ questions and concerns
are immediately resolved by clear explanation; however,
some have lingering concerns or stress as a result of the (pseudodeficiency) or highly variable age of onset, or if
experience of false-positive NBS (DeLuca et al. 2011). All there can be uncertainty as to whether two mutations are in
families therefore should be encouraged to ask any addi- cis or trans (requiring parental investigations) (Chen et al.
tional questions and be offered support from primary care 2009; Choi et al. 2010; Schmidtke and Cassiman 2010). For
providers in case of any lingering doubts or concern. Given these reasons, it is important that practitioners follow the
the wide variability in the presentation and difficulties in the guidelines of their local programs and professional organi-
diagnosis in some cases, some families will not receive a zations and stay in communication with the relevant special-
clear answer about their baby’s status after one round of ists in their region.
diagnostic testing, and during the process of ongoing evalua-
tion, families will need to be offered ongoing support in
accord with each family’s needs, typically involving direct 46.5.4 What Does Screening Test For?
communication with the same team that would be caring for
the baby if and when the baby is proven affected. The conditions for which newborns are screened vary from
Finally, the NBS program will require information from country to country and often within countries. For example,
the health care provider about results of diagnostic testing in the USA each state has authority and responsibility to
and patient outcomes in order to assure completion of the determine the conditions for which infants born in their juris-
NBS process and to provide for quality control and improve- diction will be screened, while health care professionals have
ment of the NBS systems. the responsibility to offer additional screening if the state’s
program does not offer a panel that satisfies professional
guidelines. A list is likely out of date as soon as published, as
46.5.3 Special Issues in Strategies NBS panels are currently expanding, and occasionally con-
for Testing After Abnormal NBS ditions are removed from an NBS panel if screening did not
prove successful. Table 46.4 summarizes the inborn errors of
In some cases a condition for which screening is conducted metabolism on a recommended uniform screening panel
is not expected to cause symptoms in the neonatal period, (RUSP) in the USA.
and testing after the abnormal screen is solely diagnostic; For information about the full list of primary and second-
PKU is an example of such a condition. ary conditions currently included in the recommended uni-
For other conditions, a baby may already have dangerous form screening panel (RUSP) in the USA, the reader is
laboratory abnormalities at the time that NBS abnormality is referred to the website of the Secretary’s Advisory Committee
reported. In addition to diagnostic testing, appropriate test- on Heritable Disorders in Newborns and Children
ing for these abnormalities is part of the follow-up; examples (SACHDNC) at (DHHS 2013) (http://www.hrsa.gov/adviso-
include electrolytes after abnormal NBS for CAH or for rycommittees/mchbadvisory/heritabledisorders/index.html).
MSUD and examination of urine for ketones after abnormal Demonstrating the individual states’ independence and
screen for MSUD or MMA. As discussed in the previous ability to ignore federal recommendations, Krabbe disease is
section, resources are available with guidance for analyte- already screened for in two states (MO, NY) despite the
and disease-specific follow-up. SACHDNC finding this condition to lack sufficient evidence
Diagnostic testing after screen is positive for an IEM his- in support of population screening.
torically has been to formally test for the metabolites of the
inborn error, in blood and/or urine, and to use diagnostic
enzyme assays when necessary to clarify inconclusive bio- 46.5.5 Residual NBS Samples,
chemical results. In some conditions, the biochemical phe- QC, and Research
notype may be normal at the time of follow-up. Very
long-chain acyl-CoA dehydrogenase (VLCAD) deficiency When the laboratory has completed testing for each baby,
is an important example (Boneh et al. 2006; Spiekerkoetter there may be sample remaining, typically called “residual
et al. 2010), in which the elevated levels of analytes that sample.” National and local laws and regulations that govern
make NBS possible may return to normal by several days of laboratories, including NBS laboratories, typically specify a
age, as the baby is eating normally. In such instances, an minimum retention of residual samples to assure accuracy of
enzyme assay may be useful if it can be done on accessible testing. For example, in some cases samples must be retained
sample, and molecular genetic testing can be extremely at least until the next laboratory quality control activity,
helpful when two mutations are found. But even DNA anal- while in other cases samples must be retained for a spe-
ysis can be inconclusive with respect to diagnosis and/or cific length of time. Some NBS programs are governed by
need for early treatment of disease when the mutations specific laws that address sample retention. Laboratory
found are not known to cause disease (variants of uncertain quality control and quality improvement are regular activities
significance), are associated with a normal phenotype of all laboratories and required by regulations.
Table 46.4 United States of America: IEM that are core conditions on the recommended uniform screening panel (as of April 2013)
Carbohydrate,
Organic acid Fatty acid Amino acid endocrine, or other
Core condition disorder oxidation disorder disorder disorders
Propionic acidemia X
Methylmalonic acidemia (methylmalonyl-CoA mutase) X
Methylmalonic acidemia (cobalamin disorders) X
Isovaleric acidemia X
3-Methylcrotonyl-CoA carboxylase deficiency X
3-Hydroxy-3-methyglutaric aciduria X
Holocarboxylase synthase deficiency X
ß-Ketothiolase deficiency X
Glutaric acidemia type I X
Carnitine uptake defect/carnitine transport defect X
Medium-chain acyl-CoA dehydrogenase deficiency X
Very long-chain acyl-CoA dehydrogenase deficiency X
Long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency X
Trifunctional protein deficiency X
Argininosuccinic aciduria X
Citrullinemia, type I X
Maple syrup urine disease X
Homocystinuria X
Classic phenylketonuria X
Tyrosinemia, type I X
Congenital adrenal hyperplasia X
Biotinidase deficiency X
Classic galactosemia X
Research activities are separate from laboratory quality mia. In other conditions, the use of DNA testing either as a
control and subject to additional or separate regulations in primary or secondary test in NBS will identify carriers.
most programs. Regulations vary widely with respect to what Finding carriers on NBS challenges the NBS program and
kinds of research, if any, may use residual NBS samples. Most system to provide the families affected with appropriate
research is carried out using de-identified samples. In some information and genetic counseling.
venues, use of de-identified samples may be permitted with a In addition, in some cases – where prenatal screening for
waiver of consent while in other programs such use is not carrier testing has been done – NBS may lead to recognition
permitted. Around the world, there is general agreement that of nonpaternity, causing another kind of stress on family and
use of identified human samples in research should be subject on the NBS system. When dealing with NBS results, the
to consent, with specified exceptions under the governance of health care provider should be aware of this possibility and
publicly responsible committees. There are unresolved ques- be prepared to communicate appropriately with a family. In
tions of how to proceed with consent for use of identifiable addition, the possibility that what appears to be nonpaternity
residual NBS samples, and care must be taken to work in could be the result of new mutation, uniparental isodisomy,
compliance with local laws and regulations. Transparency or other cause of discrepancy between prenatal carrier test-
and involvement of the community in decisions about research ing of parents, and NBS of the baby should be considered.
use of residual NBS samples is encouraged in all cases.
46.5.6.2 Point-of-Care Testing
Hearing screening and testing of oxygen saturation for cya-
46.5.6 Special Issues and New notic critical congenital heart defects is increasingly incor-
Challenges in NBS porated into NBS programs. Because these are not tests for
inborn errors of metabolism or performed on blood spot,
46.5.6.1 Carrier Identification they are not the subject of this chapter. However, the health
In some cases, carriers of inborn errors may be identified care practitioners and NBS policy makers should be aware
by NBS. In certain conditions, some carriers may have that the incorporation of this testing into NBS programs
elevation of analytes on NBS, as may be seen in some fatty means that the programs responsible for traditional NBS
acid oxidation disorders and in some carriers of galactose- are faced with another level of complexity to ensure that
point-of-care testing has been completed and recorded and CDC (Domestic Public Health Achievements Team) (2011) Ten great
that indicated follow-up has been completed. public health achievements – United States, 2001–2010. MMMR
Mortal Mon Rep 60:619–623
Chen B, Gagnon M, Shahangian S et al (2009) Good laboratory prac-
46.5.6.3 Whole Exome, Late Onset Conditions, tices for molecular genetic testing for heritable diseases and condi-
and Common Complex Conditions tions. MMMR Mortal Mon Rep 58(RR6):1–37
Technology continues to advance, offering both opportunity Choi JH, Velayati A, Stubblefield BK et al (2010) False-positive results
using a Gaucher diagnostic kit – RecTL and N370S. Mol Genet
and challenge. Pilot programs for NBS for future risk of dia- Metab 100(1):100–102
betes have been carried out (Barker et al. 2004), demonstrat- Comeau AM, Accurso FJ, White TB et al (2007) Guidelines for imple-
ing feasibility for testing for a common complex disorder mentation of cystic fibrosis newborn screening programs: Cystic
with variable age of onset. Whole exome and whole genome Fibrosis Foundation Workshop Report. Pediatrics 119(2):e495–e518
Davis TC, Humiston SG, Arnold CL et al (2006) Recommendations
methodology and interpretation are becoming less expensive for effective newborn screening communication: results of focus
and faster and can be carried out with increasingly smaller groups with parents, providers and experts. Pediatrics 117(5 Pt
samples. While the limitations of DNA methodology will 2):S326–S340
remain an issue and testing for metabolites should be DeLuca JM, Kearney MH, Norton SA, Arnold G (2011) Parents’ expe-
riences of expanded newborn screening evaluations. Pediatrics
expected to remain a critical element of NBS for IEM, the 128:53–61
NIH in the USA has begun funding of projects to explore Department of Health and Human Services (2013) Recommended uni-
specifically the feasibility of whole exome and/or genome form screening panel. http://www.hrsa.gov/advisorycommittees/
strategies in NBS. Issues being addressed will include both mchbadvisory/heritabledisorders/recommendedpanel/index.html.
Accessed June 2013
practical and ethical considerations, such as the increased Driscoll CJ, McPherson B (2010) Newborn screening: history, princi-
carrier detection and the detection of newborns with adult- ples and analysis. In: Driscoll CJ, McPherson B (eds) Newborn
onset disorders (such as Huntington disease) or with risk fac- screening systems: the complete perspective. Plural Publishing, San
tors for adult-onset conditions (such as breast cancer). Diego/Abingdon/Oxfordshire
Hoffmann GF, Cornejo V, Pollitt R (2010) Newborn screening
– progress and challenges. J Inherit Metab Dis 33(Suppl 2):
Conclusion S199–S200
Current NBS programs have built on more than a half- Kemper AR, Boyle CA, Dougherte D et al (2008) Long-term follow-up
century of scientific discovery, technologic progress, and after diagnosis resulting from newborn screening: statement of the
US Secretary of Health and Human Services Advisory Committee
public health commitment. NBS is a system involving on Heritable Disorders and Genetic Diseases in Newborns and
multiple participants and has achieved great success in Children. Genet Med 10(4):259–261
prevention of death and disability around the world. Kronn D, Mofidi S, Braverman N, Harris K (2010) Diagnostic guide-
Advances in medical science, as well as changes in the lines for newborns who screen positive in newborn screening. Genet
Med 12:S251–S255. doi:10.1097/GIM.0b013e3181fe5d8b
health care delivery systems around the world, challenge Maniatis AK, Taylor L, Letson GW et al (2006) Congenital hypothy-
us to continue to provide and to improve the quality NBS. roidism and the second newborn metabolic screening in Colorado,
USA. J Pediatr Endocrinol Metab 19(1):31–38
Maris JM, Woods WG (2008) Screening for neuroblastoma: a resur-
rected idea? Lance 371(9619):1142–1143
References Riley RD, Burchill SA, Abrams KR et al (2003) A systematic review
and evaluation of the use of tumour markers in paediatric oncology:
American Academy of Pediatrics Newborn Screening Authoring Ewing’s sarcoma and neuroblastoma. Health Technol Assess 7(5)
Committee (2008) Newborn screening expands: recommendations Ross LF (2008) Newborn screening for cystic fibrosis: a lesson in pub-
for pediatricians and medical homes – implications for the system. lic health disparities. J Pediatr 153(3):308–313
Pediatrics 121:192–217 Schmidt JL, Castellanos-Brown K, Childress S et al (2012) The impact
American College of Medical Genetics Newborn Screening Work of false-positive newborn screening results on families: a qualitative
Group. ACMG ACT sheets and confirmatory algorithms. http:// study. Genet Med 14(1):76–80
www.ncbi.nlm.nih.gov/books/NBK55829/. Accessed June 2013 Schmidtke J, Cassiman J-J (2010) The EuroGentest clinical utility
Andermann A, Blancquaert I, Beauchamp S, Dery V (2008) Revisiting gene cards. Eur J Hum Genet 18(9):1. doi:10.1038/ejhg.2010.85. At
Wilson and Jungner in the genomic age: a review of screening crite- http://www.eurogentest.org/professionals/public_health/
ria over the past 40 years. Bull World Health Organ 86(4):317–319. info/public/unit3/geneCards.xhtml. Accessed Oct 2012
doi:10.2471/BLT.07.050112 Spiekerkoetter U, Haussmann U, Mueller M et al (2010) Tandem mass
Barker JM, Barriga KJ, Yu L et al (2004) Prediction of autoantibody spectroscopy screening for very long-chain acyl-CoA dehydroge-
positivity and progression to type 1 diabetes: Diabetes Autoimmunity nase deficiency: the value of second-tier enzyme testing. J Pediatr
Study in the Young (DAISY). J Clin Endocrinol Metab 89: 157(4):668–673
3896–3902 Tluczek A, Orland KM, Nick SW, Brown RL (2009) Newborn screen-
Boneh A, Andresen BS, Gregersen N et al (2006) VLCAD deficiency: ing: an appeal for improved parent education. J Perinat Neonatal
pitfalls in newborn screening and confirmation of diagnosis by Nurs 23(4):326–334
mutation analysis. Mol Genet Metab 88(2):166–170 Wilson JMG, Jungner G (1968) Principles and practice of screen-
Brosco JP, Sanders LM, Dharia R et al (2010) The lure of treatment: ing for disease. WHO, Geneva. http://www.who.int/bulletin/
expanded newborn screening and the curious case of histidinemia. volumes/86/4/07-050112BP.pdf. Accessed March 2013
Pediatrics 125(3):417–419
Genetic Counseling for Inborn Errors
of Metabolism 47
Johannes Zschocke and Sigrid Tinschert

47.1 Introduction ..................................................................... 737
47.2 General Aspects of Genetic Counseling ........................ 737 The diagnosis of any inherited disease has implications not
only for the affected individual with regard to understanding
47.3 Nondirectivity .................................................................. 738
of clinical features, prognosis, possible complications, and
47.4 Genetic Counseling Within the Context long-term management, but also for other family members
of Molecular Genetic Testing ......................................... 738
who may have an increased probability of being affected
47.5 Carrier Testing ................................................................ 739 with the same or a related condition. Patients, parents, and
47.6 Molecular Genetic Testing in Children ......................... 739 other relatives need to understand the genetic basis of an
inborn error of metabolism and associated risks in the family
47.7 Prenatal Molecular Diagnosis ........................................ 740
in order to actively cope with the condition and to make deci-
47.8 Predictive Testing ............................................................ 741 sions that are right for them. Genetic counseling is the pro-
References ...................................................................................... 741 cess of providing the necessary individualized information
about a genetic condition and its implications and to assist in
decision-making in a nondirective manner (Harper 2010).
Genetic counseling therefore deals not only with the medical
aspects of a genetic disease but also with its social and psy-
chological consequences and the possibilities and strategies
for coping with genetic diseases and genetic risks. In a
genetic consultation the physician does not focus on the
patient alone but on the entire family, including other family
members who might already be affected as well as those who
might be affected in the future. Genetic counseling may
sometimes be emotionally challenging and is always time-
consuming. Frequently there are unfounded feelings of
“responsibility” and guilt for having transmitted the disease,
which need to be addressed. Sometimes more than one coun-
seling session is required in order to gradually introduce the
client to the personal significance of the various aspects of
the counseling topic.
47.2 General Aspects of Genetic

Counseling
The purpose of the genetic counseling session with regard

J. Zschocke (*) • S. Tinschert
to inborn errors of metabolism is to help the counselee to
Division of Human Genetics, Medical University Innsbruck,
Schöpfstr 41, Innsbruck 6020, Austria understand the genetic context and risks associated with
e-mail: johannes.zschocke@i-med.ac.at a particular disease. This also entails the discussion of
738 J. Zschocke and S. Tinschert
recurrence risks in future siblings of an affected child up 47.3 Nondirectivity

to the strategies, implications, and challenges of prenatal
diagnosis. The aim of many medical treatments is concurrently evident
The setting in which counseling takes place differs from to the physician and the patient, and the patient generally
country to country. In the United States, for example, clinical authorizes the doctor to decide the best course of action.
genetics and the care for children with inborn errors of Such is the case, for example, with emergency treatment
metabolism are highly integrated, while in Germany and after an accident. By contrast, some decisions with regard to
some other European countries there is limited contact genetic conditions such as the implications for future preg-
between metabolic physicians and clinical geneticists. nancies are difficult and closely tied to personal moral con-
Individual arrangements must be made in all centers to make cepts. In such situations it is a major objective of genetic
sure that patients and their families receive optimal genetic counseling to turn the patient into “a specialist for his/her
counseling services. own disease” through clarification, advice, and information.
A clear and etiologically defined diagnosis is the essential With due consideration for the medical contexts and risks,
basis for correct genetic counseling. Previous clinical reports the patient should be able to decide on the personally appro-
and other records should be obtained to accurately assess all priate course of action. Not the physician but the counselee
available information in the affected individual. Disease- decides whether or not to have predictive testing or prenatal
related genetic information in the family is collected by diagnostics done. As far as ethical considerations permit,
drawing up a pedigree tracing back at least three generations. the autonomy of the counselee should not be called into
The pedigree should include information about chronic dis- question, only challenged in order to promote
eases, occurrence of birth defects, developmental and/or consideration.
unusual behavioral profiles, unexpected deaths, miscar- An important aspect of genetic counseling therefore is its
riages, ethnic background of the family, and possible consan- nondirectivity. Genetic counseling should impart knowledge
guinity. Photographs of family members are especially useful and competence; the decision of the counselee should not be
when attempting to determine whether a particular physical influenced by the personal opinion or the judgment of the
feature may be a diagnostic clue or is part of the family’s counselor. Frequently it is difficult or impossible to counsel
phenotypic background. Clinical geneticists have a special in a completely nondirective manner; even the selection of
expertise in the assessment of morphological anomalies and terms used in the conversation may reflect the judgment of
may more easily recognize physical features that provide the counselor (grave, unfortunately, hopefully, etc.).
diagnostic clues. However, there are few inborn errors of Nevertheless, nondirectivity should remain the objective of
metabolism that manifest as dysmorphic syndromes, and the entire counseling process with regard to individual
most frequently the exact diagnosis has been made by the decisions.
time an appointment for genetic counseling is made.
Information is an essential component for coping and is
particularly relevant for rare genetic diseases for which the 47.4 Genetic Counseling Within the
general knowledge is limited. It is extremely important to Context of Molecular Genetic Testing
inform the counselee about the disease, its prognosis, and its
heritability in an intelligible and individually appropriate If clinical examination suggests a possible genetic disease
way. Important details should be presented in simple terms and in case a molecular-genetic test is available, the patient
and explained with simple comparisons. An essential com- should be informed of the implications of a positive test
ponent of genetic counseling is a detailed and individualized result before the test is done. This is the responsibility of any
counseling report addressed to the counselee. This report physician ordering such a diagnostic test. In some countries
summarizes the main aspects of the counseling session in only a geneticist is allowed to order genetic analyses. If the
plain language; a copy should be sent to the primary physi- suspected genetic disease is confirmed, the patient should be
cian. The counselee should be given contact information for offered genetic counseling to discuss the implications of the
a suitable support group for the particular condition. Many finding in detail, including the implications for other family
patients or parents initially reject such a proposal because members or future prenatal diagnostics. Failure to offer such
they feel that they have to first come to terms with their own counseling may be regarded as malpractice. If, for example,
situation and worry about being burdened with the problems after one child with a genetic disease a second affected child
of others. In the long term, however, talking and exchanging is born to a couple who did not receive formal genetic coun-
information with others who have similar experiences quite seling including a counseling letter, they could argue that
often is beneficial. In addition, psychological support should they would have requested prenatal diagnostics had they
be offered when appropriate. been adequately informed.
47 Genetic Counseling for Inborn Errors of Metabolism 739
47.5 Carrier Testing probability in a child. In case of consanguinity or if both

partners are from the same genetically homogenous small
Carrier testing is used to identify individuals who are appar- community, it is justified to test the partner of a confirmed
ently healthy but carry a pathogenic gene mutation in a het- carrier for the mutation present in his/her spouse.
erozygous state and have a risk of transmitting it (Young In X-linked conditions all daughters of an affected male
2006). It should be weighed carefully whether carrier testing are obligatory carriers. They have a 50 % probability of hav-
in a specific situation is useful and appropriate. In addition to ing an affected male offspring, independently of the carrier
individuals who are at risk of being carriers, there are obli- status of the (healthy) partner. Mothers of an affected male
gate carriers who can be identified by formal genetic reasons are not necessarily carriers if the family history is empty
alone, rendering a testing procedure unnecessary. Regardless since the mutation may have arisen de novo. However,
of the significance of carrier testing for family planning, tests X-chromosomal mutations occur with a higher probability in
may also be performed for traits which are potentially harm- the male germline. For many conditions the de novo mutation
ful for the heterozygote carriers themselves. This is most has more likely arisen in the maternal grandfather, with the
obvious in females with X-linked conditions such as orni- mother being heterozygous. Carrier testing for X-chromosomal
thine transcarbamylase (OTC) deficiency which may predis- metabolic disorders in female family members is particularly
pose heterozygous women to an acute hyperammonemic important for treatable conditions where heterozygosity is
crisis, or X-chromosomal adrenoleukodystrophy which may associated with an increased risk of acute metabolic crises
be associated with late-onset spastic paraparesis or other such as in OTC deficiency. Molecular testing is the method of
neurological symptoms. In some of these conditions, there is choice because in contrast to other (enzymatic, metabolic)
no effective preventive treatment, and testing should follow methods of carrier testing, it is independent from the effects
the guidelines for predictive testing outlined below. Carrier produced by X-chromosome inactivation.
status in other conditions may constitute a risk factor for In autosomal dominant diseases the risk for an offspring
multifactorial diseases such as vascular disease in carriers of a carrier to be affected is high and is 50 % in fully pene-
for homocystinuria. trant conditions. It is important to recognize pseudodominant
In case of autosomal recessive diseases, the parents and inheritance patterns caused by a high population prevalence
all children of an affected individual are obligatory carriers. of functional polymorphisms such as in erythropoietic proto-
The chance of being a carrier for healthy siblings of an porphyria. Carrier testing is not necessary in individuals who
affected individual is two-thirds (provided that the disease have both an affected parent and an affected child and there-
can be excluded) and 50 % for second-degree relatives; each fore are obligate carriers.
generation step further reduces the risk by 50 %. Exclusion
of carrier status in a spouse in a confirmed or likely hetero-
zygote for an inherited metabolic disease is a frequent reason 47.6 Molecular Genetic Testing in Children
for genetic counseling. The risk of being a carrier is some-
times overestimated. It may be calculated with the Hardy- Children should only undergo molecular-genetic testing if
Weinberg formula which stipulates that the carrier frequency this will be of direct and immediate medical or psychologi-
in large populations with a low rate of consanguineous mar- cal benefit to them (Borry et al. 2009; ESHG 2009).
riages is approximately the square root of the incidence mul- Molecular genetic analyses in symptomatic children as part
tiplied with 2. Thus in rare autosomal recessive diseases, an of the diagnostic workup for a genetic disease are standard
incidence of 1:10.000 translates to a calculated frequency of care. It can spare the children painful, invasive, or otherwise
heterozygotes of 1:50 in the general population. As a rule of stressful tests; may provide information on disease severity
thumb, a carrier frequency of approximately 1 % for the and prognostic factors; and facilitates genetic testing in other
more common autosomal recessive metabolic diseases (inci- family members. Predictive testing of asymptomatic chil-
dence approx. 1:40.000) and 0.5 % for the less common con- dren is also indicated if a positive test result would have
ditions (incidence approx. 1:160.000) can be assumed. immediate preventive or therapeutic consequences; this is
Carrier testing is complicated by costs and by the possibility the case, for example, in newborn screening strategies for
of identifying novel variants of uncertain clinical relevance. inborn errors of metabolism. However, predictive testing of
For large genes it may be reasonable but nevertheless reaf- children without therapeutic consequences is generally
firming to restrict the analysis to the most common muta- regarded as inacceptable since it might deprive the child of
tions in the relevant population; this may reduce the disease deciding for him/herself later on and might result in adverse
probability in a child by a factor of 5–10. Even after com- psychological or other consequences for a child tested posi-
plete sequence analysis, it is possible that a relevant mutation tive. The same applies to carrier testing in healthy siblings of
has been missed, so there is still a small remaining disease a child with an autosomal recessive condition which is often
explicitly requested by parents. It is to be preferred to let Prenatal diagnosis is a very personal decision which must
children decide themselves at a later age on how to deal with be based on the correct understanding of the risks for future
the implications of carrier diagnostics in a familial disease children. The maternal or parental decision should not be
(see above). The results of a molecular-genetic test should influenced by the values of the counseling clinician, and the
always be personally communicated to the patient (and/or consultation should be strictly nondirective. On the other
his/her parents) in a genetic counseling session. hand, the parents cannot demand from a clinician to partici-
pate in prenatal diagnosis and termination of pregnancy; no
doctor can be forced to do this against their will.
47.7 Prenatal Molecular Diagnosis Based on general considerations, four reasons for carry-
ing out prenatal diagnoses with regard to inherited metabolic
Mutation analysis in fetal DNA has become the method of disorders can be differentiated:
choice for exclusion or confirmation of inborn errors of 1. Termination of pregnancy in case the child is affected
metabolism or other monogenic disorders in a fetus during 2. Preparation of perinatal management in diseases that
pregnancy. Several aspects must be observed when molecu- require neonatal care intervention, such as intoxication-
lar prenatal diagnosis is offered to a family. type inherited disorders like organic acidurias or
The specific metabolic disease must be correctly diagnosed hyperammonemias
and the causative genotype must be identified and proven in 3. Prenatal treatment, e.g., to prevent intrauterine damage to
the family. Considering that mutation analyses have a signifi- the child such as virilization of the genitals in girls with
cant error rate of at least a few percent in many laboratories congenital adrenal hyperplasia or to anticipate complica-
(Zschocke et al. 2008), care must be taken that mutations iden- tions of pregnancy to the mother such as HELLP syn-
tified in the index patient of a family are correct and were con- drome in fetal long-chain hydroxyacyl-CoA
firmed in the parents. The assessment of the functional effects dehydrogenase (LCHAD) deficiency
of mutations/genotypes is sometimes difficult and requires 4. Psychological preparation that the expected child will be
specialist knowledge of the disease and its molecular basis. affected, particularly when invasive prenatal testing is
Care must be taken that nonfunctional or attenuated variants in carried out for other conditions such as chromosomal
the gene are recognized as such. Carrier analyses in spouses of disorders
previously known heterozygotes sometimes reveal genetic All these reasons may be valid in the individual case and
variants of unknown significance which cannot be used for need to be balanced with medical and psychological consid-
predictive testing as in prenatal diagnosis. International qual- erations as well as costs. In any case, an abnormal test result
ity assessment schemes have shown that failure to correctly generally causes considerable psychological and emotional
interpret the functional consequences of a particular genotype distress, and adequate help should be offered as part of pre-
is a major source of errors in many diagnostic molecular labo- natal genetic counseling.
ratories (Zschocke et al. 2008). Whenever possible, prenatal Whether or not termination of pregnancy is possible for a
molecular diagnostics should be considered and planned, and given disease varies from country to country; the available
genotype analyses should be completed before a pregnancy options should be clearly specified before prenatal investiga-
occurs. Prenatal testing for a large number of metabolic disor- tions are started. The decision to terminate a pregnancy is
ders without a specific family history is not a realistic option at always difficult both for the couple and the health profes-
present, but this may change with the rapid progress in next- sionals involved. Major factors in the decision include the
generation molecular techniques. severity of the disease, availability of effective treatment,
In order to enable the prospective parents to make an ade- and long-term quality of life. Some parents decide to carry a
quate decision in line with their personal circumstances and pregnancy to term when the disease is so severe that pros-
moral values, they must be counseled about the following pects of survival after birth are minimal. Many parents do not
aspects: regard termination of pregnancy as an acceptable option for
• The probability that a future child will have the disease- any condition. Availability of an accurate and reliable prena-
causing genotype tal diagnostic test is mandatory.
• The spectrum and probability of clinical symptoms and Fetal DNA is currently obtained through invasive tech-
complications that may develop in an affected child niques that have a risk of miscarriage:
• The risks of the invasive techniques for obtaining • Chorionic villus sampling (CVS) is the method of choice
fetal DNA for molecular prenatal diagnosis as it can be carried out
• The range of interventions that are medically and legally quite early in the pregnancy, from the 11th week onwards,
possible after the genetic diagnosis has been made, and allows direct extraction of fetal DNA without culture.
including termination of pregnancy The risk of miscarriage is about 0.5–1 %.
47 Genetic Counseling for Inborn Errors of Metabolism 741
• Amniocentesis (AC) is performed from the 14th week of This counseling concept was developed with major input
pregnancy onwards. It usually requires culturing of amni- by associations of Huntington’s disease patients
otic cells for up to 2 weeks for DNA extraction, and the (Huntington’s Disease Testing Committee 1994; Macleod
results of molecular analyses are therefore available et al. 2013).
5–6 weeks later in pregnancy than after CVS. The risk of The main objection of the initial counseling session is to
miscarriage is about 0.5 %. make sure the counselee is fully informed about the disease,
• Fetal cord puncture (chordocentesis) may be carried out the probability of carrying the genetic trait, the likelihood of
from the 20th week of pregnancy onwards. It has a developing symptoms, and the implications of predictive
miscarriage rate of about 1 % and is not used for routine testing. This includes aspects of heritability and diagnostic
prenatal diagnosis but may be the method of choice if spe- and therapeutic possibilities, as well as familial and social
cific fetal abnormalities are detected, e.g., by ultrasound considerations including insurance issues. All aspects are
scan in the second half of the pregnancy. also explained in a consultation letter in comprehensible lan-
The possibility of maternal cell contamination poses a guage sent to the counselee after the session. Clinical exami-
serious preanalytical risk for prenatal misdiagnosis. nation of the counselee is aimed at identifying or excluding
Therefore, testing of fetal samples for maternal cell con- early symptoms of the disease in question. For this purpose
tamination is mandatory in prenatal molecular diagnostics. the counselee is also referred to the adequate clinical special-
This generally involves the comparison of microsatellite ist after the initial meeting. In addition, the counselee is
polymorphisms (short tandem repeats, STRs) in maternal advised to have a session with a psychologist or psychother-
and fetal DNA and therefore requires a maternal blood apist prior to predictive testing, if only to establish a contact
sample for DNA extraction (Schrijver et al. 2007). which may be helpful at a later stage.
Maternal cell contamination is regarded as excluded if at The second counseling session takes place after a certain
least two STR markers gave informative results. time period (usually at least 4 weeks) and after the consulta-
Very recently, prenatal molecular diagnosis in fetal free tion with the clinical specialist and the psychotherapist. This
DNA in maternal blood (i.e., noninvasively) has become gives the counselee time to decide for him/herself whether
available for the detection of fetal chromosomal aberra- he/she wants to proceed with the test. It allows to contact
tions. It is likely that this approach will also become avail- friends, other trusted persons, or support groups. The second
able for screening for monogenic disorders. The absent session serves to clarify remaining questions. If the coun-
risk of miscarriage is a great advantage but the methodol- selee at this point wishes to proceed (and there are no acute
ogy will also create new challenges both to the pregnant contraindications such an assumed acute danger of suicide),
mothers and the society. blood samples will be taken and the test process started.
Laboratories usually require two independent samples taken
at different times or at least processed at different days in the
47.8 Predictive Testing laboratory in order to minimize the risk of sample swap or
other laboratory errors. The report is usually mailed to the
Late-manifesting genetic disease can be diagnosed before counselor in a separate closed envelope.
the first symptoms appear if molecular causes have been Communicating the test result (third counseling session)
clarified. A DNA-based test can predict with 100 % certainty should never be done by phone or by mail but always in a
whether, e. g., a severe neurodegenerative pathology without personal setting. It has proven helpful when the counselee is
current therapeutic options will occur. Neither the counselee accompanied by the spouse or another relative or friend for
nor the physician can predict the psychological and emo- the communication of a test result. Before “opening the
tional consequences for the patient and her/his relatives envelope” the physician asks one more time if the counselee
when a high certainty of developing serious untreatable is certain about hearing the test result. The reaction of a
symptoms is communicated. Often it becomes clear only in counselee to a (positive or negative) test result cannot be pre-
retrospect whether such knowledge was beneficial or coun- dicted. Therefore, a great deal of empathy on the part of the
terproductive for planning one’s life. When no therapeutic counselor and/or the accompanying person is necessary.
options exist, it is particularly important that a predictive
diagnosis involves at least a 3-step counseling process with
the following components:
1. Initial counseling session References
2. Second counseling session with drawing blood for the
Borry P, Evers-Kiebooms G, Cornel MC, Clarke A, Dierickx K (2009)
predictive test Genetic testing in asymptomatic minors: background considerations
3. Third counseling session to report the test result towards ESHG recommendations. Eur J Hum Genet 17:711–719
ESHG (2009) Genetic testing in asymptomatic minors: recommenda- Group ‘Genetic Testing Counselling’ of the European Huntington
tions of the European Society of Human Genetics. Eur J Hum Genet Disease Network (2013) Recommendations for the predictive
17:720–721 genetic test in Huntington’s disease. Clin Genet 83:221–231
Harper P (2010) Practical genetic counselling, 7th edn. Hodder Arnold, Schrijver I, Cherny SC, Zehnder JL (2007) Testing for maternal cell
London contamination in prenatal samples: a comprehensive survey of cur-
Huntington’s Disease Testing Committee (1994) International rent diagnostic practices in 35 molecular diagnostic laboratories. J
Huntington Association and the World Federation of Neurology Mol Diagn 9:394–400
Research Group on Huntington’s Chorea. Guidelines for the molec- Young ID (2006) Introduction to risk calculation in genetic counseling,
ular genetics predictive test in Huntington’s disease. J Med Genet 3rd edn. Oxford University Press, New York
31:555–559 Zschocke J, Aulehla-Scholz C, Patton S (2008) Quality of diagnostic
Macleod R, Tibben A, Frontali M, Evers-Kiebooms G, Jones A, mutation analyses for phenylketonuria. J Inherit Metab Dis
Martinez-Descales A, Roos R, Editorial Committee and Working 31:697–702
Simple Tests
48
K. Michael Gibson and Marinus Duran
A variety of rapid qualitative tests (colorimetric, dipstick, disease and the renal Fanconi syndrome. One should be
precipitate, color and smell, etc.) are useful in both specialist aware that the Fanconi syndrome includes renal glucosuria;
and nonspecialist laboratories to assist in the differential hence the presence of galactose cannot be deduced from the
diagnosis of inherited metabolic disorders. Most are limited positive Clinitest.
by some level of interference, yet these tests still have impor- Dinitrophenylhydrazine (DNPH) reacts with α-keto acids
tant utility, especially in emergency situations. to produce insoluble hydrazones, forming precipitates in
The color and odor of a patient’s urine may be a valuable urine samples (Table 48.5). Conversely, Acetest analysis
analysis for initial testing (Tables 48.1 and 48.2). Odor can (urine) complexes with urinary ketones (Bayer Corporation)
only be reliably interpreted when the urine is preserved in an (Table 48.5). Parallel use of DNPH and Acetest provides
adequate way (pH 5–7, no signs of bacterial contamination slightly more diagnostic capacity. A positive result of either
as evidenced by a negative nitrite dipstick). Alkaptonuria test will always be followed up by an immediate analysis of
patients show a rapid blackening of the urine upon standing; urine organic acids. Acetest is also frequently used for the
this process can be speeded up by adding a few drops of an home monitoring of patients with propionic and methylma-
ammonia solution to the urine test tube. lonic acidurias; it will give a good indication for the cata-
The ferric chloride test (Table 48.3) is employed to look bolic state of the patient, necessitating dietary intervention.
for the presence of oxoacids (formed in transamination or This test will be of use in establishing hypoketotic hypogly-
oxidation-reduction reactions). This test has been routinely cemia although the degree of ketonuria in several fatty acid
used in the identification of classical phenylketonuria, but oxidation disorders may be marked, especially MCAD.
several other species (in addition to the intermediates of phe- The cyanide nitroprusside test (or Brand reaction) iden-
nylalanine metabolism) react with ferric chloride to form a tifies sulfur-containing amino acids, with the formation of
number of colored complexes. An alternative for the ferric brightly colored complexes (Table 48.6). It will find its pri-
chloride test is the Phenistix dipstick. mary use in the detection of homocystinuria (both homocyste-
Reducing substances in urine (see Table 48.4; also com- ine and the cysteine-homocysteine disulfide react positively)
monly referred to the ClinitestR, Bayer Corporation) reacts
with a broad spectrum of reducing sugars in urine with the
Table 48.1 Color (urine)
formation of colored complexes (green to orange). It is com-
monly used for the detection of urine galactose on the Color Compound Disorder – source
suspicion of galactosemia in neonates with severe liver Blue Indican Hartnup disorder, severe
intestinal malabsorption
Blue/brown Homogentisic acid Alkaptonuria
Brown Methemoglobin Myoglobinuria
Red brown Hb/methemoglobin Hemoglobinuria
K.M. Gibson (*) Red Erythrocytes/lysate Hematuria
Section of Clinical Pharmacology, Red Porphyrins Porphyria
Washington State University, SAC 525M, 412 E.
Red Pyrazolones Drugs
Spokane Falls Blvd, Spokane, WA 99202-2131, USA
e-mail: mike.gibson@wsu.edu Red Phenolphthalein Chemicals
Light red Urates Physiological,
M. Duran hyperuricosuria
Red Beets Nutritional
F0-224 Meibergdreef 9, Amsterdam 1105AZ, The Netherlands
e-mail: m.duran@amc.uva.nl Yellow Riboflavin Vitamins
744 K.M. Gibson and M. Duran
Table 48.2 Odor (urine) Odor Compound Disorder – source

Musty, mousey Phenylacetic acid Classical PKU
Maple syrup or burnt 2-Oxoisocaproic acid, 2-Oxo-3- MSUD
sugar methylvaleric acid
Sweaty feet Isovaleric acid Isovaleric acidemia
Isovaleric acid 3-Hydroxy-3-methylglutaric aciduria
Idem + butyric + isobutyric acid MAD
Cat urine 3-Hydroxyisovaleric acid/ 3-Methylcrotonylglycinuria
3-methylcrotonic acid
3-Hydroxyisovaleric acid/ Multiple carboxylase deficiency
3-methylcrotonic acid
Cabbage-like 2-Oxobutyric acid Methionine malabsorption
2-Oxobutyric acid Tyrosinemia 1
Rancid butter 2-Oxo-4-methiylbutyric acid Tyrosinemia 1
Acid smell Methylmalonic acid Methylmalonic acidemia
Sulfurous Hydrogen sulfide Cystinuria
Fish market Trimethylamine/dimethylglycine Trimethylaminuria/dimethylglycinuria
Table 48.3 Ferric chloride Color Compound Disorder – source

test (urine) Blue green Phenylpyruvic acid Classical PKU
Imidazolepyruvic acid Histidinemia
Catecholamines Pheochromocytoma/ neuroblastoma
Xanthurenic acid Xanthurenic aciduria (B6 def.)
Transient blue green Homogentisic acid Alkaptonuria
Greenish gray Branched-chain oxoacids MSUD
Green p-Hydroxyphenylpyruvic acid Tyrosinemia types 1 and 2
Gray black Melanin Melanoma
Deep green Bilirubins Conjugated hyperbilirubinuria
Cherry red Acetoacetic acid Diabetic ketoacidosis, 3-oxothiolase def. + other
organic acidurias
Purple red brown 2-Oxobutyric acid Methionine malabsorption
Purple Ketones 3-Oxothiolase def.
Salicylates Drug treatment
Purple or green Phenothiazines Drug treatment
Table 48.5 Dinitrophenylhydrazine (DNPH) and Acetest (urine)

DNPH Acetest Positive compound Disorder – source
+ − Phenylpyruvic acid Classical PKU
+ − 2-Oxoisocaproic acid MSUD
Table 48.4 Reducing substances (urine)
+ − 2-Oxo-3-methylvaleric Idem
Compound Disorder – source acid
Galactose Galactosemia (+) − Imidazolepyruvic acid Histidinemia
Galactokinase def. + + Acetone 3-Oxothiolase def./
Fanconi-Bickel; Wilson disease ketosis/SCOT
Fructose Fructose intolerance deficiency
Essential fructosuria − + 2-Methylacetoacetate Propionic/MMA
academia
p-Hydroxyphenylpyruvic Tyrosinemia types 1 and 2
acid Idem 3-oxothiolase
deficiency
Homogentisic acid Alkaptonuria
− + Butanone Idem
Xylose Pentosuria
Acetoacetate SCOT deficiency,
Glucose Diabetes mellitus
ketosis
Renal Fanconi syndrome, cystinosis, etc.
+ − p-Hydroxyphe– Liver disease,
Oxalic acid Hyperoxaluria nylpyruvic acid tyrosinemia types 1
Salicylates Drug treatment and 2
Uric acid Hyperuricosuria + − 2-Oxobutyric acid Methionine
Hippuric acid Treatment with sodium benzoate/severe malabsorption
malabsorption + + Pyruvate Lactic acidosis
Ascorbic acid Excessive vitamin use + precipitate formed, − no detectable reaction
48 Simple Tests 745
Table 48.6 Nitroprusside test (urine) Table 48.7 Routine chemistry in blood (serum or plasma)
Positive compound Disorder – source Compound Disorder – source
Cystine Cystinuria Glucose ↓ Tyrosinemia type 1
Arginine Hyperargininemia (MSUD) + isovaleric acidemia
Cystine Generalized aminoaciduria (3-Methylcrotonylglycinuria)
Cystine Fanconi syndrome 3-Methylglutaconic, other types
3-Mercaptolactatecysteine- 3-Mercaptolactatecysteine- 3-Hydroxy-3-methylglutaryl-CoA synthase
disulfide disulfiduria def.
Homocysteine, mixed Homocystinuria 3-Hydroxy-3-methylglutaryl-CoA lyase def.
disulfide HIHA-syndrome (hyperinsulinism +
Idem B 12 def. and cobalamin C, D, E, G hyperammonemia)
Idem Methylenetetrahydrofolate reductase Methylmalonic acidemia
def. 2-Ketoglutarate dehydrogenase complex def.
Idem Cystathioninuria (bacterial formation Carnitine uptake defect
of Hcy) Carnitine palmitoyltransferase 1 (CPT 1)
Glutathione Glutathionuria Acylcarnitine translocase
Ketones + high creatinine Dehydration Carnitine palmitoyltransferase 2 (CPT 2)
Very long-chain acyl-CoA dehydrogenase
(VLCAD)
and cystinuria. Arginine – and at a slower rate arginino- Medium-chain acyl-CoA dehydrogenase
(MCAD)
succinic acid – will react by forming a differently colored
Long-chain 3-hydroxyacyl-CoA
product (blue/green). A concern with the Brand test is the use dehydrogenase (LCHAD)
of toxic cyanide. Mitochondrial trifunctional protein
Routine blood chemistry provides a plethora of diag- Multiple acyl-CoA dehydrogenation (MAD)
nostic insights (e.g., glucose, ammonia, blood gases, cre- CDG syndrome 1a, 1b
atinine, urea, uric acid, liver enzymes) (Table 48.7). Care Glycogen storage disease (GSD) types 0, 1, 3,
should be taken to not only interpret one clinical chemistry 6, 8, 9, 10
test result but always review the other test results which Galactosemia
may guide the diagnostic algorithm in a logical direction. Hereditary fructose intolerance
As an example a low blood glucose together with low Fructose-1,6-diphosphatase def.
Pyruvate carboxylase def.
plasma free fatty acids and the absence of ketones will
Glycerol kinase def.
most likely be the result of endocrine anomalies (hyper-
Short-chain 3-hydroxyacyl-CoA
insulinism), whereas sharply increased levels of the free dehydrogenase (SCHAD)
fatty acids generally indicate defects of the mitochondrial Ammonia ↑ Isovaleric acidemia
fatty acid beta-oxidation. MSUD
Additional colorimetric tests can be employed for 3-Methylcrotonylglycinuria
more selective identification. The Ehrlich’s test employs 3-Methylglutaconic aciduria, other types +
p-dimethylaminobenzaldehyde to assess the presence of uri- HMG-uria
nary porphobilinogen and urobilinogen, markers for the her- Biotinidase def.
itable porphyrias, but it will also react with substances such 3-Oxothiolase def.
as hydroxytryptophan and citrulline. The nitrosonaphthol test Methylmalonic acidemia
Propionic acidemia
represents a colorimetric analysis of 4-hydroxylated phenol
(l-2-Hydroxyglutaric aciduria)
acids, metabolites of tyrosine metabolism (e.g., 4-hydroxy-
Urea cycle disorders
phenylpyruvate, 4-hydroxyphenyllactate, and 4-hydroxy-
Hyperornithinemia-hyperammonemia-
phenylacetate). Artifactual results with nitrosonaphthol are homocitrullinuria
common in patients with liver disease and severe intestinal Hyperinsulinism-hyperammonemia (HIHA)
malabsorption and those receiving parenteral nutrition. The LPI
sulfite dipstick qualitatively assesses urine sulfite, indica- Carnitine uptake defect
tive of sulfite oxidase and molybdenum cofactor deficien- CPT 1
cies. It is reputedly performed in fresh urine samples and Acylcarnitine translocase def.
only few false-positive test results have been reported, a well MCAD
known one being the use of the pulmonary agent Mistabron Mitochondrial energy metabolism (MITEM)
(Mesna). Every faint-positive sulfite test result should be Severe liver disease, general
verified by urine amino acid analysis. Δ1 pyrroline-5-carboxylate synthase def.
(continued)
746 K.M. Gibson and M. Duran
Table 48.7 (continued) Compound Disorder – source

Compound Disorder – source Urea ↑ Malonyl-CoA decarboxylase def.
Blood gases MSUD Cystinosis, infantile
(acidosis) Isovaleric acidemia Cystinosis, adolescent
3-Methylcrotonylglycinuria Hyperoxaluria type 1
3-Methylglutaconic aciduria, type 1 Urea ↓ Hyperammonemia
3-Methylglutaconic aciduria, other types HHH syndrome
3-Hydroxy-3-methylglutaryl-CoA lyase def. Lysinuric protein intolerance
Biotinidase def. Uric acid ↑ MCAD
Holocarboxylase synthetase def. Hereditary fructose intolerance
Propionic acidemia GSD 1
3-Oxothiolase def. GSD 7
3-Hydroxyisobutyric aciduria Hypoxanthine phosphoribosyl transferase
Methylmalonic acidemia (HPRT)
Multiple carboxylase def. Phosphoribosyl pyrophosphate synthetase
(PRPPS)
Methylmalonic acidemia, cobalamin C and D
Barth syndrome
2-Ketoglutarate dehydrogenase complex def.
Fanconi-Bickel syndrome
Malonyl-CoA decarboxylase def.
Uric acid ↑ Molybdenum cofactor def.
Glutathione synthetase def.
Purine nucleoside phosphorylase def. (PNP)
Pyroglutamic aciduria (5-oxoprolinuria)
Xanthine oxidase/dehydrogenase def. (XDH)
Citrullinemia
Aspartate Tyrosinemia type 1
Argininosuccinic acidemia
aminotransferase, 3-Methylglutaconic aciduria, other types.
Carnitine uptake defect alanine Mevalonic aciduria
CPT 1 (renal tubular acidosis) aminotransferase
2-Ketoglutarate dehydrogenase complex def.
Acylcarnitine translocase def. (ASAT/ALAT) ↑
Fumarase def.
CPT 2
Malonyl-CoA decarboxylase def.
VLCAD
Ornithine transcarbamylase def.
MCAD
Argininosuccinic aciduria
SCAD
Arginase def.
MAD
Lysinuric protein intolerance
Glycogen storage disease (GSD) types 1 and
3 Carnitine uptake defect
Galactosemia Carnitine translocase def.
Hereditary fructose intolerance CDG syndrome Ib
Fructose-1,6-diphosphatase def. CPT 1
d-Glyceric acidemia CPT 2
Glycerol kinase def. VLCAD
MITEM MCAD
Pyruvate carboxylase def. SCAD
Succinyl-CoA: 3-oxoacid-CoA transferase LCHAD, trifunc. prot.
def. Galactosemia
Blood gases Urea cycle defects Hereditary fructose intolerance
(alkalosis) Lipoid adrenal hyperplasia Fructose-1,6-diphosphatase def.
Lactate ↑ Biotinidase def. GSD 3-7
Holocarboxylase synthetase def. Cholesterol-7a-hydroxylase def.
3-Hydroxyisobutyric aciduria 3β-Hydroxy-Δ5-C27-steroid dehydrogenase
2-Ketoglutarate dehydrogenase complex def. (3β-HSDH) def.
Fumarase def. 3-Oxo-Δ4-5β-reductase def. (5β-reductase
def.)
Fructose-1,6-diphosphatase def.
Wilson disease
l-2-Hydroxyglutaric aciduria
Zellweger spectrum disorders
Hyperammonemia
Acid lipase (Wolman disease)
LCHAD, trifunctional protein
α1-Antitrypsin def.
GSD 1, 3, 6, 9, 0
CDG-Ie
Pyruvate carboxylase def.
3-Hydroxy-3methylglutaryl-CoA
MITEM
synthase def.
Creatinine ↓ Creatine biosynthesis defects
Creatinine ↑ Cystinosis, infantile
Cystinosis, adolescent
Hyperoxaluria type 1
48 Simple Tests 747
Table 48.7 (continued) Compound Disorder – source

Compound Disorder – source Ferritin ↑ Lysinuric protein intolerance
Creatine kinase 3-Methylglutaconic aciduria type 1 Hematological Isovaleric acidemia
(CK) ↑ 3-Methylglutaconic aciduria, other types Neutropenia 3-Methylcrotonylglycinuria
Mevalonic aciduria 3-Methylglutaconic aciduria type 2
Dolicholphosphate-mannose synthase-1 def. GSD 1 B
CDG-Ic Methylmalonic acidemia
CPT 2 Propionic acidemia
VLCAD Carbamyl phosphate synthetase def.
LCHAD, trifunc. prot. Lysinuric protein intolerance
GSD 2 Hereditary orotic aciduria
GSD 3 Wilson disease
GSD 5 Thrombocytopenia Isovaleric acidemia
Myoadenylate deaminase def., AGAT def. Mevalonic aciduria
Alkaline 3β-Hydroxy-Δ5 -C27-steroid dehydrogenase 3-Methylcrotonyl-CoA carboxylase def.
phosphatase (ALP) (3β-HSDH) def. Methylmalonic acidemia
↑ 3-Oxo-Δ4-5β-reductase def. (5β-reductase Propionic acidemia
def.) Lysinuric protein intolerance
Lactate Lysinuric protein intolerance 3-Phosphoglycerate dehydrogenase
dehydrogenase GSD 5 def.
(LDH) ↑
Hb (Anemia) ↓ Tyrosinemia type 1
ALP ↓ Hypophosphatasia
Methylmalonic acidemia
Triglycerides ↓ Abetalipoproteinemia
Pyroglutamic aciduria (5-oxoprolinuria)
Hypobetalipoproteinemia
γ-Glutamylcysteine synthetase def.
Triglycerides ↑ GSD 1
Cobalamin C and D
Glycerol kinase def. (pseudo-increase)
Lysinuric protein intolerance
Lipoprotein lipase def.
Hereditary orotic aciduria
Apolipoprotein C-11 def.
Pyrimidine-5′-nucleotidase def.
Dysbetalipoproteinemia
Familial LCAT def.
Hepatic lipase def.
Wilson disease
Lecithin cholesterol acyltransferase (LCAT)
def. Acid lipase def. (Wolman disease)
Cholesterol ↓ 3-Methylglutaconic aciduria, other types 3-Phosphoglycerate dehydrogenase def.
Mevalonic aciduria Methylentetrahydrofolate reductase def.
Abetalipoproteinemia Methionine synthase def.
Hypobetalipoproteinemia Vacuolated Nearly all lysosomal storage diseases
lymphocytes on
3β-Hydroxy-Δ5 -C27-steroid dehydrogenase peripheral smear
(3β-HSDH) def.
Reticulocytes ↑ γ-Glutamylcysteine synthetase def.
3-Oxo-Δ4-5b-reductase def. (5β-reductase
def.) γ-Glutamyl transpeptidase def.
Smith-Lemli-Opitz syndrome GSD 2
Barth syndrome
CDG syndromes
Zellweger spectrum disorders References
Cholesterol ↑ Lipoprotein lipase def. This chapter draws substantially from previous chapters,
Apolipoprotein C-11 def. which the authors gratefully acknowledge: Blau N,
Dysbetalipoproteinemia Blaskovics ME, Duran M (2003) Simple tests in urine and
Hepatic lipase def.
blood. In: Blau N, Duran M, Blaskovics ME, Gibson KM
Hypercholesterolemia
(eds) Physician’s guide to the laboratory diagnosis of meta-
Defective apoB-100
bolic diseases. Springer, Heidelberg, pp 3–10; Gibson KM,
LCAT def.
Sterol 27-hydroxylase def. (cerebrotendinous
Jakobs C (2010) Biochemical studies. In: Hoffmann GF,
xanthomatosis, CTX) Zschocke J, Nyhan WL (eds) Inherited metabolic diseases, a
Myoglobin ↑ CPT 2 clinical approach. Springer, Berlin/Heidelberg, pp 263–281.
GSD 2
VLCAD
LCHAD
Amino Acids
49
Marzia Pasquali and Nicola Longo

49.1 Introduction ..................................................................... 749
Amino acids are the building block of all proteins and are
49.2 Pre-analytical Conditions ............................................... 749
49.2.1 Specimens ......................................................................... 749 therefore of vital importance for the integrity of the organism.
In addition, some amino acids play an additional important role
49.3 Patient Status/Patient Information ................................ 752
as neurotransmitters (or their precursors), modulators of neu-
49.4 Specimen Collection ........................................................ 752 rotransmitter action, precursors of hormones, cofactors, purine,
49.5 Analysis ............................................................................ 755 and pyrimidines. Amino acids, with a few exceptions, are water
soluble due to the presence of both an amino and a carboxylic
49.6 Interpretation of Amino Acids Results
and Reference Values ...................................................... 755 group that in water become ionized. Amino acids are filtered
49.6.1 Age .................................................................................... 756 by the kidney and could be easily lost in urine in the absence of
References ...................................................................................... 759 an efficient renal tubular reabsorption mechanism. There are
several amino acid transporters whose deficiency can result in
urinary loss of one or a group of amino acids and specific clini-
cal manifestations. Excess amino acids are degraded by spe-
cific sets of enzymes, thereby generating energy and urea from
the nitrogen group during catabolism. Defects in these enzymes
cause inborn errors of amino acid metabolism, almost all of
which are transmitted as autosomal recessive traits. This leads
either to the buildup of the parent amino acid or its by-products
or of the catabolic products (organic acids) depending on the
location of the enzyme block. The nitrogen group of the amino
acid upon degradation will result in the formation of ammonia
which is then removed by the urea cycle in the liver. Lack of an
enzyme or transporter in this cycle can result in hyperammone-
mia. Accurate measurement of amino acids is essential to diag-
nose disorders of amino acid metabolism and to follow
treatment in affected patients. This chapter will discuss how
amino acids are analyzed in biological fluids and the main
M. Pasquali (*) alterations encountered in common disorders.
Department of Pathology,
University of Utah and Arup Laboratories,
500 Chipeta Way, Salt Lake City, UT 84108, USA
e-mail: pasquam@aruplab.com 49.2 Pre-analytical Conditions
N. Longo
Division of Medical Genetics, 49.2.1 Specimens
University of Utah,
Amino acids can be measured in different sample types
2C412 SOM, 50N Mario Capecchi Drive,
Salt Lake City, UT 84132, USA (whole blood, plasma, cerebrospinal fluid, urine) depend-
e-mail: nicola.longo@hsc.utah.edu ing upon the reason for the amino acids analysis. Whole
750 M. Pasquali and N. Longo
Table 49.1 Amino acid concentrations in plasma (μmol/L)

Amino acid Men (n = 50)a Women (n = 15)a Adolescents (n = 80)b Children (n = 52)b
Taurine 27–95 18–66 2–90 20–120
Aspartic acid 2–9 3–6 3–15 1–17
Threonine 92–180 93–197 102–246 40–204
Serine 89–165 78–166 92–196 70–194
Asparagine 32–92 26–74 34–94 15–83
Glutamic acid 6–62 6–38 17–69 14–78
Glutamine 466–798 340–696 457–857 333–809
Proline 97–297 112–220 58–324c 40–332d
Glycine 147–299 100–384 166–330 107–343
Alanine 146–494 218–474 242–594 120–600
Citrulline 19–47 10–58 19–52c 8–47d
a-Aminobutyrate 15–35 7–35 8–36c 12–43d
Valine 179–335 172–248 155–343 132–480
Cystine 24–54 31–49 36–58c 23–68d
Methionine 13–37 14–30 13–41 3–43
Isoleucine 46–90 39–67 34–106 6–122
Leucine 133–205 98–142 86–206 30–246
Tyrosine 37–77 26–78 35–107 19–119
Phenylalanine 46–74 42–62 34–86 26–98
Ornithine 55–135 36–96 47–195 20–136
Lysine 135–243 119–203 116–276 66–270
Histidine 72–108 68–104 68–108 47–135
Tryptophan 25–65 17–53 – 12–69d
Arginine 28–96 28–108 1–81 12–112
The values represent the mean ± 2SD
a
Modified from Hagenfeldt et al. (1984)
b
Modified from Gregory et al. (1986)
c
Modified from Armstrong et al. (2007)
d
Modified from Applegarth et al. (1979)
blood spotted on filter paper is used almost universally for Amino acids can be measured also in other biological speci-
neonatal screening of inherited disorders of metabolism. mens, such as vitreous fluid (Table 49.4) (Honkanen et al. 2003;
Analysis of amino acids in plasma represents the first Bertram et al. 2008), especially in postmortem samples, when
approach to the study of a patient with a suspected disorder urine is often not available and blood is unsuitable. However, this
of amino acid metabolism (Table 49.1) (Hagenfeldt et al. type of analysis should be limited to laboratories with experience
1984; Held et al. 2011; Gregory et al. 1986; Applegarth in these studies, since postmortem changes rapidly occur even in
et al. 1979; Armstrong et al. 2007). This will identify ele- vitreous fluid. Amniotic fluid has limited value in prenatal diag-
vated phenylalanine in phenylketonuria, elevated branched- nosis for aminoacidopathies. Unlike the organic acid disorders,
chain amino acids and the presence of alloisoleucine in in most amino acid disorders the metabolites do not accumulate
maple syrup urine disease, and abnormalities of citrulline before birth. Abnormal amino acid patterns in amniotic fluid
and glutamine in different urea cycle defects. The study of have only been found in two of the urea cycle disorders, namely,
urinary amino acids is performed to screen for renal amino- argininosuccinate lyase deficiency (argininosuccinic acidemia)
acidurias, such as cystinuria, lysinuric protein intolerance, and argininosuccinate synthetase deficiency (citrullinemia).
or Hartnup disease (Table 49.2) (Parvy et al. 1988). In addi- Tables 49.1, 49.2, 49.3, and 49.4 (Hagenfeldt et al. 1984; Gregory
tion, it is a useful test for renal tubular capacity that et al. 1986; Applegarth et al. 1979; Armstrong et al. 2007; Parvy
becomes abnormal in most cases of renal Fanconi syn- et al. 1988; Honkanen et al. 2003; Bertram et al. 2008) list amino
drome. The analysis of cerebrospinal fluid (CSF), usually acid reference ranges in blood, urine, CSF, and vitreous fluid and
in addition to plasma amino acids, is necessary in the evalu- are included as examples. However, it should be noted that even
ation of patients with neurometabolic disorders, such as when using the same instrumentation, the reference values may
glycine encephalopathy or disorders of serine metabolism vary from laboratory to laboratory. Each laboratory should
(Table 49.3) (Applegarth et al. 1979). establish its own reference ranges.
49 Amino Acids 751
Table 49.2 Amino acid concentrations in urine expressed in mmol/mol creatinine

Over
Amino acid 0–1 month 1–6 months 6–12 months 1–2 years 2–4 years 4–7 years 7–13 years 13 years
Taurine 8–226 6–89 9–123 12–159 13–200 17–230 18–230 16–180
Aspartic acid 2–12 2–16 3–12 3–10 2–8 2–8 1–10 2–7
Hydroxyproline 20–320 0–143 0–22 0–13 0–13 0–13 0–13 0–13
Threonine 20–138 17–92 14–56 15–62 10–48 9–36 8–28 7–29
Serine 80–282 42–194 50–137 45–124 32–94 38–93 23–69 21–50
Asparagine 0–84 0–58 0–36 0–32 0–30 0–29 0–24 0–23
Glutamic acid 0–30 2–29 0–18 0–11 0–10 0–8 0–9 0–12
Glutamine 52–205 63–229 74–197 62–165 45–236 52–133 20–112 20–76
Proline 21–213 0–130 0–14 0–13 0–9 0–9 0–9 0–9
Glycine 283–1,097 210–743 114–445 110–356 111–326 91–246 64–236 43–173
Alanine 75–244 72–206 36–162 41–130 33–115 27–92 17–65 16–68
Citrulline 0–11 0–10 0–8 0–7 0–6 0–5 0–5 0–4
a-Aminobutyrate 0–9 0–7 0–8 0–8 0–6 0–5 0–5 0–4
Valine 3–26 4–19 6–19 7–21 6–20 3–15 3–17 3–13
Cystine 12–39 7–24 6–15 5–13 4–15 4–11 4–12 3–17
Methionine 7–27 6–22 8–29 7–29 5–21 5–20 3–17 2–16
Isoleucine 0–6 0–5 0–6 0–6 0–5 0–5 0–6 0–4
Leucine 3–25 4–12 4–16 3–17 4–18 3–13 3–16 2–11
Tyrosine 6–55 12–52 11–54 13–48 10–30 9–35 6–26 2–23
Phenylalanine 4–32 7–28 11–28 10–31 7–21 6–26 5–20 2–19
β-Aminoisobutyrate 0–87 0–216 0–226 0–206 0–175 0–59 0–85 0–91
Ornithine 0–19 0–13 0–8 0–8 0–7 0–7 0–6 0–5
Lysine 22–171 15–199 13–79 16–69 10–46 10–68 10–56 7–58
Histidine 80–295 72–342 92–278 87–287 68–255 61–216 43–184 26–153
3-Methylhistidine 20–39 19–40 20–47 22–57 20–59 21–61 18–59 19–47
Arginine 0–14 0–11 0–11 0–8 0–9 0–7 0–6 0–5
Modified from Parvy et al. (1988)
Table 49.3 Normal range of amino acid concentrations in CSF, Amino acid μmol/L (n = 120)
expressed in μmol/L, obtained by ion-exchange chromatography
Valine 7.0–37.1
Amino acid μmol/L (n = 120) Ranges represent the mean ± 2SD (Pasquali, unpublished data)
Alanine 12.5–47.3
Arginine 5.9–30.6 Table 49.4 Concentrations of amino acids in vitreous fluid (aver-
Asparagine <23.6 age ± SD, μmol/L)
Aspartate <5.8 Amino acid μmol/L (n = 17)
Citrulline <5.6 Alanine 159.5 ± 54.9
Cystine <5.0 Arginine 39.4 ± 16.3a
Glutamine 230.7–637.4 Asparagine 35.8 ± 11.6
Glutamic acid <15 Aspartate 1.4 ± 1.0
Glycine 3.1–21 GABA 0.6 ± 0.2
Histidine 5–24 Glutamate 5.2 ± 2.3
Homocysteine 0 Glutamine 1192.9 ± 404.4
Hydroxyproline <8.0 Glycine 8.5 ± 2.5
Isoleucine 1–11 Histidine 38.4 ± 10.0
Leucine 3.4–25.9 Isoleucine 37.9 ± 11.6
Lysine 7.8–40.8 Leucine 89.7 ± 28.4
Methionine 0.4–9.4 Lysine 115.4 ± 33.7
Ornithine 1.6–12.0 Methionine 22.3 ± 8.1
Phenylalanine 6.9–25.1 Phenylalanine 44.4 ± 14.2
Proline <8.0 Serine 103.9 ± 24.4
Serine 18.0–73.0 Threonine 85.5 ± 28.4
Taurine 2.7–16.2 Tyrosine 58.2 ± 18.8
Threonine 10.8–74.9 Valine 112.0 ± 4.4a
Tyrosine 5.4–23.7 Modified from Honkanen et al. (2003) and aBertram et al. (2008)
49.3 Patient Status/Patient Information For the diagnosis of most amino acid disorders, blood
specimens collected after an overnight fast are preferred
Clinical information is a necessary element for the accurate or, alternatively, several hours after a meal. Samples from
interpretation of amino acid profiles and to correctly diag- young infants should be collected immediately before the
nose patients with disorders of amino acid metabolism. next scheduled feeding (at least 2–3 h after the last feed-
Several common clinical features should prompt an investi- ing). For hyperammonemia screening, postprandial sam-
gation for a metabolic disorder. These include symptoms ples are more suited since elevation of blood ammonia
related to the CNS (lethargy, seizures, coma), GI tract may be intermittent and present only in the fed state. It is
(vomiting, poor feeding, failure to thrive), liver (hepato- not uncommon for a laboratory to receive a sample for
megaly), cardiovascular, respiratory, renal (kidney stones) amino acid analysis collected while the patient is receiv-
systems, eye (lens dislocation, retinitis pigmentosa, optic ing intravenous hyperalimentation. These samples are
atrophy), skeletal system, and skin. A positive family his- often uninterpretable as they show elevations in all the
tory (consanguineous parents, affected sibling or family amino acids present in the IV solution. If it is possible,
member) and/or a positive newborn screen result should intravenous hyperalimentation should be discontinued for
also initiate a diagnostic metabolic workup. One should at least 2–3 h prior to specimen collection. If intravenous
keep in mind that the same disease may present with differ- hyperalimentation cannot be discontinued, appropriate
ent symptoms at different ages and that inherited disorders care should be taken in drawing the sample away from the
of metabolism are not limited to infants and children, with site of entry of the solution.
late-onset diseases presenting in adulthood as well. In other The concentration of the majority of the physiological
words, a metabolic disorder should not be dismissed amino acids is the same in red blood cells and in plasma,
because of age. Routine chemistry laboratory tests, such as with exception of taurine, glutamate, aspartate, and arginino-
blood gases, pH, electrolytes, anion gap, glucose, and succinate. Thus, hemolyzed samples may show an increased
ammonia, provide clues to the kind of metabolic disorder concentration of these amino acids. The enzyme arginase
and guide the testing. These laboratory data, along with a converts arginine into ornithine and urea and is expressed in
brief clinical history, should be made available to the meta- red blood cells. Hemolysis will release arginase in the plasma
bolic laboratory for better interpretation of the results. The causing hydrolysis of arginine. Therefore, a decrease in argi-
physiological status of the patient is also important, since nine with an increase in ornithine is often seen in hemolyzed
significant variations can be seen in postprandial specimen, samples.
after prolonged fasting, and during catabolism. A list of Whole blood collected for amino acids analysis should be
medications taken at the time of specimen collection should spun down as soon as possible; the plasma should be kept
also be provided, since some may interfere with the analy- frozen during transport and until analysis is performed. For
sis or may cause alterations in the concentration of some local transport, refrigerated conditions are acceptable.
amino acids, such as increased glycine levels secondary to Improper handling of specimens can result in artifactual
valproate therapy. changes in the amino acid contents. Table 49.5 lists possible
artifacts due to collection, handling, and storage of samples.
The conversion of arginine into ornithine by arginase can
49.4 Specimen Collection be observed even in unspun samples left at room tempera-
ture, even without obvious hemolysis. Free cysteine and
The timing of the specimen collection is important in the homocysteine bind to protein and will be lost in samples that
detection of metabolic disorders. In acutely ill patients, the are not spun immediately and/or are stored for a prolonged
blood and urine specimens on admission are likely to be period of time. This results in loss of cystine and homocys-
most revealing and most appropriate for metabolic screen- tine which is evident even when samples are stored at −20 °C,
ing. It is good practice to save these specimens from all while storage at −70 °C prevents this effect. Glutamine is
patients in whom the diagnosis is unclear. For the detection unstable and breaks down with prolonged storage resulting
of amino acidopathies, this is not as critical as it is for other in increased glutamate.
metabolic disorders. For amino acid analysis, the volume of If serum is used for amino acids analysis, depending
specimen required depends on the methodology used; typi- on how it has been obtained, all the artifacts listed above
cally 200–500 μL of plasma or CSF is required, and the vol- could be present. Low serine in urine may be due to bac-
ume of urine required varies depending on creatinine terial contamination, and the presence of hydroxyproline
concentration; each laboratory should list the minimum vol- can be due to fecal contamination. Urine is not the fluid of
umes acceptable for each analysis. choice in the diagnostic investigation for an aminoacidopathy
49 Amino Acids 753
Table 49.5 Collection, handling, and storage artifacts

Factor/condition Source Amino acid(s) affected Value
Contamination, bacterial U Ala, Gly, Pro ↑H
Contamination, bacterial U Trp, aromatic amino acids, Ser ↓L
Contamination, fecal U Pro, Glu, Leu, Ile, Val, OH-pro-line ↑H
Contamination, protein U Cys ↓L
Contamination, RBC U Orn ↑H
Contamination, unwashed skin B Most amino acids ↑H
Contamination, WBC U Tau ↑H
Contamination, WBC B Asp, Glu, Tau ↑H
Hemolysis B Asp, Glu, Gly, Orn ↑H
Hemolysis B Arg, Gln ↓L
Serum vs. plasma B Serum Tau >plasma Tau
Serum vs. plasma B Serum homocysteine >plasma homocysteine
Storage U Glu, Asp, GABA ↑H
Storage U Gln, Asn, phosphoethanolamine ↓L
Storage B Gln, Cys, homocyst(e)ine ↓L
Storage B Glu ↑H
Tube artifact, thrombin B Gly ↑H
Tube artifact, EDTA B Ninhydrin-positive artifact
Tube artifact, metasulfite B S-Sulfocysteine ↑H
Unspun blood left at rm. temp. B Orn, total homocysteine ↑H
Unspun blood left at rm. temp. B Arg, Cys, homocystine ↓L
Table 49.6 Nutritional status and amino acid values

Diet, canned formula or milk U Homocitrulline ↑H
Diet, gelatin U Gly ↑H
Diet, high protein (infants) B Met, Tyr ↑H
Diet, shellfish U Taurine ↑H
Diet, white meat from fowl U Anserine, 1-methylhistidine, carnosine ↑H
Folate deficiency B Homocyst(e)ine ↑H
Kwashiorkor B Pro, Ser, Gly, Phe ↑H
Kwashiorkor B Leu, Ile, Val, Trp, Met, Thr, Arg ↓L
Obesity B Branched-chain amino acids, Phe, Tyr ↑H
Obesity B Gly ↓L
Starvation, 1–2 days (with or without vomiting) B Branched-chain amino acids, Gly ↑H
Starvation, 1–2 days (with or without vomiting) B Alanine ↓L
Vitamin B12 deficiency B Homocyst(e)ine ↑H
Vitamin B6 deficiency U Cystathionine ↑H
B blood, U urine, H high, L low
(phenylketonuria, maple syrup urine disease, homocystin- are usually reported in reference to creatinine; however, for
uria, etc.) as plasma is a better sample type. Urine amino samples very diluted or very concentrated, this correction
acids analysis is, instead, the diagnostic test when disorders may not be very accurate. The interpretation of urine amino
of amino acid transport are investigated (cystinuria, lysin- acids relies on patterns of amino acids more than on abso-
uric protein intolerance, Hartnup) or in prolidase deficiency. lute values; therefore, a careful examination of the profile
Although a random specimen is usually sufficient for diag- should lead to a correct diagnosis. Tables 49.5, 49.6, 49.7,
nostic purposes, a timed urine collection may be required and 49.8 list artifacts due to specimen collection as well as
for reabsorption studies in conjunction with a plasma sample the effect of nutritional status, illnesses, and medications on
collected at midpoint. Results of urine amino acids analysis amino acids values.
Table 49.7 Effects of illness/disease on amino acid values

Burn >20 % of surface area (0–7 days after injury) B Phe ↑H
Burn >20 % of surface area (0–7 days after injury) U Ala, Gly. Thr, Ser, Glu, Gln, Orn, Pro ↓L
Diabetes B Leu, Ile, Val ↑H
Hepatic disease B Tyr, Phe, Met, Orn, GABA ↑H
Hepatic disease B Branched-chain amino acids ↓L
Hepatoblastoma U Cystathionine ↑H
Hyperinsulinism B Leu, Ile, Val ↓L
Hypoparathyroidism, primary U All amino acids ↑H
Infection B All amino acids ↓L
Infection B Phe/Tyr ratio ↑H
Infection U All amino acids ↑H
Ketosis B Leu, Ile, Val ↑H
Ketotic hypoglycemia B Ala ↓L
Leukemia, acute U Advanced disease: all amino acids ↑H
Leukemia, acute U On therapy: Gly, Asp, Thr, Ser ↑H
Neuroblastoma U Cystathionine ↑H
Renal failure U Phe, Val ↓L
Renal failure U His ↑H
Renal failure B Phe, Cit, Cys, Gln, homocyst(e)ine ↑H
Renal failure B Leu, Val, Ile, Glu, Ser ↓L
Respiratory distress on oxygen B Cystine ↓L
Rickets U Gly ↑H
Table 49.8 Effect of medications on amino acid values

Acetaminophen U Acetaminophen-cysteine disulfide may interfere with ↑H
determination of Phe
N-Acetylcysteine U Acetylcysteine-cysteine disulfide ↑H
Ampicillin/amoxicillin U Interferes with determination of Met, Phe, argininosuccinate ↑H
Arginine infusion B Arg ↑H
Arginine infusion U Arg, Lys, Orn, Cys ↑H
Bile acid sequestrants (colestipol, niacin) B Homocyst(e)ine ↑H
Cephalexin U Ninhydrin reactive metabolite
Cyclosporin A B Total homocysteine ↑H
2-Deoxycoformycin B Homocyst(e)ine ↓L
Lysine aspirin U Lys ↑H
Methotrexate therapy B Homocyst(e)ine ↑H
Methotrexate therapy B Phe/Tyr ratio ↑H
Nitrous oxide anesthesia B Homocyst(e)ine ↑H
Oral contraceptives B Pro, Gly, Ala, Val, Leu, Tyr ↓L
Penicillamine U Penicillamine disulfide, penicillamine-cysteine disulfide ↑H
D-Phenylalanine U Phe ↑H
Tamoxifen B Homocyst(e)ine ↓L
Tetracycline, renal toxicity U All amino acids ↑H
Valproate B,U Gly ↑H
Vigabatrin/vinyl-GABA U β-Alanine, β-aminoisobutyrate, GABA ↑H
Vigabatrin/vinyl-GABA CSF GABA, β-alanine ↑H
Vigabatrin/vinyl-GABA B,U 2-Aminoadipic acid ↑H
49 Amino Acids 755
49.5 Analysis acid for argininosuccinic acid lyase deficiency), and homo-
cystine may not be sufficient to identify mild elevations of
Amino acids studies are performed for (1) diagnostic pur- these amino acids, and some patients can be missed.
poses and (2) monitoring of therapy in patients with a known Tandem mass spectrometry analysis of amino acids by
metabolic disorder. Methods for amino acids analysis should flow injection is routinely used for neonatal screening using
be sensitive (to identify even very low concentration of whole blood spotted on filter paper. Amino acids are extracted
amino acids), specific (to separate isomeric species), and from the blood spot using an organic solvent (methanol) con-
accurate (to enable monitoring of dietary therapy). taining stable-isotope-labeled amino acids. The limitation of
Paper chromatography, thin-layer chromatography, and two- this method is that isomers and isobars cannot be separated;
dimensional chromatography by high-voltage electrophoresis however, the speed of the analysis makes it a superb screen-
for amino acids screening have been used in the past but are now ing method. For diagnostic and/or monitoring purposes,
obsolete and should not be used for amino acids screening. similar methods can be used, and, to increase the specificity,
Quantitative analysis of amino acids in physiological a liquid chromatographic separation is performed prior to
fluids can be performed by ion-exchange chromatography MS/MS detection. LC-MS/MS analysis of amino acids in
(IEC), reverse-phase high-performance liquid chromatogra- physiological fluids is becoming more and more used by
phy (HPLC), gas chromatography (GC), and liquid chroma- laboratories, because of its sensitivity and increased specific-
tography/tandem mass spectrometry (LC-MS/MS). ity. Accurate quantitation by MS/MS requires the use of
IEC has been the most widely used method in clinical stable-isotope-labeled internal standards; ideally each amino
laboratories, and it is still considered the gold standard for acid quantified should have its internal standard. Tandem
amino acid analysis. With this technique, amino acids are mass spectrometry will only pick up those amino acids that
separated using a cation-exchange column and a lithium buf- have been programmed in the instrument; as a consequence,
fer system. The detection is performed by colorimetry after this technique will not detect the unusual amino acids such
post-column derivatization with ninhydrin. The adduct as cysteinylglycine (characteristic of gamma-glutamyl trans-
between ninhydrin and primary amines has a maximum peptidase deficiency). With the use of MS/MS for neonatal
absorbance at wavelength λmax = 570 nm, while the adduct screening, milder variants of metabolic disorders are now
between ninhydrin and secondary amines (hydroxyproline, identified, and the biochemical phenotype may be more sub-
proline) has a λmax = 440 nm. The samples are monitored at tle. Good communication between the testing laboratory and
both wavelengths to allow quantification of all physiological the physicians helps in clarifying these cases.
amino acids. This method is highly reproducible, with a wide The method used for quantifying amino acids in physio-
dynamic range and excellent linearity range (5–3,000 μmol/L). logical fluids should have a wide dynamic range, high sensi-
The disadvantage of the method is the long analysis time tivity, enough to measure concentrations as low as a few
(90–150 min) and the lack of specificity. In fact several micromoles/liter, and high upper limit of linearity, to accu-
metabolites other than amino acids, including medications rately quantify high concentrations of amino acids for diet/
and dietary supplements, react with ninhydrin and coelute therapy monitoring. Citrulline, alloisoleucine, argininosuc-
with amino acids. This is particularly challenging when eval- cinic acid, and free homocystine are examples of amino
uating urine samples. Depending on the methodology used, acids requiring high sensitivity, with citrulline and arginino-
special processing of the sample is required to identify and succinic acid requiring also high upper limit of linearity. It is
quantify certain amino acids. A classic example is arginino- important to be able to detect even trace amounts of these
succinic acid. With IEC the free acid elutes in the region of amino acids to reach a correct diagnosis. LC-MS/MS-based
neutral amino acids, often coeluting with tyrosine or leucine, methods are usually highly sensitive and specific; however,
depending on the buffer system and column used. Conversion their dynamic range may not be as good as IEC.
of the free acid into anhydrides by boiling the deproteinized
sample increases sensitivity and allows more accurate results.
HPLC-based methods usually require a pre-column 49.6 Interpretation of Amino Acids Results
derivatization step using reagents reacting with the primary and Reference Values
or secondary amino group of amino acids to form derivatives
that can be detected with fluorescent, UV, or electrochemical Clinical information, age of the patient, diet, and medication
detectors. The analysis time of these methods is shorter than are critical to provide an accurate interpretation for meta-
ion-exchange chromatography, but sample preparation is bolic testing in general, including amino acids analysis. The
more laborious and the derivatives may be instable. In addi- interpretation of amino acid profiles relies on pattern recog-
tion, the sensitivity for some critical amino acids, such as nition; therefore, laboratories should be familiar with meta-
alloisoleucine (the diagnostic amino acid for maple syrup bolic disorders and the changes observed in presence of a
urine disease), argininosuccinic acid (the diagnostic amino metabolic block.
49.6.1 Age metabolic decompensation, the pattern of branched-chain

amino acids will show a disproportionately high leucine
The normal range of amino acids changes with age, and compared to isoleucine and valine and a markedly reduced
appropriate reference ranges are essential for accurate inter- alanine in addition to the presence of alloisoleucine. With
pretation. Renal tubular function is not mature in many ion-exchange chromatography, alloisoleucine coelutes with
infants who can have generalized aminoaciduria until several cystathionine, and laboratories using this method should
months of age. Homocitrulline in the urine of infants is usu- develop protocols to distinguish the two amino acids, such as
ally derived from diet and is only rarely due to a metabolic the evaluation of the ratio of the absorbance at 570 and
disorder. Taurine is normally excreted in large amounts in 440 nm, that is much lower for cystathionine as compared to
the first week of life and decreases thereafter. However, the alloisoleucine.
urine from infants less than 1 year of age often contains tau- Secondary amino acid changes can be a clue to other
rine from breast milk and/or taurine-supplemented formula, types of metabolic disorders such as galactosemia, organic
and the amount can fluctuate widely depending on the time acidopathies, and disorders of pyruvate metabolism.
of urine collection in relation to the time of feeding. Taurine Gross elevations of many amino acids, particularly gluta-
is not detected in urine from infants fed unsupplemented for- mine and alanine in blood, have been reported in moribund
mulas. Taurine is a constituent of several energy drinks and children; however, elevations of the branched-chain amino
can increase in adolescents and young adults consuming acids, citrulline and arginine, can be secondary to hypoxia
these products. Administration of parenteral amino acid and liver failure. Postmortem blood shows similar but more
solutions can also cause altered blood amino acid patterns. pronounced amino acid changes (Table 49.7).
The urinary excretion of glycine is quite variable. It can Citrulline is the amino acid critical in identifying disor-
be of dietary origin (e.g., gelatin) as well as secondary to ders of the urea cycle. It is absent or markedly reduced in
medication (e.g., valproate). Persistent isolated hyperglycin- proximal blocks of the urea cycle (NAGS, CPS1, OTC defi-
uria with normal plasma glycine levels suggests familial imi- ciencies); it is markedly elevated in citrullinemia type I
noglycinuria that is usually a benign variant. Urine histidine (argininosuccinate synthase deficiency) and, to a lower
is increased during pregnancy. The concentration of plasma extent, in argininosuccinic aciduria (argininosuccinate lyase
amino acids is low compared to normal controls during preg- deficiency); and it is moderately but variably elevated in
nancy, especially in the last trimester, due to hemodilution. citrullinemia type II (citrin deficiency). Citrulline can also be
In young infants, particularly in preterm infants, the used to assess functional enterocyte mass in patients with
quantity and quality of protein feeding has a direct effect necrotizing enterocolitis, short bowel syndrome, and intesti-
on the plasma amino acid concentrations. The postprandial nal transplant. Very low levels of citrulline can be seen in
rise of total amino acids is more pronounced in infants on these patients, usually with a completely normal concentra-
higher protein feeding. Transient hypertyrosinemia and tion of all other amino acids, including glutamine.
hypermethioninemia can result from excessive protein Several medications can affect the levels or interfere with
load such as with the use of non-infant milk. The same pat- quantitation of amino acids in blood and urine (Table 49.8).
tern of elevation can be seen in premature infants or infants The use of disulfide agents, such as penicillamine, can result
with immature hepatocellular function. Table 49.6 lists the in the formation of unusual amino acids whose quantitation
most common effects of diet and nutritional status on can help in determining compliance with therapy.
amino acid values. Table 49.9 lists the amino acids and the disorders in which
Circadian rhythm is a physiological basis for higher the amino acid level is abnormal. An abnormal concentration
amino acid concentrations, up to 10–15 %, in the blood in the of an amino acid may be suggestive of several different
afternoon. A mild generalized increase in urine amino acids inborn errors. Conversely, some disorders are characterized
is a relatively common finding in hospitalized children. by abnormalities in several different amino acids, and analy-
Branched-chain amino acids are elevated in blood of patients sis of the pattern of variation is more important that analysis
with maple syrup urine disease; however, in these patients of a single amino acid level in establishing a diagnosis. This
the presence of alloisoleucine allows the correct interpreta- table serves as a quick guide to more detailed information
tion of the results. Vomiting and starvation of 1–2 days’ found in other chapters in this book.
duration may cause mild elevations (two- to threefold) of the The authors acknowledge the contribution of Dr. Vivian
plasma branched-chain amino acids, and this should not be Shih to the previous edition of this book, on which portions of
mistaken as a disease pattern. In a patient with MSUD and this chapter are based.
49 Amino Acids 757
Table 49.9 Pathologic conditions associated with abnormal amino acids concentrations
Amino acid Source Disorder(s) Value
All amino acids U Classic galactosemia ↑H
All amino acids U Renal Fanconi syndrome ↑H
All amino acids U Tyrosinemia type I ↑H
All amino acids U Hereditary fructose intolerance ↑H
All amino acids U Lowe syndrome ↑H
All amino acids U Vitamin d-dependent rickets ↑H
All amino acids U Mitochondrial disorders ↑H
Neutral amino acids U Hartnup disorder ↑H
Alanine P Lactic acidosis, disorders of pyruvate metabolism, mitochondrial ↑H
disorders, hyperammonemic syndromes
Alanine P Maple syrup urine disease ↓L
β-Alanine P/U β-Alaninemia ↑Η
β-Alanine CSF GABA transaminase deficiency ↑H
β-Alanine U Pyrimidine disorders, methylmalonate semialdehyde dehydrogenase ↑H
deficiency
Alloisoleucine P/U/CSF Maple syrup urine disease, E3 deficiency ↑H
α-Aminoadipic U α-Aminoadipic/α-ketoadipic aciduria ↑Η
β-Aminoisobutyric acid U β-Alaninemia, β-aminoisobutyric acid aminotransferase deficiency ↑Η
(benign)
δ-Aminolevulinic acid U Tyrosinemia type I, porphyria ↑H
Arginine U Cystinuria, dibasic aminoaciduria, lysinuric protein intolerance ↑H
Arginine P Arginase deficiency ↑H
Arginine P HHH syndrome, ornithine aminotransferase deficiency, urea cycle ↓L
defects (except arginase deficiency)
Argininosuccinate P/U/CSF Argininosuccinate lyase deficiency ↑H
Aspartic acid U Dicarboxylic aminoaciduria ↑H
Aspartic acid U Pyruvate carboxylase deficiency type B ↓L
Aspartylglucosamine P/U Aspartylglucosaminidase deficiency ↑H
Carnosine U Carnosinemia ↑H
Citrulline P Citrullinemia type I (argininosuccinate synthase deficiency), ↑H
citrullinemia type II (citrin deficiency), argininosuccinate lyase
deficiency, pyruvate carboxylase deficiency type B
Citrulline P Δ-Pyrroline-5-carboxylate synthase deficiency, lysinuric protein ↓L
intolerance, NAGS, CPS, OTC deficiencies, mitochondrial disorders
Cystathionine P/U Cystathionase deficiency ↑H
Cystine U Cystinuria, hyperlysinemia, hyperornithinemia ↑H
Cystine P Molybdenum cofactor deficiency, sulfite oxidase deficiency ↓L
Formiminoglutamic acid (FIGLU) U Formiminoglutamic aciduria ↑H
GABA P/U β-Alaninemia ↑Η
GABA P/U/CSF GABA transaminase deficiency ↑H
Glutamic acid U Dicarboxylic aminoaciduria ↑H
Glutamic acid P Glutamic acidemia, glutamine synthase deficiency ↑H
Glutamine P/U/CSF Urea cycle defects ↑H
Glutamine P Glutamine synthase deficiency, propionic acidemia, methylmalonic ↓L
acidemia, maple syrup urine disease
Glycine P/U/CSF Glycine encephalopathy, propionic acidemia, methylmalonic ↑H
acidemia, d-glyceric aciduria
Glycine U Familial renal iminoglycinuria, hyperprolinemia type I and II ↑H
Glycine P/CSF Serine deficiency disorders ↓L
Glycylproline U Prolidase deficiency ↑H
Hawkinsin U Hawkinsinuria ↑H
Histidine P/U Histidinemia ↑H
Homoarginine P/U Hyperlysinemia ↑H
(continued)

Amino acid Source Disorder(s) Value
Homocarnosine CSF Homocarnosinosis ↑H
Homocitrulline P/U HHH syndrome, saccharopinuria ↑H
Homocyst(e)ine P/U Cystathionine β-synthase deficiency, cobalamin disorders, folate ↑H
disorders
Homocyst(e)ine P Methionine adenosyltransferase deficiency, S-adenosylhomocysteine ↑H
hydrolase deficiency, glycine-N-methyltransferase deficiency,
adenosine kinase deficiency
Homocysteine-cysteine disulfide P Cystathionine β-synthase deficiency ↑H
Homocysteine P Molybdenum cofactor deficiency, sulfite oxidase deficiency ↓L
Hydroxylysine U Hydroxylysinuria ↑H
Hydroxyproline U Familial renal iminoglycinuria, hyperprolinemia type I and II ↑H
Hydroxyproline P/U Hydroxyprolinemia ↑H
Imidodipeptides U Prolidase deficiency ↑H
Isoleucine P/U Maple syrup urine disease, E3 deficiency ↑H
Leucine P/U Maple syrup urine disease, E3 deficiency ↑H
Lysine U Cystinuria, lysinuric protein intolerance, dibasic aminoaciduria ↑H
Lysine P/U Hyperlysinemia, saccharopinuria ↑H
Lysine P HHH syndrome, ornithine aminotransferase deficiency, lysinuric ↓L
protein intolerance
Lysine P Urea cycle defects, pyruvate carboxylase deficiency type B ↑H
Methionine P/CSF Homocysteine remethylation disorders ↓L
Methionine P/U Cystathionine β-synthase deficiency, hypermethioninemias ↑H
Methionine sulfoxide P Cystathionine β-synthase deficiency, hypermethioninemias ↑H
Ornithine U Cystinuria, dibasic aminoaciduria, hyperlysinemia, lysinuric ↑H
protein intolerance
Ornithine P HHH syndrome, ornithine aminotransferase deficiency ↑H
Ornithine P Δ-Pyrroline-5-carboxylate synthase deficiency ↓L
Phenylalanine P/U Phenylketonuria, hyperphenylalaninemias, pterin disorders ↑H
Phenylalanine P Tyrosinemia type I ↑H
Phosphoethanolamine U Hypophosphatasia ↑H
Pipecolic acid P Hyperlysinemia, antiquitin deficiency (pyridoxine-responsive seizures) ↑H
Pipecolic acid U Hyperprolinemia type II ↑H
Pipecolic acid P/U Peroxisomal disorders ↑H
Proline P Δ-Pyrroline-5-carboxylate synthase deficiency ↓L
Proline U Familial renal iminoglycinuria ↑H
Proline P/U Hyperprolinemia type I and II ↑H
Saccharopine P/U Saccharopinuria ↑H
Sarcosine P/U Sarcosinemia, mitochondrial disorders, glutaric acidemia type II ↑H
Serine P/CSF Serine deficiency disorders ↓L
S-Sulfocysteine P/U Molybdenum cofactor deficiency, sulfite oxidase deficiency ↑H
Taurine U Molybdenum cofactor deficiency, sulfite oxidase deficiency, ↑H
β-alaninemia
Threonine P/CSF Pyridoxal phosphate-dependent seizures, citrullinemia type II ↑H
(citrin deficiency)
Tryptophan U Tryptophanuria ↑H
Tyrosine P/U Tyrosinemia type I, II, III, transient tyrosinemia of the newborn ↑H
Tyrosine P Phenylketonuria, pterin disorders ↓L
Valine P/U Maple syrup urine disease, E3 deficiency, hypervalinemia ↑H
P plasma, U urine, CSF cerebrospinal fluid, H high, L low
49 Amino Acids 759
References Hagenfeldt L, Bjerkenstedt L, Edman G, Sedvall G, Wiesel FA (1984)

Amino acids in plasma and CSF and monoamine metabolites in
CSF: interrelationship in healthy subjects. J Neurochem
Applegarth DA, Edelstein AD, Wong LT, Morrison BJ (1979) Observed
42:833–837
range of assay values for plasma and cerebrospinal fluid amino acid
Held PK, White L, Pasquali M (2011) Quantitative urine amino acid
levels in infants and children aged 3 months to 10 years. Clin
analysis using liquid chromatography tandem mass spectrometry
Biochem 12:173–178
and aTRAQ reagents. J Chromatogr B Analyt Technol Biomed Life
Armstrong M, Jonscher K, Reisdorph NA (2007) Analysis of 25 under-
Sci 879:2695–2703
ivatized amino acids in human plasma using ion-pairing reversed-
Honkanen RA, Baruah S, Zimmerman MB, Khanna CL, Weaver YK,
phase liquid chromatography/time-of-flight mass spectrometry.
Narkiewicz J, Waziri R, Gehrs KM, Weingeist TA, Boldt HC, Folk JC,
Rapid Commun Mass Spectrom 21:2717–2726
Russell SR, Kwon YH (2003) Vitreous amino acid concentrations in patients
Bertram KM, Bula DV, Pulido JS, Shippy SA, Gautam S, Lu MJ,
with glaucoma undergoing vitrectomy. Arch Ophthal 121:183–188
Hatfield RM, Kim JH, Quirk MT, Arroyo JG (2008) Amino-acid
Parvy PR, Bardet JI, Rabier DM, Kamoun PP (1988) Age-related refer-
levels in subretinal and vitreous fluid of patients with retinal detach-
ence values for free amino acids in first morning urine specimens.
ment. Eye 22:582–589
Clin Chem 34:2092–2095
Gregory DM, Sovetts D, Clow CL, Scriver CR (1986) Plasma free
amino acid values in normal children and adolescents. Metabolism
35:967–969
Organic Acids
50
Isabel Tavares de Almeida and Marinus Duran

50.1 Introduction........................................................................ 761
Organic acids are low molecular weight (mass <300) organic
50.2 Preanalytical Conditions ................................................... 764
substances. They have one or more carboxylic acid groups
50.3 Analysis ............................................................................... 765 and may have keto or hydroxy groups which make them
50.4 Interpretation/Reference Values....................................... 769 polar. They do not have an amino group that distinguishes
50.5 Differential Diagnosis ........................................................ 771
them from amino acids.
In general, the physiological C2–C10 organic acids are
References ...................................................................................... 772 weak acids which implies that they are fully ionised at pH
>4, i.e. values observed in plasma or urine. Consequently,
the organic acids are strongly hydrophilic, enabling their
rapid excretion into the urine.
Fatty acids with chain length >C8 such as lauric, palmitic
and oleic acids are nonpolar organic acids, which are bound
to plasma proteins and are not excreted into the urine. These
nonesterified fatty acids (NEFA) are generally seen as the
free fatty acid (FFA) pool.
The majority of organic acids are originated from the
intermediary metabolism of various cellular components,
such as amino acids, carbohydrates, lipids and steroids.
Physiologically, organic acids are often present as their coen-
zyme A esters and good examples are propionyl-CoA and
isovaleryl-CoA, although some acids are always seen in the
free form, such as pyruvic and 2-ketoglutaric acids.
Accumulation of organic acids in the blood (‘acidemia’)
will cause a shift in blood pH to lower values resulting in
acidosis. The so-called metabolic acidosis is accompanied
by an increased anion gap. Calculation of the anion gap or
the base excess will give an idea of the concentration of the
excess of organic acid(s).
Organic acidemias (or acidurias) are inborn errors of
I. Tavares de Almeida (*)
Metabolism and Genetics, iMed.UL, metabolism (IEM) which are biochemically characterised by
Faculdade de Farmácia da Universidade de Lisboa, the accumulation, in body fluids, of organic acids which can
Av. Prof. Gama Pinto, Lisboa 1649-003, Portugal be accompanied with specific acylcarnitines and/or acylgly-
e-mail: italmeida@ff.ul.pt
cines or other conjugates. Formation of acylcarnitines and
M. Duran acylglycines can be regarded as a process aimed at restoring
Lab Genetic Metabolic Diseases,
the coenzyme A homeostasis, at the same time being a pro-
Academic Medical Center, Meibergdreef 9,
Amsterdam 1105AZ, The Netherlands cess of detoxification of the toxic organic acids, similar to
e-mail: m.duran@amc.uva.nl the formation of acyl glucuronides. The accumulating
762 I. Tavares de Almeida and M. Duran
organic acids may be either the ‘normal’ physiological known organic acidurias, including those accompanied by
metabolites in enhanced concentration (cf. methylmalonic abnormalities in the amino acid profile, is given in Table 50.1.
acid) or unusual secondary organic acids formed by alterna- The key organic acids that should not be missed in the bio-
tive enzyme reactions (cf. methylcitrate and/or isovalerylgly- chemical diagnosis of these disorders are also shown in
cine) or a combination of both. A synopsis of the currently Table 50.1.
Table 50.1 The organic acidurias and their key metabolites. All disorders are grouped according to the metabolic pathway in which the primary
defect can be found. Disorders in which amino acid changes occur are also included
Affected pathway No Defect Key metabolite(s)
Leucine 1 Branched-chain 2-ketoacid dehydrogenase (MSUD) 2-Ketoisocaproic acid
2-Hydroxyisocaproic acid
2-Ketoisovaleric acid
2-Hydroxyisovaleric acid
2-Keto-3-methylvaleric acid
2-Hydroxy-3-methylvaleric acid
Leucine 2 Isovaleryl-CoA dehydrogenase Isovalerylglycine
3 3-Methylcrotonyl-CoA carboxylase 3-Methylcrotonylglycine
4 Biotinidase 3-Hydroxyisovaleric acid
3-Hydroxypropionic acid
Lactic acid
5 Holocarboxylase synthase 3-Hydroxyisovaleric acid
3-Hydroxypropionic acid
Lactic acid
6 3-Methylglutaconic aciduria type 1 3-Methylglutaconic acid
3-Methylglutaric acid
7 3-Methylglutaconic aciduria type 2 (Barth syndrome) 3-Methylglutaconic acid
8 3-Methylglutaconic aciduria type 3 (Costeff syndrome) 3-Methylglutaconic acid
9 3-Methylglutaconic aciduria type 4 (unknown, mito?) 3-Methylglutaconic acid
10 3-Hydroxy-3-methylglutaryl-CoA lyase 3-Hydroxy-3-methylglutaric acid
3-Methylglutaconic acid
11 Mevalonate kinase Mevalonic acid + lactone
Isoleucine 12 2-Methylbutyryl-CoA dehydrogenase 2-Methylbutyrylglycine
13 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase 2-Methyl-3-hydroxybutyric acid
Tiglylglycine
14 β-Ketothiolase 2-Methyl-3-ketobutyric acid
2-Methyl-3-hydroxybutyric acid
Tiglylglycine
15 Propionyl-CoA carboxylase Methylcitric acid
3-Hydroxypropionic acid.
16 Methylmalonyl-CoA epimerase/mutase/Cbl A, B, C, Methylmalonic acid
D, F/SUCLA 2 Methylcitric acid
Valine 17 Isobutyryl-CoA dehydrogenase Isobutyrylglycine
18 3-Hydroxyisobutyryl-CoA hydrolase NONE, cf. hydroxyisobutyrylcarnitine
19 3-Hydroxyisobutyrate dehydrogenase 3-Hydroxyisobutyric acid
20 Methylmalonic semialdehyde dehydrogenase Methylmalonic acid
(continued)
50 Organic Acids 763

Lysine 21 2-Ketoadipic acid dehydrogenase (?) 2-Ketoadipic acid
2-Hydroxyadipic acid
22 Glutaryl-CoA dehydrogenase Glutaric acid
3-Hydroxyglutaric acid
23 Glutaryl-CoA oxidase Glutaric acid
Proline 24 Δ-Pyrroline-carboxylate dehydrogenase Pyrrole-5-carboxylglycine
Phenylalanine 25 Phenylalanine hydroxylase 2-Hydroxyphenylacetic acid
Phenyllactic acid
Phenylacetic acid (−glutamine)
Tyrosine 26 Alkaptonuria Homogentisic acid
27 Fumarylacetoacetase (type I) 4,6-Diketoheptanoic acid
(= succinylacetone)
28 Hawkinsin 4-Hydroxycyclohexanecarboxylic acid
29 Tyrosine aminotransferase (type II) 4-Hydroxyphenyllactic acid
4-Hydroxyphenylpyruvic acid
30 4-Hydroxyphenylpyruvate dioxygenase (type III) 4-Hydroxyphenyllactic acid
4-Hydroxyphenylpyruvic acid
31 Aromatic amino acid decarboxylase (AADC) 3-Methoxy-4-hydroxyphenyllactic acid
TCA cycle 32 Fumarase Fumaric acid
33 2-Ketoglutarate dehydrogenase 2-Ketoglutaric acid
34 SUCLA 2 Methylmalonic acid
Succinic acid
γ-Glutamyl cycle 35 Glutathione synthase Pyroglutamic acid
36 5-Oxoprolinase Pyroglutamic acid
Histidine 37 Formiminotransferase Hydantoin-5-propionic acid
Miscellaneous 38 Malonyl-CoA decarboxylase Malonic acid
Methylmalonic acid
39 Aspartoacylase N-Acetylaspartic acid
40 Aminoacylase 1 N-Acetylamino acids
41 Succinic semialdehyde dehydrogenase 4-Hydroxybutyric acid + lactone
4,5-Dihydroxyhexanoic acid,
3,4-Dihydroxybutyric acid
42 l-2-Hydroxyglutaric acid dehydrogenase l-2-Hydroxyglutaric acid + lactone
43 d-2-Hydroxyglutaric acid dehydrogenase d-2-Hydroxyglutaric acid + lactone
44 Isocitrate dehydrogenase 2 d-2-Hydroxyglutaric acid + lactone
45 d-Glyceric acid kinase d-Glyceric acid
46 d-Glyceric acid dehydrogenase l-Glyceric acid
Oxalic acid
47 Alanine-glyoxylate aminotransferase (peroxisomal) Glycolic acid
Oxalic acid
48 Generalised peroxisomal defect (Zellweger) 2-Hydroxysebacic acid
Epoxydicarboxylic acids
49 Glycerol kinase Glycerol
50 Glycogenoses, gluconeogenesis defects, mitochondrial Lactic acid
respiratory chain defects 2-Ketoglutaric acid
other TCA intermediates
3-Hydroxybutyric acid
3-Ketobutyric acid
Glycerol (phosphate)
(continued)

Fatty acid β-oxidation 51 Short-chain acyl-CoA dehydrogenase Ethylmalonic acid
Methylsuccinic acid
52 Medium-chain acyl-CoA dehydrogenase Hexanoylglycine
Suberylglycine
Dicarboxylic acids
5-Hydroxyhexanoic acid
Phenylpropionylglycine
53 Very long-chain acyl-CoA dehydrogenase Dicarboxylic acids (+unsaturated)
54 Multiple acyl-CoA dehydrogenation defect Dicarboxylic acids
(glutaric aciduria type II) Short-chain acylglycines
Ethylmalonic acid
d-2-Hydroxyglutaric acid
Glutaric acid
55 Long-chain 3-hydroxyacyl-CoA dehydrogenase 3-Hydroxyadipic acid + lactone
3-Hydroxydicarboxylic acids
Dicarboxylic acids
56 Short-chain 3-hydroxyacyl-CoA dehydrogenase l-3-Hydroxybutyric acid
3-Hydroxyglutaric acid
Historically, the classical organic acidurias were charac- of precursors cannot be quantitated as an independent vari-
terised by huge excretions of organic acids which could easily able. The formation of secondary metabolites of the key
be picked up by simple gas chromatographic/mass spectro- organic acid, e.g. propionylcarnitine, methylcitrate and pro-
metric (GC/MS) analysis. Nowadays, the biochemical genet- pionylglycine in propionic acidemia patients makes a reli-
ics laboratory needs a detailed quantitative GC/MS system able balance study even more difficult.
for urine organic acids diagnosis which must include the Urine samples of neonates generally are more diluted
recognition of small increases of specific organic acids. These than those of older children or adults. In order to achieve
data turn to be a valuable diagnostic tool. In this respect the organic acid chromatograms that can be visually compared,
European external quality assurance organisation ERNDIM the starting amount of urine prior to extraction will be
(www.erndim.org) facilitates both qualitative and quantitative aimed at a fixed amount of creatinine present in the sample.
programmes. The establishment of the correct conditions depends on the
sensitivity of the analytical methods/equipment used;
therefore, each laboratory should have its own protocol
50.2 Preanalytical Conditions regarding that issue.
Diet and drugs are the main disturbing factors of the
A random urine sample is the preferred material for the eval- organic acid profile and may cause serious interpretative dif-
uation of organic acid profiles. The timing of the urine col- ficulties. The best known dietary disturbances are those
lection is generally not important. However, the clinical caused by the use of medium-chain triglyceride-containing
signs of the patient may give some guidance, as an example, diets giving rise to the excretion of C6–C10 dicarboxylic acids
patients with fasting intolerance are best investigated using as well as (ω-1)-hydroxy acids. Also partially hydrolyzed
an early-morning specimen. Whenever an organic acid proprotein sources such as the Nutramigen infant formula may
file is unclear and does not allow a solid diagnosis, the use of pose problems due to the interfering components/additives,
in vivo loading tests is advocated because these tests have e.g. dioctyl phthalate which appears in the organic acid chro-
proven to substantially enhance the organic acid excretion matogram. Various dietary carbohydrates do not survive the
(Ofman et al. 2003). food processing steps and may give rise to artefacts such as
Timed urine collections such as the 24-h urine are mainly furane-2,5-dicarboxylic acid, 2,4-dihydroxybutyric acid or
used in the follow-up of patients, e.g. in the process of treat- glucosan.
ment monitoring. In this respect it may be quite difficult to The exact knowledge of the diet and the drugs, including
relate the excretion of the key organic acid to the dietary vitamers and natural supplements, used by the patients is
intake of the precursor amino acid(s) because the contribu- therefore essential for the correct interpretation of the organic
tion of endogenous stores and intestinal bacterial production acid profiles.
50.3 Analysis The organic acids – now sitting in an organic solvent –

can be concentrated by evaporating the solvent in a stream of
Prior to the analysis, the organic acids must be isolated from nitrogen at modestly elevated temperatures.
the sample’s matrix; the compounds of interest are then con- Gas chromatography requires the analytes to be volatile,
centrated and further transformed in derivatives suitable for i.e. they must be immediately evaporated in the injection
gas chromatography analysis. In order to check the proce- port. Organic acids do not fulfil this requirement: they are as
dure, an internal standard (non-physiological organic acid) is such not volatile enough. Derivatisation of the acid (COOH),
added to the urine, such as phenylbutyric acid or pentadeca- the hydroxy (C-OH) and the keto (C=O) groups increases
noic acid. In native urine (pH 5–7), the acids are present as their volatility. The larger the molecule, the later it elutes
salts (Na+, K+) and cannot be extracted with an organic sol- having higher retention indices. The most widely used deri-
vent. Acidification of the urine (pH 1–2) causes protonation vatisation procedure is the construction of a trimethylsilyl
of the acid, and then the protonated acid can be extracted with (TMS) derivative. This transforms the molecule into a more
an apolar organic solvent. Ethyl acetate or diethyl ether is the balloon-like structure which can easily be volatilized and
most widely used. Extraction recoveries depend on the polar- moves faster along the column. An exhaustive list of organic
ity of the acid: the more hydroxyl groups, the less recovery. acids as trimethylsilyl/methoxime derivatives sorted accord-
Keto acids tend to be unstable; the keto group can be pro- ing to their gas chromatography elution order is displayed in
tected, prior to the extraction, by formation of an oxime Table 50.2. The retention indices may vary slightly and are
through reaction with hydroxylamine or methoxyamine; an column and instrument specific.
example is succinylacetone (4,6-diketoheptanoic acid, the One of the disadvantages of the trimethylsilyl derivative is
key tyrosinemia type 1 metabolite). its extensive fragmentation in the mass spectrometer, thereby
Table 50.2 Gas chromatography elution order (relative to the internal standard 3-phenylbutyric acid) of organic acids and molecular mass of the
trimethylsilyl/ethoxime derivatives
Organic acid Mol. mass (derivatised) Ret. indicesa
2-Hydroxyisobutyric acid – 2TMS 248 0.3756
Phenol –TMS 166 0.3881
Lactic acid – 2TMS 234 0.3993
Glyoxylic ethoxime I 189 0.4326
Glycolic acid – 2TMS 220 0.4430
Pyruvic acid – ethoxime 203 0.4927
2-Hydroxybuytyric acid – 2TMS 248 0.5009
3-Hydroxypropionic acid – 2TMS 234 0.5413
p-Cresol – TMS 180 0.5417
3-Hydroxybutyric acid – 2TMS 248 0.5531
2-Hydroxyisovaleric acid – 2TMS 262 0.5535
3-Hydroxyisobutyric acid – 2TMS 248 0.5588
2-Ketoisovaleric acid ethoxime I 231 0.5849
Urea peak 1 276 0.5934
Oxalic acid – 2TMS 234 0.5912
2-Methyl-3-hydroxybutyric acid – 2TMS 262 0.6223
3-Ketobutyric acid ethoxime I 217 0.6166
3-Hydroxyisovaleric acid – 2TMS 262 0.6223
3-Ketobutyric acid (enol) – 2TMS 246 0.6427
3-Ketobutyric acid ethoxime II 217 0.6481
2-Ethylhydracrylic acid (2-ethyl-3-hydroxypropionic acid) – 2TMS 262 0.6634
2-Hydroxyisocaproic acid – 2TMS 276 0.6673
2-Hydroxy-2-methylvaleric acid – 2TMS 276 0.6715
3-Hydroxyvaleric acid – 2TMS 262 0.6720
4-Hydroxybutyric acid – 2TMS 248 0.6816
Methylmalonic acid – 2TMS 262 0.6858
2-Keto-3-methylvaleric acid ethoxime I 245 0.6879
Malonic acid – 2TMS 248 0.6909
(continued)

2-Ketoisocaproic acid (enol) – 2TMS 274 0.6940
2-Keto-3-methylvaleric acid ethoxime II 245 0.7013
Octanoic acid – TMS 216 0.7036
2-Ketoisocaproic acid ethoxime II 245 0.7259
Benzoic acid – TMS 194 0.7282
Glycerol – 3TMS 194 0.7282
3-Hydroxyhexanoic acid – 2TMS 276 0.7723
Ethylmalonic acid – 2TMS 276 0.7779
Phosphoric acid – 3TMS 314 0.8135
Glyceric acid – 3TMS 322 0.8208
Phenylacetic acid – TMS 208 0.8262
2,3-Dihydroxybutyric acid I – 3TMS 336 0.8302
Succinic acid – 2TMS 262 0.8375
Uracil-2TMS 256 0.8396
2,3-Dihydroxybutyric acid II – 3TMS 336 0.8475
2-Methylsuccinic acid – 2TMS 276 0.8449
5-Hydroxyhexanoic acid – 2TMS 276 0.8540
Fumaric acid – 2TMS 260 0.8773
3-Keto-depakine ethoxime I 273 0.8930
Urea peak 2 – 2TMS 204 0.9058
3-Keto-depakine ethoxime II 273 0.9150
Thymine – 2TMS 272 0.9209
Penicillamine 231 0.9408
Glutaric acid – 2TMS 276 0.9633
3-Methylglutaric acid – 2TMS 290 0.9827
3-Phenylbutyric acid (Internal Standard) – TMS 236 1
3-Methylglutaconic acid (I) – 2TMS 288 1.0052
Phenoxyacetic acid – TMS 224 1.0281
Mandelic acid – 2TMS 296 1.0490
Malic acid – 3TMS 350 1.0613
3-Methylglutaconic acid (II) – 2TMS 288 1.0670
Mevalonic acid (lactone) – TMS 202 1.0678
Isobutyrylglycine – TMS 217 1.0734
3-Hydroxy-3-methylglutaric acid (I) – 3TMS 378 1.0783
Adipic acid – 2TMS 290 1.0955
Mevalonic acid – 3TMS 364 1.1078
7-Hydroxyoctanoic acid – 2TMS 304 1.1164
Isovalerylglycine – TMS 303 1.1215
3-Methyladipic acid – 2TMS 304 1.1266
2-Propylglutaric acid (valproate) – 2TMS 318 1.1285
5-Hydroxymethyl-2-furanecarboxylic acid – 2TMS 286 1.1629
3-Hydroxyglutaric acid – 3TMS 364 1.1676
2-Hydroxyglutaric acid – 3TMS 364 1.1716
2-Hydroxyphenylacetic acid – 2TMS 296 1.1719
Phenyllactic acid – 2TMS 310 1.1721
Phenylpyruvic acid ethoxime I 279 1.1906
4,6-Diketoheptanoic acid (oxime) (succinylacetone) – TMS 227 1.2493
Phenylpyruvic acid ethoxime II 279 1.2502
3-Hydroxy-3-methylglutaric acid (II) – 3TMS 378 1.1841
Pyroglutamic acid – 2TMS 273 1.1971
(continued)

Pimelic acid – 2TMS 304 1.2118
4-Hydroxybenzoic acid – 2TMS 282 1.2347
2-Ketoglutaric acid ethoxime – 2TMS 333 1.2398
4-Hydroxyphenylacetic acid – 2TMS 296 1.2446
3-Methylcrotonylglycine – TMS 229 1.2866
Tiglylglycine – TMS 229 1.2894
Octenedioic acid – 2TMS 316 1.3038
Furane-2,5-dicarboxilic acid – 2TMS 300 1.3086
Suberic acid – 2TMS 318 1.3194
Orotic acid – 3TMS 372 1.3641
Hexanoylglycine – TMS 245 1.3654
Cis-aconitic acid – 3TMS 390 1.3915
Vanillic acid – 2TMS 312 1.0415
2-Furoylglycine – TMS 241 1.4033
N-acetylaspartic acid – 2TMS 319 1.3939
Homovanillic acid – 2TMS 326 1.4192
Azelaic acid – 2TMS 332 1.4235
3,6-Epoxy-tetradecanedioic acid – 2TMS 332 1.4241
Citric acid – 4TMS 480 1.4413
Methylcitric acid I – 4TMS 494 1.4505
Methylcitric acid II – 4TMS 494 1.4583
Isocitric acid – 4TMS 480 1.4594
3-Hydroxyphenyl hydracrylic acid – 3TMS 398 1.4640
Homogentisic acid – 3TMS 384 1.4714
Decenedioic acid – 2TMS 344 1.4965
3-Hydroxysuberic acid – 3TMS 406 1.5047
Vanilmandelic acid – 3TMS 414 1.5225
4-Hydroxyphenyllactic acid – 3TMS 398 1.5225
Sebacic acid – 2TMS 346 1.5227
4-Hydroxyphenylpyruvic acid ethoxime I 367 1.5269
Acetaminophen 223 1.5887
4-Hydroxyphenylpyruvic acid ethoxime II 367 1.5901
Hippuric acid – TMS 251 1.6180
Pantothenic acid 435 1.6634
3-Hydroxysebacic acid – 3TMS 434 1.6863
Indole-3-acetic acid – TMS 247 1.7034
2-Hydroxyhippuric acid – 2TMS 339 1.7960
Phenobarbital 232 1.8571
5-Hydroxyindole-3-acetic acid – 3TMS 407 1.8717
Trichloroethylglucuronide 612 1.8891
5-Hydroxyindole-3-acetic acid – 2TMS 335 1.9502
Suberylglycine – 2TMS 375 1.9962
4-Cresolglucuronide 572 2.0091
Vanilloylglycine – 2TMS 369 2.0882
Dioctyl phthalate 390 2.1099
Phenylacetylglutamine – TMS 318 2.1919
a
Retention indices relative to that of the internal standard 3-phenylbutyric acid using a CP Sil19 CB column
Table 50.3 ‘Metabolic’ and ‘neuropathic’ organic acidurias and the occurrence of acylcarnitines as an aid in the biochemical diagnosis
Metabolic Organic acids (u) Acylcarnitines (p) Neuropathic Organic acids (u) Acylcarnitines (p)
Methylmalonic acidemia +++ +++ Glutaric acid uria type 1 0/+++ 0/+
Propionic acidemia +/+++ +++ l-2-OH-glutaric aciduria ++ 0
Isovaleric aciduria +++ +++ d-2-OH-glutaric aciduria ++ 0
3-Methylcrotonyl-CoA +++ +++ Fumaric aciduria ++ 0
carboxylase deficiency SUCLA 2 deficiency + +
Biotinidase deficiency 0/+++ 0/+++ N-Acetyl aspartic aciduria +++ 0
3-Hydroxy-3-methylglutaryl- +++ +++ 2-Ketoglutaric acid uria +/++ 0
CoA lyase deficiency
Ketothiolase deficiency +++ + 4-OH-butyric aciduria 0/++ 0
5-Oxoprolinuria +++ 0 d-Glyceric aciduria ++ 0
Ketosis, gluconeogenesis 0/+++ 0 2-Methyl-butyryl-CoA + 0-tr
dehydrogenase deficiency
Alkaptonuria +++ 0 2-Methyl-3- + 0-tr
hydroxybutyryl-CoA
dehydrogenase deficiency
Mevalonic aciduria 0/+++ 0 Lactic acidemias 0/+++ 0
2-Ketoadipic acid uria ++ 0
The number of symbols (+) represents the increasing severity of the biochemical abnormalities
hampering the easy use of selected ion monitoring (see many labs still prefer the FID for quantitative analyses, e.g.
below). This can be overcome by constructing the tertiary for the monitoring of treatment. Accuracy of the organic acid
butyldimethylsilyl derivatives which give a simple fragmen- analysis in the lower concentration range can be improved by
tation with M-57 as the most abundant peak in the mass spec- using a stable isotope dilution assay. This is based on the
trum, a perfect starting point for selected ion monitoring. addition of a stable isotope-labelled (13C or 2H) internal stan-
The detector of the gas chromatograph historically used dard which behaves identically in all steps of the analysis,
was the flame ionisation detector; nowadays it was replaced i.e. extraction, derivatisation and chromatographic separa-
by a mass spectrometer. All separated compounds enter the tion. A range of applications has been developed, a.o. succi-
ion source. They are ionised (obtain a positive electric nylacetone for the follow-up of tyrosinemia type 1 patients,
charge) and fragmented and then deflected in an electromag- mevalonic acid for hyper-IgD patients and methylmalonic
netic field. Variation of the electromagnetic field will cause acid for vitamin B12-related disorders.
the passage of ions with increasing mass to pass the ion exit Historically, organic acid analysis was always performed
slit and hit the ion collector (detector). The fragments of any by GC/MS which is still the most widely used technique.
given compound are a fingerprint and give the unique identi- However, the recent availability of electrospray tandem mass
fication of the compound. spectrometers (MS/MS) in many labs has resulted in replace-
Every modern GC/MS instrument has library search facili- ment of GC/MS by MS/MS for specific applications, in par-
ties. This gives for each peak the most likely structure. ticular for methylmalonic acid (Blom et al. 2007) and
Interpretation of the library data should be done with care: the succinylacetone (Sander et al. 2006). There are even organic
predicted structure should always be compared with the posi- acid screening methods using MS/MS nowadays (Al-Dirbashi
tion in the chromatogram. Again, small molecules elute early, et al. 2007) which are rapid and sensitive but lack the ability
and large molecules elute later. There are even sophisticated of finding novel metabolites. Further developments in this
methods for the simultaneous deconvolution, identification and area will certainly emerge.
quantitation of organic acids using a dedicated library of mass One should not forget the importance of MS/MS analysis
spectra and a list of retention indices. One of these systems is of plasma, blood spot or urine acylcarnitines. This approach
called AMDIS (Automated Mass Spectral Deconvolution and gives instantaneous results and is, therefore, applicable to
Identification System), originally developed by the National newborn screening. Unfortunately, only half of the organic
Institute of Standards and Technology (NIST) for chemical acidemias are accessible by MS/MS analysis of acylcarni-
weapons convention treaty compliance (Halket et al. 1999). tines, amongst which a fair number of treatable disorders
Several experienced labs have developed search routines in (Table 50.3). It has become good laboratory practice to per-
which the mass spectrometer automatically checks for the pres- form simultaneous GC/MS of urine organic acids and MS/
ence of all diagnostic organic acids listed in Table 50.1. MS of plasma acylcarnitines in any acutely sick patient in
The linearity of the mass spectrometer total ion current whom an organic acidemia is suspected.
response is reputedly less extended than that of the tradi- The here-described analytical method does not exclu-
tional flame ionisation detector (FID). This is the reason why sively detect organic acids; other low-molecular substances
Table 50.4 ‘Normal’ organic acids in the urine of subjects referred for selective screening of IEM on a normal diet, without any medication and
without signs of intestinal disease
Reference values (mmol/mol creat)
Compound 0–4 months 4 months–2 years 2–10 years >10 years
Glycolic acid 13–129 32–162 48–164 23–146
Lactic acid – <200 <85 <50
Oxalic acid 140–360 <160 <125 <70
3-Hydroxypropionic acid 0–38 10–44 4–30 4–23
3-Hydroxy(iso)butyric acid 0–38 20–118 4–19
3-Hydroxyisovaleric acid 2–47 10–54 10–66 6–49
Methylmalonic acid 1–11 2–13 1–4 0–4
Ethylmalonic acid 0–15 0–15 0–9 2–10
Succinic acid 40–125 44–79 5–81 <16
Phosphoric acid var. – – –
Glutaric acid 0–11 2–15 1–10 1–4
Adipic acid 2–27 0–25 2–10 1–7
2-Hydroxyglutaric acid 6–67 11–49 6–35 4–16
3-Hydroxy-3-methylglutaric acid 15–105 13–49 6–27 3–11
2-Ketoglutaric acid 100–500 60–120 <80 <80
4-Hydroxyphenylacetic acid – – – <60
Homovanillic acid 3–20 2–19 1–14 1–5
N-Acetylaspartic acid 0–92 0–56 0–39 0–11
Suberic acid 1–15 2–15 1–10 1–6
Cis-aconitic acid var. – – –
Citric acid var. – – –
Hippuric acid var. – – –
The lower and upper reference values represent the 5th and 95th percentiles as used in the authors’ laboratories
The age dependency of the excretion pattern is clear, albeit not so prominent for the main urinary organic acids such as glycolic acid and
3-hydroxypropionic acid. An influence of diet and intestinal composition cannot be excluded in these instances. Var variable peak size may be
prominent in the chromatogram
may also be picked up. Important findings for the biochemi- as well as the volume of urine that was taken for the analy-
cal genetics lab are the pyrimidines (uracil, thymine, dihy- sis. The laboratory should aim at producing chromatograms
drouracil, dihydrothymine) associated with inborn errors of showing the compounds listed on Table 50.4 as moderate
the pyrimidine breakdown pathway and orotic acid, a marker peaks in order not to lose sensitivity and on the other hand
of urea cycle defects. In addition, polyols such as glycerol not to get lost in a forest of tiny, non-informative peaks. We
and butanediol may appear in the organic acid chromato- have always made a profit by analysing true patient samples
gram as well as larger, carbohydrate-related substances such of published cases; you must keep in mind that authors of
as glucosan and glucuronides. the relevant papers will often be willing to share their expe-
rience with less experienced scientists. Participation in the
qualitative ERNDIM programmes will eventually result in
50.4 Interpretation/Reference Values the collection of a series of reference patient samples for
private use. The easiest diagnoses rely on the massive
Hundreds of substances are excreted into the urine of healthy excretion of one or a few pathognomonic metabolites.
and diseased subjects, amongst which a large number of Good examples are the mut0 methylmalonic acidemia and
organic acids. The normal excretion profile depends on the the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase
age of the patient, his diet and clinical status (fasting, exer- deficiency.
cise, etc.). It is therefore essential to gain experience in pat- It is necessary to be aware that not all elevated organic
tern recognition in order to provide a reliable descriptive acids can be related to an inherited disorder (Kumps et al.
interpretation of the organic acid profile. This is one of the 2002). Table 50.5 summarises the most important non-
major tasks of the Biochemical Genetics Laboratory. genetic anomalies currently observed in the organic acid pro-
A distinct set of organic acids will always be detectable filing. The contribution of the diet, the gut bacterial action
in urine, even in healthy subjects (Table 50.4). This is, of and the antiepileptic medication is most pronounced and
course, depending on the sensitivity of the analytical system should always be taken in mind.
Table 50.5 Non-IEM organic Compound Condition

acids in urine as well as dietary/
Aromatic acids (4-hydroxyphenyl) Gut bacterial action
drug/bacterial artefacts
Mandelic acid Albumin infusion
d-Lactic acid Short bowel syndrome
d-2-Hydroxyisocaproic acid Short bowel syndrome
d-Phenyllactic acid Short bowel syndrome
3-Hydroxyisovaleric acid Valproate medication
Glutaric acid Gut bacterial action
3-Hydroxypropionic acid Gut bacterial action
Methylmalonic acid Vitamin B12 deficiency
Ethylmalonic acid Vitamin B2 deficiency
C10>C8>C6 dicarboxylic acids MCT diet
7-Hydroxyoctanoic acid MCT diet
3-Hydroxydicarboxylic acids Coeliac disease
Succinic acid 2-Ketoglutarate decomposition.
Glycolic acid Ethylene glycol poisoning
Pyroglutamic acid Glutamine decomposition
Flucloxacillin toxicity
Di-(2-ethylhexyl)phthalate Nutramigen feeding
Pregestimil feeding
Furane-2,5-dicarboxylic acid Heated sugars
Furoylglycine Heated sugars
4-Hydroxycyclohexanecarboxylic acid Food processing
Homovanillic acid Neuroblastoma
Vanilmandelic acid Neuroblastoma, phaeochromocytoma
N-Acetyltyrosine Parenteral feeding
5-Hydroxyindoleacetic acid Carcinoid syndrome
Valproate metabolites Depakine therapy
2-Hydroxyhippuric acid Salicylate ingestion
Ethosuximide metabolites Antiepileptic therapy
Keppra metabolites Antiepileptic therapy
Phenytoin metabolites Antiepileptic therapy
Abnormalities associated with the inherited organic aci- accumulation in the various organic acidemias. It can be
demias may be overwhelming, such as in most cases of understood that a fair proportion of the organic acidemias can
isovaleric or methylmalonic acidemias. On the other hand, reliably be diagnosed by acylcarnitine analysis, either in urine
there are many defects showing only marginally increased or in a dried blood spot, thereby facilitating the large scale
organic acids. Well-known examples are the 2-methyl- newborn screening of these disorders. Unfortunately several
3-hydroxybutyryl-CoA dehydrogenase deficiency (Ofman of the so-called neurometabolic organic acidurias are not
et al. 2003), mevalonate kinase deficiency, glutaric acidemia accessible by standard newborn screening methods and
type 1 and 4-hydroxybutyric acidemia. In those cases it is remain to be diagnosed by the classical GC/MS methods.
of great importance to have an accurate quantitative organic The primary accumulating organic acid (or coenzyme A
acid analysis with one’s own well-defined normal ranges. ester) may be the diagnostic hallmark by itself, as exemplified
Sometimes even a trace of a key metabolite may be diag- by pyroglutamic acid in oxoprolinase deficiency (Larsson et al.
nostic as evidenced by some cases of tyrosinemia type I hav- 1981) and homogentisic acid in alkaptonuria. It should be noted
ing only traces of succinylacetone in their urine (Haagen and that pyroglutamic acid can arise from glutamine as an artefact
Duran 1987). Patients with episodic fever and the hyper-IgD during storage; it can also be present in the urine of critically ill
syndrome may have urinary mevalonic acid levels which are patients who are being treated with flucloxacillin and
just twice the upper reference level of 0.8 mmol/mol creati- paracetamol. This combination of drugs can be misleading.
nine (Houten et al. 1999). Over the years experience has been gained about multiple
A number of the organic acid coenzyme A esters will read- secondary enzyme reactions using the accumulating organic
ily form carnitine esters as a mechanism for replenishing free acid as a substrate and giving rise to characteristic diagnostic
CoA stores and as a tool for detoxifying the potential toxic secondary metabolites (Table 50.6). Knowledge of biochemical
acyl-CoAs. Table 50.3 shows the magnitude of acylcarnitine pathways as well as enzyme biochemistry will be of great
Table 50.6 Alternative metabolism of key metabolites in the organic acidemias giving rise to (characteristic) secondary metabolites
Key metabolite Metabolic process Secondary metabolite
Isovaleryl-CoA β-Oxidation 3-Hydroxyisovaleric acid
Isovaleryl-CoA ω-Oxidation 4-Hydroxyisovaleric acid
Hexanoyl-CoA (ω-1)-Hydroxylation 5-Hydroxyhexanoic acid
3-Methylcrotonyl-CoA Glycine conjugation 3-Methylcrotonylglycine
Propionyl-CoA Carnitine conjugation Propionylcarnitine
Isovaleryl-CoA Glutamic acid conjugation Isovalerylglutamate
Octanoic acid Glucuronic acid conjugation Octanoylglucuronide
Propionyl/CoA Ketone formation 3-Ketovaleric acid
Propionyl-CoA Citric acid cycle interference Methylcitric acid
2-Ketoadipic acid Decarboxylation Glutaric acid
3-Methylglutaconyl-CoA Reduction 3-Methylglutaric acid
2-Hydroxyglutaric acid Lactone formation 2-Hydroxyglutaric acid lactone
Tiglyl-CoA Carboxylation 2-Methylglutaconic acid
help in putting the identities of the secondary metabolites ago. This should always be kept in mind when finding abnor-
into place. Sometimes the production of secondary metabo- mal levels of this substance. Over the years all inherited
lites masks the presence of the primary metabolite such as defects of methylmalonyl-CoA metabolism, including the
observed in methylmalonic acidemia (Tavares de Almeida epimerase defect (Bikker et al. 2006), have been unravelled
et al. 1991). Unfortunately, not a single laboratory will now- as well as defects of every single step of the interconversion
adays be able to assess the accumulation of free propionic of B12 vitamers. Measurement of plasma homocysteine is an
acid in propionic acidemia as this encompasses the long- undisputed prerequisite when interpreting abnormal methyl-
forgotten analysis of volatile fatty acids (Duran et al. 1973). malonate levels in urine or plasma. Furthermore, SUCLA2
In this respect both propionic acidemia (Przyrembel et al. deficiency as well as malonyl-CoA decarboxylase must be
1979) and isovaleric acidemia (Loots 2009) are notorious for taken in consideration in the presence of mild MMA levels.
their wealth of secondary metabolites. It is still a matter of 3-Hydroxyisobutyric acid, a regularly occurring organic
debate whether the occurrence of potential toxic secondary acid, is theoretically associated with a defect of
metabolites plays a role in the development of specific symp- 3-hydroxyisobutyrate dehydrogenase (Loupatty et al. 2006).
toms in individual patients. Metabolomics studies of groups Thus far no proven case has been published, although the
of patients may be helpful in answering these queries defect does exist (R.J. Wanders, personal communication
(Reinecke et al. 2012). 2013). This acid has been associated with defective methyl-
malonate semialdehyde dehydrogenase deficiency but also
with unexplained metabolic disease (Ko et al. 1991)
50.5 Differential Diagnosis 3-Methylglutaconic acid is one of the most puzzling
organic acids. Apart from its occurrence in the distal defects
Some organic acids can be regarded as characteristic of more of leucine breakdown, it appears in many conditions without
than one inborn error; in those cases a careful evaluation of a solid biochemical explanation. Think of Barth syndrome,
the complete profile is warranted. A synopsis of the problem Costeff syndrome, various defects of the mitochondrial
cases is given below. respiratory chain, Smith-Lemli-Opitz syndrome, unspecified
3-Hydroxyisovaleric acid is the primary marker of mal- neonatal metabolic disease (Hammond and Wilcken 1984)
functioning 3-methylcrotonyl-CoA carboxylase, a biotin- and neurological disease (Greter et al. 1978).
dependent enzyme. Consequently it will be elevated in all Glutaric acid, the hallmark of glutaryl-CoA dehydroge-
defects of biotin metabolism/transport. In addition, it appears nase deficiency, has been observed in ketotic patients with
in proximal and distal defects of the leucine degradation such gastroenteritis, also in HMG-CoA lyase deficiency during
as 3-methylglutaconyl-CoA hydratase deficiency, HMG-CoA decompensation. In these conditions its bacterial origin is a
lyase deficiency and isovaleric acidemia. SUCLA2-defective possibility. Another cause of elevated glutaric acid may be
patients also excrete this metabolite (Morava et al. 2009), and nonenzymatic decarboxylation of 2-ketoadipic acid. Multiple
there have been rumours about its presence in mitochondrial acyl-CoA dehydrogenation defect (formerly known as glu-
disease. Finally, valproate medication results in a marked taric aciduria type 2) and glutaryl-CoA oxidase deficiency
elevation of 3-hydroxyisovaleric acid. should also be considered.
Methylmalonic acid had been known as a marker of vita- 2-Hydroxyglutaric acid can occur in two configurations,
min B12 deficiency, both acquired and inherited, many years i.e. the l- and the d-form. Both need to be reconverted to
2-ketoglutaric acid by specific enzymes, and the d-form also Finally one should realise that organic acid analysis as a
occurs in the multiple acyl-CoA dehydrogenation defect tool for diagnosing inborn errors will always be carried out
(MADD). Separation of the isomers using a stereospecific in conjunction with complementary analyses such as those of
reagent will give proof of the identity. Separation of 2- and acylcarnitines and amino acids. Only an integrated view of
3-hydroxyglutaric acid, which occurs in glutaric aciduria the disturbed metabolism will enable the assessment of a
type 1, short-chain 3-hydroxyacyl-CoA dehydrogenase presumptive diagnosis, which will then be confirmed by
deficiency and CPT1 deficiency, may be troublesome. Their molecular or enzyme studies.
mass spectra of the TMS derivatives are, however, quite dif-
ferent showing an important fragment at m/z = 247 for the
2-hydroxyacid and a fragment at m/z = 259 for the
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aciduria. The most prominent 3-hydroxydicarboxylic acid is the
cycle. Acta Paediatr Scand 70:301–308
3-hydroxyadipic acid (C6), which may be converted into a lac- Loots DT (2009) Abnormal tricarboxylic acid cycle metabolites in iso-
tone having a molecular mass of 216 when trimethylsilylated. valeric acidemia. J Inherit Metab Dis 32:403–411
Loupatty FJ, van der Steen A, Ijlst L et al (2006) Clinical, biochemical, Reinecke CJ, Koekemoer G, van der Westhuizen FH (2012)
and molecular findings in three patients with 3-hydroxyisobutyric Metabolomics of urinary organic acids in respiratory chain deficien-
aciduria. Mol Genet Metab 87:243–248 cies in children. Metabolomics 8:264–283
Morava E, Steuerwald U, Carrozzo R et al (2009) Dystonia and deaf- Sander J, Janzen N, Peter M et al (2006) Newborn screening for hepa-
ness due to SUCLA2 defect: clinical course and biochemical torenal tyrosinemia: tandem mass spectrometric quantification of
markers in 16 patients. Mitochondrion 9:438–442 succinylacetone. Clin Chem 52:482–487
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Przyrembel H, Bremer HJ, Duran M et al (1979) Propionyl-CoA van Maldegem BT, Duran M, Wanders RJA et al (2006) Clinical, bio-
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catabolism at all levels. Eur J Pediatr 130:1–14 A dehydrogenase deficiency. JAMA 23:943–952
Acylcarnitines
51
Dietrich Matern

51.1 Introduction .................................................................... 775
51.2 Carnitine and Acylcarnitines ........................................ 776 Carnitine and its esters are physiologically present in all
biological fluids, but carnitine is most abundant in tissues
51.3 Indications for an Acylcarnitine Analysis .................... 776
with high energy requirements, particularly skeletal and car-
51.4 Methods ........................................................................... 778 diac muscle. In 1973 the first two clinically relevant disorders
51.5 Specimen ......................................................................... 778 affecting this pathway were described: primary carnitine defi-
ciency and carnitine palmitoyltransferase type II deficiency
51.6 Plasma and Serum ......................................................... 778
(DiMauro and DiMauro 1973; Engel and Angelini 1973). To
51.7 Dried Blood and Dried Bile Spots................................. 778 date, more than 20 different enzyme deficiency states affecting
51.8 Urine ................................................................................ 779 fatty acid transport and mitochondrial β-oxidation (FAO) are
51.9 Fibroblast Culture Medium .......................................... 779 known and additional enzymes involved in this pathway are
still being discovered (Bosch et al. 2011; Rinaldo et al. 2002).
51.10 Interpretation and Reference Ranges .......................... 779
Carnitine is involved as a detoxifying agent in branched-
References .................................................................................... 783 chain amino acid metabolism. Most of the classic organic
acidurias are associated with secondary carnitine deficiency.
In fact, the clinical utility of acylcarnitine analysis was first
identified for several organoacidopathies, and urine was the
preferred specimen. However, plasma became the specimen
of choice because acylcarnitine profiles are less complex in
plasma than in urine, and because the sensitivity of acylcar-
nitine analysis is higher when plasma is analyzed, especially
for the diagnosis of long-chain FAO disorders (Millington
et al. 1992). Today, urine acylcarnitine analysis is limited to
specific analytes where diagnostic value is added to the met-
abolic workup of patients with organic acidemias but incon-
clusive or borderline abnormal urine organic acid and plasma
acylcarnitine profiles (Ensenauer et al. 2004; Oglesbee et al.
2007; Tortorelli et al. 2005). Blood dried on filter paper is
analyzed for newborn screening and, together with bile, in
the postmortem evaluation of cases of sudden and unex-
pected death (Rinaldo et al. 2004). Cell-free supernatant of
amniotic fluid is used for the prenatal diagnosis of selected
inborn errors of metabolism (Rinaldo et al. 2001).
D. Matern Cultured fibroblasts or amniocytes can be probed with
Division of Laboratory Genetics, Departments of Laboratory FAO substrates and carnitine. Cell cultures deficient of an
Medicine and Pathology, Medical Genetics, and Pediatric
FAO enzyme will accumulate specific acylcarnitine species
and Adolescent Medicine, Mayo Clinic College of Medicine,
200 First Street SW, Rochester, MN 55902, USA when incubated with substrates such as palmitate, allowing
e-mail: matern@mayo.edu for the diagnosis of FAO disorders (Roe et al. 2001; Shen
776 D. Matern
Carnitine Acylcarnitine
CH3 H CH3 H
O O
+ +
H 3C N CH2 C CH2 C H3C N CH2 C CH2 C
O− O−
CH3 OH CH3 O
C O
Fig. 51.1 Structures of carnitine and acylcarnitine. The R represents the acylcarnitine species with up to 18 carbons which are typically the target
of an acylcarnitine analysis
et al. 2000; Young et al. 2003). Modifications of this assay activated long-chain fatty acids is accomplished by carni-
system have also been developed for the diagnosis of defects tine palmitoyltransferase type I (CPT-I) (Fig. 51.1). The acyl
affecting the metabolism of branched-chain amino acids and group of the activated fatty acid (fatty acyl-CoA) is trans-
for the study of peripheral blood mononuclear cells (Schulze- ferred by CPT-I from the sulfur atom of CoA to the hydroxyl
Bergkamen et al. 2005). group of carnitine. Carnitine-acylcarnitine translocase
Acylcarnitine analysis is almost exclusively performed by (CACT) then transfers the long-chain acylcarnitines across
tandem mass spectrometry (MS/MS) using stable isotope- the inner mitochondrial membrane, where CPT-II reverses
labeled internal standards that allow quantitation of acylcar- the action of CPT-I by formation of acyl-CoA and release of
nitine species. However, to provide meaningful results to free carnitine.
referring health care providers, it is critical to complement In pathologic conditions, such as FAO disorders or organic
analytical proficiency with in-depth interpretation of results acidemias due to acyl-CoA dehydrogenase deficiencies, the
as is true for many other examples of complex metabolic functions of carnitine as regulator of substrate flux and
profiles. energy balance across cell membranes and as modulator of
intracellular concentrations of free CoA become crucial. In
such conditions acyl-CoAs accumulate inside the mitochon-
51.2 Carnitine and Acylcarnitines drial matrix, and carnitine is utilized to shuttle these com-
pounds out of the mitochondria as acylcarnitines, thereby
Carnitine, l-3-hydroxy-4-(trimethylammonium)butyrate, is restoring free CoA.
a water-soluble, trimethylammonium derivative of γ-amino- Carnitine and its esters are present in all biological fluids
β-hydroxybutyric acid which is formed from trimethyllysine albeit very low in the CSF and depending on the enzyme
via γ-butyrobetaine (Vaz and Wanders 2002) (Fig. 51.1). defect, a particular acylcarnitine pattern becomes apparent
Carnitine originates to about 75 % from dietary intake of where those acylcarnitine species serving as direct substrates
meat, fish, and dairy products containing proteins with tri- for the defective enzyme accumulate disproportionately to
methyllysine residues. Under normal conditions, endoge- the down- and upstream metabolites (Table 51.1).
nous synthesis from lysine and methionine plays a minor
role. Carnitine is excreted in urine and bile as free carnitine
or as conjugated carnitine esters. Adequate intracellular lev- 51.3 Indications for an Acylcarnitine
els of carnitine depend on diet, endogenous synthesis, reab- Analysis
sorption, and cellular uptake.
Under physiologic conditions, carnitine is primarily Acylcarnitine analysis has proven a useful tool in the evalu-
required to shuttle long-chain fatty acids across the inner ation of patients at risk for inborn errors of fatty acid oxida-
mitochondrial membrane for fatty acid β-oxidation and tion and for organic acidemias that are primarily due to
products of peroxisomal β-oxidation to the mitochondria for defects in branched-chain amino acid metabolism. Given the
further metabolism in the citric acid cycle (Vaz and Wanders diverse clinical presentation of these conditions, acylcarni-
2002). Acylcarnitines (the carnitine esters) are formed tine analysis has become an integral part of the biochemical
by conjugating acyl-CoA moieties to carnitine which for genetic laboratory investigation of a large number of patients.
51 Acylcarnitines 777
Table 51.1 Clinically relevant acylcarnitine species (as butyl esters) included in a typical acylcarnitine analysis and their relevance when
abnormally elevated (unless otherwise noted)
Acylcarnitine species Disorder
C0 Free carnitine Carnitine supplementation (deficiency if low)
C2 Acetyl- Carnitine supplementation or ketosis (deficiency if low)
C3 Propionyl- PA, MMA, SUCLA2 (Carrozzo et al. 2007), treatment with heptanoic acid
FIGLU (Malvagia et al. Glutamate formiminotransferase deficiency
2006)
C4 Butyryl-/Isobutyryl- SCAD deficiency, IBDH deficiency, GA-2, EE
C5:1 Tiglyl- BKT, MCC, MHBD
C5 Isovaleryl-/2- IVA, SBCAD deficiency, GA-2, EE, antibiotics- or cream-derived artifact
Methylbutyryl-/Pivaloyl- (Abdenur et al. 1998; Boemer et al. 2014), treatment with heptanoic acid
C4-OH 3-Hydroxy butyryl- SCHAD deficiency, ketosis
C6:1 3-Methylglutaconyl- MGA
C6 Hexanoyl- MCAD deficiency, MKAT deficiency, GA-2
C5-OH 3-Hydroxy isovaleryl- Biotinidase deficiency, HMG, MCC, MCD, MGA
2-Methyl 3-hydroxy butyryl- BKT, MHBD
Benzoyl- Treatment with Na-benzoate
Dextrose (fragment) Sample contamination with dextrose
C7 Heptanoyl- Treatment with heptanoic acid
Phenylacetyl- Treatment with phenylacetic acid
Salicylyl- Treatment with salicylic acid
C8 Octanoyl- MCAD deficiency, M/SCHAD deficiency, MKAT deficiency, GA-2
C3-DC Malonyl- Malonic aciduria
C8-OH 3-Hydroxy octanoyl- MKAT deficiency
C10:2 Decadienoyl- DCR deficiency
C10:1 Decenoyl- MCAD deficiency
C10 Decanoyl- MCAD deficiency, GA-2
C4-DC Methylmalonyl-/succinyl- MMAa, SUCLA2 (Carrozzo et al. 2007)
C5-DC Glutaryl- GA-1
C10-OH 3-Hydroxy decanoyl- M/SCHAD deficiency, MKAT deficiency
C12 Dodecanoyl- GA-2
C6-DC 3-Methylglutaryl- HMG
C12-OH 3-Hydroxy dodecanoyl- LCHAD/TFP deficiency
C14:1 Tetradecenoyl- CACT deficiency, CPT type II deficiency, GA-2, VLCAD deficiency, LCHAD/TFP
deficiency
C14 Tetradecanoyl- (myristoyl-) CACT deficiency, CPT type II deficiency, GA-2, VLCAD deficiency, LCHAD/TFP
deficiency
C14-OH 3-Hydroxy tetradecanoyl- LCHAD/TFP deficiency
C16 Hexadecanoyl- (palmitoyl-) CACT deficiency, CPT type II deficiency, VLCAD deficiency, LCHAD/TFP deficiency;
CPT type Ib
C16:1-OH 3-Hydroxy hexadecenoyl- Antibiotics-derived artifact (Vianey-Saban et al. 2004)
C16-OH 3-Hydroxy hexadecanoyl- LCHAD/TFP deficiency
C18:2 Octadecadienoyl- (linolyl-) CACT deficiency, CPT type II deficiency, VLCAD deficiency, LCHAD/TFP deficiency
C18:1 Octadecenoyl- (oleyl-) CACT deficiency, CPT type II deficiency, VLCAD deficiency, LCHAD/TFP deficiency;
CPT type Ib
C18 Octadecanoyl- (stearyl-) CACT deficiency, CPT type II deficiency, VLCAD deficiency, LCHAD/TFP deficiency
C18:1-OH 3-Hydroxy octadecenoyl- LCHAD/TFP deficiency
C18-OH 3-Hydroxy octadecanoyl- LCHAD/TFP deficiency
BKT β-ketothiolase, CACT carnitine-acylcarnitine translocase, CPT carnitine palmitoyltransferase, DCR 2,4-dienoyl-CoA reductase, EE ethylma-
lonic encephalopathy, FIGLU formininoglutamate, GA-1 glutaric acidemia type I (glutaryl-CoA dehydrogenase deficiency), GA-2 glutaric acide-
mia type II (multiple acyl-CoA dehydrogenase deficiency), HMG 3-hydroxy 3-methylglutaryl-CoA lyase deficiency, IBDH isobutyryl-CoA
dehydrogenase, IVA isovaleric acidemia (isovaleryl-CoA dehydrogenase deficiency), LCHAD long-chain 3-hydroxy acyl-CoA dehydrogenase,
MCAD medium-chain acyl-CoA dehydrogenase, MCC 3-methylcrotonyl-CoA carboxylase deficiency, MCD multiple carboxylase (holocarboxyl-
ase) deficiency, MGA 3-methylglutaconic aciduria type I (3-methylglutaconyl-CoA hydratase deficiency), MMA methylmalonic acidemias, MHBD
2-methyl 3-hydroxy butyryl-CoA dehydrogenase, PA propionic acidemia (propionyl-CoA carboxylase deficiency), SCAD short-chain acyl-CoA
dehydrogenase, SCHAD short-chain 3-hydroxy acyl-CoA dehydrogenase, SUCLA2 APF-forming succinyl-CoA synthetase, TFP mitochondrial
trifunctional protein, VLCAD very long-chain acyl-CoA dehydrogenase
a
Respective analyte is not consistently elevated in MMA
b
CPT type I is suggested by low concentrations of long-chain acylcarnitine species and relatively high free carnitine
778 D. Matern
Table 51.2 Indications for acylcarnitine analysis residue derivatized with either n-butanol HCl or n-methanol
Clinical signs and symptoms Routine laboratory findings HCl yielding the acylcarnitines for analysis by flow injection
Respiratory distress Acidosis ESI-MS/MS. Following analysis, a graphical acylcarnitine pro-
Lethargy Ketosis
Coma Hypoglycemia
file is generated which can be interpreted qualitatively. (Semi-)
Recurrent vomiting Hyperammonemia quantitative calculation of the concentration of each individual
Failure to thrive Elevated liver enzymes acylcarnitine species is based on the abundance of the assigned
Feeding difficulty Elevated creatine kinase internal standard (Smith and Matern 2010).
Apnea Other abnormal laboratory findings
Hypotonia Dicarboxylic aciduria (excluding
Bradycardia dietary MCT)
Ventricular arrhythmias Hydroxydicarboxylic aciduria 51.5 Specimen
Cardiomyopathy Abnormal acylcarnitines by
Hepatic steatosis newborn screening
Hepatomegaly
A variety of body fluids can be used for acylcarnitine analysis,
Fatty acid profile suggestive of a
Encephalopathy FAO disorder but testing of plasma or whole blood spotted on filter paper is
Seizures Family history of most common. All sample types, except for dried blood and bile
Dystonia Affected sibling(s) spots and fibroblast cultures which can be sent at room tempera-
Myopathy Sudden unexplained death or
Rhabdomyolysis
ture, should be kept frozen until analysis. Reliable results, par-
SIDS in sibling(s)
Renal tubular acidosis Maternal pregnancy ticularly for short-chain acylcarnitine species, for any sample,
Polycystic kidneys complications (AFLP, liquid or dried on filter paper, cannot be achieved following
Reye or Reye-like syndrome HELLP) long-term storage at ambient temperatures (Matern et al. 1999).
Apparent Life Threatening
Event/“Near-miss” SIDS
51.6 Plasma and Serum

Other laboratory studies that should be considered in such
patient evaluations include urine organic acid and acylgly- Heparinized plasma is the preferred specimen for acylcarni-
cine, as well as plasma free fatty acid analyses. With the tine analysis, but EDTA plasma and serum are also accept-
additional application to newborn screening, among esoteric able. Hemolyzed or lipemic specimens can also be analyzed
tests acylcarnitine analysis has the highest sample through- without negative impact on sensitivity or specificity. An
put. Aside from clinical and newborn screening indications, amount of 100 μL is typically sufficient material to conduct
a relevant family and/or prenatal history and sudden unex- the analysis and repeat it at least once if necessary. Most
pected death also represent valid reasons to pursue an acyl- informative results are generally achieved when samples are
carnitine analysis (Table 51.2). An unequivocal indication as obtained during acute illness. Because inborn errors of
a test to monitor treated patients has not yet been established, metabolism are traditionally not entered early into differen-
although it is frequently being performed for this purpose. tial diagnostic considerations, sample collection should
alternatively be timed before a meal, preferably after an
overnight fast. A prolonged fasting challenge, however,
51.4 Methods should not routinely be undertaken as these require close sur-
veillance typically not possible in an outpatient setting.
Several techniques have been described to differentiate and
eventually quantify specific carnitine esters. These include gas
chromatography-mass spectrometry (GC-MS), thin layer chro- 51.7 Dried Blood and Dried Bile Spots
matography (TLC) and radioisotopic exchange/HPLC, liquid
chromatography (LC) MS, and LC-MS/MS. However, the pre- Acylcarnitine analysis has been introduced into newborn
dominant method applied is flow injection analysis MS/MS screening laboratories in the late 1990s and is now part of
using triple quadrupole analyzers combined with electrospray almost all newborn screening programs. Because newborn
ionization (ESI). The advantage of this approach is its sensitiv- screening tests make use of dried blood spots (DBS) collected
ity that allows for simple but efficient preparation procedures of after a heel prick on the second to fifth day of life, the DBS is
small sample volumes and fast analytical times, therefore the most common specimen used for acylcarnitine analysis. In
providing for a rapid throughput of large numbers of samples. In addition, many biochemical genetics laboratories offer clinical
a typical setup, acylcarnitines are extracted from the sample by testing of acylcarnitines in DBS for patients at any age. As is
mixing with methanol or an acidified acetonitrile solution con- true for plasma samples, the most informative results are
taining isotopically labeled acylcarnitines of various chain obtained when blood samples are collected during acute ill-
lengths at defined concentrations as internal standards. ness or at least prior to a meal. Blood should be obtained by
Following centrifugation, the supernatant is evaporated and the capillary stick of well-perfused skin (heels in young infants or
Table 51.3 Disorders detectable by the in vitro probe assay

Fatty acid β-oxidation disorders Short-chain acyl-CoA dehydrogenase (SCAD) deficiency
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency
Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency
Mitochondrial trifunctional protein (TFP) deficiency
Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency
Carnitine palmitoyltransferase type II (CPT-II) deficiency
Carnitine-acylcarnitine translocase (CACT) deficiency
Organic acid disorders Electron transfer flavoprotein (ETF) deficiency, ETF-dehydrogenase deficiency (multiple acyl-CoA
dehydrogenase deficiency, MADD; glutaric acidemia type II)
Isovaleric acidemia
Short-branched-chain acyl-CoA dehydrogenase deficiency (SBCAD deficiency; 2-methylbutyrylglycinuria)
Isobutyryl-CoA dehydrogenase (IBD) deficiency
fingers) and free dripping of a few drops of blood directly on results (Browning et al. 2005). Enzyme assays are limited to
the filter paper card. For postmortem analysis, blood and bile one enzyme per assay, and molecular assays for common
are collected at the latest at the time of autopsy. mutations are limited by the frequent occurrence of com-
Following complete drying at room temperature for at least pound heterozygous patients with an uncommon, private
3 h, the sample can be sent ambient. While diagnostic results mutation that must be distinguished from unaffected carriers.
can be obtained in most cases of medium- and long-chain FAO The in vitro probe assay offers screening for several defects
defects even after prolonged storage time at room temperature, of FAO and organic acid metabolism under controlled labo-
samples should be stored frozen (with desiccant) because par- ratory conditions using fibroblast cultures (Table 51.3). The
ticularly short-chain acylcarnitine species are not reliably mea- principle of this assay relies on the assumption that skin
surable several months after collection (Matern et al. 1999). At fibroblasts of patients affected with relevant conditions will
least one blood or bile spot with a diameter of 1 cm should be accumulate certain acylcarnitine species reflecting the meta-
collected to allow for any necessary repeat testing which usu- bolic defect when the cell medium is supplemented with a
ally requires only a DBS punch of 3 mm in diameter. long-chain fatty acid, branched-chain amino acids, and
l-carnitine. An acylcarnitine analysis can be performed in
the post-incubation cell medium by tandem mass spectrom-
51.8 Urine etry as for the other sample types (Smith and Matern 2010).
Fibroblasts are typically grown from a small skin biopsy
While initially the favored specimen, urine acylcarnitine anal- collected during an outpatient visit or as part of a planned sur-
ysis is the least appropriate when a FAO disorder is under diag- gical procedure following routine culturing techniques. Cell
nostic consideration. Long-chain acylcarnitines are typically cultures may also derive from umbilical cord or, for prenatal
bound to plasma albumin and are not excreted by the kidney. diagnostic purposes, from amniocytes obtained by amnio-
Urine is collected from patients suspected to have an organic centesis. Samples should be sent at ambient temperatures.
acidemia preferably during an acute metabolic decompensa-
tion. As this is often not possible, an early morning specimen
should be collected. The minimum volume of urine is 1 mL 51.10 Interpretation and Reference Ranges
which allows for acylcarnitine and creatinine analysis; the lat-
ter is essential to normalize quantitative acylcarnitine results. The laboratory director, typically a board certified clinical bio-
The sample should be sent frozen and without preservatives. chemical geneticist or equivalent, reviews all profiles and pro-
vides an interpretation based on pattern recognition and not on
single abnormal values (Tables 51.1, 51.4, 51.5, 51.6, and 51.7).
51.9 Fibroblast Culture Medium Simple reporting of numeric results is not appropriate because
most physicians are not familiar with pattern recognition. A com-
Most FAO disorders present similarly, and their biochemical prehensive interpretation takes into consideration any available
diagnosis can be difficult because common metabolite clinical and dietary information and other laboratory results and
screens, such as urine organic acids, plasma acylcarnitines, provides possible differential diagnoses, recommendations for
and fatty acids, are influenced by dietary factors and the additional biochemical testing, and confirmatory studies if indi-
clinical status of the patient (Van Hove et al. 2000) leading to cated, as well as contact information for the laboratory director in
incomplete diagnostic information or even false-negative case the referring physician has additional questions.
780 D. Matern
Table 51.4 Reference range for acylcarnitine species (as butyl esters) Table 51.5 Reference range of acylcarnitine species (as butyl esters)
in plasma (nmol/mL) in dried blood spots of adult controls (in nmol/mL)
≤7 days 8 days–7 years >7 years Blood spots (n = 99)
Acylcarnitine species (n = 36) (n = 139) (n = 140) Acylcarnitine species 1st percentile 99th percentile
C0 6.08–22.21 6.29–27.73 5.55–20.88 C0 11.16 −40.08
C2 2.14–15.89 2.00–27.57 2.00–17.83 C2 5.77 −20.23
C3 0.07–0.54 0.08–1.77 0.10–0.87 C3 <2.82
C4 0.06–0.45 0.06–1.05 0.04–0.82 C4 <0.43
C5:1 0.00–0.04 0.00–0.10 0.00–0.10 C5:1 <0.05
C5 0.06–0.37 0.05–0.62 0.06–0.50 C5 <0.30
C4-OH 0.01–0.12 0.01–0.50 0.01–0.17 C4-OH <0.11
C6 0.01–0.13 0.01–0.22 0.02–0.16 C6 <0.14
C5-OH 0.01–0.07 0.01–0.11 0.00–0.09 C5-OH <0.61
C7 0.00–0.04 0.00–0.04 0.00–0.05 C6-OH <0.06
C6-OH 0.00–0.07 0.00–0.18 0.00–0.08 C8:1 <0.26
C8:1 0.03–0.47 0.02–0.90 0.04–0.87 C8 <0.25
C8 0.03–0.18 0.01–0.44 0.02–0.77 C3-DC <0.06
C3-DC 0.01–0.08 0.00–0.13 0.00–0.25 C10:2 <0.04
C10:2 0.01–0.10 0.00–0.11 0.00–0.25 C10:1 <0.47
C10:1 0.03–0.24 0.01–0.45 0.03–0.46 C10 <0.35
C10 0.02–0.26 0.02–0.90 0.03–0.87 C4-DC <0.66
C4-DC 0.00–0.04 0.00–0.04 0.00–0.04 C5-DC <0.07
C10:1-OH 0.00–0.11 0.00–0.11 0.00–0.12 C12:1 <0.30
C5-DC 0.00–0.05 0.00–0.09 0.00–0.10 C12 <0.28
C12:1 0.01–0.18 0.00–0.36 0.00–0.34 C12:1-OH <0.06
C12 0.02–0.17 0.02–0.34 0.01–0.25 C12-OH <0.03
C12:1-OH 0.00–0.06 0.00–0.05 0.00–0.08 C14:2 <0.14
C12-OH 0.00–0.05 0.00–0.08 0.00–0.07 C14:1 <0.31
C14:2 0.01–0.08 0.01–0.12 0.01–0.17 C14 <0.41
C14:1 0.00–0.15 0.01–0.34 0.00–0.23 C14-OH <0.12
C14 0.01–0.10 0.01–0.14 0.00–0.11 C16:1 <0.10
C14:1-OH 0.01–0.05 0.00–0.17 0.00–0.12 C16 <1.72
C14-OH 0.00–0.03 0.00–0.04 0.00–0.07 C16:1-OH <0.05
C16:1 0.01–0.14 0.00–0.20 0.00–0.09 C16-OH <0.04
C16 0.06–0.35 0.04–0.51 0.03–0.22 C18:2 <0.48
C16:1-OH 0.00–0.77 0.00–0.35 0.00–0.05 C18:1 <1.49
C16-OH 0.00–0.09 0.00–0.06 0.00–0.05 C18 <0.93
C18:2 0.01–0.11 0.00–0.30 0.02–0.23 C18:2-OH <0.07
C18:1 0.02–0.24 0.03–0.44 0.02–0.38 C18:1-OH <0.06
C18 0.01–0.09 0.01–0.11 0.01–0.13 C18-OH <0.02
C18:2-OH 0.00–0.03 0.00–0.05 0.00–0.05
C18:1-OH 0.00–0.02 0.00–0.03 0.00–0.05
C18-OH 0.00–0.02 0.00–0.04 0.00–0.02
Table 51.6 Percentile ranks of acylcarnitine Blood spots (n = 6,077) Bile spots (n = 5,272)
species (as butyl esters) in postmortem dried
blood and bile spots (in nmol/mL) Acylcarnitine species 5th percentile 95th percentile 5th percentile 95th percentile
C0 31.7 – 47.3 −376.2
C2 27.3 −214.7 38.1 −335.3
C3 <11.4 <12.4
C4 <17.9 <7.8
C5:1 <0.23 <0.68
C5 <2.20 <3.43
C4-OH <7.56 <3.07
C6 <2.27 <2.93
C5-OH <1.04 <1.31
C7 <0.17 <0.92
C6-OH <0.57 <1.19
C8:1 <0.79 <28.5
C8 <0.82 <5.41
C3-DC <0.47 <1.41
C10:2 <0.12 <2.89
C10:1 <0.18 <7.89
C10 <0.54 <4.93
C4-DC <1.06 <1.20
C10:1-OH <0.13 <2.89
C5-DC <0.28 <1.13
C12:1 <0.12 <5.56
C12 <0.53 <5.66
C12:1-OH <0.13 <2.04
C12-OH <0.17 <1.10
C14:2 <0.44 <4.74
C14:1 <0.24 <6.11
C14 <0.46 <2.42
C14:1-OH <0.10 <1.52
C14-OH <0.08 <0.58
C16:1 <0.28 <1.98
C16 <2.05 <3.19
C16:1-OH <0.13 <0.86
C16-OH <0.11 <0.74
C18:2 <0.713 <2.35
C18:1 <2.04 <3.58
C18 <1.62 <3.36
C18:2-OH <0.13 <0.59
C18:1-OH <0.15 <0.76
C18-OH <0.11 <0.68
782 D. Matern
Table 51.7 Reference range for urine acylcarnitines (as butyl esters)
based on samples with normal organic acid results (n = 40) be submitted to the biochemical genetics laboratory
Acylcarnitine species (nmol/mL) with information regarding the clinical context during
C0 0.35–31.60 which the sample was collected. The laboratory must
C2 <16.46 be aware of the fact that carnitine deficiency states can
C3 <1.20 cause seemingly normal acylcarnitine profiles due to
C4 <2.74 the lack of carnitine as substrate for carnitine palmito-
C5:1 <0.34 yltransferases. Accordingly, it is crucial to review the
C5 <1.53 complete profile and to initiate follow-up when even
C4-OH <0.26
borderline elevated acylcarnitines are noted in the pres-
C6 <0.16
ence of abnormally low free acetylcarnitine. If clini-
C5-OH <0.52
cally indicated, a repeat sample should be collected as
C6-OH <0.32
C8:1 <4.30
early as 24 h after l-carnitine supplementation.
C8 <0.61 While MS/MS allows for unequivocal identifi-
C3-DC <0.50 cation of most metabolites, there are a few excep-
C10:2 <0.48 tions (Table 51.1). In particular, the short-chain
C10:1 <0.65 acylcarnitines of 4 and 5 carbons represent more
C10 <0.21 than one analyte. C4-Acylcarnitine is known to be a
C4-DC <0.57 mixture of butyrylcarnitine derived from fatty acid
C10:1-OH <0.26 metabolism and isobutyrylcarnitine derived from the
C5-DC (C10-OH) <0.37 metabolism of valine (Oglesbee et al. 2007). C4-OH
C12:1 <0.07 acylcarnitine can be a mixture of the d-3-OH-butyr-
C12 <0.19 ylcarnitine, associated with ketosis; l-3-OH-butyr-
C6-DC <0.81 ylcarnitine, associated with SCHAD deficiency; or
C12:1-OH <0.27 3-OH-isobutyrylcarnitine, characteristic of the valine
C12-OH <0.16
degradation defect 3-OH-isobutyryl-CoA hydrolase
C14:2 <0.02
deficiency. C5-Acylcarnitine is a mixture of isova-
C14:1 <0.21
lerylcarnitine and 2-methylbutyrylcarnitine derived
C14 <0.39
from leucine and isoleucine degradation, respec-
C14:1-OH <0.14
C14-OH <0.09
tively (Ensenauer et al. 2004; Matern et al. 2003).
C16:1 <0.04 Samples of patients treated with antibiotics contain-
C16 <0.18 ing pivalic acid (e.g., pivampicillin) may contain
C16:1-OH <0.02 pivaloylcarnitine, another C5 species. Several other
C16-OH <0.05 metabolites are also nonspecific markers for several
C18:2 <0.02 disorders. For example, C5-OH acylcarnitine, which
C18:1 <0.02 represents 3-hydroxyisovalerylcarnitine and 2-methyl
C18 <0.05 3-hydroxybutyrylcarnitine, can be elevated in seven
C18:2-OH <0.01 different organic acidemias. The interpretation of ele-
C18:1-OH <0.03 vated C5-OH acylcarnitine in newborns or breast-fed
C18-OH <0.02 infants is further complicated by the fact that it can
indicate maternal 3-methylcrotonylglycinuria while
the infant is only an unaffected carrier (Gibson et al.
Pitfalls 1998). Differentiation is of clinical importance and
The ordering physician must be aware of the limita- most efficiently achieved by urine acylglycine and
tions of a laboratory test and should consider involving organic acid analyses.
the biochemical genetics laboratory in discussions The long-chain FAO disorders of CACT deficiency
regarding the most appropriate diagnostic workup of and CPT-II deficiency cannot be differentiated because
patients. both cause accumulation of the same long-chain acyl-
Pitfalls in acylcarnitine analysis of plasma, serum, blood carnitine species which is explained by the fact that
spots, and bile spots neither enzyme is involved in the chain-shortening
Acylcarnitine profiles are dependent on the clinical sta- action of FAO. Isolated LCHAD deficiency and com-
tus of the patient at the time of sample collection (Van plete mitochondrial TFP deficiency also cannot be dif-
Hove et al. 2000). Accordingly, all samples should ferentiated by routine acylcarnitine analysis (Van Hove
et al. 2000). When such profiles are encountered, tions but be targeted to specific diagnostic consider-
delineation of the correct defect is only possible by ations, in particular organic acidemias. A role of urine
either specific enzyme assay in cell cultures or molecu- acylcarnitine analysis for the diagnosis of FAO disor-
lar genetic analysis of the relevant genes. MCAD and ders has not been established.
MAD defects require the identification of medium- Pitfalls in acylcarnitine analysis of post-incubation
chain acylcarnitines. In this respect it is important to fibroblast culture medium
realize that patients on a medium-chain triglyceride- The in vitro probe assay is performed under standard-
containing diet may accumulate C8 and C10 carnitine, ized conditions and is independent of the patient’s sta-
potentially obscuring the diagnosis. tus at the time the skin biopsy is obtained. The
Isolated elevations of propionylcarnitine (C3) are accumulation of acylcarnitines in this assay system
not specific for propionic acidemia but also observed appears to be dependent on potentially present residual
in methylmalonic acidemias of various etiologies. enzyme activity and therefore provides information
Because methylmalonylcarnitine (C4-DC) is not con- regarding the severity of an enzyme deficiency state
sistently elevated in methylmalonic acidemias, eluci- for some disorders such as VLCAD and SCAD defi-
dation of the correct diagnosis requires at a minimum ciencies. Furthermore, as is true for acylcarnitine anal-
urine organic acid analysis. ysis of other sample types, TFP and isolated LCHAD
Another antibiotic that may cause problems in the deficiencies as well as CPT-II and CACT deficiencies
interpretation of butylated acylcarnitines is cefotaxime cannot be differentiated. Finally, the analysis of fibro-
(Vianey-Saban et al. 2004). This antibiotic or metab- blasts does not allow for the diagnosis of enzyme defi-
olites thereof reveal itself by acylcarnitine analysis ciency states that are not expressed in this tissue (e.g.,
(following derivatization to butyl esters) at m/z 470 the liver-specific SCHAD deficiency).
which otherwise is considered to represent the mono-
unsaturated form of 3-hydoxyhexadecenoylcarnitine
(C16:1-OH). In poorly resolved scans, this may be dif-
ficult to differentiate from m/z 472 which is a marker
for LCHAD and TFP deficiencies. However, whereas
m/z 472 (C16-OH) is more abundant than C16:1-OH in References
these FAO disorders, the profile of a patient treated
with cefotaxime usually reveals an m/z 470 to m/z Abdenur JE, Chamoles NA, Guinle AE, Schenone AB, Fuertes AN
(1998) Diagnosis of isovaleric acidaemia by tandem mass spec-
472 ratio that is greater than 1. Furthermore and in
trometry: false positive result due to pivaloylcarnitine in a newborn
contrast to cefotaxime treatment, both LCHAD and screening programme. J Inherit Metab Dis 21:624–630
TFP deficiencies are usually accompanied by eleva- Boemer F, Schoos R, de Halleux V, Kalenga M, Debray F-G (2014)
tions of other long-chain species (Table 51.1) (Van Surprising causes of C5-carnitine false positive results in newborn
screening. Mol Genet Metab (in press)
Hove et al. 2000).
Bosch AM, Abeling NG, Ijlst L, Knoester H, van der Pol WL, Stroomer
Formiminoglutamate (FIGLU), a marker for gluta- AE, Wanders RJ, Visser G, Wijburg FA, Duran M, Waterham HR
mate formiminotransferase deficiency, is revealed in (2011) Brown-Vialetto-Van Laere and Fazio Londe syndrome is
acylcarnitine profiles by a peak with m/z 287 (Malvagia associated with a riboflavin transporter defect mimicking mild
MADD: a new inborn error of metabolism with potential treatment.
et al. 2006). In poorly resolved acylcarnitine profiles,
this peak may be confused with iso-/butyrylcarnitine Browning MF, Larson C, Strauss A, Marsden DL (2005) Normal acyl-
(m/z 288). To avoid the incorrect interpretation of acylcarnitine levels during confirmation of abnormal newborn screening
carnitine profiles, the analysis is best performed in in long-chain fatty acid oxidation Defects. J Inherit Metab Dis
28:545–550
product scan mode as opposed to multiple reaction
Carrozzo R, Dionisi-Vici C, Steuerwald U, Lucioli S, Deodato F, Di
monitoring (MRM) mode. Giandomenico S, Bertini E, Franke B, Kluijtmans LA, Meschini MC,
Pitfalls in acylcarnitine analysis of urine Rizzo C, Piemonte F, Rodenburg R, Santer R, Santorelli FM, van Rooij
Aside from the above mentioned potential problems A, Vermunt-de Koning D, Morava E, Wevers RA (2007) SUCLA2
mutations are associated with mild methylmalonic aciduria, Leigh-like
such as the inability to discriminate isomers and inter-
encephalomyopathy, dystonia and deafness. Brain 130:862–874
fering metabolites of antibiotics, urine also often con- DiMauro S, DiMauro PM (1973) Muscle carnitine palmityltransferase
tains a variety of nonidentified substances. This makes deficiency and myoglobinuria. Science 182:929–931
the interpretation of urine acylcarnitine profiles inher- Engel AG, Angelini C (1973) Carnitine deficiency of human skeletal
muscle with associated lipid storage myopathy: a new syndrome.
ently more complex and overinterpretation must be
Science 179:899–902
avoided. Therefore, urine acylcarnitine analysis should Ensenauer R, Vockley J, Willard JM, Huey JC, Sass JO, Edland SD,
not be included in the first line of screening investiga- Burton BK, Berry SA, Santer R, Grunert S, Koch HG, Marquardt I,
Rinaldo P, Hahn S, Matern D (2004) A common mutation is
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associated with a mild, potentially asymptomatic phenotype in acyl-CoA dehydrogenase deficiency: aspects of substrate specificity
patients with isovaleric acidemia diagnosed by newborn screening. and correlation with clinical phenotype. Clin Chim Acta 312:55–67
Am J Hum Genet 75:1136–1142 Schulze-Bergkamen A, Okun JG, Spiekerkotter U, Linder M, Haas D,
Gibson KM, Bennett MJ, Naylor EW, Morton DH (1998) Kohlmuller D, Mayatepek E, Schulze-Bergkamen H, Greenberg
3-methylcrotonyl-coenzyme a carboxylase deficiency in amish/ CR, Zschocke J, Hoffmann GF, Kolker S (2005) Quantitative acyl-
mennonite adults identified by detection of increased acylcarnitines carnitine profiling in peripheral blood mononuclear cells using in
in blood spots of their children. J Pediatr 132:519–523 vitro loading with palmitic and 2-oxoadipic acids: biochemical con-
Malvagia S, La Marca G, Casetta B, Gasperini S, Pasquini E, Donati firmation of fatty acid oxidation and organic acid disorders. Pediatr
MA, Zammarchi E (2006) Falsely elevated C4-carnitine as expres- Res 58:873–880
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spectrometry newborn screening. J Mass Spectrom 41:263–265 Qumsiyeh M, Kahler SG, Chen YT, Van Hove JL (2000)
Matern D, Strauss AW, Hillman SL, Mayatepek E, Millington DS, Acylcarnitines in fibroblasts of patients with long-chain
Trefz FK (1999) Diagnosis of mitochondrial trifunctional protein 3-hydroxyacyl-CoA dehydrogenase deficiency and other fatty acid
deficiency in a blood spot from the newborn screening card by tan- oxidation disorders. J Inherit Metab Dis 23:27–44
dem mass spectrometry and DNA analysis. Pediatr Res 46:45–49 Smith EH, Matern D (2010) Acylcarnitine analysis by tandem mass spec-
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Prospective diagnosis of 2-methylbutyryl-CoA dehydrogenase defi- (2005) The urinary excretion of glutarylcarnitine is an informative
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Lysosomals
52
Giancarlo la Marca

52.1 Introduction ..................................................................... 785
Lysosomal storage diseases (LSDs) are a group of more than
52.2 Preanalytical Conditions ................................................ 787
52.2.1 Specimens ......................................................................... 787 50 inherited metabolic disorders that result from defective
52.2.2 Chitotriosidase in Plasma .................................................. 787 lysosomal acid hydrolysis of endogenous macromolecules
causing their accumulation (Winchester et al. 2000). A classi-
52.3 Analysis ............................................................................ 787
52.3.1 Mucopolysaccharides in Urine.......................................... 787 fication based on the nature of the primary stored material
52.3.2 Oligosaccharides in Urine ................................................. 788 involved is reported in Table 52.1. Actually it is well known
52.3.3 Free and Total Sialic Acid in Urine................................... 790 that all LSDs are not simply due to this storage, but they result
52.3.4 Enzymatic Tests in Dried Blood Spots.............................. 791
from the perturbation of complex cell-signaling mechanisms.
References ...................................................................................... 793 Generally lysosomal storage disorders occur when lysosomes
do not function properly. All lysosomal storage diseases are
single-gene/single-enzyme disorders resulting in lysosomal
dysfunction. Various metabolic substrates are involved: lipids,
glycoproteins, and mucopolysaccharides. Most of LSDs are
autosomal recessively inherited disorders, with only three
exceptions: Fabry disease and Hunter disease (MPS II) are
X-linked recessive, while Danon disease is X-linked domi-
nant. To date, in the absence of newborn screening programs,
true LSD incidence is not known. Although some pilot proj-
ects reported a higher incidence (Spada et al. 2006; Chien
et al. 2008), individual LSDs occur with an incidence of less
than 1:100.000, while as a group the incidence is about 1:7,700
(Meikle et al. 1999). Comprehensive quali/quantitative analy-
sis of the entire panel of accumulation products is still not
available due to the complexity and chemical diversity of the
storage products involved in LSDs.
Important technological advances in the biological sci-
ences over the past few years have forged a new era of
research including the emerging field of systems biology and
in particular metabolomics. Metabolomics aims at the com-
prehensive and qualitative identification and quantification
of all metabolites in a biological system. Although in very
G. la Marca recent years many papers have been published with very
Department of Neurosciences, Psychology,
high potentiality, there is no single instrument platform/ana-
Pharmacology and Child Health,
University of Florence, Florence, Italy lytical technique that currently can analyze all metabolites
involved in LSDs, and the metabolomics approach is still not
Newborn Screening, Clinical Chemistry and Pharmacology Lab,
Meyer Children’s Hospital, Florence, Italy widely available. This means that LSDs analysis is generally
e-mail: g.lamarca@meyer.it performed on a single category of molecules with classical
786 G. la Marca
Table 52.1 Classification of lysosomal storage disorders

Alternative titles Enzyme deficiency OMIM
Mucopolysaccharidoses
52.1 MPS type I Hurler syndrome, Scheie Alpha-l-iduronidase 252800
syndrome
52.2 MPS type II Hunter syndrome Iduronate 2-sulfatase 309900
52.3 MPS type IIIA Sanfilippo A syndrome Heparan N-sulfatase 252900
52.4 MPS type IIIB Sanfilippo B syndrome Alpha-N-acetylglucosaminidase 252920
52.5 MPS type IIIC Sanfilippo C syndrome Acetyl-CoA: alpha-glucosaminide 252930
acetyltransferase
52.6 MPS type IIID Sanfilippo D syndrome N-Acetylglucosamine 6-sulfatase 252940
52.7 MPS type IVA Morquio A syndrome N-Acetylgalactosamine-6-sulfate sulfatase 253000
52.8 MPS type IVB Morquio B syndrome Beta-galactosidase 253010
52.9 MPS type VI Maroteaux-Lamy syndrome N-Acetylgalactosamine-4-sulfatase 253200
52.10 MPS type VII Sly syndrome Beta-glucuronidase 253220
52.11 MPS IX Natowicz syndrome Hyaluronidase 601492
Glycoproteinoses
52.12 Aspartylglucosaminuria Glycosylasparaginase 208400
52.13 Fucosidosis Alpha-l-fucosidase 230000
52.14 Galactosialidosis Goldberg syndrome Cathepsin A/beta-galactosidase/neuraminidase 256540
52.15 α-Mannosidosis Alpha-mannosidase 248500
52.16 β-Mannosidosis Beta-mannosidase 248510
52.17 Mucolipidosis I Sialidosis Type II Alpha-neuraminidase 256550
52.18 Mucolipidosis II I-cell disease N-Acetylglucosamine-1-phosphotransferase 252500
52.19 Mucolipidosis III Pseudo-Hurler polydystrophy N-Acetylglucosamine-1-phosphotransferase 252600
52.20 Schindler disease Neuroaxonal dystrophy Alpha-N-acetylgalactosaminidase 609241
Sphingolipidoses
52.21 Fabry disease Anderson-Fabry disease Alpha-galactosidase A 301500
52.22 Farber disease Farber lipogranulomatosis N-Acylsphingosine amidohydrolase 613468
52.23 Gaucher disease Acid beta-glucosidase 230800
52.24 GM1 gangliosidosis Beta-galactosidase-1 230500
52.25 Krabbe disease Globoid cell leukodystrophy Galactosylceramidase 245200
52.26 Metachromatic leukodystrophy Arylsulfatase A 250100
52.27 Multiple sulfatase deficiency Austin disease 272200
52.28 Niemann-Pick disease type A Acid sphingomyelinase 257220
52.29 Niemann-Pick disease type B Acid sphingomyelinase 607616
52.30 GM2-gangliosidosis type II Sandhoff disease Beta-hexosaminidase 268800
52.31 GM2-gangliosidosis type I Tay-Sachs disease Alpha-hexosaminidase 272800
Glycogen storage disease
52.32 Glycogen storage disorder type II Pompe disease Acid-alpha-glucosidase 232300
52.33 Glycogen storage disorder type IIb Danon disease LAMP-2 300257
Lysosomal transport defects
52.34 Cystinosis Cystinosin 219800
52.35 Mucolipidosis IV Sialolipidosis Mucolipidin 252650
52.36 Sialuria Salla disease Vesicular excitatory amino acid transporter 604369
(VEAT)
Others
52.37 Neuronal ceroid lipofuscinosis Cathepsin D 610127
52.38 Niemann-Pick disease type C NPC1 protein 257220
52.39 Lysosomal acid lipase deficiency Wolman disease Lysosomal acid lipase 278000
52.40 Pycnodysostosis Cathepsin K 265800
52 Lysosomals 787
biochemical tests ranging from thin-layer chromatography, 2. From venous blood collection
HPLC, electrophoresis, enzyme assays with fluorometric/ Apply one single drop of blood and follow the previous
spectrophotometric detection, and liquid chromatography- indications.
tandem mass spectrometry (LC-MS/MS). In addition, after a 3. From blood collection tubes
diagnosis has been made by biochemical means, mutation Apply 50 μL of blood from a heparinized or EDTA tube
analysis may be performed. and follow the previous indications.
52.2 Preanalytical Conditions 52.2.2 Chitotriosidase in Plasma
52.2.1 Specimens For this analysis 1 mL of heparinized blood is needed.

Samples should be kept refrigerated at +4 °C for the trans-
52.2.1.1 Glycosaminoglycans in Urine port. Do not bring the blood in direct contact with ice to
Urine samples should be collected for 24 h, when possible. avoid hemolysis.
But considering that prolonged urine collections are quite
difficult especially in newborns or very young babies, a ran- 52.2.2.1 Enzyme Assays in Leukocytes
dom sample, preferably the first morning voiding, is accept- For most assays 5–15 mL of heparinized or EDTA blood at room
able (not less than 20 mL). By that it is possible to achieve temperature is needed. It is important to use a needle with less
reliable results similar to those obtained in a 1-day collec- chance of hemolysis. The separation of leukocytes from whole
tion. Urines must be kept frozen until analysis, without pre- blood must be performed within 24 h of collection. A leukocytes
servatives. Samples should be shipped on dry ice for the pellet is stable at −20 °C until the day of the analysis.
transport; alternatively, heat inactivation with the use of thi-
omersal will allow the shipment of urine samples at ambient 52.2.2.2 Enzymatic Assays from Fibroblasts
temperature. All fibroblast cultures are generally derived from forearm skin
biopsies. For most tests one 75 cm2 flask is sufficient. In some
52.2.1.2 Oligosaccharides in Urine cases four 75 cm2 flasks are required due to the wide variations
Urine samples collected for 24 h are mandatory. The mini- in activity depending on the confluence and passage number.
mum volume of urine is 10 mL and the storage temperature A fibroblast pellet is stable at −20 °C until the day of the assay.
must be −20 °C until analysis. No preservatives are required.
52.2.2.3 Other Specimens
52.2.1.3 Analysis of Free and Total Sialic Acid Amniotic fluid, vitreous fluid, chorionic villi, or cultured
Urine samples should be collected for 24 h when possible amniocytes can be used in some isolated cases.
and stored refrigerated (2–8 °C) until the analysis.
52.2.1.4 Enzymatic Assays from Dried 52.3 Analysis

Blood Spot
1. From the heel for newborn screening purposes: the gen- 52.3.1 Mucopolysaccharides in Urine
eral instructions for screening tests should be followed. It
is important to apply the blood only on one side of the The mucopolysaccharidoses are a group of LSDs character-
filter paper and not to apply multiple layers of blood ized by an accumulation of glycosaminoglycans (GAGs)
drops on to the same circle. Avoid touching the area within the lysosomes. Each disorder is due to a single-enzyme
within the circles on the filter paper section before, dur- defect affecting the degradation of glycosaminoglycans. The
ing, and after collection of the specimen. Do not allow correlation between biochemical findings and enzyme defect
any material to come into contact with the specimen card is reported in Table 52.2, and the correlation between structure
before or after use. Cards must be dried at room tempera- and catabolism is reported and in Fig. 52.1. Several methods
ture for at least 90 min. If the analysis is performed after have been developed for the assay of the urinary GAGs.
20 days from the drawing of the blood, the storage tem- The first approach is an Alcian blue (tetravalent cationic
perature must be +4 °C or −20 °C. Most enzymes are dye) spot test, which semiquantitatively estimates the amount
stable at room temperature in the first 20 days. of total mucopolysaccharides present in the urine. Because the
788 G. la Marca
Table 52.2 Classification of the mucopolysaccharidoses

Mucopolysaccharidoses Glycosaminoglycans excreted in urine Defective enzyme Inheritance
MPS type I H/S DS; HS Alpha-l-iduronidase AR
MPS type II DS; HS Iduronate sulfatase XR
MPS type IIIA HS Heparan N-sulfatase AR
MPS type IIIB HS Alpha-N-acetylglucosaminidase AR
MPS type IIIC HS Acetyl-CoA: alpha-glucosaminide acetyltransferase AR
MPS type IIID HS N-Acetylglucosamine 6-sulfatase AR
MPS type IVA KS; C 6-S N-Acetylgalactosamine-6-sulfate sulfatase AR
MPS type IVB KS Beta-galactosidase AR
MPS type VI DS N-Acetylgalactosamine-4-sulfatase AR
MPS type VII DS; HS, C 4,6-S Beta-glucuronidase AR
MPS IX Hyaluronan Hyaluronidase AR
DS dermatan sulfate, HS heparan sulfate, KS keratan sulfate, C 6-S chondroitin 6-sulfate, C 4,6-S chondroitin 4,6-sulfate, AR autosomal recessive,
XR X-linked recessive
complex formation with Alcian blue depends on the sulfation definitive diagnosis enzyme activity test is needed.
patterns, keratan sulfate fragments with a low degree of sulfa- Appropriate enzyme sources are summarized in Table 52.4.
tion may escape detection; Morquio patients can be alterna- Considering that no homogeneous data of the different
tively detected by their excretion of chondroitin 6-sulfate. enzyme activity are present in literature, normal values are
Cetylpyridinium chloride (CPC) is generally used to extract not referred.
urinary GAGs prior to analysis. Quantitation of the GAGs can
be performed by the carbazole method determining uronic
acid formation following acid hydrolysis of the GAGs. 52.3.2 Oligosaccharides in Urine
Another simple test is based on the modification of
dimethylmethylene (DMB) assay (de Jong et al. 1992). Deficiency of a lysosomal enzyme required for glycoprotein
It allows the measurement of all GAGs with an equal degradation causes the accumulation of the corresponded
response for all urinary GAGs. By using any method, unmetabolized products with the resulting symptoms of a
false-negative results rarely occur, especially in MPS I, storage disorder. Normal urine contains different sugars
III, IV, VI, and VII. Many cases of false-positive results including a series of oligosaccharides depending on different
have been reported in literature. Some of these concerned factors (diet, medication, blood group type, etc.), but their
non-mucopolysaccharidoses with normal total concentra- excretion is very low.
tions of urinary GAGs but isolated increments such as in A one-dimensional thin-layer chromatography on 20 × 20
multiple sulfatase deficiency (HS), GM1 (KS), Morquio IV silica plates was developed 40 years ago (Palo and Savolainen
A and B (KS), and fucosidosis (KS). A new DMB-based 1972). Other authors (Humbel and Collart 1975; Friedman
method named GAG-Test® has been recently reported with a et al. 1978; Sewell 1981; Tsai and Marshall 1979) have
claimed sensitivity of 100 % (Lage et al. 2011). In addition, extended this approach, formerly developed to detect aspar-
GAGs can be quantified and analyzed by NMR (Guerrini tylglucosaminuria, to other defects. This author performs the
et al. 2005), MS (Saad and Leary 2005), LC/MS (Kuberan Humbel and Collart test with slight modifications: urine
et al. 2002), electrophoretic methods (Volpi and Maccari samples containing about 15 μg of creatinine are analyzed by
2006), and HPLC (Studelska et al. 2006). Reference values TLC on a 20 × 20 cm pre-coated silica gel plate using a
of urine GAGs are reported in Table 52.3. freshly prepared mobile phase containing n-butanol, glacial
A new screening test able to quantify glycosaminogly- acetic acid, and distilled water (10:5:5 v/v/v) and stained by
cans in urine by a fast LC-MS/MS method has been recently spraying orcinol 400 mg/dL in sulfuric acid 10 %. Spots cor-
developed for selective MPS I, MPS II, and MPS VI muco- responding to oligosaccharides are visualized by heating the
polysaccharidoses (Auray-Blais et al. 2011). The test is per- plate at 130 °C for about 10 min. This method is suitable for
formed on random urine samples and the concentration screening purposes, although a specific oligosaccharide pat-
expressed as a ratio to creatinine for normalization purposes. tern has been reported for many diseases (Sewell 2008).
Dermatan sulfate (DS) and heparan sulfate (HS) are mea- Normal neonatal urine may reveal multiple bands; therefore,
sured as derivatives by using calibration curve in urine, after an alternative method or a repetition of TLC test at the age of
a chemical degradation step (methanolysis). This assay can 6 months is needed. In some cases an intense band corre-
be considered a useful screening test for the future. For the sponding to a pentasaccharide may be present in a newborn
52 Lysosomals 789
2
1
a H2C O SO3–
CH2OH COOH
COOH –O S O O O
3 O
O
Glucuronate
NAcGal NAcGal
Glucuronate O O O
O
NHAc NHAc
4 3
3
b 1
CH2OH COOH CH2OH
–O S
O 3 O O
O
O –O S O
3 Glucuronate
COOH NAcGal NAcGal
O O O
Iduronate
O
NHAc
NHAc 4 3
–O S O
3 6
5 7
c COOH
H2C O SO3– CH2OH
O O O
O
Glucosamine NAcGlc
COOH Glucuronate
O O O
Iduronate O
–O S NH NH A c
3 9 3
–O S 6
3 O
8
5
11
10
d CH2OH
H2C O SO3– H2C O SO3–
CH2 O SO3– O O O
O
Galactose NAcGlc
NAcGlc
Galactose O O O
O
AcHN 4 12 NHAc
12
Fig. 52.1 NAcGlc N-acetylglucosamine, NAcGal N-acetylgalactosamine. (II); (6) a-l-iduronidase (I); (7) N-acetylglucosamine 6-sulfatase (III D);
(a) Chondroitin 4,6-sulfate; (b) dermatan sulfate; (c) heparan sulfate; (d) (8) heparan N-sulfatase(III A) followed by acetyl-CoA: a-glucosaminide
keratan sulfate. (1) N-Acetylgalactosamine 4-sulfate (VI); (2) acetyl transferase; (9) a-N-acetyl-glucosaminidase (III B); (10) N-acetyl-
N-acetylgalactosamine 6-sulfate (IV A); (3) β-glucuronidase (VII); (4) galactosamine 6-sulfatase (IV A); (11) N-acetyl-glucosamine 6-sulfatase
β-hexosaminidase (GM2-Sandhoff disease); (5) iduronate 2-sulfatase (III D); (12) β-galactosidase-1 (GMi-gangliosidosis, IV B)
790 G. la Marca
Table 52.3 GAGs reference ranges in urine

Alcian bluea DMB-Tris CPC/carbazole GAG-Test®
Age (years) (mg/mmol creatinine) (mg/mmol creatinine) (μmol uronic acid/mmol creatinine) (mg/mmol creatinine)
<0.1 10–40 26.1 ± 8.1
<0.3 10–35
<0.5 10–30 33.6 ± 9.2
<1 5–25 23.3 ± 4.1 17.8 ± 8.2 17.1 ± 7.7
<2 19.5 ± 5.2 14.6 ± 6.1
<3 5–20 14.5 ± 3.4 12.0 ± 4.2
<5 2–15 11.0 ± 1.7 10.7 ± 6.7
<6 8.3 ± 2.2 10.3 ± 3.7
<7 9.3 ± 1.8
<9 8.4 ± 1.6
<10 6.2 ± 1.9
<14 7.0 ± 1.8 4.6 ± 2.6
<15 2–12 7.0 ± 4.4
<18–20 4.1 ± 1.3 3.2 ± 1.6
>18–20 1–5 3.3 ± 0.9 1.6 ± 1.2 3.2 ± 1.6
Table 52.4 Source material for MPS enzymatic assays

Disorder Enzyme defect Material
Postnatal Prenatal
52.1 Alpha-l-iduronidase P, WBC, FB, LYM CV, CCV, CAFC
52.2 Iduronate 2-sulfatase P, WBC, FB, LYM CV, CCV, CAFC
52.3 Heparan N-sulfatase WBC, FB, LYM CV, CCV, CAFC
52.4 Alpha-N-acetylglucosaminidase P, WBC, FB, LYM CV, CCV, CAFC
52.5 Acetyl-CoA: alpha-glucosaminide acetyltransferase WBC, FB, LYM CV, CCV, CAFC
52.6 N-Acetylglucosamine 6-sulfatase WBC, FB, LYM CV, CCV, CAFC
52.7 N-Acetylgalactosamine-6-sulfate sulfatase WBC, FB, LYM CV, CCV, CAFC
52.8 Beta-galactosidase P, WBC, FB, LYM CV, CCV, CAFC
52.9 N-Acetylgalactosamine-4-sulfatase WBC, FB, LYM CV, CCV, CAFC
52.10 Beta-glucuronidase P, WBC, FB, LYM CV, CCV, CAFC
52.11 Hyaluronidase WBC, FB, LYM CV, CCV, CAFC
P plasma, WBC white blood cells, LYM lymphocytes, FB fibroblasts, CV chorionic villi, CAFC cultured amniotic fluid cells, CCV cultured chorionic villi
that is on parenteral nutrition; this pattern is not indicative of To date, in any case, a confirmatory enzyme assay must
disease. A modification of Humbel and Collart TLC method be performed in serum, fibroblasts, or leucocytes for defini-
has been proposed by Abeling et al. (1996) to distinguish tive diagnosis. Appropriate enzyme sources are summarized
urine of breast-fed newborns from that of α-mannosidosis in Table 52.5.
and β-mannosidosis patients. In case of unclear patterns
alternative and more selective methods must be performed.
Novel useful semiquantitative method based on tandem mass 52.3.3 Free and Total Sialic Acid in Urine
spectrometry for the diagnosis of the oligosaccharidurias has
been reported (Ramsay et al. 2005). Patients from I cell, Sialic acid (SA) or N-acetylneuraminic acid (NANA) repre-
mucolipidoses types II and III, alpha-mannosidosis, Pompe, sents the N- or O-terminal monosaccharide of complex gly-
GM1 gangliosidosis, GM2 type II, sialidosis, alpha-fucosido- coproteins and glycolipids. Sialic acids often represent part
sis, Gaucher, and SADS (sialic acid storage disorders) have of antigenic determinants of glycolipids or glycoproteins.
been analyzed successfully. This new procedure seems to be Urine total SA is increased in some lysosomal disorders such
sensitive, robust, automatable, and faster than classical TLC. as sialidosis, galactosialidosis, Kanzaki disease, and muco-
When stable isotope-labeled internal standards become com- lipidosis II and III. It has been reported that an increase in
mercially available, this method will be considered a power- urine total SA is also found in individuals with chronic glo-
ful tool for quantitative determination of oligosaccharides in merulonephritis, rheumatoid arthritis, lupus erythematosus,
urine. and pseudohypoparathyroidism and in diabetic patients. In
52 Lysosomals 791
Table 52.5 Source material for Disorder Enzyme defect Material

oligosaccharidurias enzymatic
Postnatal Prenatal
assays
52.12 Glycosylasparaginase WBC, FB CV, CAFC
52.13 Alpha-l-fucosidase P, WBC, FB CV, CAFC
52.14 Cathepsin A/beta-galactosidase/neuraminidase WBC, FB CV, CAFC
52.15 Alpha-mannosidase P, WBC, FB CV, CAFC
52.16 Beta-mannosidase P, WBC, FB CV, CAFC
52.17 Alpha-neuraminidase FB, WBCa CV, CCV, CAFC
52.18 N-Acetylglucosamine-1-phosphotransferase FB CV, CCV, CAFC
52.19 N-Acetylglucosamine-1-phosphotransferase FB CCV, CAFC
52.20 Alpha-N-acetylgalactosaminidase WBC, FB CV, CAFC
52.24 Beta-galactosidase-1 WBC, FB CV, CAFC
52.25 Galactosylceramidase WBC, FB CV, CAFC
52.30 Beta-hexosaminidase P, WBC, FB CV, CAFC
52.31 Alpha-hexosaminidase P, WBC, FB CV, CAFC
a
Fresh material, not frozen
P plasma, WBC white blood cells, FB fibroblasts, CV chorionic villi, CAFC cultured amniotic fluid cells,
CCV cultured chorionic villi
Table 52.6 Free and total sialic Age-related normal range of free sialic acid in urine Age-related normal range of total sialic acid in urine
acid reference ranges in urine
Age (years) Mean ± 2SD (mmol/mol creatine) Age (years) Mean ± 2SD (mmol/mol creatine)
<0.5 45 ± 38 <0.5 156 ± 120
<1 32 ± 28 <1 100 ± 70
1–3 29 ± 21 1–3 90 ± 70
3–5 21 ± 13 3–5 63 ± 40
5–10 15 ± 12 5–10 51 ± 37
10–20 9±8 10–20 34 ± 30
>20 7±6 >20 31 ± 25
Modified from van der Ham (2007)
all these diseases, the excretion of bound SA is lower than in using isotope dilution technique and 8-point calibration
conditions involving the neuraminidase enzyme. About curve. Total SA analysis was performed after a hydrolysis
40 % of SA is in the free form. Urine-free SA can be step in sulfuric acid at 80 °C for 1 h. The method is rapid
increased in infantile sialic acid storage disease (ISSD), sial- (6 min), semiautomatic, specific, robust, and sensitive and
uria, and Salla disease. Although the excretion of free SA therefore can be considered suitable for high-throughput
seems to be higher in ISSD than in Salla disease and sialuria, analysis. Reference ranges of urinary free and total sialic
a distinction between these forms of sialuria cannot be made acid are reported in Table 52.6.
on the basis of the sialic acid excretion. Many methods have
been described for the determination of both free and bound
SAs. In many laboratories the measurement of urinary SA is 52.3.4 Enzymatic Tests in Dried Blood Spots
performed by using a modified colorimetric assay (thiobarbi-
turic acid test) developed to eliminate the ubiquitary interfer- Chamoles et al. (2001) have demonstrated that many lyso-
ent 2-deoxyribose. As an alternative method Okamura-Oho somal enzymes are still active in rehydrated dried blood
et al. (1984) proposed an enzymatic procedure based on spots. A direct multiplex assay of lysosomal enzymes in
cleavage on NANA by aldolase to N-acetylmannosamine dried blood spots by tandem mass spectrometry has been
and pyruvic acid with following spectrophotometric deter- developed based on these observations (Li et al. 2004). The
mination (340 nm) with NADH and lactic dehydrogenase. use of mass spectrometric techniques has shown advantages
For the measurement of free sialic acid, an incubation step over fluorometric or spectrophotometric assays in the simul-
with neuraminidase is performed. In recent years, some taneous quantification of several markers (Gelb et al. 2006).
authors (van der Ham et al. 2007) proposed a LC-MS/ In fact, the incorporation of a chromophore or fluorophore
MS-based method for the assay of total and free sialic acid. into the substrate can cause false-negative results. Conventional
For free SA assay the procedure required a filtration step fol- methods of quantifying enzymatic activity, such as spectro-
lowed by a dilution. Quantitative results were obtained by photometry and fluorometry, have the limitations of specificity
792 G. la Marca
Table 52.7 Enzyme activities from dried blood spot

Enzyme Healthy newborns n = 15,000 Healthy adults n = 500 Heterozygotes Affected patients
Median High value Low value Median High value Low value High value Low value n High value n
GAA 11.56 27.38 2.31 6.56 21.88 1.86 4.85 2.72 30 0.98 24
ABG 11.65 47.41 2.20 14.40 38.33 1.72 8.89 3.4 14 0.45 14
GLA 11.95 36.60 1.13 21.20 49.02 5.71 5.87 1.01 28 0.40 33
ASM 2.43 19.00 0.56 1.78 32.40 0.42 n.a n.a n.a 0.08 6
GALC 1.41 5.64 0.50 3.38 10.07 1.01 n.a n.a n.a 0.20 8
IDUA 19.12 69.60 4.60 n.a n.a n.a n.a n.a n.a 2.85 4
GAA acid-alpha-glucosidase, ABG acid beta-glucosidase, GLA alpha-galactosidase A, GALC galactosylceramidase, IDUA alpha-l-iduronidase
Table 52.8 Enzyme activities from dried blood spot

Enzyme Control newborns (n) Patients (n)
Median High value Low value Median High value Low value
IDS 9.10 (75) 16.00 4.80 0.29 (14) 0.52 0.17
ASB 7.40 (89) 16.90 1.40 0.12 (1) 0.12 0.12
GALNS 1.84 (30) 4.37 0.64 0.014 (9) 0.26 0.006
IDS iduronate 2-sulfatase, ASB N-acetylgalactosamine-6-sulfatase, GALNS N-acetylgalactosamine-4-sulfatase
and limited capacity for multiplexing. This limitation is not (la Marca et al. 2009; Metz et al. 2011). For the quantitation
present if an immune quantification of lysosomal proteins is measurements, the tandem mass spectrometer was operated
performed (Fuller et al. 2011), although this led to at least one in multiple reaction monitoring (MRM) mode.
false-negative MPS II. The activities of GAA, ABG, GLA, ASM, GALC, and
Based on the work of Chamoles and Gelb, several experi- IDUA enzymes in healthy newborn (48–72 h of life) and
mental approaches have been developed for LSDs using adult noncarriers, heterozygous carriers, and affected patients
enzymatic assay followed by fluorometric or tandem mass are reported in Table 52.7.
spectrometric methods in order to detect the product of one Considering GLA enzyme activity, the heterozygous car-
specific enzyme (Spada et al. 2006; Blanchard et al. 2008) or riers presented lower levels than in control subjects but still
by tandem mass spectrometry to quantify multiple enzymes well distinguished from the affected patients. The high activ-
simultaneously (Zhang et al. 2008). ity level of heterozygous carriers was 27.2 % (for GLA) and
Recently, a new and simplified liquid chromatography- 73.9 % (for GAA) of the median activity in healthy adults,
tandem mass spectrometry-based method to perform multi- but the range of activities partially overlapped. Lack of
ple enzyme analysis on dried blood spots has been published GALC and ASM and IDUA heterozygous carriers of DBS
(la Marca et al. 2009). This method takes only 4 min as anal- did not allow to achieve the related data. For GAA and ASM,
ysis run-time and does not involve any time-consuming sam- the median enzyme activity in healthy adults was lower than
ple preparation step after the enzymatic reaction. in healthy newborns, but the higher ends of the activity range
The entire assay comprises a 2-day process. On the first are quite similar, except for GALC. The lower end of the
day, the samples are punched and the enzymatic reaction is activity range is similar, except for GALC and GLA. In both
started. The protocol for the incubation of DBS with enzyme cases, activities were higher (five times for GLA; two times
reagent cocktails was adapted from Zhang et al. (2008) for for GALC) than in healthy newborns. For IDUA the lower
Gaucher (ABG) , Niemann-Pick A and B (ASM), Pompe activity level in healthy newborns is 1.6 times higher than the
(GAA), and Fabry (GLA) and from Blanchard et al. (2008) highest level in affected patients. The activities of all the six
for MPS I (IDUA). The protocol for Krabbe (GALC) was enzymes in all samples were measured from a single 3.2 mm
slightly modified from Metz et al. (2011). On the second day, DBS per enzymatic reaction. Methodologies hereby
combining the six reaction mixtures and the centrifugation described enable the monitoring and the quantifying of 6
requires 15 min. The incubation for GALC enzyme reaction enzymes’ activity without any purification steps and in one
was performed separately. single “4-min” analytical run and should be good candidates
Initial analytical protocols proposed time-consuming for implementation in routine clinical screening and quanti-
offline sample preparation such as LLE and SPE (Li et al. fication environments. The screening panel for LSDs was
2004). To date, most published papers reported the use of an recently expanded and currently includes MS/MS assays for
analytical column in conjunction with an online cleanup col- MPS II (Wolfe et al. 2011), IVA (Khaliq et al. 2011), and VI
umn which improves a multiplex approach by separating (Duffey et al. 2010). The activities of IDS, ASB, and GALNS
several enzymatic products from residual substrates are reported in Table 52.8.
52 Lysosomals 793
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the rapid detection of mucopolysaccharidoses: GAG-test(®). J Clin
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Li Y, Scott CR, Chamoles NA et al (2004) Direct multiplex assay of
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lysosomal enzymes in dried blood spots for newborn screening.
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Meikle PJ, Hopwood JJ, Clague AE et al (1999) Prevalence of lyso-
urinary glycosaminoglycans by LC-MS/MS in mucopolysacchari-
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Metz TF, Mechtler TP, Orsini JJ et al (2011) Simplified newborn
Blanchard S, Sadilek M, Scott CR et al (2008) Tandem mass spectrom-
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application to screening newborns for mucopolysaccharidosis I.
Okamura-Oho Y, Yamanaka T, Suzuki Y et al (1984) A simple enzy-
Clin Chem 54:2067–2070
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Chamoles NA, Blanco M, Gaggioli D (2001) Fabry disease: enzymatic diag-
diagnosis of disorders of sialic acid metabolism. Clin Chim Acta
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Chien YH, Chiang SC, Zhang XK et al (2008) Early detection of Pompe
Palo J, Savolainen H (1972) Thin-layer chromatographic demonstration
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de Jong JG, Wevers RA, Liebrand-van Sambeek R (1992) Measuring
Ramsay SL, Meikle PJ, Hopwood JJ et al (2005) Profiling oligosac-
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Sewell AC (1981) Simple laboratory determination of excess oligosac-
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Proton NMR Spectroscopy of Body
Fluids 53
Udo Engelke, Angelina Goudswaard, and Ron Wevers

53.1 Introduction...................................................................... 795
The laboratory diagnosis of inherited metabolic diseases can-
53.2 Background of Proton NMR Spectroscopy ................... 795
not always be achieved by analysis of amino acids, organic
53.3 Potential of the Method ................................................... 797 acids, and other small molecules alone. Often, however, addi-
53.4 Indications for NMR Spectroscopy ................................ 797 tional investigations also do not lead to the diagnosis while
there is a strong suspicion of a metabolic disease. In such
53.5 Sample Choice, Sample Preparation,
and Measurement ............................................................ 797 cases 1H-NMR spectroscopy of body fluids has been applied
as a complementary technique, in fact a last resort to find the
53.6 Spectrum Interpretation ................................................. 797
diagnosis (Engelke et al. 2007a, b, 2008; Moolenaar et al.
53.7 Quantification................................................................... 798 1999, 2001a, b). 1H-NMR spectroscopy of body fluids
53.8 NMR Spectroscopy and Inborn Errors requires minimal or no sample pretreatment. The resulting
of Metabolism ................................................................... 799 spectra show the majority of proton-containing compounds.
References .................................................................................... 800 They provide an overall view on metabolism. This holistic
view makes NMR spectroscopy a cornerstone of
metabolomics. In the diagnostics of hereditary metabolic
diseases, this is a major advantage compared to other tech-
niques. 1H-NMR spectroscopy of body fluids may be consid-
ered as an alternative analytical approach for diagnosing
known but also as yet unknown inborn errors of metabolism.
53.2 Background of Proton

NMR Spectroscopy
The principle of nuclear magnetic resonance (NMR) is based

on the behavior of atoms placed in a strong magnetic field.
Superconducting magnets provide such a strong, extremely
homogenous magnetic field (Fig. 53.1). The nuclei of most
atoms (e.g., protons) possess a property known as spin.
Inside the magnet and under special conditions, these spin-
ning charged particles generate resonances depending on
the chemical environment of the compound; a proton of a
U. Engelke • A. Goudswaard • R. Wevers (*) compound leaves a characteristic fingerprint behind in the
1
Department of Laboratory Medicine, Laboratory of Genetic, H-NMR spectrum (Fig. 53.2). Signals can be attributed to all
Endocrine and Metabolic Diseases (HP 830), proton-containing small molecules. Several spectral param-
Radboud University Medical Center,
eters are useful for body fluid analysis. These are the peak
9101, 6500 HB Nijmegen, The Netherlands
e-mail: udo.engelke@radboudumc.nl; ag_goudswaard@hotmail.com; area, the chemical shift (resonance position), and the splitting
ron.wevers@radboudumc.nl of resonances. The peak area is important for quantification.
796 U. Engelke et al.
Fig. 53.1 Left: standard NMR sample tube with 5 mm diameter. The magnet and Bruker Avance console. The instrument is configured for
sample volume needed is 650 μl. Right: NMR setting in Nijmegen: the study of metabolomics
500 MHz NMR spectrometer composed of an 11.7 T superconducting
HO H2N Alanine Lactic acid

OH OH CH3 CH3
H3 C CH C H3C CH C
O O
Lactic acid Alanine
Lactic acid Alanine

CH CH
4.50 4.25 4.00 3.75 2.00 1.75 1.50 1.75
Fig. 53.2 1H-NMR spectrum of lactic acid and alanine illustrating the fingerprint of both molecules
The technique provides reliable quantitative data about of one or more peaks (splitting) under the influence of their
proton-containing metabolites in the sample. Sensitivity is chemical environment. Singlet, doublet, triplet, quartet, or
in the low micromolar range. The resonance position and the multiplet resonances may occur. Figure 53.2 gives a sche-
splitting pattern help in identifying the compound and give matic presentation of the NMR spectra of lactate and alanine
structural information on the molecule. The signal from a to illustrate that molecules with a high degree of similarity
particular proton or group of equivalent protons may consist can be observed as separate resonances in the spectrum.
53 Proton NMR Spectroscopy of Body Fluids 797
The methyl groups of lactate and alanine appear as doublet 53.5 Sample Choice, Sample Preparation,
resonances. The proton from their methylene groups gives and Measurement
a quartet in the spectrum. Multiplicity is determined by the
number of protons attached to neighboring carbon atoms. Traditionally urine has been the body fluid of choice to find
The doublets originating from the methyl protons of lactic the way towards the diagnosis in inborn errors of metabo-
acid and alanine have a slightly dissimilar resonance position lism. Urine NMR spectra are very complex. Moreover, some
(0.10 ppm) caused by differences in the chemical environ- metabolites are rapidly excreted but others remain pref-
ment of the methyl group in the two molecules (NH2 group erentially in the blood. In relevant cases it is advised also
versus OH group). This explains how the two substances, in to investigate the plasma. It may be necessary to use CSF
spite of their structural similarity, can be distinguished in the in diseases affecting the central nervous system (Mochel
spectrum. Peak areas in the NMR spectrum are proportional et al. 2009, 2010). Minimal sample volumes are 1 ml for all
to the concentration of a metabolite. The areas of the doublet body fluids. For a proper interpretation of the spectrum, the
and the quartet resonances in Fig. 53.2 have a three to one request to do NMR spectroscopy should include information
ratio, because three protons contribute to the signal of the on the medication. As the technique requires no derivatiza-
methyl group against one proton of the methylene group. tion or extraction, there is no loss of metabolites in sample
pretreatment, and the sample can even be used a second time
for follow-up investigations. Sample preparation is limited
53.3 Potential of the Method to the removal of proteins from plasma and CSF samples
on a 10 kD filter, pH standardization of samples, and addi-
Proton NMR spectroscopy of body fluids is a powerful new tool tion of an internal standard. The measurement itself requires
in diagnosing known and novel inborn errors of metabolism. adjustment per sample of the magnetic field in order to
Potentially it may replace some of the conventional diagnostic achieve good homogeneity (“shimming”). Automatic sam-
techniques in metabolic screening laboratories in the future. At ple changers and automatic shimming programs take care
the moment the technique is used mainly diagnostically in those of efficient measurement of larger series of samples. The
cases where the metabolic specialist is convinced that a patient actual measurement takes approximately 30 min. The signal
suffers from a metabolic disease but cannot find the diagnosis is recorded by the computer and can be further analyzed later
with conventional techniques (Table 53.1). An important advan- on by means of software programs that have been especially
tage of NMR spectroscopy over conventional techniques that designed for this purpose.
are being used in basic screening of hereditary diseases is the
nonselective character of the technique.
53.6 Spectrum Interpretation
53.4 Indications for NMR Spectroscopy In complex matrices like body fluids, many resonances will
be present in the NMR spectrum. The spectrum provides an
Optimal results from NMR analysis of body fluids require overall view on metabolism. Basically, all proton-containing
close cooperation between the referring clinician or chemist small molecules (MW < 500) can be observed with proton
and the NMR spectroscopy group. Detailed information on NMR spectroscopy. Under the conditions used, however, –
the patient and the medication of the patient should be pro- NH2 and –COOH protons may actually be NMR invisible
vided to result in an optimal interpretation of the NMR data. as they exchange rapidly. Protons from proteins and from
It is possible for clinicians and (clinical) chemists to send us molecules that are protein bound are also largely invisible.
samples of relevant patients (Table 53.1). It is impossible to give a full list of metabolites that can
Table 53.1 Indications for in vitro 1H-NMR spectroscopy

Clinical Biochemical
1. Two or more children in the same family with unexplained 1. Abnormal unknown metabolite observed repeatedly in body fluids with
similar clinical signs and symptoms other technique
2. Unusual body odor (e.g., fish odor) 2. Confirmation of a special diagnosis with an independent technique is
needed
3. An unknown metabolite is observed using in vivo NMR 3. Reliable quantification of a metabolite that is difficult to quantify
spectroscopy of the brain or other organ otherwise is needed
4. Any well-defined clinical syndrome with unknown etiology 4. For one or more metabolites not leading to a diagnosis. Consistent
abnormal results are found using conventional screening techniques
Fig. 53.3 500 MHz 1H-NMR

spectrum of CSF. The lower Glucose Lactic acid
panel magnifies the region
between 1.6 and 0.8 ppm of the
spectrum and shows more details.
500 MHz 1H-NMR spectrum of
CSF: The upper panel shows the
Acetic acid
chemical shift region between Glucose
4.6 and 0.8 ppm
Lactic acid
Creatinine
Creatine Citric acid
myo-
Glutamine
Inositol
4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm
Lactic acid
Alanine
Leucine/Isoleucine
Threonine
Valine
2-Oxoiso-
3-Hydroxybutyric
valeric acid
acid
13C Lactic
acid 2-Hydroxyiso-
valeric acid
1.5 1.4 1.3 1.2 1.1 1.0 0.9 ppm
be observed in body fluid NMR spectra. For various body magnifies part of the spectrum showing more details of that
fluids, partial lists are available in literature (Lehnert and specific part.
Hunkler 1986; Wevers et al. 1994, 1995). Typically spectra
from urine samples contain well over 300 resonances, while
the number of resonances in a CSF sample is limited to 53.7 Quantification
approximately 100. At least 53 compounds were identified
in CSF samples (Wishart et al. 2008). From NMR spectra Quantification of a metabolite can be performed by adding an
it could be concluded that 3-hydroxyisovaleric acid turned internal standard to the sample (plasma and CSF). In practi-
out to be a normal constituent of CSF samples. In this way cally all studies, trimethylsilyl-2,2,3,3-tetradeuteropropionic
1
H-NMR spectroscopy provides new insight. Figure 53.3 acid (TSP), which provides a singlet resonance from 9
shows the 1H-NMR spectrum of CSF. The lower panel equivalent methyl protons, is used as such. Metabolites
in urine can be expressed per creatinine without using an Several novel diseases were discovered by body fluid
internal standard. For various compounds good correla- NMR spectroscopy. These are dimethylglycine dehydroge-
tions have been obtained with conventional techniques. nase deficiency (OMIM 605850) (Moolenaar et al. 1999),
The sensitivity of 1H-NMR spectroscopy generally is in the 3-ureidopropionase deficiency (OMIM 613161) (Moolenaar
low micromolar range but depends on the strength of the et al. 2001b), cerebellar ataxia with elevated CSF free sialic
magnetic field. It depends on the number of protons con- acid (CAFSA) (Mochel et al. 2009), and severe hypomyelin-
tributing to the resonance(s) and on the multiplicity of the ation with elevated CSF N-acetylaspartylglutamate (Wolf
resonance. Furthermore, the region of the spectrum where et al. 2004). In other cases NMR spectroscopy was involved
the compound resonates and the measuring time play a role. in the elucidation of the molecular defect, e.g., ribose-5-
Examples of the sensitivity level that can be reached are phosphate isomerase deficiency (OMIM 608611) (Moolenaar
(600 MHz apparatus and 30 min measuring time per sam- et al. 2001a; Huck et al. 2004) and aminoacylase 1 deficiency
ple): 2 μmol/l for betaine (singlet, 9 protons), 10 μmol/l for (OMIM: 609924) (Engelke et al. 2008). For most diseases,
lactic acid (doublet, 3 protons), and 30 μmol/l for glucose the NMR spectrum confirms the disease at metabolite level.
(doublet, 1 proton). For molecules where the detection relies Sometimes, NMR spectroscopy revealed novel findings in
on a multiplet resonance, the sensitivity may be significantly known IEM such as new biomarkers or unexpected
lower. A representative coefficient of variation has a value of metabolite observations, e.g., erythronic acid in transaldol-
2.5 % (1.2 mmol alanine/l). ase deficiency (OMIM 606003) (Engelke et al. 2010a), uro-
canylglycine in urocanic aciduria (OMIM 276880) (Engelke
et al. 2010b), and CSF cis 3-methylglutaconic acid in
53.8 NMR Spectroscopy and Inborn 3-methylglutaconic aciduria type I (OMIM 250950) (Engelke
Errors of Metabolism et al. 2006). The characteristic metabolites in some diseases
cannot be picked up with conventional screening techniques
NMR spectroscopy on body fluids can be successfully (e.g., dimethylglycine in dimethylglycine dehydrogenase
applied to diagnose more than 80 of the known inborn deficiency) or can only be detected with techniques that are
errors of metabolism including most organoacidurias, not commonly used in metabolic screening laboratories (the
aminoacidurias, and diseases in purine and pyrimidine detection of arabitol and ribitol requires GC of monosaccha-
metabolism. Several defects in lipid metabolism can be ride and polyols).
diagnosed using blood plasma lipid extracts (Oostendorp Figure 53.4 shows 1H-NMR spectra of urine samples
et al. 2006). A list of diseases that can be diagnosed using from three different IEM (Fig. 53.4a–c). The characteristic
NMR spectroscopy is available in the “Handbook of 1H- metabolic profile for each disease can be observed in the
1
NMR spectroscopy in inborn errors of metabolism: body H-NMR spectra. The examples of these diseases clearly
fluid NMR spectrum and in vivo MR spectroscopy.” The illustrate the advantage of the nonselective character of NMR
handbook is free and available to download from the web- spectroscopy and the power of the overall view on metabo-
site www.bodyfluidnmr.nl. NMR spectroscopy can be lism that it provides. Conventional techniques used in the
used to find inborn errors of metabolism characterized by screening for inborn errors of metabolism provide informa-
the presence of metabolites that cannot be detected with tion on roughly 15–20 % of metabolites known to play a role
conventional screening methods. Trimethylaminuria or in human metabolism. Considering that most of the other
“fish odor syndrome” may serve as an example (Abeling metabolites actually contain protons that make them visible
et al. 1995; Podadera et al. 2005; Maschke et al. 1997). It with NMR, it is to be expected that NMR spectroscopy will
is a hereditary disease based on an enzyme deficiency in be able to open new perspectives for the field of inborn errors
the liver due to defects in the FMO3 gene. Trimethylamine of metabolism. In case a high concentration of an unknown
(TMA) derives from bacteria that produce it from dietary metabolite is found with conventional screening techniques,
choline. There is an enzyme in the liver that oxidizes proton NMR spectroscopy may help to identify the com-
TMA efficiently. TMA and trimethylamine N-oxide pound. The 1H-NMR spectrum contains structural informa-
(TMAO) are both excreted in the urine. TMA has the tion on the molecules in the sample. Of course medication
smell of rotten fish, causing a social problem for the will first have to be excluded as a source. In such cases the
patient. 1H-NMR spectroscopy can measure TMA and one-dimensional NMR spectrum already may help to iden-
TMAO simultaneously to prove this deficiency at the tify the unknown. Additional two-dimensional NMR tech-
metabolic level (Podadera et al. 2005; Maschke et al. niques are available and may be used for further structure
1997). analysis (Engelke et al. 2005).
Cr 3
4
2.10 2.00
7.50 5.00 2.50
Cr 9
8 6
7
b
7.50 5.00 2.50 4.25 4.15
11
Cr
10
6.90 6.70
7.50 5.00 2.50
Cr
7.50 5.00 2.50
Fig. 53.4 500 MHz 1H-NMR spectra (measured at pH 2.50) of urine proportional to the concentration. For urine, the singlet of creatinine at
samples from patients with different IEM (a, aminoacylase I deficiency; 3.13 ppm (Cr) can be used as a concentration reference. Peak assign-
b, transaldolase deficiency; c, alkaptonuria) with three small sections ments: creatinine [CH3] (Cr), N-acetylalanine (1), N-acetylglutamic
(black square) expanded to allow more detailed analysis and interpreta- acid (2), N-acetylglutamine (3), N-acetylglycine (4), N-acetylmethionine
tion. Spectrum d is from a healthy individual. Each peak represents (5), sedoheptulose (6, 7), creatinine [CH2] (8), erythronic acid (9), and
protons in a different chemical environment. The peak area is directly homogentisic acid (10, 11)
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SH, Wevers RA (2005) Dimethyl sulfone in human cerebrospinal Verlagsgesellschaft, Heilbronn
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MRI and In Vivo Spectroscopy
of the Brain 54
Alessandro Burlina and Renzo Manara

54.1 Introduction ..................................................................... 803
Magnetic resonance is an essential tool to study brain dis-
54.2 Brain MRI........................................................................ 803
54.2.1 Diffusion-Weighted Imaging (DWI) ................................. 804 eases. Even though the diagnosis of inherited metabolic dis-
eases is achieved by means of biochemical and genetic tools,
54.3 White Matter Abnormalities .......................................... 809
brain magnetic resonance imaging (MRI) is useful for detect-
54.4 Inherited Metabolic Strokes ........................................... 811 ing brain complications (i.e., stroke-like episodes for propi-
54.5 Proton MRS ..................................................................... 812 onic acidemia or MELAS) and for the follow-up of brain
lesions load (i.e., white matter abnormalities in Fabry dis-
References ...................................................................................... 814
ease). In specific diseases, like Canavan disease and creatine
deficiency syndromes (AGAT deficiency and creatine trans-
porter defects), diagnosis can be made with in vivo proton
magnetic resonance spectroscopy (MRS).
It is not possible, within the limited space of a single
chapter, to describe in detail all the MRI and in vivo MRS
findings reported in the literature for many inherited meta-
bolic diseases. We, therefore, decided to discuss those dis-
eases with characteristic MRI and/or MRS findings (i.e.,
stroke-like lesions, thalamic involvement, diagnostic metab-
olites) that are detectable with routinely used MR scanners.
54.2 Brain MRI
In this chapter, we do not go into detail regarding sequences

for image acquisition. Nevertheless, we recommend that
for each patient, FLAIR images and T1, T2 and diffusion-
weighted images (DWI) should always be acquired. Indeed,
there are characteristic findings that can only be recognized
in specific sequence: for example, the pulvinar sign in
Fabry disease can be detected in T1 not in T2 sequence
A. Burlina (*) (Burlina et al. 2008) (Fig. 54.1). There are MR images
Neurology Unit, St. Bassiano Hospital,
which can be characteristic for specific inherited metabolic
via dei Lotti 40, 36061 Bassano del Grappa, Italy
e-mail: alessandro.burlina@aslbassano.it diseases (i.e., adrenoleukodystrophy), but brain MRI find-
ings can show large variation among patients and different
R. Manara
Neuroradiology, University of Salerno, kinds of lesions are possible according to the age (e.g.,
Via S. Allende 1, 84081 Baronissi, Italy white matter abnormalities in Fabry disease are usually not
804 A. Burlina and R. Manara
Fig. 54.1 Brain MRI showing the signal abnormality

pattern of the pulvinar in a male adult Fabry patient. The
pulvinar doesn’t display signal abnormalities on the fast
spin-echo T2-weighted image, while it appears
hyperintense on the spin-echo T1-weighted image; in this
patient the pulvinar was also hypointense on gradient
echo T2-weighted and susceptibility-weighted images
Table 54.1 Inherited metabolic diseases with “stroke-like episodes”

present in childhood; Fellgiebel and Ginsberg 2006) and (inherited metabolic strokes)
gender (e.g., the pulvinar sign is much less frequent and Homocystinuria, MTHFR, and cobalamin defects
has a faint signal in females; Burlina et al. 2012). Fabry disease
Furthermore, sometimes mild phenotypes can show severe Organic acidurias (methylmalonic aciduria, propionic aciduria,
neuroradiological findings and vice versa, like in glutaric isovaleric aciduria, glutaric aciduria type I)
aciduria type I (Fig. 54.2). Moreover, new neuroradiologi- MELAS and other mitochondrial encephalopathies
cal findings are also discovered in well-established diseases OTC deficiency and other urea cycle disorders
(Manara et al. 2012). Therefore, paradoxically, heterogene- Menkes disease
ity is the “rule” for brain MR imaging in the field of inborn Sulfite oxidase deficiency
errors of metabolism (glutaric aciduria type I is a paradig- CDG type Ia
matic disease; Harting et al. 2009; Citton et al. 2012). Multiple acyl-CoA dehydrogenase deficiency
3-Methylcrotonyl-CoA carboxylase deficiency
The most characteristic neuroradiological findings detect-
Purine nucleoside phosphorylase deficiency
able in inborn errors of metabolism are white matter abnor-
3-Hydroxymethylglutaryl-CoA lyase deficiency
malities and inherited metabolic strokes (Table 54.1); skull
Familial hypercholesterolemia
abnormalities, subdural collections, brain, and vessels mor- Cerebrotendinous xanthomatosis
phological changes (Figs. 54.3 and 54.4) (Table 54.2); thala- Tangier disease
mus lesions (Table 54.3), cerebellar lesions (Table 54.4); and
basal ganglia lesions (Table 54.5).
the field of inherited metabolic diseases, is not well estab-
lished. However, we recommend to perform DWI when study-
54.2.1 Diffusion-Weighted Imaging (DWI) ing patients with acute lesions and inherited myelinopathy
(Figs. 54.5 and 54.6) (Manara et al. 2009; Cakmakci et al.
This imaging sequence can easily be obtained when perform- 2010; Citton et al. 2012). In Table 54.6 inherited metabolic
ing conventional MRI. Despite its wide use for other neuro- diseases with white matter abnormalities, detected by DWI,
logical disorders (i.e., stroke, abscess), the utility of DWI, in are reported.
54 MRI and In Vivo Spectroscopy of the Brain 805
Fig. 54.2 A 30-year-old man affected with GA type 1 (with no cognitive lower panel: white matter abnormalities on T1-, diffusion-, and T2-
impairment): upper panel: temporal pole hypoplasia and batwing weighted axial images showing the peculiar strip-like involvement of
appearance due to poor opercularization; basal ganglia signal abnormalities; the genu of the corpus callosum
Fig. 54.3 MPS II (Hunter disease) male subjects

showing some typical skull and brain MRI features such
as the J-shaped pituitary sella, middle cranial fossa
multilobulated closed meningoencephalocele, and
enlarged Virchow-Robin spaces in the corpus callosum
and in the posterior regions, with sieve-like appearance
Fig. 54.4 A 20-day-old boy affected with Zellweger

disease: perisylvian polymicrogyria is evident on both
coronal (upper row) and axial (lower row) T2-weighted
images
Table 54.2 Inherited metabolic diseases with skull abnormalities, Table 54.3 Inherited metabolic diseases with thalamic lesions
subdural collections, brain, and vessels morphological changes
Disease MRI findings
Disease MRI findings GM1 gangliosidosis T1-weighted hyperintensity (especially in
Mucopolysaccharidoses J-shaped sella, closed type 1)
meningoceles, enlarged Virchow- GM2 gangliosidosis T1-weighted hyperintensity (especially in
Robin spaces, cerebral atrophy/ Sandhoff disease)
communicating hydrocephalus Fabry disease Pulvinar T1-weighted hyperintensity in
Peroxisomal disorders Perisylvian polymicrogyria, males (may occur in females, less
germinolytic cysts hyperintense)
Leigh syndrome (neonatal Triventricular hydrocephalus, Fucosidosis T1-weighted hyperintensity, T2-weighted
form) hypoplasic cerebellum hypointensity
Pyruvate carboxylase deficiency Subependymal cysts Canavan disease T2-weighted hyperintensity in the thalamus
Glutaric aciduria type I Poor opercularization (“batwing” (and globus pallidus)
appearance), subdural collections Wilson disease T2-weighted hyperintensity
d-2-hydroxyglutaric aciduria Batwing appearance, subdural
collections, incomplete gyration,
especially in the posterior regions Table 54.4 Inherited metabolic diseases with cerebellar lesions
Menkes disease Increased vessel tortuosity,
subdural collections Disease Anatomical involvement
Methylenetetrahydrofolate Hydrocephalus, microgyria, Leigh syndrome (neonatal form) Cerebellar white matter
reductase deficiency (MTHFR) dilated cerebral vessels Cerebrotendinous xanthomatosis Dentate nucleus calcifications,
Isolate sulfite oxidase Cystic-like diffuse white matter cerebellar atrophy
deficiency lesions (neonatal hypoxia-like) l-2-hydroxyglutaric aciduria Dentate nucleus, cerebellar
atrophy, especially in the vermis
Table 54.5 Inherited metabolic diseases with basal ganglia lesions

Disease MRI findings
GM2 gangliosidoses T1-weighted hyperintensity,
especially in Sandhoff disease
Fucosidosis T1-weighted hyperintensity and
T2-weighted hypointensity in the
globus pallidus
Glutaric aciduria type I T2-weighted hyperintensity in the
basal ganglia
Canavan disease T2-weighted hyperintensity in the
globus pallidus (and in the thalamus)
l-2-hydroxyglutaric aciduria T2-weighted hyperintensity in the
basal ganglia
Isolate sulfite oxidase T2-weighted hyperintensity in the
deficiency globus pallidus
Wilson disease T2-weighted hyperintensity in the
basal ganglia
Fig. 54.5 Brain MRI of a 10-year-old child affected with classical quadrigemina, mamillary bodies, and symmetric medial portion of both
MSUD during metabolic decompensation: FLAIR images (first row) thalamus and hypothalamus, more evident on diffusion-weighted
and diffusion-weighted (second row) images showing Wernicke-like images (From Manara et al. (2012), with permission of Springer Verlag)
signal abnormalities involving the periaqueductal gray, lamina
Fig. 54.6 Brain MRI of a newborn affected with classical MSUD dur- (third row) images showing diffuse brain edema, more evident on diffu-
ing metabolic decompensation: T2-weighted (first row), diffusion- sion-weighted imaging at the level of myelinated regions; hypointen-
weighted (second row), and apparent diffusion coefficient (ADC) map sity on ADC maps is consistent with intramyelinic edema
Table 54.6 Inherited metabolic disease with white matter abnormali-

ties detected by DWI
Adrenoleukodystrophy
Canavan disease
Galactosemia
Glutaric aciduria type I
l-2-hydroxyglutaric aciduria
Leigh disease, MELAS
Maple syrup urine disease
Megalencephalic leukoencephalopathy
Menkes disease
Metachromatic leukodystrophy
Pelizaeus-Merzbacher disease
Phenylketonuria
Rhizomelic chondrodysplasia punctata
Tay-Sachs disease
Fig. 54.7 A 25-year-old man affected with phenylketonuria: despite early dietary restriction, supratentorial white matter involvement is evident
on FLAIR imaging and T2- and diffusion-weighted imaging
54.3 White Matter Abnormalities ticular region (i.e., globus pallidus, U fibers), while retarded
myelination can occur because of malnutrition, hormonal
Brain white matter is frequently involved in inherited meta- imbalances, and exposure to toxins (alcohol, anticonvulsants).
bolic diseases. CNS myelination process starts at 4th month of Roughly speaking, myelin abnormalities in inherited meta-
gestation and is almost completed at the end of 2nd year of bolic diseases involve the white matter in a symmetrical man-
life. The myelination process starts from caudal regions (spi- ner (Figs. 54.7–54.10). In some diseases there is a selective
nal cord) and proceeds towards rostral regions (brain). In the vulnerability of specific white matter bundles (i.e., Canavan
brain, myelination usually spreads from central to peripheral disease, Pelizaeus-Merzbacher disease). Myelin disorders and
areas, even though the cortical spinal tracts and optic radia- their MRI findings are described in detail in van der Knaap
tions are already myelinated at birth. White matter lesions can and Valk (2005).
involve different areas due to the selective vulnerability of par-
Fig. 54.8 Ethylmalonic aciduria in a 4-year-old boy: sagittal and axial T1-weighted images show the severe white matter signal abnormalities
involving the genu and the splenium of the corpus callosum and the posterior part of the centrum semiovale bilaterally
Fig. 54.9 A 11-year-old affected with X-linked adrenoleukodystrophy with characteristic symmetric white matter involvement of the posterior
regions on T2-weighted and T1-weighted images; peripheral contrast enhancement is also evident
Fig. 54.10 A 13-month-old boy affected with GM1 type

1: basal ganglia signal abnormality with diffuse
hypomyelination on axial T1-weighted and T2-weighted
images
54.4 Inherited Metabolic Strokes atherothrombotic process has been reported, the involvement
of brain vessels is rare (it has been reported for classic homo-
Stroke-like episodes are reported in many inherited meta- cystinuria, and basilar dolichoectasia is an important anatom-
bolic disorders, and MRI (including DWI) is necessary to ical finding in Fabry disease), and sometimes it is difficult to
confirm the brain area involved (Fig. 54.11). match the anatomic area of infarction with the distribution of
Stroke comprises one or more neurological deficits due to a specific brain vascular territory. Therefore, strokes occur-
sudden abnormal blood supply. It usually depends on an ath- ring in inborn errors of metabolism are frequently called
erothrombotic mechanism which can involve the brain ves- “stroke-like episodes” to emphasize the metabolic mecha-
sels or the cardiac circulation. Ultrasound investigations and nism and/or the irregular vascular territory involvement, like
neuroimaging of vessels can show the stenotic vessel in the in MELAS (Fig. 54.12) (Sproule et al. 2013). The term “met-
extracranial or in the intracranial circulation. Neuroimaging, abolic stroke” has also been applied to stroke occurring in
CT, or MRI is able to visualize the infarct zone corresponding organic acidurias and urea cycle disorders (Thompson et al.
to the distribution of blood supply in the brain. 1989; Chakrapani et al. 2002; Sperl et al. 1997). We believe
Strokes occurring in inherited metabolic diseases usually that the pathomechanism of stroke in inherited metabolic dis-
lack some or all features mentioned above. No evidence of eases justifies the definition of “inherited metabolic strokes.”
Fig. 54.11 Brain MRI during a stroke-like episode

(inherited metabolic stroke) of a 12-month-old girl
affected with late-onset isolated sulfite oxidase
deficiency. Bilateral selective involvement of the globus
pallidus (white arrows) is evident on T2-weighted and
T1-weighted images; on diffusion-weighted imaging and
apparent diffusion coefficient (ADC) map images the
lesions present with ring-like signal characteristics
consistent with cytotoxic edema, especially on the left
side (From Del Rizzo et al. (2013), with permission of
Elsevier)
Fig. 54.12 A 10-year-old boy affected with MELAS:

FLAIR axial images disclose several bilateral cortical-
subcortical and basal ganglia lesions, more evident on the
right hemisphere, without vascular territory distribution.
Note the midline shift due to severe parenchymal edema
and an old lesion in the left occipital lobe due to a
previous metabolic stroke episode
Basal ganglia are frequently involved in inherited metabolic taurine, and mobile lipids (Burlina et al. 2000). Table 54.7
strokes, especially in organic acidurias (Figs. 54.13 and 54.14) reports the assignments of the most prominent signals which
(Thompson et al. 1989; Burlina et al. 2001, 2003; Chakrapani can be identified and quantified in an in vivo proton MR
et al. 2002; Harting et al. 2009). The stroke can be unilateral or spectrum (according to Burlina et al. 2000; Bittsansky et al.
bilateral; it may selectively involve a specific nucleus (i.e., cau- 2012).
date, putamen, globus pallidus) or more nuclei at the same time. In clinical practice, the most common modality of quanti-
Table 54.1 lists inherited metabolic diseases which can be tation of proton brain MR spectra is the calculation of the
complicated with strokes. peak ratios relative to creatine, which is the metabolite,
among those visible, that has the most stable concentration.
Therefore, this is not an absolute quantitation, but the ratio
54.5 Proton MRS measurement assumes the Cr = 1.
In the brain, the final product of failed oxidative metabo-
Proton MRS has been extensively applied for the investiga- lism is lactate. Its presence in a cerebral MR spectrum can be
tions of inherited metabolic diseases (Frahm and Hanefeld expression of ischemia/hypoxia, abnormalities in the Krebs
2007; Barker et al. 2010; Cakmakci et al. 2010; Prust et al. cycle, and induction of glycolysis. Therefore, the lactate
2011). For a 1.5 T magnet, the concentration limit of protons peak is usually below the level of detectability under normal
that can be detected is about 0.5–1.0 mM. Therefore, there conditions.
are only few metabolites which are well resolved in a proton The signal at 2.01 ppm is assigned to the well-resolved
brain spectrum: lactate (Lac), N-acetylaspartate (NAA), cre- peak of NAA (acetyl moiety), with an unresolved contribu-
atine plus phosphocreatine (Cr), choline (Cho), myo-inositol tion (at the field strength of 1.5 T) from the acetyl group of
(mI), and glutamate plus glutamine (Glx). Other resonances N-acetylaspartylglutamate. The acetylated amino acid NAA
can contribute to the in vivo spectrum, like scyllo-inositol, and the dipeptide NAAG are part of a tri-cellular metabolic
Fig. 54.13 Methylmalonic aciduria in a 12-year-old boy with symmetric globus pallidus chronic lesions on T1-weighted, T2-weighted, and
FLAIR axial images
Fig. 54.14 A 2-year-old girl affected with propionic aciduria during subacute decompensation with basal ganglia signal abnormalities on T2-weighted
images; diffusion-weighted imaging and apparent diffusion coefficient (ADC) map disclose the cytotoxic-like involvement of the globus pallidus
Table 54.7 The major signals detected in an in vivo proton spectrum NAA-NAAG system that can occur over minutes up to sev-
eral months (Baslow and Burlina 2012). While MRS NAA
Metabolite Resonance (ppm) Chemical group
Lactate 1.31 Methyl group
measurement may reflect neuron abundance and/or viability,
NAA (and NAAG) 2.01 (and 2.04) Acetyl group
the reduction of its peak is less specific for the identification
Creatine 3.03 Methyl group of a neurometabolic disorder. Nevertheless, specific meta-
Choline 3.21 Trimethyl amine groups bolic failures of the NAA-NAAG system have been reported,
Myo-inositol 3.5–3.6 From all 6 hydrogen atoms as in Canavan disease (increased levels of NAA in brain;
Glx 2.1–2.4, 3.7 Methine and methylene groups increased levels of NAAG in CSF and urine) and in hypo-
Glx = glutamate + glutamine acetylaspartia (absence of brain NAA-NAAG signal; absence
of NAA-NAAG in CSF, normal levels of NAA-NAAG in
urine) (Grodd et al. 1990; Austin et al. 1992; Barker et al.
unit including neurons, oligodendrocytes, and astrocytes. The 1992; Burlina et al. 1999; Martin et al. 2001; Boltshauser
NAA-NAAG metabolic system can be considered as a bio- et al. 2004). Figure 54.15 shows a proton spectrum of a patient
marker for brain cells functionality. Changes in NAA-NAAG with Canavan disease. Salla disease is another unique inher-
can reflect normal physiological variability within the ited metabolic disorder with a high NAA signal at 2.01 ppm,
Fig. 54.15 Canavan disease in a 15-month-old boy. Long TE MR spectroscopy shows the increased concentration of NAA in brain parenchyma,
while T2-weighted axial images disclose a diffuse and severe white matter involvement
even though in this case the peak reflects the accumulation of reported for guanidinoacetate methyltransferase deficiency
free sialic acid (N-acetylneuraminic acid) rather than elevated and alpha-mannosidosis (Stöckler et al. 1996; Avenarius
levels of NAA (Varho et al. 1999). et al. 2011).
The creatine resonance is the sum of creatine and phos-
phocreatine. It is a reliable marker of intact cerebral energy
metabolism. The creatine peak shows regional differences:
gray matter exhibits higher content in comparison with white References
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1764–1782 Neurology 52:1668–1672, Erratum in Neurology 53:1162
SSIEM Classification of Inborn Errors
of Metabolism 55
Johannes Zschocke
Content 55.1 Introduction

55.1 Introduction ...................................................................... 817
Inborn errors of metabolism that belong to the same bio-
chemical pathway often have similar clinical features, are
detected by the same diagnostic procedures (basic and spe-
cial diagnostic tests), and are treated according to similar
principles of emergency intervention and long-term manage-
ment. These clinical aspects are reflected in the SSIEM clas-
sification of inborn errors of metabolism which groups the
large number of individual conditions according to their
respective biochemical pathways or disease groups as well
as common pathophysiological mechanisms. The SSIEM
classification can be used for learning and understanding
inborn errors of metabolism, provides a framework for text-
books and is expected to form the basis of the classification
of inherited metabolic disorders in the forthcoming 11th edi-
tion of the international classification of diseases (ICD11).
J. Zschocke
Division of Human Genetics,
Medical University Innsbruck, Schöpfstr 41,
6020 Innsbruck, Austria
e-mail: johannes.zschocke@i-med.ac.at
818 J. Zschocke
Disease group/disease Gene name OMIM number

1. Disorders of amino acid and peptide metabolism
1.1. Urea cycle disorders and inherited hyperammonaemias
1.1.1. Carbamoylphosphate synthetase I deficiency CPS1 237300
1.1.2. N-acetylglutamate synthetase deficiency NAGS 237310
1.1.3. Ornithine transcarbamylase deficiency OTC 311250
1.1.4. Citrullinaemia type 1 ASS 215700
1.1.5. Argininosuccinic aciduria ASL 207900
1.1.6. Argininaemia ARG1 207800
1.1.7. HHH syndrome SLC25A15 238970
1.1.8. Citrullinaemia type 2 SLC25A13 603859
1.1.9. Hyperinsulinemic hypoglycaemia and hyperammonaemia GLUD1 138130
1.2. Organic acidurias
1.2.1. Glutaric aciduria
1.2.1.1. Glutaric aciduria type I GCDH 231670
1.2.1.2. Glutaric aciduria type III C7orf10 231690
1.2.2. Propionic aciduria PCCA 232000
PCCB
1.2.3. Methylmalonic aciduria
1.2.3.1. Methylmalonyl-CoA mutase deficiency MUT 251000
1.2.3.2. Methylmalonyl-CoA epimerase deficiency MCEE 251120
1.2.4. Isovaleric aciduria IVD 243500
1.2.5. Methylcrotonylglycinuria MCCC1, MCCC2 210200
1.2.6. Methylglutaconic aciduria
1.2.6.1. Methylglutaconic aciduria type I AUH 250950
1.2.6.2. Methylglutaconic aciduria type II, Barth syndrome TAZ 302060
1.2.6.3. Methylglutaconic aciduria type III, Costeff syndrome OPA3 258501
1.2.6.4. Methylglutaconic aciduria type IV various 250951
1.2.6.5. Methylglutaconic aciduria type V DNAJC19 610198
1.2.7. 3-Hydroxy-3-methylglutaric aciduria HMGCL 246450
1.2.8. 2-Methylbutyric aciduria ACADSB 610006
1.2.9. 2-Methyl-3-hydroxybutyric aciduria, HSD10 disease HSD17B10 300438
1.2.10. 3-Oxothiolase deficiency ACAT1 203750
1.2.11. Isobutyric aciduria ACAD8 611283
1.2.12. Methacrylic aciduria HIBCH 250620
1.2.13. 3-Hydroxyisobutyric aciduria ? 236795
1.2.14. Methylmalonate semialdehyde dehydrogenase deficiency ALDH6A1 614105
1.2.15. l-2-hydroxyglutaric aciduria L2HGDH 236792
1.2.16. d-2-hydroxyglutaric aciduria
1.2.16.1. d-2-hydroxyglutarate dehydrogenase deficiency D2HGDH 600721
1.2.16.2. Mitochondrial isocitrate dehydrogenase deficiency IDH2 613657
1.2.17. Aminoacylase deficiency
1.2.17.1. Aminoacylase 1 deficiency ACY1 104620
1.2.17.2. Aminoacylase 2 deficiency ASPA 271900
1.2.18. Methylmalonate semialdehyde dehydrogenase deficiency MMSDH 603178
1.3. Disorders of the metabolism of branched-chain amino acids not classified as organic
acidurias
1.3.1. Branched-chain amino acid transferase 238340
1.3.2. Maple syrup urine disease
1.3.2.1. BCKD E1 alpha subunit of deficiency BCKDHA 248600
1.3.2.2. BCKD E1 beta subunit of deficiency BCKDHB 248600
1.3.2.3. Dihydrolipoamide branched-chain transacylase deficiency DBT 248610
1.4. Disorders of phenylalanine or tyrosine metabolism
1.4.1. Phenylalanine hydroxylase deficiency PAH 261600
1.4.2. Tyrosinaemia type II TAT 276600
55 SSIEM Classification of Inborn Errors of Metabolism 819

1.4.3. 4-hydroxyphenylpyruvate dioxygenase deficiency HPD
S Tyrosinaemia type III 276710
S Hawkinsinuria 140350
1.4.4. Alkaptonuria HGD 203500
1.4.5. Tyrosinaemia type I FAH 276700
1.5. Disorders of the metabolism of sulphur amino acids
1.5.1. Methionine adenosyltransferase I/III deficiency MAT1A 250850
1.5.2. Glycine N-methyltransferase deficiency GNMT 606664
1.5.3. S-adenosylhomocysteine hydrolase deficiency AHCY 180960
1.5.4. Cystathionine beta-synthase deficiency CBS 236200
1.5.5. Cystathionase deficiency CTH 219500
1.5.6. Isolated sulfite oxidase deficiency SUOX 272300
1.5.7. Methionine synthase deficiency-cblG MTR 250940
1.5.8. Methionine synthase reductase deficiency-cblE MTRR 236270
1.6. Disorders of histidine, tryptophan or lysine metabolism
1.6.1. Histidinaemia HAL 235800
1.6.2. Urocanase deficiency UROC1 276880
1.6.3. Glutamate formiminotransferase deficiency FTCD 229100
1.6.4. Tryptophanaemia TDO2?
1.6.5. Hyperlysinaemia AASS
1.6.5.1. Hyperlysinaemia type I 238700
1.6.5.2. Saccharopinuria 268700
1.6.6. 2-Aminoadipic aciduria ? 204750
1.6.7. 2-Oxoadipic aciduria ? 245130
1.6.8. Hydroxykynureninuria KYNU? 236800
1.6.9. Hydroxylysinuria ? 236900
1.7. Disorders of serine, glycine or glycerate metabolism
1.7.1. Phosphoglycerate dehydrogenase deficiency PGDH 606879
1.7.2. Phosphoserine phosphatase deficiency PSPH 172480
1.7.3. Phosphoserine aminotransferase deficiency PSAT 610992
1.7.4. Nonketotic hyperglycinaemia
1.7.4.1. P protein deficiency GLDC 238300
1.7.4.2. T protein deficiency AMT 238310
1.7.4.3. H protein deficiency GCSH 238330
1.7.5. Sarcosinaemia SARDH 268900
1.7.6. d-glyceric aciduria GLYCTK 220120
1.8. Disorders of ornithine or proline metabolism
1.8.1. Ornithine aminotransferase deficiency OAT 258870
1.8.2. Hyperprolinaemia type I PRODH 239500
1.8.3. Hyperprolinaemia type II ALDH4A1 239510
1.8.4. Hypoprolinaemia, cutis laxa, autosomal recessive, type IIIa ALDH18A1 219150
1.8.5. Cutis laxa, autosomal recessive, type IIb/IIIb PYCR1 612940
614438
1.9. Disorders of amino acid transport
1.9.1. Lysinuric protein intolerance SLC7A7 222700
1.9.2. Cystinuria SLC3A1 220100
SLC7A9
1.9.3. Cystinuria-hypotonia syndrome (contiguous gene defect) n/a 606407
1.9.4. Hartnup disease SLC6A19 234500
1.9.5. Iminoglycinuria SLC36A2 242600
1.9.6. Lowe syndrome OCRL 309000
1.10. Other disorders of amino acid metabolism
1.10.1. Glutamine deficiency, congenital GLUL 610015
820 J. Zschocke

1.11. Disorders of the gamma-glutamyl cycle
1.11.1. Glutathionuria GGT1 231950
1.11.2. Cysteinylglycinase deficiency DPEP1?
1.11.3. Oxoprolinuria OPLAH 260005
1.11.4. Gamma-glutamylcysteine synthetase deficiency GCLC 230450
1.11.5. Glutathione synthetase deficiency GSS 266130
1.12. Other disorders of peptide metabolism
1.12.1. Prolidase deficiency PEPD 170100
1.12.2. Carnosinaemia CNDP1? 212200
1.12.3. Homocarnosinosis ? 236130
1.13. Other disorders of amino acid and protein metabolism
2. Disorders of carbohydrate metabolism
2.1. Disorders of galactose metabolism
2.1.1. Classical galactosaemia GALT 230400
2.1.2. Galactokinase deficiency GALK1 230200
2.1.3. Uridine diphosphate galactose-4-epimerase deficiency GALE 230350
2.2. Disorders of fructose metabolism
2.2.1. Essential fructosuria KHK 229800
2.2.2. Hereditary fructose intolerance ALDOB 229600
2.3. Disorders of pentose metabolism
2.3.1. Essential pentosuria DCXR 260800
2.3.2. Ribose-5-phosphate isomerase deficiency RPIA 608611
2.3.3. Transaldolase deficiency TALDO1 606003
2.4. Disorders of glycerol metabolism
2.4.1. Glycerol kinase deficiency GK 307030
2.4.2. Complex glycerol kinase deficiency due to contiguous gene deletion n/a 300679
2.5. Disorders of glyoxylate metabolism
2.5.1. Primary hyperoxaluria type I AGXT 260000
2.5.2. Primary hyperoxaluria type II GRHPR 260000
2.6. Disorders of glucose transport
2.6.1. Glucose transporter 1 deficiency (blood-brain barrier) SLC2A1 606777
2.6.2. Glucose transporter 2 deficiency SLC2A2 227810
S Fanconi-Bickel syndrome 261670
2.6.3. Glucose/galactose malabsorption SLC5A1 606824
2.7. Disorders of gluconeogenesis
2.7.1. Fructose-1,6-bisphosphatase deficiency FBP1 229700
2.7.2. Pyruvate carboxylase deficiency PC 266150
2.7.3. Phosphoenolpyruvate carboxykinase deficiency PCK1 261650
2.8. Glycogen storage disorders
2.8.1. Glycogen storage disease type 1a, von Gierke G6PC 232200
2.8.2. Glycogen storage disease type 1b, von Gierke SLC37A4 232220
2.8.3. Glycogen storage disease type II, Pompe GAA 232300
2.8.4. Glycogen storage disease type III, Cori AGL 232400
2.8.5. Glycogen storage disease type IV, Andersen GBE1 232500
2.8.6. Glycogen storage disease type V, McArdle PYGM 232600
2.8.7. Glycogen storage disease type VI, Hers PYGL 232700
2.8.8. Glycogen storage disease type VII, Tarui PFKM 232800
2.8.9. Glycogen storage disease type IX
2.8.9.1. Hepatic phosphorylase kinase deficiency PHKA2 306000
2.8.9.2. Hepatic and muscle phosphorylase kinase deficiency PHKB 261750
2.8.9.3. Hepatic phosphorylase kinase deficiency with cirrhosis PHKG2 613027
2.8.9.4. Muscle phosphorylase kinase deficiency PHKA1 300559
2.8.9.5. Cardiac muscle phosphorylase kinase deficiency PRKAG2 261740

2.8.10. Glycogen storage disease type X PGAM2 261670
2.8.11. Glycogen storage disease type XI SLC2A2 227810
2.8.12. Glycogen storage disease type XIV PGM1 612934
2.8.13. Glycogen storage disease type XV GYG1 612934
2.8.14. Glycogen storage disease type 0a, liver GYS2 240600
2.8.15. Glycogen storage disease type 0b, muscle GYS1 611556
2.8.16. Other glycogen storage disease
2.8.16.1. Muscle LDH deficiency LDHM 612933
2.8.16.2. Aldolase A deficiency ALDOA 611881
2.8.16.3. Beta-enolase deficiency ENO3 612932
2.8.16.4. Phosphoglycerate kinase deficiency PGK1 300653
2.8.17. Unspecified glycogen storage disease
2.9. Other carbohydrate disorders
2.9.1. Lactose intolerance LCT 223000
2.9.2. Disaccharide intolerance 1 SI 222900
2.9.3. Trehalase deficiency TREH 612119
3. Disorders of fatty acid and ketone body metabolism
3.1. Disorders of lipolysis
3.2. Disorders of carnitine transport and the carnitine cycle
3.2.1. Carnitine transporter deficiency SLC22A5 212140
3.2.2. Carnitine palmitoyltransferase I (CPTI) deficiency CPT1A 255120
3.2.3. Carnitine acylcarnitine translocase deficiency SLC25A20 212138
3.2.4. Carnitine palmitoyltransferase II (CPTII) deficiency CPT2 255110
3.3. Disorders of mitochondrial fatty acid oxidation
3.3.1. Very long-chain acyl-CoA dehydrogenase deficiency ACADVL 201475
3.3.2. Mitochondrial trifunctional protein deficiency HADHA 143450
HADHB
3.3.3. Medium-chain acyl-CoA dehydrogenase deficiency ACADM 201450
3.3.4. Short-chain acyl-CoA dehydrogenase deficiency ACADS 201470
3.3.5. 3-alpha-hydroxyacyl-CoA dehydrogenase deficiency HADH 231530
3.3.6. Multiple acyl-CoA dehydrogenase deficiency
3.3.6.1. Electron transfer flavoprotein deficiency, alpha chain ETFA 231680
3.3.6.2. Electron transfer flavoprotein deficiency, beta chain ETFB 130410
3.3.6.3. ETF-ubiquinone oxidoreductase deficiency ETFDH 231675
3.4. Disorders of ketone body metabolism
3.4.1. 3-Hydroxy-3-methylglutaryl-CoA synthase deficiency HMGCS2 600234
3.4.2. Succinyl-CoA:3-oxoacid-CoA transferase (SCOT) deficiency SCOT 245050
3.4.3. Cytosolic acetoacetyl-CoA thiolase deficiency ACAT1 100678
3.5. Other disorders of fatty acid and ketone body metabolism
3.5.1. Malonyl CoA decarboxylase deficiency MLYCD 248360
4. Disorders of energy metabolism
4.1. Disorders of pyruvate metabolism
4.1.1. Pyruvate dehydrogenase complex deficiency 312170
4.1.1.1. Pyruvate dehydrogenase E1α subunit deficiency PDHA1 312170
4.1.1.2. Pyruvate dehydrogenase E1β subunit deficiency PDHB 179060
4.1.1.3. Dihydrolipoyl transacetylase deficiency DLAT 245348
4.1.1.4. Dihydrolipoyl dehydrogenase deficiency DLD 248600
4.1.1.5. Pyruvate dehydrogenase E3-binding protein deficiency PDHX 245349
4.1.1.6. Pyruvate dehydrogenase kinase deficiency PDK1-4 266200
4.1.1.7. Pyruvate dehydrogenase phosphatase deficiency PDP1-2, PDR 608782
4.1.1.8. Pyruvate dehydrogenase deficiency, unspecified 312170
4.2. Disorders of the citric acid cycle
4.2.1. 2-Oxoglutarate dehydrogenase deficiency OGDH 203740
DLST
4.2.2. Fumarase deficiency FH 136850
822 J. Zschocke

4.3. Mitochondrial respiratory chain disorders
4.3.1. Respiratory chain disorders caused by mutations of mtDNA
4.3.1.1. Large-scale single deletion of mtDNA
4.3.1.1.1. Pearson syndrome 557000
4.3.1.1.2. Kearns Sayre syndrome 530000
4.3.1.1.3. Chronic progressive external ophthalmoplegia (CPEO) with n/a
mitochondrial myopathy [onset after 20 years]
4.3.1.2. Point mutations of mtDNA
4.3.1.2.1. Mitochondrial encephalomyopathy lactic acidosis and stroke-like 540000
episodes, MELAS
4.3.1.2.2. Myoclonic epilepsy associated with ragged red fibres, MERRF 545000
4.3.1.2.3. Neuropathy ataxia and retinitis Pigmentosa, NARP 551500
4.3.1.2.4. Leber hereditary optic neuropathy, LHON 535000
4.3.1.2.5. Maternally Inherited Leigh syndrome, MILS 256000
4.3.1.2.6. Sporadic Leigh syndrome 256000
4.3.1.2.7. Maternally inherited mitochondrial dystonia 500001
4.3.1.2.8. Maternally inherited mitochondrial cardiomyopathy n/a
4.3.1.2.9. Maternally inherited mitochondrial myopathy n/a
4.3.1.2.10. Maternally inherited deafness and diabetes, MIDD 520000
4.3.2. Respiratory chain disorders caused by mutations of nuclear DNA
4.3.2.1. Mitochondrial DNA depletion syndromes
4.3.2.1.1. Alpers-Huttenlocher syndrome POLG 203700
4.3.2.1.2. Hepatocerebral DGUOK, MPV17, PEO1 251880
4.3.2.1.3. Myopathic TK2 609560
4.3.2.1.4. Encephalomyopathy with methylmalonic aciduria SUCLA2 612073
4.3.2.1.5. Fatal infantile lactic acidosis with methylmalonic aciduria SUCLG1 245400
4.3.2.1.6. Encephalomyopathic with renal tubulopathy RRM2B 612075
4.3.2.1.7. Childhood-onset autosomal dominant optic atrophy OPA1 165500
4.3.2.1.8. Mitochondrial neurogastrointestinal encephalopathy, MNGIE ECGF1 603041
4.3.2.2. Multiple mtDNA deletion syndromes
4.3.2.2.1. Progressive external ophthalmoplegia autosomal dominant (PEOA)
4.3.2.2.1.1. PEOA1 POLG 157640
4.3.2.2.1.2. PEOA2 ANT1 609283
4.3.2.2.1.3. PEOA3 PEO1 609286
4.3.2.2.1.4. PEOA4 POLG2 610131
4.3.2.2.1.5. PEOA5 RRM2B 613077
4.3.2.2.2. Progressive external ophthalmoplegia autosomal recessive (PEOB) POLG 258450
4.3.2.2.3. Sensory ataxic neuropathy, dysarthria and ophthalmoparesis, SANDO POLG 607459
4.3.2.2.4. Optic atrophy 1 and deafness OPA1 125250
4.3.2.3. Leigh syndrome, LS SDHA, SURF1 COX10, 256000
COX15 LRPPRC PDSS2
COQ2
4.3.2.4. Ubiquinone (CoQ10) deficiency (non-LS) APTX PDSS1 CABC1 607426
4.3.2.5. Growth retardation, aminoaciduria, cholestasis, iron overload, lactic BCS1L 603358
acidosis and early death (GRACILE) syndrome
4.3.2.6. Renal tubulopathy, encephalopathy and liver failure BCS1L 124000
4.3.2.7. Cardio-encephalopathy with hyperammonaemia TMEM70 604273
4.3.2.8. Exercise intolerance with lactic acidosis
4.3.2.8.1. Complex I deficiency; riboflavin responsive ACAD9 611126
4.3.2.8.2. Complex I and II deficiency ISCU 255125
4.3.2.9. Isolated oxidative phosphorylation defects with variable phenotype
(not classified elsewhere)
4.3.2.9.1. Complex I structural subunit gene defect NDUFV1, NDUFV2, n/a
NDUFS1, NDUFS2,
NDUFS3, NDUFS4,
NDUFS6, NDUFS7,
NDUFS8, NDUFA1,
NDUFA2, NDUFA11

4.3.2.9.2. Complex I assembly gene defect C20orf7, NDUFAF1, n/a
NDUFAF2, NDUFAF3,
NDUFAF4, C80orf38,
NUBPL, FOXRED1
4.3.2.9.3. Complex II structural subunit gene defect SDHA, SDHB, SDHC, n/a
SDHD
4.3.2.9.4. Complex II assembly gene defect SDHAF1 n/a
4.3.2.9.5. Complex III structural subunit gene defect UQCRB, UQCRQ n/a
4.3.2.9.6. Complex III assembly gene defect n/a
4.3.2.9.7. Complex IV structural subunit gene defect COX6B1 n/a
4.3.2.9.8. Complex IV assembly gene defect SCO1, SCO2, SURF1, n/a
COX10, COX15, TACO1,
FASTKD2
4.3.2.9.9. Complex V structural subunit gene defect ATP5E n/a
4.3.2.9.10. Complex V assembly gene defect ATPAF2, TMEM70 n/a
4.3.2.10. Mitochondrial protein translation defects
4.3.2.10.1. Combined oxidative phosphorylation defect 1, COXPD1 EFG1 609060
4.3.2.10.2. Combined oxidative phosphorylation defect 2, COXPD2 MRPS16 610498
4.3.2.10.3. Combined oxidative phosphorylation defect 3, COXPD3 TSFM 610505
4.3.2.10.4. Combined oxidative phosphorylation defect 4, COXPD4 TUFM 610678
4.3.2.10.5. Combined oxidative phosphorylation defect 5, COXPD5 MRPS22 611719
4.3.2.10.6. Combined oxidative phosphorylation defect 6, COXPD6 AIFM1 300816
4.3.2.10.7. Combined oxidative phosphorylation defect 7, COXPD7 C10ORF65 613559
4.3.2.10.8. Myopathy, lactic acidosis and sideroblastic anaemia 1, MLASA1 PUS1 600462
4.3.2.10.9. Acute infantile liver failure TRMU 613070
4.3.2.10.10. Leukoencephalopathy with brainstem and spinal cord involvement DARS2 611105
and lactate elevation, LBSL
4.3.2.10.11. Pontocerebellar hypoplasia type 6 RARS2 611523
4.3.2.10.12. Myopathy, lactic acidosis and sideroblastic anaemia 2, MLASA2 YARS2 613561
4.3.3. Respiratory chain deficiencies with no known genetic basis
4.3.3.1. Complex I deficiency 252010
4.3.3.2. Complex II deficiency 252011
4.3.3.3. Complex III deficiency 124000
4.3.3.4. Complex IV deficiency 220110
4.3.3.5. ATP synthase deficiency 604273
4.3.3.6. Combined respiratory chain deficiency n/a
4.4. Mitochondrial membrane transport disorders
4.4.1. Mitochondrial substrate carrier disorders
4.4.1.1. Mitochondrial phosphate carrier deficiency SLC25A3 600370
4.4.1.2. Mitochondrial aspartate glutamate carrier 1 deficiency SLC25A12 603667
4.4.1.3. Mitochondrial glutamate carrier 1 deficiency SLC25A22 609302
4.4.1.4. Mitochondrial carrier, haem biosynthesis, sideroblastic anaemia SLC25A38 610819
4.4.2. Mitochondrial protein import disorders
4.4.2.1. Mohr-Tranebjaerg syndrome TIMM8A 300356
4.5. Unspecified mitochondrial disorders
4.5.1. Leigh syndrome with no known genetic or respiratory chain deficiency 256000
4.5.2. Ethylmalonic encephalopathy ETHE1 602473
4.5.3. Anaemia, sideroblastic and spinocerebellar ataxia, ASAT ABCB7 301310
4.6. Disorders of creatine metabolism
4.6.1. Creatine transporter deficiency SLC6A8
4.6.2. Guanidinoacetate methyltransferase deficiency GAMT 612736
4.6.3. Arginine: glycine amidinotransferase deficiency GATM 612718
4.7. Other disorders of energy metabolism
5. Disorders in the metabolism of purines, pyrimidines and nucleotides
5.1. Disorders of purine metabolism
5.1.1. Primary idiopathic gout 138900
5.1.2. Familial juvenile hyperuricaemic nephropathy UMOD 162000
5.1.3. Adenylosuccinate lyase deficiency ADSL 103050
824 J. Zschocke

5.1.4. AICAR transformylase deficiency ATIC 601731
5.1.5. Adenosine deaminase deficiency ADA 102700
5.1.6. Deoxyguanosine kinase deficiency DGUOK 251880
5.1.7. Myoadenylate deaminase deficiency AMPD1 102770
5.1.8. Lesch-Nyhan syndrome HPRT 308000
5.1.9. Adenine phosphoribosyl transferase deficiency APRT 102600
5.1.10. Phosphoribosyl pyrophosphate synthetase 1 defects PRPS1 311850
5.1.11. Inosine triphosphatase deficiency ITPA 147520
5.1.12. Adenosine deaminase superactivity ?
5.1.13. Purine nucleoside phosphorylase deficiency PNP 164050
5.1.14. Mitochondrial ribonucelotide reductase subunit 2 deficiency RRM2B 604712
5.1.15. Xanthinuria type I XDH 278300
5.1.16. Xanthinuria type II ? 603592
5.1.17. Thiopurine S-methyltransferase deficiency TPMT 610460
5.2. Disorders of pyrimidine metabolism
5.2.1. Orotic aciduria UMPS 258900
5.2.2. Pyrimidine-5-nucleotidase deficiency NT5C 266120
5.2.3. Dihydroorotate dehydrogenase deficiency DHODH 263750
5.2.4. Uridine-5′-monophosphate hydrolase superactivity NT5C3 266120
5.2.5. Thymidine phosphorylase deficiency TYMP 131222
5.2.6. Thymidine kinase 2 deficiency TK2 609560
5.2.7. Dihydropyrimidine dehydrogenase deficiency DPYD 274270
5.2.8. Dihydropyrimidinase deficiency DPYS 222748
5.2.9. Beta-ureidopropionase deficiency UPB1 613161
5.2.10. Hyper-beta-alaninaemia ? 237400
5.2.11. Beta-aminoisobutyrate-pyruvate transaminase deficiency ? 210100
5.3. Disorders of nucleotide metabolism
5.3.1. Aicardi-Goutières syndrome (AGS)
5.3.1.1. AGS1 TREX1 225750
5.3.1.2. AGS2 RNASEH2B 610181
5.3.1.3. AGS3 RNASEH2C 610181
5.3.1.4. AGS4 RNASEH2A 610181
5.3.1.5. AGS5 SAMHD1 612952
5.3.2. RNASET2-deficient cystic leukoencephalopathy RNASET2 612951
6. Disorders of the metabolism of sterols
6.1. Disorders of sterol biosynthesis
6.1.1. Mevalonate kinase deficiency MVK 610377
6.1.2. Smith-Lemli-Opitz syndrome DHCR7 270400
6.1.3. X-linked dominant chondrodysplasia punctata 2 EBP 302960
6.1.4. Congenital hemidysplasia with ichthyosiform erythroderma and limb defects NSDHL 308050
6.1.5. Desmosterolosis DHCR24 602398
6.1.6. Lathosterolosis SC5DL 607330
6.1.7. Greenberg skeletal dysplasia LBR 215140
6.1.8. Antley-Bixler syndrome
6.1.8.1. Antley-Bixler syndrome with disordered steroidogenesis POR 201750
6.1.8.2. Antley-Bixler syndrome type without disordered steroidogenesis FGFR2 207410
6.1.9. Sterol-C4-methyl oxidase deficiency SC4MOL 607545
6.2. Disorders of bile acid biosynthesis
6.2.1. 3- β-hydroxysterol Δ5-oxidoreductase/isomerase deficiency HSD3B7 607765
6.2.2. Δ4-3-oxysterol 5β-reductase deficiency AKR1D1 235555
6.2.3. Oxysterol 7-alpha-hydroxylase deficiency CYP7B1 613812
6.2.4. Cholesterol 7-alpha-hydroxylase deficiency CYP7A1 118455
6.2.5. Cerebrotendinous xanthomatosis CYP27A1 213700
6.2.6. Bile acid amidation defect BAAT 607748

6.3. Disorders of bile acid metabolism and transport
6.3.1. Bilirubin UDP-glucuronosyltransferase 1 deficiency UGT1A1
6.3.2. Byler disease ATP8B1
6.3.3. Progressive familial intrahepatic cholestasis type 2 ABCB11
6.3.4. Progressive familial intrahepatic cholestasis type 3 ABCB4
6.4. Other disorders in the metabolism of sterols
6.4.1. X-linked ichthyosis STS 308100
7. Disorders of porphyrin and haem metabolism
7.1.1. Acute neuropathic porphyrias
7.1.1.1. Acute intermittent porphyria HMBS 176000
7.1.1.2. Variegate porphyria PPOX 176200
7.1.1.3. Hereditary coproporphyria CPOX 121300
7.1.1.4. Acute hepatic porphyria ALAD 612740
7.1.2. Porphyrias with erosive photodermatosis
7.1.2.1. Porphyria cutanea tarda UROD 176100
7.1.2.2. Congenital erythropoietic porphyria UROS 263700
7.1.3. Porphyrias with acute painful photosensitivity
7.1.3.1. Erythropoietic protoporphyria FECH 177000
7.1.3.2. X-linked dominant protoporphyria ALAS2 300752
7.1.3.3. X-linked sideroblastic anaemia (XLSA) ALAS2 300751
8. Disorders of lipid and lipoprotein metabolism
8.1. Inherited hypercholesterolaemias
8.1.1. Disorder of low-density lipoprotein receptor LDLR 143890
8.1.2. Sitosterolaemia ABCG5 210250
ABCG8
8.2. Inherited hypertriglyceridaemias
8.2.1. Familial chylomicronaemia 238600
8.2.1.1. Familial lipoprotein lipase deficiency LPL 238600
8.2.1.2. Familial apolipoprotein C-II deficiency APOC2 207750
8.2.2. Familial hypertriglyceridaemia APOA5, LIPI 238600
8.3. Inherited mixed hyperlipidaemias
8.3.1. Familial dysbetalipoproteinaemia APOE 107741
8.3.2. Familial combined hyperlipoproteinaemia USF1 602491
8.3.3. Hepatic lipase deficiency LIPC 614025
8.4. Disorders of high-density lipoprotein metabolism
8.4.1. Apolipoprotein A-I deficiency APOA1 604091
8.4.2. Tangier disease ABCA1 205400
8.4.3. Lecithin cholesterol acyltransferase deficiency LCAT
8.4.3.1. Fish-eye disease 136120
8.4.3.2. Norum disease 245900
8.4.4. Familial hyperalphalipoproteinaemia CETP
8.5. Inherited hypolipidaemias
8.5.1. Familial abetalipoproteinaemia MTTP 200100
8.5.2. Familial hypobetalipoproteinaemia APOB 144010
8.5.3. Anderson disease SAR1B
8.6. Other disorders of lipid and lipoprotein metabolism
8.6.1.1. Sjøgren-Larsson syndrome ALDH3A2 270200
8.6.1.2. Pancreatic triacylglycerol lipase deficiency PNLIP 614338
8.6.1.3. Pancreatic colipase deficiency CLPS 120105
8.7. Unspecified disorders of lipid and lipoprotein metabolism
826 J. Zschocke

9. Congenital disorders of glycosylation and other disorders of protein modification
S CDG
9.1. Disorders of protein N-glycosylation
9.1.1. Phosphomannomutase 2 deficiency PMM2 601785
9.1.2. Phosphomannose isomerase deficiency MPI 602579
9.1.3. Glucosyltransferase 1 deficiency ALG6 603147
9.1.4. Mannosyltransferase 6 deficiency NOT56L 601110
9.1.5. Mannosyltransferase 8 deficiency ALG12 607143
9.1.6. Glucosyltransferase 2 deficiency ALG8 608104
9.1.7. Mannosyltransferase 2 deficiency ALG2 607906
9.1.8. UDP-GlcNAc:Dol-P-GlcNac-P transferase deficiency DPAGT1 608093
9.1.9. Mannosyltransferase 1 deficiency HMT1 608540
9.1.10. Mannosyltransferase 7–9 deficiency DIBD1 608776
9.1.11. Flippase of Man5GlcNAc2-PP-Dol deficiency RFT1 611633
9.1.12. N-acetylglucosaminyltransferase deficiency MGAT2 602616
9.1.13. Glucosidase 1 deficiency GLS1 606056
9.1.14. TUSC3-CDG TUSC3 601385
9.1.15. SRD5A3-CDG SRD5A3 612379
9.2. Disorders of protein O-glycosylation
9.2.1. O-xylosylglycan synthesis deficiencies
9.2.1.1. Multiple exostoses type I EXT1 133700
9.2.1.2. Multiple exostoses type II EXT2 133701
9.2.1.3. Beta-1,4-galactosyltransferase 7 deficiency B4GALT7 604327
9.2.2. O-N-acetylgalactosaminylglycan synthesis deficiencies
9.2.2.1. Polypeptide N-acetylgalactosaminyltransferase deficiency GALTNT3 601756
9.2.3. O-xylosyl/N-acetylgalactosaminylglycan synthesis deficiencies SLC35D1 610804
9.2.4. O-mannosylglycan synthesis deficiencies
9.2.4.1. Protein-O-mannosyltransferase 1 deficiency POMT1 607423
9.2.4.2. Protein-O-mannosyltransferase 2 deficiency POMT2 613150
9.2.4.3. Protein-O-mannose beta-1,2-N-acetyglucosaminyltransferase deficiency POMGNT1 606822
9.2.4.4. Fukutin deficiency FKTN 607440
9.2.4.5. Fukutin-related protein deficiency FKRP 606596
9.2.4.6. N-acetylglucosaminyltransferase-like protein deficiency LARGE 603590
9.2.4.7. O-fucose-specific beta-1,3-N-acetylglucosaminyltransferase deficiency SCDO3 602576
9.2.4.8. O-fucose-specific beta-1,3-N-glucosyltransferase deficiency B3GALTL 610308
9.3. Disorders of glycosphingolipid and glycosylphosphatidylinositol anchor
glycosylation
9.3.1.1. Lactosylceramide alpha-2,3-sialyltransferase deficiency SIAT9 609056
9.3.1.2. Phosphatidylinositolglycan, class M deficiency PIGM 610273
9.4. Disorders of multiple glycosylation and other glycosylation pathways
9.4.1. GDP-Man:Dol-P mannosyltransferase deficiency DPM1 603503
9.4.2. Lec35 deficiency MPDU1 608799
9.4.3. Beta-1,4-galactosyltransferase 1 deficiency B4GALT1 607091
9.4.4. UDP-GlcNAc epimerase/kinase deficiency GNE 600737
9.4.5. CMP-sialic acid transporter deficiency SLC35A1 605634
9.4.6. GDP-fucose transporter deficiency SLC35C1 605881
9.4.7. Dolichol pathway deficiencies
9.4.7.1. Dolichol kinase deficiency DK1 610768
9.4.8. Conserved oligomeric Golgi (COG) complex deficiency
9.4.8.1. Component of COG complex 7 deficiency COG7 606978
9.4.9. V-ATPase deficiencies
9.4.9.1. V0 subunit A2 of vesicular H(+)-ATPase deficiency ATP6V0A2 611716
9.4.9.2. COPII component SEC23B SEC23B 610512

9.5. Disorders of protein ubiquitinylation
9.6. Other disorders of protein modification
10. Lysosomal disorders
10.1. Mucopolysaccharidoses
10.1.1. MPS I, Hurler, Scheie disease IDUA 252800
10.1.2. MPS II, Hunter disease IDS 309900
10.1.3. MPS III, Sanfilippo disease 252900
10.1.3.1. MPS IIIA, Sanfilippo A disease SGSH 252900
10.1.3.2. MPS IIIB, Sanfilippo B disease NAGLU 252920
10.1.3.3. MPS IIIC, Sanfilippo C disease HGSNAT 252930
10.1.3.4. MPS IIID, Sanfilippo D disease GNS 252940
10.1.4. MPS IV, Morquio disease 253000
10.1.4.1. MPS IVA, Morquio A disease GALNS 253000
10.1.4.2. MPS IVB, Morquio B disease GLB1 253010
10.1.5. MPS VI, Maroteaux-Lamy disease ARSB 253200
10.1.6. MPS VII, Sly disease GUSB 253220
10.1.7. MPS IX, Natowicz HYAL1 601492
10.2. Oligosaccharidoses
10.2.1. Alpha-d-mannosidosis MAN2B1 248500
10.2.2. Beta-d-mannosidosis MANBA 248510
10.2.3. Sialidosis NEU1 256550
10.2.4. Aspartylglucosaminuria AGA 208400
10.2.5. Fucosidosis FUCA1 230000
10.2.6. Schindler disease NAGA 104170
10.3. Sphingolipidoses
10.3.1. GM1-gangliosidosis GLB1 230500
10.3.2. GM2-gangliosidosis
10.3.2.1. GM2-gangliosidosis 0-variant, Sandhoff disease HEXB 268800
10.3.2.2. GM2-gangliosidosis B-variant, Tay-Sachs disease HEXA 272800
10.3.2.3. GM2-gangliosidosis AB-variant GM2A 272750
10.3.3. Gaucher disease GBA 230800
10.3.4. Krabbe disease GALC 245200
10.3.5. Metachromatic leukodystrophy ARSA 250100
10.3.6. Prosaposin deficiency PSAP
10.3.6.1. Saposin A deficiency 611722
10.3.6.2. Saposin B deficiency 249900
10.3.6.3. Saposin C deficiency 610539
10.3.6.4. Saposin D deficiency 611721
10.3.7. Fabry disease GLA 301500
10.3.8. Farber disease ASAH 228000
10.3.9. Niemann-Pick disease type A or B SMPD1 257200
10.3.10. Niemann-Pick disease type C
10.3.10.1. Niemann-Pick disease type C1 NPC1 257220
10.3.10.2. Niemann-Pick disease type C2 NPC2 607625
10.4. Ceroid lipfuscinoses, neuronal (CLN)
10.4.1. CLN1, Santavuori-Haltia disease PPT1 256730
10.4.2. CLN2, Jansky-Bielschowsky disease TPP1 204500
10.4.3. CLN3, Batten Spielmeyer-Vogt disease CLN3 204200
10.4.4. CLN4A, Kufs disease recessive type CLN6 204300
10.4.5. CLN4B Kufs disease dominant type DNAJC5 162350
10.4.6. CLN5 Finnish variant CLN5 256731
10.4.7. CLN6 CLN6 601780
10.4.8. CLN7 MFSD8 610950
10.4.9. CLN8, Northern epilepsy type CLN8 600143
10.4.10. CLN9 ? 609055
10.4.11. CLN10 CTSD 610127
828 J. Zschocke

10.5. Lysosomal export disorders
10.5.1. Cystinosis CTNS 219800
10.5.2. Salla disease/infantile sialic acid storage disease SLC17A5 269920
10.6. Other lysosomal disorders
10.6.1. Mucolipidosis II, I-cell disease GNPTAB 252500
10.6.2. Mucolipidosis III, Pseudo-Hurler polydystrophy GNPTG 252605
10.6.3. Mucolipidosis IV MCOLN1 252650
10.6.4. Multiple sulphatase deficiency SUMF1 272200
10.6.5. Wolman/cholesterol ester storage disease LIPA 278000
10.6.6. Pompe disease, GSD type II GAA 232300
10.6.7. Sialuria GNE 269921
10.6.8. Danon disease LAMP2 300257
10.6.9. Cathepsin-related disorders 265800
10.6.9.1. Galactosialidosis PPGB 256540
10.6.9.2. Papillon-Lefèvre syndrome CTSC 245000
10.6.9.3. Pycnodysostosis CTSK 265800
10.6.10. Hermansky-Pudlak syndrome HPS1 203300
11. Peroxisomal disorders
11.1. Disorders of peroxisome biogenesis PEX1, various other PEX
genes
11.1.1. Zellweger spectrum disorder, severe form 214100
11.1.2. Zellweger spectrum disorder, attenuated form 214100
11.1.2.1. Neonatal adrenoleukodystrophy 202370
11.1.2.2. Infantile Refsum disease 266510
11.2. Rhizomelic chondrodysplasia punctata
11.2.1. Rhizomelic chondrodysplasia punctata type 1 PEX7 215100
11.2.2. Rhizomelic chondrodysplasia punctata type 2 GNPAT 222765
11.2.3. Rhizomelic chondrodysplasia punctata type 3 AGPS 600121
11.3. Disorders of peroxisomal alpha, beta and omega oxidation
11.3.1. X-linked adrenoleukodystrophy ABCD1 300100
11.3.2. Peroxisomal acyl-CoA oxidase 1 deficiency ACOX 264470
11.3.3. Peroxisomal d-bifunctional protein deficiency HSD17B4 261515
11.3.4. Sterol carrier protein deficiency SCP2 613724
11.3.5. Alpha-methylacyl-CoA racemase deficiency AMACR 604489
11.3.6. Refsum disease PHYH 266500
11.4. Other peroxisomal disorders
11.4.1. Primary hyperoxaluria type I AGXT 259900
11.4.2. Acatalasaemia CAT 115500
11.4.3. Mulibrey nanism TRIM37 253250
12. Disorders of neurotransmitter metabolism
12.1. Disorders in the metabolism of biogenic amines
12.1.1. Tyrosine hydroxylase deficiency TH 191290
12.1.2. Aromatic l-amino acid decarboxylase deficiency DDC 608643
12.1.3. Dopamine beta-hydroxylase deficiency DBH 223360
12.1.4. Monoamine oxidase MAOA 300615
12.2. Disorders in the metabolism of gamma-aminobutyrate
12.2.1. Succinic semialdehyde dehydrogenase deficiency ALDH5A1 271980
12.2.2. GABA transaminase deficiency ABAT 137150
12.3. Other disorders of neurotransmitter metabolism
13. Disorders in the metabolism of vitamins and (non-protein) cofactors
13.1. Disorders of folate metabolism and transport

13.1.1. Hereditary folate malabsorption SLC46A1 229050
13.1.2. Cerebral folate deficiency due to FOLR1 deficiency FOLR1 613068
13.1.3. Dihydrofolate reductase deficiency DHFR 613839
13.1.4. Methylenetetrahydrofolate reductase deficiency MTHFR 236250
13.2. Disorders of cobalamin absorption, transport and metabolism
13.2.1. Intrinsic factor deficiency GIF 609342
13.2.2. Enterocyte intrinsic factor receptor deficiency 261100
13.2.2.1. Intrinsic factor receptor deficiency due to CUBN mutations CUBN 602997
13.2.2.2. Intrinsic factor receptor deficiency due to AMN mutations AMN 605799
13.2.3. Haptocorrin deficiency TCN1 189905
13.2.4. Transcobalamin II deficiency TCN2 275350
13.2.5. Defect in adenosylcobalamin synthesis-cbl A MMAA 251100
13.2.6. Defect in adenosylcobalamin synthesis-cbl B MMAB 251110
13.2.7. Combined defect in adenosylcobalamin and methylcobalamin synthesis-cbl C MMACHC 277400
13.2.8. Defect in adenosylcobalamin and/or methylcobalamin synthesis-cbl D MMADHC 277410
13.2.9. Combined defect in adenosylcobalamin and methylcobalamin synthesis-cbl F LMBD1 277380
13.2.10. Transcobalamin receptor (TCblR/CD320) defect CD320 606475
13.3. Disorders of pterin metabolism
13.3.1. Guanosine 5 triphosphate cyclohydrolase I deficiency GCH1 233910
13.3.2. 6-Pyruvoyl-tetrahydropterin synthase deficiency PTS 261640
13.3.3. Sepiapterin reductase deficiency SPR 612716
13.3.4. Quinoid dihydropteridine reductase deficiency QDPR 261630
13.3.5. Pterin 4 carbinolamine dehydratase deficiency PCBD1 125310
13.4. Disorders of vitamin D metabolism and transport
13.5. Disorders of biotin metabolism
13.5.1. Biotinidase deficiency BTD 253260
13.5.2. Holocarboxylase synthetase deficiency HLCS 253270
13.6. Disorders of pyridoxine metabolism
13.6.1. Pyridoxine-dependent seizures ALDH7A1 266100
13.6.2. Pyridoxamine 5′-oxidase deficiency PNPO 610090
13.7. Disorders of thiamine metabolism
13.7.1. Thiamine-responsive megaloblastic anaemia syndrome SLC19A2 249270
13.7.2. Biotin-responsive basal ganglia disease SLC19A3 607483
13.7.3. Microcephaly, Amish type SLC25A19 607196
13.8. Disorders of molybdenum cofactor metabolism
13.8.1. Molybdenum cofactor deficiency 252150
13.8.1.1. Mo cofactor deficiency, complementation group A MOCS1 603707
13.8.1.2. Mo cofactor deficiency, complementation group B MOCS2 603708
13.8.1.3. Mo cofactor deficiency, complementation group C GEPH 603930
13.9. Other disorders of vitamins and cofactors
13.9.1. TTP1 deficiency TTPA 277460
13.9.2. Vitamin K epoxide reductase deficiency VKORC1 607473
13.9.3. Retinol binding protein deficiency RBP4 180250
13.9.4. Pantothenate kinases deficiency PANK2 234200
14. Disorders in the metabolism of trace elements and metals
14.1. Disorder of copper metabolism
14.1.1. Menkes syndrome ATP7A 309400
14.1.1.1. Occipital horn syndrome 304150
14.1.2. Wilson disease ATP7B 277900
830 J. Zschocke

14.2. Disorder of iron metabolism
14.2.1. Hereditary haemochromatosis
14.2.1.1. Hereditary haemochromatosis type 1 HFE 235200
14.2.1.2. Hereditary haemochromatosis type 2 HFE2, HAMP 235200
14.2.1.3. Hereditary haemochromatosis type 3 TFR2 235200
14.2.1.4. Hereditary haemochromatosis type 4 SLC11A3 235200
14.2.2. Acoeruloplasminaemia CP 604290
14.3. Disorder of zinc metabolism
14.3.1. Acrodermatitis enteropathica ZIP4 201100
14.3.2. Hyperzincaemia and hypercalprotectinaemia 194470
14.4. Disorder of phosphate, calcium and vitamin D metabolism
14.5. Disorder of magnesium metabolism
14.5.1. Hypermagnesaemia
14.5.2. Primary hypomagnesaemia
14.5.2.1. Hypomagnesaemia type 1, intestinal TRPM6 602014
14.5.2.2. Hypomagnesaemia type 2, renal FXYD2 154020
14.5.2.3. Hypomagnesaemia type 3, renal CLDN16 248250
14.5.2.4. Hypomagnesaemia type 4, renal EGF 611718
14.5.2.5. Hypomagnesaemia type 5, renal with ocular involvement CLDN19 248190
14.5.2.6. Hypomagnesaemia type 6, renal CNNM2 613882
14.5.2.7. Gitelman syndrome SLC12A3
14.5.3. Secondary hypomagnesaemia
14.5.4. Hypomagnesaemic tetany
14.6. Disorders in the metabolism of other trace elements and metals
15. Disorders and variants in the metabolism of xenobiotics
15.1. Disorders and variants of cytochrome P450-mediated oxidation
15.2. Disorders and variants of other enzymes that oxidise xenobiotics
15.2.1. Trimethylaminuria FMO3 602079
15.2.2. Dimethylglycinuria DMGDH 605850
15.3. Disorders and variants of xenobiotics conjugation
15.4. Disorders and variants of xenobiotics transport
Disorder Index
A AIP. See Acute intermittent porphyria (AIP)

AARF domain-containing kinase 3 (ADCK3) deficiency, 235 AKU. See Alkaptonuria (AKU)
ABCB4 deficiency, 557, 559, 564, 571 Alanine-glyoxylate aminotransferase (AGT), 376, 466, 467, 471,
ABCB11 deficiency, 557, 564, 571 763, 772
ABCC2 deficiency, 557, 564 Alanine-glyoxylate aminotransferase deficiency (peroxisomal),
Abetalipoproteinemia (MTP), 674, 678 467, 763
ABS. See Antley-Bixler syndrome (ABS) ALDOA deficiency. See Aldolase A (ALDOA) deficiency
ACAD9 deficiency. See Acyl-CoA dehydrogenase 9 (ACAD9) Aldoketoreductase 2 deficiency, Backdoor pathway defect, 605
deficiency Aldolase A (ALDOA) deficiency, 269, 821
N-Acetyl-alpha-d-glucosaminidase deficiency, 450 Aldolase B (ALDOB) deficiency, 268, 820
N-Acetylaspartic aciduria, 143 Aldosterone synthase deficiency, 605
Acetyl-CoA alpha-glucosaminide acetyltransferase deficiency, 450 ALG1-CDG. See Mannosyltransferase 1 deficiency congenital
N-Acetylgalactosamine-4-sulphatase deficiency, 450 disorders of glycosylation (ALG1-CDG)
N-Acetylgalactosamine-6-sulphatase deficiency, 450 ALG 2-CDG. See Mannosyltransferase 2 deficiency congenital
N-Acetylglucosamine-1-phosphotransferase (alpha/beta) disorders of glycosylation (ALG 2-CDG)
deficiency, 402 ALG3-CDG. See Mannosyltransferase 6 deficiency congenital
N-Acetylglucosamine-1-phosphotransferase (gamma) deficiency, 402 disorders of glycosylation (ALG3-CDG)
N-Acetylglucosamine-6-sulphatase deficiency, 450 ALG6-CDG. See Glucosyltransferase 1 deficiency, congenital
N-Acetylglucosaminyltransferase 2 congenital defects of glycosylation disorders of glycosylation (ALG6-CDG)
(MGAT2-CDG), 484–486, 489 ALG 8-CDG. See Glucosyltransferase 2 deficiency, congenital
N-Acetylglucosaminyltransferase 2 deficiency-CDG-IIa, 486, 496 disorders of glycosylation (ALG 8-CDG)
N-Acetylglutamate synthase deficiency, 49, 51, 715, 818 ALG9-CDG. See Mannosyltransferase 7-9 deficiency congenital
Achondrogenesis type IA, 508 disorders of glycosylation (ALG9-CDG)
Acid beta-glucosidase deficiency, 786 ALG11-CDG. See Mannosyltransferase 4-5 deficiency congenital
Acid ceramidase deficiency, 405 disorders of glycosylation (ALG11-CDG)
Acrodermatitis enteropathica, 623, 625, 626, 629–632, 830 ALG 12-CDG. See Mannosyltransferase 8 deficiency congenital
Action myoclonus-renal failure syndrome, 402, 406, 407, 412, 426 disorders of glycosylation (ALG 12-CDG)
Acute infantile liver failure, 341, 353, 823 Alkaptonuria (AKU), 23, 25, 27, 28, 30, 31, 743, 744, 763, 768, 770,
Acute intermittent porphyria (AIP), 541, 542, 544, 546, 549, 825 800, 819
Acyl-CoA dehydrogenase 9 (ACAD9) deficiency, 249, 250, 255, Alkyldihydroxyacetonephosphate (alkyl-DHAP) synthase deficiency,
342, 354 385, 388
Adenine phosphoribosyl transferase deficiency, 643, 649, 658, Alpers-Huttenlocher syndrome, 341, 822
659, 824 Alpha-amino adipic semialdehyde (AASA) dehydrogenase deficiency,
Adenosine deaminase deficiency, 643, 648, 658, 659, 824 181
Adenosine kinase deficiency, 34, 35, 40, 41, 758 Alpha-galactosidase A deficiency, 402, 407
Adenosine monophosphate deaminase deficiency, 643, 648, 658 Alpha-iduronidase deficiency, 450
Adenosylcobalamin and methylcobalamin synthesis defect Alpha-l-fucosidase deficiency, 437, 439, 786, 791
-cblC, 207, 212 Alpha-mannosidase B deficiency, 439
-cblD-MMA/HC, 207, 212 Alpha-methylacetoacetic aciduria, 106, 116, 136
-cblF, 207, 212 Alpha-methylacyl-CoA racemase (AMACR)deficiency, 377, 384, 387,
-cblJ, 207, 212 394, 556, 557, 562, 574, 828
Adenosylcobalamin synthesis defect Alpha-N-acetylgalactosaminidase deficiency
-cbl A, 207, 211, 829 type I, 439
-cbl B, 207, 211, 829 type II, 439
-cblD-MMA, 207, 211 type III, 439
Adenylosuccinate lyase (ADSL) deficiency, 643, 647, 658, 710, 823 5-alpha-reductase type II deficiency, 605, 610
AGC1 deficiency. See Aspartate/Glutamate transporter 1 (AGC1) AMACR deficiency. See Alpha-methylacyl-CoA racemase (AMACR)
AGT. See Alanine-glyoxylate aminotransferase (AGT) 2-Aminoadipic semialdehyde synthase deficiency, 693, 700
AHCY deficiency, 42, 45 Aminoglycoside induced deafness, 342, 355
AICAribosiduria, 643 Amish infantile epilepsy, 485
AICAR transformylase/IMP cyclohydrolase deficiency, 643, 647 Amish lethal microcephaly, 315
DOI 10.1007/978-3-642-40337-8, © Springer-Verlag Berlin Heidelberg 2014
832 Disorder Index
Amish microcephaly, 227, 229 Beta-aminoisobutyrate-pyruvate transaminase deficiency, 644, 652,

Amnionless deficiency, 207 824
Amylo-1,6-glucosidase (debrancher) deficiency, 269 Beta-enolase deficiency, 269, 821
Analphalipoproteinemia/hypoalphalipoproteinemia, 686 Beta-galactosidase deficiency, 438, 450
Andersen disease, 820 Beta-galactosidase-1 deficiency, 401
Androgen insensitivity syndrome, 605, 611 Beta-1,4-galactosyltransferase 1 deficiency, 487, 504, 826
Angiokeratoma, 407, 408, 412, 441, 442 Beta-1,4-galactosyltransferase 7 deficiency, 486, 499, 826
ANGPTL3 deficiency, 674, 688 Beta-glucuronidase deficiency, 450
Antiquitin (ALDH7A1) gene defect, 180 Beta-ketothiolase, 361, 710, 711
Antley-Bixler syndrome (ABS), 587, 588, 594, 596, 604, 611, 824 Beta-ketothiolase deficiency, 104, 106, 129, 132, 137, 362, 368, 728,
AOA1. See Ataxia oculomotor apraxia 1 (AOA1) 734, 768
APOA1. See Apolipoprotein A-I (APOA1), deficiency Beta-mannosidosis, 437–441, 445–447, 786, 790
APOC2. See Apolipoprotein C-II deficiency (APOC2) 5-beta-reductase deficiency, 557, 568, 573
APOE. See Dysbetalipoproteinemia (APOE) Beta-ureidopropionase deficiency, 644, 652, 824
Apolipoprotein A-I (APOA1) B4GALT1-CDG, 484, 485, 487, 489
deficiency, 675, 680, 683, 688, 689, 825 B4GALT7-CDG, 484, 486
structural mutations, 675 B3GALTL-CDG, 484, 485, 487
Apolipoprotein C-II deficiency (APOC2), 675, 681, 683, 688, 689 d-Bifunctional protein deficiency, 375, 377, 387, 394, 557
Apparent mineralocorticoid excess, 601, 603, 605, 609 Bilateral striatal necrosis (SLC25A19), 227–231, 345, 829
Aprataxin (APTX) deficiency: secondary coenzyme Q10 deficiency, Bile acid amidation defect, 557, 824
235 Bile acid-CoA:aminoacid N-acyl transferase deficiency, 377
Arginase 1 deficiency, 49 Bile acid-CoA ligase deficiency, 556, 557, 563, 574, 575
Arginine:glycine amidinotransferase deficiency, 530, 531, 823 Biotinidase deficiency, 219–224, 702, 710, 716, 724, 734, 768, 777,
Argininemia, 49, 53 829
Argininosuccinate lyase deficiency, 49, 728, 750, 756, 757 Branched-chain alpha-keto acid dehydrogenase complex (BCKDC)
Argininosuccinate synthetase deficiency, 49, 750, 756, 757 deficiency, 104, 106, 124, 818
Argininosuccinic aciduria, 49, 52, 715, 734, 746, 750, 756, 818 B12-responsive MMA and homocystinuria, 205–207
ARH. See Autosomal recessive hypercholesterolemia (ARH) Brown-Vialetto-Van Laere syndrome, 233–235, 237, 239, 242
Aromatase deficiency, 601, 603, 605, 611 Byler disease, 825
Aromatic l-amino acid decarboxylase deficiency, 515–517, 519, 828
Arterial tortuosity syndrome, 268, 275
Arts syndrome. See Phosphoribosyl pyrophosphate synthetase 1 defects C
Arylsulphatase A deficiency, 401 CABC1/ADCK3 deficiency, 235, 238, 242
Arylsulphatase B deficiency. See Maroteaux - Lamy disease Canavan disease, 148, 151, 154, 413–146, 803, 806, 807, 809,
Aspartate/glutamate transporter 1 (AGC1) deficiency, 85, 87, 93, 95, 813, 814
96, 98 Carbamoyl phosphate synthetase I (CPS1) deficiency, 49, 51,
Aspartylglucosaminidase deficiency, 757 58–61, 756
Aspartylglucosaminuria, 437, 439, 440, 445–447, 786, 788, 827 Carbamyl phosphate synthetase (CPS) deficiency, 715, 757
Ataxia oculomotor apraxia 1 (AOA1), 235, 238 Carnitine acylcarnitine translocase deficiency (30 patients), 250, 252
ATP8B1 deficiency, 557, 563, 571 Carnitine palmitoyl-CoA transferase 1 deficiency, 250
ATP synthase deficiency, 104, 341, 349, 823 Carnitine palmitoyl-CoA transferase 2 deficiency, 250
ATP6VOA2-CDG, 488 Carnitine palmitoyltransferase 1(CPT I) deficiency, 248, 250, 252,
Atransferrinemia, 634, 635, 638 728, 772, 821
Autosomal dominant GTPCH deficiency, 20 Carnitine palmitoyltransferase 2 (CPT II) deficiency, 249, 250, 253,
Autosomal dominant hypercholesterolemia, 673, 674, 677, 683, 689 260, 262, 728, 775, 779, 782, 783, 821
type 2 (binding-defective apo B), 674 Carnitine transporter deficiency, 248, 250, 252, 260, 262, 821
type 3 (PCSK9 gain-of-function), 674 Carnitine uptake defect (CUD), 250, 726–728, 745, 746
type B. See Familial defective apolipoprotein B (APOB) Carnosinase deficiency, 693, 699
Autosomal recessive hypercholesterolemia (ARH), 674, 678, 683, Carnosinemia and homocarnosinosis, 693, 694
686, 687, 689 cblG deficiency, 206, 207
CDG-Ia, 486, 490
CDG-Ib, 486, 491
B CDG-Ic, 486, 491
BAAT deficiency. See Peroxisomal bile acid-CoA amino acid CDG-Ig, 486, 492
transferase (BAAT) deficiency CDG-Ih, 486, 493
Ballinger-Wallace syndrome, 340 CDG-Ii, 486, 493
Barth syndrome, 104, 106, 111, 124, 126, 128, 129, 132, 136, 711, CDG-IIa, 486, 496
746, 747, 762, 771 CDG-IIb, 486, 497
Bassen-Kornzweig syndrome, 674 CDG-IId, 487, 491
Batten Spielmeyer-Vogt disease, 402 CDG-IIe, 487, 503, 746
BCAA aminotransferase deficiency, 106, 107, 128, 129 CDG-IIf, 487, 504
BCAA transaminase, 106 CDG-IIg, 487, 492
BCKDC deficiency. See Branched-chain alpha-keto acid CDG-IIh, 487, 493
dehydrogenase complex (BCKDC) deficiency CDG-Ij, 486, 494
Beta-alanine alpha-ketoglutarate transaminase deficiency, 644, 652 CDG-Ik, 486, 494
CDG-IL, 486, 495
Disorder Index 833
CDG-Im, 487 Combined MMA and MA, 106, 121–122, 129

CDG-In, 486, 496 Combined oxidative phosphorylation defect
CDG-Ip, 486 1, 341, 349, 823
Cerebral folate deficiency, 168–170 2, 341, 349, 823
Cerebral folate deficiency due to FOLR1 autoantibodies, 168, 169, 3, 341, 349, 823
173, 175, 177, 829 4, 341, 350, 823
Cerebroside sulfatase activator deficiency, 401 5, 341, 350, 823
Cerebrotendinous Xanthomatosis, 557, 576, 747, 804, 806, 824 6, 341, 350, 823
Ceroid lipofuscinosis, neuronal 7, 341, 350, 823
1, 402, 416, 424, 426 Combined saposin deficiency, 402, 405, 412, 426
2, 402, 417, 424, 426 Combined semialdehyde dehydrogenase deficiency, 106
3, 402, 417, 426 Combined xanthine oxidase and aldehyde oxidase deficiency, 643, 649, 658
4A, 402, 418, 426 Complex I assembly disorder, 250, 255
4B, 402, 418, 426 Complex I deficiency, 340, 348, 823
5, 402, 419, 426 Complex II deficiency, 340, 348, 823
6, 402, 419, 426 Complex III deficiency, 340, 348, 823
7, 403, 420, 426 Complex IV deficiency, 341, 349, 823
8, 403, 420, 426 Complex V deficiency, 341
9, Northern epilepsy variant, 403, 421, 426 Component of COG complex
10, 403, 421, 424, 426 1 deficiency, 487, 506, 826
11, 403, 422 5 deficiency, 487, 507
12, 403, 422 6 deficiency, 488, 508
13, 403, 422 7 deficiency, 487, 506, 826
14, 403, 423 8 deficiency, 487, 507, 826
cathepsin D-deficient, 403 Congenital adrenal hyperplasia, 602, 605, 703, 724, 734, 740
congenital, 400, 401 Congenital cataracts, hearing loss, and low serum copper and
Parry type. See 4B ceruloplasmin, 625
CESD. See Cholesteryl ester storage disease (CESD) Congenital dyserythropoietic anemia type II, 509
CETP. See Cholesteryl ester transfer protein deficiency (CETP) Congenital erythropoietic porphyria, 541, 542, 544, 546, 553, 825
Chaperone-activity of BC1 complex-like, CABC1 Congenital hemidysplasia with ichthyosiform erythroderma and limb
deficiency, 235 defects, 588, 824
Childhood-onset autosomal dominant optic atrophy, 340, 347, 822 Congenital hypophosphatasia, 180, 181, 184, 186, 187, 189
CHILD syndrome, 586, 588, 592, 596 Congenital pernicious anemia, 207
Cholesterol 7α-hydroxylase deficiency, 556, 557, 561, 574, 824 Congenitial alloimmune hepatitis, 635
Cholesterol side-chain cleavage deficiency, 605, 607 Conradi-Hünermann syndrome, 586, 588
Cholesteryl ester storage disease (CESD), 402, 405, 431 Constitutional AMP-activated protein kinase activation, 269, 283
Cholesteryl ester transfer protein deficiency (CETP), 674, 679, 683, COPII component SEC23B deficiency, 488, 509, 826
688, 689 Coproporphyrinogen oxidase deficiency, 543
Chondrodysplasia punctata 2 CoQ2 deficiency, 235, 238
female, 588, 591 CoQ6 deficiency, 235, 238
male, 588, 592 CoQ9 deficiency, 235, 238
Chondrodystrophy, hydropic and prenatally lethal. See Greenberg Cori disease, 820
skeletal dysplasia Corpus callosum agenesis with dysmorphism
Chondroitin sulfate synthase 1 deficiency, 486, 498 and fatal lactic acidosis, 341
CHSY1-CDG, 484, 486 Corticosterone methyl oxidase deficiency, 601, 603, 605, 609
Citrin deficiency, 49, 56, 85, 703, 728, 756–758 Cortisone reductase deficiency, 603, 605, 610
Citrullinemia type I, 49, 52, 734, 756, 757 Costeff syndrome, 104, 106, 113, 124, 126, 128, 129, 132, 136, 762,
Citrullinemia type II, 48, 49, 54, 58–61, 728, 756–758 771, 818
CK syndrome, 586, 588, 593, 596, 598 CPS deficiency. See Carbamyl phosphate synthetase (CPS) deficiency
Classical citrullinemia. See Citrullinemia type I CPS1 deficiency. See Carbamoyl phosphate synthetase I (CPS1)
Classical homocystinuria, 33–35 deficiency
Classical phenylketonuria, 3, 743, 744 Creatine transporter deficiency, 530, 532, 823
CLN8 disease CrT or SLC6A8 deficiency, 529, 530, 534, 537
late infantile variant, 403 Cubilin deficiency, 207
progressive epilepsy with mental retardation, 403 Cutis laxa, autosomal recessive, type IIA; wrinkly skin syndrome, 819
CLN5 Finnish variant, 827 Cu(2+)-transporting ATPase, alpha polypeptide (ATP7A) deficiency, 624
CLN6 late infantile variant, 402 CYP51 deficiency, 587, 588
CLN7 Turkish variant, 403 Cystathionase deficiency, 34, 35, 39, 41, 819
CMP-sialic acid transporter deficiency, 487, 504, 826 γ-Cystathionase deficiency, 693
Cobalamin R binder protein deficiency. See Haptocorrin deficiency Cystathionine beta-synthase deficiency, 33–35, 39, 703, 728, 758, 819
Coenzyme Q10 deficiency, 235, 342, 354, 358 Cystathionine gamma-lyase deficiency, 35
COG1-CDG, 484, 487 Cystathioninuria, 693, 696, 702, 745
COG5-CDG, 484, 487 Cysteinyl-glycinase deficiency, 618, 661, 820
COG6-CDG, 484, 488 Cysteinyl leukotriene synthase 4 deficiency, 618
COG7-CDG, 484, 487 Cystinuria, 34, 85–89, 93, 94, 96–98, 700, 744, 745, 750, 753, 757,
COG8-CDG, 484, 487 758, 819
834 Disorder Index
Cytochrome P450, family 51 deficiency, 588 Ehlers-Danlos syndrome, progeroid form.

Cytosolic acetoacetyl-CoA thiolase deficiency, 362, 365, 821 See Beta-1,4-galactosyltransferase 7 deficiency
Electron transfer flavoprotein deficiency, electron transfer defect, 821
Electron transfer flavoprotein dehydrogenase deficiency, electron
D transfer defect, 251
Danon disease, 785, 786, 828 Elevated lipoprotein(a) (LPA), 675, 682, 683, 688, 689, 778
Decaprenyl diphosphate synthase (DPS) deficiency, 235 Encephalomyopathy
Decreased thiopurine tolerance, 644, 654 with methylmalonic aciduria, 315, 341, 822
7-Dehydrocholesterol reductase deficiency, 588 with renal tubulopathy, 341
3-beta-Dehydrogenase deficiency, 557 respiratory failure and lactic acidosis, 341
Delta-aminolevulinate dehydratase deficiency, 542, 545 Episodic ataxia due to EAAT1 glutamate transporter defect, 87, 90
Deoxyguanosine kinase deficiency, 642, 644, 653, 658, 824 Episodic ataxia type 6, 86
Desmosterolosis, 586–588, 593, 596, 599, 824 Erythroid 5-aminolevulinate synthase
Desmosterol reductase deficiency, 588 deficiency, 542
DHCR7 deficiency, 586, 588, 599, 824 gain of function, 542
DHDPSL deficiency, 466 Erythropoietic protoporphyria, 541, 543, 544, 548, 550, 553, 725, 739
D-2-hydroxyglutarate dehydrogenase deficiency, 145, 818 Essential fructosuria, 268, 277, 744, 820
D-2-hydroxyglutaric aciduria Estrogen receptor defect, 605
type I, 143, 145, 147, 149, 154 Estrogen resistance, 603, 604
type II, 143, 145, 147, 149, 154 ETHE1 deficiency, 158
Dibasic aminoaciduria type I, 86, 87, 90, 93, 94, 96 Ethylmalonic aciduria, 105, 133, 138, 143, 161, 162, 205–207, 250,
Dicarboxylic aminoaciduria, 85–87, 89, 93, 94, 96, 757 313, 315, 320, 693, 704, 743, 810, 813, 818
Digenic hyperbilirubinaemia, rotor type. Ethylmalonic encephalopathy, 157–162, 704, 728, 777, 823
See OATP1B1 and OATP1B3 disease Exostosin 1 congenital defects of glycosylation (EXT1-CDG),
Dihydrofolate reductase deficiency, 168, 169, 172, 175, 176, 829 483–486
Dihydrolipoyl dehydrogenase deficiency, 305, 821 Exostosin 2 congenital defects of glycosylation (EXT2-CDG),
Dihydrolipoyl transacetylase deficiency, 305, 821 483–486
Dihydroorotate dehydrogenase deficiency, 643, 650, 824 Exostosin 1 deficiency, 486, 498
Dihydropteridine reductase deficiency, 4, 6–7, 12, 17, 19, 829 Exostosin 2 deficiency, 486, 498
Dihydropyrimidinase deficiency, 644, 652, 659, 824
Dihydropyrimidine dehydrogenase deficiency, 644, 651, 657, 659, 824
Dihydropyrimidinuria, 644 F
2,8-Dihydroxyadeninuria, 643 Fabry disease, 399, 402, 407–408, 412, 424, 426, 428–430, 729, 785,
Dimethylglycine dehydrogenase deficiency, 693, 799 786, 803, 804, 806, 811, 827
Dimethylglycinuria, 693, 695, 699, 744, 830 Familial combined hyperlipidemia, 675, 681, 683, 688, 689
Dolichol kinase congenital defects of glycosylation (DK1-CDG), 484, Familial combined hypolipidemia (ANGPTL3), 674, 679, 683, 688,
487, 489 689
Dolichol kinase deficiency, 487, 505, 826 Familial defective apolipoprotein B (APOB), 672–674, 677, 678, 683,
Dolichol phosphomannose 1 (DPM1-CDG), 484, 487, 489 686, 687, 689, 825
Dolichol phosphomannose 3 (DPM3-CDG), 484, 487, 489 Familial hyperalphalipoproteinemia type 1, 674
Dolichol phosphomannose utilization congenital defects of Familial hypercholesterolemia heterozygous (LDLR), 674, 676, 677,
glycosylation (MPDU1-CDG), 484, 487, 489 683, 685–687, 689, 825
Dol-P-Man synthase 1 deficiency. See GDP-Man:Dol-P Familial hypercholesterolemia homozygous (LDLR), 674, 676, 677,
mannosyltransferase subunit 1 deficiency - CDG Ie 683, 685–687, 689, 825
Dol-P-Man synthase 3 deficiency. See GDP-Man:Dol-P Familial hyperchylomicronemia, 686
mannosyltransferase 3 deficiency Familial hypotransferrinemia, 634, 635
Dol-P-Man utilization 1 deficiency, 487, 503 Familial LCAT deficiency
Dopamine beta-hydroxylase deficiency, 517, 520, 828 complete, 675, 680, 683, 688, 689
Dopamine transporter deficiency, 516, 517, 521 partial, 675, 680, 683, 688, 689
Dopamin-serotonin vesicular transport defect, 516, 517, 521, 526 Familial protein intolerance. See Lysinuric protein intolerance
Dopa-responsive dystonia (DRD), 3, 4, 7, 10, 17, 20 Fanconi-Bickel syndrome, 265, 266, 268, 275, 295, 746, 820
Doss porphyria, 541, 542, 545 Farber disease, 402, 403, 405, 413, 426, 431, 786, 827
D4-3-Oxosteroid-5β-reductase deficiency, 557, 560, 573 Farber lipogranulomatosis, 402, 786
DPAGT1-CDG, 484, 486, 489 Fatal infantile lactic acidosis, 341, 822
DPS deficiency. See Decaprenyl diphosphate synthase (DPS) Fazio-Londe syndrome, 233–235, 237, 239, 242
deficiency Fellman disease, 634, 635
DRD. See Dopa-responsive dystonia (DRD) Fellman syndrome, 340
Dubin-Johnson syndrome, 557, 575, 618 Ferrochelatase deficiency, 543
Dynamin-like protein 1 deficiency, 377, 388 Finnish lethal neonatal metabolic syndrome, 635, 827
Dysbetalipoproteinemia (APOE), 675, 681, 683, 688, 689, 747 Fish eye disease, 675, 825
Dystonia deafness syndrome, 313, 340 Fish odor syndrome, 693, 699, 799
Flavin-containing monooxygenase 3 (FMO3) deficiency, 693, 699,
799, 830
E Flippase of Man5GlcNAc2-PP-Dol deficiency-CDG-In, 484, 486,
Early fatal progressive hepatoencephalopathy, 341 496, 826
Early infantile epileptic encephalopathy 3 (EIEE3), 87, 96, 341, 353 Folate receptor alpha (FRα) deficiency, 167–169, 171, 175, 176, 710
Early onset steroid-resistant nephrosis with sensorineural deafness, Forminoglutamic aciduria (FIGLU), 693, 757
235 5-Formyltetrahydrofolate cycloligase, 169
Disorder Index 835
Fructokinase deficiency (FK-D), 267–269 mild, 662, 664, 665

Fructose-1,6-bisphosphatase deficiency, 268, 278, 820 severe, 662, 664, 665
Fructose-1-phosphate aldolase deficiency, 267, 268 Glutathionuria, 618, 661–663, 665, 745, 820
O-Fucose-specific beta-1,3-N-acetylglucosaminyltransferase GLUT1 deficiency, 268, 286
deficiency, 487, 501, 826 GLUT2 deficiency, 268
O-Fucose-specific beta-1,3-N-glucosyltransferase deficiency, 487, GLUT10 deficiency, 268
501, 826 d-Glycerate dehydrogenase deficency, 466, 467
Fucosidosis, 437–441, 444–446, 786, 788, 806, 807, 827 Glycerate kinase deficiency, 267, 268, 278
O-Fucosylglycan synthesis (LFNG-CDG), 484, 485, 487 d-Glyceric acidaemia, 267, 268, 297, 746
Fumarase deficiency, 313–315, 317, 319–321, 821 Glycerol kinase deficiency, isolated, 693, 696
Fumaric aciduria, 315, 768 Glycine encephalopathy, 63, 81, 750, 757
Fumarylacetoacetase deficiency, 25, 728 Glycine N-methyltransferase deficiency, 33, 35, 38, 703, 758, 819
Glycogen branching enzyme deficiency, 268
Glycogen storage disease
G type 0a, 268, 821
GABA transaminase deficiency, 65, 69, 74, 81, 82, 717, 757, 828 type 0b, 268, 278, 821
Galactocerebrosidase deficiency, 401, 424, 426, 728 type I a, 268, 279
Galactokinase deficiency (GALK-D), 266, 268, 276, 728, 820 type I non-a, 268, 280
Galactosaemia, 266–268, 276, 289, 295, 820 type II a, 268, 281
Galactose-1-phosphate uridyltransferase deficiency, 266, 268, 728 type II b, 269
Galactosialidosis, 399–401, 403, 404, 408, 409, 426, 431, 438, 444, type III, 282, 820
711, 786, 790, 828 type IV, 268, 279, 820
GALTNT3-CDG, 486 type V, 269, 282, 820
Gamma-glutamylcysteine synthetase deficiency, 662, 664, 820 type VI, 269, 282, 820
Gamma-glutamyl transferase deficiency, 662, 663 type VII, 269, 284, 820
Gamma-glutamyl transpeptidase deficiency, 556, 559–564, 580, type IX a-c, 269
617–621, 661–663, 666, 668, 747, 755 type IXd, 269, 283
Gaucher disease, 399, 401, 402, 406–407, 411, 412, 424, 426–430, type X, 269, 285, 821
446, 462, 710, 711, 786, 827 type XI, 269, 286, 820
Gaucher disease-like disorder due to saposin C deficiency, 402, 412, type XII, 269, 284
426 type XIII, 269, 285
GCS1-CDG, 484–486, 489 type XIV, 268, 279, 821
GDP-fucose transporter deficiency, 487, 504, 511, 826 type XV, 268, 279, 821
GDP-Man:Dol-P mannosyltransferase 3 deficiency, 487, 503 Glycosylasparaginase deficiency, 437, 439
GDP-Man:Dol-P mannosyltransferase subunit 1 deficiency-CDG Ie, Glycosylphosphatidylinositol deficiency, 483, 484, 826
487, 503 Glyoxylate reductase deficiency, 467, 472
Global cerebral hypomyelination, 86, 341, 353 GM2 activator deficiency, 401
due to AGC1 defect, 85, 87, 91, 93, 95 GMAP210-CDG, 484, 488
Globoid cell leukodystrophy, 401, 786 GM1-gangliosidosis, 399–401, 403, 408, 424, 426, 430, 445, 827
Glucocerebrosidase deficiency, 401 GM2-gangliosidosis
Glucocorticoid receptor defect, 605 AB variant, 401, 404, 410, 426, 827
Glucocorticoid resistance, 604, 605, 612 B-variant, 401, 827
Glucocorticoid suppressible hyperaldosteronism, 601, 603, 605, 609 O-variant, 401
Glucose-6-phosphatase deficiency, 268 GNE-CDG, 483, 487
Glucose-6-phosphate translocase deficiency, 268 GNMT deficiency, 34–36, 41, 42, 45
Glucose transporter-1 deficiency, 265, 266, 268, 274, 293–295 Goldberg syndrome, 786
Glucosidase 1 deficiency-CDG-IIb, 486 Golgi-microtubule-associated protein, 210 kD,
Glucosyltransferase 1 deficiency deficiency, 488, 508
-CDG-Ic, 486 GPHN (gephyrin) deficiency. See Molybdenum
congenital disorders of glycosylation (ALG6-CDG), 484, 486, cofactor deficiency C
489 Gracile syndrome, 634–635, 638, 822
Glucosyltransferase 2 deficiency Greenberg skeletal dysplasia, 587, 588, 594, 824
-CDG-Ih, 486 Growth retardation, aminoaciduria, cholestasis, iron overload, lactic
congenital disorders of glycosylation (ALG 8-CDG), 484, acidosis and early death (GRACILE) syndrome, 340,
486, 489 634–635, 638, 822
Glutamate formimino transferase deficiency, 693, 694, 777, 783, 819 GS deficiency, 49, 58–61
Glutamate gamma-semialdehyde synthetase. See Hypoprolinaemia GTP cyclohydrolase deficiency, 4, 6
Glutamine synthetase deficiency, 49, 54 Guanidinoacetate methyltransferase deficiency,
Glutaric acidemia type I, 144, 145, 734, 777 530, 531, 814, 823
Glutaric aciduria Gyrate atrophy of the choroid and retina, 530
type 3, 693, 695, 701
type I, 143–146, 148, 151, 154, 155, 728, 804, 806, 807, 809, 818
type II, 153, 162, 235, 250, 700, 728, 764, 818 H
type II, ETF-ubiquinone oxidoreductase deficiency, Haptocorrin deficiency, 207, 209, 829
250, 702, 821 Hartnup disorder, 85–89, 93, 94, 96, 97, 743, 757
Glutaryl-CoA dehydrogenase deficiency, 771, 777 Hawkinsinuria, 23, 25, 27, 28, 30, 757, 819
Glutaryl-CoA oxidase deficiency, peroxisomal, 693 Hemolytic anemia due to GGCS deficiency, 662
Glutathione synthetase deficiency Heparan-N-sulphatase deficiency, 450, 455, 460, 786, 788–790
836 Disorder Index
Hepatic lipase deficiency (LIPC), 674, 679, 683, 689, 825 4-Hydroxy-2-oxoglutarate aldolase deficiency (mitochondrial), 467
Hepatic uroporphyrinogen decarboxylase deficiency, 543 4-Hydroxyphenylpyruvate dioxygenase change of function, 25
Hepatocyte nuclear factor 4alpha loss-of-function mutations, 325, 327 4-Hydroxyphenylpyruvate dioxygenase deficiency, 25, 728, 819
Hepatolenticular degeneration, 625 Hydroxyprolinemia, 693, 694, 698, 728, 758
Hepatorenal tyrosinemia, 725 Hydroxyproline oxidase deficiency, 693, 698
Hereditary coproporphyria (HCP), 541–544, 547, 825 Hydroxysteroid dehdrogenase deficiency
Hereditary folate malabsorption, 167–169, 171, 175, 176, 829 17-beta-hydroxysteroid dehdrogenase IV deficiency.
Hereditary fructose intolerance (HFI), 267, 268, 277, 710, 745, 746, See D-Bifunctional protein deficiency
757, 820 3-alpha-hydroxysteroid dehydrogenase deficiency, 605
Hereditary hemochromatosis 11-beta-hydroxysteroid dehydrogenase 2 deficiency, 605
type 1, 635, 637 17-beta-hydroxysteroid dehydrogenase deficiency, 603, 605
type 2a, 635, 637 3-beta-hydroxysteroid dehydrogenase type II, 605, 608
type 2b, 635, 637 17-beta-Hydroxysteroid dehydrogenase type 10 (HSD 10) deficiency,
type 3, 635, 637 104, 106
Hereditary myopathy with lactic acidosis, 342, 354 3-beta-Hydroxysteroid-delta8, delta7-isomerase deficiency, 588
Heredopathia atactica polyneuritiformis, 377 3-beta-Hydroxysterol-delta24-reductase deficiency, 588
Hers disease, 820 Hyperammonemia-hyperornithinemia-homocitrullinuria syndrome,
Hexosaminidase A and B deficiency. See Sandhoff disease 49, 728
Hexosaminidase activator deficiency. Hyperargininemia, 745
See GM2-gangliosidosis AB variant Hyper-beta-alaninaemia, 644, 824
Hexosaminidase A deficiency. See Tay-Sachs disease Hyperbilirubinaemia, Dubin-Johnson type. See ABCC2 deficiency
HHH syndrome, 49, 53, 56, 58–61, 746, 757, 758, 818 Hyperdibasic aminoaciduria type 2, 87
HI-HA/HHF6. See Hyperinsulinism-hyperammonemia syndrome Hyperekplexia due to Gly transporter GLYT2 defect, 87, 90
Histidase deficiency, 693, 698 Hyperglycerolemia, 693
Histidine ammonia-lyase, 698 Hyper IgD syndrome, 590
Histidinemia, 693, 694, 698, 720, 744, 757 Hyperinsulinemic hypoglycaemia-3
HLAH, 636 Hyperinsulinemic hypoglycaemia-4
HMG-CoA lyase deficiency. See 3-Hydroxy-3-methylglutaryl-CoA Hyperinsulinemic hypoglycaemia-5
(HMG-CoA) lyase deficiency Hyperinsulinemic hypoglycaemia-6
HMG-CoA synthase deficiency. See 3-Hydroxy-3-methylglutaryl-CoA Hyperinsulinemic hypoglycemia-7, exercise induced, 328, 330
(HMG-CoA) synthase deficiency Hyperinsulinism-hyperammonemia syndrome, 48, 49, 324–326
HMGCS2 deficiency, 362, 821 Hyperinsulinism of infancy, 323, 325
HNF1a deficiency, 325, 327, 331 Hyperinsulinism of infancy 2, 325, 326
Holocarboxylase synthetase deficiency, 219–223, 228, 711, 716, 746, 829 Hyperlipoproteinemia
Homocystinuria type 1, 675
cblD-HC type, 206, 207, 210 type 2A, 674
cblD type, variant 1, 207 type 2B, 675
due to deficiency of MTHFR activity, 169 type 3, 675
Homogentisate 1,2-dioxygenase deficiency, 25 Hyperlysinemia and saccharopinuria, 693, 695, 700
HSD10 deficiency, 104. See also 17-beta-Hydroxysteroid Hypermethioninemia
dehydrogenase type 10 (HSD 10) deficiency due to adenosine kinase deficiency, 35
Hunter disease, 450, 453, 454, 785, 805, 827 isolated persistent. See Methionine adenosyltransferase I/III
Hurler, Scheie disease, 446, 450, 453, 454, 827 deficiency
Hyaluronidase deficiency, 450, 458, 460, 786, 788, 790 Hyperphosphatasemia-mental retardation syndrome, 485
Hydrops-ectopic calcification-moth-eaten skeletal dysplasia, 587 Hyperphosphatemic familial tumoral calcinosis, 484
11-beta-Hydrosteroid dehydrogenase type 1 defect, 605 Hyperprolinaemia, type II, 180, 181, 184–186, 819
4-Hydroxybutyric aciduria, 63, 65, 69, 772 Hyperprolinemia, type I, 819
3-beta-Hydroxy-Δ5-C27-steroid dehydrogenase/isomerase deficiency, Hypobetalipoproteinemia (APOB), 672–674, 677, 678, 683, 686, 688,
557, 559 689, 747, 825
3-Hydroxyisobutyrate dehydrogenase deficiency, 106, 117, 129, 132, Hypobetalipoproteinemia (PCSK9 loss-of-function), 674
136, 771 Hypoprolinaemia, 819
3-Hydroxyisobutyric aciduria, 105, 106, 746, 818 Hypoxanthine guanine phosphoribosyltransferase deficiency, 643, 645,
3-Hydroxyisobutyryl-CoA deacylase deficiency, 105, 106, 117, 129, 649, 658, 659
132, 136
Hydroxykynureninuria, 691, 693, 695, 701, 819
17-alpha-Hydroxylase, 604, 605, 607 I
21-Hydroxylase deficiency, 601–603, 605, 608, 614, 728 I-cell disease, 402, 405, 711, 786, 828
11-beta-Hydroxylase type I deficiency, 605, 609 Iduronate 2-sulphatase deficiency, 450, 454, 460, 786, 789, 790, 792
Hydroxymethylbilane synthase deficiency. See Acute intermittent Iminodipeptiduria, 69
porphyria Iminogycinuria, 693, 694
Hydroxymethylglutaric aciduria, 106, 129, 132, 136, 818 Inclusion body myopathy, Nonaka myopathy. See UDP-GlcNAc
3-Hydroxy-3-methyl glutaric aciduria, 106, 114, 128, 129, 131, 132, epimerase/kinase deficiency
136, 744, 818 Infantile onset multiple carboxylase deficiency, 220
Hydroxymethylglutaryl-CoA lyase deficiency, 362, 804 Infantile subacute necrotizing encephalopathy, 340
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase deficiency, 104, Inosine monophosphate dehydrogenase deficiency, 643, 648, 658
106, 124, 126, 362, 367, 369, 370, 768, 769, 771 Inosine triphosphatase deficiency, 642, 644, 654, 824
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase deficiency, Inosine-5ʹ-triphosphate pyrophosphohydrolase deficiency, 644
362, 366, 369, 370, 821 Intestinal glucose-galactose malabsorption, 265, 266, 268, 274, 293
Disorder Index 837
Intrinsic factor deficiency (IFD), 206, 207, 209, 829 L-2-hydroxyglutarate dehydrogenase deficiency, 145, 818
Isobutyryl CoA dehydrogenase deficiency, 104, 106, 117, 129, 132, L-2-hydroxyglutaric aciduria, 144, 145, 147, 149, 154, 745, 746, 806,
136, 728, 762, 777, 779 807, 809, 818
Isobutyrylglycinuria, 106 Lipoid adrenal hyperplasia, 601, 602, 605, 607, 746
Isocitrate dehydrogenase defect, 145, 306, 763, 818 Lipoprotein lipase deficiency (LPL), 672, 675, 681, 683, 685, 686,
Isolated deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase, 688, 689, 711, 825
250, 254, 255 Liver glycogen phosphorylase deficiency, 269
Isolated deficiency of long-chain 3-ketoacyl CoA thiolase (1 patient), Liver glycogen synthase deficiency, 268
250 Liver phosphorylase kinase deficiency, 269
Isolated sulfite oxidase deficiency, 188, 811, 819 LPL. See Lipoprotein lipase deficiency (LPL)
Isovaleric acidemia, 104, 106, 109–112, 114, 117, 123–125, 128, 129, 17,20-Lyase deficiency, 601, 603, 605, 610
131, 134, 135, 221, 702, 710, 728, 734, 744–747, 768, 771, Lysinuric protein intolerance, 57, 85–89, 93, 94, 96–98, 700, 746, 747,
777, 779, 804, 818 750, 753, 757, 819
Isovaleryl-CoA dehydrogenase deficiency, 104, 106, 109, 762, 777 Lysosomal acid lipase deficiency, 402, 415, 426, 431, 786
Lysosomal alpha-1,4-glucosidase deficiency, 268
Lysosomal β-mannosidase deficiency, 439
J Lysosomal CLN5 protein deficiency, 402
Jansky-Bielschowsky disease, 402, 827 Lysosomal membrane cobalamin transporter deficiency, 446
Juvenile cystinosis, 475, 479 Lysosomal palmitoyl protein thioesterase-1 deficiency, 402
Juvenile optic atrophy, 340 Lysosomal transmembrane CLN3 protein deficiency, 402
Lysosomal tripeptidyl-peptidase-1 deficiency, 402
K
Kanzaki disease, 437, 439, 442, 446, 790 M
Kearns Sayre syndrome, 340, 345, 347, 348, 822 MADA deficiency. See Adenosine monophosphate deaminase
Kelley-Seegmiller syndrome. See Hypoxanthine guanine deficiency
phosphoribosyltransferase deficiency MADD. See Multiple acyl-CoA dehydrogenase deficiency (MADD)
2-Ketoadipic and 2-aminoadipic academia, 693, 695, 701 Magnesium transporter 1 deficiency (MAGT1-CDG), 483–486, 489,
2-Ketoglutarate dehydrogenase deficiency, 64, 693 498
Ketohexokinase deficiency, 277 Malonic aciduria, 105, 106, 129, 133, 138, 777
Kinky (steely) hair disease, 625 Malonyl-CoA decarboxylase deficiency, 105, 106, 728
Krabbe disease, 401, 403, 404, 411, 424, 426, 430–431, 446, 729, 733, O-Mannose beta-1,2-N-acetyglucosaminyltransferase deficiency, 487,
786, 827 501, 826
Krabbe disease-like disorder due to saposin A deficiency, 401, 426, Mannosidosis, 439, 441
431 Mannosyltransferase 4-5 deficiency (ALG11-CDG), 484, 486, 495
Kufor-Rakeb syndrome, Parkinson disease 9, 403 O-Mannosyltransferase 1 deficiency, 487, 500, 826
Kufs disease O-Mannosyltransferase 2 deficiency, 487, 500, 826
dominant type, 402, 827 Mannosyltransferase 6 deficiency-CDG-Id, 484, 486, 491, 826
recessive type, 402 Mannosyltransferase 8 deficiency-CDG-Ig, 484, 486, 491, 492, 826
Kynureninase deficiency, 701 Mannosyltransferase 2 deficiency-CDG-Ii, 484, 486, 487, 493, 826
Mannosyltransferase 1 deficiency-CDG-Ik, 484, 486, 487, 494, 500,
503, 826
L Mannosyltransferase 7-9 deficiency-CDG-IL, 484, 486, 495, 826
Lactate dehydrogenase A deficiency, 269 Mannosyltransferase 1 deficiency congenital disorders of
Lactosylceramide alpha-2,3-sialyltransferase deficiency, 487, 502, 826 glycosylation (ALG1-CDG), 484, 486, 489
LAMP2 deficiency, 269 Mannosyltransferase 2 deficiency congenital disorders of
Lanosterol demethylase deficiency, 587, 588, 595, 596 glycosylation (ALG 2-CDG), 484, 486
Late onset multiple carboxylase deficiency, 220 Mannosyltransferase 4-5 deficiency congenital disorders of
Lathosterolosis, 587, 588, 593, 596, 824 glycosylation (ALG11-CDG), 484, 486, 489, 495
LCAT. See Lecithin cholesterol acyl transferase deficiency (LCAT) Mannosyltransferase 6 deficiency congenital disorders of
l-dopa responsive dystonia, 14, 517 glycosylation (ALG3-CDG), 484, 486, 489
Leber hereditary optic neuropathy (LHON), 243, 340, 348, 357, 822 Mannosyltransferase 7-9 deficiency congenital disorders of
Leber optic atrophy and dystonia, 340 glycosylation (ALG9-CDG), 484, 486, 489
LEC35 deficiency, 826 Mannosyltransferase 8 deficiency congenital disorders of
Lecithin cholesterol acyl transferase deficiency (LCAT), 672, 675, glycosylation (ALG 12-CDG), 484, 486, 489
680, 683, 685, 686, 688, 689, 711, 747, 825 Maple syrup urine disease (MSUD), 71, 103, 104, 106, 108, 124, 126,
Leigh syndrome, 112, 230, 234, 235, 237, 304, 307, 308, 318, 344, 127, 130, 134, 135, 138, 146, 710, 713, 724, 726, 728,
348–350, 354, 806, 822, 823 732–734, 744–746, 750, 753, 755–758, 762, 807–809, 818
with French-Canadian ethnicity, 340, 344 Maroteaux-Lamy disease, 827
Lesch-Nyhan syndrome, 641, 643, 824 MAT deficiency, 34, 35, 41, 42, 44, 45, 362, 368–370, 693
Lethal infantile mitochondrial myopathy, 340, 344 Maternally inherited deafness and diabetes, 340, 346, 822
Leukocyte adhesion deficiency, type II. See GDP-fucose transporter Maternally inherited mitochondrial dystonia, 340, 345, 822
deficiency MBD deficiency. See 2-Methylbutyryl-CoA dehydrogenase (MBD)
Leukoencephalopathy with brainstem and spinal cord involvement and deficiency
lactate elevation, 341, 354, 823 McArdle disease, 820
Leukotriene C4 (LTC4) deficiency, 617–619, 621 Medium-chain acyl CoA dehydrogenase deficiency, 153, 234,
LFNG-CDG. See O-Fucosylglycan synthesis (LFNG-CDG) 249–251, 256, 724, 728, 734, 745, 764, 777, 779, 821
LHON. See Leber hereditary optic neuropathy (LHON) Mednik syndrome, 625, 629, 631
838 Disorder Index
Megaloblastic anemia-1 cblJ type, 207

Finnish type, 439 Methylmalonyl-CoA epimerase deficiency, 693, 697, 703, 818
Norwegian type, 291 Methylmalonyl-CoA mutase deficiency, 105, 106, 216, 217, 728, 818
Megaloblastic anemia due to DHFR deficiency, 169 Mevalonate kinase deficiency, 585, 588, 590, 596, 770, 824
MEGDEL syndrome, 104, 106, 112, 124, 126, 128, 129, 132, 136 Mevalonic aciduria, 585, 588, 597, 711, 746, 747, 768
Membrane-bound dipeptidase deficiency, 617, 618, 620, 621 MGAT2-CDG. See N-Acetylglucosaminyltransferase 2 congenital
Menkes disease, 623–626, 628, 630–632, 710, 711, 804, 806, 809 defects of glycosylation (MGAT2-CDG)
Metachromatic leukodystrophy, 401, 403, 405, 411, 424, 426, 431, MHBD deficiency. See 2-Methyl-3-hydroxybutyryl-CoA
786, 809, 827 dehydrogenase (MHBD) deficiency
Metachromatic leukodystrophy-like disorder due to saposin B Miller syndrome, 643, 650
deficiency, 401, 411, 426 Mineralocorticoid resistance, 605
Methacrylic aciduria, 105, 106, 818 Mitochondrial aspartate glutamate carrier 1 deficiency, 341, 823
5,10-Methenyltetrahydrofolate synthetase deficiency, 169 Mitochondrial depletion syndrome
Methionine adenosyltransferase deficiency, 33, 34, 693, 697, 703, 758 1, 341, 351
Methionine adenosyltransferase I/III deficiency, 35, 37, 41, 728, 819 2, 341, 351
Methionine synthase deficiency-cblG, 207, 819 4A, 341, 351
Methionine synthase reductase deficiency-cblE, 206, 207, 819 3, 341, 351
Methlymalonic acidemia, TCblR type. 5, 341, 352
See Transcobalamin receptor defect 8A, 341, 352
Methylacetoacetyl-CoA thiolase deficiency, 361, 362, 366, 368–370 9, 341, 352
α-Methylacyl-CoA racemase deficiency, 377, 384, 394, 556, 557, 562, Mitochondrial DNA depletion syndrome
828 2 (Myopathic type), 642, 644, 822
2-Methylbutyryl-CoA dehydrogenase (MBD) deficiency, 104, 106, 3 (Hepatocerebral type), 644
125, 126, 137, 702 5. See SCS deficiencies SUCLA2
2-Methylbutyrylglycinuria, 104, 106, 115, 128, 129, 132, 136, 702, 8A and 8B, 644
779 9. See SCS deficiencies SUCLA2
benign, 693, 696 Mitochondrial glutamate carrier 1 deficiency, 341, 823
Methylcobalamin deficiency Mitochondrial 4-hydroxybenzoate-polyprenyltransferase deficiency, 235
cblE type, 207 Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency,
cblG type, 207 362
Methylcobalamin synthesis defect-cblD-HC, 206, 207, 210, 211, Mitochondrial myopathy, encephalopathy, lactic acidosis and
214–217 stroke-like episodes, 340, 346
Methylcrotonyl-CoA carboxylase deficiency, 104, 106, 219 Mitochondrial myopathy with diabetes mellitus, 340, 346
3-Methylcrotonyl-CoA carboxylase deficiency, 104, 106, 221, 693, Mitochondrial myopathy with reversible cytochrome c oxidase (COX)
728, 734, 747, 768, 777, 804 deficiency, 342, 354
Methylcrotonylglycinuria, 106, 109, 131, 132, 135, 818 Mitochondrial neurogastrointestinal encephalopathy syndrome
3-Methylcrotonylglycinuria, 131, 132, 691, 693, 696, 702, 711, (MNGIE), 341, 642, 644, 711, 822
744–747, 782 Mitochondrial phosphate carrier deficiency, 341, 352, 823
5,10-Methylene-tetrahydrofolate dehydrogenase deficiency, 169, 172 Mitochondrial ribonucelotide reductase subunit 2 deficiency, 824
Methylenetetrahydrofolate reductase deficiency, 167–169, 172, 176, Mitochondrial thiamine pyrophosphate carrier deficiency, 229
728, 745, 806, 829 Mitochondrial trifunctional protein deficiency, 248–250, 254, 779, 821
Methylglutaconic aciduria MNGIE. See Mitochondrial neurogastrointestinal encephalopathy
type I, 106, 110, 128, 129, 131, 132, 136, 728, 799, 818 syndrome (MNGIE)
type II, 106, 128, 129, 818 MoCo deficiency
type III, 106, 128, 129, 818 complementation group A, 193
type IV, 104, 128, 129, 132, 136, 818 complementation group B, 193
3-Methylglutaconic aciduria, 103, 104, 106 complementation group C, 193
3-Methylglutaconyl-CoA hydratase deficiency, 106, 128, 229, 771, MOCS1 deficiency, 191–194, 200, 201, 829
777 MOCS2 deficiency, 191–194, 200, 829
2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency, Mohr-Tranebjaerg syndrome, 340, 346, 823
104, 106, 115, 125, 126, 129, 132, 136, 137, 368, 762, 768, Molybdenum cofactor deficiency
770 A, 193, 197, 829
Methylmalonate semialdehyde dehydrogenase deficiency, 105, 106, B, 193, 197, 829
118, 125, 129, 132, 136, 757, 771, 818 C, 193, 198, 829
Methylmalonic acidemia, 105, 106, 119, 129, 133, 137, 138, 143, Monoamine oxidase A deficiency, 517, 520
205–207, 313, 320, 341, 704, 743, 804, 813, 818, 822 Monocarboxylate transporter gain-of-function, 325
Methylmalonic aciduria Morquio A disease, 450, 456, 827
3, 693 Morquio B disease, 450, 457, 827
cblA type, 207 MPDU1-CDG. See Dolichol phosphomannose utilization congenital
cblB type, 207 defects of glycosylation (MPDU1-CDG)
cblD-MMA type, 207 MPI-CDG. See Phosphomannose isomerase congenital defects of
vitamin B12 responsive, cblA type, 207 glycosylation (MPI-CDG)
vitamin B12 responsive, cblB type, 207 MSUD. See Maple syrup urine disease (MSUD)
vitamin B12 responsive, cblD type, 206, 207 Mucolipidosis II alpha/beta, 402, 414, 426
Methylmalonic aciduria and homocystinuria Mucolipidosis III alpha/beta, 402, 414, 426
cblC type, 206, 207 Mucolipidosis III gamma, 402, 415, 426
cblD type, 206, 207 Mucolipidosis type I, 439
cblF type, 207 Mucopolysaccharidosis type IVB variant, 450, 453, 457
Disorder Index 839
Mucosulfatidosis. See Multiple sulphatase deficiency Orotidine-5-monophosphate decarboxylase deficiency, 643

Multiple acyl-CoA dehydrogenase deficiency (MADD), 234, 235, 2-Oxoglutarate dehydrogenase deficiency, 146, 821
239, 242, 249, 250, 257, 260, 396, 700, 710, 711, 716, 772, 2-Oxoglutaric aciduria, 313–316, 319–321
777, 779, 804, 821 Oxoprolinase deficiency, 661, 662, 668, 693, 696, 702, 703, 770
Multiple cartilaginous exostoses 5-Oxoprolinase deficiency, 661, 662, 668
type I, 484 Oxoprolinuria, 661–663, 665, 667, 820
type II, 484 5-Oxoprolinuria, 661, 662, 667, 768
Multiple sulphatase deficiency, 402, 828 3-Oxothiolase deficiency, 104, 744, 818
Muscle glycogenin deficiency, 268 Oxysterol 7α-hydroxylase deficiency, 556, 557, 561, 568, 574, 824
Muscle glycogen synthase deficiency, 268
Muscle phosphofructokinase deficiency, 269
Muscle phosphoglycerate kinase deficiency, 269, 285 P
Muscle phosphoglycerate mutase deficiency, 269 Pantothenate kinase-associated neurodegeneration (PKAN), 635
Muscle phosphorylase deficiency, 269 PCSK9 deficiency with low LDL, 674, 679, 683, 688, 689
Muscle phosphorylase kinase deficiency, 269, 820 PDHC E2 deficiency, 305
Muscular dystrophy-dystroglycanopathy type A1,-type B1,-type C1. PDHC E3 deficiency, 305
See O-Mannosyltransferase 1 deficiency Pearson marrow-pancreas syndrome, 340
Muscular dystrophy-dystroglycanopathy type A2,-type B2,-type C2. Pearson syndrome, 340, 345, 711, 822
See O-Mannosyltransferase 2 deficiency Peroxisomal acyl-CoA oxidase 1 deficiency, 375, 377, 383, 387, 394,
Muscular dystrophy-dystroglycanopathy type A3,-type B3,-type C3. 828
See O-Mannose beta-1,2-N-acetyglucosaminyltransferase Peroxisomal and mitochondrial fission defect, 386
deficiency Peroxisomal bile acid-CoA amino acid transferase
Myoadenylate deaminase deficiency, 643, 824 (BAAT) deficiency, 377, 386, 388–389, 396, 556, 557, 563,
Myoclonic epilepsy associated with ragged red fibers, 340, 345, 822 567, 568, 574
Myoclonus-neuropathy syndrome, 402 Peroxisome assembly disorders, 377
Myopathic form of CoQ10 deficiency (ETFDH), 235, 239 Peroxisome biogenesis disorders (PBD), 375, 377, 389
Myopathy lactic acidosis and sideroblastic anaemia, 341, 353 Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), 323
Peters plus syndrome, 485
PGR defect, 605
N Phenylalanine hydroxylase deficiency, classic PKU, 4, 5
NAGS deficiency, 49, 58–61 PHGDH deficiency, 68
Najman-Imerslund-Gräsbeck syndrome due to AMN, 207 Phosphatidylinositolglycan, class M, deficiency, 487, 502, 826
Najman-Imerslund-Gräsbeck syndrome (IGS) due to CUBN, 207 Phosphatidylinositolglycan, class V, deficiency (PIGV-CDG), 487,
Neonatal hemochromatosis, 634, 635, 638, 639 502
Neonatal mitochondrial encephalocardiomyopathy, 104, 106, 112, Phosphoethanolaminuria, 181
126, 128, 129, 132, 136 Phosphoglucomutase 1 deficiency, 268
Neonatal myoclonic epilepsy due to mitochondrial glutamate carrier Phosphoglycerate dehydrogenase deficiency, 68, 73, 819
GC1 defect, 87, 91, 93, 95 Phosphomannomutase 2 deficiency-CDG-Ia, 486, 490
Nephopatic cystinosis. See Juvenile cystinosis Phosphomannose isomerase congenital defects of glycosylation
Neuraminidase deficiency, 437 (MPI-CDG), 484–486, 489, 511
Neurodegeneration with brain iron accumulation 1, 635, 638 Phosphomannose isomerase deficiency-CDG-Ib, 486
Neuropathy ataxia and retinitis pigmentosa, 340, 345, 822 Phosphoribosyl pyrophosphate synthetase 1
Niemann-Pick disease defects, 643, 647, 824
type A or B, 402, 405, 413, 424, 426, 446, 729, 786, 792, 827 superactivity, 647, 658, 659
type C1, 402, 416, 426, 827 Phosphoserine aminotransferase deficiency, 65, 68, 73, 81, 819
type C2, 402, 416, 426, 762, 786, 827 Phosphoserine phosphatase deficiency (PSPH),
Nonketotic hyperglycinaemia, 43, 63, 64, 68, 72, 77, 79–82, 131, 819 65, 68, 74, 81, 819
Non-nephropathic cystinosis, 477 Phytosterolemia, 675
NOT5SL-CDG. See Mannosyltransferase 6 deficiency - CDG-Id PIGM-CDG, 485, 487, 511
NSDHL deficiency, 586, 588 PIGV-CDG, 485, 487, 502
PMM2-CDG, 484–486, 489, 511
P5N deficiency. See Pyrimidine-5’-nucleotidase I deficiency
O Polypeptide N-acetylgalactosaminyltransferase 3 deficiency, 499
OATP1B1 and OATP1B3 disease, 557, 564 POMGNT1-CDG, 484, 485, 487
Occipital horn syndrome, 67, 623–625, 628, 630, 631, 829 Pompe disease, 710, 729, 786, 828
Ocular coloboma with ichthyosis, POMT1-CDG, 487
brain malformations and endocrine abnormalities. POMT2-CDG, 484, 485, 487
See Steroid 5 alpha-reductase 3 deficiency Porphobilinogen deaminase deficiency, 542
Oligosaccharyltransferase subunit TUSC 3 deficiency, 486, 498 Porphobilinogen synthase deficiency, 28
Optic atrophy 1 and deafness, 340, 347, 822 Porphyria cutanea tarda type I, II and III and hepatoerythropoietic
Organic cation carnitine transporter 2 deficiency, 248, 250, 251 porphyria, 543, 546
Ornithine aminotransferase deficiency, 530, 532, 728, 819 Porphyria variegata, 543, 544, 547
Ornithine carbamoyltransferase deficiency, 49 P450 oxidoreductase deficiency, 587, 596, 599, 605, 611
Ornithine transcarbamylase deficiency, 49, 51, 728, 739, 818 P protein/T protein/H protein deficiency, 819
Orotate phosphoribosyltransferase deficiency, 646 Prenyl diphosphate synthase
Orotic aciduria subunit 1 (PDSS1) deficiency, 235, 237
type I, 643, 650, 658, 659 subunit 2 (PDSS2) deficiency, 235, 237
type II, 643, 650, 658 Primapterinuria, 4
840 Disorder Index
Primary coenzyme Q10 deficiency, 235 RFT1-CDG, 484, 486, 489

Primary familial hypoalphalipoproteinemia, 674 Rhizomelic chondrodysplasia punctata
Primary hyperoxaluria type 1 (PEX deficiency), 385
type I, 467, 468, 820, 828 type 2, 377, 828
type II, 466–468, 820 type 3, 377, 828
type III, 467, 469 Riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency,
Progesterone resistance, 605, 612 250, 257
Progranulin deficiency, 403 Ribose-5-phosphate isomerase deficiency, 578
Progressive external ophthalmoplegia (PEO), 340, 347 Richner-Hanhart syndrome. See Tyrosinaemia type II
Progressive familial intrahepatic cholestasis Rogers syndrome, 228, 230
type 1 (PFIC1), 557 Rotor syndrome, 557, 571, 575
type 2 (PFIC 2), 557, 825 RPI deficiency, 578, 582, 583
type 3, 557, 825
Progressive myoclonic epilepsy type 3, 403
Prolidase deficiency, 66, 68, 69, 76, 81, 82, 753, 757, 758, 820 S
Proline oxidase deficiency, 66, 69, 693, 728 S-adenosylhomocysteine hydrolase (SAHH) deficiency, 34, 35, 38,
Propionic acidemia, 105, 106, 118, 129, 133, 137, 138, 221, 224, 734, 41–45, 728, 758, 819
745–747, 757, 764, 768, 771, 777, 783, 803 Salla disease, 437–440, 442, 445, 446, 786, 791, 813, 828
Propionyl-CoA-carboxylase deficiency, 105, 106, 777 Sandhoff disease, 401, 403, 404, 409, 424, 426, 431, 786, 789, 806,
Prosaposin deficiency, 402, 407, 827 807, 827
Protective protein/cathepsin A deficiency, 401, 404 Sanfilippo disease
Protein X deficiency, 305 A, 450, 453, 455, 786, 827
Proton-coupled folate transporter (PCFT) deficiency, 169 B, 450, 453, 455, 786, 827
Protoporphyrinogen oxidase deficiency, 543 C, 450, 453, 455, 786, 827
PSAT deficiency, 68 D, 450, 453, 456, 786, 827
Pseudocorpus luteum deficiency. See Progesterone resistance Santavuori-Haltia disease, 402, 827
Pseudo-Hurler polydystrophy, 402, 786 Saposin deficiency
Pseudohypoaldosteronism, 604, 605, 612, 614 A, 401, 411, 426, 431, 827
Pseudovaginal perineoscrotal hypospadia, 605 B, 401, 411, 426, 431, 827
Pterin carbinolamine-4a-dehydratase deficiency. C, 402, 406, 407, 412, 426, 827
See Primapterinuria Sarcosine dehydrogenase deficiency, 693
Pulmonary hemosiderosis, 634, 635, 638, 639 Sarcosinemia, 693, 695, 700, 758
Purine nucleoside phosphorylase deficiency, 643, 648, 658, 659, SCAD deficiency. See Short-chain acyl-CoA dehydrogenase (SCAD)
804, 824 deficiency
Pyridoxal 5ʹ-phosphate (PLP) dependent seizures, 181 Scavenger receptor B1 deficiency (SCARB1),
Pyridoxine-dependent seizures, 701, 829 674, 679, 683, 688, 689
Pyridox(am)ine 5ʹ-phosphate oxidase deficiency, 181 Schindler disease
Pyridoxine-refractory sideroblastic anemia, 341, 353 type I, 439, 441
Pyrimidine-5ʹ-nucleotidase I deficiency, 643, 650 type II. See Kanzaki disease
Pyrimidine 5ʹ-nucleotidase superactivity, 643, 650, 658 type III, 439, 442
Pyroglutamic aciduria, 711, 746, 747 Schneckenbecken dysplasia, 484
Pyrroline-5-carboxylate dehydrogenase deficiency, 66, 181, 728 SCID. See Severe combined immunodeficiency (SCID)
Pyrroline-5-carboxylate synthase deficiency, 69, 757, 758 SC4MOL deficiency. See Sterol C4-methyloxidase-like deficiency
Pyrroline-5-carboxylate synthetase (P5CS) deficiency, 48, 49, 55, 56, (SC4MOL) deficiency
58–61, 66, 81 SEC23B-CDG, 483, 484, 488
Pyruvate carboxylase deficiency, 224, 304, 305, 307, 700, 712, 728, Segawa disease, 3, 4
806, 820 Sensory ataxic neuropathy, dysarthria and ophthalmoparesis, 340, 348,
Pyruvate dehydrogenase complex deficiency 822
E1a, 305, 307 Sepiapterin reductase (SR) deficiency, 3, 4, 8, 14, 17, 20, 703, 829
E1b, 305, 307 Severe combined immunodeficiency (SCID), 169, 172, 643, 656, 728
E2, 305, 308 SGLT1 deficiency, 266, 268, 286, 288, 289
E3, 305, 308 SGLT2 deficiency, 266, 288
E3 X, 305, 308 Short-chain acyl-CoA dehydrogenase (SCAD) deficiency, 158, 249,
PDHP, 305, 309 693, 697, 704, 728, 772, 777, 779, 783, 821
Pyruvate dehydrogenase E1α subunit deficiency, 305, 821 Short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (13
Pyruvate dehydrogenase E3 binding protein deficiency, 305, 821 patients), 250
Pyruvate dehydrogenase phosphatase deficiency, 305, 321 Short chain l-3-hydroxyacyl-CoA dehydrogenase deficiency, 250,
6-Pyruvoyl-tetrahydropterin synthase deficiency, 3, 4, 6, 12, 18, 325, 330. See also Short-chain 3-hydroxyacyl-CoA
728, 829 dehydrogenase deficiency (13 patients)
Sialidosis, 437–440, 442, 444–446, 711, 786, 790, 827
Sialuria, Finnish type, 439
R Sideroblastic anemia with marrow cell vacuolization and exocrine
RCDP pancreatic dysfunction. See Pearson Syndrome
type 1, 375, 377, 388, 391 Sitosterolemia (ABCG5/ABCG8), 675, 682, 689
type 2, 377, 388 SLC25A19. See Bilateral striatal necrosis (SLC25A19)
type 3, 377 SLC35A1-CDG, 484, 487
Refsum disease (classic, adult), 377, 386 SLC6A3 deficiency, 517
Renal glucosuria, 265, 266, 268, 274, 293, 743 SLC25A13 deficiency, 49, 54
Disorder Index 841
SLC33A1 deficiency with low serum copper and ceruloplasmin, 624, TK2-related mitochondrial DNA depletion myopathy, 341, 653
625, 629 Transaldolase (TALDO) deficiency, 577, 578, 580, 582, 583, 711, 799,
SLC35C1-CDG, 484, 485, 487 800, 820
SLC35D1-CDG, 484, 487 Transcobalamin deficiency, 207, 210
Sly disease, 450, 453, 458, 460, 463, 710, 711, 827 transcobalamin I (TC I) (TCN1) deficiency, 207
Smith-Lemli-Opitz syndrome (SLOS), 585–588, 590, 596, 597, 599, transcobalamin II (TC II) deficiency, 207, 208, 829
747, 771, 824 transcobalamin III (TCN III), TCN3 deficiency, 207
Solute carrier family 17 member 5 (SLC17A5) deficiency, 446 transcobalamin receptor defect, 207, 210, 829
South African porphyria. See Porphyria variegata Transient hyperammonemia of prematurity, 49
Spastic paraplegia 5A, 557 Transient hyperammonemia of the newborn, 47–49, 54, 58–61
Sphingolipid activator protein deficiency, 400 Trans-prenyl transferase (TPT) deficiency. See Prenyl diphosphate
Sphingomyelinase deficiency, 402 synthase, subunit 1 (PDSS1) deficiency
Spondylocostal dysostosis type 3, 484–485 Trifunctional dehydrogenase/cyclohydrolase/synthetase, 169
SRB1 deficiency, 674 Trimethylaminuria, 693, 695, 699, 711, 744, 799, 830
SRD5A3-CDG, 484, 487, 489, 826 TUSC3-CDG, 483, 484, 486, 489, 826
SSADH deficiency. See Succinic semialdehyde dehydrogenase Tyrosinaemia
deficiency (SSADH) type I (TYR1), 23–26, 29–31, 819
StAR deficiency. See Steroidogenic Acute Response Protein (StAR) type II ((TYR2), 23–25, 27, 30, 31, 818
deficiency type III (TYR3), 23–25, 27, 30, 31, 819
Startle disease, familial, 87 Tyrosine aminotransferase deficiency, 25, 728
Steroid 5 alpha-reductase 3 deficiency, 487, 505 Tyrosine hydroxylase deficiency, 516–518, 828
Steroidogenic acute response protein (StAR) deficiency, 605
Sterol C5-desaturase deficiency, 588
Sterol C4-methyloxidase deficiency, 588, 596, 824 U
Sterol C4-methyloxidase-like deficiency (SC4MOL) deficiency, 587, Ubiquinone deficiency, 342, 359
588, 595, 596, 599 UDP-GlcNAc:Dol-P-GlcNac-P transferase deficiency-CDG-Ij, 486,
Sterol 27-hydroxylase deficiency, 557, 561, 568, 574, 747 494
ST3GAL5-CDG, 485, 487 UDP-GlcNAc epimerase/kinase deficiency, 487, 504, 826
Stiff-Baby syndrome. See Hyperekplexia due to Gly transporter GLYT2 UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter
defect deficiency, 487, 499
Straight-chain acyl-CoA oxidase deficiency, 377 UMP hydrolase I deficiency, 643
Streptomycin ototoxicity, 342 Uncoupling protein deficiency, 342, 355
Succinic semialdehyde dehydrogenase deficiency (SSADH), 65, 69, Uncoupling protein 2 deficiency, 325, 327
75, 80–82, 152, 153, 710, 828 Uridine diphosphate galactose-4-epimerase deficiency, 266, 268, 277,
Succinyl-CoA:acetoacetate CoA transferase deficiency. See Succinyl- 820
CoA:3-oxoacid CoA transferase deficiency Uridine-5ʹ-monophosphate hydrolase superactivity, 643, 824
Succinyl-CoA:3-ketoacid CoA transferase deficiency, 362 Uridine monophosphate synthase deficiency, 643
Succinyl-CoA:3-oxoacid CoA transferase deficiency, 361–363, 370, Urocanase deficiency, 693
710, 711, 744, 746, 821 Uroporphyrinogen cosynthase deficiency. See Congenital
Succinyl-CoA synthetase (SCS) deficiency erythropoietic porphyria
SUCLA2, 315, 318 Uroporphyrinogen III synthase deficiency, 542
SUCLG1, 315, 318
Sulphatase-modifying factor 1 (SUMF1) deficiency, 402, 405
Sulphite oxidase deficiency, 33–35, 39–41 V
Sulphocysteinuria. See Sulphite oxidase deficiency van Bogaert-Bertrand disease, 143, 145
Van Bogaert-Scherer-Epstein disease. See Sterol 27-hydroxylase
deficienc
T Very long-chain acyl CoA dehydrogenase deficiency (VLCAD),
Taffazin deficiency, 106 249–251, 253, 728, 733, 734, 745, 779
TALDO deficiency. See Transaldolase (TALDO) deficiency Vitamin B12-responsive homocystinuria, cblE type. See Methionine
Tangier disease (ABCA1), 674, 680, 683, 688, 689, 804, 825 synthase reductase deficiency-cblE
Tarui disease, 820 von Gierke disease, 820
Tay-Sachs disease, 401, 403, 404, 410, 424, 426, 786, 809, 827 V0 subunit a2 of vesicular H(+)-ATPase deficiency, 488, 508, 826
T cell immunodeficiency, 498, 643, 648, 650
TCN2 deficiency. See Transcobalamin deficiency, transcobalamin II
(TC II) deficiency W
TCR/CD320 defect, 207 Wernicke like encephalopathy and BRBG (SLC19A3), 230
Tentamy preaxial brachydactyly syndrome. See Chondroitin sulfate Wilson disease, 623–626, 630–632, 710, 711, 725, 746, 747, 806, 807,
synthase 1 deficiency 829
Testicular feminization, 605 Wolman disease, 402, 403, 405, 431, 711, 746, 747, 786
TfR2-related hereditary hemochromatosis, 634, 635, 830
Thiamine-responsive megaloblastic anemia syndrome (SLC19A2),
227–231, 711, 829 X
Thiopurine S-methyltransferase deficiency, 644, 654, 657, 659, 824 Xanthine dehydrogenase deficiency, 643, 648
THTR1 deficiency, 229 Xanthine oxidase deficiency, 649, 659
Thymidine kinase 2 deficiency, 644, 653, 824 Xanthinuria
Thymidine phosphorylase deficiency, 644, 651, 658, 824 type I, 192, 199, 643, 824
Thymine-uraciluria, 644 type II, 192, 194, 643, 824
842 Disorder Index
Xanthurenic aciduria, 693, 744 X-linked mitochondrial myopathy, 341

X-linked adrenoleukodystrophy and adrenomyeloneuropathy, 377, X-linked sideroblastic anaemia (XLSA), 825
385, 395
X-linked Charcot-Marie-Tooth disease-5, 643
X-linked creatine deficiency syndrome, 530 Z
X-linked cutis laxa, 625 Zellweger spectrum disorders, 375, 377, 383, 386, 393–395, 746, 747,
X-linked distal spinal muscular atrophy, 625, 628, 630 828
X-linked dominant protoporphyria, 825 Zinc-deficiency type (AEZ), 625
Test and Medication Index
A AICAriboside, 647
a
2-Acetamido-N-(l-aspart-4'-oyl)-2-deoxy-β-glucopyranosylamine, AICAR transformylase/IMP cyclohydrolase, 643, 645, 647
443, 444 5-ALA, 26, 546, 547, 549
ABCB11 (bile salt export pump [BSEP]), 557 5-ALA dehydratase, 545
Acetaminophen-cysteine, 754 Alanine, 50, 55, 92, 93, 124, 134, 230, 278, 288, 305, 307–309, 337,
Acetazolamide, 96, 98, 294 339, 355, 367, 376, 726, 746, 750, 751, 753, 756, 757, 796,
Acetoacetate, 26, 116, 127, 309, 357, 361, 362, 366–368, 712, 744 797
Acetoacetic acid, 744 Alanine-glyoxylate aminotransferase, 376, 466, 467, 763, 772
Acetone, 116, 361, 744 Alanylproline, 68, 76
Acetyl-CoA 5-ALA synthase 2, 545
acetyltransferase, 451 Albumin, 20, 38, 238–240, 331–333, 479, 490, 491, 493, 495, 551,
alpha-glucosaminide acetyltransferase, 450, 460, 788, 790 553, 559, 560, 563, 580, 623, 627, 653, 770, 779
alpha-N-glucosaminide-N-acetyl transferase, 455 Aldehyde oxidase, 192, 194, 643, 649, 658
synthase family 3, 106 Aldolase A, 269, 284, 821
Acetylcysteine, 552 Aldose reductase, 5
Acetylglycine, 258 Aldosterone, 602–605, 607–610, 612–614
Acetyl-lysine, 695, 700 ALD protein, 377, 388
Acetylsalicylic acid, 511, 522, 667 Alendronate, 98
Acids Alglucosidase-α, 300
ceramidase, 402, 405, 413 Alkaline phosphatase, 20, 180–182, 184, 186, 187, 384, 502, 559–561,
lipase, 402, 431, 746, 747 563, 580, 625, 629, 632, 747
sphingomyelinase, 402, 405, 431, 565, 728, 786 Alkyl-DHAP synthase, 377, 380, 382, 385, 388, 389
ACTH, 333, 334, 395, 594, 602, 603, 607–610, 612–615 Allochenodeoxycholic, 566
Acylcarnitines, 104, 108, 111, 123, 126–128, 132, 133, 153, 162, 223, Allochenodeoxycholic acid, 560, 568
234, 239, 240, 242, 247–249, 251, 252, 255, 258–262, 367, Allocholic, 560, 566
368, 699, 700, 712–714, 716, 717, 720, 723, 728, 761, 762, Allocholic acid, 568
768, 770, 772, 775–783 Allo-isoleucine, 108, 728
Acyl-CoA dehydrogenase 9, 249, 250, 255, 342 Allopurinol, 298, 642, 648, 649, 655, 657–659
Acylglycines, 127, 128, 162, 260, 368, 702, 761, 778, 782 Alpha-amino, 181
Acyl transferase, 671, 675 α-Aminobutyrate, 750, 751
Adenine, 645, 649, 654, 655 Alpha-aminosemialdehyde, 185
Adenine phosphoribosyltransferase, 643, 649, 658, 659, 824 Alpha-fetoprotein, 24, 26, 30, 653, 728
Adenosine, 34, 35, 40, 654, 655 Alpha-galactosidase, 402, 424, 728, 786, 792
deaminase, 643, 645, 648, 658, 659, 724, 824 Alpha-1,4-glucosidase, 268, 281
kinase, 34–36, 40, 41, 758, 824 Alpha-iduronidase, 450, 454, 728
monophosphate, 36 Alpha-keto acid dehydrogenase complex, 104, 106, 124, 228
monophosphate deaminase, 643, 648, 658 Alpha-ketoglutarate, 65, 326, 328
Adenosylcobalamin, 306, 703 Alpha-l-fucosidase, 439, 786, 791
Adenosylhomocysteinase, S-adenosylhomocysteine hydrolase, 35 Alpha-mannosidase, 439, 710, 786, 790, 791, 814
Adenosylhomocysteine, 33, 34 Alpha-N-acetylgalactosaminidase, 437–439, 786, 791
Adenosylmethionine, 33, 34 Alpha-N-acetylglucosaminidase, 455, 460, 786, 788, 790
Adenosyltransferase, 33–37, 41, 206, 207, 693, 697, 703, 728, 758, Alpha-neuraminidase, 409, 439, 786, 791
819 5-Alpha-reductase type II, 605, 610
Adenylosuccinate lyase, 643, 645, 647, 658, 710, 823 Alpha-2,3-sialyltransferase, 487, 502, 826
Adipate, 258 Alpha-tocopherol, 668
Adipic acid, 114, 121, 367, 766, 769 Alpha-tocopherol acetate, 394, 396, 573
Adipic semialdehyde dehydrogenase, 146 Amino acids, 5, 24, 33, 48, 63, 85, 86, 103, 153, 158, 167, 179, 195,
Adrenal androgens, 610 206, 223, 229, 234, 251, 265, 304, 314, 328, 338, 361, 377,
Agalsidase, 428, 429 405, 446, 476, 515, 533, 556, 617, 653, 661, 672, 693, 710,
AICAR, 170, 643, 645, 647, 654, 655, 658, 824 720, 743, 745, 749, 761, 775, 795, 812, 818
843
844 Test and Medication Index
Amino acid transporter, 85–98, 104, 130, 134, 266, 377, 386, 396, B
661, 693, 749, 753, 786, 818, 819 BAAT. See Bile acid-CoA: amino acid N-acyl transferase (BAAT)
2-Aminoadipate, 146, 695 Batylalcohol, 394, 395
2-Aminoadipic semialdehyde synthase, 693, 700 B12-binding alpha-globulin, 207
Aminoglycoside, 342, 355, 551 BCAA aminotransferase, 106, 107, 128, 129
2-Aminoisobutyrate, 118, 125 Beets, 743
3-Aminoisobutyric acid, 118 Benzoate, 49, 58, 61, 81, 82, 511, 715
5-Aminolevulinate synthase 2, 542 Beta-alanine, 65, 74, 80, 118, 644, 652, 699, 754, 757
5-Aminolevulinic acid, 26, 541–543, 545, 550 Beta-alanine-2-ketoglutarate transaminase, 644
3-Amino-2-piperidone, 532 Beta-aminoisobutyrate, 652, 751, 754
Ammonia, 47–56, 58–60, 73, 76, 81, 82, 97, 108–111, 113, 114, 116, Beta-aminoisobutyratepyruvate transaminase, 644, 652, 824
119–121, 127, 130–133, 137, 149, 162, 211, 221, 254–256, β-Alanine, 65, 74, 80, 118, 644, 652, 655, 757
279, 282–284, 286, 290, 307, 309, 326, 328, 350, 355, β-Aminoisobutyric acid, 757
366–368, 532, 696, 698, 712–716, 743, 745, 749, 752, 814 Beta-d-glucosidase, 424
Amnionless, 206–208 Beta-enolase, 269, 285, 821
AMP-activated protein kinase, 269, 283 Beta-galactosidase, 401, 403–405, 409, 424, 430, 438, 440, 450, 457,
Amylo-1,6-glucosidase, 269, 282 460, 786, 788, 790, 791
Amylopectin, 279 Beta-1,4-galactosyltransferase, 486, 487, 499, 504, 826
Androgen receptor, 603, 605 Beta-glucuronidase, 450, 458, 460, 786, 788–790
Androgens, 602–605, 608–611 Beta-hexosaminidase, 401, 404, 409, 410, 424, 786, 789, 791
Androstenedione, 594, 603, 608, 610, 611, 613–615 11-Beta-hydrosteroid dehydrogenase type 1, 605
Angiopoietin-like, 674 Beta-hydroxybutyrate, 255, 328
Anion gap, 108–110, 112, 113, 115–117, 119, 120, 211, 221, 368, 11-Beta-hydroxylase I/II, 605
712–714, 752, 761 3-Betahydroxysteroid dehydrogenase, 586, 588, 589, 596, 601, 602,
Anserine, 694, 699, 753 605, 608
Anti-Müllerian hormone (AMH), 611 11-Beta-hydroxysteroid dehydrogenase type 2, 603, 605
“Antioxidant cocktail,” 639 17-Beta-hydroxysteroid dehydrogenase type 3, 605, 610
Antithrombin, 504, 552 17-Beta-hydroxysteroid dehydrogenase type 10, 104, 106, 115
Apo B, 672–674, 676–683 Beta-hydroxysteroid dehydrogenase type II, 605, 608
Apocarboxylases, 220, 223, 224 Betaine, 41, 43, 44, 175, 176, 216, 217, 699, 700, 799
Apolipoprotein Betainehomocysteine methyltransferase (BMT), 36
A-I, 85, 675, 680, 683, 688, 689 Beta-ketothiolase (BKT), 104, 106, 125, 126, 129, 132, 137, 361, 362,
B, 674, 677, 683, 687 368, 710, 711, 726, 728, 734, 762, 777
C-II, 675, 681, 683, 688, 689 Beta-mannosidase, 437, 439, 786, 791
C-III, 483, 485, 506–509, 511 Beta-ureidopropionase, 644, 646, 652, 824
E, 675 BH4. See Tetrahydrobiopterin (BH4)
Apolipoprotein(a) moiety of Lp(a), 675 d-Bifunctional protein, 375, 377, 382, 387, 389, 394, 557, 568, 828
Aprataxin, 235 Bile acid(s), 375, 378, 384, 385, 389, 394, 396, 555–576, 585, 629,
APTT, 504 672, 687, 688, 711, 754, 824, 825
Arabitol, 578, 580–58, 799 Bile acid-CoA: amino acid N-acyltransferase (BAAT), 377, 386,
d-Arabitol, 579 388–389, 396, 556, 557, 563, 567, 568, 574, 824
Arginase 1, 49 Bile acid-CoA ligase, 556, 557, 563, 567, 568, 575
Arginine, 51–56, 61, 67, 81, 82, 86, 89, 90, 92, Bilirubin, 24, 276, 277, 279, 284, 285, 317, 384, 386, 509, 556,
93, 108, 156, 529, 535, 537, 539, 715, 559–561, 563, 564, 575, 580, 627, 632, 637, 653, 728, 729,
726, 745, 750–752, 754, 756, 757 732, 744, 825
Argininea, 61 Biocytin, 219, 223
Arginine/glycine amidinotransferase, 529–531, 823 Biopterin, 6–14, 21
Arginine hydrochloride, 60, 61, 216 Biotin, 60, 104, 105, 133, 136, 137, 219–224, 227, 228, 231, 303, 310,
Argininemia, 49, 53 702, 710, 711, 716, 771, 829
Argininosuccinate, 51, 52, 55, 56, 58, 59, 752, 754, 757 Biotinidase, 219–224, 228, 280, 282, 283, 287, 702, 710, 711, 716,
lyase, 48, 49, 728, 750, 756, 757 724, 726–728, 734, 745, 746, 762, 768, 777, 829
synthetase, 48, 49, 750 Bisphosphonates, 98, 427, 431
Argininosuccinic acid, 57, 71, 726, 745, 755 BKT. See Beta-ketothiolase (BKT)
Aromatic l-amino acid decarboxylase (AADC), 5, 180, 515–517, 522, Botulinum toxin, 155, 200
523, 525–527, 763 Branched-chain, 108, 146, 162, 260, 305, 379, 702, 744, 818
Arylsulfatase A, 405, 424, 490, 491, 496, 786 Bromocriptine, 20, 526, 527
Arylsulphatase A, 401, 491 Bromsulphthalein, 557, 564
Ascorbic acid, 17–19, 162, 472, 525, 668, 774 Butanone, 744
Asparagine, 75, 92, 440, 484, 751 Butyrylcarnitine, 123, 126, 256, 697, 726, 782, 783
Aspartate aminotransferase, alanine aminotransferase, 746 Butyrylglycine, 258, 697, 704
Aspartate glutamate carrier, 49, 341, 823 Butyryl-/isobutyrylcarnitine, 726
Aspartic acid, 89, 750, 751, 757
Aspartoacylase, 143–145, 151, 154, 763
Atorvastatin, 574, 687 C
ATP8B1 (type 4 P-type ATPase), 557 Cabbage-like odor, 697
ATP-binding cassette, 325 CACT. See Carnitine acylcarnitine translocase (CACT)
Test and Medication Index 845
C2 acylcarnitine, 366, 369 C4-DC succinyl-carnitine, 318

C4-acylcarnitine, 117, 162, 704, 782 CD32 receptor, 207
C5-acylcarnitine, 109, 158, 162, 606, 702, 782 Ceramides, 64, 403–407, 426
C5:1 acylcarnitine, 368 Ceruloplasmin, 64, 623–625, 627–630, 636
C6:1 acylcarnitine, 114 C26:0 fatty acid, 384
Calcium, 20, 98, 109, 181, 294, 295, 298, 299, 330, 332, 334, 465, Chenodeoxycholic acid, 555, 560, 565–568, 571–574
466, 472, 473, 479, 559, 572, 573, 622, 830 Cherry red spot, 408–410, 413, 442
Calcium (U), 275–277, 469, 470 Chitotriosidase, 407, 411, 412, 426, 428, 429, 787
Carbamazepine, 81, 82, 91, 96, 98, 431, 446 Chloride, 563, 564
Carbamoyl phosphate, 93 Chlorothiazide, 332, 334
Carbamoyl phosphate synthetase I, 48, 49, 51, 646, 728 Cholesta-8,14-dien-3β-ol, 596
Carbamylglutamate, 47, 58–61 Cholestanehexols, 565, 568
Carbamyl glutamatea, 61 Cholestane pentol glucuronides, 562
Carbidopa, 17, 18, 527 Cholestanepentols, 565, 566, 568
Carbonyl reductase (CR), 5 Cholestanepentolsc, 566
5-Carboxylate Cholestane terols, 568
Carboxylic acid, 219, 558, 761 Cholestanol, 556, 562, 566, 568, 571, 574
20-Carboxy-LTB4, 62 8(9)-Cholestenol, 586, 591, 592, 596
4-Carboxymethyl sterol, 592 Cholesterol, 20, 105, 111, 121, 134, 138, 206, 208, 280, 282, 283, 299,
Cardiolipin, 111 362, 365, 384, 405, 415, 426, 430, 490, 491, 495, 555–562,
Carnitine, 60, 81, 97, 127, 128, 130–133, 137, 144, 153–155, 162, 574, 585–599, 602, 605, 607, 671–673, 679–682, 685–688,
216, 224, 248–252, 258–263, 294, 295, 298, 313, 320, 357, 711, 746, 747, 824, 825, 828
359, 370, 381, 479, 481, 658, 691, 701, 702, 704, 711, 716, Cholesterol 7α-hydroxylase, 555–557, 561, 574, 746, 824
724, 734, 745, 746, 770, 771, 775, 776, 778, 782, 821 Cholesteryl ester transfer, 674, 679, 683, 688, 689
free, 109–111, 114, 116, 117, 135, 136, 138, 199–122, 211, 237, Cholestyramine, 571, 575, 687
239, 248, 252–259, 366, 368, 370, 777 Cholic acid, 394, 565–567, 571–574
palmitoyltransferase, 250, 777, 782 Choline, 41, 116, 168, 171, 174, 176, 380, 699, 799, 812–814
palmitoyltransferase II, 249–251, 253, 728, 745, 779, 821 Chondroitin sulfate, 456, 458
Carnitine acylcarnitine translocase (CACT), 249–252, 259–263, 726, Chondroitin sulfate synthase, 486
728, 776, 777, 779, 782, 783, 821 Cis-aconitic acid, 767, 769
Carnosinase, 693, 699 C4 isobutyryl-carnitine, 160
Catecholamines, 3, 515–518, 522, 525, 526, 624, 744 C5 isovaleryl-carnitine, 160
Cathepsin Citric acid, 114, 138, 303, 314, 578, 767, 769, 771, 776, 821
A, 401, 404, 409, 438, 786, 791 Citrulline, 50–56, 58, 59, 61, 67, 81, 92, 93, 97, 307, 309, 715, 726,
D, 400, 401, 403, 424, 786 745, 750, 751, 755–757
F, 400, 403 Citrullinea, 56
C27-bile acid, 384 Citrullinemia, 715, 746, 750
C4 butyryl-carnitine, 160 Clobazam, 98
C4–C18 acylcarnitines, 237, 239, 257 Clonazepam, 96, 98
C16–C18 acylcarnitines, 252, 253 C5 2-methylbutyryl-carnitine, 160
C2-carnitine, 258, 259 CMP-sialic acid transporter, 487, 504, 826
C4-carnitine, 249, 259 Cobalamins, 41, 43, 45, 105, 205, 207, 208, 215, 217, 710, 711, 723,
C5-carnitine, 259 725, 728, 734, 758, 804, 829
C6-carnitine, 259 Coenzyme Q10, 138, 157, 158, 162, 233–236, 241, 337, 338, 342,
C8-carnitine, 259 354, 358, 359
C10-carnitine, 259, 783 COG complex, 826
C10:1-carnitine, 259 C5-OH-acylcarnitine, 110, 111, 114, 367, 368, 696
C12-carnitine, 259 C4OH-carnitine, 259
C12:1-carnitine, 259 C5-OH-carnitine, 222
C14-carnitine, 259 C12OH-carnitine, 259
C14:1-carnitine, 259 C14OH-carnitine, 259
C16-carnitine, 259 C16:1OH-carnitine, 259
C18-carnitine, 259 C18:1OH-carnitine, 259
C18:1-carnitine, 259 C18:2OH-carnitine, 259
C18:2-carnitine, 259 C4-OH hydroxybutyrylcarnitine, 326
C5–C10 dicarboxylic acids, 257 Colestipol, 687, 754
C16–C18 hydroxyacylcarnitines, 254 Complex V, 106, 338, 341, 343, 823
C6DC acylcarnitine, 114, 367 Copper, 67, 623–632, 829
C5DC-carnitine, 259 Copper-transporting P-type ATPase, 625
C6DC-carnitine, 259 Coproporphyrin I, 548, 549, 564
C8DC-carnitine, 259 Coproporphyrin III, 545, 547–549
C10DC-carnitine, 259 Coproporphyrinogen, 543
C5DC glutarylcarnitine, 145, 150, 153, 160 Coproporphyrinogen oxidase, 543
C4-DC methylmalonyl-carnitine, 123, 126, 318 Coproporphyrins, 545, 546, 548, 549
CoQ10, 162, 233–236, 239, 240, 242, 243, 710, 711, 822 Deoxyadenosine, 648, 654, 655
CoQ6 monooxygenase, 235 Deoxycorticosterone, 607, 609, 613, 614
Cornstarch, 136, 295, 297 11-Deoxycortisol, 609
Corticosterone methyl oxidase, 605, 609 1-Deoxygalactonojirimycin, 429
Cortisol, 288, 332, 584, 602–604, 608, 612, 614, 616 Deoxyguanosine, 642, 644, 645, 653–655, 658, 824
C-peptide, 328 Deoxyinosine, 654, 655
cPMP, 192–194, 197, 198, 200, 201 Deoxythymidine, 170
14C-Propionate, 120 Deoxyuridine, 170, 651, 655
C3 propionylcarnitine, 211, 212, 214 Deprenylb, 20
Creatine, 41, 42, 44, 53, 67, 76, 81, 82, 217, 299, 338, 529–539, 791, Dermatan sulfate, 451, 452, 454, 457, 458, 462, 788, 789
813, 814 D-Erythrose-4-phosphate, 579
kinase, 34, 38, 40, 45, 110, 111, 113, 148, 234, 239, 240, 248, Desmosterol, 586, 587, 589, 593, 595, 596, 598
252–255, 257, 275, 281–286, 299, 300, 349, 354, 355, 357, Dexamethasone, 522, 604, 609, 612, 614, 616
493, 494, 496, 500, 501, 503, 504, 506, 507, 530, 590, 648, Dextromethorphan, 81, 82, 658
653, 700, 747, 778 Dextrometorphan, 43
monohydrate, 537–539 Dextrose, 130–133, 324, 716, 777
transporter, 529, 530, 532, 710, 803, 823 D-Fructose-6-phosphate, 579
Creatinine, 96, 299, 430, 460, 468, 469, 473, 479, 530–532, 534–536, D-Glyceraldehyde-3-phosphate, 70, 579
548, 571, 680, 701, 703, 745, 746, 751–753, 764, 779, 788, D-Glycerate, 278, 287, 289, 297, 466, 467
799, 800 dGuo, 648
Crotonylglycine, 366 DHEA, 594, 602, 608, 610, 611, 613–615
C14:1 tetradecenoyl-carnitine, 253 DHEAS, 608, 610, 611
Cubilin, 206–208 DHEA sulfate, 608
Cu-histidinate, 632 DHPR. See Dihydropteridine reductase (DHPR)
C6-unsaturated acylcarnitine, 111 DHT, 603, 610–615
Cyclic pyranopterin monophosphate, 191, 192, 194, D-2-Hydroxyglutaric acid, 66, 147, 150, 257, 260, 763, 764, 806, 818
197, 198, 201 Diazoxide, 263, 323, 330–334
Cyproheptadine, 517, 526, 527 Dicarboxylic, 85
Cystathionine, 33, 37, 39, 40, 696, 702, 753, 754, 756, 757 Dicarboxylic acids, 66, 116, 143, 144, 219, 237, 252, 253, 256, 260,
Cystathionine beta-synthase (CBS), 33–37, 39–41, 43, 45, 195, 196, 261, 368, 384, 764, 772
702, 703, 711, 728, 758, 819 (3-Hydroxy) Dicarboxylic acids, 254, 255, 260, 324, 764, 770, 772
Cystathionine gamma-lyase, 35, 36, 44, 195, 196 (C5–C10) Dicarboxylic acids, 257
Cysteamine, 105, 475–478, 481 Dichloroacetate, 29, 310, 359
Cysteine, 33–36, 41, 43, 96, 97, 105, 191, 192, 195, 400, 405, 620, Dihydrobiopterin, 5, 7, 8, 525
668, 702, 703, 752 7,8-Dihydrobiopterin, 9
dioxygenase, 35, 36, 196 Dihydrofolate, 70, 170
sulphinate decarboxylase, 36 Dihydrofolate reductase, 5, 70, 167–169, 172, 175, 176, 829
Cysteinylglycinase, 618, 661–663, 820 Dihydrolipoyl dehydrogenase, 305, 821
Cysteinylglycine, 620–622, 661, 755 Dihydrolipoyl transacetylase, 305, 821
Cystine, 34, 37, 39–41, 86, 89, 90, 92, 93, 96–98, 191, 192, 195, Dihydroorotate, 655
197–199, 201, 475–482, 535, 620, 700, 745, 750–752, 754, Dihydroorotate dehydrogenase, 643, 646, 650, 824
757 Dihydropteridine reductase (DHPR), 3–7, 9, 11–13, 16–21, 728, 829
l-Cystine dimethylester, 98 Dihydropyrimidinase, 644, 646, 652, 659, 824
Cystinosin, 475–478, 786 Dihydropyrimidine dehydrogenase, 642, 644, 646, 651,
Cytochrome c (Cyt c), 192, 338, 343 657, 659, 824
Cytochrome c oxidase (COX), 157, 158, 162, 239, Dihydrotestosterone, 603, 604, 610, 611, 613, 614
240, 342, 354, 358, 624 Dihydrothymine, 652, 655, 769
Cytochrome P450 oxidoreductase, 588, 589, 596, 604 Dihydrouracil, 652, 655, 769
Cytochromome P450 17-alpha-hydroxylase, 605 Dihydroxyacetone, 380
Cytosolic acetoacetyl-CoA thiolase, 362, 365, 821 Dihydroxyacetone phosphate acyltransferase, 377
2,8-Dihydroxy adenine, 649, 654, 655
1,25-Dihydroxycholecacliferol, 573
D 1,25-Dihydroxycholecalciferol, 481, 572, 573
dATP, 648 Dihydroxyphenylacetic acid, 518, 624, 630
Deacylase, 105, 106, 117, 129, 132, 136 Dihydroxyphenylserine, 516, 517, 526
Decadienoylcarnitine, 726 Dimethylglycine, 695, 699, 700, 799
Decanedioate, 258 Dimethylglycine dehydrogenase, 693, 700, 711, 799
Decanoylcarnitine, 726 2,4-Dinitrophenylhydrazine (DNPH), 122, 124, 128, 743, 744
Decaprenyl, 235, 240, 243 2,4-Dinitrophenylhydrazine test, 108
Decenoylcarnitine, 726, 783 Dino, 648
7-Dehydrocholesterol, 585–590, 596 3β,7α-diOH-5-cholenoic acida, 566
8-Dehydrocholesterol, 591, 592, 596 Diphenylhydantoin, 96
7-Dehydrocholesterol reductase, 588, 589 Diphosphate, 240, 489
Delta-aminolevulinate dehydratase, 542 2,3-Diphosphoglycerate, 284
δ-Aminolevulinic acid, 757 Disialotransferrins, 509
Delta(4)-3-oxosteroid-5β-reductase, 557 Diversion, 571, 575
dl-Dihydroxyphenylserine, 526 Flavin-containing monooxygenase, 693, 699

D-Mannoheptulose, 579 Flippase, 484, 486, 496, 826
DNPH. See 2,4-Dinitrophenylhydrazine (DNPH) Fludrocortisone, 616
Docosahexaenoic acid, 263, 378, 384, 394 Fluoxetine, 81, 98, 552
Dodecanedioate, 258 Fluvastatin, 687
Dodecanoylcarnitine, 726 Folate(s), 18, 20, 21, 41, 43–45, 69, 167–177, 212, 216, 229, 526, 575,
Dodecenoylcarnitine, 726 700, 710, 711, 753, 758, 828, 829
Dolichol, 484, 826 Folic acid, 167, 172, 175–177, 216, 217, 228, 297, 699
kinase, 487, 505 Folinic acid, 17–19, 81, 168, 169, 175–177, 180, 189, 216, 217, 526,
phosphate, 489 710, 716, 717
Dol-P-Man utilization, 487, 503 FOLR1
Dopamine, 515–517, 519, 522, 523, 525–527 blocking autoantibodies, 173
beta-hydroxylase, 515–518, 520, 522, 523, 525, 526, 624, 828 transporter, 173
transporter, 515–517, 521 Formiminoglutamic acid (FIGLU), 171, 172, 174, 693, 694, 698, 699,
Dopamine–serotonin, 3, 4, 516, 517, 521, 526 726, 727, 757, 777, 783
D-6-phosphogluconate, 579 Formiminotransferase, 693, 763
D-6-phosphoglucono-d-lactone, 579 Formylglycine generating enzyme (FGE), 402, 405
D-Ribose, 658 Formyl-5'-phosphoribosyl-5-aminoimidazole-4-carboxamide, 170
D-Ribose-5-phosphate, 579 Formyl-5'-phosphoribosylglycinamide, 170
D-Ribulose-5-phosphate, 579 5-Formyltetrahydrofolate, leucovorin, 18, 169, 526
D-Sedoheptulose, 579 Formyltetrahydrofolate synthase, 169
D-Sedoheptulose-7-phosphate, 579 Free carnitine, 123, 126, 135–138, 248, 258, 259, 262, 366, 368, 370,
D-Xylulose, 579 776, 777
D-Xylulose-5-phosphate, 579 Free fatty acids (FFA), 114, 116, 260, 261, 288, 325–327, 361, 366,
368, 369, 672, 712, 714, 716, 745, 761, 778
Fructokinase, 267, 268
E Fructose, 265–267, 270, 271, 274, 277, 278, 287, 289, 293, 295–297,
Elaprase, 461, 462 710, 714, 744–746, 757, 820
Electron transfer flavoprotein (ETF), 162, 234, 249–251, 257, 262, fructose-1,6-bisphosphatase, 268, 278, 820
700, 702, 779, 821 fructose 6-phosphate, 484
Elongation factor, 338, 341 fructose-1-phosphate aldolase, 267, 268, 277
Entacapone, 527 Fucose, 440, 487, 489, 501, 504, 511, 826
Entacaponec, 20 Fumarase, 50, 306, 313–315, 317, 319–321, 357, 746, 763, 821
Coenzyme Q10, 138, 157, 158, 162, 233–236, 241, 337, 338, 342, Fumaric acid, 121, 313, 317, 319, 320, 578, 763, 766
354, 358, 359 Fumarylacetoacetase, 25, 26, 728, 763
Epinephrine, 515–517, 520, 522, 523, 525, 552 Furane-2,5-dicarboxylic acid, 764, 770
Erythritol, 578, 580–582 Furosemide, 130, 332, 552
Erythronic acid, 578, 580–582, 799, 800 Furoylglycine, 767, 770
Estradiol, 611, 613–615
Estrogen receptor, 605
ETF. See Electron transfer flavoprotein (ETF) G
ETF-ubiquinone oxidoreductase (ETF:QO), GABA, 63–82, 86, 91, 98, 147, 192, 195, 699,
249, 250, 700, 702, 821 751, 753, 754, 757, 828
Ethanolamine, 380 GABA transaminase, 65, 69, 70, 74, 81, 82, 710, 717, 757, 828
Ethosuximide metabolites, 770 GAGs, 426, 449, 450, 454–460, 464, 787–790
Ethylhydracrylic acid, 696, 702 Galactitol, 267, 275, 276, 287, 292, 296
2-Ethylhydracrylic acid, 111, 115, 116, 123, 125, 368, 765 Galactocerebrosidase, 401, 424, 426, 728
Ethylmalonate, 125, 158 Galactokinase, 266, 268, 276, 287, 728, 744, 820
Ethylmalonic acid, 118, 121, 158, 160, 237, 249, 256, 257, 260, 355, Galactonat, 271
697, 702, 704, 728, 764, 766, 769, 770, 772 Galactose, 54, 265–268, 270, 271, 274–277, 286–289, 293, 295–297,
Exostosin, 486, 498 440, 484, 485, 489, 709–711, 714, 726, 732, 743, 744, 820
galactose-1-phosphate, 266–268, 276, 712
galactose-1-phosphate (RBC), 276, 277, 287
F galactose-1-phosphate uridyltransferase, 276, 728
Fabrazyme, 428 galactose 6-sulfatase, 452
Factor VII and X, 53 γ-Cystathionase, 35, 693, 702
Factor XI, 490, 491, 493, 495–497, 503, 507 γ-Glutamylamino acid, 663
Ferric chloride, 108, 122, 124, 128, 698, 743, 744 γ-Glutamylcysteine, 196, 661–668, 711, 747, 820
Ferritin, 20, 353, 428, 429, 545, 546, 548, 580, 633, 634, 636–639, Gamma-glutamylcysteine synthetase, 196, 661–664, 666–668, 747,
747 820
Ferrochelatase, 543 γ-Glutamyl-P, 71
FFA. See Free fatty acids (FFA) γ-Glutamylphosphate, 66
FGE. See Formylglycine generating enzyme (FGE) Gamma-glutamyl transpeptidase, 556, 559–564, 580, 617–621,
FIGLU. See Formiminoglutamic acid (FIGLU) 661–663, 666, 668, 747, 755
Filipin test, 112, 416 Gamma-glutathione synthetase, 664
Fish odor, 693, 695, 699, 700, 711, 797, 799 β-GCase, 406, 407, 427, 428
GDP-fucose transporter, 487, 504, 511, 826 all tissues, 281

GDP-Man:Dol-P mannosyltransferase, 487, 503, 826 branching enzyme, 268
GDP-mannose, 484 synthase, 268, 278
Globotriaosylceramide, 406, 407, 412 Glycogenin-1, 268
Globotriaosylsphingosine, 412 Glycolate, 466, 469–471
Glucagon, 263, 289, 328, 331–334, 552 Glycolic acid, 468, 763, 765, 769, 770, 772
Glucocerebrosidase, 401, 406 Glycosaminoglycans, 404, 414, 415, 426, 449, 450, 787, 788
Glucocorticoid receptor, 604, 605 Glycosylasparaginase, 437, 439, 446, 786, 791
Glucokinase, 325, 330, 711 Glycylproline, 68, 76, 757
Glucose Glyoxylate, 376, 465–467, 472, 820
(-galactose), 268 GM2 activator, 401, 403, 404, 409, 410
-6-phosphatase, 268, 280 GM3 ganglioside, 502
-6-phosphate, 728 GMP-lipoate, 69
-6-phosphate Golgi-microtubule-associated protein, 488, 508
dehydrogenase, 577, 662, 667, 668, 726, 728 Gonadotropins, 295, 610, 611, 615
translocase, 268 GTPCH activity, 6
transporter-1, 265, 266, 268, 274, 293–295 GTP cyclohydrolase I, 3–5, 12, 18, 728
Glucosidase 1, 484, 486, 497, 826 Guanidinoacetate, 529–534, 536, 814, 815
Glucosyltransferase, 427, 484, 486, 487, 491, 493, 501, 826 Guanidinoacetate methyltransferase, 530, 531, 814, 823
Glucotetrasaccharides, 281 Guanosine, 489, 654, 655, 829
Glucuronate sulfatase, 451
β-Glucuronidase, 450, 458, 460, 786, 788–790
GLUT1, 266, 268, 270, 274, 286, 288, 290, 292, 294, 710 H
Glutaconic acid, 146, 148 Haemoglobin, 293, 296, 580
Glutamate, 48, 50, 58, 66, 67, 86, 87, 91–93, 95, 96, 119, 180, 194, Haptocorrin, 207–209, 829
195, 249, 305, 330, 341, 617, 662, 693, 694, 698–700, 715, Haptoglobin, 497
751, 752, 777, 783, 812, 813, 819, 823 Hawkinsin, 25, 27–29, 757
aspartate transporter, 86, 87 HCS. See Holocarboxylase synthetase (HCS)
dehydrogenase-1, 325 Hemoglobin (Hb), 8, 9, 172, 230, 287, 428, 639, 658, 664, 665, 668,
transporter, 85–87, 90 728, 743, 747
Glutamic acid, 54, 89, 184, 309, 698, 726, 750, 751, 757, 771 Hemojuvelin (HJV), 634, 635
Glutamine, 48–56, 58, 59, 61, 66, 92, 93, 119, 120, 134, 305, 309, Heparan-N-sulfatase, 450, 455, 460, 786, 788–790
667, 715, 726, 750–752, 756, 757, 763, 770, 800, 812–814, Heparan sulfate (HS), 450, 451, 454–456, 458, 460, 462, 788, 789
819 Hepatic triglyceride, 674
Glutamine synthetase, 47–49, 54, 757 Hepatocyte nuclear factor 4A, 325, 327, 330
Glutarate, 144, 153, 258, 701 Hepcidin, 634, 635
Glutaric acid, 114, 121, 148, 150, 151, 237, 239, 240, 260, 701, 763, Heptacarboxyporphyrin, 545, 549, 550
768–771 Heptacarboxyporphyrinogen, 543
Glutarylcarnitine, 144, 146, 148, 150, 153 Hexacarboxyporphyrin, 549
Glutaryl-CoA Hexacarboxyporphyrinogen, 543
dehydrogenase, 144–146, 153, 249, 763, 771, 777 Hexanoylcarnitine, 726
oxidase, 376, 693, 701, 763, 771 Hexanoylglycine, 249, 256–258, 260, 764
Glutaryl-/OH decanoylcarnitine, 726 Hexasialotransferrins, 509
Glutathione, 25, 195, 239, 477, 559, 617, 618, 620, 622, 650, Hexosaminidase, 401, 403
661–668, 745 β-Hexosaminidases A and B, 404
Glutathione synthetase, 196, 661–667, 702, 703, 728, 746, 820 5HIAA. See 5-Hydroxyindoleacetic acid (5HIAA)
Glyceraldehyde, 578 High-density lipoprotein (HDL) cholesterol, 671, 672, 676–682, 686
l-Glycerate, 469, 470 Hippuric acid, 537, 744, 769
D-Glycerate dehydrogenase, 466, 467 Histidase, 693, 694, 698
Glycerate kinase, 267–269, 278 HJV. See Hemojuvelin (HJV)
D-Glycerate kinase, 278, 287, 297 HMG-CoA. See 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)
Glyceric acid, 466, 763, 766 HMGL. See 3-Hydroxy-3-methylglutaryl-CoA lyase (HMGL)
Glycerol, 277, 278, 380, 672, 696, 703, 763, 766, 769, 820 HNF1A, 325, 327, 331
kinase, 693, 703, 745–747, 763, 820 HNF4A, 325, 330
triacetate, 154, 156 Holocarboxylase synthetase (HCS), 219–223, 228, 702, 710, 711, 716,
Glycine, 36, 49, 59, 63–82, 86–88, 92, 93, 98, 108, 109, 116, 119, 734, 746, 762, 829
120, 122–124, 131, 135, 162, 181, 186, 192, 224, 257, 278, Holotranscobalamin, 205, 208–210, 212–215
368, 529–531, 537, 555, 556, 565, 566, 617, 634, 694, 697, Homoarginine, 156, 757
698, 700, 703, 704, 710, 715, 726, 728, 750–752, 756, 757, Homocarnosine, 69, 74, 694, 699, 758
771, 819, 823 Homocitrulline, 52, 53, 55, 56, 58, 695, 700, 753, 756, 758
Glycine cleavage, 63, 68, 77, 180 Homocysteine-cysteine, 172
Glycine N-methyltransferase, 33–35, 38, 703, 728, 758, 819 Homocysteine/homocyst(e)ine (Hcy), 33–35, 37–41, 43, 168–172,
Glycocholic acid, 396 174, 176, 197, 198, 201, 205, 206, 208–214, 216, 320, 697,
Glycocholic acidb, 574 699, 703, 743, 745, 751–755, 758, 771
Glycogen, 114, 265–270, 272, 273, 275, 278–287, 289–293, 295–300, Homocystinuria, 33–35, 169, 205–207, 210, 217, 667, 710, 711, 724,
304, 324, 331, 332, 367, 714, 745, 746, 786, 820, 821 725, 734, 739, 743, 745, 753, 804, 811
Homogentisate 1,2-dioxygenase, 25 7α-Hydroxy-3-oxo and 7α, 12α-dihydroxy-3-oxo-4-cholenoic acids,

Homogentisic acid, 743, 744, 763, 770, 800 560
Homovanillic acid (HVA), 5–9, 14, 16, 21, 172, 174, 181, 185, 187, 4-Hydroxy-2-oxoglutarate, 467
516–523, 525, 767, 769, 770 4-Hydroxy-2-oxoglutarate aldolase, 467
HS. See Heparan sulfate (HS) 4-Hydroxyphenylacetate, 26, 27, 29, 745
5HTP. See 5-Hydroxytryptophan (5HTP) 4-Hydroxyphenyllactate, 26, 27, 29, 745
HVA. See Homovanillic acid (HVA) 4-Hydroxyphenylpyruvate, 23, 26, 27, 29, 745
Hyaluronan, 458, 788 4-Hydroxyphenylpyruvate dioxygenase, 23–26, 28, 728, 763, 819
Hyaluronidase, 450, 458, 460, 786, 788, 790 4-Hydroxyphenylpyruvate hydroxylase, 25
Hydantoin-5-propionic acid, 694, 699, 763 p-Hydroxyphenylpyruvic acid, 744
Hydrochloride, 60, 61, 429, 517, 526, 527, 537 Hydroxyproline, 68, 71, 79, 80, 185, 186, 472, 694, 697, 698, 751,
Hydrocortisone, 615, 616 752, 755, 758
Hydrogen carrier protein (H-protein), 63, 64, 68, 819 Hydroxyproline oxidase, 693, 698
Hydrogen sulfide, 195, 744 3-Hydroxypropionic acid, 66, 118–120, 125, 205, 211–214, 221, 222,
Hydroxo-Cbl, 210, 216, 217 762, 769, 770
Hydroxocobalamin, 43, 175, 716 Hydroxypyruvate, 467
2-Hydroxyadipate, 695 3β-Hydroxysteroid
3-Hydroxyadipate, 258 delta8, delta7-isomerase, 588
4-Hydroxybenzoate-polyprenyltransferase, 235 delta14-reductase, 588, 589, 596
Hydroxybutyrate, 294, 295 delta24-reductase, 586, 588, 589, 596
3-Hydroxybutyrate, 127, 258, 262, 263, 288, 355, 357, 712 3-Hydroxysuberate, 258
3-Hydroxybutyric acid, 121, 763 5-Hydroxytryptophan (5HTP), 4, 17–20, 516, 518, 519, 522, 523, 525,
4-Hydroxybutyric acid, 63, 66, 75, 79, 80, 763 526
Hydroxybutyrylcarnitine, 256, 326, 328, 330 25-Hydroxy-vitamin D, 560, 562
3β-Hydroxy-5-cholenoic acids, 561, 565, 567, 568 Hyperammonemia, 47–61, 67, 85, 94, 96, 97, 104, 105, 127, 128, 130,
3β-Hydroxy-5-cholestenoic acid, 561, 568 132, 133, 136, 138, 143, 221, 248, 700, 710, 712, 714–715,
27-Hydroxycholesterol, 556, 558, 561, 568 740, 745, 746, 749, 752, 778
7α-Hydroxycholesterol dehydrogenase, 560 Hyperammonemia-hyperornithinemia-homocitrullinuria, 49
Hydroxycobalamin, 60, 133, 137, 703 Hyperinsulinism, 48, 249, 256, 263, 323–334, 580, 710, 712, 714,
4-Hydroxycyclohexanecarboxylic acid, 763, 770 745, 754
3β-Hydroxy-D5-C27-steroid dehydrogenase/isomerase, 557, 559 Hypogalactosylation Tf glycans, 504
3-Hydroxydecanedioate, 258 Hypoglycosylated glycoproteins, 494
3-Hydroxydicarboxylic acid, 254, 255, 326, 764, 770, 772 Hypotaurine, 35, 36, 191
3-Hydroxydodecanedioate, 258 Hypoxanthine, 191, 199, 201, 647–649, 654, 655
3-Hydroxyglutarate, 144, 153, 256, 326, 328, 330 Hypoxanthine-guanine phosphoribosyltransferase, 643, 645, 649,
D-2-Hydroxyglutarate dehydrogenase, 145, 249, 818 658, 659
l-2-Hydroxyglutarate dehydrogenase, 145
Hydroxyglutaric acid, 66, 147, 149, 150, 154, 257, 260, 710, 763, 764,
769, 771, 772 I
3-Hydroxyglutaric acid, 148, 150, 151, 249, 695, 763, 764, 772 Idebenone, 242, 243
5-Hydroxyindoleacetic acid (5HIAA), 5–9, 14, 16, 21, 172, 174, 185, Iduronate sulfatase, 451, 452, 788
516–523, 525, 770 Iduronate 2-sulfatase, 450, 454, 460, 786, 789, 790, 792
3-Hydroxyisobutyrate dehydrogenase, 105, 106, 117, 129, 132, 136, IGF1, 492
762, 771 Imidazole pyruvic acid, 694, 698
3-Hydroxyisobutyric acid, 117, 118, 762, 771 Immunoglobulin(s), 78, 171, 492, 497, 552, 590, 648
3-Hydroxyisobutyryl-carnitine, 117 Indomethacin, 481
3-Hydroxyisobutyryl-CoA, 105, 762 Inosine, 642, 654, 655, 824
3-Hydroxyisobutyryl-CoA deacylase, 105, 106, inosine-5'-triphosphate pyrophosphohydrolase, 644
117, 129, 132, 136 monophosphate dehydrogenase, 643, 645, 648, 658
2-Hydroxyisovaleric acid, 117, 123, 762 Inositol, 168, 171, 174, 176
3-Hydroxyisovaleric acid, 109–112, 114, 123, 125, 221, 696, 702, Insulin, 59, 130, 131, 133, 135, 249, 263, 266, 288, 295, 299, 323,
744, 762, 769–771, 798 324, 328, 330–334, 481, 551, 552, 686, 712, 715, 716
3-Hydroxykynurenine, 695, 701 Insulin receptor, 325, 331
27-Hydroxylase, 557, 558, 561, 562, 567, 568, 747 Interlipid(s), 130
20-Hydroxy-LTB4, 620 Intrinsic factor, 206–209, 829
Hydroxylysine, 144, 146, 758 Iron, 20, 98, 340, 346, 348, 550, 551, 553, 623, 633–639, 822, 830
3-Hydroxy-2-methylbutyric acid, 368, 369 Iron transport protein, 635
3-Hydroxy-3-methylglutaric acid, 114, 123, 124, 367, 762, 766, 769 Isobutyryl-CoA dehydrogenase, 104, 106, 117, 132, 136, 728, 762,
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA), 361, 431, 576 777, 779
3-Hydroxy-3-methylglutaryl-CoA lyase (HMGL), Isobutyrylglycine, 117, 123, 125, 258, 696, 702, 762, 766
57, 106, 114, 361, 362, 367, 369, 370, 711, 745, 746, 762, Isocitrate dehydrogenase 2, 145, 763
768, 769, 771 Isocoproporphyrin, 545, 546, 550
3-Hydroxy-3-methylglutaryl-CoA synthase 2, 362 Isoleucine, 92, 93, 103–139, 305, 308, 328, 362, 366, 368, 369, 702,
4-Hydroxy-6-methyl-2-pyrone, 366, 367 726, 728, 750, 751, 756, 758, 762, 782, 798
3-Hydroxy-n-butyric acid, 109–112, 114, 116, 119, 121, 148, 211, Isovaleric acid, 109, 123, 744
212, 222, 326, 366, 368, 695, 696 Isovaleryl-CoA dehydrogenase, 104, 106, 109, 762, 777
Isovalerylglycine, 109, 123, 124, 160, 162, 258, 702, 762, 766 Lipids, 45, 63, 114, 132, 229, 233, 234, 239, 240, 266, 294, 394, 395,
Isovaleryl-/2-methylbutyrylcarnitine, 726 399, 400, 405, 556, 561, 587, 602, 617, 618, 642, 668,
671–673, 678, 685–687, 761, 785, 799, 812, 825
lipid-linked Glc1Man9GlcNAc2, 493
K lipid-linked GlcNAc2, 494
Keppra, 770 lipid-linked Man7GlcNA2, 492
Keratan sulfate, 408, 451, 456, 457, 788, 789 lipid-linked Man5GlcNAc2, 491, 496, 503
Ketoacidosis, 104, 105, 107–110, 113, 115–117, 119–121, 124, 304, lipid-linked Man9GlcNAc2, 491, 494, 503
311, 331–332, 361, 367, 370, 703, 744 lipid-linked Man3GlcNAc2 and Man4GlcNAc2, 495
3-Ketoacyl-CoA, 250, 255, 728 lipid-linked oligosaccharides (LLO), 485, 505, 509, 511
2-Ketoglutarate dehydrogenase, 64, 306, 693, 701, 745, 746, 763 Lipoic acid, 82, 294, 304, 311, 359
Ketohexokinase, 277 Lipoprotein, 333, 576, 671–689, 825
Ketones, 104, 114, 116, 119–121, 127, 128, 131–134, 143, 247, 248, Lipoprotein lipase (LPL), 672, 675, 681, 683, 685, 686, 688, 689, 747,
251–257, 260, 278, 280, 282, 283, 288, 292–295, 297, 811, 825
307–310, 316, 325–328, 361–370, 657, 694, 712–714, 716, Lithium citrate, 144, 154, 156
732, 733, 745, 771, 772, 821, 743744 L-methylmalonyl-CoA, 206, 703
Kuvan, 10, 20 Long-chain acylcarnitines, 240, 248, 252, 255, 261, 262, 354, 366,
Kynureninase, 693, 701, 702 716, 776, 777, 779, 782
Kynurenine, 695, 701 Long-chain enoyl-CoA hydratase (LCEH), 250, 251
Long-chain 3 hydroxyacyl-CoA dehydrogenase (LCHAD), 248–251,
254, 259–263, 710, 711, 726, 728, 740, 745–746, 764, 777,
L 779, 782, 783
Lactate, 50, 91, 93, 109, 111–113, 115–117, 119–121, 123–125, Long-chain 3-ketoacyl-CoA thiolase, 250, 255
131–134, 143, 149, 157, 158, 160, 162, 180, 185, 211, 222, Lorenzo’s oila, 394, 395
230, 238–240, 252, 263, 265, 274, 278–280, 282–284, 286, Lovastatin, 576, 687
288–290, 296, 297, 299, 303, 305–310, 313, 316–318, 320, Low-density lipoprotein (LDL)
330, 337, 339, 341, 344–355, 357, 359, 367, 368, 386, 495, cholesterol, 405, 671–673, 676–679, 681, 682, 686, 687
525, 651, 653, 691, 712–714, 716, 728, 746, 796, 797, 812, receptor, 674, 825
813, 823 receptor associated protein, 674
Lactate dehydrogenase (LDH), 50, 172, 269, 286, 290, 357, 747, 821 LPL. See Lipoprotein lipase (LPL)
Lactic acid, 221, 223, 287–289, 319, 320, 712, 762, 763, 765, 769, LTB4, 617, 618, 620, 621
770, 796, 797, 799 LTC4, 617–621
Lactic acidosis, 64, 113–115, 131, 132, 136, 138, 160, 162, 181, 227, LTD4, 617, 618, 621
228, 234, 235, 238, 254, 255, 267, 303–305, 307–309, 314, LTE4, 617, 618, 620, 621
316–318, 340–342, 346, 353, 354, 358, 359, 634, 638, 664, Lumizyme, 300
716, 744, 757, 822, 823 Lyase, 601, 603–605, 610
Lactosylceramide, 502 Lysine, 55, 86, 89, 90, 92, 93, 97, 144–146, 149, 154–156, 180, 183,
Lamotrigine, 81, 82, 431 189, 219, 220, 309, 314, 376, 478, 532, 535, 537, 539, 695,
Lanosterol, 587, 588, 596 700, 701, 726, 750, 751, 754, 758, 763, 776, 819
Lanosterol 14-alpha demethylase, 588 Lysosomal export of cobalamin, 206
L-arginine hydrochloride, 60, 216
Laronidase, 461, 462
Lathosterol, 586, 587, 593, 595, 596 M
L-carnitine, 97, 103, 126, 132, 133, 135–138, 155, 216, 217, 263, 370, Magnesium transporter, 484, 486, 498
716, 779, 782 Malate dehydrogenase, 50, 306
LCAT. See Lecithin-cholesterol acyl transferase (LCAT) Malic acid, 121, 147, 766
LCAT activity, 680, 685 Malonic acid, 121–123, 125, 763, 765
LCEH. See Long-chain enoyl-CoA hydratase (LCEH) Malonic semialdehyde, 70
LCHAD. See Long-chain 3 hydroxyacyl-CoA dehydrogenase Malonylcarnitine, 121–123, 126, 726, 783
(LCHAD) Malonyl-CoA decarboxylase, 105, 106, 121, 728, 746, 763, 771, 821
LDH. See Lactate dehydrogenase (LDH) Malonyl-/OH octanoylcarnitine, 726
L-Dopa, 4, 14, 17–20, 446, 516, 517, Mandelic acid, 766, 770
519, 520, 522, 523, 525–527 Mannoheptulose, 578, 580–582
L-Dopa Carbidopa, 4, 17–20, 526 Mannose, 405, 406, 483, 484, 487, 501, 511, 826
Lecithin-cholesterol, 672, 675 Mannose 1-phosphate, 489
Lecithin-cholesterol acyl transferase (LCAT), 672, 675, 680, 683, 685, Mannose 6-phosphate, 405, 406, 511
686, 688, 689, 711, 747, 825 Mannosyltransferase, 484, 486, 487, 491–495, 500, 503, 826
Leucine, 92, 93, 103–139, 221, 249, 305, 308, 323, 324, 326, 328, MAO-A, 516, 517, 520, 522, 523, 525–527
330, 361, 362, 366, 367, 369, 702, 710, 726, 750, 751, 755, Medium-chain acyl CoA, 153, 234, 239, 249–251, 256, 724, 728, 734,
756, 758, 762, 771, 782 745, 764, 777, 779, 821
Leukotriene C4 synthase, 618, 619 Medium-chain and long-chain acylcarnitines, 366
Leukotriene E4, 590 Medium-chain dicarboxylic acids, 326
Levodopa, 17, 515, 516, 526, 527 Melanin, 744
L-2-hydroxyglutaric acid (L2HG), 144, 145, 147, 149, 150, 152, 153, Melatonine, 20
763 3-Mercaptolactatecysteine disulfide, 745
Lipase, 299, 402, 405, 415, 426, 431, 679, 685, 746, 747, 786 α-Mercaptopropionylglycine, 97
Metanephrine, 518, 520, 522, 523, 525 5-Methyl-THF, 73, 167, 168, 171, 172, 175, 177
e
Methacrylic acid conjugates, 117 4-Methylumbelliferyl-α-d-mannopyranoside, 443
c
Methacrylic aciduria, 105, 106, 818 4-Methylumbelliferyl-α-l-fucoside, 443
g
Methemoglobin, 743 4-Methylumbelliferyl-α-N-acetylgalactosaminide, 443, 444
h
Methenyl-tetrahydrofolate cyclohydrolase, 169, 170 4-Methylumbelliferyl-α-neuraminide, 443
f
5,10-Methenyltetrahydrofolate synthetase, 169 4-Methylumbelliferyl-β-d-mannopyranoside, 443
Methionine, 24, 26, 33–45, 54, 92, 93, 105, 114, 118, 122, 138, 168, Metronidazole, 133, 137, 157, 158, 162
169, 172, 174–176, 191, 195, 196, 205–207, 211–216, 529, Mevalonate, 239
697, 699, 703, 711, 716, 723, 726, 728, 744, 747, 750, 751, Mevalonate kinase, 585, 586, 588–590, 596, 762, 770, 824
758, 776 Mevalonic acid, 586, 590, 595, 596, 762, 766, 768, 770
adenosyltransferase, 33–37, 41, 695, 697, 703, 728, 758, 819 Mevinolin, 576
sulfoxide, 697, 758 MHPG, 518–520, 523, 525
synthase, 5-methyltetrahydrofolate-homocysteine Microsomal triglyceride transfer protein, 674
methyltransferase, 207 Midazolepyruvic acid, 744
synthase reductase, 5-methyltetrahydrofolate-homocysteine Miglustat, 407, 427–431
methyltransferase reductase, 207 Mineralocorticoid, 599, 601–605, 609, 616
Methotrexate, 18, 19, 754 Mitochondrial deoxyguanosine kinase, 644
3-Methoxy-4-hydroxyphenylglycol, 518, 523, 528 Mitochondrial ribonucleotide reductase, 644, 653
3-Methoxytyramine, 518, 520, 522, 523, 525 Monoamine oxidase A, 515–517, 520
3-Methoxytyrosine, 181, 185, 187 Monocarboxylate transporter 1, 325
2-Methylacetoacetate, 368, 369, 744 Monophosphate, 36, 50, 170, 191, 192, 194, 197, 198, 201, 228, 577,
2-Methylacetoacetic acid, 116 643, 645, 648, 658, 824
Methylacetoacetyl-CoA thiolase, 361, 362, 366, 368–370 Monosialotransferrins, 509
α-Methylacyl-CoA racemase, 377, 384, 387, 394, 556, 557, 562, 568, 5MTHF, 8, 169, 172–177
574, 828 Muscle glycogen phosphorylase, 269
4-Methyl and 4,4-dimethyl sterols, 592, 593, 595 Myocytes, 281
2-Methylbutyric acid, 115 Myoglobin, 279, 282–286, 300, 747
2-Methylbutyrylcarnitine, 115, 726, 782 Myozyme, 300
2-Methylbutyryl-CoA dehydrogenase, 104, 106, 115, 699, 702, 762
2-Methylbutyrylglycine, 115, 123, 125, 160, 162, 696, 702, 762
Methylcitric acid, 119, 120, 205, 211–214, 222, 725, 762, 767, 771 N
Methylcobalamin, 36, 169, 172, 175, 177, 206–208, 211, 212, 829 NaBicarbonate, 130, 131, 133
3-Methylcrotonic acid, 744 N-Acetyl-alpha-dglucosaminidase, 450
3-Methylcrotonylcarnitine, 110 N-acetylaspartate, 65, 73, 87, 91, 93, 111, 114, 119, 120, 144, 146,
Methylcrotonyl-CoA carboxylase, 106, 110, 219, 221, 224 151, 153, 154, 363, 812
3-Methylcrotonyl-CoA carboxylase, 104, 128, 129, 221, 224, 693, N-acetylaspartic acid, 143, 148, 150, 763, 767, 769
702, 728, 734, 747, 762, 771, 777, 804 N-acetylcysteine, 42, 157, 158, 162, 583, 661, 662, 667, 668, 764
3-Methylcrotonylglycine, 110, 114, 135, 221–224, 367, 696, 702, 762, N-acetyl galactosamine, 295, 487, 499
767, 771 N-Acetylgalactosamine-4-sulfatase, 450, 457, 460, 786, 788, 790, 792
Methylene-tetrahydrofolate, 63, 65, 69, 172 N-Acetylgalactosamine-6-sulfatase, 438, 450, 456, 786, 788–790, 792
5,10-Methylenetetrahydrofolate dehydrogenase, 169 N-Acetylgalactosaminyltransferase, 486, 499, 826
5,10-Methylenetetrahydrofolate reductase, 169 N-Acetylglucosamine-1-phosphotransferase, 402, 405, 786, 791
3-Methylglutaconic acid, 111–114, 123, 124, 349, 351, 352, 355, 367, N-Acetylglucosamine-6-sulfatase, 450, 456, 786, 788–790
762, 766, 771, 799 N-acetylglucosaminidase, 455, 460, 786, 788, 790
Methylglutaconic aciduria, 104, 106, 110, 113, 128, 129, 131, 132, N-acetylglucosaminyltransferase, 484, 486, 487, 496, 501, 826
136, 728, 745–747, 762, 777, 799, 818 N-acetylglutamate synthase, 48, 49, 51, 715, 818
3-Methylglutaconyl-CoA hydratase, 104, 106, 111, 128, 129, 771, 777 N-Acetyl-l-glutamate, 50
3-Methylglutaric acid, 104, 106, 111, 113, 114, 121, 124, 128, 129, Naglazyme, 461, 462
131, 132, 136, 367, 762, 766, 771 N-butyl deoxynojirimycin, 427, 429
Methylglutarylcarnitine, 726 N-Carbamoyl-β-alanine, 652, 655
3-Methylhistidine, 699, 751 N-Carbamoyl-β-aminoisobutyrate, 655
2-Methyl-3-hydroxybutyric acid, 111, 116, 728, 762, 765, 818 N-Carbamoyl-β-aminoisobutyric acid, 652
2-Methyl-3-hydroxy-butyrylcarnitine, 115, 116 N-Carbamylglutamate, 49, 60, 133, 715
Methylmalonate semialdehyde dehydrogenase, 105, 106, 118, 125, Neopterin, 6–9, 12–14, 21, 525
129, 132, 136, 751, 771, 818 Neuronal glycine transporter, 86, 87
Methylmalonic acid, 120–122, 128, 169, 172, 205, 206, 208–213, 215, Neutral amino acids, 86–89, 755, 757
314, 318–320, 352, 697, 703, 725, 744, 762, 763, 765, 768–771 Neutropenia, 104, 109–111, 116, 118, 120, 133, 136, 280, 298, 317,
Methylmalonic semialdehyde dehydrogenase, 762 332, 511, 628, 711, 747
Methylmalonylcarnitine, 120, 726, 783 Niacin, 574, 687, 701, 754
Methylmalonyl-CoA epimerase, 693, 697, 703–704, 762, 818 Nicotinamide, 96, 97, 251, 701
Methylmalonyl-CoA mutase, 105, 106, 120, 206, 215–217, 703, 716, Nifedipine, 332, 334
728, 734, 818 Nitisinone, 23, 24, 26, 30, 31
Methylsuccinate, 258 Nitric oxide synthase, 3, 50
Methylsuccinic acid, 160, 162, 697, 704, 764, 766 Nitrogen scavenger, 47–49, 59
b
5-Methyltetrahydrofolate, 14, 18, 36, 44, 65, 176, 207, 208, 526 p-Nitrophenyl-α-l-fucopyranoside, 443, 444
5-Methyltetrahydrofolatehomocysteine methyltransferase, 36 27-Nor-cholestanepentol(s), 565, 568
Norepinephrine, 51–517, 519–523, 525, 624, 630 P

Normetanephrine, 520, 523, 525 Palivizumab, 300
Normosang, 551 Palmitoyl protein thioesterase, 400–402, 424
NPC1 protein, 402, 786 Palmitoyl protein thioesterase-1, 402
O Panhematin, 551
Octanoylcarnitine, 256, 259, 726, 727, 770, 777, 780–783 Pantothenate kinase, 635, 829
Octanoyl-mtACP, 69 P450 aromatase, 605
OCTN2. See Organic cation carnitine transporter 2 (OCTN2) P450 11-beta-hydroxylase type 1, 605
Octreotide, 328, 331–334 PBG. See Porphobilinogen (PBG)
O-Fucose-specific beta-1,3-Nacetylglucosaminyltransferase, P5CS. See Pyrroline-5-carboxylate synthetase (P5CS)
487, 501, 826 PEG-ADA, 658
OH butyrylcarnitine, 726, 782 Pegloticase, 657
OH hexadecenoylcarnitine, 726 Penicillamine, 97, 631, 754, 756, 766
OH isovalerylcarnitine, 368, 726, 727, 777, 780–782 d-Penicillamine, 94, 632
5-OH-Me-uracil, 355 Pentacarboxyporphyrin, 549
OH palmitoylcarnitine, 726, 727, 777, 780–783 Pentacarboxyporphyrinogen, 549
17-OH-pregnenolone, 594, 608 Pentasialotransferrins, 509
17-OH-progesterone, 594, 608–611, 614, 615 Peptidase D, 68, 69, 617, 618, 620–622, 661, 662, 747, 755
Oligosaccharides, 288, 295, 297, 399, 408–410, Pergolide, 526
414, 415, 426, 437, 438, 440, 445, 446, 450, 484, 485, 505, Peroxins, 377, 382
509, 511, 787–790 Peroxisomal acyl-CoA oxidase 1, 375, 383, 387, 393, 395, 828
Oligosaccharyltransferase, 484, 486, 498 Perseitol, 578, 580–582
O-Mannose beta-1,2-Nacetyglucosaminyltransferase, 487 Persulphide, 159
O-Mannosyltransferase, 487, 500, 826 Phe loading test, 7, 8, 14
3-O-Methyldopa, 516, 518–520, 522, 523, 525, 526 Phenobarbitone, 180, 575
OPA3 protein, 106 Phenolphthalein, 743
Organic cation carnitine transporter 2 (OCTN2), 248, 250, 251, 258, Phenothiazines, 90, 551, 744
259, 261–263 Phenylacetic acid, 744, 763
Ornithine, 48, 50, 53, 55, 56, 58, 61, 81, 82, 85, 86, 89, 90, 92, 93, Phenylalanine, 3–21, 24, 29–31, 92, 93, 720, 725, 726, 728, 743, 750,
529, 530, 532–538, 715, 726, 750–752, 758, 819 751, 758, 763, 818
aminotransferase, 50, 57, 67, 530, 728, 732, 757, 758, 819 Phenylalanine hydroxylase, 4, 5, 17–18, 20, 720, 728, 763, 818
aspartate, 537, 538 Phenylbutyrate, 47–49, 59, 61, 138, 715
δ-Ornithine aminotransferase, 50 Phenylpropionylglycine, 258, 260, 764
ornithine transcarbamylase (OTC), 48, 49, 51, 56, 58–61, 646, 703, Phenylpyruvic acid, 5, 744, 763, 766, 767
710, 715, 727, 728, 739, 746, 756, 757, 804, 818 Phenytoin, 332, 431, 770
ornithine transporter (ORNT1), 49, 50 Phosphate, 228, 229, 249, 267, 275, 276, 289, 295, 341, 351, 377, 384,
Orotate, 55, 646, 655, 714 476, 479, 481, 499, 530, 577, 578, 646, 710, 716, 823, 830
Orotic acid, 51–53, 56, 58, 86, 90, 93, 98, 172, 643, 650, 712, 728, Phosphatidylcholine (PC), 41, 44, 557, 559
767, 769 Phosphatidylinositolglycan, 485, 487, 502, 826
Orotidine, 50, 55, 650, 655 Phosphoenolpyruvate, 485, 487, 502, 826
Orotidine monophosphate, 50 Phosphoethanolamine, 403, 753, 758
Osmolality, 108, 130, 131, 133, 134 Phosphoethanolaminuria, 181
Oxalate, 465–467, 469–473 Phosphofructokinase, 269, 284
Oxalic acid, 468, 469, 744, 763, 765, 769, 772 Phosphoglucomutase, 268, 272, 279
Oxipurinol, 648, 649, 659 Phosphoglucomutase 1, 268
2-Oxoadipate, 695 Phosphoglycerate
2-Oxobutyric acid, 744 dehydrogenase, 64, 68, 73, 819
7αOH-3-oxo-4-cholenoic acidb, 566 kinase, 269, 285, 821
18-Oxocortisol, 609, 614 mutase, 269, 285
2-Oxoglutarate dehydrogenase, 3-Phosphohydroxypyruvate, 70
104, 144, 146, 314, 315, 320, 357, 821 Phospholipids, 229, 376, 380, 556, 559
2-Oxoglutaric acid, 308, 313–317, 319–321, 578 Phosphomannomutase 2, 484, 486, 490, 826
2-oxo-4-hydroxybutanoic, 70 Phosphomannose isomerase, 484–486, 491, 509, 511, 826
3-oxo-4-hydroxybutanoic, 70 5'-Phosphoribosyl-5-aminoimidazole-4-carboxamide, 170
4-oxo-6-hydroxyheptanoate, 26 5'-Phosphoribosylglycinamide, 170
2-Oxoisocaproic acid, 744 Phosphoribosyl pyrophosphate, 647
2-Oxo-4-methiylbutyric acid, 744 Phosphoribosyl pyrophosphate synthetase 1, 643, 647, 658, 659, 824
2-Oxo-3-methylvaleric acid, 744 Phosphoribosyl pyrophosphate synthetase (PRPPS), 643, 645, 746
5-Oxoprolien, 663 Phosphorylase, 269, 282, 283, 296, 643–646, 648, 651, 658, 659, 746,
Oxoprolinase, 693, 696, 702–703, 770 804, 824
5-Oxoprolinase, 661–663, 666, 668, 763 Phosphorylase kinase, 269, 283, 332, 820
5-Oxoproline, 25, 27, 661, 662, 664, 665, 667, 696, 702 3-Phosphoserine, 70
3-Oxothiolas, 106 Phosphoserine aminotransferase (PSATI), 64, 65, 68, 70, 78, 79, 710
3-Oxothiolase activity, 104, 116, 744, 745, 818 Phosphoserine phosphatase (PSPH), 64, 65, 68, 70, 74, 81, 710, 819
Oxysterol 7α-hydroxylase, 555–557, P450 21-hydroxylase, 605
561, 567, 568, 574, 824 Phytanic acid, 76, 378, 381, 384–386, 388, 389, 393–395
Phytanoyl-CoA hydroxylase, 377, 381–383 Pyrimidine-5'-nucleotidase I, 643, 650, 747

Phytomenadione, 394, 396, 573 Pyrimidine nucleotides, 650
Pipecolic acid, 183, 186, 188, 384, 758 Pyroglutamic acid, 661–663, 702, 703, 711, 726, 746, 747, 763, 766,
Plasmalogens, 229, 375, 380, 385, 388, 389, 393–395 770
Polymerase gamma, 338, 340, 341 Δ-1-Pyrroline-5-carboxylate (P5C), 66, 67, 71, 78, 81, 82, 181, 184,
Porphobilinogen (PBG), 24, 26–29, 541, 542, 550, 745 186, 697, 698
deaminase, 542, 549 synthetase, 71
synthase, 24, 26–29, 542 Pyrroline-5-carboxylate, 47, 48, 184, 728
Porphyrin I, 549 Pyrroline-5-carboxylate dehydrogenase, 66, 181, 728
Porphyrins, 541, 542, 545–548, 550–553, 743, 825 Pyrroline-5-carboxylate synthetase (P5CS), 47–50, 55, 58–61, 66, 67,
Porphyrins type I, 546 69, 71, 79–81
Potassium, 97, 295, 325, 330, 332, 476, 479, 481, 607–609, 612, Pyruvate, 35, 38, 50, 57, 60, 64, 70, 104, 146, 159, 219, 223, 229,
616, 716 230, 303–311, 316, 327, 328, 330, 332, 337–339, 345,
channel tetramerization domain-containing protein 7, 403 351, 355, 357, 358, 652, 700, 701, 712, 728, 744–746,
citrate, 97, 298 756–758, 806
P450 oxidoreductase (POR), 587–589, 596, 599, 601, 604, 605, 611 carboxylase, 57, 219, 221, 224, 303–311, 700, 712, 728, 745, 746,
P-protein, 63, 64, 68 757, 758, 806, 820
Pramipexole, 526 dehydrogenase, 64, 77, 104, 146, 228, 303–311, 338, 357, 358,
Pravastatin, 687 728, 821
Prednisolone, 616 dehydrogenase phosphatase, 305, 821
Prednisone, 616 6-Pyruvoyl-tetrahydropterin synthase (PTPS), 3, 4, 6, 12, 13, 18, 728,
Pregnanediol, 587, 594 829
Prenyl diphosphate synthase, 235, 237
Primapterin, 4, 7, 9, 12
Pristanic acid, 378, 379, 381, 382, 384–387, 389, 393, 574 R
Progesterone, 551, 604, 605, 607, 608, 610, 612–614 Receptor, 43, 65, 81, 98, 167–169, 171, 175, 176, 192, 194, 198,
Progesterone receptor, 605 206–210, 325, 330–332, 382, 402, 428, 476, 516, 517, 526,
Progranulin, 400, 403 551, 553, 555, 587, 602–604, 634, 636, 672–674, 679, 683,
Prolactin, 6–8, 495, 516, 519, 521, 523, 525 685, 688, 710, 724, 728, 825, 829
Prolidase, 66, 68, 69, 76, 79–82, 710, 753, 757, 758, 820 Receptor 2, 634, 635
Proline, 50, 55, 56, 58, 63–82, 92, 181, 185, 186, 305, 309, 537, 693, Renin, 604, 608, 609, 612
694, 697, 698, 726, 728, 750, 751, 755, 758, 763, 816, 819 Replagal, 428
Proline oxidase, 66, 69, 693, 697, 698 Reticulocytes, 231, 284, 285, 664, 665, 668, 747
Propionylcarnitine, 119, 120, 122, 211–214, 716, 725, 726, 764, 771, Rhodamine B, 462
783 Rhodanese, 159
Propionyl-CoA carboxylase, 105, 106, 119, 219, 221, 223, 762, 772, Ribitol, 578–582, 799
777 Riboflavin, 145, 154–156, 162, 189, 233–243, 249–251, 261–263,
Propionyl-CoA carboxylase deficiency, 105, 106, 777 359, 716, 743, 772, 822
Propionylglycine, 97, 119, 123, 223, 258, 260, 764 Riboflavina, 155
Proprotein convertase subtilisin/kexin type 9, 674 Riboflavin transporter 2, 235
Protein 3, 559, 674 Ribose-5-phosphate isomerase, 577–579, 581, 582, 710, 799, 820
Protein kinase, 269, 283, 332 Rifampicin, 571, 575
Prothrombin, 24, 28, 38, 40, 386, 559–561, 563, 580, 627, 678 Ropinirole, 527
Prothrombin ratio, 386, 559–561, 563, 627, 678 Rosuvastatin, 687
Protoporphyrin, 545, 548–550, 553, 636
IX, 541, 545, 547–549
IX-Zn, 545 S
Protoporphyrinogen oxidase, 543 Saccharopine, 146, 695, 700, 758
Protoporphyrin-Zn, 545 S-Adenosylhomocysteine, 34–41, 43, 44, 196, 531, 728, 758, 819
Protoprophyrin, 543 S-Adenosylmethionine (SAM), 35, 37–41, 176, 193, 195, 206,
Protoprophyrinogen, 543 211–213, 215, 531, 697, 703
PRPP synthetase activity, 647 SAdo, 654, 655
PSATI. See Phosphoserine aminotransferase (PSATI) SAICArc, 654
Pseudouridine, 655 SAICAriboside, 647
P450 side-chain cleavage, 605 Salicylates, 744
Pterin-4a-carbinolamine dehydratase, 4, 7, 19 Saposin A, 401, 404, 411, 426, 431, 827
PTPS activity, 6 Saposin A-D, 402, 405
Purine nucleoside phosphorylase, 643, 645, 648, 658, 659, 746, 804, Saposin B, 401, 405, 411, 426, 431, 827
824 Saposin C, 402, 406–407, 412, 426, 827
Pyrazolones, 743 Sapropterin, 4, 10, 20
Pyridoxal phosphate, 35, 36, 64, 717 Sarcosine, 40, 538, 693, 695, 699–701, 758
Pyridoxal 5'-phosphate (PLP), 168, 175, 178–182, 184, 187, 195 Sarcosine dehydrogenase, 93, 700
Pyridoxine, 41, 43, 66, 77, 78, 179–181, 184, 186, 188, 189, 201, 341, SCAD. See Short-chain acyl CoA dehydrogenase (SCAD)
353, 466, 471, 472, 526, 527, 537, 701, 710, 716, 717, 829 S-2-carboxypropyl-cysteamine, 117, 123, 125
Pyridoxine hydrochloride, 537 S-2-carboxypropyl-cysteine, 117, 123, 125
Pyridox(am) ine 5'-phosphate oxidase, 180, 181, 184, 185, 195 Scavenger receptor B1, 674, 679, 683, 688, 689
SCHAD. See Short-chain 3-hydroxyacyl-CoA dehydrogenase Succinyl-CoA: 3-oxoacid CoA transferase (SCOT), 361, 362,
(SCHAD) 367–370, 711, 744, 746, 821
SCOT. See Succinyl-CoA: 3-oxoacid CoA transferase (SCOT) Succinyl-/methylmalonylcarnitine, 726
Sebacic acid, 114, 121, 367, 767 4-Sulfatase, 450, 452, 460, 486, 788, 790, 792
Sedoheptitol, 560, 581, 582 Sulfatides, 403, 405, 411, 415, 424, 426
Sedoheptulose, 476, 577, 578, 580–582, 800 Sulfite, 188, 191–198, 200, 201, 745
Sedoheptulose-7-P, 578, 580–582 Sulfite dipstick, 200, 745
Selegiline, 20, 526, 527 Sulfocysteine, 197
Sepiapterin, 3, 4, 8, 14, 17, 20, 703, 829 Sulphide-CoQ reductase, 159, 162
Sepiapterin reductase, 3–5, 8, 14, 17, 20, 703, 829 Sulphite, 33–35, 39–41, 45, 159, 200, 657
Serine, 35, 63–82, 92, 93, 180, 267, 297, 382, 484, 634, 750–752, Sulphite oxidase, 159, 162
757, 758 Sulphocysteine, 33, 39, 40
Serotonin, 3, 4, 20, 333, 515–518, 521, 522, 525–527 Sulphur dioxygenase, 158, 159
Sertraline, 20, 517, 526, 527 Sulthiame, 96
Short-chain acyl CoA dehydrogenase (SCAD), 158, 162, 249–251,
256, 259, 261, 262, 602, 693, 697, 704, 726, 728, 746, 764,
772, 777, 779, 783, 821 T
Short-chain glycine conjugates, 257 Taffazin, 106
Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), 153, Taliglucerase, 427
249–251, 256, 259, 261–263, 330, 711, 745, 764, 772, 777, Taurine, 33, 35, 81, 82, 191, 195,
782, 783 197, 198, 201, 555, 556, 559, 565, 566, 568, 750–753, 756,
Sialic acid, 404, 437–447, 483–485, 489, 504, 787, 790–791, 799, 758, 812
814, 826, 828 Taurineg, 199
Sialin, 437–440, 446 Taurotetra-[tri/penta]-hydroxycholestanoic acids, 562
Sialini, 443 Testosterone, 481, 602–604, 608, 610–615
Sialotransferrins, 490–509, 511, 653 Tetradecanedioylcarnitine, 726
Simvastatin, 588, 687 Tetradecanoylcarnitine, 726
Sitosterols, 675, 682, 683, 686, 689, 825 Tetradecenoylcarnitine, 726
SLC6A3 transporter, 517 Tetrahydrobiopterin (BH4), 3–21, 168, 172, 174, 515, 516,
SLC6A8 transporter, 530 525, 720
SLC46A1 transporter, 169 Tetrahydrobiopterin (BH4) loading test, 6, 7, 11, 17, 20
Sodium, 59–61, 81, 96, 108, 130, 132, 216, 261, 266, 274, 295, 297, Tetrahydrocortisol, 609, 610, 614
298, 358, 359, 476, 479, 481, 537, 545–547, 551, 604, Tetrahydrocortisone, 609, 610, 614
607–609, 612–614, 667, 697, 715, 716 Tetrahydrofolate, 63, 70, 170, 208
benzoate, 47, 48, 59–61, 81, 96, 97, 132, 133, 216, 537, 715, 744 Tetrahydroxycholestanoic acids, 565, 568
benzoatea, 61 Tetrasaccharide, 497
bicarbonate, 358, 359, 481, 667, 716 Tetrasialotransferrins, 509
PBAa, 61 Thiamin, 124, 359
PBA/sodium phenylacetate, 60 Thiamine, 104, 131, 134, 227–231, 304, 310, 710, 711, 716, 829
Sodium-dependent, 86, 87, 270 monophosphate, 228
Sodium-dependent glucose transporter-2, 266, 268 Thiaminee, 134
Sorbitol, 278, 287 Thiamine pyrophosphate (TPP), 227–229, 304
Sphingomyelinase, 285 Thiamine triphosphate (ThTp), 228
S-Sulfocysteine, 191, 192, 195, 196, 200, 753, 758 Thiolase, 393, 568
S-Sulfocysteined, 199 Thiopurines, 642, 644, 654, 657, 659
Statins, 97, 298, 586, 599, 687, 688 Thiopurine S-methyltransferase, 644, 654, 659, 824
Steroid 5 alpha-reductase, 487, 505 Thiosulfatee, 199
Steroids, 81, 82, 429, 586, 602, 607, 615, 761 Thiosulphate, 34, 43, 45, 158–160, 162
Sterol Threonine, 54, 92, 93, 105, 122, 138, 180, 181, 185, 187, 484, 702,
C5-desaturase, 588 726, 750, 751, 758
C4-methyloxidase, 588, 589, 596 Thrombocytopenia, 24, 68, 76, 97, 104, 109, 110, 116, 118, 120, 133,
27-hydroxylase, 557, 558, 561, 567, 568, 747 228, 230, 232, 345, 406, 411–413, 427, 492, 493, 578, 580,
Suberate, 258 590, 627, 747
Suberic acid, 114, 121, 367, 769 THTR1 transporter, 229
Suberylglycine, 258, 260, 764, 767 Thymidine, 65, 167, 651, 655
Subfamily member A1, 674 Thymidine kinase 2, 341, 642, 644, 653, 824
Subtilisin/kexin type 9, 674 Thymidine phosphorylase, 642, 644, 646, 651, 658, 824
Succinate, 91, 194, 239, 240, 306, 313, 314, 320, 358, 359 Thymine, 644, 651, 655, 766, 769
Succinate-CoA ligase, 315, 341 Thyroxin-binding globulin, 490, 491, 497
Succinic acid, 70, 121, 317, 319, 763, 766, 769, 770 Tiglylcarnitine, 115, 116, 123, 126, 726
Succinic semialdehyde, 65, 69, 70, 75, 81, 153, 763, 828 Tiglylglycine, 115, 116, 119, 123, 125, 221, 368, 369, 762, 767
Succinic semialdehyde dehydrogenase, 65, 69, 70, 75, 81, 153, Tiopronina, 97
763, 828 TPP. See Thiamine pyrophosphate (TPP)
Succinylacetone, 24, 26–31, 723, 725, 726, 763, 765, 766, 768, 770 T-protein, 63, 64, 68, 819
Succinyladenosine, 647 Tranilast, 657
Succinyl-CoA, 107, 146, 205, 206, 313–315, 320, 703, 708, 777 Transaldolase, 577–580, 582, 583, 799, 800, 820
Transaminases, 24, 50, 65, 69, 70, 74, 81, 82, 106, 248, 252–257, 386, Uroporphyrins, 548, 549
490–492, 494, 496, 503, 505–508, 556, 562, 563, 575, 590, Urothione, 197, 198
625, 632, 644, 652, 701, 710, 717, 728, 757, 824, 828 Ursodeoxycholic acid, 552, 553, 565, 571, 574, 575
Transcobalamin, 207–210, 829 Ursodeoxycholic acida, 574
Transferrin, 68, 296, 483, 485, 506, 507, 511, 545, 633, 635–638
Transmembrane CLN3 protein, 400, 402
Transporter-1, 85–87, 228, 265, 266, 268, 274, 293–294, 711 V
Tranylcypromide, 526 Vacuolated lymphocytes, 281, 408, 409, 418, 440, 442, 747
Trientine, 631, 632 Valine, 92, 93, 103–139, 305, 308, 328, 726, 728, 750, 751, 756, 758,
Triglycerides, 20, 280, 282, 283, 294, 298, 299, 405, 415, 671, 762, 782
676–682, 686, 703, 714, 747 Valproate, 64, 78, 79, 81, 155, 294, 359, 431, 709, 752, 754, 756, 766,
Triglycerides, pseudo, 696 770, 771
Trihexyphenidyl, 155, 526, 527 Valproic acid, 98
(25R)-3α,7α,12α-Trihydroxy-5β-cholestan-26-oic acid (THCA), 562 Vanillactic acid, 518, 519, 522, 523, 525
Trimethoprim-sulfamethoxazoles, 18, 19 Vanillinlactic acid, 185
Trimethylamine, 695, 699, 700, 799 Vanillylmandelic acid (VMA), 518, 520, 523, 525
3β,7α,12α-triOH-5-cholenoic acida, 566 Very long-chain acyl-CoA dehydrogenase (VLCAD), 249–251, 253,
7α,12α-triOH-3-oxo-4-cholenoic acidb, 566 258–263, 704, 726, 728, 733, 734, 745, 747, 764, 777, 779,
Tripeptidylpeptidase-1, 400–402, 424 783, 821
Trisialotransferrins, 509 Very-long-chain fatty acids (VLCFA), 378, 384–389, 393–395, 629,
Tryptophan hydroxylase, 3 729
Tyrosine, 3, 10, 11, 17–19, 23–31, 723, 725, 726, 750, 751, 755, 758, 763 Vesicular H(+)-ATPase subunit a2, 488
aminotransferase, 23–26, 728, 763 Vesicular monoamine transporter 2, 517
hydroxylase, 5, 515, 516, 518, 828 Vesicular transport, 516, 517, 521, 526
Tyrosine-3-hydroxylase, 3, 517 Vitamin
A, 386, 394, 560, 563, 573, 678
B6, 33, 175, 176, 179–189, 299, 471, 476, 526, 537, 538, 629,
U 710, 753
Ubiquinone, 162, 233, 234, 243, 251, 342, 359, 822 B12, 20, 176, 205–217, 710, 716, 723, 753, 768, 770, 771
Ubiquinone-50, 590 binding protein, 207
UDP-Gal epimerase, 277 C, 44, 297, 639, 661, 662, 667, 668
UDP-GlcNAc epimerase/kinase, 487, 504, 826 D, 98, 205, 298, 299, 386, 396, 553, 563, 572, 574, 672
Uncoupling protein 2, 325, 327, 330, 343 E, 162, 386, 394, 396, 560–563, 573, 661, 662, 667,
Uracil, 55, 651, 655, 769 668, 678
Urated, 654 K, 295, 296, 387, 394, 396, 571, 573, 829
Urates, 743 VLCAD. See Very long-chain acyl-CoA dehydrogenase (VLCAD)
Urea, 47–53, 59–61, 96, 98, 468, 469, 537, 646, 680, 709, 715, 724, VLCFA. See Very-long-chain fatty acids (VLCFA)
732, 745, 746, 749, 750, 752, 756, 804, 811, 818 VMA. See Vanillylmandelic acid (VMA)
Uric acid, 39, 109–111, 116, 119, 120, 191, 193, 197, 198, 200, 201,
275, 277, 278, 280, 282–284, 289, 298–300, 331, 473, 476,
479, 642, 647–650, 656, 657, 696, 712–714, 716, 717, X
744–746 Xanthine, 191, 193, 194, 197–201, 648, 649, 654, 657, 659
Uridine, 50, 655, 658 Xanthine dehydrogenase, 643, 648
diphosphate galactose-4-epimerase, 266, 268, 277, 820 Xanthurenic acid, 695, 701, 744
monophosphate, 50 Xylitol, 658
monophosphate synthase, 643 Xylose, 744
Uridine-5'-monophosphate hydrolase, 643, 824 Xylulose, 578, 581
Uridyltransferase, 266, 268, 276
Urocanase, 693, 694, 698, 819
Urocanic acid, 694, 698 Z
Urocanoylglycine, 694 Zinc, 98, 446, 548, 550, 553, 623–632, 830
Uroporphyrinogen acetate, 631, 632
decarboxylase, 543, 549 protoporphyrin, 548–550
III synthase, 542 sulphate, 631, 632
SignSymptoms Index
A Anterior eye chamber anomalies, 485, 501

Abasia, 148 Anteriorly placed anus, 501
Abdominal, 294, 324, 427, 541, 542, 544, 673 Anteverted nares, 591–593
Abdominal distension, 180, 334, 415, 499 Antley-Bixler syndrome, 587, 588, 594, 596, 600, 611, 824
Abdominal pain/paina, 277, 288, 289, 333, 351, 407, 412, 545–547, Anxiety, 65, 75, 417, 732
550, 627, 637, 651, 681, 687, 710 Aortic insufficiency, 414, 504, 506
Abducted thumbs, 498 Apathy, 209, 210, 625, 629
Abnormal behavior, 34, 67, 193 Apnea, 72, 91, 96, 108, 112, 172, 188, 193, 197, 198, 200, 221, 304,
Abnormal jitter, 493 401, 454, 457, 461, 497, 778
Absent head control, 54, 619 Arachnodactyly, 39, 275, 491, 499, 594
Absent puberty, 496 Arched palate, 497, 595
Absent pubic and axillary hair, 603, 611 Arcus cornealis, 676–678, 680, 681
Absent uterus, 611 Areflexia, 350, 651
Absent/weak tendon reflexes, 38 Arrhythmia, 109, 111, 118, 247–249, 252, 254, 255, 261, 263, 279,
Acanthocytosis, 678 386, 388, 417, 710, 716, 778
Accelerated growth, 65, 74, 608, 609 Arterial ruptures, 628
Achondrogenesis, 508 Arthralgia, 585, 637
Acrocyanosis, 157, 158 Arthritis, 25, 27, 30, 413, 464, 790
Adducted thumbs, 65, 73 Ascites, 350, 490, 627
Adiposity, 280, 282, 283 edema, 408
Adrenal calcification, 415 oedema, 560
Adrenal hyperplasia, 601, 602, 605, 607–610, 724, 734, 746 Astasia, 148
Adrenal insufficiency, 385, 387, 394, 395, 562, 580, 594, 607–609, 711 Asterixis, 51, 53
Agenesis, 64, 67, 72, 185, 307, 341, 498, 590, 592, 593 Ataxia, 51–54, 64, 65, 72, 73, 75, 85–87, 89, 90, 93, 94, 96, 98, 108–110,
corpus callosum, 105, 117, 185, 304, 307, 308, 349, 492, 500, 501, 113, 116–119, 144, 148, 149, 171, 173, 210, 211, 221, 222,
586, 587, 590, 592, 593 234, 235, 238, 274, 304, 339, 340, 344–348, 350–352, 354,
Akinesia, 422 383, 386–388, 408–411, 416–423, 441, 442, 491, 496, 521,
Alopecia, 107, 109, 221, 222, 591, 592, 629 561, 581, 585, 627, 647, 664, 678, 710, 822, 823
Alopecia areata, 497 cerebellar, 65, 144, 234, 235, 387, 388, 503, 561, 578, 590, 799
Altered consciousness, 109, 317, 732 Atherosclerosis, severe, 415
Ambiguous genitalia, 587, 591, 601, 602, 604, 607, 608, 610, 611 Athetosis, 110, 227, 317, 519
Amblyopia, 508 Atlantoaxial instability, 453, 454, 456, 457, 461
Anaemia, 181, 206, 207, 214, 234, 266, 292, 298, 577, 583 Atrophic scars, 499
haemolytic, 211, 212, 274, 276, 284, 285, 577, 580, 627 Atrophy, 6, 7, 39, 65, 67, 72–75, 104, 108, 110–115, 118–121, 144,
megaloblastic, 206, 209–212, 650, 829 148, 168, 171, 173, 180, 198, 201, 210–212, 222, 228, 234,
Anemia, 65, 68, 104, 168, 169, 176, 177, 228, 230, 231, 428, 544, 237, 279, 304, 313, 317, 320, 339, 340, 345, 347, 348, 350,
550, 553, 585, 634, 639, 661, 699 353, 354, 386, 388, 400, 405, 408–410, 412, 414, 416–423,
dyserythropoietic, 509 441, 468, 469, 491, 493–496, 498, 502, 503, 505–507, 530,
hemolytic, 639, 661, 662, 664–666 532, 561, 581, 624, 625, 628–631, 638, 647, 651, 653, 724,
hypochromic, 353, 545, 546, 634, 638 752, 822, 826
macrocytic, 113, 210, 231, 345 gyrate of choroid and retina, 532
megaloblastic, 65, 73, 168, 169, 171, 172, 206, 209–212, 214, Attention deficit disorder, 38, 88
227–230, 650, 699, 711, 829 Attention deficit hyperactivity, 64
microcytic, 634 Attention disorder, 75
hypochromic, 353, 545, 546 Autism, 5, 169, 317, 531, 532, 647, 651
nonhemolytic, 711 Autistic spectrum disorder, 115, 121, 171
sideroblastic, 341, 345, 353, 542, 544, 545, 711, 823, 825 Autonomic symptoms, 14
Angiokeratoma, 407, 408, 412, 430, 441, 442 Axial hypotonia, 6, 8, 39, 65, 144, 148, 160, 316, 318, 350, 491, 494,
Anomalous pulmonary venous return, 593 495, 504, 521, 590
Anorectal anomalies, 502 Axonal motor neuropathy, 67
Anorexia, 209, 276, 277, 333, 490, 502, 629, 651 Axonal sensory motor, 75, 507
Anosmia, 386, 388, 395, 396 Axonal sensory motor polyneuropathy, 65, 73, 108
857
858 SignSymptoms Index
B cardiomegaly, 368
Basal ganglia dilated, 104, 111, 114, 117, 118, 255, 281, 367, 503, 505
abnormalities, 112, 113, 115, 116, 118, 119, 320, 531, 631, 810, 813 hypertrophic, 111, 278, 279, 281, 283, 324, 348–350, 352, 353,
calcifications, 7 412, 440, 442, 492, 506
lesions, 111, 113–116, 119–121, 211, 230, 627, 711, 804 Carotid/femoral bruits, 676–678, 681, 682
Beaked nose, 497 Carpal tunnel syndrome, 453, 454, 457, 461
Behavior Cataract, 55, 65, 67, 73, 75, 112, 267, 275, 276, 295, 298, 347, 384,
aggressive, 38, 75, 171, 417, 455, 456, 520, 590 385, 388, 393–396, 493, 494, 500, 501, 505, 532, 556, 561,
difficulties, 75, 112, 385 562, 585–588, 590–593, 595, 625, 627, 629
psychotic, 8, 89 posterior subcapsular, 532
Behavioral abnormalities, 34, 422, 588, 593 retinitis pigmentosa glaucoma, 384
Behavioral disorder, 256, 416–418, 420–422, 453, 454, 520 Cerebellar, 34, 65, 75, 233, 234, 354, 407, 507, 561, 662, 804, 806
Big open mouth, 496 abnormalities, 110, 111, 446, 500, 501, 505
Bilateral sensory hearing loss, 112 ataxia, 65, 144, 235, 387, 388, 410, 578, 799, 823
Bilateral striatal necrosis, 227–230, 345 atrophy, 38, 39, 65, 112, 168, 171, 234, 412, 416–423, 506, 507,
Bile duct proliferation, 561, 563 629, 806
Bitemporal narrowing, 593 hypoplasia, 39, 54, 75, 91, 113, 490, 492, 493, 590, 647
Bladder diverticula, 624, 628 and pontine hypoplasia, 34
Bleeding, 31, 161, 280, 427, 428, 496, 504, 559, 561, 562, 573, 578, vermis agenesis, 65, 74, 498
638, 678 Cerebral
tendency, 280, 496, 678 atrophy, 65, 75, 108, 110, 111, 113, 114, 119, 120, 198, 210–212,
Blindness, 148, 345, 346, 387, 401, 411, 507, 530, 532, 647 320, 353, 354, 493, 495, 496, 503, 629, 631, 651, 653, 806
cortical, 197, 198, 284 and cerebellar atrophy, 39, 112, 168, 171, 412, 416–423, 506, 507,
Blisters, 27, 544, 546, 547, 550 629
hepatosplenomegaly, 27 cortical malformations, 500, 501, 508
Bone dysplasia, 462 infarction, 114, 367, 412
Bone pain, 406, 411, 412, 468, 469, 499 neocortical dysplasia, 384, 593
Brachydactyly, 498, 501 palsy, 4, 8, 14, 317, 649
Bradykinesia, 7, 518, 521 white matter involvement, 65, 144, 384, 387, 399, 578
Brain atrophy, 115, 201, 400, 405, 495, 506 Cerebrocostomandibular-like syndrome, 506
Brain dysgenesis, 304 Cervical
Brain edema, 48, 108, 116, 118, 119, 130, 143, 294, 483, 715, 808 compressive myelopathy, 490, 591
cytotoxic, 108 myelopathy, 453, 454, 456, 457, 461
Brain oedemaa, 276 stenosis, 385
Bridging fibrosis, 560, 561, 563 Cherry red spot, 408–410, 413, 442
Broad alveolar ridges, 591, 593 “Cherubic” face, 111
Bulbar dysfunction, 222, 345, 493, 521 Choanal atresia, 594
Buphthalmos, 500, 501 Cholestasis, 35, 53, 54, 267, 333, 340, 346, 393, 553, 556, 557,
Burst suppression, 64, 72, 91, 181, 497 559–563, 565, 569, 571, 574, 575, 634, 635, 638, 822, 825
Cholestatic jaundice, 416, 557, 564, 578, 711
Chondrodysplasia punctata, 375, 377, 385, 389, 391, 393–395, 586,
C 588, 591, 592, 594, 598, 809, 824, 828
Cabbage-like odor, 697 Chondro-osseous, 587, 594
Cachexia, 351 Chondrosarcoma, 498
Calcifications, 7, 25, 115, 388, 407, 415, 499, 586–588, 591, 592, Chorea, 64, 65, 72, 113, 148, 519, 521
594, 806 Choreoathetosis, 6, 115, 118, 119, 149, 316, 318, 409, 502, 649
Calcified aortic valve, 677 Chorioretinal degeneration, 529, 530, 532, 537
Calcinosis cutis, 468, 469 Chronic, 24, 26, 49, 60, 65, 67, 68, 73, 89, 97, 104, 108, 118, 130,
Callosum, 34, 64, 67, 72, 76, 105, 117, 180, 185, 198, 304, 305, 307, 134, 135, 143, 157, 158, 160–162, 175, 180, 210, 211, 228,
308, 341, 349, 414, 492, 500, 501, 586, 587, 590, 592, 593, 242, 249, 313, 317, 321, 349, 354, 407, 410, 412, 427, 429,
805, 810 431, 442, 465, 466, 468, 469, 471, 476, 479, 496, 517, 542,
Camptodactyly, 67, 493 543, 550, 553, 580, 625, 633, 634, 639, 649, 651, 659, 672,
Cardiac 700, 738, 790, 813, 814, 822
anomalies, 35, 40, 212, 386, 501, 580, 590, 592, 593 Chronic aphthous ulceration, 111
malformations, 40, 211, 501, 580, 590, 592, 593 Cleft palate, 279, 502, 587, 591–593
Cardiac arrest, sudden, 278 Clinodactyly, 498
Cardiac arrhythmias, 109, 111, 252, 254, 255, 261, 388, 417, 716 Clitoral hypertrophy, 580, 594, 608
Cardiac failure, 281, 331, 430, 710 Clonic seizures, 67
Cardiac preexcitation syndrome, 281, 283 Closure of fontanels, 493, 508
Cardiomegaly, 349, 368 Clots, stroke, 111
Cardiomyopathy, 64, 104, 105, 109, 111, 113, 115, 116, 118–121, Club foot, 384, 491, 492, 499, 591, 593
130, 132, 138, 145, 149, 210–212, 230, 234, 237, 238, 247, Clumsiness, 416, 627
248, 252–255, 257, 262, 263, 266, 279, 282, 291, 300, CNS ataxia, 52–54, 73, 89, 90, 110, 113, 116, 118, 119, 149, 171, 173,
345–349, 354, 358, 388, 396, 408, 414, 417, 446, 453, 454, 211, 222, 238, 274, 344–348, 350–352, 354, 383, 408, 410,
457, 461, 468, 469, 490, 504, 509, 578, 580, 633, 634, 637, 411, 416–423, 441, 442, 491, 496, 521, 561, 581, 590, 627,
778, 822 647, 664, 678
SignSymptoms Index 859
Coagulopathy, 24, 31, 38, 181, 353, 387, 389, 493, 573, 627, 710, 711 Degenerative hip dysplasia, 453, 454, 456–458, 461
Coarse facial features, 317, 408, 409, 414, 415, 440–442, 453–458, 463 Dehydration, 105, 130, 131, 133–135, 210, 211, 221, 274, 293, 304,
Cobblestone lissencephaly, 500, 501 370, 476, 607–609, 659, 710, 745
Cognitive decline, 105, 400, 401, 418, 421, 422, 562 Delayed puberty, 563
Cognitive dysfunction, 37, 113, 238, 416, 417, 422, 478, 521, 593 Dementia, 110, 209, 210, 212, 345, 348, 385, 400, 401, 409, 410, 556,
Colitis, 158, 663 561, 571, 574, 638, 710
Collodion skin, 411 Demyelination, 34, 37, 168, 173, 386, 387, 394, 431, 490, 493, 495,
Coloboma, 493, 505, 650, 651 561, 814
Color vision deficit, 347 Dental caries, 277
Coma, 48, 51–54, 64, 130, 221, 228, 324, 332, 545–547, 710, 752, 778 Dental defects, 479, 499
hyperammonemic, 89 Depression, 64, 347, 417, 625, 629, 638
lethargy, 108, 109, 114, 116, 118, 119, 121, 252–254, 257, 366, 368 Dermatitis, 595, 623, 625, 629, 631, 632
Conduction deficits, 248, 347, 348, 358 psoriasiform, 595
Confusion, episodic, 51–54 Developmental delay, 14, 24, 30, 34, 35, 37–40, 51–55, 64,
Congenital 65, 67, 68, 94, 105, 109, 117, 118, 120, 121, 127,
anomalies, 249, 257, 261, 386 144, 145, 148, 149, 168, 185, 210–212, 221, 222,
encephalopathy, 421, 541 234, 256, 304, 307–309, 314, 320, 344, 348, 351,
heart defects, 210, 228, 385, 578, 719, 734 362, 383, 387, 394, 395, 401, 406, 442, 493, 497,
Connective tissue abnormalities, 628 516, 519, 521, 530–532, 534, 561, 562, 585–588,
Consciousness disturbance, 54, 197, 198 590, 592, 595, 647, 648, 650, 697, 699, 701, 702
Constipation, 476, 532, 545–547, 550 Developmental regression, 168, 169, 171, 172, 209, 221, 339, 416,
Contractures, 67, 75, 112, 300, 385, 388, 394, 395, 414, 415, 453, 454, 417, 419–421, 423, 502
457, 458, 461, 490, 494, 507, 587 Development delay, 15, 611
Convulsions, 34, 55, 65, 114, 191, 193, 304, 325, 326, 628, 710 Deviations of fingers, 498
Corneal clouding, 75, 284, 408, 414, 441, 442, 453, 456–458, 461, Diabetes, 4, 19, 230, 331, 340, 346, 428, 481,
593, 664, 680 673, 711, 735, 754, 822
Corneal cystine crystals, 475, 477, 479 Diabetes mellitus, 227, 228, 230, 231, 266, 323, 333, 340, 346, 367,
Corneal erosion, 222 476, 478, 509, 633, 691, 744
Cornea verticillata, 412 type 2, 327, 355
Coronal clefts, 385 Diabetes MODY3-like, 7
Coronary artery disease (CAD), 453, 461, 646, 679, 680, 682, 687 Diaphragm dysfunction, 479
Coronary atherosclerosis, 677 Diarrhea, 89, 134, 135, 168, 171, 351, 453–456, 461, 473, 490, 491,
Corpus, 604 504, 511, 517, 585, 590, 663, 696
Corpus callosum, 34, 64, 67, 72, 76, 105, 117, 180, 185, 198, 304, chronic, 158, 160, 496, 625
305, 307, 308, 341, 349, 414, 492, 500, 501, 586, 587, 590, Diarrhoea, 158, 162, 274, 280, 288, 289,
592, 593, 805, 810 298, 333, 334, 366, 383, 384, 556,
Cortical atrophy, 173, 414, 494, 496, 502, 532 562, 563, 625, 628, 629, 650, 651, 659
Cortical-subcortical atrophy, 6, 7, 65, 73, 112, 304, 414 chronic, 160, 210
Course, episodic, 317 Digestive signs, 711
Coxa valga, 67 Dimethylsulphide, 37
Cramps, 281, 283, 290, 354, 538, 648, 711 Diminished visual activity, 384
Craniosynostosis, 587, 594, 604 Dimorphism, 545
Cryptorchidism, 112, 493, 501, 580, 591, 592, 607, 608, 610, 611 Dislocation, 34, 67, 75, 90, 197, 198, 414, 497, 752
Cushing stigmata, 612 Diurnal fluctuation of symptoms, 7, 8
Cutaneous nodules and swelling, 458 Doll-like face(ies), 280, 282, 283, 409
Cutis laxa, 67, 68, 75, 275, 485, 508, 580, 625, 628, 819 Drooling, 6, 394, 461, 518, 519, 521, 627
Cystic hygroma, 594 Drowsiness, 94
Drug reactions, 497
Dry skin, 395, 505, 508
D Ductus arteriosus, 580, 593
Dandy-Walker malformation, 314, 586, 592 Dwarfism, 492, 499, 506, 507, 585, 597
Deafness Dysarthria, 8, 90, 115, 148, 149, 340, 348, 411, 416, 417, 422, 519,
conductive, 498, 650 521, 522, 627
sensorineural, 104, 112, 228, 234, 235, 237, 238, 313, 314, 316, Dysdiadochokinesis, 37, 521
318, 346, 347, 351, 352, 355, 383, 385, 387, 441, 461, 498, Dyskinesia, 7, 17, 169, 173, 521, 527
563, 591, 638, 647 Dysmetria, 37
Death, 6, 7, 34, 67, 90, 96, 144, 168, 181, 192, 193, 195, 210, 227, Dysmorphia, 387
228, 231, 238, 247, 248, 252, 276–279, 281, 283, 284, 300, Dysmorphic features, 67, 76, 105, 111, 117, 121,
304, 314, 317, 321, 340, 344–346, 348–353, 357, 368, 386, 197, 198, 212, 256, 284, 304, 307, 317,
388, 400, 401, 411, 428, 492, 495, 505, 508, 580, 594, 618, 353, 384–386, 490, 500, 501, 503, 578,
619, 634, 638, 717, 719, 720, 732, 735, 738, 775, 778, 822 580, 587, 590, 593, 619, 647, 650, 652
Decerebrate posture, 148 Dysmorphism, 68, 94, 112, 117, 249, 304, 341, 344, 349, 399, 453,
Decreased body height, 118, 120, 496, 501, 504, 506 491–497, 499, 501, 502, 504–06, 508, 587, 593
Decreased cortical sulci, 115 Dysmyelination, 386, 653
Deep tendon reflexes, 209, 210, 413, 678 Dysostosis multiplex, 399, 408, 409, 415, 440–442,
Deficient ossification, 508 453–458, 490
Dysphagia, 7, 156, 288 Facial weakness, 493

Dysphasia, 561 Failure to thrive, 23–25, 27, 28, 38, 51, 53, 55, 67, 104, 105, 107–110,
Dysplastic ears, 496, 594 112, 113, 116–119, 121, 127, 160, 168, 171, 181, 209–212,
Dystonia, 3, 4, 7, 10, 20, 37, 65, 67, 75, 108, 112, 115, 117–121, 144, 221, 255, 256, 267, 277–279, 304, 307–309 316–318, 345,
148, 149, 155, 160, 221, 228, 230, 231, 234, 238, 274, 308, 348, 350–352, 368, 384, 386, 387, 390, 413, 415, 468, 469,
314, 316–318, 320, 340, 344–346, 352, 408–410, 416, 417, 475, 476, 479, 490, 492, 495, 496, 502, 505, 506, 508, 530,
419, 420, 446, 516–519, 521, 627, 652, 710, 778, 822 531, 563, 564, 585, 595, 609, 617–619, 625, 629, 635,
Dystonic posturing, 304 650–653, 696, 752, 778
Dystrophic nails, 591, 592 acidosis, 23, 27
Dystrophic thumbs, 316 Fanconi syndrome, 28, 292, 346, 475–477, 479, 480, 656, 711,
743–745, 750, 757
and renal, 711
E Fatigue, 281, 637, 700
Early death, 34, 181, 276–279, 281, 283, 284, 340, 346, 411, 492, 495, Fat pads, 484, 495
505, 508, 638, 822 Features, 14, 39, 67, 68, 76, 104, 105, 111, 117, 121, 161, 162, 168,
Ectopia lentis, 39 169, 176, 197, 198, 201, 210, 212, 221, 222, 228, 256, 266,
Ectopic calcifications, 499 274, 276–279, 284, 304, 307 317, 320, 328, 338, 353, 382,
Edema, 34, 108, 130, 133, 198, 228, 350, 408, 409, 492, 493, 508, 384–388, 400, 406–409, 414, 415, 438, 440–442, 446, 449,
538, 548, 550, 578, 585, 808, 811 453–458, 461, 462, 479, 490, 500, 501, 503, 517, 519, 521,
generalized, 350, 493 529, 530, 539, 569, 578, 580, 585, 587, 590, 593, 619, 647,
Elongation, 275 650, 652, 657, 659, 673, 710, 737, 738, 752, 805, 811, 817
Emotion ability, 411 Febrile episodes, 711
Enamel hypoplasia, 508 Feeding
Encephalocele, 500, 501 difficulties, 6, 39 72, 74, 75, 107–109, 111, 112, 117–119, 121,
Encephalomyopathy, 129, 234, 314, 315, 320, 341, 822 148, 172, 197, 198, 212, 238, 305, 317, 318, 352, 358, 411,
Encephalopathic crisis, acute, 51–54, 108, 109, 148, 211, 230, 507 413, 492–496 503, 506, 518, 521, 591, 628, 651, 653
Encephalopathy habits, 277
acute, 109, 117–120, 230, 257 protein aversion, 51–53
epileptic, 54, 87, 96, 168, 181, 341, 353, 717 Female external genitalia, 602, 603, 607
episodic, 14, 239 Femoral bowing, 594
progressive, 91, 619, 628, 653 Fertility, 608, 609
Endometrium, 604, 612 Fetal hydrops, 317, 409, 458, 490, 580, 587, 594, 711
Endothelial dysfunction, 642 Fever, 97, 180, 189, 227, 292, 358, 407, 411, 429, 473, 506, 770
Epicanthal folds, 386, 591 Fibrosis, 34, 68, 188, 282, 283, 407, 429, 466, 470, 478, 491, 560,
Epilepsy 561, 563, 564, 577, 578, 582, 634, 637, 638, 653, 719, 724,
generalized, 228, 345 725, 728
intractable, 54, 169, 230, 351, 496, 502, 511 Finger anomalies, 506, 507
Epileptic encephalopathy, 96, 168, 181, 717 Fingers, overlapping, 497, 499
Epileptic seizures, 177, 405, 440, 441, 446, 491, 518, 552 Fish odor, 693, 695, 699, 700, 711, 797, 799
Epiphyseal and periarticular calcific stippling, 384, 385, 387 Fits, 110, 573, 650
Epstein-Barr virus infection, 498 Flared nostrils, 304
Equinovarus, 7, 499 Flexion contractures of fingers, 507
Erythema, 588, 592, 595 Foam cells, 409, 411–413, 416, 672
necrotizing, 54 Focal epilepsy, 230
Erythematous, papular eruptions, 68, 76 Focal segmental glomerulosclerosis, 346
Erythroderma, 503, 505, 561, 586–588, 629, 824 Foetal hydrops, 34, 38, 41
Erythrodontia, 544 Follicular atrophoderma, 591
Everted lower lip, 496 Fontanel enlarged, 384
Excess skin, 493 Foot deformity, 497, 620
Exercise intolerance, 111, 234, 255, 256, 347, 353, 354, 520, 648, 822 Frequent infections, 168, 171, 629
Exertion intolerance, 267, 278, 279, 282–286 Frontal bossing, 35, 40, 304, 499, 595
Exophthalmia, 499–501 Frontotemporal atrophy, 115, 121
Exostosis, 628 Functional joint impairment, 498
External genitalia, 602, 603, 607, 615
Extrapyramidal movement disorder, 65, 113, 148, 154, 155, 210, 211,
417, 418, 422 G
Extrapyramidal signs, 112, 118, 119, 212, 638 Gait disturbance, 171, 172, 408, 411, 415–417, 422, 503
Eye movements, 181 Gallstones, 284, 548, 556, 561, 562, 564, 565
abnormal, 8, 172, 411, 412, 519, 651, 653 Gastric tube feeding, 431, 492, 497, 502
Gastroesophageal reflux, 317, 496, 501
Gastrointestinal bleeding, 496
F Gastrointestinal dysmotility, 8, 112, 347, 351, 358, 492, 651, 653
Facial dysmorphism, 68, 94, 112, 249, 344, 349, 399, 449, 453, Gastrointestinal side effect, 18, 19, 333, 428
491–495, 497, 499, 501, 502, 504–506, 508, 587, 593 Gastroparesis, 651
Facial dysmorphism, mild, 344 Gelastic cataplexy, 416
Facial stigmata, 275, 279 Genital ambiguity, 603, 604, 608, 609, 611, 612
Genital hypoplasia, 492, 496, 594 Holoprosencephaly, 590

Genitalia, 587, 591, 601–604, 607, 608, 610, 611, 615 Hydrocephalus, 64, 72, 172, 180, 415, 453, 454, 461, 500, 501, 592,
Genu valgum, 39, 453, 456, 457, 461, 479 703, 711, 806
Giant cell hepatitis, 559–562, 564 Hydronephrosis, 501, 594
Gingival hypertrophy, 408, 414 Hydrops, 34, 38, 41, 279, 317, 409, 453, 458, 490, 499, 578, 580, 587,
Glaucoma, 384, 453, 457, 461, 500, 501 594, 711
Glomerulonephritis, 90, 97, 790 Hydroureter, 501
Glomerulopathy, 24, 234 Hyperactivity, 38, 64, 65, 72, 75, 90, 388, 455, 456, 650, 651
Glomerulosclerosis, 280, 346 Hyperekplexia, 85–87, 90, 93, 95, 96, 98, 198
Glossitis, 222 Hyperelastic/loose skin, 67, 499
Glucose intolerance, 231, 288, 611 Hyperesthesia, 545–547
Growth hormone deficiency, 367, 503, 505 Hyperexcitability, 65, 73, 401
Growth retardation, 55, 67, 73, 97, 111, 234, 254, 298, 314, 323, 340, Hyperfiltration, 275, 280
346, 349, 385, 401, 409, 468, 469, 501, 508, 518, 578, 580, Hypergonadotropic, 118, 276, 295, 490
591, 593, 623, 638, 822 Hyperinsulinemic, 711, 818
Gum hypertrophy, 497 Hyperinsulinism, 48, 249, 256, 263, 323–334, 580, 710, 712, 714,
Gynecomastia at puberty, 610 745, 754
Gyration, 64, 806 Hyperkeratosis, 24, 27, 503, 508, 629
palms and soles, 27
Hyperkinesia, 107, 412, 516, 517, 519, 521
H Hyperlaxity joints, 78
Haematuria, 649 Hyperlipidaemia, 298, 556, 561, 574, 825
Haemolysis, 300, 624, 627 Hypernasal speech, 521
Haemolytic anaemia, 266, 577, 580, 627 Hyperostosis, 499
Haemolytic uraemic syndrome, 212 Hyperphalangism, 498
Hair abnormality, 505 Hyperpigmentation, 327, 633, 637
Hallucinations, 417, 550 Hyperreflexia, 91, 198, 493, 495
Handwriting, 627 Hypersplenism, 68, 427
Headache, 37, 144, 148, 358, 517, 562, 607, 609 Hypertelorism, 68, 317
migraine like, 346 Hypertension, 64, 90, 298, 383, 406, 427, 428, 545–547, 550, 551,
Head circumference, 5, 65, 209, 228, 464 564, 580, 583, 601, 603, 604, 607, 609, 612, 642, 657, 710
Head control, 54, 519, 521, 619 arterial, 275
Head-retraction reflexa, 90 Hyperthermia, 521
Hearing impairment, 35, 492, 495, 502 Hyperthyroidism, 562
Hearing loss, 112, 173, 222 Hypertonia, 5–8, 39, 65, 74, 108, 109, 116, 118, 119, 171, 210, 211,
sensorineural, 40, 115, 118, 228 221, 345, 491, 494, 495, 519, 521, 651, 664, 700
Heart block, 283 extremities, 6, 491, 495, 521
Heart failure, 111, 171, 228, 388 Hypertrichosis, 331, 334, 544, 546, 550
HELLP syndrome, 249, 254, 711, 740, 778 Hypertrophic, 112, 248, 278, 279, 281, 283, 291, 324, 348–350, 352,
Hemihypertrophy, 324 353, 407, 412, 440, 442, 492, 506
Hemiplegic migraine, 90 Hyperventilation, 267
Hemolytic anemia, 639, 661, 662, 664–666 Hypodense white matter, 651
Hemolytic uremic syndrome, 169, 172, 711 Hypogenesis, 185, 307, 590, 592, 593
Hemophagocytic, 89, 97 corpus callosum, 185, 307, 590, 592, 593
Hemorrhagic diarrhoea, chronic, 157, 160 Hypoglycemia, 104, 105, 111, 114, 127, 131–133, 136, 138, 247–249,
Hemosiderosis, 346, 634, 635, 638, 639 252–257, 263, 267, 303, 309, 314, 319, 321, 367, 492, 517,
Hepatomegaly, 34, 38, 45, 109, 110, 112, 114, 116, 118–120, 248, 526, 711–714, 716, 743, 754, 778
265, 267, 275, 278, 280–283, 324, 366–368, 383, 387, 414, Hypogonadism, 65, 73, 118, 276, 295, 347, 478, 481, 490, 580,
478, 493, 495–497, 506, 507, 710, 752, 778 633, 637
Hepatomegalya, 276, 277 Hypogonadotropic hypogonadism, 580
Hepatopathy, 27, 248, 279, 368, 635, 637, 710 Hypo/hypertonia, 8, 108, 109, 116, 118, 119, 211
myoclonic epilepsy, 339 Hypokinesia, 7, 8, 304, 307, 345, 417, 423, 497, 519, 521
Hepatosplenomegaly, 89, 317, 351, 353, 399, 405, 406, 408–416, phenotype, 494
427, 440–442, 446, 449, 453, 454, 457, 458, 461, 503, Hypolobated, 594
506, 544, 560, 561, 577, 578, 580, 585, 587, 590, 627, Hypomimia, 521
633, 653, 680, 696 Hypomyelination, 34, 63, 78, 85–87, 91, 93, 95, 171, 341, 353, 414,
Hernia(s), 55, 67, 75, 90, 409, 414, 440–442, 453, 454, 457, 458, 415, 629, 799, 810
461, 628 Hypopigmentation, 5, 595
diaphragmatic, 275 Hypopigmented hair, 6, 7
Hiccupping, 64 Hypoplasia, 34, 38, 54, 64, 72, 74, 77, 91, 113, 144, 148, 173, 456,
Hiccups, 72 457, 490, 492–494, 496, 498, 499, 508, 580, 586, 587,
High-pitch crying, 191, 198 590–594, 602, 647, 703, 805, 823
Hip dislocation, 67, 75, 90, 414 of pons, 38
Hip dysplasia, 453, 454, 456–458, 461 Hypoplasia/cortical atrophy, 173, 494
Hirschsprung, 591 Hypoplastic corpus callosum, 34
Hoarseness, 413 Hypoplastic labia majora, 497, 594
Hypoplastic long bones, 592 J

Hypoplastic nails, 492, 502, 592 Jaundice, 31, 54, 284, 383, 386, 387, 389, 413, 509, 556, 557,
Hyporeflexia, 502, 506 559–564, 574, 575, 580, 627, 653, 664, 667, 711
Hypospadias, 112, 492, 591–594 cholestatic, 416, 557, 564, 578
Hypotension, 197, 198, 334, 481, 517, 526 Joint contractures, 75, 385, 414, 415, 453, 454, 457, 458, 461, 490, 587
orthostatic, 520, 628 Joint laxity, 55, 67, 75, 275, 453, 456, 457, 499, 508, 624
Hypothalamic dysfunction, 562
Hypothermia, 238, 304, 517, 520, 628, 653
during crisis, 108, 109, 120 K
Hypothyroidism, 475, 476, 478, 481, 493, 562, 580, 673, 719, 720, Kearns-Sayre syndrome (KSS), 340, 345, 347, 348, 358, 710, 822
723–725, 728, 731 Kinky hair, 625, 628
Hypotonia, 7, 14, 34, 38, 39, 41, 64, 65, 67, 72, 74, 75, 91, 94, 104, KSS. See Kearns-Sayre syndrome (KSS)
107, 145, 149, 161, 185, 209–212, 253, 256, 304, 305, Kyphoscoliosis, 461, 498
307–309, 313, 314, 317, 320, 344, 345, 348–354, 383, Kyphosis, 39, 411, 453, 454, 456, 457, 461, 497, 505
386–388, 401, 405, 408–410, 412, 413, 442, 490–495, 497,
502, 503, 505–508, 516, 518, 519, 521, 530, 578, 580,
585–587, 590, 592, 593, 595, 619, 647, 651–653, 778, 819, L
664702 Lack of pubertal, 607
axial, 6, 8, 144, 148, 160, 316, 318, 350, 491, 494, 495, 504, 521, Lacrimation, 27
590 Language difficulties, 8, 37, 75, 416, 421–423
mild, 7 Large-and medium-size arteries, 275
muscular, 108, 109, 111, 112, 114–118, 120, 121, 132, 143, 144, Large ears, 67
148, 197, 198, 221, 252–255, 257, 274, 278, 279, 281, 383, Laryngeal and tracheal calcification, 587, 594
387, 410, 414, 415, 505, 517, 590, 617, 618 Laxity, 63, 67, 75, 624
Hypotoniaa, 518 Leber amaurosis-like, 648
Hypovolaemic shocka, 74 Leber hereditary optic neuropathy, 340, 348, 822
Hypoxia, 287, 413, 756, 812 Left ventricular non-compaction, 111
Hypsarrhythmia, 64, 65, 72, 73, 91 Leigh-like syndrome, 160, 314
Leigh syndrome, 112, 230, 233–235, 237, 304, 307, 308, 318, 340,
344, 348–350, 354, 806, 822, 823
I Lennox-Gastaut syndrome, 65, 73
Ichthyosiform erythroderma, 505, 586–588, 591, 824 Lens, 292
Ichthyosiform skin lesions, 586, 592 changes, 281
Ichthyosis, 385, 386, 388, 394–396, 415, 503, 505, 586, 592, 598, 618, dislocation, 34, 197, 198, 752
625, 629, 825 Lethargy, 48, 64, 72, 74, 81, 210, 304, 324, 518, 696, 710
Immunodeficiency, 169, 441, 498, 650 coma, 108, 109, 114, 116, 118, 119, 121, 252–254, 257, 366, 368,
Immunodeficiency, severe combined (SCID), 169, 172, 643, 648, 752, 778
656, 724 Leucoencephalopathy, 577, 578, 581, 651
Impaired ejaculation, 520 Leucopenia, 97, 580
Incomplete epiphyseal closure, 603, 611 Leukodystrophy, 64, 148, 385, 387, 396, 401, 403, 405, 408, 409, 411,
Infections, 16, 31, 68, 109, 117–120, 132, 133, 136, 221, 229, 230, 424, 426, 431, 786, 809, 827
242, 247–249, 257, 293, 295, 296, 298, 300, 304, 332, 370, progressive, 348
385, 395, 466, 473, 480, 511, 551, 552, 623, 625, 634, 659, Leukoencephalopathy, 110, 341, 351, 354, 809, 823, 824
661, 714, 715, 719, 754 Leukopenia, 109, 334, 345, 627
Infertility, 478, 603, 604, 612 Limb defects, 586, 588, 592, 824
Inflammatory bowel disease, 280, 298, 466 Lipemia retinalis, 681
Inguinal hernias, 67, 461 Livedo reticularis, 234, 468, 469
Insomnia, 519 Liver adenoma, 280
Intellectual disability, 65, 113, 168, 169, 384, 529, 530, 532, 534, 586, carcinoma, 280, 282
593 Liver carcinoma, 26, 280
Interictal nystagmus, 90 Liver cirrhosis, 276, 277, 279, 411, 413, 509, 553, 559, 563, 564, 580,
Intersexuality, 602, 615 633, 637
Interstitial changes, 90, 413 Liver cirrhosisa, 276, 277
Intestinal dysmotility, 591 Liver disease, 23, 24, 28, 29, 34, 35, 45, 387, 544, 553, 561, 563, 571,
Intestinal pseudo obstruction, 185, 351, 625, 629, 651, 711 573–575, 583, 624, 634, 639, 703, 723, 743–745, 814
Intrauterine cardiomyopathy, 254 Liver dysfunction, 24, 38, 40, 53, 54, 110, 112–114, 116–119, 121,
Intrauterine growth retardation, 55, 67, 254, 314, 323, 346, 349, 401, 212, 239, 248, 252–255, 257, 316, 318, 348, 350, 354, 366,
578, 580, 638 455–457, 490, 508, 548, 564, 591, 627, 629
Inverted nipples, 492, 493, 495, 496 Liver failure, 26, 30, 31, 255, 341, 349, 351, 353, 354, 384, 387, 575,
Iridodonesis, 39 580, 583, 631, 634, 710, 715, 756, 814, 822, 823
Irritability, 5, 209, 317, 324, 411, 502, 518, 519, 527, 590, 625, 627, acute, 51–53, 580, 627, 711
629, 710 Reye-like, 256, 326
episodic, 108 Liver failurea, 276, 277
Ischaemic heart disease, 561 Liver fibrosis, 34, 68, 188, 283, 407, 429, 466, 478, 491, 560, 561,
Itching, 386, 550, 559, 563, 564, 631 563, 577, 578, 634, 637, 638, 653, 719, 724, 725
eczematous lesions, 76 biliary cirrhosis, 282, 564
Liver steatosis, 54, 345, 653 315, 326, 385, 386, 388, 401, 416, 421, 423, 440, 491–496,
Long eye lashes, 497 503, 505–508, 586–588, 590, 592, 593, 595, 617–619, 651,
Long philtrum, 304 653, 663, 829
Loose stools, 134, 135, 333, 394, 575 Micrognathia, 68, 491, 591–593
Loss of central vision, 281, 345, 348 Microhaemorrhages, 160, 161
Loss of hair, 107 Micromelia, 50, 85
Loss of night vision, 386 Micropenis, 492, 580
Loss of peripheral retinal pigment, 281 Microphthalmia, 500, 501, 505, 591
Loss of speech, 121 Midface hypoplasia, 499, 591, 592, 594
Loss of very early milestones, 148 Midline brain malformations, 505
Low birth weight, 210, 212, 345 Migraine, ocular, 121
Low body temperature, 119 Mild dysmorphic features, 67, 111, 121
Lung bleeding, 638 Miller syndrome, 643, 650
Lung disease, 411, 427, 454, 456, 457, 461 Minor contralateral bone, 592
Lung infiltrates, 413 MODY, 326
Lungs, 98, 299, 300, 411, 413, 427, 447, 454, 456, 457, 461, 555, Motor impairment, 618, 620
594, 634 Motor neuropathy, 67, 545–547, 550
Lymphadenopathy, 413 Motor retardation, 148, 285, 414, 415, 647, 651
Lymphedema, 68, 76, 442 Movement
Lymphocyte growth, 496 abnormal, 39, 496, 627
Lymphohistiocytosis, 89, 97 decreased spontaneously, 107
Lymphopaenia, 648 disorder, 113, 115, 116, 144, 155, 171, 209, 221, 274, 293, 294,
352, 400, 401, 416–423, 517, 521, 526, 529–531, 537
Multifocal epilepsy, 64, 65, 72, 73
M Muscle cramps, 281–284, 290, 354, 538, 648
Macrocephaly, 40, 144, 148, 149, 237, 317, 409, 410, 441, 442, 453, Muscle crampsa, 279, 282, 283
454, 458, 493, 508, 587, 593 Muscle crampsb, 284–286
Macroglossia, 281, 324, 354, 408, 414 Muscle dystrophy, 504
Macrophage activation, 89, 446, 711 Muscle pain, 110, 283, 358, 545–547, 710
Macrosomia, 324–327, 330 Muscle paina, 282
Macrothrombocytopenia, 504 Muscle painb, 284–286
Macular dystrophy, 346, 419 Muscle wasting, 504
Macular hypoplasia, 592 Muscle weakness, 8, 38, 110, 172, 234, 237–239, 249, 257, 267, 278,
Maculopathy/retinopathy, 212, 416 279, 282–284, 299, 344, 349, 351, 354, 358, 409–411, 479,
Malabsorption, 167–169, 171, 175, 176, 206, 265, 266, 268, 274, 503, 530, 532, 628, 653
275, 293, 394, 466, 556, 573–575, 590, 651, 678, 743–745, progressive, 40, 347
820, 829 Muscle weaknessb, 284–286
Malar flush, 39, 76 Muscular atrophy, 279, 410, 624, 625, 628, 630, 724
Male genital hypoplasia, 492, 496, 594 Muscular dystrophy, 497, 500, 501, 703
Malignant hepatoma, 564 Muscular hypotonia, 34, 41, 91, 132, 143, 144, 148, 221, 252–255,
Malnutrition, 42–45, 156 257, 383, 387, 401, 410, 414, 415, 505, 530, 618
chronic, 317, 651, 809 Musty odor, 711
Malrotation, 492, 501 Myasthenic syndrome, 493, 494
Maple syrup odor, 711 Myelination delay, 34, 38, 144
Marfanoid features, 39 Myelopathy, 37, 211, 212, 453, 454, 456, 461, 490, 561, 591
Megacisterna magna, 39 Myocardial infarct, 561, 672
Megalencephaly, 410 Myocardial ischemia, 676–679, 681, 682, 687
Megalocornea, 410 Myoclonic epilepsy, 86, 87, 93, 95, 168, 339, 340, 345, 400–403,
Memory problems, 121 416–420, 423, 441, 822
Mental deficiency, 385 Myoclonic epilepsyb, 91
Mental deterioration, 408–410 Myoclonic seizures, 64, 65, 181, 304, 401
Mental development, 4, 366, 368 Myoclonus, 65, 405–408, 412, 416–422, 426
Mental retardation, 5, 6, 17, 23, 24, 27, 28, 39, 64, 66–68, 81, 90, 94, Myoglobinuria, 234, 249, 267, 304, 308, 650, 711, 743
104, 142, 148, 149, 171–173, 180, 185, 209, 211, 231, 234, Myokymia, 94
237, 256, 274, 276, 278, 281, 284, 304, 313, 314, 326, Myopathy, 34, 38, 111, 233, 234, 248, 252–255, 257, 261, 267, 304,
345–347, 351, 383, 403, 414, 415, 440–442, 461, 494, 517, 308, 340–342, 344–349, 351, 353, 354, 476, 479, 585, 648,
534, 556, 578, 618, 625, 628, 647, 650–652, 662, 663, 649, 651, 662, 664, 703, 778, 822, 823
698–700, 720 peripheral, 479
mild, 90, 113, 520 Myopia, 34, 39, 281, 496, 501, 504, 508, 530, 532
Mental retardationa, 518
Mental retardationb, 89
Metabolic stroke, 109, 112, 118, 119, 210, 211, 217, 804, 811–812 N
Metaphyseal and epiphyseal dysplasia, 385 Nasal congestion, 519, 521
Microadenomatosis, 323 Nasal hypoplasia, 499, 592
Microcephaly, 5–7, 35, 65, 67, 73–75, 91, 117, 121, 168, 171–173, Nasal secretion, 521
197, 198, 227–230, 256, 274, 278, 303, 304, 307, 308, 313, Nausea/vomiting, 90, 289, 332, 333, 545–547, 550
Neoplasm, 498 Orthostatic, 517, 520, 526, 628

Nephrocalcinosis, 26, 298, 465, 466, 468, 469, 471, 479, 580 acrocyanosis, 157, 160, 161
Nephrolithiasis, 197, 198, 294, 466, 468, 469, 479 cyanosis, 711
Nephromegaly, 275 Ossification, 499, 508
Nephropathy, 234, 295, 298, 656, 657, 823 Osteochondroma, 498
Nephrotic syndrome, 234, 237, 238, 241, 684, 711 Osteodystrophy, 313, 314, 316, 414, 479
Nerve conductive velocity, 411, 415 Osteopenia, 67, 75, 118, 120, 280, 282, 384, 406, 428, 479, 490, 493
Nesidioblastosis, 323 Osteoporosis, 34, 39, 67, 75, 89, 98, 295, 411, 427, 428, 479, 497,
Neuroaxonal dystrophy, 438, 441, 786 508, 562, 595, 703
Neurodegeneration, 191, 192, 194, 195, 201, 234, 242, 311, 399–431, Otitis media, 76, 414, 415, 453, 454, 457, 461
582, 635, 638 Ovarian cysts, 607, 611
Neurodegenerative disease, 400, 416–423 Ovarian failure, 276
Neurogenic bladder, 506, 507
Neurological deterioration, 4, 320, 411, 415, 430, 431, 475, 476, 624, 710
Neurological symptoms, 11, 24, 35, 63, 104, 105, 143, 151, 211, 216, P
316–318, 394, 395, 399, 400, 427, 428, 437, 511, 516, 526, Pain, 24, 30, 110, 248, 283, 288, 289, 333, 351, 358, 395, 406, 407,
544, 551, 562, 617, 618, 624, 627, 661, 662, 664, 739 411, 412, 427, 428, 468, 469, 499, 515, 545–547, 550–552,
Neurologic crises, 24, 31 627, 637, 651, 681, 687, 710
Neurologic deterioration, 72, 161, 171, 350, 352 Palmoplantar hyperkeratosis, 508
Neurologic dysfunction, 210, 212, 255, 303, 376, 395 Pancreatic dysfunction, 345, 478
Neuropathic pain, 395, 412 Pancreatic failure, 353
Neuropathy Pancreatitis, 108, 109, 114, 118, 119, 131, 133, 211, 280, 367, 556,
axonal motor, 67, 75, 507 563, 671, 681, 687, 711
myelinating, 497, 651 Pancytopenia, 109, 168, 171, 172, 176, 209–211, 411, 413, 468, 469, 711
peripheral, 171, 172, 227, 237, 238, 248, 249, 254, 281, 304, 307, PAP. See Pulmonary alveolar proteinosis (PAP)
318, 344, 351, 383, 387, 388, 478, 503, 538, 550, 578, 618, Paresis, 53, 386, 551, 561
680, 710 Parkinsonism, 7, 8, 347, 561
sensorimotor, 384, 387 hypokinetic, 307, 521
sensory, 44, 189, 340, 347, 348, 532, 822 Patent ductus arteriosus, 580, 593
sensory axonal, 347, 348 Pathological fractures, 406, 411, 412, 427, 468, 469
Neutropenia, 104, 109–111, 116, 118, 120, 133, 136, 280, 298, 317, Pectus excavatum, 497, 498
332, 511, 628, 711, 747 Pectus excavatum/carinatum, 275
Neutrophilia, 504 Peculiar facies, 628
No visual contact, 619 Perceptive hearing loss, 385
Nystagmus, 37, 65, 73, 90, 107, 110, 113, 148, 210–212, 234, 304, Pericardial effusion, 490, 495
350, 351, 442, 494, 503, 505, 587, 651, 653 Perinatal bleeding diathesis, 504
Periodic limb movement during slip, 90
Periodontitis, 504
O Peripheral neuropathy, 227, 248, 249, 304, 387, 388, 478, 538, 578,
Obesity, 234, 237, 355, 358, 611, 673, 753 618, 710
Obstructive sleep apnea, 454, 457, 461 Pes cavus, 39, 562
Obstructive uropathy, 89, 506 Pes equinovarus, 7
Ocular flutter, 521 Pes planus, 67, 75, 479, 503
Ocular hypertelorism, 68 Petechiae, 158, 160, 161, 711
Oculogyric crises, 14, 516 Phalangeal duplications, 498
Oculomotor apraxia, 238, 407 Phalangeal malformations, 502, 594
Oculomotor dyspraxia, 75 Photodermatitis, 94, 96, 97
Odontoid hypoplasia, 456, 457 Photophobia, 27, 90, 476, 478, 479
Odor Photosensitivity, 89, 541, 544, 545, 548, 825
acrid, 110 Pigmentary retinopathy, 254, 345, 347, 417–421, 468, 469, 490, 500,
of sweaty feet, 104, 109, 127, 711, 744 501, 562, 648, 664, 678
Odorous breath due to, 37 Pigment gallstones, 548
Oligohydramnios, 578, 580 Pitched cry, high, 74
Omphalocele, 594 Platyspondyly, 499, 587, 594
Ophthalmoparesis, 347–349, 358 Pneumonia, 6, 7, 148, 210, 358, 563, 599
Ophthalmoplegia, 228, 339, 340, 344, 346, 347, 350–352, 407, 653 otitis, 385
Opisthotonous, 148 Polycystic ovaries, 610
Opisthotonus, 108, 198, 221 Polydactyly, 586, 587, 591, 592
Optic atrophy, 72, 104, 110, 113, 118, 148, 222, 228, 234, 237, 317, 340, postaxial, 591, 593, 594
345, 347, 348, 350, 388, 408, 416–421, 423, 441, 468, 469, Polyneuropathy, chronic, 73, 228
491, 493, 495, 498, 503, 505, 561, 581, 638, 647, 651, 752, 822 Polyuria, 367, 475, 476, 479
Optic nerve atrophy, 144, 148, 234, 339 Pontobulbar palsy, 234, 237, 239
Optic neuropathy, 105, 120, 210, 211, 227, 230 Poor, 17, 33, 43, 47, 64, 65, 67, 153, 157, 162, 181, 200, 205, 210,
Oral ulcerations, 280 221, 261, 310, 324, 387, 393, 409, 462, 472, 519, 521, 716,
Organomegaly, 324, 428 752, 805, 806
Orobulbar dysfunction, 197, 198 visual fixation, 386, 494, 519
Porphyria-like neurological crisis, 24, 26, 31 Renal Fanconi syndrome, 292, 346, 475, 476, 479, 480, 743, 744, 750,
Portal hypertension, 383, 564 757
Posterior fossa malformations, 64 Renal hypoplasia, 591–593
Preaxial brachydactyly, 498 Renal hypoplasia/agenesis, 591–593
Prematurity, 3, 49, 54, 181, 185, 492 Renal osteodystrophy, 479
Progeroid appearance, 55, 67 Renal salt loss, 612
Progeroid face, 499 Renal tubular acidosis, 252, 627, 711, 746, 778
Progressive renal impairment, 120, 465, 466 Renal tubulopathy, 24, 26, 112, 120, 238, 275, 277, 341, 348, 350,
Proliferation, 239, 240, 561, 563, 564, 587, 594 352, 493, 506, 580, 653, 822
Protein intolerance, 57 85, 86, 89, 94 proximal, 345, 352, 638
Proteinuria, 97, 206, 209, 240, 407, 409, 412, 428, 460, 479, 490, 504 Renal tubulopathya, 277
Pseudoacinar transformation, 560 Respiratory distress, 111, 114, 116, 754, 778
Pseudopuberty, 610 Respiratory failure, 34, 54, 64, 186, 193, 341, 350, 504, 538,
Pseudotumor cerebria, 276 551, 590
Psychiatric signs, 710 Respiratory infections, 76, 304
Psychiatric symptoms, 39, 81, 172, 211, 212, 314, 410, 417, 418, 550, Respiratory insufficiency, 38, 41, 90, 112, 119, 120, 181, 237, 497
627, 710 Restrictive lung disease, 411, 454, 456, 457, 461
Psychosis, 66, 89, 410, 411, 416, 441, 618, 620, 663, 664 Retardation, 5, 6, 17, 23, 24, 27, 28, 39, 55, 64, 66–68, 80, 89, 90, 94,
dementia, 209, 210 97, 104, 111, 113, 143, 147–149, 171–173, 180, 185, 211,
Ptosis, 358, 517 231, 234, 237, 254, 256, 274, 276, 278, 281, 284, 298, 304,
Ptosis of eyelid, 346, 347, 351, 517, 519–521, 591–593, 653 313, 314, 323, 326, 340, 345–347, 349, 351, 383, 385, 401,
Ptosis of eyelida, 518 403, 409, 414, 415, 440–442, 461, 468, 469, 475, 476, 494,
Puberty, delayed, 478, 505, 607 501, 508, 517, 518, 520, 534, 556, 578, 580, 591, 593, 618,
Pulmonary alveolar proteinosis (PAP), 90, 97, 98 623, 625, 628, 638, 647, 650–652, 662, 663, 698–700, 720,
Pulmonary edema, 255 822
Pulmonary hypertension, 64, 406, 427, 428, 710 psychomotor, 6, 8, 34, 39, 65, 72–76, 107–110, 112–116, 118, 119,
Pulmonary hypoplasia, 591, 593 160, 173, 198, 209, 228, 235, 304, 316–318, 344, 348, 349,
Pulmonary interstitial, 413 351–354, 401, 411–413, 415, 490–496, 498–504, 506–508,
Pulmonary lobation, 591 580, 581, 585, 587, 590, 618–620, 625, 629, 647, 652, 663,
Pulmonary stenoses, 275 664, 703
Punctate calcifications, 388, 592 psychomotorb, 91
of epiphyses, 586, 591 and regression, 110, 112, 117, 453–456, 458
Pyloric stenosis, 591 Retinal detachment, 492, 532
Pyramidal signs, 53, 75, 116, 157, 235, 238, 307, 316–318, 407, 478, Retinal dystrophy, 285, 394, 417–422, 453, 454, 461, 495, 503
521, 561, 649 Retinitis pigmentosa, 340, 345, 384, 386, 388, 396, 752, 822
Pyramidal syndrome, 67 Retinopathy, 210, 212, 248, 249, 254, 261–263, 345, 347, 387, 388,
416–421, 468, 469, 478, 490, 500, 501, 562, 638, 648,
664, 678
Q Reye-like, 248, 255, 256, 304, 326, 701, 702, 778
Quadriplegia, 200, 304 Rhabdomyolysis, 247–249, 252–255, 257, 260, 262, 265, 299, 349,
384, 387, 710, 778
Rheumatological, 648
R Rhizomelia, 75, 591
Radiohumeral, 498, 594, 604 Rhizomelic
Radiolucent metaphyseal bands, 468, 469 shortness, 587, 594
Radioulnar synostosis, 498, 499, 594, 604 Rhizomesomelia, 593
Radius dislocation, 497 Rickets, 24, 26, 68, 275, 386, 475–477, 479, 481, 556, 559, 560, 563,
Rash, eczematous, 6, 7 572, 573, 754, 757
Recurrent bacterial infections, 661, 662, 664 Rigidity, 7, 115, 304, 418, 422, 521
Recurrent infections, 173, 280, 496, 504, 506, 508, 638, 647, 650 Rigidityc, 518
Recurrent otitis media, 76, 414, 415, 453, 454, 457, 461
Recurrent skin infections, 504
Recurrent vomiting, 711, 778 S
Reduced glomerular filtration rate, 120 Sagging cheeks, large fontanel, 67
Regression Scoliosis, 7, 39, 458, 479, 497, 501, 507, 591, 594
motor, 112, 143, 317, 401, 556, 578 Scotomata, 347
psychomotor, 115, 160, 238, 344, 348, 350, 400 ‘Second wind’ phenomenon, 282–284
Renal colic, 468, 469, 649, 663 Seizures
Renal cysts, 249, 384, 386, 493, 495 complex partial, 417, 420, 421
Renal dysgenesis, 580, 591, 594 febrile, 110, 185
Renal enlargement, 26, 280, 490 intractable, 74, 353
Renal failure, 103, 105, 130, 137, 210, 234, 275, 299, 406, 409, 428, myoclonic, 6, 172, 412, 416, 417, 419, 420, 423
465, 466, 472, 481, 648, 649, 659, 673, 711, 754 myoclonic-astatic, 171
chronic, 26, 89, 118, 210, 211, 349, 354, 412, 442, 468, 469, 471, pharmacoresistant, 39, 185
476, 479, 580, 649, 659 tonic-clonic, 171, 197, 198, 416–422
end stage (ESRF), 90, 408, 428, 465, 466, 472, 475 Seizuresa, 410
Self-injury, 590, 595 Stature

Self-mutilation, 642, 649, 656, 659 short, 75
Sensorineural deafness, 104, 228, 235, 313, 314, 387, 461 tall, 611
Sensory disturbances, 386, 395 Steatorrhea/steatorrhoea, 333, 334, 386, 415, 556, 559, 563
Sepsis, 24, 31, 111, 112, 276, 295, 687 Stenosis, 385, 591, 594, 677
Severe combined immunodeficiency (SCID), 169, 172, 643, 648, Stenotic canals, 592
656, 724 Sternal bulging, 456, 457
Severe multisystem disease, 238 Sternal deformities, 39
Sexual dysfunction, 385, 388, 395 Stiffness, 90, 95, 96, 98, 286
Short clavicle, 499 Stillbirth, 508, 565, 594
Shortening of long bones, 385, 499, 586 Stomatitis, 171, 222, 234
Short fingers or toes, 498 Strabismus, 38, 75, 490–492, 494, 495, 503, 507, 508, 587, 591
Short metacarpals/metatarsals, 498, 587 coagulopathy, 34
Short palpebral fissures, 497 Stridor, 221, 710
Short stature, 67, 105, 275, 280, 282–284, 345–348, 440–442, 453, inspiratory, 222, 495
456–458, 586–588, 595, 608, 609 Stroke, 39, 109, 111, 112, 114, 118, 210, 211, 217, 228, 239, 407, 428,
Sideroblasts, 545 671, 672, 804, 811, 812
Skeletal abnormalities, 34, 437, 461, 586, 587, 599, 611 cerebellar, 354
Skeletal deformations, 498 ischaemic, 275
Skeletal dysplasia, 385, 453, 492, 587, 588, 594, 824 Stroke-like episode(s), 51–53, 238, 339, 340, 346, 359, 384, 387, 478,
Skeletal hypomineralisation, 186 490, 503, 511, 710, 803, 804, 811, 812, 822
Skeletal malformations, 386, 388, 604 Subcapsular cataracts, 67
Skeletal myopathy, 261 Subcutaneous fat distribution, 490
Skeletal myopathy/muscular hypotonia, 252–255, 257 Subcutaneous nodules, 413
Skin blisters, 546 Subdural hematoma, 711
Skin fragility, 544, 546 Sudden death, 6, 7, 228, 247, 252, 281, 300, 317, 368
Skin lesions, 23, 24, 30, 68, 221, 586, 592 Sudden hearing loss, 710
Skin rash, 5, 222, 286, 334, 711 Sudden infant death, 90, 96, 717
Skin scarring and mutilation, 546 Sudden visual loss, 710
Skin ulceration, 76 Swallowing difficulties, 148, 453–456, 478, 519
Sleep disturbance, 14, 75, 173, 455, 461, 518 Sweating, 519, 521
Slender fingers, 501 Sweatinga, 518
Small ilia, 499 Sweaty feet odor, 711
Somatic development, delayed, 603, 611 Symmetric extension in the legs, 619
Somnolence, 48, 652, 696 Symphalangism, 498
Sparse hair, 67, 75, 89 Symptoms, 642, 662, 676, 694, 709, 732, 739, 752, 771, 778, 788, 797
Spasms, 64, 172, 221, 228, 304 Syncope, 278, 428, 517, 520
Spastic diplegia, 53, 648 Syndactyly, 498, 586, 591–593
Spasticity, 7, 64, 65, 72, 112, 113, 115, 144, 148, 149, 155, 161, 304, Synostosis, 498, 499, 594, 604
308, 314, 353, 354, 388, 395, 401, 408–411, 415–421, 431, Synovitis, 453
440–442, 581, 618, 628, 649, 662, 703
limbs, 110, 350
Spasticityc, 91 T
Spastic paraparesis, 384, 562, 739 Tachycardia, 228, 428, 545–547, 550, 551
Spastic paraplegia, 113, 173, 556, 557, 561 Tachypnea, 210, 221, 278, 280, 304
Spastic pareses, 383, 385 Tachypnoea, 368
Spastic paresis/pyramidal signs, 53, 561 Teeth malposition, 497
Spastic tetraplegia, 73, 160, 197, 198, 411 Telangiectasia, 76, 408
Speech delay, 173, 278, 313, 317, 416, 417, 419–421, 423, 529–532 Teleangiectases, 275
Speech disorder, 257 Temperature instability, 6, 48, 51–54, 518, 519
Speech disturbances, 408–410, 414, 415, 627, 652 Temporal optic nerve pallor, 347
Sphincter control problems, 385 Tendon reflexes, 38, 209, 210, 304, 386, 413, 503, 628, 678
Spike wave discharges, 7, 75 decreased, 75, 354, 490, 619
Spinal cord increased, 7, 8, 37, 74, 171, 353, 653
compression, 457 Tetraparesis, 65, 160, 228, 648
myelopathy, 561 Tetraplegia, 73, 160, 197, 198, 441, 647
Spinocerebellar ataxias, 94 Thin body habitus, 593
Splenomegaly, 68, 76, 267, 279, 406, 427, 478, 495, 509, 564, 650 Thoracic hypoplasia, 499
Spondyloepiphyseal dysplasia, 453 Thrombocytopenia, 24, 68, 76, 97, 104, 109, 110, 116, 118, 120, 133,
Sporadic tonic seizures, 91 228, 230, 332, 345, 406, 411–413, 427, 492, 493, 578, 580,
Startle reflex, 90, 95 590, 627, 747
Startle response, 90, 95 Thromboembolic episodes, 172
exaggerated, 408–410, 441 Thromboembolism, 39, 43, 44, 711
Thromboses, 491 Vertebral anomalies, 490, 497, 501, 591, 592

infarcts, 39 Vertical gaze palsy, 416
Thrombosis, 34, 279, 485, 496, 502, 672 Vertigo, 144, 148
Tiny face, 499 Virilization, 601–604, 608, 610, 611, 615, 740
Toe syndactyly, 586, 591–593 Visceral calcifications, 499
Tonsils, enlarged, 680 Vision
Tortuosity, 55, 268, 275, 624, 806 decreased, 115
Tortuous arteries, 628 impaired, 51, 53, 173, 211, 212, 317, 346, 351, 352, 408–410, 478,
Tremor, 7, 37, 149, 171, 304, 503, 521, 562, 627 496, 503, 638
Tremora, 518 progressive loss, 562, 647
Truncal ataxia, 117 tunnel, 532
Tubular acidosis, 711 Vision loss, 347, 385, 413, 416–423
Tubulointerstitial nephritis, 120 Volvulus of the stomach, 496
Tubulopathy, 341, 583, 634, 635, 717 Vomiting, 5, 48, 51–54, 67, 89, 90, 104, 105, 111, 118–121, 131,
Twitching, 193, 197, 198 132, 148, 180, 185, 210, 211, 221, 239, 249, 289, 304,
307, 332, 334, 344, 346, 351, 353, 366, 367, 415, 473,
476, 490, 491, 502, 545–547, 550, 551, 651, 709, 711,
U 716, 752, 753, 756, 778
Unrest, 173, 551 cyclic, 257
Upper airway obstruction, 454, 457, 461 episodic, 107–109, 114, 116–118, 495, 607, 609, 696
Urinary infections, 89, 468, 469, 628, 649 Vomitinga, 276, 277
Urolithiasis, 89, 298, 465, 466, 647–650, 657, 659, 663
cystine stones, 89
Urolithiasisa, 89 W
Uterine muscle stiffnessa, 286 Walker-Warburg syndrome, 500
White matter lesions, 117, 367, 407, 408, 428, 806, 809
Wide nasal bridge, 304
V Wolf-Parkinson-White syndrome, 112
Vacuolar lymphocytes, 441 Wormian bones, 67, 75
Vacuolated lymphocytes, 281, 408, 409, 418, 440, 442, 747 Wrinkly skin, 55, 67, 506
Vacuolating, 34, 108
myelopathy, 37
Vacuolization, 345 X
Valvular thickening, 408, 414, 453, 454, 456–458 Xanthelasma, 676–678, 680–682
Valvulitis, mitral, 27, 55, 89, 222 Xanthomas, 681, 682
Vegetative statec, 91 eruptive, 681
Ventricular arrhythmia, 249, 279, 778 tendon, 561, 676–678
Ventricular septal defect, 492, 493, 496, 506, 587, 592
Ventriculomegaly, 198, 593

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Physician’s Guide to the

Diagnosis, Treatment, and

Physician’s Guide to the

ISBN 978-3-642-40336-1 ISBN 978-3-642-40337-8 (eBook)

Library of Congress Control Number: 2014933397

© Springer-Verlag Berlin Heidelberg 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

The Editors take this opportunity to acknowledge Dr. Milan Blaskovics,

Oxford, UK James Leonard

Heidelberg, Germany Nenad Blau

Signs and Symptoms

Body fluids, cells, tissues, etc., are defined as:

Age groups are defined as:

Normal Values/Pathological Values/Differential Diagnosis

Diagnostic Flow Chart

Treatment and Follow-Up

Part I Amino Acids

1 Disorders of Phenylalanine and Tetrahydrobiopterin Metabolism. . . . . . . . . . . 3

Part II Organic Acids

7 Disorders of Leucine, Isoleucine, and Valine Metabolism . . . . . . . . . . . . . . . . . . 103

Part III Vitamins, Cofactors, and Metals

10 Disorders of Folate Metabolism and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . 167

14 Biotin Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Part IV Energy Metabolism

17 Mitochondrial Fatty Acid Oxidation Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 247

24 Peroxisomal Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Part VI Selected Disorder

31 Neurotransmitter Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

32 Creatine Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529

Part VII General Subjects and Profiles

45 Emergency Diagnostic Procedures and Emergency Treatment . . . . . . . . . . . . . 709

Ivo Barić Division for Metabolic Diseases, Department of Pediatrics,

Bernd Hoppe Division of Pediatric Nephrology, Department of Pediatrics,

Dietrich Matern Division of Laboratory Genetics, Departments of Laboratory Medicine

Jan Smeitink Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders,

Mirjam M. C. Wamelink Metabolic Unit, Department of Clinical Chemistry,

17βHSD 17-beta-hydroxysteroid dehydrogenase

ACTH Adrenocorticotropic hormone

AMACR 2-methylacyl-CoA racemase

BCAT2 Branched-chain amino acids transporter

CHI Congenital hyperinsulinism

CPT I Carnitine palmitoyltransferase I

DHCA Dihydroxycholestanoic acid

ETF Electron transfer flavoprotein

GNMT Glycine N-methyltransferase

HSD10 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency

MCT-1 Monocarboxylate transporter

MRI Magnetic resonance imaging

PCT Porphyria cutanea tarda

PPT1 Lysosomal palmitoyl protein thioesterase-1

SDH Hyperlysinemia and saccharopinuria

Contents 1.1 Introduction

1.3 Metabolic Pathway

Fig. 1.1 Biosynthesis and GTP

1.4 Signs and Symptoms

Table 1.1 Phenylalanine hydroxylase deficiency, classic PKU

Table 1.2 GTP cyclohydrolase deficiency

Table 1.3 6-Pyruvoyl-tetrahydropterin synthase deficiency

Table 1.4 Dihydropteridine reductase deficiency

Table 1.4 (continued)

Table 1.5 Pterin-4a-carbinolamine dehydratase deficiency

Table 1.6 Dopa-responsive dystonia