Genetics of Eye

Genetics of Eye
Diseases
An Overview
Chitra Kannabiran
123
Genetics of Eye Diseases
Chitra Kannabiran
Genetics of Eye Diseases

An Overview
Chitra Kannabiran
Kallam Anji Reddy Molecular Genetics Laboratory
L V Prasad Eye Institute (LVPEI)
Hyderabad
India
ISBN 978-981-13-7145-5 ISBN 978-981-13-7146-2 (eBook)

https://doi.org/10.1007/978-981-13-7146-2
© Springer Nature Singapore Pte Ltd. 2019

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Preface
The Genetics of Eye Diseases is written as a broad overview of the field that attempts
to capture salient aspects pertaining to the current state of knowledge in several
major eye diseases. Since the subject of eye diseases is very vast, there are diseases
that could not be included here, though they may be important causes of visual
impairment and blindness and of scientific interest to many in the field. Knowledge
of the genetic and molecular basis of various eye diseases has accumulated through
studies done across the world over several decades on clinically defined families
and populations, using methods of gene identification, and also of studying protein
function, that have been continuously evolving over time. Thus, I have tried to pres-
ent the genetics of each eye disease through a discussion of the discoveries made
and the approaches used to arrive at the same. It has been said that every revolution
in science is a revolution in method. Hence, looking at the methods used, however
cursorily, can be a key to understanding the evolution of the field as a whole. Though
I had to be necessarily selective (and brief) about the content, hopefully the major
trends and features of the genetics of each of the diseases are captured. In fact, a
major dilemma in writing the various chapters in the book has been in deciding
which aspects to present and in how much detail. The chapters are organized
disease-wise, and within each, there are sections on specific genes. The book is
aimed at a more specialized reader and presupposes knowledge of the basic princi-
ples of genetics, which are not dealt with here. My journey in the genetics of eye
diseases has been considerably enriched by collaborations and insights from several
colleagues, both clinical and research faculty at LVPEI. I am especially thankful to
Dr. Gullapalli N. Rao for the many valuable opportunities to learn and grow in this
field. The idea of putting together this book came from my publishers, and I am
thankful to particularly Mr. Naren Agarwal and Mr. Athiappan Kumar for their
patience and persistence and in continually nudging me on to finish this despite
many interruptions during the process. I am indebted to Ms. Sabera Banu, our
librarian, for very readily and promptly fetching me the vast amount of literature as
and when needed.
Hyderabad, India Chitra Kannabiran
v
Introduction and Scope of the Book
The genetics of eye diseases is a field that encompasses a large number of indepen-
dent areas, each of which is diverse and vast in its scope. It includes several diseases
that affect each part of the eye, each disease in turn being defined by multiple genetic
bases and pathways that lead to development of the specific disease. This book
attempts to examine the genetics of several (though not all) major eye diseases with
reference to the genes involved, genetic variants thereof, mechanisms of pathogen-
esis, and animal models that have been studied. The basic principles, especially in
relation to the methods used, in discovering the genetics of these diseases are high-
lighted, but the content is aimed at providing an overview of the current status in the
field. There is also a specific mention of genetics of disorders that are substantially
characterized in Indian populations, but in some of the disorders, data on genetics is
relatively little to devote a whole section to this aspect.
The spurt in the knowledge and understanding of human genetic diseases in gen-
eral began with the availability of the sequence of the human genome in the public
domain since the last two decades. Over this period, the identification of disease
genes has been gained considerable momentum. This is especially true for the rare
inherited familial diseases in which mutations in a single gene of major effect lead
to the disease phenotype. The transformations in the field of human genetics that
began with the sequence of the first human genome were equally enabled by rapid
changes and innovations in sequencing technology. The advent of next-generation
sequencing (NGS) made it possible to complete the sequencing of an entire genome
in a day. At inception, next-generation sequencers had a capacity that was thousands
of times that of the capillary-based conventional sequencers, which worked by
Sanger’s sequencing method. Over time, there have been successive improvements
in the capacity of NGS machines, and the output of sequence data is now in the
range of gigabases (109 bases) to terrabases (1012 bases) per run. Apart from the
huge increase in throughput and reduction in time, this trend has been accompanied
by decreasing costs and thus given rise to the idea of “personal genomes.” By 2015,
commercial NGS had arrived at a price of $1000 for a human genome sequence, and
costs have been further declining since then.
These developments have accelerated the pace of genetic discovery for human
diseases. The terrain of NGS also extends to complex diseases wherein genome
sequencing is employed to detect rare variants in the genome that give rise to phe-
notypes such as changes in gene expression or in transcription factor binding, and
vii
viii Introduction and Scope of the Book
such rare variants are tagged by more common single-nucleotide polymorphisms

(SNPs). Thus, the applications of whole exome sequencing as well as high-
throughput “multiplexed” sequencing of several genes in parallel have resulted in
several discoveries of new disease genes as well as in a more comprehensive
appraisal of the role of known genes in the pathogenesis of many eye diseases.
In this scenario, we are still in the midst of a continuous and robust growth in the
understanding of the genetics of various eye diseases. Due to the vast and evolving
nature of the subject areas involved, the purpose of the text is to highlight the first
discoveries in each of the fields as well as a few representative studies that have
dealt with the aspects mentioned above. Hopefully, the salient features and key
trends of the genetics in relation to each disease and gene that is included here are
captured.
Contents
1 Genetics in Corneal Diseases�� 1

1.1 Corneal Dystrophies�� 1
1.1.1 Introduction and Classification �� 1
1.1.2 Epithelial and Subepithelial Corneal Dystrophies�� 4
1.1.3 Corneal Stromal Dystrophies�� 7
1.1.4 Corneal Dystrophies Associated with Mutations
in the Transforming Growth Factor Beta-Induced
(TGFBI) Gene �� 9
1.1.5 Corneal Endothelial Dystrophies�� 16
1.2 Keratoconus�� 23
1.2.1 Mapped Loci for Keratoconus�� 24
1.2.2 Genes Associated with Keratoconus �� 25
References�� 25
2 Genetics in Cataracts�� 31
2.1 Congenital Cataracts �� 31
2.1.1 Genetics of Congenital Cataract �� 32
2.1.2 Mutations in Alpha-Crystallins�� 34
2.1.3 Mutations in Beta-Crystallins �� 35
2.1.4 Mutations in Gamma-Crystallins�� 41
2.1.5 Mutations in Genes Encoding Lens Membrane
Proteins and Gap Junctions�� 44
2.1.6 Mutations in Genes Encoding Transcription Factors�� 51
References�� 53
3 Genetics of Ectopia Lentis�� 61
3.1 Genetics of EL�� 61
3.2 Genetics of Isolated EL�� 62
References�� 63
4 Genetics of Glaucoma�� 65
4.1 Primary Congenital Glaucoma�� 65
4.1.1 Genetics of PCG �� 66
4.2 Primary Open-Angle Glaucoma (POAG) �� 69
4.2.1 Genetics of POAG�� 69
ix
x Contents
4.3 Primary Angle Closure Glaucoma (PACG)�� 75

4.3.1 Genetics of PACG �� 75
References�� 76
5 Hereditary Retinal Degenerations�� 81
5.1 General Features of Major Forms of Non-syndromic
Retinal Dystrophy �� 82
5.1.1 Retinitis Pigmentosa �� 82
5.1.2 Leber Congenital Amaurosis�� 82
5.2 Homozygosity Mapping in Retinal Disorders�� 84
5.3 Genes Involved in Phototransduction �� 89
5.3.1 Rhodopsin �� 89
5.3.2 Phosphodiesterase 6�� 91
5.3.3 Guanylate Cyclase 2D (GUCY2D) �� 95
5.4 Genes Encoding Structural and Membrane Proteins�� 99
5.4.1 RDS (Retinal Degeneration Slow)�� 99
5.4.2 Retinal Outer Segment Membrane Protein 1 (ROM1)�� 103
5.4.3 ATP-Binding Cassette Subfamily A Member 4
Protein (ABCA4) Gene�� 104
5.4.4 Crumbs Homolog 1 (CRB1)�� 108
5.5 Genes Encoding Splicing Factors �� 111
5.5.1 Precursor RNA Processing Factor 31 (PRPF31; RP11)�� 111
5.5.2 Precursor RNA Processing Factor 6 (PRPF6)�� 112
5.5.3 Precursor RNA Processing Factor 4 (PRPF4)�� 113
5.5.4 Precursor RNA Processing Factor 8 (PRPF8; RP13)�� 113
5.5.5 Pre-mRNA Processing Factor 3
(PRPF3; HPRP3, RP18)�� 114
5.5.6 Pim1-Associated Protein Gene (PAP1; RP9)�� 114
5.5.7 Small Nuclear Ribonuclear Protein 200 (SNRNP200)�� 115
5.6 Genes Encoding Ciliary/Centrosomal Proteins�� 115
5.6.1 Retinitis Pigmentosa 1 (RP1)�� 115
5.6.2 Leber Congenital Amaurosis 5 (LCA5)�� 117
5.6.3 Topoisomerase 1 Binding RS-Like Protein
Gene (TOPORS)�� 118
5.6.4 FAM161A�� 119
5.6.5 Retinitis Pigmentosa 3 (RP3)/Retinitis Pigmentosa
GTPase Regulator (RPGR) �� 121
5.6.6 Centrosomal Protein 290 KDa (CEP290)�� 124
5.7 Genes Involved in the Metabolism of Retinoids�� 125
5.7.1 Cellular Retinaldehyde Binding Protein Gene
(CRALBP, RLBP1)�� 125
5.7.2 Retinal Pigment Epithelial 65 KDa Protein
(RPE65) Gene �� 127
5.8 Genes Encoding Transcription Factors �� 133
Contents xi
5.8.1 Nuclear Receptor Subfamily 2 Group E Member 3

(NR2E3, PNR)�� 133
5.8.2 Neural Retina Leucine Zipper (NRL)�� 136
5.9 Genes Involved in Various Other Pathways�� 139
5.9.1 RP25/Eyes Shut (EYS)�� 139
5.9.2 Retinitis Pigmentosa 2 (RP2)�� 142
5.10 Genetics of Usher Syndrome: A Form of Syndromic Retinitis
Pigmentosa�� 145
5.10.1 Usher Syndrome 1�� 146
References�� 158
Genetics in Corneal Diseases
1
1.1 Corneal Dystrophies
1.1.1 Introduction and Classification
Corneal dystrophies are hereditary disorders that are generally bilateral, involving
the formation of opacities in one or more layers of the cornea. The opacities cause
blurring of vision, and when this occurs to a significant extent, a corneal graft to
replace the cornea, or involving replacement of one or more layers of the cornea, is
required to restore vision. An essential part of the traditional definition of corneal
dystrophy includes the absence of systemic or environmental factors in the etiology
of these diseases. The most common method of classification of corneal dystrophies
is an anatomical one, based on the layer(s) of the cornea that are affected; thus,
corneal dystrophies are grouped as epithelial and subepithelial, Bowman layer, stro-
mal, Descemet membrane, and endothelial dystrophies. However, a critical evalua-
tion of the literature in the field by a committee of experts has brought out many
limitations in this system. These are as given below.
1. There were discrepancies between the conventional text book definition of a

corneal dystrophy and the phenotypes actually observed in affected patients and
families.
An example of this is the postulation that the presence of corneal crystals is
required to make the diagnosis of Schnyder crystalline corneal dystrophy
(SCCD). Examination of patients in large families with SCCD has shown that
crystals are not always present in affected individuals (Weiss et al. 2008).
Another such example is found in the case of Avellino corneal dystrophy (ACD),
also known as granular corneal dystrophy (GCD) type II, which was historically
defined as having features of both granular and lattice corneal dystrophies. This
implies the presence of lattice-type (linear) and granular (rounded) opacities as
seen clinically, each distinguishable by characteristic histopathologic staining
properties. However, careful examination of multiple patients from affected
© Springer Nature Singapore Pte Ltd. 2019 1

C. Kannabiran, Genetics of Eye Diseases, https://doi.org/10.1007/978-981-13-7146-2_1
2 1 Genetics in Corneal Diseases
families revealed that the opacities are very variable on clinical examination.
Thus the definition of this entity as combined granular-lattice dystrophy may be
misleading. This is also true of the name “Avellino” corneal dystrophy for this
disease; it was named on the basis of initial observations of patients from the
Avellino province of Italy. Though some affected families have been reported
trace their ancestry to this region, this is not the case with all families having this
disorder.
2. The hereditary nature of some corneal dystrophies was not known in the absence
of a family history. They could represent degenerative conditions rather than
dystrophies.
Some forms of corneal dystrophy are extremely rare, have been reported in
very few patients, and often in the absence of a family history. In the pre-genomic
era, one could not confirm the genetic basis and ascertain if the disease in such
cases is in fact, hereditary. This made it difficult to differentiate a corneal dystro-
phy from other non-hereditary or idiopathic conditions with similar or overlap-
ping manifestations. This situation is illustrated by the corneal diseases, Central
Cloudy Dystrophy of Francois (CCF) and Vogt posterior crocodile shagreen. The
two conditions are indistinguishable based on phenotype. CCF is often asymp-
tomatic and nonprogressive. Most cases reported have no familial inheritance
documented, though there are some reports of familial disease in the literature
(Stratchan 1969; Bramsen et al. 1976). Based on clinical features alone, there is
insufficient evidence to distinguish whether those patients without a family his-
tory in fact have CCF or another condition (Weiss et al. 2015).
3. The definition of a corneal dystrophy as unrelated to environmental or systemic
factors is not necessarily true. Such dystrophies include Schnyder corneal dys-
trophy, which is associated with hypercholesterolemia and a form of macular
corneal dystrophy, in which antigenic keratan sulfate may be detected in serum.
4. It is unclear whether certain corneal disorders fall within the category of corneal
dystrophies or not. These include cornea plana, a hereditary nonprogressive dis-
ease that has a genetic basis. Are ecstatic diseases such as keratoconus, corneal
dystrophies? Keratoconus has a genetic basis only in a small percentage of cases,
with the remaining cases being sporadic, and associated with other predisposing
factors such as eye rubbing.
5. Two different dystrophies that may be partly similar, but are in fact separate enti-
ties, have been confused with one another. This phenomenon is encountered with
the superficial stromal dystrophies, Reis-Bücklers corneal dystrophy, and Thiel
Behnke corneal dystrophy. They have been interchanged in the literature, with
Thiel Behnke corneal dystrophy being labeled as Reis-Bücklers corneal
dystrophy.
6. Unusual forms of corneal dystrophy were reported, that could not be grouped
with the existing types. In addition there may be overlaps in phenotype between
different dystrophies, as well as variant or unusual phenotypes for a given entity.
7. The anatomical classification is not an accurate characterization of corneal dys-
trophies since several dystrophies affect multiple layers of the cornea. For exam-
ple, macular corneal dystrophy affects the stroma and endothelium, and some of
1.1 Corneal Dystrophies 3
the TGFBI-induced corneal dystrophies such as Reis-Bücklers corneal dystro-

phy affects multiple layers including the Bowman’s layer, superficial layer, and
deep stroma.
In order to address the shortcomings of the conventional classification, a newer

classification system for corneal dystrophies has been recommended in an effort to
streamline and categorize known corneal dystrophies and incorporate any new enti-
ties that are discovered, in a harmonized manner. The International Committee for
Classification of Corneal Dystrophies (IC3D) was constituted, which consisted of
experts in corneal diseases from several different countries, to identify problems in
naming and classification of corneal dystrophies (Weiss et al. 2008). The IC3D
made an effort to standardize the nomenclature for corneal dystrophies, based on
the available evidence of clinical and histopathologic features as well as genetics.
The corneal dystrophies were divided into four categories based on the level of dif-
ferent types of evidence available for a dystrophy (shown in Box 1.1).
Box 1.1 Classification of Corneal Dystrophies

Category 1: A well-defined corneal dystrophy in which the gene has been
mapped and identified, and the specific mutations are known.
Category 2: A well-defined corneal dystrophy that has been mapped to one
or more specific chromosomal loci, but the gene(s) remains to be identified.
Category 3: A well-defined corneal dystrophy in which the disorder has
not yet been mapped to a chromosomal locus.
Category 4: This category is reserved for a suspected, new, or previously
documented corneal dystrophy, although the evidence for it, being a distinct
entity, is not yet convincing.
The first set of IC3D recommendations as reported in 2008 included the anatomi-
cal grouping of corneal dystrophies, based on the corneal layer that was chiefly
affected. However, in view of the fact that several dystrophies actually involve mul-
tiple layers of the cornea rather than being limited to one layer, a further revision to
this was brought out by the IC3D in 2015, in which the anatomical basis of grouping
corneal dystrophies was recognized as insufficient (Weiss et al. 2015). In this ver-
sion, the two noncellular layers—the Bowman’s layer and Descemet membrane—
are excluded as entities. The anatomical classification was modified to include the
following: epithelial-subepithelial dystrophies, epithelial-stromal TGFBI dystro-
phies, and stromal and endothelial dystrophies. Thus the updated classification
regrouped the TGFBI dystrophies as a separate category, since both the epithelial
and stromal layers of the cornea are involved.
However, with the ability to directly sequence the genomes of patients to find the
disease genes using NGS, genetic mapping is no longer necessary; hence the cor-
neal diseases that have been mapped but with the gene not identified—i.e., belong-
ing to category 2 in the classification system—are decreasing in number. As per the
criteria given above, the dystrophies belonging to categories 1 and 2, which have
been defined to some extent in terms of their genetics, will be discussed here.
1.1.2 Epithelial and Subepithelial Corneal Dystrophies
1.1.2.1 Meesmann Corneal Dystrophy (MECD)
Manifestations and Clinical Features

The most common manifestation of MECD is blurred vision with impairment in
visual acuities being mild to severe. The onset of the disease is at birth or in early
childhood, though patients may not experience any symptoms until later in life
since the disease progresses with age. MECD was first reported by Meesmann and
Wilke (1939). A further description of MECD was given by Stocker and Holt
(1954–1955). They examined a group of 200 individuals that were direct descen-
dants of Moravian settlers in North Carolina, of whom 20 were affected. Most
patients were reported to experience mild loss of vision with visual acuities of
around 20/50, with few individuals being severely affected. Other subjective symp-
toms of MECD include occasional itching, burning, and watering of the eyes.
Patients may often be asymptomatic.
The clinical and ultrastructural features of the cornea in MECD have been eluci-
dated from studies of affected families. The appearance of the cornea on slit lamp
examination in diffuse illumination shows haze, and the anterior surface may be
studded with grayish, punctate opacities. In addition, serpiginous grayish-white
lines arranged in whorls are seen in some parts of the cornea. All changes in MECD
are located anterior to the Bowman’s membrane. The space between the Bowman’s
membrane and the surface of the epithelium shows diffuse opacities and several
white dots, while the layers posterior to the Bowman’s membrane are normal. A
notable feature in the histopathology of the corneal sections is the presence of pro-
trusions, described as pedunculated excrescences, in the Bowman’s membrane.
Light microscopy shows variability in the thickness and organization of the epithe-
lial layers of the cornea. Notable also are the cysts present in the epithelium, which
stain positive with periodic acid-Schiff (PAS) reagent, and are resistant to diastase
and neuraminidase. Granules present within the cysts and within epithelial cells
stain positively for acid mucopolysaccharides and colloidal iron. The epithelial
basement membrane is thickened and appears different in composition between its
anterior and posterior aspects, upon electron microscopy (Fine et al. 1977).
Genetics
The underlying genes for MECD were mapped and identified simultaneously by
two groups. MECD is brought about by mutations in the genes KRT3 and KRT12
encoding the cornea-specific keratins keratin-3 and keratin-12 (K3 and K12, respec-
tively). The keratin genes encoding K3 and K12 were postulated as likely candi-
dates for the disease (Irvine et al. 1997). The bases for such a postulation were the
following:
(a) That the two keratins were expressed specifically in the superficial layers of the
corneal epithelium, which is also the affected tissue in MECD.
(b) MECD is a dominantly inherited disorder, and since cytokeratins exist as poly-
meric structures, dominant mutations were a likely mechanism for the disease
and are commonly known in other diseases involving keratins.
(c) The phenotypes of K12-knockout mice with loss of cytokeratin were associated
with extreme fragility of the corneal keratocytes.
(d) Changes at the subcellular level in the epithelial cells in MECD were similar to
those of other keratin disorders.
Since the loci for the keratin genes were not precisely known at the time, the
authors mapped the loci for the two genes KRT3 and KRT12 onto chromosomes 12q
and 17q, respectively, using radiation hybrid panels. Linkage analysis in the affected
families mapped the disease to chromosome 17q21.2, which is the locus for the
KRT12 gene in one family, and to the KRT3 locus in two separate families.
Heterozygous missense changes in K3 and K12 were identified in German and
northern Irish families. Mutation of Arg135Thr (arginine-135 to threonine) was
identified in the German family originally described by Meesmann. In addition, in
the two remaining families, they found a mutation of Glu509Lys in KRT3 and
Val143Leu in KRT12.
In a parallel study, the KRT12 gene was mapped, and its organization was deter-
mined. Essentially the human KRT12 cDNA from a corneal epithelial cDNA library
was used as a probe to screen a human genomic library for isolating the KRT12
gene. The gene was localized to chromosome 17 by fluorescence in situ hybridiza-
tion (FISH). A screen for mutations in four affected families showed mutations of
Arg135Gly, Arg135Ile, Leu140Arg, and Trp429Asp in these families. Various mis-
sense mutations in KRT3 and KRT12 genes have since been reported in families
with MECD from populations across the world (Nishida et al. 1997) (see Table 1.1).
1.1.2.2 G
elatinous Drop-Like Corneal Dystrophy (GDLD; MIM
#204870 [Also Known as Subepithelial Amyloidosis,
Primary Familial Amyloidosis])

GDLD is a very rare disorder with autosomal recessive inheritance. It has a reported
incidence of 1 in 300,000 in Japan (Kinoshita et al. 2000), and very few cases have
been reported throughout the world. The initial symptoms include blurred vision,
foreign body sensation, and intense photophobia. The cornea shows raised yellow-
ish lesions that are described as mulberry-shaped. These are formed due to massive
subepithelial deposits of amyloid, leading to severe impairment of vision with onset
in the first decade of life. The condition is treated by lamellar keratoplasty, but the
deposits can recur within the grafted cornea. Microscopic changes observed in
GDLD consist of an irregular corneal epithelium with edematous cells, disruption
of the Bowman’s membrane, and eosinophilic deposits in the anterior stroma. The
deposits stain with Congo red with birefringence under polarized light,
Table 1.1 Keratin gene mutations in MECD

Mutation in cDNA Amino acid Country of Reference
(Keratin gene) change origin of
patient
c.1525G>A (KRT3) Glu509Lys Northern Irvine et al. (1997)
Ireland
c.451G>C (KRT12) Val143Leu Northern Irvine et al. (1997)
Ireland
c. 428 G>C (KRT12) Arg135Thr Germany
c. 427A>G (KRT12) Arg135Gly Nishida et al. (1997)
c. 428 G>T (KRT12) Arg135Ile Japan
c. 443T>G (KRT12) Leu140Arg
c. 1286A>C (KRT12) Tyr429Asp
c.1300A>G (KRT12) Ile426Val USA Coleman et al. (1999)
c.410T>C (KRT12) Met129Thr America Corden et al. (2000)
c.428G>C (KRT12) Arg135Thr Germany
c. 429A>C (KRT12) Arg135Ser Japan Yoon et al. (2004)
c. 1222ins27 (KRT12) – Europe
c.1289G>C (KRT12) Arg430Pro USA Sullivan et al. (2007)
c.1493A>T (KRT3) Glu498Val Poland Szaflik et al. (2008)
c.1298T>G (KRT12) Leu433 Arg Japan Seto et al. (2008)
c. 409A>G (KRT12) M et 129V al Germany Clausen et al. (2010)
c. 395T>C (KRT12) Leu132Pro UK Hassan et al. (2013)
c. 250C>T (KRT3) Arg84Trp USA Chen et al. (2015)
c.1288_1293delinsAGCC Arg430_Arg43 USA
CT (KRT12) 1delinsSerPro
The table shows the details of mutations (with residues affected in cDNA and protein) reported in
the literature in the keratin genes KRT3 and KRT12, in association with MECD. Rows with KRT3
mutations are shaded
characteristic of amyloid. Electron microscopy (EM) shows the amyloid deposits

just beneath the basal layer of the epithelium. The epithelium lacks a basal mem-
brane in the region of the amyloid, and collagen fibrils appear to be absent in this
region (Weber and Babel 1980). In addition, scanning electron microscopy of cor-
neas from patients with GDLD shows irregularity of the epithelial cells, which are
more elongated than the normal epithelial cells, easily detached from the surface of
the cornea, and with abnormal spaces or gaps in between cells (Kinoshita et al.
2000).
Genetics
Since GDLD is a rare disease with autosomal recessive inheritance, it was mapped
by combining ten families which were all consanguineous, consisting of a total of
13 affected and 11 unaffected members. The approach used was linkage mapping
with microsatellite markers located throughout the genome, combined with homo-
zygosity mapping. In the latter approach, one looks for homozygous regions that are
common among all affected individuals. The extremely rare nature of the disease
also made it likely that the same locus was involved in all families, which were
Japanese in origin—this made it possible to combine data from these families and
thus map the disease locus. The locus for GDLD was thus mapped to a 2.6-cM
interval on chromosome 1p (Tsujikawa et al. 1998). Analysis of 20 families showed
pathogenic mutations in the M1S1 gene [chromosome 1, surface marker 1; also
designated as TACSTD2 (tumor-associated calcium signal transducer 2); TROP2]
located in this region. M1S1 encodes a gastrointestinal tumor-associated antigen,
whose function is not well-understood. The protein has a signal sequence, a poten-
tial transmembrane domain, an EGF-like repeat, and a thyroglobulin repeat. Sixteen
of 20 Japanese families tested showed a common mutation consisting of a C>T
transition at nucleotide 352, predicting a truncation of the protein (Q118X). This
mutation occurred in a shared haplotype background in the aforementioned fami-
lies, suggesting that they had a common ancestry. Mutations in M1S1 have been
found in families with GDLD in various populations tested.
1.1.2.3 Lisch Epithelial Corneal Dystrophy (MIM 300778)

Lisch epithelial corneal dystrophy was described by Lisch and coworkers in 1992, as
a “band-shaped and whorled microcystic dystrophy of the corneal epithelium.” The
cornea shows gray, band-shaped, and feathery opacities that sometimes have whorled
patterns. The clinical and histological features of Lisch epithelial corneal dystrophy
were documented based on examination of eight individuals from four families and
recognized as distinct from the two other epithelial dystrophies—epithelial basement
membrane dystrophy and Meesmann’s dystrophy (Lisch et al. 1992). The band-
shaped opacities were evident in the paracentral cornea on direct slit lamp examina-
tion. On retroillumination, these opacities were seen to consist of clear, densely
crowded, intraepithelial blisters extending in some instances to the corneoscleral
limbus. Visual acuity was occasionally affected and treated by epithelial abrasion.
Genetics
Lisch epithelial corneal dystrophy was mapped to chromosome Xq22.3 in a large
family of 48 members. At the same time, linkage to the keratin genes K3 and K12,
associated with Meesmann corneal dystrophy, was excluded. Direct screening for
mutations in the hotspots in the K3 and K12 genes, also did not provide evidence for
involvement of the keratin genes in Lisch epithelial corneal dystrophy; these genetic
analyses demonstrated conclusively that Lisch epithelial corneal dystrophy was a dis-
tinct entity from Meesmann dystrophy, and not a variant of it, despite certain pheno-
typic similarities such as microcystic epithelial lesions, between the two dystrophies.
1.1.3 Corneal Stromal Dystrophies
1.1.3.1 Macular Corneal Dystrophy (MCD, MCDC1 [OMIM 217800])

MCD is a bilateral corneal disorder with fine, punctate corneal opacities developing
within the first decade of life. Opacities are at first located in the superficial layers
of the corneal stroma, and as the disease progresses, extend to the entire thickness
of the cornea, involving the central and peripheral cornea. Ultrastructural as well as
histochemical studies by Klintworth and Vogel (1964) showed the presence of
deposits and vacuoles within corneal fibroblasts. The deposits were identified on the
basis of staining with various dyes as acid mucopolysaccharide. These intracellular
accumulations are found in the cisternae of the endoplasmic reticulum and also
within cytoplasmic vacuoles. The deposits in MCD involve the Descemet mem-
brane and the corneal endothelium in addition to the corneal stroma. The fine struc-
ture of the collagen lamellae is largely intact. At least three different
immunophenotypes are recognized in MCD, based on the reactivity of the patients’
serum and corneal tissue to an antibody against sulfated epitopes on KS (Yang et al.
1988). MCD type I is characterized by an absence of antigenic KS in the cornea and
serum of patients; MCD type II has detectable antigenic KS in corneas and normal
or slightly reduced KS in serum; and MCD type Ia has an absence of antigenic KS
reactivity in serum and detectable KS only in corneal keratocytes. These different
immunophenotypes are, however, clinically indistinguishable. MCD type I is by far,
the most predominant type in different populations studied, with very few patients
being reported so far with types Ia and II.
Genetics
The involvement of keratan sulfate (KS) in the pathology of MCD was evident from
biochemical studies of MCD corneas using organ culture of corneas from patients
affected with MCD and those of unaffected controls (Hassell et al. 1980). There was
a lack of formation of mature keratan sulfate proteoglycan (KSPG) in corneas
affected with MCD. Specifically, the defect appeared to be an absence of sulfate
residues in the carbohydrate side chains from proteoglycan in the MCD-affected
corneas; second, the oligosaccharides in the MCD corneas were smaller than those
in the keratan sulfate side chains present in the normal control corneas. Subsequently,
the genetic locus for MCD types I and II was mapped to chromosome 16q22 in
families of American and Icelandic origin (Vance et al. 1996). Mutations in the gene
for carbohydrate sulfotransferase-6 (CHST6), located in the mapped interval, were
identified in both MCD types I and II by Akama et al. (2000). MCD type I is associ-
ated with mutations in the coding regions of CHST6, while in MCD type II, dele-
tions and rearrangements occur in the upstream region of the gene. The CHST6 gene
codes for the corneal N-acetyl glucosamine-6-O-sulfotransferase enzyme, respon-
sible for sulfation of C6 of N-acetyl glucosamine to form KS. KS is a major corneal
glycosaminoglycan and a component of the proteoglycans lumican, keratocan, and
mimecan. KS is hydrophilic and is required for maintaining proper hydration of the
cornea by imbibing water. The mutations associated with MCD result in loss of
function of CHST6, thereby leading to a failure of synthesis of KSPG in the cornea.
Several studies have found mutations in the CHST6 gene in MCD patients from dif-
ferent regions, indicating that the same locus is responsible for MCD in different
populations from across the world including Asian, North American, and European
(Sultana et al. 2003; Iida-Hasegawa et al. 2003; Aldave et al. 2004). A large number
of mutant alleles have been identified so far indicating the high degree of mutational
(allelic) heterogeneity in MCD. Overall, about 200 families with MCD from popu-
lations in different parts of the world have been analyzed for mutations in CHST6,
and over half of these are from India. The mutational spectrum in these different
studies consists of a predominance of missense mutations, found in over half of the
patients with MCD (Sultana et al. 2005). One-third of mutations are null mutations,
being either nonsense mutations, deletions, insertions or indels. Other types of
changes appear to be less frequent, being reported in very few cases. Also, due to the
occurrence of MCD mostly in consanguineous and inbred families, more than 90%
of all patients are homozygous for the mutations, while the rest are compound
heterozygotes.
1.1.3.2 F
leck Dystrophy (Central Cloudy Dystrophy of Francois;
Francois-Neetens Fleck Corneal Dystrophy; CFD, MIM
121850)
Manifestations
This disorder was originally described in two publications in French by Francois
(1956) in familial and sporadic cases, and further by Francois and Neetens (1957),
as central speckled corneal dystrophy. The cornea has lesions appearing on the slit
lamp, as cloudy gray areas (flecks) with indefinite structure and margins. The opaci-
ties may appear in childhood, are nonprogressive, and are located in the posterior
central stroma, with normal corneal stroma in between the lesions. Visual acuity is
not affected in most of the cases reported. The corneal epithelium and endothelium
are normal and the stroma is of normal thickness.
Genetics
Transmission of CFD is autosomal dominant, and the locus was mapped to a 24 cM
interval on chromosome 2q35 by linkage analysis of four affected families. Disease-
associated mutations in the PIP5K3 gene (also known as PIKFYVE; phosphoinosit-
ide kinase, FYVE-type zinc finger containing) were found in the families originally
mapped and in additional families with this phenotype. The PIP5K3 gene encodes
a member of the phosphoinositide 3-kinase family, responsible for the synthesis of
phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. This molecule is impli-
cated in regulating both endosomal sorting and transport of proteins from the early
endosomes to the trans-Golgi network (Rutherford et al. 2006).
1.1.4 C
orneal Dystrophies Associated with Mutations in the
Transforming Growth Factor Beta-Induced (TGFBI) Gene
The major dystrophies of the corneal stroma that have autosomal dominant inheri-
tance include the various types of lattice corneal dystrophy (LCD) and granular cor-
neal dystrophy (GCD). The LCDs and the GCDs are distinguished from each other
by the clinical appearance of the corneal opacities and the histopathologic appear-
ance and staining of the deposits in the stroma. The LCDs are characterized by the
presence of linear network of fine, branching opacities within the stroma. They typi-
cally arise within the first two decades of life and are slowly progressive, with an
increase in the number and density of the opacities with age. The GCDs manifest as
dot-like, rounded opacities that are described as having the appearance of bread-
crumbs, also developing during the first to second decades of life. By histopathology,
the deposits in LCD stain positive for amyloid, and are detectable with the Congo red
stain, giving a characteristic birefringence when viewed under polarized light. The
deposits in GCD are hyaline in nature, and are identifiable with a special stain,
Masson trichrome, giving a red color with this dye. Yet another variety of autosomal
dominant stromal dystrophy that has distinct histopathologic properties is Avellino
corneal dystrophy (ACD), which shows the presence of both amyloid and Masson-
positive deposits in the stroma. It therefore shares features of both LCD and GCD
and is also referred to as combined granular-lattice corneal dystrophy or as GCD type
2. It derives its name from the province of Avellino in Italy, as it was initially
described in families originating from Avellino. However, it has since been recog-
nized that this corneal dystrophy is found in other parts of the world as well and is
not unique to Avellino. The current designation for this dystrophy is GCD type II.
There are two other types of autosomal dominant stromal dystrophy that involve
the superficial layers, including the epithelium, Bowman’s layer, and anterior
stroma. These are Reis-Bücklers corneal dystrophy (RBCD or CDRB) and Thiel-
Behnke corneal dystrophy (TBCD or CDTB). They are similar in that the superficial
cornea is affected in both, and have thus been confused with each other, the two
names having been interchanged in the literature. However, the two disorders are
distinct in their clinical and microscopic features. The corneal deposits in RBCD
and TBCD show different properties on light and electron microscopy (see follow-
ing sections).
The genetic locus for GCD, LCD as well as ACD was mapped to the same region
on chromosome 5q by Stone and coworkers. Eight families, four with ACD, and two
each with LCD and GCD, including a total of 114 affected individuals, were
included in a linkage study using markers throughout the genome. The locus in all
eight families was mapped to chromosome 5q31, with significant linkage to the
same locus in each of the corneal dystrophies (Stone et al. 1994). The locus for
GCD type 1 was independently identified from the mapping of the disease in a
Danish pedigree of seven generations with over 100 individuals by Eiberg and
coworkers (Eiberg et al. 1994). The same locus was also confirmed in a separate
study on a family with RBCD consisting of 22 members with 11 affected persons.
These data suggested that the dystrophies that are grouped as LCD and GCD are in
fact allelic disorders, possibly arising from different mutations in the same gene
(Small et al. 1996).
The gene at the chromosome 5q31 locus for the abovementioned corneal dystro-
phies was isolated by physical mapping and cDNA selection. A YAC clone was
constructed encompassing the disease interval that was mapped in the various fami-
lies with LCD and GCD. Various cDNAs were screened for hybridization to the
sequences in the YAC clone, and analysis of the positive clones thus obtained identi-
fied a cDNA for BIGH3. Based on its localization to chromosome 5q31 and its
specific expression in the corneal epithelium and keratocytes, it was considered as a

suitable candidate gene for the corneal stromal dystrophies LCD and GCD. Screening
of the BIGH3 cDNA in frozen corneal tissue and in cultured cells of a patient with
GCD type I (CDGG1), led to the detection of a C to T change at cDNA position
1710, predicting a missense change of arginine 555 to tryptophan (Arg55Trp). None
of the unaffected family members of the 150 members of the normal control popula-
tion tested carried this change, thus confirming it as a disease-associated mutation.
Further mutations associated with the other types of dystrophies in this group
including LCD type I (CDL1), ACD, and CDRB were discovered upon sequencing
of the exons of the BIGH3 gene. The mutation of c417C>T leading to a change of
arginine-124 to cysteine (Arg124Cys) was found to be associated with CDL1, and
the identical mutation occurred in 15 CDL1 families tested. Two unrelated patients
with ACD showed a change of G to A at c418, corresponding to a change of argi-
nine-124 to histidine (Arg124His). A three-generation family with CDRB was
tested for mutations, and a change of arginine-555 to glutamine (Arg555Gln) was
detected in all affected individuals in the family but not in unaffected controls or in
those affected with the other forms of LCD. Thus, this study established the two
groups of mutations at Arg124 and Arg555 to be associated, each mutation with a
specific form of LCD or GCD tested. Moreover, the mutations were associated to a
particular disorder across multiple families. It must be noted here that the designa-
tion of “CDRB,” is, in retrospect, a misnomer since it has been established by vari-
ous studies that the Arg555Gln mutation occurs in patients with CDTB.
1.1.4.1 Transforming Growth Factor-Induced (TGFBI) Gene

The TGFBI gene encodes an extracellular matrix protein (TGFBIp), which is about
68 kilodaltons in size. Various designations have been used in literature to refer to
the TGFBIp. It is known as keratoepithelin, based on the idea that it is produced by
the corneal epithelium. However, it is now known that TGFBIp is produced by vari-
ous tissues apart from epithelia. TGFBIp is alternatively referred to as Arg-Gly-Asp
(RGD) collagen-associated protein “RGD-CAP” and MP78/70 (a component of
elastin-associated microfibrils). It was first isolated from a cDNA library from an
adenocarcinoma cell line, after treatment of the cells with transforming growth fac-
tor beta (TGF-beta), by Skonier and coworkers. The cDNA, designated as beta ig-h3
(BIGH3), encoded a protein of 683 amino acids, with an amino-terminal secretory
sequence, a carboxy-terminal RGD (Arg-Gly-Asp) motif that binds to integrins, and
four fasciclin-like domains that have homology to the fasciclin-1 of Drosophila
(Skonier et al. 1992). The expression of the BIGH3 gene was found to be induced
severalfold upon treatment of cells with TGF-beta and was thus designated as
BIGH3 (TGF-beta-induced gene h3). The BIGH3 cDNA was independently iso-
lated from the nonpigmented ciliary epithelial cDNA library, and the analysis of its
sequence showed it to be identical to the gene reported by Skonier and coworkers.
The BIGH3 mRNA was detected at a high level in the corneal epithelium, and the
protein was localized by immunodetection, to the surface of corneal epithelial cells.
A striking feature was that its expression is specific to the cornea among all the
ocular tissues (Escribano et al. 1994).
There are two arginine residues, Arg124 and Arg555, which are mutational
hotspots in the TGFBIp, associated with lattice corneal dystrophy (LCD) and granu-
lar corneal dystrophy (GCD) (Munier et al. 1997). Specific missense changes
involving these mutational hotspots are each associated with a particular form of
LCD or GCD in patients across different populations.
1.1.4.2 G
ranular Corneal Dystrophy Type III (Reis-Bücklers
Dystrophy; Cornea Dystrophy of Bowman Layer Type
I (CDB1; CDRB); Geographic Corneal Dystrophy; OMIM
608470)

Reis-Bücklers corneal dystrophy (CDRB) arises in the first decade of life as super-
ficial reticular opacities in the central cornea. It is associated with epithelial ero-
sions, leading to pain, redness, and photophobia. It is accompanied by reduced
vision in the second and third decades of life with a superficial corneal haze and an
irregular corneal surface. A clinico-pathologic study of CDRB in a large family and
review of the cases from various studies by Rice et al. (1968) found recurrent attacks
of pain and photophobia due to epithelial erosions, which were more frequent early
in life. The symptoms are evident from infancy, with recurrent attacks until the sec-
ond or third decades, after which they may become infrequent or cease altogether.
The region of the Bowman’s layer has opacities extending up to 2–3 mm from the
limbus. The opacification appears to progress with age.
Histopathological staining properties of the deposits in CDRB are those of gran-
ular corneal dystrophy, and stain positively with Masson trichrome, giving a char-
acteristic red color. They appear as rod-shaped bodies on electron microscopy. They
are seen predominantly in the anterior stroma.
Genetics
CDRB is associated with a specific mutation in the TGFBI (transforming growth
factor beta-induced) gene, which has a c.371G>T change leading to a missense
substitution of arginine-124 to leucine (Arg124Leu) in patients with this form of
corneal dystrophy, as observed in different populations. Other mutations in TFBIp,
such as p.Phe540del and p.Gly623Asp, have been associated with CDRB in the
literature. The Phe540del was reported in patients from Sardinia in Italy (Rozzo
et al. 1998). However, phenotype data as reported for patients with these two muta-
tions are not confirmatory for CDRB (Kannabiran and Klintworth 2006).
1.1.4.3 T hiel-Behnke Corneal Dystrophy [(CDTB, CDB2); Curly Fiber

Dystrophy; MIM 602082]
CDTB is another form of corneal dystrophy that involves the superficial cornea,
with epithelial erosions and honeycomb-shaped opacities in the subepithelial region.
It has been confused with CDRB in the literature due to the superficial location of
the corneal deposits. However, the deposits in CDTB do not stain red with Masson
trichrome, thus distinguishing it from CDRB. There is also an absence of amyloid
in the deposits. The cornea is characterized by a layer of fibrous tissue described as

a “sawtooth-like configuration” in the subepithelial region between the epithelium
and stroma and the presence of “curly fibers” beneath the corneal epithelium that
are only evident by transmission electron microscopy (Küchle et al. 1995). In addi-
tion, the epithelium shows vacuolization and degenerative changes, an absent base-
ment membrane, and mostly an absent Bowman’s layer. Since the major confirmatory
findings that are distinctive of CDTB rely on electron microscopy of the cornea, the
literature shows that this entity has often been confused with other corneal disorders
based on clinical evaluations, and these include CDRB as mentioned above, as well
as another entity, epithelial recurrent erosion dystrophy (ERED).
Genetics
CDTB is associated with the specific TGFBI mutation leading to a missense change
of arginine-555 to glutamine (Arg555Gln; R555Q), involving one of the two muta-
tional hotspots of the TGFBIp. The mutation was first reported by Munier et al.
(1997), although the disorder associated with this mutation was mistakenly desig-
nated by these authors as Reis-Bücklers corneal dystrophy. The same misdiagnosis
of CDRB was reported in another study of two families in which patients had differ-
ent types of corneal opacities and different mutations in keratoepithelin. One patient
with a corneal phenotype described as “honeycomb-shaped” opacities had the
Arg555Gln mutation, whereas a second patient had “geographic opacities” along
with recurrent epithelial erosions and progressive subepithelial opacification—a
novel mutation of Arg124Leu in keratoepithelin was identified in the second case.
The two mutations of Arg555Gln and Arg124Leu were considered as causing two
different variants of CDRB (Okada et al. 1998).
The assignment of a locus on chromosome 10, for CDTB was again, in retro-
spect, based upon a misidentification of the disease in a family in which the affected
members were reported to have honeycomb-shaped corneal opacities and the cor-
neal deposit-shaped like “curly fibers” upon EM (Yee et al. 1997). However, a sub-
sequent re-evaluation of clinical and microscopic features of the disease in this
family indicated that the phenotype was not consistent with CDTB, and hence the
chromosome 10q23 region is no longer considered as an authentic locus for this
disorder. The disease linked to the 10q locus is in fact has also been reported in
several families and is designated as ERED (see Box 1.2).
Box 1.2 Epithelial Recurrent Erosion Dystrophy (ERED)

ERED is a phenotype mapped to chromosome 10q23 by Yee et al. (1997). It
was designated as TBCD, but the diagnosis was revised upon re-evaluation of
the data.
The phenotype was in fact found to be highly variable in the original fam-
ily mapped to chromosome 10—the opacities ranged from “honeycomb”-like
reticular opacities consistent with Thiel-Behnke dystrophy in some to granu-
lar type of opacities in others and to small superficial vesicles in younger
subjects.
The corneal manifestations include early recurrent erosions, with diffuse

subepithelial opacification and scarring.
The gene responsible for the disease in this family was identified by whole
exome sequencing, as the COL17A1 gene.
A synonymous variant in COL17A1, c.3156C>T was found to be the
pathogenic variant in ERED. It interferes with correct splicing and thereby
has a possible pathogenic effect.
The ERED phenotype is reported to be common among families from
Sweden.
The mutation at this site is recurrent as reported in other unrelated families
with the same phenotype, including families from New Zealand, Australia,
and United Kingdom.
(Jonsson et al. 2015; Oliver et al. 2016).
1.1.4.4 G
ranular Corneal Dystrophies Types I and II
(MIM # 121900; 607541)

Granular corneal dystrophy (GCD) type I (also known as classic GCD, corneal
dystrophy Groenouw type I, CDGG1) develops within the first to second decades of
life, appearing as rounded, discrete opacities resembling bread crumbs or snow-
flakes, in the central cornea. The opacities do not usually extend to the peripheral
cornea. The region between the opacities is clear. GCD type I may present with only
a few opacities in childhood and vision is not affected, but they may become more
numerous and dense in older patients, sometimes merging with each other. The
deposits extracellular and accumulate in the superficial and mid-stroma; they are
eosinophilic in nature and stain positively with Masson trichrome, giving a bright
red color. The differences between the lesions in various stromal dystrophies can be
distinguished in corneal sections by routine histopathological techniques when
viewed under the light microscope (Jones and Zimmerman 1961). The deposits in
GCD appear as an aggregate of hyaline granules.
Granular corneal dystrophy type II (GCD type II) is also known as Avellino cor-
neal dystrophy or combined lattice-granular dystrophy. It is very variable in mani-
festation, but clinical signs that are noted as characteristic of GCD type II are
rounded grayish-white opacities in the anterior stroma, linear opacities in the deeper
stroma, and anterior stromal haze (Ferry et al. 1997). Granular deposits appear ear-
lier and are seen more commonly. Lattice lesions are present in some patients with
granular lesions. The extent of granular- and lattice-shaped opacities shows wide
variation within families, with younger members showing only granular opacities,
and lattice lines generally appearing in older individuals. Occasional histopatho-
logical data available in these families include the presence of granular material
staining with Masson trichrome at the level of the Bowman’s membrane, superficial
and mid-stroma, and fusiform deposits that are positive for Congo red stain in the
mid- and posterior stroma (Folberg et al. 1988; Rosenwasser et al. 1993). This
disorder was initially discovered in Italian patients from Avellino district in Italy but
has since been found to occur in patients from various regions of the world. It is the
most common form of stromal dystrophy in Japan and accounts for about 70% of all
autosomal dominant stromal dystrophies that are associated with mutations in the
TGFBI gene (Fujiki et al. 2001).
Genetics
Both types I and II of GCD are associated to specific hotspot mutations in the
TGFBI gene. In addition, mutant protein, TGFBIp, is present in the deposits in GCD
type I as seen by reactivity to a specific antibody (Klintworth 2009). GCD type I or
CDGG1 is associated with a missense change arginine-555 to tryptophan
(Arg555Trp), due to substitution of c1710C>T, while CDGG2 (GCD type II or
ACD) is due to a mutation of Arg124His in the same gene (Korvatska et al. 1998).
These same mutations have also been identified in patients with GCD from various
populations (reviewed by Kannabiran and Klintworth 2006).
1.1.4.5 Lattice Corneal Dystrophy (LCD)

The lattice corneal dystrophies are a group of autosomal dominant disorders that are
characterized by the presence of amyloid in the corneal stroma. As the name sug-
gests, the opacities in the LCDs are typically seen as a linear branching network that
is radially oriented. The deposits are fusiform in shape and stain positively for amy-
loid. They have a characteristic appearance upon staining with Congo red and show
apple-green birefringence when viewed in polarized light. Lattice corneal dystro-
phy type I (LCD type I; CDL1; MIM 122200) is the first form of LCD to be
described. It arises in the first to second decades of life as a fine, linear network of
branching opacities in the anterior stroma. LCD type I is associated with mutation
at one of the two hotspots of the TGFBI gene-arginine-124 to cysteine (Arg124Cys).
It was first reported by Munier and coworkers (Munier et al. 1997). Since then, LCD
type I has been found to be associated with this mutation in various other regions
across the world.
Apart from LCD type I, other forms of LCD are recognized based on their clini-
cal and histopathological features. These include LCD types II, III, IIIA, and
IV. Among these, LCD type II (MIM 105120) involves systemic amyloidosis and is
also known as Meretoja syndrome. It is associated with mutations in the gelsolin
gene and will not be discussed here as it is not part of the group of TGFBI-associated
corneal dystrophies. Several mutations in TGFBI have been discovered till date for
the LCD types III and IV, which show some mutational heterogeneity as compared
with LCD type I. The range of mutations associated with these types of LCD
involves residues in the fourth Fas1 domain of the TGFBI protein; these residues are
located in exons 11–14 of the gene.
LCD type III/IIIA is characterized by thick, ropy linear opacities in the mid-
stroma, corneal erosions, and a late onset of disease. An affected family that was
designated as type III, originally described by Hida et al. (1987), had members in
only one generation affected, and was considered to have autosomal recessive inher-
itance. The genetic basis for LCD type III is unknown. A related variant of LCD,
termed LCD type IIIA (CDL3A; MIM 608471), has been described, having an
autosomal dominant mode of inheritance and associated with mutations in TGFBI.

A few different mutations are reported from patients with LCD type IIIA, and these
include the missense changes of proline-501 to threonine (Pro501Thr), asparagine-
622 to lysine (Asn622Lys), and a single base deletion leading to frameshift at
valine-627 (c.1879delG; Val627fs).
LCD type IV is a subtype of LCD with deep stromal opacities, in contrast to the
opacities in the anterior stroma in other forms of LCD. It is characterized by a late-
onset of disease, in about the fifth to sixth decades of life. Only a few reports of
patients with these features are available so far, and mutations in these patients are
leucine-527 to arginine (leu527Arg) and arginine-544 to serine (Arg544Ser).
Other unusual phenotypes of LCD have been reported and are associated with
different mutations in TGFBI. These disorders have clinical features that do not fit
the abovementioned types and hence cannot be strictly classified as any of those
types. Some of the phenotypes of “atypical” LCD are described as “intermediate,”
with features overlapping both types I and IIIA with respect to the appearance of the
lattice lines, age of onset, etc. These are designated as LCD types I/III and have
been identified in patients with mutations in TGFBI other than those in the Arg124
and Arg555 residues. Thus the mutations of asparagine-622 to histidine (Asn622His)
and histidine-626 to arginine (His626Arg) are reported in patients with LCD type 1/
III involving an asymmetric progression of the opacities. Other mutations reported
for the “intermediate” forms of LCD type I/type III include glycine-623 to aspartic
acid (Gly623Asp) and histidine-626 to proline (His626Pro) (Munier et al. 2002).
1.1.5 Corneal Endothelial Dystrophies
1.1.5.1 Congenital Hereditary Endothelial Dystrophy (CHED)

CHED involves bilateral, diffuse corneal edema manifesting at birth or early child-
hood. Based on studies in families, autosomal dominant and recessive inheritances
were recognized for CHED, and attempts were made to document clinical differ-
ences between the dominant and recessive forms (Kirkness et al. 1987). The essen-
tial manifestations of CHED are bilateral corneal edema within the first year of life,
without any other abnormality in the anterior segment. Microscopic changes include
abnormal irregularly shaped endothelial cells, atrophy of the epithelium, subepithe-
lial fibrosis, and loss of the Bowman’s membrane. The Descemet’s membrane is
markedly thickened (18–20 μM as compared with a normal thickness of 7–8 μM)
with the formation of a posterior collagenous layer in some cases. The endothelium
shows loss of cells and degeneration. Electron microscopy reveals thickening of the
Descemet’s membrane especially in the region of the posterior non-banded zone
and the formation of a fibro-collagenous layer.
Genetics
Autosomal dominant CHED (AD-CHED; CHED1) and posterior polymorphous
corneal dystrophy (PPCD) were mapped to the pericentromeric region of chromo-
some 20 by different studies, demonstrating overlapping regions on chromosome
20 for the two disorders (Toma et al. 1995). Due to an overlap of the clinical char-
acteristics of AD-CHED with PPCD, it was suggested that AD-CHED is not a
distinct entity and that the corneal disease in families described as AD-CHED
might actually be posterior polymorphous corneal dystrophy (PPCD) (Aldave
et al. 2013). A subsequent re-evaluation of the original British family with
AD-CHED extended the original pedigree (Davidson et al. 2016). Clinical mani-
festations included corneal haze and photophobia within 1 year of age, in the
absence of raised intraocular pressure or iris abnormalities. A whole genome
sequencing approach in this region of chromosome 20 led to identification of the
gene as OVOL2 (ovo-like 2), encoding a zinc finger transcription factor which
regulates mesenchymal-to-epithelial transition (MET). Analyses of the British
family along with Czech families with the same phenotype showed that mutations
in the promoter of the OVOL2 gene are associated with the CHED phenotypes in
these cases. The effect of the promoter mutations is a deregulation of its activity,
with the putative consequence of abnormal gene expression during corneal
development.
The locus for AR-CHED (CHED2; MIM 217700) was excluded from the locus
for CHED1/PPCD on chromosome 20q and mapped to an 8 cM interval on chromo-
some 20p in a large consanguineous family of Irish descent (Hand et al. 1999).
Subsequently, the CHED2 locus was refined to a 2 Mb region on chromosome 20p,
and mutations in the SLC4A11 gene (solute carrier family 4, member 11), which
encodes a sodium-borate cotransporter [designated alternatively as bicarbonate
transporter-related protein-1 (BTR1); sodium-coupled borate cotransporter 1
(NABC1)] were found in association with AR-CHED in families from Myanmar,
Pakistan, and India (Vithana et al. 2006). There is extensive mutational heterogene-
ity in SLC4A11. Close to 80 different mutations in SLC4A11 are reported in litera-
ture (listed by Kodaganur et al. 2013).
1.1.5.2 Posterior Polymorphous Corneal Dystrophy (PPCD)

PPCD manifests with the fundamental defect in the endothelial cells, which present
with an epithelial-like morphology. Though there is clinical heterogeneity in PPCD,
characteristic features are the presence of bullous lesions on the posterior corneal
surface, diffuse stromal and epithelial edema, thickened Descemet’s membrane, iris
adhesions to the cornea, and associated glaucoma. The age at onset may be very
variable, with complaints of a painless decrease in vision occurring anytime between
the first to the fifth decades of life. The degree of visual loss is also variable and can
range from mild to severe loss of vision (Krachmer 1985). In severe cases, scarring
and degenerative changes occur with calcific and lipid degeneration, obscuring the
view of the posterior cornea.
Genetics of PPCD
PPCD is transmitted as an autosomal dominant disorder that is genetically
heterogeneous with three loci known so far. These are PPCD1 in the pericentro-
meric region of chromosome 20, PPCD2 on chromosome 1p34, and PPCD3 on
chromosome 10.
Various studies mapped the PPCD1 locus to chromosome 20, within an interval
of 1.8 Mb (Héon et al. 1995; Gwilliam et al. 2005; Yellore et al. 2007). However,
screening of the exonic regions of all genes in this mapped region failed to identify
the causative variants for PPCD at this locus (Aldave et al. 2013). The PPCD1 gene
was identified eventually by the method of whole genome sequencing (WGS) in a
study on British and Czech families with PPCD. The pathogenic sequence variants
were found in the promoter of the Ovo-Like 2 (OVOL2) gene (Davidson et al. 2016).
Essentially, analysis of noncoding regions such as intergenic and promoter regions
of genes in the critical interval for PPCD on chromosome 20 revealed disease-
associated mutations in the promoter of the OVOL2 gene in multiple families. The
effect of these PPCD-associated mutants of OVOL2 was demonstrated to be an
increase in the promoter activity by cloning and expression of the promoter mutants
in cell lines.
The OVOL2 gene encodes a transcription factor that belongs to a large family of
proteins with homology to Drosophila Ovo. The OVOL (Ovo-like) proteins in ver-
tebrates are found to share a common domain with a set of four highly conserved
C2H2-type zinc finger motifs, each of which is 23–24 amino acids long. OVOL2 is
implicated in mesenchymal-to-epithelial transition (MET) via repression of ZEB1
activity. Note that this process of MET is the converse of epithelial to mesenchymal
transition (EMT), which is stimulated by ZEB1 activity. Expression of the OVOL2
gene is not detected in the normal human corneal endothelium in the fetal or adult
stages or in stromal fibroblasts. On the other hand, it is reported to be expressed in
epithelial tissues from various organs including the human cornea (Li et al. 2002).
These observations imply a pathogenic mechanism in PPCD in which the OVOL2
promoter mutations found in affected individuals lead to an increased and deregu-
lated expression of OVOL2 in the corneal endothelium, along with an increased
repression of ZEB1. Loss of ZEB1 expression in the PPCD corneas and an increase
in OVOL2 would be expected to lead to epithelization of the endothelial cells,
which is the phenotype of PPCD corneas.
The COL8A2 gene has been reported as the PPCD2 locus, but there is not much
evidence available so far to support the role of COL8A2 in PPCD, since variants
were reported in only two affected individuals (Biswas et al. 2001); further, it is
unclear whether these variants are pathogenic. Subsequent studies on PPCD patients
did not find any disease-associated variants in COL8A2 (Kobayashi et al. 2004;
Yellore et al. 2005).
PPCD3 was mapped to chromosome 10p11 in a multigenerational family
(Shimizu et al. 2004). Evaluation of the ZEB1 gene, considered as a positional can-
didate for PPCD3, showed nonsense and frameshift mutations in five families with
PPCD, including the original family that was mapped to PPCD3 locus (Krafchak
et al. 2005). Several mutations in ZEB1 are reported in patients from other popula-
tions (Table 1.2); in these cases as well, there are predominantly nonsense and
frameshift mutations. The ZEB1 gene encodes the two-handed zinc- finger home-
odomain transcription factor 8 (TCF8/ZEB1), which induces epithelial to mesen-
chymal transition (EMT) in various cell types, and is thought to promote tumor
invasion and metastasis by inducing EMT.
Table 1.2 Mutations identified in PPCD3

TCF8/ZEB1 mutation (cDNA) TCF8/ZEB1 mutation (amino acid) Reference
c.2916-17delTG Ser972fsX56 Krafchak et al. (2005)
c.1332-35delCAAT Ile444fsX22
c.1350C>T Gln451X
c.1578-79insG Gly524fsX2
c.2184G>T E728X
c.2324-2325dupA E776fs Liskova et al. (2007)
c.2157C>G Y719X
c.1124delT F375fs
c.1387_1390delCCTT P463fs
c.953_954insA Gln310fsX26 Aldave et al. (2007)
c.1506dupA Gln495fsX10
c.1592delA Glu523fsX4
c.3012_3013delAG Thr996fsX8
c.58C>T Gln12X
c.664C>T Gln214X
c.997C>T Arg325X
c.26T>G Met1Arg
The table lists several mutations identified in the ZEB1 gene in PPCD3 families. The mutations are
all shown as cDNA and amino acid changes. References of the studies that reported the mutations
are in the right-hand column
1.1.5.3 Fuchs Endothelial Corneal Dystrophy (FECD)

Fuchs endothelial corneal dystrophy is the most common form of endothelial dys-
trophy in various parts of the world. It has a prevalence ranging from about 3% in
the Japanese (Kitagawa et al. 2002) to 9% in the Icelandic population (Zoega et al.
2006). It has a female preponderance in all populations studied. It is characterized
by the progressive loss of corneal endothelial cells, irregularities in the size and
shape of remaining cells, and the development of “guttae” or excrescences in the
Descemet’s membrane. With increasing loss of endothelial cells, there is a concomi-
tant increase in the formation of guttae. The endothelium eventually loses its ability
to pump excess water from the cornea, thereby leading to edema, corneal clouding,
and loss of vision. FECD progresses through four clinical stages that are graded
according to severity. In stage 1 FECD patients are asymptomatic but have guttae
that are non-confluent and visible on microscopy. Stage 2 consists of more confluent
guttae, loss of endothelial cells, and changes in the size and shape of the cells. Stage
3 is accompanied by the appearance of corneal edema due to loss of endothelial
function. Stage 4 involves chronic edema, scarring, and opacity of the cornea,
accompanied by loss of vision. FECD is treated by corneal transplantation or by
Descemet’s stripping endothelial keratoplasty (DSEK). FECD is most commonly a
late-onset disease developing in the fifth decade or after, and about 50% of cases
show familial disease. It is autosomal dominant with incomplete penetrance. The
late-onset form of FECD is genetically heterogeneous, and multiple loci are associ-
ated to the disease. A rarer early-onset FECD, with onset in infancy, or within the
first decade of life, is a familial disease with autosomal dominant inheritance.
Table 1.3 Types of FECD and their genetic loci

OMIM
nomenclature Chromosome Locus/gene Onset
FECD1 1p34.3 FECD1/PPCD2/COL8A2 Infancy
FECD2 13pter-q12.13 FECD2/gene not known Late-onset
FECD3 18q21.2 FECD3/TCF4 (SEF2, ITF2, PTHS) Late-onset
FECD4 20p13 FECD4/CHED2/ CDPD/SLC4A11 (BTR1, Late-onset
NABC1)
FECD5 5q33.1-q35.2 FECD5/gene not known Late-onset
FECD6 10p11.22 FECD6/PPCD3/ZEB1 (TCF8, NIL2A) Late-onset
FECD7 9p24.1-p22.1 FECD7/gene not known Late-onset
FECD8 15q25.3 FECD8/AGBL1 (CCP4) Late-onset
The data above is taken from OMIM [http://omim.org/]. The numbers FECD1-8 refer to phenotype
designations of different forms of the disease. Different designations of the same locus are sepa-
rated by a slash “/.” Alternative symbols for the genes in column 3 are given in parentheses
Various forms of early- and late-onset FECD have been reported, and each form
is associated with a particular genetic locus (shown in Table 1.3).
Genetics of FECD
Early-Onset FECD
The locus for early-onset FECD (FECD1) was mapped to chromosome 1p34-p32 in
a pedigree with autosomal dominant transmission of the disease. The mapped interval
of about 7–8 cM included the gene for the alpha 2 chain of type VIII collagen
(COL8A2) (Biswas et al. 2001), which was considered as a positional candidate for
FECD1 since this form of collagen is a component of the Descemet’s membrane. A
mutation of pGln455Lys was detected in the family that was originally mapped, as
well as in two other families with early-onset FECD, and in one family with
PPCD. Three of the four families with this mutation were from Northern England and
shared a common haplotype at this locus. In addition, screening of the COL8A2 gene
in a series of probands with familial and sporadic forms of FECD revealed missense
mutations in about 8% of 116 probands tested (Biswas et al. 2001), although some
missense changes (such as Arg155Gln and Arg434His) that were detected in sporadic
cases were not confirmed to be pathogenic since they were also found in normal con-
trols. Overall, mutations in COL8A2 appear to be associated with early-onset FECD
that is distinct in its phenotype from the more common, late-onset, sporadic form.
Late-Onset FECD
Genes that have been evaluated for disease-associated variants in late-onset FECD
are SLC4A11, ZEB1, and TCF4. As seen from the foregoing section, mutations in
one gene can be associated with more than one type of corneal endothelial dystro-
phy, thus suggesting that the different CEDs are allelic conditions having a spec-
trum of phenotypes, rather than being genetically separate entities. Based on this
rationale, genes already known to have mutations in one CED have been explored
as candidate genes in other CEDs.
Table 1.4 Mutations in the SLC4A11 gene in AR-CHED families from India
Amino acid
Mutation (cDNA) change Comments References
c.2240+1G>A – North Indian families. Splice site Paliwal et al.
change detected in 1 family (2010)
c.2470G>A Val824Met North Indian. Detected in four
families
c.1156T>C Cys386Arg North Indian. Detected in two
families
c2518-c2520 delCTG Leu840del North Indian. One family
c.1831T>C Cys611Arg South Indian consanguineous Kodaganur
c.1249G>A Gly417Arg families. Gly417Arg found in two et al. (2013)
c.2170C>G His724Asp families. Others in one family each
c.785C>T Thr262Ile
c.2606G>A Arg869His
c.427G>A Glu143Lys Arg755Trp found in two families Ramprasad
c.1156T>C Cys386Arg et al. (2007)
c.2263C>T Arg755Trp
c.2264G>A Arg755Gln
c.2318C>T Pro773Leu Found in one family each Hemadevi
c.2618T>C Leu873Pro et al. (2008)
c.478G>A Ala160Thr
c.1156T>C Cys386Arg
c.859_862delGAGA E287fsX21 Kumar et al.
insCCT (2007)
c.2014_2016delTTC F672del
Several mutations found in representative studies on Indian families are shown in the above table
Pathogenic variants in the SLC4A11 gene are reported in 10% or less of patients
with late-onset FECD in patients from various populations including Chinese,
Indian, and European (FECD4 in Table 1.4) (Vithana et al. 2008; Riazuddin et al.
2010a; Soumittra et al. 2014; Hemadevi et al. 2010).
Mutations of the ZEB1 gene (FECD6 in Table 1.3) are reported in 1–2% of
patients with late-onset FECD. All ZEB1 mutations so far associated with FECD are
missense changes (Mehta et al. 2008; Riazuddin et al. 2010a, b). In contrast, ZEB1
mutations associated with PPCD are predicted to encode proteins with truncation or
premature termination. These observations have led to a model of genotype-
phenotype correlation in which ZEB1 mutations with ostensibly milder effects
(leading to proteins with reduced or partially impaired activity such as missense
mutants) lead to the milder phenotype of FECD, and those of severe impact (pro-
ducing null alleles or complete absence of function) are associated with PPCD
which has a severe phenotype.
In addition, there is possibly another locus for late-onset FECD (FECD7) on
chromosome 9p as suggested by a study of a large family, in which about half of all
affected individuals carried a missense allele in ZEB1, glutamine-840 to proline
(Gln840Pro; Q840P) (Riazuddin et al. 2010b). Analysis on a conditional model,
based on the premise that another disease locus is involved in individuals lacking a
ZEB1 mutation, yielded significant linkage to a locus on chromosome 9p. The gene
in the chromosome 9p locus is not yet identified.
The transcription factor 4 (TCF4, E2-2; MIM 602272) gene encodes the E2-2
protein which is a member of a family of ubiquitous transcription factors, the
basic helix-loop-helix (bHLH) proteins, involved in cell growth and differentia-
tion, and particularly, in EMT. A genome-wide association study on European
subjects with FECD (designated FECD3) found significant association with a
region on chromosome 18q21.2 that spans the TCF4 gene. Particularly, four
SNPs were independently associated with FECD3 (rs17595731, rs613872,
rs9954153, and rs2286812) (Baratz et al. 2010). The association of SNPs at this
locus was also validated in another large study (case-control) population, as
well as by family-based linkage studies (Li et al. 2011). Meta-analysis of data
from several individual association studies of FECD3 confirmed significant
association with the aforementioned SNPs in TCF4- (Li et al. 2015). By implica-
tion from these data, it is possible that regulatory pathways influenced by E2-2
are disrupted in the disease process in FECD.
Another marker that is associated with FECD3, also located within the TCF4
gene, is a trinucleotide repeat expansion containing CTG repeats (CTG18.1),
reported to be unstable in about 3% of the normal population. This repeat sequence,
present in the third intron of TCF4, is in linkage disequilibrium with an SNP,
rs613872. Association of CTG18.1 with FECD was first demonstrated in Caucasian
patients, and a strong association was observed with FECD of repeat lengths over a
threshold of 50 repeat units—about 79% of cases had a repeat length of over 50
repeats, while 3% of normal controls showed repeats that crossed this threshold
(Wieben et al. 2012). The effect of the triplet repeat expansion in FECD was further
established in a large study of over 500 cases. A threshold of 40 repeats or above
was found in about 60% of cases and 3% of controls, all heterozygotes for the
expansion. Two percent of cases and none of the controls were homozygous for the
repeats. Disease severity was correlated to the presence of the repeat allele and its
dosage, i.e., homozygotes for the repeat allele were more severely affected than
heterozygotes (Vasanth et al. 2015).
As shown in Table 1.3, there are three mapped loci for FECD (i.e., FECD2,
FECD5, and FECD7), but the genes involved in these cases are not known as yet.
1.1.5.4 G enetics of Corneal Endothelial Dystrophies in Indian

Patients
Among the CEDs, autosomal recessive CHED (AR-CHED; CHED2) is extensively
studied in Indian families. The mapping as well as identification of disease-
associated mutations was facilitated by the availability of several affected families,
many of which were consanguineous. Data available so far suggests that AR-CHED
is associated with a single locus on chromosome 20p13, and mutations in the sodium
bicarbonate transporter-like protein gene (solute carrier family 4, member 11;
SLC4A11) are responsible for the phenotype. Linkage mapping of families with
AR-CHED was used to map the disease gene by combined analysis of several
1.2 Keratoconus 23
families, since each family is typically small with one to two affected individuals,
and lacks sufficient power for detecting linkage. Pooling of families was also fea-
sible since almost all families with AR-CHED appear to map to the same locus,
without evidence of heterogeneity.
The CHED2 locus was mapped to a 1.3 Mb (2 cM) interval on chromosome
20p13 by linkage analysis of 16 consanguineous families from South India (Jiao
et al. 2007). Screening of the SLC4A11 gene in the mapped interval revealed
disease-associated changes in 12 families. The mutations are distributed through-
out the length of the gene and include missense, nonsense, and frameshift muta-
tions. Further analysis of 42 families showed extensive mutational heterogeneity,
with all the abovementioned types of mutations being equally prevalent (Sultana
et al. 2007). Attempts to find correlations between genotype and phenotype, i.e.,
types of mutations with respect to their predicted consequence or their location
in the protein with the clinical and histopathological characters of the disease in
the patients having particular mutations, such as age at first corneal graft, post-
operative visual acuity, and thickness of the cornea and Descemet membrane,
showed that no correlations could be found. Mutational heterogeneity in
AR-CHED was observed in patients from various regions of India; some repre-
sentative examples of mutations reported in Indian patients are shown in Table 1.4
(see also Sultana et al. 2007).
1.2 Keratoconus
Keratoconus involves corneal thinning (ectasia) leading to conical protrusion of the

cornea, with changes in refractive powers, astigmatism, and diminished vision. The
onset is usually in the second to third decades, and it often occurs as a sporadic
disease. It can present in more subtle forms that show changes in corneal topogra-
phy, which are not readily diagnosed by clinical examination. The designation of
“forme fruste keratoconus” is applied when one eye is affected, and the fellow eye
displays changes in corneal topography with no evident clinical signs. The term
“keratoconus suspect” implies that one eye has only topographic changes and the
fellow eye is normal.
Differences in the prevalence of keratoconus in males versus females have been
noted, but these patterns are not consistent between different studies (Georgiou
et al. 2004; Li et al. 2004). Environmental risk factors that predispose to develop-
ment of keratoconus are well known. They include eye rubbing, atopy, contact lens
wear, and oxidative damage triggered by UV exposure. It is a disease that has a
complex and multifactorial etiology, and both environmental and genetic factors are
thought to play a role. The magnitude of each type of factor may differ between
individuals. Thus, environmental influences may be predominant in some individu-
als with keratoconus, while genetics may be the major contributory factor in others.
A genetic etiology in keratoconus is suggested by its higher prevalence in Asians
than in Caucasians (Pearson et al. 2000), and also a higher risk among first-degree
relatives of affected individuals, as compared with the general population. Further
evidence for a genetic contribution to the disease is provided by twin studies show-
ing a greater concordance of keratoconus in mono- versus dizygotic twins (Chang
and Chodosh 2013).
Essentially, about 15% or fewer of keratoconus patients have an affected family
member (Wheeler et al. 2012). Familial disease shows Mendelian transmission, with
autosomal dominant or recessive modes of inheritance. However, keratoconus as it
commonly occurs has a complex multifactorial etiology and affects mostly sporadic
(isolate) cases. Genetic studies in families and sporadic cases with keratoconus have
used linkage mapping, screening of candidate genes, and genome-wide association
studies (GWAS) to map susceptibility loci. GWAS is a model-free approach in which
no assumptions are made about the underlying genes or pathways involved in the
disease. These studies may provide leads for further evaluation of genes based on the
association signals obtained (Burdon et al. 2011; Li et al. 2012).
1.2.1 Mapped Loci for Keratoconus
Keratoconus is genetically heterogeneous, and a large number of loci have been

mapped in affected families. Some loci have been confirmed independently in sepa-
rate studies and are mostly associated with autosomal dominant keratoconus (shown
in Table 1.5; Wheeler et al. 2012). The keratoconus-affected families that are
mapped belong to various ethnicities including European, Asian, Australian, and
Middle Eastern. In addition, non-Mendelian inheritance is reported in several fami-
lies; association with the disease has been mapped to various chromosomes includ-
ing 4, 5, 9, 12, and 14 using nonparametric methods on sib pairs (Li et al. 2006).
Evidence for digenic inheritance involving loci on chromosomes 1 and 8 was found
in an Australian pedigree (Burdon et al. 2008). Genes for the mapped loci are not
known, except VSX1 for KTCN1.
Table 1.5 Mapped loci for keratoconus

Locus Inheritance Gene References
20p11.21 AD Visual system homeobox Héon et al. (2002)
gene 1 (VSX1)
16q22.3-q23.1 AD Not known Tyynismaa et al. (2002)
3p14-q13 AD Not known Brancati et al. (2004)
2p24 AD Not known Hutchings et al. (2005)
5q14.1-q21.3a AD Not known Tang et al. (2005) and
Bisceglia et al. (2009)
9q34 Complex Not known Li et al. (2006)
13q32 AD Not known Gajecka et al. (2009)
14q24.3 AD Not known Liskova et al. (2010)
1p36 and 8q13 Digenic Not known Burdon et al. (2008)
The details of various loci mapped so far are shown, with the references of the same
The locus on chromosome 5q was mapped independently in two studies
a
References 25
1.2.2 Genes Associated with Keratoconus
The VSX1 gene (visual system homeobox, gene 1; also known as RINX {retinal
inner nuclear layer homeobox}) encodes a paired-like homeodomain protein
expressed in the neonatal cornea, as well as the retina, in the inner nuclear layer. It
was first identified to have mutations in keratoconus and posterior polymorphous
dystrophy (Héon et al. 2002); missense changes were detected in two out of 66
patients with keratoconus. Subsequent studies have also reported VSX1 mutations in
keratoconus but in very few cases (Bisceglia et al. 2005; Dash et al. 2010).
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Genetics in Cataracts
2
A cataract is a lens opacity, which can develop at any stage of life from infancy to
late adulthood. According to the age at onset, cataracts may be congenital or infan-
tile (at birth or within 1 year of life), juvenile (in childhood or adolescence), prese-
nile (in adulthood, before 45 years of age), or senile (after 45 years of age).
2.1 Congenital Cataracts
Congenital cataracts can be hereditary in nature or secondary to various insults in

intrauterine and early postnatal period. The latter include congenital rubella infec-
tion, diarrhea or dehydrational crises, and trauma. Hereditary cataracts constitute
between 10 and 30% of congenital cataracts in various populations and are reported
to be within the same range in India (Eckstein et al. 1996). Congenital hereditary
cataracts are most commonly inherited as Mendelian traits, with autosomal domi-
nant, autosomal recessive, or in rare cases, X-linked transmission. They are highly
heterogeneous both clinically and genetically. They are described according to the
shape and location of the opacity—i.e., pulverulent, stellate, coralliform, cerulean,
pouch-like, etc. based on shape and polar, subcapsular, lamellar, nuclear, sutural, or
total cataract based on location within the lens. Mutations in the same genes give
rise to cataracts with diverse morphologies, and conversely, similar phenotypes of
cataract can result from mutations at different loci. Though hereditary congenital
cataracts may be associated with various birth defects and developmental syn-
dromes, they occur in isolation in the majority of cases (Shiels and Hejtmancik
2015). Some major syndromes involving congenital cataracts are listed below:
• Chondrodysplasia syndrome
• Conradi-Hünermann syndrome
• Down syndrome (Trisomy 21)
• Ectodermal dysplasia syndrome
• Galactosemia

32 2 Genetics in Cataracts
• Marfan syndrome
• Hallermann-Streiff syndrome
• Lowe syndrome
• Marinesco-Sjögren syndrome
• Pierre-Robin syndrome
• Trisomy 13
2.1.1 Genetics of Congenital Cataract
Genes associated with congenital cataracts are summarized in Table 2.1, and pro-
teins encoded by these genes are functionally diverse and include lens structural
proteins/crystallins, gap junction proteins, transcription factors, membrane proteins,
water channels, heat shock proteins, etc. Mutations in over 45 genes are known to
be associated with familial congenital cataracts till date. Details of mutations in
each of the genes associated with human hereditary cataracts have been catalogued
in the CatMap database [available at http://cat-map.wustl.edu/; accessed November
2018].
Mutations in genes encoding crystallins are a predominant cause of familial con-
genital cataracts. The crystallins constitute the most abundant soluble proteins in the
lens, making up over 90% of lens soluble proteins. They are divided into three major
groups—alpha (α-), beta (β-), and gamma (γ-) crystallins, which are found in all
vertebrates. The α-crystallins are members of the heat shock family of proteins and
consist of two subunits—αA- and αB-crystallins, encoded by separate genes. Both
αA- and αB-crystallins are expressed in lens as well as in other non-lenticular tis-
sues (Hejtmancik et al. 2015). The beta- and gamma-crystallins belong to a super-
family and are made up of a common structural unit known as the Greek key motif.
Each Greek key motif is a four-stranded antiparallel β-sheet structure. Two such
motifs make up a globular domain. The β-crystallins consist of the acidic (βA1/A3,
βA2, and βA4) and basic (βBl, βB2, and βB3) β-crystallins, based on the overall
charge on the protein. Each of the proteins is encoded by a separate gene except for
βA1/A3-crystallin, which arises from a single gene with two different initiation
codons. The β-crystallin proteins exist as oligomers ranging from 40 to 200 kDa.
The γ-crystallins are monomeric proteins of about 20 kDa and consist of seven
members encoded by separate genes. They are the γA- to γF-crystallins and
γS-crystallin. The genes for γA-γF-crystallins are located as a cluster on chromo-
some 2, while that for γS-crystallin is on chromosome 3. Though the γ-crystallins
possess the conserved structural unit, i.e., the two globular domains, they differ
from the β-crystallins in that the linker peptide between the two domains is folded.
This leads to an intramolecular interaction between the globular domains, and to the
compact, monomeric structure of γ-crystallins. They are present in the lens nucleus,
where they are packed at high densities, making up the hardest part of the lens. The
γS-crystallin is highly conserved in evolution and considered as having features of
both the γ-crystallins and the β-crystallins, although it has more sequence identity
with the γ-crystallins than the beta-crystallins. It is found in more hydrated regions
2.1 Congenital Cataracts 33
Table 2.1 Mutations in crystallin genes associated with congenital cataracta, b

Chromosomal
Gene locus Inheritance Mutation Phenotype Reference
Alpha 21q22 AD Arg116Cys Microphthalmia, Litt et al.
A-crystallin microcornea (1998)
AR Trp9Ter Not available Pras et al.
(2000)
Alpha 11q22.3– AD Arg120Gly Posterior polar Berry et al.
B-crystallinb q23.1 cataract (2001)
Beta A3/ 17q11–12 AD c.215+1G>A Zonular-sutural Padma et al.
A1-crystallin (IVS3 cataract (1995) and
+1G>A) Kannabiran
et al. (1998)
Beta 2q35 AD Val50Met Multifocal Reis et al.
A2-crystallin congenital (2013)
cataract,
incomplete
penetrance
Beta 22q12.1 AD Phe94Ser Lamellar cataract Billingsley
A4-crystallin Microphthalmia et al. (2006)
Gamma 2q33 AD g.67delG Lamellar, anterior Al-Fadhli
B-crystallin (intron 1) and polar, and et al. (2012)
g.167delC complete
(exon 2) cataracts
Gamma 2q33 AD Thr5Pro Coppock-like Lubsen
C-crystallin cataract et al. (1987)
and Héon
et al. (1999)
Gamma 2q33 AD Arg15Cys Progressive, Stephan
D-crystallin early-onset et al. (1999)
punctuate
cataract
Arg58His Aculeiform Héon et al.
cataract; same (1999)
mutation found in
three families
with the
phenotype
a
Mutations in this table refer to the first mutations reported in each gene, with the references for the
same in the last column. Missense mutations are designated by amino acid residues affected, while
deletion and intronic changes are designated according to genomic DNA or cDNA residues involved.
AD and AR indicate autosomal dominant and autosomal recessive inheritance, respectively
b
Also reported to have missense mutation in a family with desmin-related myopathy (Vicart et al.
1998). No mutations are known in the gamma A-crystallin gene in human cataracts at the time of
compiling this data. The genes for gamma E (CRYGEP1) and gamma F (CRYGFP1)-crystallins are
pseudogenes
of the lens as opposed to the other gamma-crystallins, which are found in the hard-
est and most compact regions that have a high refractive index. Thus γS-crystallin is
found in the cortical regions and even in the epithelium. In contrast to the embryonic
expression of the gamma A-F crystallin genes, gamma S-crystallin is expressed
relatively late, and appears in the adult lens, in the secondary fibers. It is also
expressed in the retina; both the transcript and protein are detected in mouse and
bovine tissues, albeit at a later postnatal stage than in the lens (Sinha et al. 1998).
2.1.2 Mutations in Alpha-Crystallins
2.1.2.1 Alpha A-Crystallin (CRYAA)

Mutation in the αA-crystallin gene was first reported in a large pedigree with con-
genital zonular central cataracts (Litt et al. 1998). Microcornea and microphthalmia
were present in some affected individuals in the family, as well as amblyopia, stra-
bismus, and glaucoma. Thirteen affected and 11 unaffected individuals from 4 gen-
erations were tested for linkage at candidate regions for various cataract genes; the
cataracts in this family showed linkage to chromosome 21q22.3. The CRYAA gene
for αA-crystallin present within the mapped interval was screened and found to
have an Arg116Cys change that completely cosegregated with the cataracts in the
family. The mutation is predicted to cause loss of a positive charge and gain of a
sulfhydryl group and thus a change in the conformation of the protein. The
Arg116Cys mutation is highly recurrent and is reported in several families across
the world. Thus, the Arg1167 residue possibly represents a mutational hotspot.
Microcornea seems to be a frequently associated feature of the phenotypes associ-
ated to this mutation, with the cataracts being very heterogeneous in their morphol-
ogy. An Indian family of four generations, with all ten affected members having
congenital “fan-shape” cataract and microcornea, was found by linkage mapping
and candidate gene screening, to have the Arg116Cys mutation in αA-crystallin
(Vanita et al. 2006a). Apart from differences in phenotype between families with the
same mutation, there is a range of intrafamilial heterogeneity in the cataract pheno-
types as well. Such variability within a family with asymmetry in the cataract
between the two eyes of an individual is reported in a study of a four-generation
Chinese family with congenital nuclear cataract with microcornea (Wang et al.
2012). Yet another example of extensive phenotype variation in individuals with the
Arg116His mutation in αA-crystallin is a four-generation family from Chile, in
which cataracts ranged from anterior polar, cortical, embryonal, and fan-shaped to
anterior subcapsular, thus displaying a range of phenotypes within the family
(Richter et al. 2008). In addition to the above, another feature documented in
patients with the same mutation is the presence of iris coloboma in a four-generation
French family (Beby et al. 2007).
Another mutation in CRYAA that is recurrent in several families from different
regions is the Arg12Cys. It was reported in an Indian family of two affected mem-
bers with dominant cataract, microcornea, and microphthalmia (Devi et al. 2008).
Interestingly, microcornea is again a feature in several patients with this mutation,
from different countries and ethnicities, with variation in the cataract phenotype.
The cataracts in a Chinese family with the Arg12Cys mutation ranged from lamellar
to nuclear to dot-like opacities, thus displaying intrafamilial heterogeneity in the
phenotype; here again, microcornea was present, with microphthalmia in two of the
subjects (Zhang et al. 2009b). The phenotype of congenital or developmental cata-

ract with microcornea is regarded as a distinct syndrome, referred to as congenital
cataract-microcornea syndrome (CCMC), and presents within a subset of cases with
autosomal dominant cataracts. However, apart from variation in the cataract pheno-
types in these patients, the definition of microcornea itself may be variable in differ-
ent studies. The phenotype of cataract-microcornea syndrome is genetically
heterogeneous, and mutations in different genes associated with familial cataract
are implicated in this phenotype. These include crystallin genes CRYAA, CRYBB1,
CRYGD, connexin (GJA8) gene, and MAF (transcription factor) gene, in addition to
the CRYAA mutations mentioned above (Hansen et al. 2007).
2.1.3 Mutations in Beta-Crystallins
2.1.3.1 Acidic Beta-Crystallins
BetaA3/A1-Crystallin
Mutation of the βA3/A1-crystallin (CRYBA3) gene in human cataract was first
reported in an Indian family consisting of four generations, having congenital cata-
ract with an autosomal dominant transmission. Examination of 24 out of 48 mem-
bers of this family revealed an autosomal dominant transmission of cataract, with 10
individuals affected. The cataracts consisted of anterior and posterior Y-shaped
sutural opacities, a pulverulent (dustlike opacities) fetal nucleus, and a zonular
opacity of about 4 mm in diameter (designated as CCZS; Basti et al. 1996), with
some variation in the cataract phenotype within the family. The cataract locus in this
family was mapped by traditional linkage analysis, in which markers at loci for
genes known to be important for lens physiology were tested for linkage to the cata-
ract phenotype. This study mapped the cataract to a 17 centimorgan (cM) interval of
chromosome 17 (Padma et al. 1995), a region that included the locus for the betaA3/
A1-crystallin (CRYBA1) gene. Screening of the CRYBA1 gene revealed the muta-
tion of G to A at the first base of the third intron of the βA3/A1crystallin gene
{cDNA position c.215+1G>A), associated with this cataract phenotype (Kannabiran
et al. 1998). It is located in the highly conserved splice donor site “GT” dinucleotide
at the exon-intron junctions of most eukaryotic genes, and change of the sequence
of the dinucleotide is expected to generally disrupt splicing of the adjoining exon.
In this case, further investigation of the effect of the mutation was carried out by
expression of the mutant gene in transgenic mice. Analysis of the βA3/A1 crystallin
mRNA and protein in the mouse lenses revealed that the mutation led to missplicing
and skipping of the third and fourth exons predicting the deletion of one of the
globular domains in the βA3/A1 crystallin protein. However, the level of the mutant
protein in the transgenic mouse lenses was much found to be lower than the wild
type protein derived from the endogenous mouse Cryba1 gene. The mouse lenses
displayed no evident cataract phenotype. In an effort to overexpress the mutant
Cryba1 to levels similar to the wild type Cryba1, the cDNA for the mutant βA3/A1
crystallin (c.97_357del) was cloned into a transgenic expression vector with a
chicken CRYBB1 promoter and overexpressed in transgenic mice (Ma et al. 2016).
The considerably higher levels of the mutant protein achieved by this method also
led to a phenotype of disorganization of lens fibers, rupture of the lens capsule, and
vacuolization of the lens, thus manifesting with loss of transparency.
Interestingly, the same splice donor dinucleotide at the exon 3-intron 3 junction
in the CRYBA3 gene appears to be a site of recurrent mutation since mutations at
this location have been in several families with congenital cataract from different
populations. The phenotypes of cataract associated with the mutation are variable in
different families that have been described. Some examples of such families are
given below.
1. A Brazilian family with the c.215+1G>A mutation had pulverulent nuclear

opacities, in addition to sutural opacities, “star-shaped” or radial opacities in the
posterior embryonal nucleus, and cortical opacities (Bateman et al. 2000).
2. The mutation of c.215+1G>A was also identified in an Australian family with
cataract of variable severity ranging from mild opacities that were asymptom-
atic, to severe disease requiring surgery in childhood. Similar to the phenotypic
variation in the Brazilian family mentioned above, the opacities are described as
a spectrum of Y-sutural opacities, mild opacification of the fetal nucleus, and
cortical opacities (Burdon et al. 2004a).
3. There are two families reported with the same splice site mutation, from the UK
(Gillespie et al. 2014).
4. Two South Indian families with lamellar and floriform cataracts, respectively
(Devi et al. 2008), were identified to have the c.215+1G>A change. Intrafamilial
variability of the phenotype was observed in the Indian families.
5. The c.215+1G>A mutation is also reported in several Chinese families with con-
genital cataract, from different parts of China. A family of 4 generations, with 13
individuals studied (Gu et al. 2010), had cataracts consisting of posterior polar
opacities which developed within the first decade of life. A second Chinese fam-
ily with the same mutation consisted of four generations, with nine affected
members having Y-sutural opacities and nuclear and cortical opacities (Zhu et al.
2010). The opacities in this family were described as progressive, with cortical
opacities appearing later. Yet another family, also of Chinese ethnicity, including
members of five generations with Y-sutural opacities, was found to have the
c.215G>A mutation in CRYBA3 (Yang et al. 2011c). Thirteen members of the
family were studied, and seven were affected with cataracts within the first few
years of life. Evidently, there is phenotypic variability of cataract among families
with this mutation; it is also associated with lamellar cataract (Sun et al. 2011)
and with polymorphic cataracts (very variable cataract phenotypes) in a family
of seven affected and four unaffected (Yu et al. 2012).
A different mutation, IVS3+2 T→G, located in the same splice site at the junc-
tion of exon3-intron 3 in the CRYBA3 gene was identified in another family, of
Chinese origin. The family included 15 individuals from 4 generations of whom 7
were affected with nuclear cataracts (Yang et al. 2012).
Yet another mutation in bA3/A1-crystallin that is also highly recurrent consists

of a 3 bp deletion leading to deletion of the glycine residue at 291 (c.272_274delGAG;
Gly91del). There are at several different reports of this mutation being identified in
separate families with cataract. Nine members of a five-generation family of north-
eastern Chinese origin including eight affected were evaluated, and linkage analysis
of candidate gene loci for cataract showed linkage to the bA3/A1 locus on chromo-
some 17. Sequencing of the bA3/A1-crystallin in the mapped locus revealed a 3 bp
in-frame deletion leading to a mutation of Gly91del (Yang et al. 2011b). Two more
occurrences of the same mutation in Chinese families with autosomal dominant,
nuclear, congenital cataracts were reported by Sun et al. (2011). Another phenotype
of cataract associated with the mutation of Gly91del in bA3/A1-crystallin is con-
genital nuclear lactescent cataract in a Swiss family of 5 generations and 15 affected
individuals (Ferrini et al. 2004). This cataract was noted to not affect the lens sutures
and was hence designated as “suture-sparing.” The Gly91del mutation was also
associated with autosomal dominant lamellar cataract in a five-generation British
family (Reddy et al. 2004). The mutation is presumed to lead to high molecular
weight aggregates in the lens.
In contrast to the Gly91del deletion mentioned in the previous paragraph, which
is an in-frame deletion involving one amino acid, another small deletion of two base
pairs in bA3−/A1-crystallin—c.590–591delAG—is also associated with cataract.
The consequence from such a deletion is expected to be a frameshift at amino acid
197. This mutation was identified in a four-generation Chinese family in which five
affected individuals had congenital cataract (Zhang et al. 2014).
Although the majority of mutations in bA3-crystallin are of dominant inheri-
tance, autosomal recessive transmission has been reported in certain cases, with
homozygosity at the disease locus. An example of this is a family of Middle-Eastern
origin with total cataract and a deletion of 3 bp, c.585_588del (p.195_196del in the
protein) (Khan et al. 2015). It is noteworthy that almost all of the foregoing muta-
tions (except the Gly91del) involved frameshifts in the bA3-crystallin protein due to
conserved splice site mutations or deletions in the coding region, thus predicting the
formation of truncated proteins. Another change that may well have a pathogenic
effect on splicing is a single-base substitution close to the splice donor site at the
exon boundary, c.213C>T, leading to a synonymous change (Gly71Gly). Though it
does not change the protein sequence, it is predicted to cause splicing alterations at
the donor site (Gillespie et al. 2014). There are relatively few reports of missense
mutations in the bA3-crystallin gene in families with cataract. A mutation in this
category is the Ser209Trp mutation in a patient with bilateral congenital cataract
and cardiomyopathy. The latter was regarded as an incidental finding by the authors
since there was no prior association of myopathy with CRYBA1 mutations.
Beta A2 and Beta A4-Crystallins

Involvement of βA2-crystallin (CRYBA2) in human cataract is infrequent as com-
pared with the CRYBA3/A1 as has been reported so far, and there are very few
instances of mutations in this gene, all in families with autosomal dominant con-
genital cataract. A mutation of c.148G>A, p.(Val50Met) in CRYBA2 was in a
four-generation family included in a study involving mostly Caucasian patients. The

phenotype here is described as multifocal congenital cataract with eccentric pupils
and incomplete penetrance. All seven affected individuals in the family except two
had congenital cataracts (Reis et al. 2013). Another occurrence of a mutation in
CRYBA2 was reported in a study on a series of sporadic cases with congenital cata-
ract. One patient out of 74 that were included in the study had a mutation of
Phe63Ser, predicted as damaging due to the nonconservative nature of the substitu-
tion, involving replacement of the bulky aromatic phenylalanine residue with a
small, polar amino acid, serine (Li et al. 2016).
In the case of βA4-crystallin gene (CRYBA4) as well, there are fewer known
mutations in families with congenital cataract as reported till date, relative to βA3/
A1-crystallin. A phenylalanine to serine mutation in CRYBA4 is associated with
autosomal dominant congenital lamellar cataract in an Indian family (Billingsley
et al. 2006). The family comprised of 4 generations with 13 affected members, and
a missense change of phenylalanine-94 to serine (Phe94Ser) in CRYBA4 was found
to be the basis for the cataract. The predicted consequence of this change is that it
leads to introduction of a polar residue into the hydrophobic core of the protein and
thus destabilizes its native conformation. In addition, mutations of the CRYBA4
gene may be associated with microphthalmia and microcornea, suggesting that
mutations inactivating this gene may affect eye development. A mutation of leucine-
69 to proline (Leu69Pro), again affecting a highly conserved residue, was detected
in a heterozygous individual with bilateral cataracts and microphthalmia, with sec-
ondary enophthalmia (eyes sunken in the sockets). Another example of a mutation
in CRYBA4 is a change of glycine-64 to tryptophan (Gly64Trp), discovered in two
affected individuals with congenital cataract and microcornea from a Chinese fam-
ily in which nine members are evaluated. The substitution involves the replacement
of a small amino acid with a bulky one and is predicted to destabilize the beta-sheet
structure in which glycine-64 is located (Zhou et al. 2010a).
2.1.3.2 Basic Beta-Crystallins

The basic β-crystallins consist of three major species in the human lens—they are
the βB1-, βB2-, βB3-crystallin genes (designated as CRYBB1, CRYBB2, and
CRYBB3, respectively) and are conserved in vertebrates as several of these genes
have orthologs that have been identified in other species. The genes for the three
basic beta-crystallins are located in a cluster on chromosome 22. The CRYBB2 has
a pseudogene (CRYBB2P1) that is also located in the same region of chromosome
22. Each of the basic beta-crystallin genes has six exons, and as with all β-crystallins,
each exon encodes a single Greek key motif of the protein. Two such motifs make
up a single globular domain in the protein tertiary structure. Mutations in the vari-
ous genes encoding the basic beta-crystallin proteins have been described till date
in several families with hereditary cataracts from different parts of the world.
Crystallin Beta B1
The βB1-crystallin gene is associated with both dominant and recessive cataracts.
The association of a mutation in the CRYBB1 gene with hereditary familial
cataract was first reported in a study from the USA, on a four-generation family of
12 members with autosomal dominant congenital cataract consisting of fine pul-
verulent (dustlike) opacities in the fetal nucleus of the lens. The cataract locus was
mapped in this family to chromosome 22q, in the vicinity of the CRYBB1 and
CRYBA4 genes (Mackay et al. 2002). Screening of the CRYBB1 gene identified a
C-to-T substitution leading to a stop codon at glycine-220 (Gly220X) as the patho-
genic mutation in the family. The stop codon predicts a truncation of the protein
within the fourth Greek key motif. A different type of mutation in the CRYBB1
gene, having the opposite result as compared to a premature truncation, is the
mutation of X235R (stop codon at 235 to arginine), in which the mutation leads to
an elongation of the reading frame due to a translational read-through at the stop
codon. This mutation has been associated with autosomal dominant congenital
cataract and microcornea in a family from the UK, in which the affected individu-
als had nuclear cataract with cortical and polar opacities. The mutation here
involved a T>C change converting the nonsense codon (TGA) to arginine (CGA)
and thereby resulted in an elongated reading frame with 26 additional amino acids
(Willoughby et al. 2005).
There are several reports of CRYBB1 gene mutations in familial congenital cata-
racts, and while there are a few instances of truncating mutations either due to
frameshift or nonsense mutations, most mutations known till date are missense
changes involving amino acid substitutions throughout the protein. An interesting
trend is that the recessive mutations appear to be frequently those that truncate the
protein within the Greek key motifs or alter residues in the amino terminal region.
On the other hand, the dominantly inherited mutations tend to be missense changes
throughout the protein or truncations toward the C-terminal region, beyond the
Greek key motifs. The following examples of mutations in CRYBB1 in families
with recessive congenital cataracts illustrate this pattern. Mutation of the initiation
codon of βB1-crystallin gene has been reported in a consanguineous family with
autosomal recessive cataract, of Somali origin (Meyer et al. 2009). The cataracts in
this family, ranging from dense nuclear cataracts to mild pulverulent opacities,
were mapped by genome-wide homozygosity analyses to the β-crystallin gene
cluster on chromosome 22. A mutation converting the methionine-1 residue to
lysine (Met1Lys) in CRYBB1 was associated with the cataract in this family. A
change at methionine-1 would be expected to affect its translation and possibly lead
to an absence of protein. Another mutation in CRYBB1 affecting the protein
sequence near the amino-terminus, also associated with recessive cataract, is a
frameshift at asparagine-58 due to a single-base deletion at c.171 (Asn58Thrfs*107).
It appears to be highly recurrent in the Middle-Eastern families and represents an
ancestral mutation in this population. The mutation was found in a study of 16
affected offspring from 10 Arab families with central pulverulent cataract. All the
affected members had the mutation against a shared haplotype of markers linked to
the disease gene suggesting a founder effect of the mutation (Khan et al. 2012). A
subsequent study on families from the Middle East detected this mutation in 11
families, making up about 15% of mutations in the 74 families from Saudi Arabia
(Patel et al. 2017).
Several missense mutations in CRYBB1 are reported in families with dominant

cataract, and in some cases, the cataract is associated with microcornea. Among
these is a missense change of valine-96 to phenylalanine (Val96Phe) associated with
autosomal dominant cataract with microcornea in a Caucasian family (Reis et al.
2013). Mutations of Ser228Pro, Arg230Cys, and Arg233His have been reported in
patients with dominant cataracts from Chinese families (Wang et al. 2011; Sun et al.
2017).
Crystallin Beta B2
The association of the crystallin βB2 (CRYBB2) gene with congenital cataract was
first made in a study of autosomal dominant cerulean cataract in a large family of
159 individuals. The cataracts in this family manifested as peripheral blue flakes
and central spoke-like opacities and were described as mild. Vision was normal to
mildly reduced until adulthood in all the affected persons except one offspring of a
consanguineous marriage of two affected first cousins, who had dense cataracts,
microcornea and microphthalmos. Genetic analysis of a branch of the family con-
sisting from the USA, of about 35 members, mapped the locus to chromosome 22,
near the β-crystallin gene cluster (Kramer et al. 1996).
Mutation of the CRYBB2 gene in the cerulean cataract in the abovementioned
family was demonstrated by Litt and coworkers (Litt et al. 1997), and a nonsense
codon at glutamine 155 (Gln155Ter) leading to premature termination of the protein
was identified in the CRYBB2 gene. The Gln155Ter mutation is reported in several
other families from across the world and is associated with different cataract pheno-
types including cerulean, polymorphic, and pulverulent cataracts. Thus, the
Gln155Ter was also found in a Swiss family of 3 generations of 16 members, 10 of
whom were affected with cerulean cataracts mapped to chromosome 22, at the locus
for CRYBB2 (Gill et al. 2000). The same mutation in CRYBB2 is also associated
with diverse forms of cataracts in several families including (a) autosomal dominant
cataract in a 5-generation Chinese family of 41 individuals, described as progressive
polymorphic congenital cataract in 17 of the members – essentially the cataract
phenotypes were very variable between members of the family and were evident
from birth (Yao et al. 2005); (b) cataract involving gene conversion between the
CRYBB2 gene and its pseudogene CRYBB2P1, a phenomenon reported in an Indian
family with cerulean and punctate opacities [see below], and also in a Chilean fam-
ily; (c) a 5-generation Chinese family with “progressive polymorphic congenital
coronary cataracts” (Li et al. 2008); and (d) a 3-generation Indian family with corti-
cal and pulverulent opacities (Devi et al. 2008). There are at least a dozen separate
records of families with the Gln155Stop mutation in the Cat Map database, suggest-
ing that it is one of the most highly recurrent crystallin gene mutations reported.
The phenomenon of gene conversion is a different mutational mechanism that is
generally found with genes associated with congenital cataracts. Gene conversion
involving the CRYBB2 gene and its pseudogene CRYBB2P1 is found to occur in two
families reported from different regions. Gene conversion occurs between two
sequences that are located close to each other and have very similar sequences.
Thus, there is a nonreciprocal transfer of genetic material from a “donor” sequence
to a highly similar “acceptor.” In the gene conversion involving CRYBB2 and

CRYBB2P1, segments of the gene and its pseudogene are exchanged during DNA
replication and crossing over. In a study of an Indian family with sutural, punctate,
and cerulean opacities, the cataract locus was mapped to chromosome 22, and
sequencing of the CRYBB2 gene in this region showed two sequence changes—
aGln155Stop (Q155X) mutation due to a C>T change at position 475 and another
silent change at position 483, also a change of C>T (Vanita et al. 2001). The possi-
bility of gene conversion was considered as a mutational mechanism by the authors
in view of the high degree of similarity between CRYBB2 and CRYBB2P1 and the
short distance of about 220 kb between them. Comparison of the two sequences
showed that the two mutations detected in the CRYBB2 gene were in fact present in
the normal sequence of the pseudogene CRYBB2P1 at these positions. Thus, the
CRYBB2 gene in this family apparently carried a short stretch (about 100 bp) of
pseudogene sequence in the region, which included the two sequence changes. This
finding of a short segment of “replaced” DNA in the CRYBB2 gene was compatible
with gene conversion as the underlying mechanism. Surprisingly, the same two
variations at nucleotides 475 and 483, representing gene conversion between
CRYBB2 and CRYBB2P1, were also found in a study on an unrelated family from
Chile, in which cataracts were in the form of pulverulent embryonal, cortical, and
subcapsular opacities (Bateman et al. 2007).
Animal Models of Cataract from Mutant Beta B2-Crystallin

A well-studied mouse model for cataract is the Philly mouse, which develops cata-
ract within a few weeks of birth. Analysis of the mouse lenses showed that lens fiber
formation is defective and a β-crystallin protein of 27 kDa is missing from the lenses
(Nakamura et al. 1988). The confirmation of the mutant gene as betaB2-crystallin
and the details of the mutation in the Philly mouse were obtained by analysis of the
lens cDNA of the Philly mouse in relation to normal mice. Total lens cDNA from
both species was cloned into a lambda gt11 cDNA library and probed with a rat
βB2-crystallin cDNA probe. Comparison of the cDNA sequences showed a deletion
of 12 nucleotides in the Philly strain, leading to premature termination of the coding
sequence. The effect of the 12-nucleotide deletion on the protein is a deletion of 4
amino acids located within motif 4 of βB2-crystallin near the C-terminus. A com-
parison of the mutant βB2-crystallin protein with the X-ray crystallographic struc-
ture of the normal bovine βB2 protein suggests that the consequence of an in-frame
deletion in this region is the introduction of nonconservative changes in the amino
acid residues that are normally involved in beta-sheet structure. The structure of the
mutant protein is thus predicted to be affected by irregular packing, eventually
resulting in its instability (Chambers and Russell 1991).
2.1.4 Mutations in Gamma-Crystallins
The gamma-crystallin genes (CRYGA-CRYGF) are located on chromosome 2q33 in

a cluster, and the genes CRYGA-CRYGD encode the corresponding the γ-crystallins
A-D, while CRYGE and CRYGF are pseudogenes. Among the genes for γ-crystallins,
there are several reports of mutations in the γC and γD-crystallin genes associated
with human cataract, in families from across the world. Mutations in the γS-crystallin
are also known to occur in a few families with congenital cataract. Many of the
mutations in CRYGC and CRYGD genes are missense changes; some are highly
recurrent, with the same mutation being reported in several unrelated families.
2.1.4.1 Mutations in CRYGC

The mutational spectrum of γC-crystallin consists largely of missense and stop
mutations. Mutations with premature terminations comprise about one-third of all
reported mutations in γC. As in the case of γD-crystallin, there are recurrent muta-
tions in γC-crystallin as well. Among them, the missense change of arginine-168 to
tryptophan (Arg168Trp) in γC-crystallin is reported in two separate families from
India and in one family from Mexico. While the cataract in the two Indian studies
was lamellar, in the Mexican family, it was nuclear cataract (Santhiya et al. 2002;
Devi et al. 2008; Gonzalez-Huerta et al. 2007). In the latter cases, the patients were
reported to have additional features of iris atrophy and nystagmus.
A missense mutation in γC-crystallin is also associated with a form of congenital
cataract referred to as Coppock-like cataract (CCL), appearing as dustlike opacities
in the fetal nucleus. This cataract was mapped to the γ-crystallin gene cluster on
chromosome 2 (Lubsen et al. 1987), and its molecular basis further attributed to
sequence changes in the pseudogene for γE-crystallin, involving the promoter at or
near the TATA box. These changes were considered to increase the expression of the
pseudogene several-fold, raising the level of expression of γE-crystallin. Thus, the
mechanism of CCL in this family was proposed to be the reactivation of the pseu-
dogene for γE-crystallin, with production of an aberrantly expressed protein, which
is normally absent in the lens (Brakenhoff et al. 1994). However, a later re-evaluation
of the CCL locus in the original family that was studied showed that the sequence
changes reported in the pseudo-γE crystallin promoter were not specific to the dis-
ease, as they occurred at an appreciable frequency in the normal population as well.
A change of threonine-5 to proline (Thr5Pro) in the γC-crystallin gene was identi-
fied upon screening of the gamma-crystallin genes in the mapped region and further
confirmed as the pathogenic change in this particular cataract (Héon et al. 1999).
Termination mutations in γC-crystallin are sometimes associated with more
severe phenotypes that include developmental anomalies affecting the globe or
other eye structures in association with congenital cataract. Congenital cataract with
microcornea constitutes one such entity that is associated with truncating mutations
in γC-crystallin. It is a genetically heterogeneous condition, being associated with
mutations in various genes including crystallin and gap junction protein genes
(Hansen et al. 2007). There are families reported in which other developmental
abnormalities occur in addition to cataract and microcornea, in association with
mutations in γC-crystallin. Thus, mutation of tryptophan-157 to Stop (Trp157Stop)
is associated with nuclear cataracts and microcornea in Chinese families (Guo et al.
2012; Zhang et al. 2009a). Another termination mutation, Tyr139Stop, was identi-
fied in a family with a syndrome of cataract, microcornea, microphthalmia, and
congenital glaucoma (Reis et al. 2013). Mutation of cysteine-42 to alanine with
frameshift and termination after 60 amino acids (p.Cys42Alafs*60; here, a frame-
shift is predicted at the 42nd codon due to a single-base deletion) was associated
with a severe form of cataract in which the patient had nystagmus and amblyopia in
a Korean family (Kondo et al. 2013). Linkage analysis of several cataract-associated
loci, followed by screening of candidate genes, showed an insertion of 5 bases in the
CRYGC gene in a large pedigree of 47 members with 17 affected with zonular pul-
verulent cataract, with the obvious consequence of a frameshift in the messenger
RNA sequence (Ren et al. 2000). A notable feature of this family was the incom-
plete penetrance and variable expressivity of the phenotype. There were mutation
carriers with no cataracts detectable even by clinical examination and others who
were severely affected in infancy.
2.1.4.2 Mutations in CRYGD

A missense mutation of proline-24 to threonine (Pro24Thr) in the CRYGD
(γD-crystallin) gene is the most frequent mutation till date in the gamma-crystallins
and has been identified in several families from various parts of the world, including
India, China, the Middle East, Europe, North and South America, and Australia.
This mutation is designated as Pro23Thr in some publications. It was first reported
in an Indian family with congenital lamellar cataract (Santhiya et al. 2002). There
are at least 15 different entries of the same mutation as listed in the CatMap data-
base [available at http://cat-map.wustl.edu/; accessed 2018], and a striking aspect of
these is that the cataract in at least half of the probands with this mutation, repre-
senting different populations, is reported as coralliform in its morphology.
Other mutations in γD-crystallin which are recurrent though to a lesser extent
have been identified in two to three families each, in different studies. These include
arginine-15 to cysteine (Arg15Cys), arginine-37 to serine (Arg37Ser), and arginine-
140 to stop (Arg140Stop). The Arg15Cys mutation was found in association with
autosomal dominant juvenile-onset punctate cataract in a three-generation family.
Cataracts in this family developed within the first year of life and not at birth
(Stephan et al. 1999). The Arg37Ser mutation in γD-crystallin is associated with an
unusual phenotype of crystal deposits in the lens. Aspirated lens material showed
prismatic, birefringent crystals. Analysis of the crystals by protein electrophoresis,
amino-terminal peptide sequencing, and mass spectrometry by MALFI-TOF identi-
fied the protein in the crystals as γD-crystallin (Kmoch et al. 2000). The same muta-
tion is also reported in a Chinese family with autosomal dominant congenital
cataract, described as “nuclear golden crystal cataract” with punctate opacities (Gu
et al. 2005). In the Chinese family, however, the crystalline nature of the lens opac-
ity was described on the basis of clinical examination and not by direct analysis of
the lens material. Yet another mutation involving the same amino acid is Arg37Pro,
reported in a Chinese family with autosomal dominant congenital nuclear cataract.
Although this mutant protein did not form crystals, the substitution of proline for
arginine is predicted to increase the surface hydrophobicity of the protein without
any obvious effect on its secondary structure. The effect would be to reduce its solu-
bility, as is also the case for other mutant γD-crystallins involving substitutions at
proline-23 and arginine-37 (Evans et al. 2004). The Arg140Stop mutation men-
tioned above was first reported in an Indian family with congenital nuclear cataract
(Devi et al. 2008). Similar phenotypes of nuclear cataract are also reported in an
Ashkenazi Jewish family and a Chinese family, associated with the same mutation
in γD-crystallin (Reis et al. 2013; Zhai et al. 2014).
2.1.4.3 Mutations in CRYGS

The gamma S-crystallin gene is located on chromosome 3q27, as compared to the
genes for other γ-crystallins consisting of γA- to γE-crystallin are located in a clus-
ter on chromosome 2. Gamma S-crystallin shares the three-exon structure of the
other gamma-crystallin genes but has a longer first intron that may have a short
region that is transcribed through alternative splicing. The CRYGS gene expressed
at high levels in the adult lens, particularly in the cortex. The expression of CRYGS
is thus distinct from other gamma-crystallin genes, which are expressed in high
concentrations in the lens nucleus and during embryonic development. As per the
conserved domain structure of the βγ-crystallins, the γS-crystallin has a two-domain
structure, with each domain formed from two Greek key motifs. The Greek key
motif is each made up from four antiparallel β-sheets.
The involvement of γS-crystallin in human cataract was first reported in a
6-generation Chinese family with autosomal dominant cataract in 14 patients that
were studied. The opacities were cortical, being located in the anterior or posterior
cortex, but not involving the fetal nucleus. The cataracts were progressive in nature,
and vision was not affected until 8 years of age or above (Sun et al. 2005). The locus
in this family was mapped to chromosome 3, and a Gly18Val change in the
γS-crystallin was found to be the pathogenic mutation. A few other mutations in
CRYGS have been documented in families with congenital or juvenile cataracts.
Two cataract-associated mutations in the CRYGS gene include missense changes of
Ser39Cys and Val42Met in families from Southern and Northern India, respectively
(Devi et al. 2008; Vanita et al. 2009). Variability in the cataract phenotype was noted
in the family from South India. An opalescent cataract that was denser in the nuclear
region of the lens was noted in the North Indian family.
2.1.5 M
utations in Genes Encoding Lens Membrane Proteins
and Gap Junctions
Lens membrane proteins include those that are involved in cell-cell transport, sig-
naling, sorting, etc. Genes encoding various membrane proteins such as gap junc-
tion proteins (connexins), aquaporins, receptor tyrosine kinases, the endosomal
sorting complex, and mitochondrial membrane proteins have been implicated in
congenital cataracts.
2.1.5.1 Connexin Genes

Among the lens membrane proteins, a number of mutations in the gap junction
genes are associated with autosomal dominant as well as recessive congenital
cataracts. Gap junctions are important in mediating the transport of water, small
molecules and ions between cells, and therefore critical in lens development and
homeostasis. They are made up of connexin proteins, of which there are three in the
lens-Cx43, Cx46, and Cx50, encoded by the GJA1, GJA3, and GJA8 genes, respec-
tively. These three genes differ in their spatial and temporal expression in the lens.
Cx43 is expressed in the epithelium but not in the fibers, Cx46 is expressed during
differentiation of fiber cells but is absent from the epithelium, whereas Cx50 is pres-
ent in both types of cells, appearing first in the epithelium and later on in the devel-
oping fibers (Mathias et al. 2010). The Cx50 protein was designated in earlier
studies as MP70, identified as a protein expressed in lens fiber cell membranes
(Kistler et al. 1985). Each connexin molecule has four transmembrane segments,
two extracellular loops connecting these segments, and one cytoplasmic loop. Each
has different gating properties and shows differences in permeability from the other
types. The connexins assemble into hexamers to form “connexons” or hemichan-
nels. A gap junction is formed by two connexons from neighboring cells, docking
together.
Mutations in genes encoding Cx46 (GJA3) and Cx50 (GJA8; protein also known
as MP70) shown in Table 2.2 largely consist of missense mutations across the length
of the connexin protein, with a few frameshift mutations. The latter have been found
in families with autosomal recessive cataracts. Thus, it is possible that the mecha-
nism of disease in autosomal recessive inheritance entails a loss of function due
with instability of either mRNA or protein having a premature termination codon.
Mechanisms of disease in autosomal dominant cataract might involve dominant
negative effects of mutant proteins. In such a situation, the mutant connexin proteins
interfere with the formation of gap junctions by the normal protein subunits. Another
way in which dominant mutations could lead to disease is by haploinsufficiency—
i.e., one copy of the normal allele is not sufficient for maintaining gene function.
Specific effects of missense mutations may consist of inability to form gap junc-
tions, altered gating properties, and retention of mutant proteins with accumulation
in the endoplasmic reticulum (Mathias et al. 2010).
Evidence for the involvement of a connexin gene in human cataract first came
from the study of a six-generation English family with zonular pulverulent cataract
(designated as CZP1) which was mapped to the Duffy blood group antigen locus
on chromosome 1. The CZP1 locus was refined by linkage analysis to a chromo-
some 1q region, which included the GJA8 gene (Shiels et al. 1998). A proline-88
to serine (Pro88Ser) change in connexin-50, involving a nonconservative substitu-
tion, was identified as the cause of cataract in this family. Subsequently, another
locus for zonular pulverulent cataract was mapped on chromosome 13 (CZP3) in
two separate pedigrees with dominant congenital cataracts in multiple generations,
and the connexin gene present within this interval, gap junction protein alpha-3
gene (GJA3, encoding the connexin 46 protein), was found to have pathogenic
mutations in both families that mapped to CZP3 (Mackay et al. 1999). The muta-
tions involved an insertion of C at nucleotide 1167 of GJA3, with frameshift at
codon 380 in one family and a missense substitution of asparagine to serine
(Asn63Ser) in a second family.
There are several mutations in both the GJA8 and GJA3 genes that have been
associated with dominant or recessive cataracts in families from various popula-
tions, as shown in the Table 2.2.
Mutations in GJA8/Connexin 50 in Indian Families

A number of mutations in GJA8 have been associated with autosomal dominant and
recessive cataracts in Indian families. In some instances, the mutation was identified
in sporadic cases, and no family members were available for the study.
Autosomal Dominant Cataracts

Mutations in GJA8 in autosomal dominant cataracts have been associated with con-
genital cataract in South Indian families. Missense substitutions of Cx50 were first
reported in two families with autosomal dominant cataracts with microcornea.
These substitutions were Val44Glu and Arg198Gln and involved nonconservative
changes in the Cx50 protein. In one family the probands had total cataract, while the
second family had developmental, posterior subcapsular cataract (Devi and
Vijayalakshmi 2006). The residues involved are located in the first transmembrane
domain and second extracellular loop, respectively, of the connexin 50 protein.
Studies from North India have identified mutations in Cx50, mostly missense
changes, segregating with various cataract phenotypes in families with autosomal
dominant cataracts. These include Trp45Ser and Val79Leu, located in the first and
second transmembrane domains, respectively, of Cx50. The associated phenotypes
consisted of a jellyfish-like cataract and microcornea in the family with Trp45Ser
and a “full moon”-like cataract in association with mutation of Val79Leu (Vanita
et al. 2006b, 2008a, b). Another mutation in Cx50, affecting the second α-helical
transmembrane domain, is the mutation of Pro88Gln, which occurred in a family
with a cataract in the fetal nucleus of the lens. This cataract was described as semi-
opaque, with feathery opacities in between the Y sutures. However, heterogeneity in
the associated phenotype has been noted with this mutation, which is also reported
in a family with lamellar pulverulent cataract in a British family (Arora et al. 2006).
Missense change of Pro199Ser located in the second extracellular loop in Cx50
was associated with a dominantly transmitted cataract that was congenital in onset
and affecting both parent and child of a family from South India (Ponnam et al.
2013). This substitution is probably damaging to the protein according to protein
prediction tools. Analysis of a series of 30 cases with congenital cataract revealed
one patient with lamellar cataract and nystagmus, to be heterozygous for a change
of Leu281Cys in Cx50. The residue affected is located in the C-terminal intracel-
lular domain of the protein, and leucine is a conserved amino acid at this position;
the substitution is predicted as damaging, thus supporting the idea that it is patho-
genic (Kumar et al. 2011).
Autosomal Recessive Cataracts

Apart from autosomal dominant cataracts, mutation in GJA8 may also lead to
autosomal recessive cataracts. This was demonstrated in a consanguineous fam-
ily from South India, with two affected offspring out of three, having total
cataracts and nystagmus (Ponnam et al. 2007). The mutation in this family
involved an insertion of one base at codon 203, leading to frameshift in the sec-
ond extracellular domain of Cx50 protein. Thus, it would be expected to cause
premature truncation of the protein and thereby lead to loss of function, due to an
unstable mRNA or protein. Though missense changes in GJA8 are by and large
the major type of mutation in dominant cataracts, a missense change may be
associated with recessive cataract. An example of this is a valine-196 to methio-
nine (Val196Met) substitution in a single affected individual born of a consan-
guineous union; the diagnosis of cataract was made in the second year of life
(Ponnam et al. 2013). The prediction of the impact of this substitution on the
protein using various predictive software tools suggested that it may be damaging
to the protein.
Mutations in GJA3 in Indian Families

As highlighted in Table 2.2, there are several families of Indian origin that have
GJA3 mutations. Analysis of 60 unrelated families from South India showed two
probands to be heterozygotes for changes in the GJA3 gene encoding connexin-46
(Cx46) (Devi et al. 2005). One family among these is reported to have a variable
cataract, both in age at onset and morphology. It was associated with Val28Met mis-
sense change that is predicted to lie in the first transmembrane domain of Cx46. A
second missense change in the same gene is Arg76Gly in a patient with total cata-
ract, again involving a nonconservative substitution from a polar, charged residue,
arginine, to an uncharged polar amino acid, glycine, which is also much smaller in
size. A mutation at the same position with perhaps a milder effect in arginine-76 to
histidine (Arg76His) was associated with pulverulent opacities and incomplete pen-
etrance in an Australian family. The cataract in this family was described as “faint
lamellar nuclear opacity surrounding pulverulent nuclear opacities,” and most of the
patients in this family did not require surgery until the second decade or after
(Burdon et al. 2004b).
Various other missense changes in GJA3/Cx46 are reported to be associated with
familial cataracts. These include the following:
(a) Missense mutation is a change of Arg33Leu located in the first transmembrane

domain of the Cx46 peptide, found in a 4-generation family with 18 members
affected with granular opacities in the embryonal nucleus (Guleria et al.
2007a).
(b) A second mutation of Thr87Met in GJA3, identified in a family with two
affected members with an autosomal dominant cataract described as “pearl
box” cataract. The opacities involved both the embryonal and fetal nuclei sepa-
rated by a clear space. The opacities were dot-like and more elongated in the
periphery of the lens (Guleria et al. 2007b).
(c) A mutation of thronine-19 to methionine (Thr19Met), associated with a pheno-
type of posterior polar cataract in a family, diagnosed at age of 9 years in the
proband, and observed to cosegregate with disease in this family (Santhiya et al.
2010).
2.1.5.2 Lens Intrinsic Membrane Protein 2 (LIM2, MP19, MP20)

The lens intrinsic membrane protein 2 (LIM2) is also known as MP20, or MP19,
and is one of the most abundant proteins in the lens fiber cell membranes. The LIM2
protein has four membrane-spanning domains, two extracellular loops, short intra-
cellular N and C-termini, and a short intracellular loop.
Table 2.2 Mutations in GJA8 and GJA3 in families with congenital cataract
No. Gene Mutation Phenotypes and References

associated features
1 GJA3 Asn63Ser Pulverulent cataract Mackay et al. (1999)
2 GJA3 p.S380QfsX87 Pulverulent cataract
3 GJA3 Pro187Leu Zonular Rees et al. (2000)

pulverulent cataracts
4 GJA3 Phe32Leu Nuclear pulverulent Jiang et al. (2003)
5 GJA3 Arg67His Lamellar nuclear and Burdon et al. (2004)
pulverulent
6 GJA3 Pro59Leu Nuclear punctate Bennett et al. (2004)
7 GJA3 Asn188Thr Nuclear pulverulent Li et al. (2004)
cataract
8 GJA3 Trp45Ser Nuclear cataract Ma et al. (2005)
9 GJA3 Val28Met Variable Devi et al. (2005)
10 Arg76Gly Total
11 GJA3 Asp3Tyr Zonular pulverulent Addison et al. (2006)
12 GJA3 Leu11Ser Congenital “ant-egg” Hansen et al. (2006)
phenotype
13 GJA3 Thr87Met “Pearl box” cataract Guleria et al. (2007b)
14 GJA3 Arg33Leu Granular, embryonal Guleria et al. (2007a)
cataract
15 GJA3 Thr19Met Posterior polar cataract Santhiya et al. (2010)
16 GJA3 Val44Met Congenital nuclear Zhou et al. (2010b)
cataract
17 GJA3 Asp47Asn Congenital nuclear Yang et al. (2011a)
cataract
18 GJA3 Pro187Ser Nuclear pulverulent Ding et al. (2011)
cataract
19 GJA3 Asn188Ile Nuclear coralliform Zhang et al. (2012)
cataract; blue punctate
opacities in cortical region
20 GJA3 Phe206Ile Nuclear cataract Wang and Zhu (2012)
21 GJA3 Gly143Arg Coppock-like cataract Zhang et al. (2012)
22 GJA3 Met1Val Anterior polar cataract Kumar et al. (2013)
23 GJA3 Asn55Asp – Hu et al. (2014)
24 GJA3 Gly143Glu Nuclear cataract Yuan et al. 2015.

Table 2.2 (continued)
No. Gene Mutation Phenotypes and References
associated features
25 GJA8 Glu48Lys Zonular, nuclear Berry et al. (1999)
pulverulent
26 GJA8 Val64Gly Nuclear cataract Ma et al. (2005)
27 GJA8 Pro88Gln Lamellar pulverulent Arora et al. (2006)
28 GJA8 Val44Glu Total cataract with Devi and Vijayalakshmi
microcornea (2006)
29 GJA8 Arg198Gln Developmental cataract
30 GJA8 Val79Leu “Full moon” with Y- Vanita et al. (2006b)
sutural opacities
31 GJA8 Thr203AsnfsX47 Total cataract Ponnam et al. (2007)
32 GJA8 ins776G Triangular nuclear Schmidt et al. (2008)
cataract
33 GJA8 Asp47Asn Nuclear pulverulent Arora et al. (2008)
cataract
34 GJA8 Ser278Phe Nuclear pulverulent Yan et al. (2008)
cataract
35 GJA8 Trp45Ser “Jellyfish-like” cataract Vanita et al. (2008a)
36 GJA8 Pro88Gln “ Balloon-like" cataract Vanita et al. (2008b)
with Y-sutural opacities
37 GJA8 Ile31Thr Congenital nuclear Wang et al. (2009)
cataract
38 GJA8 Arg198Trp Congenital cataract- Hu et al. (2010)
microcornea syndrome
39 GJA8 Ser258Phe Congenital nuclear Gao et al. (2010)
cataract
40 GJA8 Glu201Lys Perinuclear cataract Su et al. (2013)
41 GJA8 Asp47His Nuclear and zonular Li et al. (2013)

pulverulent
42 GJA3 1361insC, Coralliform cataracts Zhou et al. (2013)
p.Ala397Glyfs×71
43 GJA8 Pro88Thr Total cataract Ge et al. (2014)
44 GJA8 Leu7Pro Not known Mackay et al. (2014)
45 GJA8 His98Pro Not known
46 GJA8 Val44Ala Suture-sparing nuclear Zhu et al. (2014)

cataract
47 GJA8 His277Tyr Pulverulent nuclear Chen et al. (2015)
cataracts
48 GJA8 c.426_440del15 Nuclear cataracts Min et al. (2016)
49 GJA8 Pro59Ala Nuclear Yu et al. (2016)
50 GJA8 Arg67His Lamellar cataract with

punctate opacities
The above table lists the details of several mutations reported in the GJA8 and GJA3 genes encod-
ing Connexin-50 and Connexin-46, respectively. The shaded rows refer to mutations identified in
Indian families
The importance of the LIM2 gene for lens transparency was brought out by a
cataractous mouse strain created by chemical mutagenesis. A strain of mice desig-
nated as To3 (total opacity 3) had autosomal dominant cataracts that were evident in
the mutant heterozygotes but had a severe phenotype in homozygous mice which
had very dense cataracts and microphthalmia. All homozygotes were viable and
fertile. The To3 locus was mapped to mouse chromosome 7 very close to the Lim2
gene (Kerscher et al. 1996). The human chromosome that is orthologous to this
region is chromosome 19. Histology of the lenses of homozygous mice showed a
disorganized structure of lens fibers, vacuolated lenses, and rupture of the lens cap-
sule. In addition, the eyes of the homozygous To3 mice had small vitreous cavities
and a markedly small eye size. The mutation in the Lim2 gene in the To3 mice was
identified by PCR amplification of the genomic DNA of homo- and heterozygous
To3 mouse strains as well as normal mice. The entire Lim2 gene was amplified so as
to cover all five exons and the intervening introns, and overlapping PCR products
generated were analyzed by cloning and sequencing. A missense change of glycine-
15 to valine was observed in the To3/To3 homo- and To3/+ heterozygous mice
(Steele et al. 1997). This change is located in the alpha-helical region of the first
transmembrane segment of the protein and is predicted to create a turn in the
alpha-helix.
LIM2 Mutations in Cataracts

There are very few mutations reported in the LIM2 gene in families with cataracts.
These are all missense mutations, and in three cases, these were found in association
with adult-onset (presenile or senile) cataracts. Familial transmission in congenital
and presenile cataracts reported so far is autosomal recessive. The first report of a
LIM2 gene mutation in cataract was obtained from a study of an Iraqi-Jewish fam-
ily, having three affected children of a first-cousin marriage, reported with presenile
cataract of autosomal recessive inheritance. The first visual symptoms in three
affected offspring, described as having cortical pulverulent cataracts, appeared in
adulthood between the second and fifth decades of life. Evaluation of markers at
various cataract loci corresponding to genes associated with human and mouse cata-
racts suggested that markers on chromosome 19, close to the LIM2 gene, cosegre-
gated with the cataract phenotype in the family. A mutation leading to a
nonconservative change of Phe105Val in LIM2 was identified as the pathogenic
basis of the cataract in this family (Pras et al. 2002). The mutation involves a residue
in the third transmembrane segment of the protein and is thought to result in a mild
reduction in the activity of the protein, thereby associated with a milder form of
cataract with late-onset. In contrast, the Lim2 mutation in the To3 mouse is a non-
conservative change replacing a small amino acid for a bulky one.
LIM2 mutations are associated with congenital cataracts, and the mutations in
these cases are predicted to have a severe impact on the protein. One such mutation
is a missense change of Gly154Glu involving a nonconservative change located in
the fourth transmembrane region of LIM2, identified in an Indian family with auto-
somal recessive congenital cataracts. The affected offspring were born of a consan-
guineous marriage and had dense cataracts reportedly in the first few years of life,
with nystagmus and amblyopia (Ponnam et al. 2008). Another example of a LIM2
mutation associated with congenital cataract also consists of a missense change of
glycine-78 to aspartic acid (Gly78Asp). In this case as well, both the mutation and
the cataract phenotype of the patients may be categorized as severe. The mutation
Gly78Asp was detected in a family of Pakistani origin, in which all four offspring
of a consanguineous union were affected at birth with dense cataracts and had nys-
tagmus and amblyopia. Linkage analysis in this family mapped the disease onto a
region of several centimorgans on chromosome 19, which contained over 100 genes,
among which the LIM2 gene was an obvious candidate gene for the cataract pheno-
type. The nature of the mutation of Gly78Asp in LIM2 in the family is a nonconser-
vative substitution, involving replacement of a small neutral amino acid with a
larger, charged amino acid. The residue is located in the second transmembrane
region of LIM2 and is predicted to alter the topology of the protein and thus render
it nonfunctional (Irum et al. 2016).
Apart from the abovementioned, two heterozygous changes in the LIM2 gene,
one missense (Met23Leu) and one synonymous, were detected in a screen of
patients with age-related cataracts (Zhou et al. 2011).
2.1.6 Mutations in Genes Encoding Transcription Factors
Congenital cataracts have been mapped to genes that encode transcription factors
important for development of the lens and for regulating the expression of lens pro-
teins. These genes include MAF, PITX3, FOXE3, and PAX6. In many of these cases,
the phenotypes associated with such genes include one or more developmental
anomalies such as microphthalmia, microcornea, and anterior segment dysgenesis,
in addition to cataract.
2.1.6.1 MAF
MAF (musculoaponeurotic fibrosarcoma) is a transcription factor family containing
a basic leucine zipper (bZIP) domain. The MAF gene was originally identified as a
proto-oncogene. It binds to a target DNA sequence known as a MaF-responsive ele-
ment (MARE) that resembles the AP1 site, both as homodimers and heterodimers.
The lens-specific isoform L-Maf closely resembles other large Maf proteins, with a
high degree of similarity especially among the DNA-binding domains of these
proteins.
The Maf protein from lens (L-Maf) was identified by screening for proteins
bound to a consensus oligonucleotide having a lens-specific enhancer sequence ele-
ment (termed α-CE2) present in the alpha-crystallin promoter of chicken. A lens-
specific cDNA expression library prepared from chick embryonic lens was screened
with oligonucleotide probes containing the α-CE2 enhancer element in order to
isolate cDNAs for proteins that bound to the enhancer. The transcript thus obtained
was found to be expressed exclusively in embryonic lenses, in the lens placode at
very early stages of lens development. It was 3.6 kb long and encoded a predicted
protein of 286 amino acids with sequence motifs that were typical of the maf
proto-oncogene family. This factor was thus named as L-Maf (lens specific) and
found to be a regulator of alpha-crystallin gene expression (Ogino and Yasuda
1998). Through its effects on the lens gene expression, L-Maf appears critical for
the process of lens differentiation. Indeed, it was found that the expression of L-Maf
in neural retinal cells from chick embryos could induce them to differentiate to lens
fiber cells through the upregulation of various lens-specific genes.
Outside the eye, MAF is expressed in several tissues such as bone, cartilage,
nervous system, lymphocytes, heart intestine, kidney, liver, lung, muscle, etc.
The association of MAF with ocular disease in humans was made in a study of
two families (Jamieson et al. 2002). In one family of three generations, affected
members had varying phenotypes that ranged from the milder end, of just juvenile-
onset cataract in some individuals, to severe ones of cataract with anterior segment
dysgenesis and microphthalmia in others. The former category of affected individu-
als with just congenital cataracts were found to have a balanced translocation
between chromosomes 5 and 16, while those that had more severe phenotypes
including developmental delay had an unbalanced translocation. The cloning of the
translocation and sequencing of the junction regions revealed that the chromosome
16 breakpoint disrupts the regulatory region of MAF. In a second family analyzed
in the same study, affected members in three generations had cortical or nuclear
pulverulent opacities, associated with a mutation leading to substitution arginine-
228 to proline in MAF (Arg228Pro). The mutation is located in the DNA-binding
domain. Microcornea and colobomas were present in some of the affected individu-
als in addition to cataracts. Subsequent studies have reported several more muta-
tions in MAF, with the majority being missense changes. Most of these mutations
are located in the region coding for the b-ZIP domain of MAF, and a few are located
in the N-terminal region, upstream of the transactivating domain (Anand et al.
2018). Mutations in MAF are also associated with developmental syndromes such
as Ayme-Gripp syndrome which includes defects in other organs in addition to cata-
ract. These appear to be located in the upstream region of the gene, coding for the
N-terminal part of the protein. The mutations associated with cataract are located in
the C-terminal region of the protein, in the bZIP domain of MAF. No mutations are
known so far to localize to the transactivation domain, possibly because such muta-
tions may be lethal. As in the case of other transcription factor genes, MAF gene
mutations are also associated with other ocular defects, such as colobomas, glau-
coma, microcornea, microphthalmia and Peter’s anomaly.
Animal Models of Maf Deficiency

The importance of L-Maf on lens development and maturation is further empha-
sized by studies on Maf deficient mice having a replacement of the Maf gene with a
LacZ by homologous recombination. Absence of Maf in these mice led to defects in
the maturation of lens fiber cells, along with a reduction in the size of the lens and a
severe reduction in the levels of alpha and beta-crystallins (Ring et al. 2000). A dif-
ferent type of mouse model having a mutation that changed a specific residue in Maf
was created by a random ENU mutagenesis by Perveen and coworkers. The change
involved substitution of aspartic acid-90 for valine (Asp90Val; D90V) located in the
References 53
transctivation domain of Maf. Mice homozygous for the Asp90Val change dis-
played isolated cataracts, in contrast to the other developmental defects and extra-
ocular phenotypes shown by the total loss of function alleles of Maf. The Asp90Val
substitution in Maf protein was found to lead to an increased activity from Maf-
responsive promoters as compared with the wild type Maf, by transient expression
in cell lines (Perveen et al. 2007).
Another ENU mutant affecting the Maf gene is the Arg291Gln mutation, known
as the “opaque flecks in lens” (Ofl). The mutation is located in the basic domain of
the Maf protein, analogous to some mutations identified in humans, such as the
Arg288Pro and Arg291Gln substitutions. The Arg291Gln mutation in the Ofl het-
erozygous mice causes pulverulent cataracts, while mice homozygous for the same
mutation display renal abnormalities and early lethality. Other ocular phenotypes
involving anterior segment anomalies in addition to cataract have been noted in
some strains of heterozygous Ofl mice produced in certain genetic backgrounds
suggesting a modifier effect on the phenotype that is strain-dependent (Lyon et al.
2003).
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Genetics of Ectopia Lentis
3
Ectopia lentis (EL) is the dislocation of the lens from its normal position, which is
maintained by the zonular filaments. Lens subluxation refers to a partial displace-
ment of the lens. Lens displacement can be associated with various complications
such as refractive error, diplopia, cataract, leakage of lens proteins into the vitreous
with consequent inflammation of the vitreous, and chorioretinitis. Displacement
into the anterior chamber may result in papillary block and glaucoma. EL can arise
due to hereditary factors or may be secondary to various conditions including
trauma, large eyeball, cataracts, pseudoexfoliation, etc. Hereditary EL can be iso-
lated or associated with systemic manifestations. It is most commonly found in
Marfan syndrome and also in syndromes such as Weill-Marchesani and Ehlers-
Danlos and some metabolic disorders.
3.1 Genetics of EL
EL is a principal feature of Marfan syndrome, an autosomal dominant systemic

disorder involving the connective tissue of the eye, bone, and cardiovascular sys-
tems. Manifestations include skeletal abnormalities such as bone overgrowth with
abnormally long and slender digits (arachnodactyly), chest deformities, scoliosis,
and rib overgrowth. In addition, aortic root aneurysm and mitral valve prolapse
occur in patients with Marfan syndrome, leading to increased morbidity and mortal-
ity. Lens subluxation occurs in 50% or more of patients with Marfan. Disorders
such as mitral valve prolapse can occur in isolation, without ectopia lentis. The
foregoing manifestations all involve the connective tissue and may occur either sin-
gly or in combination with each other. They form a continuum of phenotypes desig-
nated as “MASS” (mitral valve, aorta, skeleton, and skin are affected). They may
not be clearly distinguishable entities, especially in the absence of molecular or
biochemical diagnosis. Another factor that can complicate the diagnosis is the fact
that all manifestations may not develop at the same point of time—e.g., defects in
mitral valve and aorta may appear only after the age of 20 years, since the

62 3 Genetics of Ectopia Lentis
cardiovascular system is not fully developed before this. Therefore, a diagnosis of

isolated ectopia lentis cannot be made in patients who are under 20 years of age. In
this situation, the presence of ectopia lentis in combination with a known FBN1
mutation that is previously associated with aortic dilatation and dissection may be
used to assign a diagnosis of Marfan syndrome. Thus, the genotype-phenotype cor-
relation, as documented for a particular mutation, may aid in the diagnosis in subse-
quent patients. The importance of molecular genetics in establishing a diagnosis
here is underlined in the criteria given under the Ghent nosology as revised in 2010
(Loeys et al. 2010; Chandra et al. 2015).
Marfan syndrome is most commonly transmitted through autosomal dominant
inheritance, as seen in 75% of cases; 25% of patients have de novo mutations (Sadiq
and Vandeveen 2013). The fibrillin family in mammals consists of the FBN1, FBN2,
and FBN3 genes encoding three fibrillin proteins. Apart from being key structural
components of the extracellular matrix (ECM), fibrillins aid in sequestering trans-
forming growth factor β and related proteins known as the latent TGF-beta binding
proteins (LTBP1-4). Fibrillin 1, a 350 kDa glycoprotein, is the most abundant pro-
tein in the zonular filaments. It is encoded by the FBN1 gene on chromosome
15q21.1, made up of 65 exons. The fibrillin 1 protein contains 47 epidermal growth
factor (EGF)-like domains; these are described as “beads on a string.” Fibrillin mol-
ecules can associate with each other into microfibrils that can form one-, two-, or
three-dimensional structures. The beads are 50 nm apart, while the diameter of the
microfibril is 10–15 nm (Davis et al. 2002).
The FBN1 gene shows a high degree of mutational heterogeneity in the fibrilli-
nopathies. Over 600 mutations are reported till date in the FBN1 gene and are
recorded in a database (UMD FBN1). The mutation spectrum consists mainly of
missense mutations, which make up about two-thirds of the total, the remaining
being nonsense, splice-site, and insertion/deletion mutations (Robinson et al. 2006).
3.2 Genetics of Isolated EL
Autosomal dominant isolated ectopia lentis is reported in families that do not show
the other features of Marfan syndrome (OMIM 129600). It is in some cases caused
by mutations in the FBN1 gene, and rigorous exclusion of other manifestations that
are part of Marfan syndrome must be carried out to make the diagnosis. A point
mutation in FBN1 was first reported in a family of four generations with autosomal
dominant EL (Lönnqvist et al. 1994).
Autosomal recessive ectopia lentis is associated with mutations in the ADAMTSL4
(a disintegrin-like and metalloproteinase with thrombospondin-like 4 gene on chro-
mosome 1q21.4) (Ahram et al. 2009). The ADAMTSL4 gene [alternative symbol
TSRC1] belongs to a family of secreted glycoproteins that are located within the
extracellular matrix (ECM). The ADAMTSL proteins have a modular structure with
resemblance to the thrombospondin-like domains in the ADAMTS proteases, but
unlike these, they have no protease domain. They may have structural or regulatory
functions in the ECM (Apte 2009). Mutations in the ADAMTSL4 gene have been
References 63
identified in families with ectopia lentis from various regions including Europe and
the Middle East.
There are few reports on the genetics of EL or Marfan syndrome in Indian
patients. Isolated ectopia lentis was mapped to chromosome 15q in an Indian family
of four generations having 27 affected members. Sequencing of the FBN1 gene
showed a mutation of c718T>C leading to substitution of Arg240Cys (Vanita et al.
2007).
References
Ahram D, Sato TS, Kohilan A, Tayeh M, Chen S, Leal S, et al. A homozygous mutation
in ADAMTSL4 causes autosomal-recessive isolated ectopia lentis. Am J Hum Genet.
2009;84:274–8.
Apte SS. A disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1
motif (ADAMTS) superfamily: functions and mechanisms. J Biol Chem. 2009;284:31493–7.
Chandra A, Patel D, Aragon-Martin JA, Pinard A, Collod-Béroud G, Comeglio P, et al. The revised
Ghent nosology; reclassifying isolated ectopia lentis. Clin Genet. 2015;87:284–7.
Davis EC, Roth RA, Heuser JE, Mecham RP. Ultrastructural properties of ciliary zonule microfi-
brils. J Struct Biol. 2002;139:65–75.
Loeys BL, Dietz HC, Braverman AC, Callewaert BL, De Backer J, Devereux RB, et al. The
revised Ghent nosology for the Marfan syndrome. J Med Genet. 2010;47(7):476–85. https://
doi.org/10.1136/jmg.2009.072785.
Lönnqvist L, Child A, Kainulainen K, Davidson R, Puhakka L, Peltonen L. A novel mutation of
the fibrillin gene causing ectopia lentis. Genomics. 1994;19:573–6.
Robinson PN, Arteaga-Solis E, Baldock C, Collod-Béroud G, Booms P, De Paepe A, et al. The
molecular genetics of Marfan syndrome and related disorders. J Med Genet. 2006;43:769–87.
Sadiq MA, Vandeveen D. Genetics of ectopia lentis. Semin Ophthalmol. 2013;28:313–20.
Vanita V, Singh JR, Singh D, Varon R, Robinson PN, Sperling K. A recurrent FBN1 mutation in an
autosomal dominant ectopia lentis family of Indian origin. Mol Vis. 2007;13:2035–40.
Genetics of Glaucoma
4
Glaucoma refers to a group of heterogeneous conditions involving optic neuropa-

thy, with progressive loss of retinal ganglion cells (RGCs). The death of RGCs gives
rise to a characteristic cupping of the optic nerve head, with thinning of the neuro-
retinal rim and an increased cup-disc ratio. Loss of RGCs eventually leads to a loss
of visual fields, which may progress to blindness. Raised intraocular pressure is one
of the many risk factors for glaucoma, although it is not necessarily a feature of all
forms of glaucoma. There are broadly two forms of glaucoma—primary and sec-
ondary. The primary glaucomas include primary open-angle glaucoma (POAG),
primary angle closure glaucoma (PACG), and primary congenital glaucoma (PCG).
They are non-syndromic and not causally associated with other factors such as
trauma or systemic diseases. PACG results from the closure of the angle between
the iris and cornea, while POAG involves a block in the trabecular meshwork due to
malfunction. PCG (also known as buphthalmos) is a rare form of developmental
glaucoma which occurs due to defects in the development of the trabecular mesh-
work (TM), in the absence of any other ocular anomalies.
4.1 Primary Congenital Glaucoma
Primary congenital glaucoma (PCG) represents less than 5% of all forms of glau-
coma and has a higher prevalence in communities where inbreeding is practiced.
These include sections of the Indian population, Saudi Arabians, and Slovak gyp-
sies (prevalence of about 1:2500–3000); it is much less frequent in Western popula-
tions (prevalence 1:20,000–30,000) (Dandona et al. 1998; Cascella et al. 2015). It
develops at birth or shortly afterward, and its manifestations include elevated intra-
ocular pressure (IOP), corneal epithelial edema, enlargement of the globe (buph-
thalmos), increased corneal diameter, Haab’s striae or breaks in the Descemet’s
membrane, optic nerve cupping, blepharospasm, and photophobia.
It is inherited as an autosomal recessive trait, and loci for PCG are designated as
GLC3; four loci have been mapped in this group so far.

66 4 Genetics of Glaucoma
4.1.1 Genetics of PCG
4.1.1.1 GLC3A/Cytochrome P4501B1 (CYP1B1) Gene

The GLC3A locus was mapped by linkage analysis to chromosome 2p21 in 17 fami-
lies of Turkish origin, and 11 of the families showed linkage of the disease pheno-
type to this region. Positional cloning of the mapped interval led to the identification
of the CYP1B1 gene as the GLC3A gene and three mutations leading to frameshift
were identified in five families with PCG that were linked to the GLC3A locus
(Sarfarazi et al. 1995; Stoilov et al. 1997). The same locus was independently
mapped to chromosome 2p21 in several consanguineous Saudi Arabian families. In
this population, an approach of homozygosity mapping with highly polymorphic
markers spread throughout the genome was employed. Analysis of pooled DNA
samples from a subset of families showed homozygosity for an informative marker
in the chromosome 2p21 locus. Further linkage analysis of over 200 individuals
from 25 Saudi families revealed significant linkage to 2p21, mapping the disease to
a 5 cM interval in this region (Bejjani et al. 1998). Analysis of the CYP1B1 gene
showed three missense changes in 24 of the 25 families that were mapped to the
GLC3A locus. An additional feature in this study was that a subset of individuals in
11 families, though unaffected, shared the disease haplotypes with their affected
siblings, suggesting non-penetrance of the disease gene in these individuals.
Mutations in CYP1B1 have been reported in PCG-affected families from various
populations, including Indian, Brazilian, Japanese, Saudi Arabians, and Slovaks,
with differences in the prevalence of mutations between populations (reviewed by
Kaur et al. 2011). However, there is genetic heterogeneity in PCG as suggested by
the absence of linkage to the GLC3A locus in a proportion of families. A second
locus, GLC3B on chromosome 1p36, was mapped in some GLC3A-negative fami-
lies (Sarfarazi et al. 1995). The GLC3B gene is not identified as yet.
The CYP1B1 gene encodes a protein belonging to the cytochrome P450 family of
proteins, which constitute a superfamily of monooxygenases. There are over 50 mem-
bers of the cytochrome P450 family in humans. All cytochromes have heme as the
prosthetic group. They are named on the basis of their characteristic absorbance at
450 nm and are mostly membrane-bound proteins expressed within the endoplasmic
reticulum and mitochondria. They are involved in the activation/inactivation of metab-
olites and xenobiotics, commonly through oxygenation. These processes may result in
either detoxication or in the formation of toxic compounds. The human Cyp1b1 protein
has 543 amino acids and is made up of three regions: the membrane-bound N-terminal
region of 53 residues, a proline-rich hinge region made of 10 residues, and the 480 resi-
due long cytosolic globular domain. It has high activity toward endogenous steroids
and polycyclic aromatic hydrocarbons. It is widely expressed in several extrahepatic
tissues, but not detected in the liver. It is expressed at higher levels in fetal as compared
to adult human eyes, and the protein is detectable in the corneal epithelium, kerato-
cytes, and iris stromal cells (Zhao et al. 2015). Knockdown of Cyp1b1 in mice led to
phenotypes that were apparently normal in the gross structure of the eye and intraocu-
lar pressure but had defects detectable by electron microscopy in the Schlemm’s canal,
iris, and trabecular meshwork, similar to those in PCG patients (Libby et al. 2003).
4.1 Primary Congenital Glaucoma 67
Mutations in CYP1B1 in Indian PCG Patients

Mutations in CYP1B1 in association with PCG were first identified in consanguine-
ous Indian families (Panicker et al. 2002), with affected individuals being homozy-
gous or compound heterozygous for various missense and termination mutations.
The prevalence of PCG in Southern India appears relatively high partly due to its
association with consanguinity and has been studied extensively to characterize the
genetic spectrum of the disease. Analysis of over 140 Indian families suggests that
certain mutations are more frequent in these patients (summarized in Table 4.1).
Mutations that appear to be highly recurrent (i.e., Pro129Leu, Glu229Lys,
Arg368His, Arg390Cys, Gly61Glu, and 367insA) have a combined frequency of
about 30% in PCG-affected families from India. Among them, Arg368His is the
predominant allele, found in almost half of all families with mutations (Reddy et al.
2003). The overall frequency of all CYP1B1 mutations in PCG patients from India
is about 40%, and the majority of mutations are missense changes. Deletions and/or
frameshifts occurred in fewer patients (Reddy et al. 2004). Analysis of haplotypes
Table 4.1 CYP1B1 mutations in Indian patients

CYP1B1 mutation (amino
acid) No. of families References
Leu24Arg 1/50 Tanwar et al. (2009)
Gly61Glu 4/143 Panicker et al. (2002) and Reddy et al. (2003)
Leu77Pro 1/64 Reddy et al. (2004)
Ala115Pro 1/64 Reddy et al. (2004)
Met132Arg 1/64 Reddy et al. (2004)
Gln144Pro 1/64 Reddy et al. (2004)
Phe160Leu 1/50 Tanwar et al. (2009)
Pro193Leu 2/138 Reddy et al. (2003)
Pro193Leu+Glu229Lys 1/5 Panicker et al. (2002)
Glu229Lys 6/138 Reddy et al. (2003)
Ter@223 2/143 Panicker et al. (2002) and Reddy et al. (2003)
9/50a Tanwar et al. (2009)
Ser239Arg 2/64 Reddy et al. (2004)
Gly329Asp 1/50 Tanwar et al. (2009)
Arg368His 25/143 Panicker et al. (2002) and Reddy et al. (2003)
1/8 Yang et al. (2017)
Arg390Cys 5/138 Reddy et al. (2003)
Arg390His 8/50a Tanwar et al. (2009)
Pro400Ser 1/8 Yang et al. (2017)
Pro437Leu 1/64 Reddy et al. (2004)
Gly466Asp 1/64
Gly466Val 1/8 Yang et al. (2017)
Arg469Trp 2/8 Yang et al. (2017)
The data in the table shows representative CYP1B1 mutations in Indian PCG patients, though it is
not an exhaustive list
a
These figures include homo- and heterozygous patients. The denominators represent the total
number of patients screened
of intragenic polymorphisms in CYP1B1 in PCG patients with CYP1B1 mutations

(CYP1B1 (+)), PCG patients without CYP1B1 mutations (CYP1B1 (+)), and nor-
mal controls suggested that the frequencies of various haplotypes are significantly
different between the three groups. The predominant haplotypes were different in
CYP1B1 (+) and CYP1B1 (−) subgroups of patients, denoting mutation carriers and
noncarriers, respectively. The common haplotype in the CYP1B1 (+) subgroup was
four times more frequent in this subgroup of patients as compared to normal con-
trols and threefold more prevalent in CYP1B1 (+) than in the CYP1B1 (−) patients.
Further, specific SNP haplotype backgrounds are shared by patients with a particu-
lar mutation, even across different populations. These observations have suggested
that the mutations originated from a common founder (Chakrabarti et al. 2006).
CYP1B1 mutations obtained in a study of eight PCG families from Southern India
are also depicted in the Table 4.1 (Yang et al. 2017).
The mutational pattern obtained on PCG patients from North India shows
certain similarities and certain differences from the population in Southern
India. Screening of fifty probands with PCG in a study conducted in Northern
India showed about 46% of patients to have CYP1B1 mutations (Tanwar et al.
2009), similar to patients from Southern India (previous para). The Arg368His
mutation is found to be less frequent in North Indian patients, with a frequency
of 8% (4 patients positive out of 50 tested) as compared with the data on Southern
Indian patients, in which 16% of patients screened, i.e., 23 out of 138 patients,
were positive for this mutation. Screening for the six most prevalent mutations
reported by Reddy et al. (2004), in the North Indian population of PCG patients,
showed that two of these, i.e., mutations of Ter@223 and Arg390His, were also
frequent in these cases; the two mutations accounted for about 16% and 18% of
cases if one considers both homo- and heterozygotes. In this study, three novel
mutations in CYP1B1 were also found, viz., leucine-24 to arginine (Leu24Arg),
phenylalanine-190 to leucine (Phe190Leu), and glycine-329 to aspartic acid
(Gly329Asp).
4.1.1.2 The GLC3C/LTBP2 (Latent Transforming Growth Factor Beta

Binding Protein 2) Gene
Further evidence for a third PCG locus (designated GLC3C) was obtained in some
families showing absence of linkage to the GLC3B region (Akarsu et al. 1996).
Mapping of the GLC3C locus to chromosome 14q24 led to the identification of the
gene as LTBP2 (latent transforming growth factor beta binding protein 2) as the
GLC3C gene. Mutations in the LTBP2 (GLC3C) gene were identified in PCG cases
from two populations. One study involved Iranian families (Narooie-Nejad et al.
2009). The second study included Pakistani families mapped to the GLC3C locus,
as well as 15 families, all CYP1B1-negative, of European gypsies (Ali et al. 2009).
Interestingly, several of the gypsy families and a Pakistani family were found to
have the same mutation, c.895C→T (Arg299Stop), present within a common hap-
lotype of intragenic SNPs at the GLC3C locus, suggesting that the mutation has a
common ancestral origin. The Romas are a nomadic group of gypsies with an esti-
mated population of a few million, dispersed throughout Europe. Linguistic,
4.2 Primary Open-Angle Glaucoma (POAG) 69
anthropological, and genetic evidences suggest that Roma gypsies originated from
an ancestral population in the Indian subcontinent about 1000 years ago, more prob-
ably from northwestern India, based on mitochondrial haplogroup affinity
(Mendizabal et al. 2011). They may therefore be genetically related to Pakistanis,
who also share geographical boundaries and ancestry with this region.
4.2 Primary Open-Angle Glaucoma (POAG)
Primary open-angle glaucoma is the most common type of glaucoma in some popu-
lations such as Caucasians and Africans and is characterized by an open or normal
iridocorneal angle, with normal or raised intraocular pressure (IOP). The mecha-
nism of retinal ganglion cell death is largely unknown, but there are several risk
factors that are associated with POAG. These include age, raised IOP, family his-
tory, gender, ethnicity, and myopia. It is found to be more prevalent and shows rapid
progression in Africans as compared with Caucasians, Hispanics, and Asians
(Rotchford et al. 2003; Zhang et al. 2012). Systemic diseases such as diabetes and
hypertension have been associated with POAG, the latter being associated with high
tension glaucoma (Dielemans et al. 1995; Zhou et al. 2014). There are conflicting
reports on the association of gender with POAG, some population-based studies
showing a higher prevalence in males while others have found no difference (Abu-
Amero et al. 2015). A positive family history increases risk of POAG to about five-
to tenfold. Moderate to high myopia confers a two- to threefold higher risk of
developing POAG. The only modifiable risk factor is IOP, and reduction of IOP is
considered to slow the progression of the disease even if it is not elevated above the
“normal” range.
4.2.1 Genetics of POAG
POAG is most commonly a genetically complex disorder, but a fraction of patients

have a highly heritable disease with familial transmission. Studies on large affected
families with POAG have resulted in the identification of at least 20 loci that were
mapped by linkage studies, designated as GLC1A-P (shown in Table 4.2). The dif-
ferent forms of disease reflect varying magnitudes of genetic effects involved in the
causation of POAG, ranging from alleles with small effects to those with large
effects and of low and high penetrance. The alleles with high penetrance are gener-
ally present only in diseased individuals and are deleterious to the protein function.
Mutations in several genes are considered as “causative” for POAG, i.e., alterations
in these genes are sufficient to give rise to the disease, with little influence of other
genes or environmental factors. This also implies that they have high penetrance and
are likely to severely affect gene function. They fit in with the traditional definition
of a “disease-causing variant” (DCV), which was defined by the following crite-
ria—(a) the variant alters the myocilin amino acid sequence, (b) is present in one or
more glaucoma subjects, (c) is present in less than 1% of the general population,
Table 4.2 Genes and loci associated with POAG

Locus Chromosome Inheritance Gene Comments References
GLC1A 1q31 AD MYOC Juvenile-onset Stone et al.
open-angle glaucoma (1997)
(JOAG) and adult-onset
POAG
GLC1B 2cen-q13 Linkage analysis in six Stoilova et al.
families (1996)
GLC1C 3q21-24 Single large family Wirtz et al.
from North America (1997)
GLC1D 8q23 Single family Trifan et al.
(1998)
GLC1E 10p15-14 OPTN Normal tension Sarfarazi et al.
glaucoma in British (1998) and
family Rezaie et al.
(2002)
GLC1F 7q35-36 ABS10 Single family from Wirtz et al.
Oregon, USA. ABS10- (1999) and
Ankyrin repeats and Pasutto et al.
suppressor of cytokine (2012)
signaling box-
containing protein 10
GLC1G 5q22.1 WDR36 Mapping of seven Monemi et al.
families; gene involved (2005)
in T-cell activation
GLC1H 2p15-16 Not known Adult-onset POAG Suriyapperuma
mapped in seven et al. (2007)
families
GLC1I 15q11-q13 Complex Not known Mapped in 17/86 Allingham
multiplex families with et al. (2005)
early adult-onset POAG and Wiggs
et al. (2000)
GLC1J 9q22 AD JOAG Wiggs et al.
GLC1K 20p12 AD JOAG (2004)
GLC1L 3p21-22 Not known Tasmanian family with Baird et al.
POAG; second locus (2005)
mapped in addition to
MYOC in same family
GLC1M 5q22-32 AD Not known JOAG in five- Pang et al.
generation Filipino (2006)
family
GLC1N 15q22-24 AD Not known JOAG Wang et al.
(2006)
GLC1O 19q13 AD NTF4 Normal and high Pasutto et al.
tension POAG; (2009)
missense changes in
1.7% of cases
GLC1P 12q14 AD Duplication Low tension glaucoma Fingert et al.
of 600 kb (2011)
spanning
four genes
Shown above are various loci for POAG known so far with details of genes/loci that are associated.
The comments noted are certain details of the first studies (shown under References in the right
hand column) that reported the respective loci
and (d) is absent in normal controls (Fingert et al. 1999). The genes that have
glaucoma-associated variants in this category include myocilin (MYOC), optineurin
(OPTN), WD repeat domain 36 (WDR36), cytochrome P4501B1 (CYP1B1), neuro-
trophin 4 (NTF4), ankyrin repeat, and SOCS box-containing 10 (ASB10) (reviewed
by Abu-Amero et al. 2015).
Support for genetic factors in complex forms of POAG comes from various
studies that have examined heritable nature of POAG itself as well as of various
clinical traits that are altered in POAG. For example, quantitative traits related to
POAG such as intraocular pressure (IOP) or vertical cup-disc ratio (VCDR), as
inferred from twin and sibling studies, are heritable, suggesting that they are influ-
enced by genes (Chang et al. 2005; Toh et al. 2005). Traits such as these, which are
associated with a disease and have a strong genetic component, are otherwise
known as endophenotypes. Thus, some independent endophenotypes for POAG,
apart from IOP and VCDR, are the disc area, cup area, and central corneal thick-
ness (CCT). Genetic studies on complex forms of POAG have used candidate gene-
based association studies as well as GWAS. These studies detect low-penetrance
variants which are often polymorphisms present in the normal population and
thereby do not conform to the traditional definition of a “disease-causing variant”
(DCV) as noted above.
4.2.1.1 Myocilin/TIGR
The first gene to be identified for familial open-angle glaucoma is the TIGR (tra-
becular meshwork-induced glucocorticoid response) gene, also known as myocilin
(MYOC), mapped to the GLC1A locus in a large family of 37 members with auto-
somal dominant JOAG, using linkage analysis with short tandem repeat polymor-
phisms to chromosome 1q21-31 (Sheffield et al. 1993). The linkage of JOAG to this
locus on chromosome 1 was also made independently in another large family of
Caucasian descent. Thirty individuals were examined. The pedigree showed an
autosomal dominant transmission of the disease, and age at diagnosis in the affected
persons was during the first to second decades of life (Richards et al. 1994). Genetic
studies on open-angle glaucoma from various populations mapped the disease to the
same locus, thereby confirming that both JOAG (juvenile open-angle glaucoma,
diagnosed in childhood or early adulthood) and the late-onset form designated as
COAG (chronic open-angle glaucoma, diagnosed after 40 years of age) share the
same etiology.
The gene for COAG and/or JOAG representing the GLC1A locus was identified
by Stone and coworkers. The GLC1A locus was mapped to chromosome 1q by link-
age and haplotype analysis of several families, and suitable candidate genes were
selected from the interval for screening. A gene that was expressed in the trabecular
meshwork, known as the trabecular meshwork-induced glucocorticoid response
(TIGR) gene or myocilin (MYOC), mapping to the same locus, was considered as a
potential candidate gene for POAG. Analysis of the TIGR/MYOC gene for muta-
tions in the POAG families showed pathogenic variants in some families. Three
mutations were found, among the 13 probands tested (Stone et al. 1997), thus estab-
lishing MYOC as the gene for POAG.
The MYOC cDNA was independently isolated from a human ciliary body library,
and the gene was found to be expressed in the iris, ciliary body, trabecular mesh-
work, heart, and muscle tissue (Ortego et al. 1997). The myocilin protein has two
major domains—the myosin-like domain in the N-terminus and the olfactomedin
homology domain in the C-terminus. The myosin-like domain has periodic repeats
of leucine and arginine residues arranged into a leucine zipper-like motif.
Mutations in the myocilin gene are found in 2–4% of glaucoma patients of dif-
ferent populations, familial and isolate cases from North America (including
Caucasians and African-Americans), Australia, and Japan (Fingert et al. 1999). In
general, myocilin mutations are largely missense mutations, making up >80% of the
total. Frameshift, indel or nonsense mutations each contribute to 5% or less of cases.
Almost 90% of all mutations reside in the third exon of the myocilin gene, which
encodes the olfactomedin-like domain. The most common mutation is Gln368Stop
found in about 1.6% of probands with POAG from across multiple ethnicities and
occurred in all groups except the Japanese (Fingert et al. 1999). Details of MYOC
gene mutations reported in literature and their phenotypes are recorded in a data-
base (www.myocilin.com; accessed November 2018). Over 200 variants in the
myocilin gene coding regions and promoter are documented, of which about 40%
are characterized as DCVs. The analysis of data obtained across multiple studies
shows evidence of genotype-phenotype correlations. Taking into consideration
weighted phenotypes (i.e., a phenotype associated with each mutation was given
weight according to the number of patients studied in each case), genotype-
phenotype analyses indicate the association of specific mutations with either early
or later onset of disease. However, all mutations are found to reach 100% penetrance
by about 75 years of age (Hewitt et al. 2008). In addition, the prevalence of the com-
mon myocilin mutations shows ethnicity-dependent variations. Meta-analysis of
several studies done in different populations suggests that Gln368Stop is predomi-
nant in Caucasian patients, while Thr353Ile and Arg46Stop appear to be more prev-
alent in Asian populations (Cheng et al. 2012).
Genetics of POAG in Indian Populations

The genetics of POAG in Indian patients has been approached by looking for asso-
ciations of candidate gene loci with the disease. Among these, the myocilin gene
(MYOC) has been widely studied, for POAG-associated changes in patients from
various parts of India. Data reported from studies across the country are summa-
rized in Table 4.3. Association of a missense change of glutamine-48 to lysine
(Gln48His; Q48H) with POAG in Indian patients is evident from various studies.
Strikingly, this mutation is recurrent in POAG-affected individuals from different
regions of India as shown (Table 4.3). Among patients from Eastern India, this
mutation was detected in 3 out of 56 cases evaluated (Mukhopadhyay et al. 2002).
A study of 200 patients from Southern India by Chakrabarti and coworkers shows a
frequency of 2% for the Gln48His mutation for POAG. Interestingly, apart from
POAG, the same mutation in MYOC is found to occur in about 2.5% of patients with
PCG as well, although some affected individuals were heterozygotes for the muta-
tion. In addition, a digenic mechanism of disease involving both MYOC and CYP1B1
Table 4.3 Mutations in the myocilin gene in Indian patients with POAG
No. of patients MYOC mutations (no. of cases/
Reference screened Methods used families)
Mukhopadhyay et al. 56 Sequencing Gln48His (3); Pro370Leu (1)
(2002)
Kanagavalli et al. 107 SSCP, sequencing Gly367Arg (1), Thr377Met (1)
(2003)
Sripriya et al. (2004) 100 Sequencing Gln48His (2)
Chakrabarti et al. 200 PCR-RFLP and Gln48His (4)
(2005) sequencing
Bhattacharjee et al. 315 Sequencing Gln48His (3), Thr256Met (1),
(2007) Thr353Ile (1), Pro370Leu (1),
Gln368Stop (2), Gln399Aspa
(1), Ala427Thr (2)
Kumar et al. (2007) 251 PCR-RFLP, SSCP, Gln48His (2)
sequencing
Rose et al. (2007) 200 SSCP and Ser331Thr (1), Pro370Leu (1),
(2011) sequencing Gln48His (2), Thr353Ile/
Asn480Lysb (1)
Banerjee et al. 765 Sequencing Gln48His (7), R125fsX158 (1),
(2012) D273fsX344 (1), Gln368Stop
(3), Pro370Leu (1), Gly399Asp
(1), Ala427Thr (2), Thr256Metc
(2), Ser331Leuc (1)
Details of several representative studies on Indian patients with POAG from different regions are
shown above. The references denote the first author and year of publication for each of the studies.
The figures in parentheses in the last column indicate the numbers of patients or families having
each of the mutations
PCR polymerase chain reaction, SSCP single-strand conformation polymorphism, RFLP restric-
tion fragment length polymorphism
a
Found in homozygous individual
b
These two mutations occurred in a compound heterozygous individual
c
Predicted as benign changes
genes has been proposed, based on compound heterozygosity for the Gln48His
mutation in MYOC and Arg368His mutation in the CYP1B1 gene, detected in pro-
bands with PCG (Chakrabarti et al. 2005). Together with the absence of the
Gln48His mutation in a normal control population, these data point to a causative
role of the myocilin gene in PCG. The mutation is reported to have a frequency of
about 2% among patients with POAG and JOAG in another study from Southern
India as well, based on screening of 100 patients (Sripriya et al. 2004). Frequencies
of the other myocilin mutations obtained in each study can also be gleaned from the
table, which shows the number of mutation-positive patients as well as the total
numbers screened. These data suggest that the mutations shown have frequencies of
about 2–4% in Indian POAG patients, based upon only studies that have included
100 patients or more for screening since these estimates would be more reliable than
the frequencies of mutations obtained in smaller samples. Other mutations that
appear to be recurrent in Indian patients with POAG, although to a lesser extent than
Gln48His, are Gln368Stop and Pro370Leu.
4.2.1.2 Optineurin Mutations in POAG

The GLC1E locus was originally mapped in a family of 46 individuals including 15
affected, with chronic primary open-angle glaucoma having characteristic clinical
features of glaucoma, including an abnormal appearance of the optic nerve head,
visual field loss, and large cup-disc ratios (Sarfarazi et al. 1998). In addition, a dis-
tinctive feature of the family was the intraocular pressure, which was normal in all
affected members, with values below 22 mmHg. The disease was mapped to chro-
mosome 10p15-p14, and the optineurin (OPTN) gene was a strong candidate gene
within the mapped region due to its retinal expression. A missense change of glu-
tamic acid-50 to lysine (Glu50Lys; E50K) was detected in affected members of the
family, and further evaluation showed the association of four different OPTN muta-
tions in about 16% of familial POAG (Rezaie et al. 2002). The role of optineurin
appears to be limited to a specific subtype of POAG, namely, familial, normal ten-
sion POAG. Screening a large series of both normal and high tension glaucoma
cases, primarily Asian and Caucasian, failed to detect any variants that showed sig-
nificant associations with the disease (Alward et al. 2003).
Optineurin Mutations in Indian Patients

There are few reports of optineurin mutations in Indian POAG patients in literature,
with conflicting evidence on associations of the reported mutations with disease.
Potentially pathogenic mutations detected in Indian patients include arginine-545 to
glutamine (Arg545Gln) in a study which involved Indian patients from various parts
of the country, and a novel missense change of threonine-202 to arginine (Thr202Arg;
c.915C>G) in exon 7 identified in one heterozygous patient with POAG from
Southern India. The Arg545Gln mutation was detected at a frequency of 3% in a
population of 200 POAG patients but absent in controls. Though this variant seems to
have very low to negligible frequencies in various studies from the USA and UK, it
is reported as more prevalent in East Asian populations, with frequencies in the range
of 5–7%, even in normal populations from these regions. A similar phenomenon is
found with the Met98Lys (M98K) variant, which was detected to be associated with
open angle in some studies (Sripriya et al. 2006) but not in others (Mukhopadhyay
et al. 2005; Kumar et al. 2007). In normal populations with a higher prevalence of
Met98Lys alleles, there was no observed significance in its association with POAG.
Thus the differences with regard to the association of optineurin variants with
glaucoma are likely to be due to differences in the frequency of the relevant OPTN
alleles in different normal populations. They also need to be interpreted in light of
the sample sizes of patients and controls recruited, which range from below 50 to
over 1000 subjects in different studies. The association derived from the analysis of
smaller samples may not be robust, and validations of the associations may require
larger groups of cases and controls. There could be other reasons for the observed
variability in associations of a particular allele with disease, including differing
definitions of disease between studies, differences in ethnicity between patient pop-
ulations screened, and between ethnicities of case and control groups used to deter-
mine the association. Overall, existing evidence suggests that optineurin mutations
play a limited role in the pathogenesis of POAG in Indian patients.
4.3 Primary Angle Closure Glaucoma (PACG) 75
The pathological consequences of certain optineurin mutations have been inves-

tigated in an in vitro system by heterologous expression in suitable cell lines of
optineurin cDNAs containing selected mutations associated with glaucoma. OPTN
wild type and mutant cDNAs were evaluated in cultured cell lines by transient
expression assays. The OPTN cDNA carrying the substitution of methionine-98 to
lysine (M98K) variant apparently induced death of retinal ganglion cells (RGCs)
upon transient expression, but did not have this effect in other cell types tested. The
magnitude of cell death with this mutant was somewhat higher than that with other
OPTN mutants, or with the wild type protein, and was dependent on caspases (Sirohi
et al. 2013). Effects of induction of cell death have been attributed to the glutamic
acid-50 to lysine substitution (E50K) mutant of OPTN as well. The cell death in
response to the OPTN cDNA bearing the (E50K) mutant was found specifically in
the RGC5 cell line, which is presumably of retinal origin, but was not observed in
other cell lines such as HeLa and COS cells. It is found to be mediated by oxidative
stress, as shown by a partial protection from cell death induced by the OPTN E50K
protein, after the addition of various antioxidants into the cells. It is thought to occur
via cell death pathway that is distinct from apoptosis (Chalasani et al. 2007).
4.3 Primary Angle Closure Glaucoma (PACG)
PACG involves the obstruction to the outflow of aqueous humor due to the apposition
of the peripheral iris against the trabecular meshwork and a narrow anterior chamber
angle. Like POAG, it is accompanied by high intraocular pressure, with eventual
progressive loss of optic nerve axons and retinal ganglion cells. It is much more com-
mon in Asians as compared with Europeans and has a larger proportion of affected
females than other forms of glaucoma (Quigley and Broman 2006). Anatomic fea-
tures characteristic of certain ethnic groups such as East Asians are recognized as
predisposing factors for PACG. These include a short axial length, small corneal
diameter, shallow anterior chamber, and an anteriorly placed lens. The association of
PACG with older age and female gender may be explained by a decrease in anterior
chamber depth in these groups. PACG is a multifactorial disease, and genetic effects
contribute to its etiology. Observations that point to the involvement of genes in the
development of PACG are (1) an increased risk of the disease among siblings and
first-degree family members of affected individuals and (2) differences in heritability
of PACG between mono- and dizygotic twins (Ahram et al. 2015).
4.3.1 Genetics of PACG
The genetics of PACG has been explored through associations with specific candi-
date loci as well as genome-wide association studies (GWAS). Two GWAS on mul-
tiethnic cohorts of patients with PACG revealed the potential involvement of novel
genes in the pathogenesis of this complex disease. A GWAS study on PACG in
populations from different Asian countries (Singapore, Hong Kong, India, Malaysia,
and Vietnam), as well as Saudi Arabia and the UK, revealed association to SNPs at
genome-wide significance in the pleckstrin homology domain containing A7 pro-
tein (PLEKHA7) gene, collagen type XI alpha 1 chain (COL11A1), and in an inter-
genic region on chromosome 9q (Vithana et al. 2012). The PLEKHA7 gene on
chromosome 11p15 encodes an adherens junction protein that is thought to have a
role in fluid flow through the Schlemm’s canal. COL11A1 encodes one of the alpha
chains of collagen type XI. It is expressed in the trabecular meshwork cells.
Mutations in this gene are also associated with various diseases such as Stickler and
Marshall syndromes, two related disorders that manifest with defects in the audi-
tory, orofacial, and ocular tissues.
The involvement of the abovementioned loci was confirmed in another GWAS
on patients from 24 countries across Asia, Europe, and America involving over
10,000 cases (Khor et al. 2016). Apart from these, SNPs at five other loci (thus, a
total of eight loci) showed significant associations with the disease; the associated
SNPs localize to genes involved in a range of functions such as cell-cell adhesion,
glycosylation, neurotransmission, and others.
4.3.1.1 Genetics of PACG in Indian Patients

Associations of SNPs with PACG obtained in various populations were tested in a
population from Southern India which included cases with PAC (primary angle clo-
sure), PACS (primary angle closure suspect), and PACG. These included three loci
previously associated with PACG, namely, the intergenic region of PCMTD1-ST18,
PLEKHA7, and COL11A1 genes. Association of the SNP at the PCMTD1-ST18
locus alone was detected in this population, but not with SNPs at the other two loci
(Duvesh et al. 2013).
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Hereditary Retinal Degenerations
5
Hereditary retinal diseases are a group of very heterogeneous disorders involving

the retinal or choroidal tissue or both. Retinal disorders are classified broadly
according to the retinal layers primarily involved and whether they are stationary
or progressive. Thus there are the rod-dominated diseases (such as retinitis pig-
mentosa (RP) and night blindness), cone-dominated disease (such as cone and
cone-rod dystrophies and macular degenerations), generalized photoreceptor dys-
trophies (involve both types of photoreceptor), and vitreoretinal diseases (affect-
ing the vitreous and retina). These entities are further grouped according to whether
they are stationary or progressive and, within each of these categories, into syn-
dromic and non-syndromic disorders (Berger et al. 2010). The syndromic retinal
dystrophies involve other organs in addition to the retina. Genetics plays a major
role in further grouping of these diseases, based on the inheritance mode (autoso-
mal dominant, recessive, X-linked, digenic, mitochondrial, and simplex diseases)
and, further, according to their molecular etiologies, depending on the genetic
mutations involved. Among the so-called “monogenic” diseases, the diseases
which primarily affect the photoreceptors are a predominant group. The disorders
that are dealt with in the following sections include two major forms of non-syn-
dromic retinal dystrophy—retinitis pigmentosa (RP) and Leber congenital amau-
rosis (LCA)—that have overlaps in their underlying genetic bases. The aspects of
genetics of RP and LCA are arranged according to the cellular pathways in which
various genes function. In addition, a subsequent section on a syndromic form of
RP—Usher syndrome—deals with the genetics of the different types of Usher
syndrome.

82 5 Hereditary Retinal Degenerations
5.1 eneral Features of Major Forms of Non-syndromic

G
Retinal Dystrophy
5.1.1 Retinitis Pigmentosa
Retinitis pigmentosa (RP) is one of the major forms of photoreceptor dystrophies

and has a prevalence of about 1 in 4000 in different parts of the world (reviewed in
Kannabiran 2008). In India, the prevalence of RP is estimated to be about 1:1000 in
epidemiological studies from South India, thereby indicating a higher prevalence
than reported in the Western world (Dandona et al. 2001; Sen et al. 2008). RP typi-
cally has an onset in the first to second decades of life with initial symptoms of night
blindness, or reduced visual acuity, with progressive loss of visual fields, and dimi-
nution of vision, leading to blindness. Changes in the clinical appearance of the
retina include attenuated retinal vessels, the presence of pigmentary deposits in the
retina, pallor of the optic disc, and degeneration of the RPE. Electroretinography
shows extinguished rod and cone responses. Despite these characteristic changes,
RP is extremely clinically heterogeneous, with variability between families and
even between members of the same family in its onset, rate of progression, severity
of the disease, and the clinical appearance of the retina.
The group of disorders represented by RP has a high degree of genetic heteroge-
neity. Various modes of inheritance are observed in affected families, as mentioned
above, with about 30–40% being simplex cases with no affected family member.
Simplex cases could represent de novo mutations that have arisen in the germ line
or in the embryo, or they could be instances of autosomal recessive inheritance with
a single affected offspring. In the latter, parents and all previous generations may be
heterozygous carriers and therefore not affected with the disease. Homozygosity or
compound heterozygosity for the mutant allele occurs with a 25% chance in the
offspring of such carriers. Hence only one offspring may be affected in many of the
families with AR inheritance, thus presenting as an isolate case.
In addition, there are several syndromic forms of RP which involve organs other
than the retina. The relative proportions of different modes on inheritance in fami-
lies with RP tend to vary between populations (reviewed by Kannabiran 2008).
Among the non-syndromic forms of RP, autosomal recessive RP (ARRP) is the
most heterogeneous, and over 60 genes are identified or mapped for this group of
disorders. For autosomal dominant RP (ADRP), about half as many genes are
known to have disease-associated mutations. There are relatively few genes associ-
ated with X-linked RP, with three genes known as of date. Details of the numbers of
loci and genes for each form of RP, and details thereof, can be obtained from the
Retinal Information Network (RetNet) web site (available at https://sph.uth.edu/
retnet/). The database is updated as new genes are discovered.
5.1.2 Leber Congenital Amaurosis
Leber congenital amaurosis (LCA) is a form of severe visual impairment within the
first year of life, with congenital nystagmus, absent pupil reflexes (amaurotic
5.1 General Features of Major Forms of Non-syndromic Retinal Dystrophy 83
pupils), and a normal appearance of the retina or late pigmentation. It was first
described by Theodore Leber in 1869. It is characterized by attenuated electroreti-
nographic (ERG) responses, and the critical distinction of LCA from various
related retinal diseases such as RP is the onset at birth and documented loss of
vision and ERG responses within the first year of life. It is thus the most severe
form of retinal dystrophy and is considered to make up about 5% of all retinal dys-
trophies. Clinically, the retina is very variable in LCA patients, and the phenotype
may overlap with other retinal disorders that occur in early childhood. The appear-
ance of the retina in LCA can range from fairly normal to the presence of extensive
changes due to loss and disorganization of the photoreceptor cells and the underly-
ing blood vessels. A characteristic feature of LCA is the presence of the eye poking
reflex (known as the oculodigital sign of Franceschetti). LCA is often accompanied
by other ocular abnormalities such as keratoconus, cataracts, high refractive errors,
and atrophic lesions of the macula. In certain cases, complicated forms of LCA are
found, with associated developmental delay and mental retardation. The pathogen-
esis of LCA involves an absence or a deficit of photoreceptor cells; this is consid-
ered to be due to either a defect in their development or to a very early degeneration
of these cells.
LCA is mostly inherited in an autosomal recessive mode, with rare cases of auto-
somal dominant disease. Despite having predominantly one form of inheritance,
LCA is genetically very heterogeneous. Autosomal recessive LCA is associated
with mutations in over 20 genes as identified till date. Three loci are identified for
the dominant form of LCA. In addition, it shows clinical heterogeneity, and certain
unique features in the appearance of the retina have been associated with mutations
in specific genes (Shukla et al. 2012). Based on such shared features among groups
of patients with mutations in the same gene, genotype-phenotype correlations have
been derived for the genetic subtypes of LCA. Similar to RP, LCA can occur as a
syndromic or non-syndromic disorder, the former type involving other organs apart
from the retina.
Populations having a high prevalence of consanguinity tend to have a higher
frequency of recessive disorders as compared to outbred populations. In such fami-
lies, the spouses are related to each other, and therefore there is a higher chance of
both being carriers of the same disease allele, through inheritance from a common
ancestor. Hence, a child of two carrier parents related to each other inherits such an
identical (disease-causing) allele from each parent with a 25% chance and is thereby
homozygous for the allele. In such offspring, the homozygosity extends to a larger
region of the genome flanking the disease gene, up to several megabases in length.
This is because this entire region is inherited from a common ancestor by both par-
ents, who are related to each other through this ancestor (separated by one genera-
tion in the case of uncle-niece and two generations in the case of first-cousin
marriages). This phenomenon is known as homozygosity by descent (HBD) or
autozygosity, since it is derived from a single ancestor. Thus, one can map the dis-
ease gene for rare, autosomal recessive diseases in such families, by exploiting the
presence of HBD. It increases the power of mapping, such that three offspring of a
consanguineous marriage provide sufficient power for mapping the disease gene
(Lander and Botstein 1987; Farral 1993).
5.2 Homozygosity Mapping in Retinal Disorders
Autozygosity mapping is used in the identification of disease genes through conven-

tional approaches such as marker-based linkage analysis. Both microsatellite mark-
ers and SNPs have been employed to detect autozygosity (see Box 5.1). While a
microsatellite marker has more power to detect a homozygous region due to its
greater polymorphism than a SNP, the advantage of the SNPs is that they are much
more numerous and occur at higher density. With the information collected by the
two large-scale genome projects—the International HapMap project in the earlier
stages and, subsequently, the 1000 Genomes Project—the discovery of novel SNPs
and other types of variations was greatly accelerated (The 1000 Genomes Project
Consortium 2015). There are about ten million SNPs estimated to be present in the
human genome. Apart from SNPs, structural polymorphisms consisting of inser-
tion-deletion (indel) sequences also occur throughout the genome, and these are
identified and catalogued by the use of high throughput sequencing methods. Over
one million indels are documented. For the purpose of homozygosity mapping how-
ever, SNPs have been a useful tool since they are present at a high density in the
genome, combined with the ease of genotyping them through the use of rapid, large-
scale methods such as SNP arrays or genome sequencing. SNP-based approaches
enable the mapping and identification of disease-associated genomic variations
within the autozygome of consanguineous families, particularly in autosomal reces-
sive diseases. The application of this approach, however, has been wider, and it is
being used in mapping of genes in complex diseases as well (Abu-Safieh et al.
2013). Since the disease loci are not yet identified in a substantial number of patients
with RP in different populations, especially for ARRP, the number of RP-associated
genes is likely to go up as more genes are identified. This process is being facilitated
by the application of next-generation sequencing technologies which enable the
sequencing of the whole genome or the exome of an individual. Thus, one can cir-
cumvent the tedious and time-consuming process of screening a large number of
genes individually in groups of patients. There is enormous functional diversity in
the genes that are associated with different forms of RP and retinal dystrophies such
as LCA. Categories of various RP genes (and their encoded proteins) are shown in
the table based on their known or assumed functions—some of the pathways in
which these genes function are the phototransduction cascade, mRNA splicing,
transcription, protein degradation, metabolism of glucose (energy generation), reti-
noid cycle, signaling, lipid biosynthesis, and several more. As can be seen from
Table 5.1, some genes with disease-associated mutations identified in families with
RP are not yet characterized for their function. Hence the underlying mechanism by
which mutations in these genes lead to disease is not yet understood.
Box 5.1 Homozygosity Mapping

The fraction of the genome that is expected to be HBD, also referred to as the
autozygome, in offspring of consanguineous marriages, is given by the coeffi-
cient of inbreeding. It represents the probability that a person receives an iden-
tical pair of alleles from both parents, originating from one common ancestor.
5.2 Homozygosity Mapping in Retinal Disorders 85
For a first-cousin marriage, the coefficient of inbreeding is 1/16, for second

cousins it is 1/32, and for an uncle-niece marriage, it is 1/8. In other words, the
probability for homozygosity by descent at any given locus in first-cousin
marriages is 1/16, 1/32 for a second-cousin marriage, and so on. For a reces-
sive disease locus, the probability of HBD is equal to 1.
Thus, looking for shared regions of HBD among affected offspring of con-
sanguineous marriages represents a powerful approach to narrowing down
and identifying the disease locus. The more recent the mutation, the longer
would be the region of homozygosity at that locus, as compared with an
ancient mutation that has been passed down through many generations.
In contrast, homozygosity by state (HBS) or identity by state (IBS) is a
condition in which there are identical alleles at a given locus, but these have
arisen independently in the population.
Table 5.1 Genes associated with RP and LCA

Function or pathway Gene name (symbols) Associated disorders
Phototransduction Rhodopsin (RHO) Autosomal dominant retinitis
cascade pigmentosa (ADRP), autosomal
recessive retinitis pigmentosa
(ARRP)
Phosphodiesterase 6A (PDE6A) ARRP
Phosphodiesterase 6B (PDE6B) ARRP
Phosphodiesterase 6G (PDE6G) ARRP
Guanylyl cyclase 2D (GUCY2D) Leber congenital amaurosis (LCA),
dominant cone-rod dystrophy
(CRD)
Cyclic nucleotide gated channel ARRP
alpha1 subunit gene (CNGA1)
Cyclic nucleotide gated channel
beta 1 subunit gene (CNGB1)
Retinal S antigen (SAG) ARRP
Retinoid Cellular retinaldehyde binding ARRP, recessive Bothnia
metabolism and protein (CRALBP, RLBP1) dystrophy; recessive retinitis
transport punctata albescens (RPA),
recessive Newfoundland rod-cone
dystrophy (NFRCD)
Retinol dehydrogenase 12 ADRP, LCA, recessive childhood-
(RDH12; LCA13) onset severe retinal dystrophy
Retinal pigment epithelium 65 ARRP, LCA
KDa protein (RPE65)
Lecithin retinol acyltransferase ARRP, LCA
(LRAT; LCA14)
Retinal G protein-coupled ARRP, LCA
receptor (RGR)
Retinol-binding protein 3 (RBP3) ARRP
(continued)
Structural and Retinal degeneration slow (RDS) ADRP, digenic RP, dominant
membrane proteins, macular dystrophy
transporters ATP-binding cassette subfamily A ARRP, Stargardt’s disease,
member 4 (ABCA4) age-related macular degeneration
(AMD)
Bestrophin 1 (BEST1) ADRP, vitelliform macular
dystrophy, autosomal dominant
vitreoretinochoroidopathy
(ADVIRC), autosomal recessive
bestrophinopathy
Solute carrier family 7 member ARRP
14 (SLC7A14)
Clarin 1 (CLRN1) Recessive Usher syndrome (Usher
syndrome 3), ARRP
Rod outer segment membrane Digenic RP, ADRP
protein 1 (ROM1)
Heparan acetyl-CoA alpha- ARRP
glucosaminide
N-acetyltransferase (HGSNAT)
Carbonic anhydrase 4 (same as ADRP
progressive rod-cone
degeneration; PRCD)
Tubby-like protein 1 (TULP1; LCA, ARRP
LCA15)
mRNA splicing Pre-mRNA processing factor 3 ADRP
(PRPF3)
Pre-mRNA processing factor 4 ADRP
(PRPF4)
(PRPF6)
(PRPF8)
(PRPF31)
Small nuclear ribonucleoprotein ADRP
200 (SNRNP200)
DEAH-box helicase 38 (DHX38) ARRP
Polynucleotide ADRP
adenylyltransferase (PAP1)/RP9
Transcription Cone-rod homeobox (CRX) ADRP, dominant CRD, dominant
factors LCA
Nuclear receptor subfamily 2 ADRP, ARRP, enhanced S-cone
group E member 3 (NR2E3) syndrome (ESCS), Goldmann-
Favre syndrome (GFS)
Neural retina leucine zipper ADRP, ARRP
(NRL)
Neuronal differentiation 1 ARRP
(NEUROD1)
Zinc finger protein 408 (ZNF408) Dominant familial exudative
vitreoretinopathy (FEVR); ARRP
5.2 Homozygosity Mapping in Retinal Disorders 87
Zinc finger protein 513 (ZNF513) ARRP
Protein degradation Kelch-like family member 7 ADRP
(KLHL7)
Glucose metabolism Hexokinase 1 (HK1) ADRP
and energetics Isocitrate dehydrogenase 3B ADRP
(IDH3B)
Nicotinamide mononucleotide LCA
adenylyl transferase 1 (NMNAT1;
LCA9)
Cilia/centrosomal Centrosomal protein 290 KDa LCA (syndromic and
proteins (CEP290) non-syndromic)
Retinitis pigmentosa GTPase X-linked retinitis pigmentosa
regulator (RPGR) (XLRP)
Retinitis pigmentosa 1(RP1) ADRP, ARRP
Retinitis pigmentosa 1 like 1 ARRP, dominant occult macular
(RP1L1) dystrophy
Retinitis pigmentosa 2 (RP2)a XLRP
Topoisomerase binding arginine/ ADRP
serine-rich protein (TOPORS)
Family with sequence similarity ARRP
161 member A (FAM161A)
Tetratricopeptide repeat ARRP, Bardet-Biedl syndrome
domain-containing protein 8 (BBS)
(TTC8)
Kizuna (KIZ) ARRP
Intraflagellar transport protein ARRP, recessive BBS
172 (IFT172)
ADP-ribosylation factor-like 2 ARRP
binding protein (ARL2BP)
Spermatogenesis-associated ARRP, LCA
protein 7 (SPATA7)
Orofaciodigital syndrome 1 XLRP
(OFD1)
Male germ cell-associated kinase ARRP
(MAK)
Never in mitosis gene A-related ARRP
kinase 2 (NEK2)
Retinitis pigmentosa GTPase LCA
regulator-interacting protein 1
(RPGRIP1/LCA6/CORD13)
Lebercilin (LCA5) LCA
Chromosome 2 open reading ARRP
frame 71 (c2orf71; BBS21;
CORD16; RP64)
Chromosome 8 open reading ARRP, CRD, recessive BBS
frame 37 (c8orf37)
(continued)
Immune response Semaphorin 4A (SEMA4A) ADRP, dominant CRD
Chaperone Aryl hydrocarbon interacting LCA
protein-like 1 (AIPL1)
Kinases and Mer tyrosine kinase proto- ARRP, recessive CRD
signaling proteins oncogene (MERTK)
Lipid metabolism Ceramide kinase-like (CERKL) ARRP, recessive CRD
Mevalonate kinase (MVK)
Nucleotide Inosine monophosphate ADRP, ARRP
metabolism dehydrogenase 1 (IMPDH1)
Extracellular matrix Eyes shut homolog (EYS) ARRP
and cell-cell Crumbs homolog 1 (CRB1) ARRP, LCA, Coats-like exudative
interaction vasculopathy
Usher syndrome 2A (USH2A) Usher syndrome, ARRP
Interphotoreceptor matrix ARRP
proteoglycan 2 (IMPG2)
Protein synthesis tRNA nucleotidyl transferase 1 ARRP
and modification (TRNT1)
ATP-GTP binding like 5 (AGBL5)
Protein O-mannose beta-1,2-N-
acetylglucosaminyltransferase
(POMGNT1)
Function not known Retinal degeneration 3 (RD3) LCA
The table lists several genes in which mutations are reported in patients with RP, LCA, or other
forms of retinal disease in some cases, as shown in the right column. They are grouped according
to known or putative functions, shown on the left
a
The RP2 protein is found in the connecting cilium and is also reported to be on the inner side of
the plasma membranes in vesicles (see text)
The following sections are concerned with a brief discussion of several genes
that have mutations in patients with RP and LCA. Certain key aspects concerning
the original identification of the gene in relation to the disease, pathogenic impact,
and cellular functions are dealt with. Due to the vast amount of literature that is
available for retinal dystrophies and their associated genes, the subject matter cov-
ered in this chapter is necessarily selective, and does not attempt to include all the
genes identified till date. Emphasis is given to the original discoveries of the asso-
ciation of a particular gene with disease, along with a review of selected aspects of
each of the genes mentioned, including mutational surveys, focusing on larger stud-
ies which apply to any population, unusual or unique aspects of their mutational
patterns, and the biological attributes and functions of each gene, as evident from
studies done in vitro or in animal models. While knowledge of the genetics of retinal
diseases in Indian patients is very sparse in comparison to the overall literature in
the field, a brief mention of representative studies done in India has been included.
For the genetics of RP and LCA, the text is arranged gene-wise and not under dif-
ferent forms of inheritance or of disease, since some genes are associated with more
than one form of retinal dystrophy, or with more than one type of inheritance—i.e.,
5.3 Genes Involved in Phototransduction 89
autosomal dominant and autosomal recessive disease. Needless to say, simplex

forms of RP, though placed in a different category due to the absence of any family
history, in fact can have genetic overlap with recessive, dominant, or X-linked
RP. Simplex RP involves a substantial fraction of patients.
A subsequent section deals with syndromic RP, particularly on the molecular
genetics of Usher syndrome, which is the major form of syndromic RP. Again, syn-
dromic RP includes other entities such as Bardet-Biedl syndrome and Refsum dis-
ease that are not dwelt upon here.
5.3 Genes Involved in Phototransduction
This section is concerned with the biology and genetics of genes that encode pro-
teins involved in the phototransduction pathway, the primary process in the visual
response of photoreceptors. There are several genes that function in this pathway, in
which mutations are associated with RP and related diseases. Important aspects of
their discovery, biological properties, and genetics are presented here.
5.3.1 Rhodopsin
Associated disorders—autosomal dominant RP, autosomal recessive RP.
5.3.1.1 Cloning and Isolation of the RHO Gene

The first gene to be discovered to have pathogenic mutations in retinitis pigmentosa
is the gene for rhodopsin, which is the visual pigment in the rods in the retina.
Rhodopsin is an opsin protein that has retinal (or retinaldehyde, which is a form of
vitamin A) as a chromophore. On exposure to light, isomerization of retinal in the
rhodopsin molecule from the 11-cis to the all-trans form results in a conformational
change that is transmitted from the retinal molecule to the opsin apoprotein, trigger-
ing a series of chemical reactions that eventually lead to a neuronal impulse with
hyperpolarization at the photoreceptor membrane. These impulses are then trans-
mitted through second-order neurons in the retina to the optic nerve. The human
RHO gene was first isolated and sequenced on the basis of its homology with the
bovine rhodopsin gene. A bovine cDNA probe was used to isolate the human gene
from a human germ cell genomic DNA library. The organization of the human RHO
gene and its sequence were determined by comparison of sequences of the human
genomic fragments with the bovine rhodopsin cDNA clone. This was feasible due
to the high degree of homology (over 90%) between the coding regions of the
bovine and human rhodopsin genes (Nathans and Hogness 1984).
5.3.1.2 Mutations in the Rhodopsin Gene

The rhodopsin locus was mapped in a large Irish pedigree with a rod-cone type of
RP. Affected members had early onset of visual loss, with pigmentary deposits in
the mid-peripheral retina, thinning of the RPE, attenuation of retinal vessels, and
pallor of the optic disc. The disease in this pedigree was mapped onto chromosome
3q by linkage analysis, thus placing it in the same chromosomal region as the genes
for rhodopsin and the retinol-binding proteins, RBP1 and RBP2 (McWilliam et al.
1989). A mutation in the rhodopsin gene, consisting of a missense change of pro-
line-23 to histidine (Pro23His), was found to segregate with the disease in this pedi-
gree. As shown in another study that closely followed, the same substitution of
Pro23His appears fairly frequent in patients with ADRP from North America, being
detected in 17 patients in a series of 148 unrelated patients tested, amounting to a
frequency of about 12% (Dryja et al. 1990). The mutation involves a non-
conservative substitution of a proline residue that is conserved at this position in
members of the opsin family of proteins and G protein-coupled receptors. Thus,
such a mutant protein would be expected to be defective in its function.
Mutations in the rhodopsin gene are reported in a sizeable proportion of patients
with ADRP in North America. About 6% of patients are positive for a mutation
involving codon 347 of RHO, which codes for proline. Two mutations at this codon
are associated with ADRP—proline to leucine (Pro347Leu) and proline to serine
(Pro347Ser) substitutions. However, mutation of Pro23His is probably the most fre-
quent mutation. This was confirmed in another study on the same population, which
found a frequency of 12% for the Pro23His mutation and 6% for the codon 347
mutations (Dryja et al. 1991).
Apart from ADRP, mutation of the rhodopsin gene is also associated with auto-
somal recessive RP. As might be expected, the recessive allele is a null mutation
resulting from a nonsense codon at 249, thus leading to loss of function (Rosenfeld
et al. 1992). This is in contrast to the rhodopsin mutations reported in families with
ADRP, which are, by and large, missense changes; in these cases, the basis for
pathogenicity is likely to be a dominant negative effect of the mutant allele. A domi-
nant negative effect implies that the mutant version of the protein interferes with the
function of the normal protein. This may particularly occur in proteins that associate
into higher-order complexes of two or more subunits, either involving the same
subunit (homomeric) or of different subunits (heteromeric).
The relatively higher frequency of rhodopsin mutations and the high prevalence
of specific mutations in populations from North America as mentioned in the pre-
ceding paragraph facilitated comparisons of the phenotypes comprising clinical and
visual parameters of groups of patients with the same mutation versus those without
it. As a result, genotype-phenotype correlations were made from such studies. An
analysis of a group of 17 unrelated patients with the same rhodopsin mutation
Pro23His, for their visual parameters in comparison with a large control group of
131 patients with ADRP without this mutation, suggested that patients with
Pro23His mutation retained better visual acuities and ERG responses on an average
at comparable ages than those without the mutation (Berson 1990). Similar analyses
have been reported on patients with other RHO mutations such as the proline-347-
leucine mutation (Berson et al. 1991). Despite these correlations, it is evident that
there is extensive clinical heterogeneity in RP, even between members of the same
family. An example of such heterogeneity reported for the same mutation, Pro23His
in rhodopsin, is a form of RP known as sectoral RP. In this condition, there is an
asymmetric involvement of the retina, in contrast to typical RP, which manifests

symmetrically throughout the retina. In sectoral RP, recognized as a distinct clinical
entity, one or more quadrants of the retina are affected by the disease while the
remainder is normal. A screen for mutation Pro23His in a series of ADRP-affected
families showed that two out of five families with autosomal dominant sectoral RP
carried this mutation (Heckenlively et al. 1991).
5.3.2 Phosphodiesterase 6
The phosphodiesterase 6 enzyme is involved in hydrolysis of cGMP in the photore-

ceptors during the transduction of light signals. The hydrolysis of cGMP by this
enzyme in response to light leads to a fall in cGMP levels in the photoreceptor. Thus
the cGMP-gated sodium ion channels (known as cyclic nucleotide gated channels
(CNGC)) on the outer segment membranes close due to fall of cGMP levels. This
process arrests the influx of sodium ions into the cells and thus results in hyperpo-
larization of the outer segment (i.e., the membrane becomes more negative, with a
potential of −65 mV). In the dark, the photoreceptor cells are depolarized and have
a potential of around −40 mV. Phosphodiesterase 6 has three types of subunits, two
large alpha and beta subunits and two small gamma subunits; each of the three sub-
units is encoded by a separate gene, PDE6A, PDE6B, and PDE6G, respectively. It
is thus a heteromeric enzyme, and the subunits α, β, and Ƴ are about 88 kDa, 84 kDa,
and 10 kDa, respectively, in the bovine protein.
5.3.2.1 Phosphodiesterase 6A
Associated disorders—autosomal recessive RP.
Cloning and Isolation of PDE6A

The cDNA for PDE6A was isolated from both bovine and human retinal cDNA
libraries by using oligonucleotide probes designed from peptide sequences of the
phosphodiesterase protein purified from bovine retina. The isolated cDNA was
sequenced and identified as the alpha subunit based on the available sequence of the
same subunit from earlier studies (Pittler et al. 1990). The human PDE6A cDNA
was obtained by screening a human retinal cDNA library with a probe made from
the bovine cDNA. Both the human and bovine cDNAs contain open reading frames
encoding a protein of 859 amino acids, 94% of which are identical in the two
species.
Animal Models of Pde6a

Mouse models having mutations in the Pde6a gene have been created by chemical
mutagenesis with ethylnitrosourea. Two strains of mice with missense mutations in
the catalytic domain of the Pde6a molecule, affecting residues 670 and 685 of the
protein, showed retinal degeneration phenotypes (Sakamoto et al. 2009). Loss of the
outer nuclear layer (ONL) was significant by 2 weeks of age, and there was evi-
dence of apoptosis by standard markers. Rearing the animals in the dark appeared
to slow the progression of the disease, both in terms of preservation of the ONL and
in the extent of ERG responses obtained.
Mutations in PDE6A
Mutations in PDE6A are found in a very low percentage of patients with RP as
shown first in a study by Huang and coworkers, who screened most of the exons of
the PDE6A gene (19 out of 22 exons) in over 300 patients, including those with
dominant and recessive RP. They identified PDE6A gene mutations in two families,
suggesting a frequency of <1% (Huang et al. 1995). Screening of about 160 patients
with recessive RP in North America in a subsequent study found a frequency of
mutations of approximately 3–4% (Dryja et al. 1999).
5.3.2.2 Phosphodiesterase 6B (PDE6B)

Cloning and Isolation of Human PDE6B

The search for another gene, the one for Huntington’s disease, led to the cloning of
the human PDE6B gene, since the two genes map within the same genetic interval
on chromosome 4p16 (Weber et al. 1991). This genomic segment overlapping the
HD gene was cloned into cosmid vectors. Sequences of the genomic inserts in the
subclones from this segment showed high degree of similarity with the bovine and
mouse Pde6b cDNA sequences in the GenBank database. The structure and organi-
zation of the human PDE6B were thus determined by alignment of the genomic
sequences with the bovine and mouse cDNAs. Twenty-two exons were mapped on
to the human PDE6B gene. Northern blot analysis using a probe specific for exonic
regions of PDE6B indicated highly specific expression in the retina, with two tran-
scripts of 3.5 and 4.5 kilobases. The gene was localized at the telomeric end of
chromosome 4p.
Animal Models of Pde6 Knockdown

Evidence that the loss of function of this gene may lead to retinal degeneration came
from an inbred strain of mice, known as the rd mouse, in which the Pde6b gene was
identified as the defective locus (Pittler and Baehr 1991). The rd mice showed an
autosomal recessive form of retinal degeneration and included inbred laboratory
strains of mice from across different parts of Europe. Homozygous rd mice showed
complete loss of rods by postnatal day 20, and biochemical analyses of the retinas
indicated an increase in cGMP levels and an absence of phosphodiesterase activity,
along with a corresponding reduction in the mRNA level for the beta subunit of
PDE in the rd retina. On the other hand, the heterozygous mutant mice had reduced
ERG responses, but their retinas appeared normal upon histological evaluation. The
genetic defect in the Pde6b gene was found to be a truncating mutation in exon 7,
leading to loss of function. This provided a basis for the biochemical changes that
were observed in the mutant animals, since cGMP is not degraded due to lack of
phosphodiesterase activity, thereby accumulating and leading to the development of
retinal degeneration in this strain of mice.
A different strain of mice with retinal degeneration due to disruption of the

Pde6b locus is the rd1 mouse. The genetic aberration in this strain is the insertion of
a provirus, the xenotropic murine leukemia virus (Xmv-28) into the genome of
inbred strains of mice. The site of integration of the Xmv-28 in the mouse genome
is within the first intron of the Pde6b gene, thereby disrupting its transcription. The
retinal degeneration phenotype in these strains, referred to as the “rodless retina,”
was found to completely coincide with the presence of the integrated provirus at this
locus. The levels of the Pde6b transcript in these mice were strikingly low, and the
transcript size was abnormal (Bowes et al. 1993).
The rd10 mouse is another recombinant inbred line that has retinal degeneration
due to a missense mutation (arginine-560 to cysteine) in exon 13 of the Pde6b locus
(reviewed by Chang et al. 2007). These mice manifest with early-onset retinal
degeneration and sclerotic retinal vasculature. Retinal degeneration, starting first in
the central retina and evident at about 2 weeks after birth, leads to the complete loss
of the outer nuclear layer at 60 days. A notable feature of this strain is that retinal
degeneration is delayed when the mice were reared in the dark. The responses of
photoreceptors, as recorded by electroretinography, show a faster decline of rod
responses as compared with cones.
As compared with the murine models described above, a large animal model for
RP associated with the Pde6b locus is a naturally occurring canine breed, the Irish
setter dog, with phosphodiesterase 6B deficiency. Several dogs of this breed have a
retinal dystrophy that is described as rod-cone dysplasia 1 (rcd1). In this disorder,
photoreceptor cells degenerate before reaching maturity. In Irish setter dogs affected
with rcd1, a recessive mutation causes degeneration of the retinal photoreceptors,
with early and predominant loss of rod cells, which occurs before the age of 20 weeks,
while a small number of cones remain. Histological examination of retinas of
affected dogs at 8 and 12 weeks of age revealed changes in the retinal layers as a
result of the disease. The photoreceptor layers were thinner than normal, and rods
were lost early in the disease, while cones were relatively intact. The outer segments
of both rods and cones were abnormal in shape (Aquirre et al. 1978). Similar bio-
chemical changes as those seen with rd mice described above were also observed in
the retinas of rcd1 dogs. Their retinas showed elevated levels of cGMP, reported to
be about tenfold higher than controls, associated with a defect in phosphodiesterase
activity, before the onset of degeneration. This is due to a failure to hydrolyze cGMP
in the diseased retinas. As a consequence of the disease process in the rcd1 dogs, the
retinas showed a severely reduced level of PDE6B mRNA from early postnatal life.
The PDE6B mutation in Irish setter dogs consists of a nonsense mutation at
codon 807 arising due to a single base (G>A) transition at cDNA position 2420
(Suber et al. 1993). Analysis of 436 Irish setter dogs from North America for the
presence of the codon 807 mutation suggested a high frequency of carriers of about
8% in this canine population (Aguirre et al. 1999).
Pathogenic Mutations in PDE6B

The effect of the loss of function of the PDE6B gene on the retina was established
in two animal models, as outlined in the previous paragraphs. The involvement of
the PDE6B gene in human RP has been established from the study of families with
ARRP, although only a few families are known to date that are found to have muta-
tions in the PDE6B gene.
The first evidence for an association of PDE6B mutation with ARRP in humans
came from a screen of 92 patients. This was carried out by two approaches—(1)
screening for the presence of any large deletions or rearrangements by Southern
blots on genomic DNA of patients, which were probed with the PDE6B cDNA, and
(2) screening for small mutations within exonic regions by single-strand conforma-
tion polymorphism (SSCP) analysis. Four individuals from separate families were
found to carry pathogenic changes in this gene, including splice site substitutions
and nonsense and missense changes, thus amounting to a frequency of about 4% for
mutations in the PDE6B gene in patients from North America (McLaughlin et al.
1995).
5.3.2.3 Phosphodiesterase 6 Gamma Subunit (PDE6G)

Mapping and Isolation of the PDE6G Gene

The PDE6G gene encodes the gamma subunit of the phosphodiesterase 6 enzyme,
which is inhibitory on the activity of the enzyme and is therefore important in regu-
lating its activity. On the basis of this function, and the prior knowledge of associa-
tion of mutations in the PDE6A and PDE6B genes encoding the alpha and beta
subunits, respectively, with RP, PDE6G was also a significant candidate gene for
RP. The human PDE6G cDNA was isolated from a retinal cDNA library cloned into
the bacteriophage lambda vector, using a mouse Pde6Ƴ cDNA as probe, based on
the high degree of similarity between human and mouse cDNA (Tuteja et al. 1990).
The human cDNA of about 1 kb in length has about 90% identity with the ortholo-
gous mouse and bovine cDNAs. By hybridization of its cDNA to human metaphase
chromosomes, the human PDE6G gene was assigned to chromosome 17q21.
The organization of the PDE6G gene and investigation of its possible role in reti-
nal disease in humans were first reported by Hahn and coworkers. The PDE6G
cDNA was used to screen a human genomic DNA library in bacteriophage lambda,
and the positive genomic clones sequenced to obtain the partial sequence and exon-
intron structure of the gene. It contains four exons that are distributed over about 6.5
kilobases of genomic DNA. Screening of over 700 patients from North America
with RP and related retinal disorders, for mutations in this gene, did not detect any
disease-associated variants (Hahn et al. 1994). These and other findings from
Southern blot analysis of DNA from a few hundred retinal disease patients also
from North America, for detecting genomic changes in the PDE6G gene, turned out
negative for pathogenic mutations, suggesting that alteration of PDE6G is an
extremely rare cause of RP in these populations.
Animal Model for Knockdown of PDE6G

A targeted knockdown of the Pde6g gene in mice was achieved by deletion of two
exons of the gene. Heterozygous knockout mice were normal by histology and
physiology of the retina, but homozygous knockout mice showed profound struc-
tural and functional defects in the retina. Electroretinographic responses were extin-
guished with a progressive decline in response from 2 weeks onward, and no
detectable response after 3 months of age (Tsang et al. 1996). By histology, the
photoreceptor outer segments were disorganized and were lost in the first 2 postna-
tal weeks. This was followed by the loss of nuclei of the photoreceptors, more
marked in the central retina. The photoreceptors were completely lost by about
8 weeks of age. The retina showed an increase of cGMP levels relative to those of
normal mice at about 2 weeks of age, and this increase preceded the degeneration of
photoreceptors. These observations reflected an overall loss of phosphodiesterase
activity in the Pde6−/− retinas.
Mutations in PDE6G
The identification of PDE6G mutations in retinal dystrophy in humans was demon-
strated in an extended family of Arab-Israeli origin, in which the affected members
had a severe form of early-onset RP. Homozygosity mapping of genomic regions
using high-density SNP arrays was employed to map the disease locus in the two
nuclear families within this extended kindred. The mapping analysis showed a sin-
gle region of about 4 Mb shared by all affected members. Screening of the PDE6G
gene, mapped within the shared region of homozygosity in the family, detected a
splice donor mutation at the +1 position of the intron, c.187+1G>T, carried by all
four affected individuals, which segregated with the disease in the entire family. The
pathogenicity of this change is predictable with a high probability since it involves
the highly conserved splice junction. In addition, the inactivating effect of this
sequence change was experimentally confirmed in cell lines. The splice junction
mutation was indeed found to result in an aberrantly spliced transcript and thus
amount to a loss of function (Dvir et al. 2010). Available literature till date has no
other reports of families with a mutation in PDE6G, suggesting that it is probably a
very rare cause of RP.
5.3.3 Guanylate Cyclase 2D (GUCY2D)
Associated disorders—LCA, dominant cone-rod dystrophy.
5.3.3.1 Cloning and Isolation of GUCY2D

The guanylate cyclase 2D (GUCY2D) gene encodes retinal guanylate cyclase-1
(also known as RetGC1; GC1), which is an enzyme responsible for synthesis of
cGMP in the retinal photoreceptors. During the phototransduction reaction, cGMP
is hydrolyzed by cGMP phosphodiesterase, leading to closure of the cGMP-gated
ion channels, with consequent reduction of calcium ions and hyperpolarization of
the photoreceptors. Recovery from light exposure requires the resynthesis of cGMP,
so as to permit the photoreceptors to respond to further stimulation by light and
initiate a fresh cycle of phototransduction. Thus the retinal guanylate cyclases play
a critical role in ensuring the continuous supply of cGMP required for this process.
Vertebrate photoreceptors have two retinal guanylyl cyclases. The human cDNA
clone for the first retinal guanylyl cyclase (GC) to be studied, RetGC1, was isolated
by Shyjan et al. (1992). They used degenerate oligonucleotide primers correspond-
ing to conserved sequences of various GCs to amplify sequences from human
genomic DNA by PCR. By subcloning and sequencing these various amplified frag-
ments, they discovered a fragment with a predicted protein sequence that appeared
related to but separate from other GC enzymes known until then. Screening of vari-
ous human cDNA libraries using the cloned genomic PCR product as a probe led to
isolation of a cDNA from a retinal library, of about 3 kb in length with a predicted
peptide of 1051 amino acids. Thus the RetGC1 cDNA was identified, and further
analysis of the encoded peptide sequence identified motifs that corresponded to
extracellular, transmembrane, and intracellular domains of the protein. The RetGC1
transcript is expressed solely in the outer nuclear layer and inner segments in retinal
sections (Shyjan et al. 1992).
The deduced amino acid sequences from the human RetGC cDNA had a high
degree of similarity to the amino acid sequences of the bovine RetGC enzyme puri-
fied from rod outer segment membranes. Due to this similarity in the protein
sequences, the human cDNA sequence for RetGC1 was instrumental in the design
of suitable probes for isolation of the bovine cDNA. Probes generated by PCR from
the human cDNA library were then used to isolate the RetGC cDNA clone from
bovine retinal cDNA. The bovine cDNA thus isolated was about 4 kilobases long,
coding for a protein of 1054 amino acids (Goraczniak et al. 1994). Similar to the
human, its expression pattern was highly specific for the retina and not detectable in
other tissues tested.
The cDNA for yet another retinal guanylyl cyclase was isolated by screening a
human retinal cDNA library under low stringency conditions, using probes corre-
sponding to homologous domains of various guanylyl cyclases. The enzyme iso-
lated by this process was named as RetGC2. Essentially the transcript for RetGC2
has an identical expression pattern as RetGC1. It was detectable specifically in the
outer nuclear layer and the inner segments of the photoreceptors, and a similar pat-
tern of expression was displayed by the protein. The RetGC2 protein was character-
ized using antibodies raised against its peptide sequences, as a 115 kilodalton
protein, expressed in the photoreceptor membranes (Lowe et al. 1995).
5.3.3.2 Animal Models for GUCY2D

Several animal models have been generated over the last few decades for studying
the effects of RETGC1 deficiency. These knockdown models have also been sub-
jected to gene replacement, thereby serving as models for the absence of RETGC1
as well as proof of concept for gene replacement therapy. An avian model of a
RETGC1 defect is the GUCY1*B chicken, which has a naturally occurring com-
plex (deletion-insertion) mutation in the gene encoding RETGC1 (Ulshafer and
Allen 1985; Semple-Rowland et al. 1998). The mutant gene behaves as a null allele
producing no detectable protein. Cyclic GMP levels in the photoreceptors of the
chickens immediately after hatch were found to be about one-fifth to one-tenth of
normal, and they were blind at birth, with non-recordable ERG responses. Similar
to the pattern of the human LCA1 disease, cones degenerated first in the GUCY1*B
chicken, and loss of photoreceptors proceeds from the central to the peripheral ret-
ina (reviewed by Boye 2015). Gene replacement of the bovine ReTGC1 cDNA in a
lentiviral vector in 2-day chick embryos led to some restoration of vision in the
treated chicks and a partial recovery of ERG responses.
An animal model for deficiency of the retinal guanylate cyclase gene was created
in a mouse by creating a knockout of the Gucy2e gene (the murine homolog of
human GUCY2D) by insertion of a neomycin resistance cassette into the gene cod-
ing sequences, to produce a null mutant (Yang et al. 1999). This engineered mouse
model of GC1 knockout (GC1KO) has a truncated GC1 gene and no detectable
presence of the protein, although the retinal guanylate cyclase 2 (GC2) is expressed.
The notable feature was that cone photoreceptors degenerated rapidly, beginning in
the first few weeks of life. Rods do not degenerate and continue to respond to light,
though this is at a fraction of the wild type response. The rod response is attributed
to the functional GC2 enzyme in these mice. ERG responses in the knockout ani-
mals showed a reduction in rod responses and an absence of cone responses within
2 months of age. Experimental gene therapy in this animal model was successful in
restoring cone viability and function when the transgene was from the same species,
that is, the murine Gucy2e cDNA was able to restore cone function in the GC1KO
mouse, but not the bovine cDNA (Boye et al. 2011). Further, the rescue of the retinal
defects in the GC1KO mouse model was sustained up to 1 year after treatment using
gene delivery with two types of adeno-associated virus (both AAV5 and AAV8) vec-
tors. The AAV8 vector carrying the human GUCY2D gene under the human rhodop-
sin kinase promoter was able to rescue the defect in the GC1KO mice, indicating the
therapeutic potential of this mode of gene replacement.
Another knockout model consisting of a double knockout mouse with deletion of
both GC1 and GC2 loci was created with a view to determining the individual roles
of GC1 and GC2 in phototransduction. The double knockouts were generated by
cross-breeding of two single knockout strains of mice, GC1−/−and GC2−/−, that had
knockouts of the GC1 and GC2 loci, respectively. Comparison of the phenotypes of
this model with the GC1−/− (GC1 KO) mice mentioned above suggests that GC2
maintains rod function to an appreciable degree and prevents rod degeneration. The
double knockout mouse retina showed a total absence of both rod and cone activity
and a phenotype of recessive rod-cone dystrophy, thereby resembling LCA (Baehr
et al. 2007).
5.3.3.3 Pathogenic Mutations in GUCY2D

GUCY2D (i.e., RETGC1) was the first gene to be mapped for LCA, and the cor-
responding locus was designated as LCA1. Fifteen affected multiplex families
were studied in order to map the gene for LCA in them. The families were of
Maghrebian (North African) and French origins. Linkage and homozygosity map-
ping of five families from North Africa, all having autosomal recessive inheritance
of the disease and multiple affected children, localized the LCA1 gene to chromo-
some the distal short arm of chromosome 17, with linkage to a marker on 17p
(Camuzat et al. 1995). Patients recruited in this study had characteristic signs of
LCA which were the presence of visual loss at birth or within the first few months
of life, roving eye movements, eye poking, nystagmus, extinguished ERG
responses, and an inability to visually follow objects. They were also excluded
from having other systemic diseases or syndromes which shared features of
LCA. Genetic heterogeneity of LCA was evident from the fact that the locus
mapped to the LCA1 locus in the five Maghrebian families, whereas the French
families did not show linkage of the disease with this locus. The mapped region of
chromosome 17 contains various potential candidate genes for LCA including the
gene for retinal guanylate cyclase (RETGC1, GUCY2D). The exon-intron struc-
ture of the human GUCY2D gene was deduced by comparison of its cDNA
sequence with the mouse ortholog Gce, having a known exon-intron structure
made up of 20 exons. Screening of the coding regions of the GUCY2D gene in the
LCA1 families revealed missense and truncating mutations in four separate fami-
lies, which accounted for the segregation of disease in the respective families
(Perrault et al. 1996). Thus mutations in the GUCY2D gene were established as a
cause of LCA in a subset of cases.
GUCY2D is one of the genes with the highest frequency of mutations in LCA,
based on studies in various populations. Mutations in this genes accounted for over
20% of LCA patients in a large study which screened patients from various parts of
the world including America, Europe, Asia, and Africa (Perrault et al. 2000; Hanein
et al. 2004). Patients with the LCA1 form of disease manifest the characteristic
signs of LCA including visual loss within the first year of life, with loss of visual
acuity, diminished or absent ERG responses, nystagmus, and the eye poking (ocu-
lodigital) reflex. Characteristic signs of LCA1 disease are a congenital cone-rod
type of dystrophy that is non-progressive, with photophobia and high hypermetro-
pia (Perrault et al. 1999). However, the appearance of the retina is clinically nor-
mal. High-resolution imaging of the retina using optical coherence tomography
(OCT) has been employed to examine the structure of the retinal layers in these
patients. OCT imaging of GUCY2D-mutant retinas shows that the organization of
retinal layers is fairly normal even in patients over 50 years of age. Loss of photo-
receptors corresponding to a reduction in the thickness of the outer nuclear layer
(ONL) has been observed mainly around the fovea in the central retina, with the
peripheral retina being fairly comparable in thickness to normal retinas (Jacobson
et al. 2013). Substantial degree of rod function may be retained in patients with
mutations in GUCY2D as measured by ERG, consistent with the structural integ-
rity of rods observed by imaging. Only the central retina is found to be affected due
to loss of cones at the fovea. In contrast with the rods, cones are severely affected
in these cases with the corresponding functional defects of loss of visual acuity and
color vision.
The same chromosomal locus at 17p was also mapped in an entirely different
retinal disease, autosomal dominant cone-rod dystrophy. In this case, linkage map-
ping was performed on a large family of four generations having several members
affected with a disorder known as central areolar cone-rod dystrophy. The disease in
this family was characterized by an early onset of vision loss, especially affecting
central vision, followed by loss of peripheral vision, photophobia, and atrophy of
5.4 Genes Encoding Structural and Membrane Proteins 99
the macula; the affected persons presented with a retinal appearance known as
“bull’s eye maculopathy.” The mapping of the locus in this family was carried out
through a genetic analysis with markers throughout the genome but selected based
on their positions corresponding to specific disease loci that were already known.
This study showed significant linkage on chromosome 17 and mapped the disease
locus to a region of 8 cM on chromosome 17p12-13; the locus is designated as
CORD6 (cone-rod dystrophy 6). The CORD6 region overlapped with and included
the LCA1 locus within it (Kelsell et al. 1997). The family was therefore screened for
mutations in the GUCY2D gene, because it was within the mapped interval and,
importantly, was already known to have mutations associated with a retinal disease.
A missense change of glutamic acid-837 to aspartic acid (Gly837Asp; E837D) was
found in the affected members who were heterozygotes for the missense allele. In
addition, it was absent in the unaffected members of the same family mapped to
CORD6 and in a normal control population, thus indicating it to be a pathogenic
change. In addition, missense changes E837D, as well as R838C (Arg838Cys; argi-
nine-838 to cysteine), were detected in a few more families with the cone-rod dys-
trophy phenotype.
5.3.3.4 Mutations in GUCY2D in Indian Patients

There are few reported studies on the frequency of mutations in GUCY2D in Indian
patients with LCA and mostly involve a few patients; thus one cannot estimate the
frequency of mutations in any of these studies. A study from Aravind Eye Hospital
in Madurai, Southern India, reported the screening of 25 LCA patients with a com-
mercially available mutation chip, the microarray-based Asper LCA chip (Asper
Ophthalmics, Estonia). The chips consist of glass slides coated with a dense array
of oligonucleotides corresponding to short segments of LCA genes that contain
known mutations. The LCA chip used consisted of a total of 784 known variations
from 15 LCA genes. Three patients were found positive for GUCY2D mutations
thus suggesting a frequency of 10% of cases in this study (Verma et al. 2013).
However, a more reliable mutation frequency in Indian populations will have to
await more data covering a larger number of patients.
5.4 Genes Encoding Structural and Membrane Proteins
This section concerns several genes that encode structural and membrane proteins
in the photoreceptors. They include genes that are required for morphogenesis and
organization of the photoreceptors (CRB1, RDS) as well as transporter proteins
(ABCA4).
5.4.1 RDS (Retinal Degeneration Slow)
Associated disorders—digenic and dominant RP, dominant macular dystrophies,

dominant cone-rod dystrophy.
The RDS gene is also known as Peripherin 2 (PRPH2) and is required for the
proper formation of the discs in the photoreceptor outer segments. The RDS gene
encodes a transmembrane glycoprotein present in the rim of the discs in outer seg-
ments. It was the second gene that was discovered to be associated with ADRP, after
the rhodopsin gene. The name of this locus is derived from the phenotype of an
inbred strain of mouse bearing a mutant gene, known as the rds (retinal degenera-
tion slow) mouse originally described by Van Nie et al. (1978). The rds mouse strain
is also known as rd2 since it is the second mouse strain to be identified with a purely
retinal disease phenotype. The gene was named as retinal degeneration slow since
the progression of the disease was much slower in this strain, than in the rd1
mouse—the only other mouse model known until then. The rd1 mouse showed a
relatively rapid course of retinal degeneration.
The mutant gene in the rds mouse has an insertion of about 10 kb of exogenous
sequences in one of its exons, thus giving rise to an abnormal transcript and protein.
Mice that are homozygous for the mutant rds gene show abnormal development of
photoreceptors that is evident by 5 weeks of age, with a slow degeneration of the
photoreceptors which progresses to completion by 1 year of age. The underlying
mutation in the Rds mouse was mapped to chromosome 17 and thus provided the
locus for the gene.
5.4.1.1 Mapping and Isolation of RDS

The photoreceptor-specific character of the mutant phenotype as well as the expres-
sion pattern of the gene paved the way to its cloning. The isolation of the Rds mRNA
was achieved by identifying cDNA clones that were present in a library from a
normal retina but absent in corresponding tissues of a retinal degeneration mouse
(the rd1 mouse) which lacks all photoreceptors. Analysis of several such transcripts
led to the isolation of the Rds mRNA; it was further conclusively identified as being
the Rds transcript by its altered size in the rds−/− mouse. The Rds clones from nor-
mal retinas consisted of two transcripts of 1.6 and 2.7 kb, while the abnormal tran-
script in the rds−/− mouse retina was about 12 kb in size. The Rds cDNA sequence
isolated from the normal mouse retina encodes a 346 amino acid protein with sev-
eral putative transmembrane regions (Travis et al. 1989). The protein probably
exists as a dimer linked by disulfide bonds and is non-covalently associated with the
Rom1 protein, based on co-immunoprecipitation of both the proteins together by
the use of antibody to either one. They form homo- and heterotetramers as well as
higher-order complexes in the rod outer segments in vivo. The photoreceptor-
specific expression of the RDS protein was established by the analysis of a retinal
degeneration mouse model, referred to as the rd mouse. The suitability of this mouse
model in studying expression of Rds lay in the fact that homozygous rd/rd mice
showed complete degeneration of rods by 1 month and had only a few cones remain-
ing. The other retinal neurons were intact in these mice. It must be noted here that
the retinal degeneration in this mouse mapped to a different locus and not to the Rds
locus. Expression of the Rds protein was studied by immunologic detection in
Western blots of retinal extracts of rd/rd mice with complete retinal degeneration
and those without retinal degeneration (with photoreceptors intact) using
Rds-specific antibodies. This study confirmed that Rds protein expression was
highly specific to the photoreceptors since the photoreceptorless retina did not show
any detectable Rds protein in the Western blot. Rds is a glycosylated membrane
protein, and the monomer is about 38 kDa, that appears to form a dimer of about
70 kDa linked by disulfide bonds. By immunohistochemistry, it is found to be pri-
marily located in the photoreceptor outer segments, specifically along the discs of
the OS (Travis et al. 1991b; Connell et al. 1991). Notably, there is a high degree of
similarity between the sequences of mouse and bovine peripherin (~92%) such that
antibodies raised against the protein of one species cross-reacted with the other.
The characterization of the phenotype of the rds mouse and its associated gene
led to the investigation of the human RDS gene as a candidate gene for human reti-
nal dystrophies. The mapping and isolation of the human RDS gene and cDNA
closely followed the identification of the mouse gene. Travis and coworkers used
the Rds cDNA clone from mouse to probe a human retinal cDNA library and thus
isolated the human cDNA for RDS. The encoded protein was similar in length to the
mouse protein and over 90% identical to it in its amino acid sequence. The human
RDS gene was assigned to chromosome 6 and further localized to chromosome
6p12 by in situ hybridization of the human RDS cDNA onto human chromosomes
(Travis et al. 1991a).
5.4.1.2 Animal Models for RDS

The rds mouse has been extensively studied as a model for human RP. The degen-
eration in the rds mouse occurs initially in the peripheral retina and progresses
toward the center, with loss of cones by 12 months. The key defect in the rds−/− mice
appears to be the failure to form normal outer segments and an absence of discs in
the outer segments. In contrast, the inner segments are relatively normal (reviewed
by Stuck et al. 2016). The histological defects in the retina are evident by the third
postnatal week, and by 1 year, there is a loss of all photoreceptors, except for a
single layer of cone cells. In place of outer segment discs, the rds−/− mice were
observed to have extracellular vesicles that contained arrestin and rhodopsin, sug-
gesting that they represented unformed or defective OS discs. Though the rds mutant
was thought to be recessive in nature, heterozygous mice also displayed some
abnormalities in the outer segment, which were milder than in homozygous ani-
mals. This difference in the degree of severity in phenotype of heterozygote and
homozygote mice is consistent with a dosage effect of the mutant gene, with haplo-
insufficiency being the ostensible mechanism of the disease.
There are several other mouse models for RDS knockdown, and both transgenic
and knockout models have been generated. Missense mutations in the RDS gene,
associated with ADRP in humans, have been expressed as transgenes in mice in
order to study their effects. An example of a transgenic mouse model with a mis-
sense mutation is the Pro216Leu (P216L) transgenic mice. These mice have the
transgene expressed in either the rds−/− or rds+/− background. They display a faster
rate of degeneration of the retina and dysplasia of the outer segments of the photo-
receptors. The mechanism of the rds gene defect in these mice includes both haplo-
insufficiency and a dominant negative effect of the missense mutation. Another such
missense mutation expressed in transgenic mice is the Cys214Ser (C214S) mutation

in RDS. Here, too, the mutant protein is observed at very low levels compared with
the wild type, and haploinsufficiency is the proposed mechanism of the disease.
Another interesting mouse model of RDS-associated disease is a model for
digenic RP, caused by mutations of the RDS and ROM1 genes. Digenic RP is very
rare, and several families with this form of RP have affected members who are
double heterozygotes for a missense mutation Leu185Pro in the RDS gene, and a
null mutation in the ROM1 gene. Transgenic mice with the same alleles in Rds and
Rom1 were created to study the effects of the two heterozygous mutant genes on the
retina. This digenic model exhibited a faster rate of retinal degeneration than the
monogenic control mice. The retinas of the digenic mutant mice manifest with a
shortening and disorganization of the outer segments, loss of the outer nuclear layer,
and diminished ERG responses corresponding to photoreceptor hyperpolarization
as compared to controls or singly heterozygous mice (Kedzierski et al. 2001).
Apart from RP, other phenotypes such as macular degeneration, associated with
RDS mutations, have been recreated using transgenic mice. The mutation of
Arg172Trp (R172W), found in families with macular degeneration, expressed in
transgenic mice, shows varying degrees of severity which correlated with the level
of expression of the mutant protein as compared to wild type. Transgenic mice
which expressed lower levels of the mutant transgene had milder manifestations of
the disease, with a later onset, while mice with a higher level of transgene expres-
sion showed a more early onset of the disease and a severe phenotype (Ding and
Naash 2006).
5.4.1.3 Mutations of the RDS Gene in Families

with Retinal Dystrophies
The evidence for the RDS gene as a cause of retinal diseases in humans was estab-
lished in a study of over 100 families each with autosomal dominant and recessive
RP. There were no large gene deletions or rearrangements identified in any of the
families using Southern blot analyses. On the other hand, screening of exons for
small mutations by the method of single-strand conformation polymorphism (SSCP)
followed by sequencing in these families revealed pathogenic mutations in four
separate families with dominant RP. These comprised two missense mutations and
one deletion, which were not found in a control population (Kajiwara et al. 1991).
The mapping of the RDS locus was also achieved through another independent
route, involving the study of a large Irish pedigree with a delayed onset of visual
loss. Although ERG responses were below normal in the affected members of the
family by about 5 years of age, visual defects were apparent much later and were
observed only in adulthood. Night blindness developed in the fourth decade, and
further visual loss was evident by the fifth decade of life. Genotyping of 72 mem-
bers of this family mapped the disease locus to chromosome 6p, thereby co-
localizing it with the RDS gene. RDS was an obvious candidate for the retinal
disease in this family, again based on the phenotype of the rds mouse. A three-base
pair deletion was found in the RDS gene in this family, leading to loss of two con-
served cysteine residues in the protein (Farrar et al. 1991a, b).
Though the original associations of RDS were made with ADRP in the two
studies mentioned in the previous paragraph, mutations in the RDS gene are also
associated with a variety of other retinal diseases. These disorders primarily
involve the central retina and include phenotypes such as cone-rod dystrophy and
various forms of macular dystrophy. Phenotypic heterogeneity associated with
RDS gene mutations may thus extend across the spectrum of retinal dystrophies,
including both rod-cone and cone-rod disorders. Diverse forms of retinal diseases
including RP and macular dystrophies have been documented in association even
with the same mutation in the RDS gene, within a family (Weleber et al. 1993).
Besides these, RDS mutations are also associated with digenic RP in combination
with a mutation in the ROM1 gene (see below). Retinitis punctata albescens (RPA),
a retinal disease characterized by the occurrence of yellow-white deposits in the
retina, has been associated with a null mutation resulting from a 2 bp deletion in
the RDS gene (Kajiwara et al. 1993). A patient with RPA was characterized clini-
cally as having punctate deposits along with diminished vision, atrophy around the
fovea, constriction of the retinal vessels, pigmentary clumps in the retina, and optic
disc pallor.
Another group of retinal disorders associated with different mutations in RDS
is classified as pattern dystrophy. These diseases, known variously as “butterfly
dystrophy” or “foveomacular dystrophy,” are autosomal dominant diseases
which consist of abnormal pigmentary deposits at the level of the RPE, some-
times appearing in the form of wings or arms, similar to a butterfly in shape. A
mutation at codon 167 changing glycine to aspartic acid (Gly167Asp) in the RDS
gene was identified in a three-generation family with this disease; there were 24
members of which 11 were affected with butterfly dystrophy (Nichols et al.
1993). Another mutation that has been associated with pattern dystrophy in a
family is a frameshift mutation due to an insertion of 4 bp (Keen et al. 1994).
Since RDS mutations are associated with many different forms of central retinal
dystrophies, this locus has a relatively high mutation frequency among patients
with retinal dystrophies. In fact, about 10% of families with various macular
dystrophies were attributable to RDS mutations as reported in a study of 76
families (Kohl et al. 1997). For ADRP, the frequency of RDS mutation appears
to be lower and is estimated to be 4% based on the analysis of a series of 170
patients from North America, tested for mutations by PCR and direct sequencing
(Sullivan et al. 2013).
5.4.2 Retinal Outer Segment Membrane Protein 1 (ROM1)
Associated disorders—digenic RP, dominant RP.

The ROM1 (retinal outer segment membrane protein 1) gene encodes a 37 KDa
protein having partial identity in its amino acid sequence to the peripherin/RDS
protein and like the latter is thought to function in the formation of the outer seg-
ments of photoreceptors. Both proteins are located in the rim of outer segment discs
and are thought to non-covalently associate with each other.
5.4.2.1 Isolation and Cloning of ROM1

The ROM1 cDNA was isolated by differential hybridization of a human retinal
cDNA library against total bovine retina cDNA, in order to detect evolutionarily
conserved retina-specific clones (Bascom et al. 1992). One of the clones isolated by
this method encoded a previously unknown protein, which was designated as
ROM1. The complete cDNA of about 1.4 kilobases encoded a protein of 351 amino
acids. The ROM1 transcript constituted one of the most abundant transcripts in the
retina.
5.4.2.2 Mutations in ROM1

Mutations in both genes together, RDS and ROM1, give rise to a rare form of digenic
RP. A study of three large pedigrees with RP revealed a missense mutation in the
RDS locus, present in all affected as well as in some of the unaffected members of
the family. The pattern of transmission of the disease in the pedigree was thus
unusual in that it deviated from an autosomal dominant mode of transmission. The
“deviations” included the segregation of the mutation as mentioned above, in both
affected and unaffected individuals, the presence of affected individuals who were
offspring of unaffected parents, and transmission of the disease from affected par-
ents to less than 50% of offspring. The similarity of the ROM1 gene with RDS in its
organization and expression pattern led to the idea that it could be a candidate for
“digenic” RP in the abovementioned families. Mutations were indeed discovered in
the ROM1 gene in all the families, which in combination with the RDS mutations
provided an explanation for the inheritance of the disease (Kajiwara et al. 1994).
Apart from digenic RP, mutations in the ROM1 gene alone may give rise to domi-
nant RP, though this appears to be very rare. A screen of over 250 probands with
autosomal dominant and simplex RP revealed about 1% of patients to have muta-
tions in the ROM1 gene (Bascom et al. 1995).
5.4.3 A
TP-Binding Cassette Subfamily A Member 4 Protein
(ABCA4) Gene
Associated disorders—autosomal recessive RP, recessive cone-rod dystrophy,

Stargardt’s macular dystrophy, age-related macular degeneration.
The ABCA4 gene (formerly known as ABCR (ATP-binding cassette, retinal)),
encodes the Rim protein. The protein is so-called because it is expressed in the rim
of the discs in the outer segments of photoreceptors. It belongs to a superfamily of
membrane proteins having at least 50 members that have common structural motifs
known as ATP-binding cassettes (ABC). The ABC proteins basically act as trans-
porters for various types of molecules such as peptides, lipids, drugs, amino acids,
ions, saccharides, and lipopolysaccharides. Several of the members of the ABC
gene family are associated with various diseases. A well-known example is the pro-
tein product of the cystic fibrosis gene, a chloride ion channel known as ABCC7.
The ABCA4 protein is a transporter which functions in the removal of all-trans reti-
nal (ATR) from the discs in the photoreceptor outer segments. Removal of ATR
occurs in the form of its conjugate with phosphatidylethanolamine—known as

N-retinylidene phosphoethanolamine. N-retinylidene PE is transported from the
luminal to the cytoplasmic side of the OS discs by the ABCA4 or Rim protein. The
Rim protein was first identified as a 220 KDa protein that is abundantly expressed
in the rim of the ROS of frogs and mammals (Illing et al. 1997).
5.4.3.1 Cloning of the ABCA4 cDNA

The mammalian cDNA for ABCA4 was isolated from a bovine retinal cDNA expres-
sion library by the use of monoclonal and polyclonal antibodies against the Rim
protein, to obtain a cDNA clone of about 1.8 Kb in length. This represented a partial
cDNA of ABCA4, and hence specific oligonucleotide probes designed from the
above cDNA sequence were then used to rescreen the library, in order to isolate
cDNA clones that cover the entire ABCA4 cDNA. Such a screen resulted in the
identification of several overlapping clones together making up an open reading
frame (ORF) of over 6.8 kb. The ORF predicted an encoded protein of 2280 amino
acids of molecular mass 257 KDa. The cDNA clones isolated by this process were
confirmed to indeed code for the major 220 KDa Rim (ABCA4) protein, since the
predicted protein sequence of the cDNA was identical with the sequences of the
actual peptides obtained from the native 220 KDa protein purified from the ROS
membranes.
The sequence of the ABCA4 or Rim protein is organized into two halves that are
structurally similar. Each half contains a hydrophobic region and an ATP-binding
cassette. The hydrophobic regions contain transmembrane segments, with 12 such
regions predicted on the basis of amino acid motifs. The ATP-binding cassette is a
domain that is common to the superfamily of ABC proteins. Overall, the protein
shows a high degree of similarity with various other members of the ABC family,
with over 60% identity in the sequence of the ABC domains between members of
this family. The ABCA4 protein is expressed in the rod outer segments, and also in
cones of the retina (Molday et al. 2000). Immunoelectron microscopy showed the
protein to be localized at the periphery of the ROS near the rim of the outer segment
discs, in close proximity with the plasma membrane of the OS (Illing et al. 1997).
Apart from the 12 predicted transmembrane regions, the protein has two ABC
motifs, and the amino- and carboxy-termini are on the cytoplasmic side of the
membrane.
5.4.3.2 ABCA4 and Its Association with Stargardt’s Disease

Stargardt’s disease is a juvenile-onset, autosomal recessive disorder involving pro-
gressive loss of central vision, yellow flecks around the macula, RPE atrophy, and
pigmentary changes in the macular region. The onset of vision loss is usually in the
first to second decades of life. Night vision and peripheral vision are generally not
affected. Stargardt’s is frequently associated with a condition known as fundus fla-
vimaculatus, characterized by the presence of yellow spots around the macula, and
the two are often regarded as the same disorder (Noble and Carr 1979).
Electroretinography shows cone function to be selectively affected. The genetic
locus for Stargardt’s disease was first mapped in eight multiplex families of French
and Moroccan origins (Kaplan et al. 1993). All families displayed symptoms of
early-onset vision loss affecting the macula, pigmentary changes in the macula with
a normal peripheral retina and vasculature, central visual field loss, color vision
defects, and the presence of a “dark choroid” throughout the posterior pole, visible
on fluorescein angiography. The typical appearance of the choroid is thought to be
because of the presence of lipofuscin deposits in the RPE, which block the normal
autofluorescence of the choroid. The gene for Stargardt’s disease was mapped in the
abovementioned families to chromosome 1p21-p13, within an interval of about
4 cM. This locus was confirmed by a subsequent analysis of 47 additional families
by linkage and physical mapping, which located the gene within the same interval
of chromosome 1 (Anderson et al. 1995). The same locus on chromosome 1 was
also mapped in a Spanish family with autosomal recessive RP, in which six out of
seven siblings were affected with an atypical form of RP, and was designated as
RP19. The clinical phenotype consisted of night blindness as the initial symptom,
subsequent loss of visual acuity, pallor of the optic disc, pigmentary deposits in the
peripheral and central retina, RPE atrophy, and attenuation of the retinal blood ves-
sels. The distinctive aspect of the disease reported in this family was atrophy of the
choriocapillaris (Martínez-Mir et al. 1997).
The mapping of the ABCA4 gene, however, was also achieved via an independent
route from that of mapping the disease locus in Stargardt’s disease. The ABCA4
gene was first identified as a retinal EST in human retinal cDNA libraries by
Allikmets and coworkers, as part of a study on ABC transporters, and found to be
very specific to the retina in its expression pattern (Allikmets et al. 1997b). Using
mapping techniques based on radiation hybrids between human and hamster chro-
mosomes (Box 5.2), the ABCA4 gene was then mapped to chromosome 1p13-21,
thus co-localizing it with the Stargardt’s disease locus. Based on its genomic posi-
tion, and its highly specific expression in the retina, ABCA4 was considered a good
candidate gene for Stargardt’s disease. Screening of the ABCR gene revealed muta-
tions in over half the families that were tested. These consisted of missense, splice
site, and frameshift mutations, distributed throughout the length of the gene. The
majority of mutations constitute missense changes. Consistent with its autosomal
recessive inheritance, patients are either compound heterozygous or homozygous
for mutations.
Box 5.2 Radiation Hybrid Mapping

Radiation hybrid mapping is a method of physically mapping the genome to
obtain relative orders of markers.
Markers distributed over several megabases of DNA can be mapped to a
resolution of a few hundred kilobases.
Cell fusion is carried out between the human (donor) and rodent (recipient)
cells using the Sendai virus.
Chromosomes are eliminated from the fusion cell such that only one
selected chromosome remains.
The chromosome of interest is then treated with X-rays to shear it into

small pieces.
The ends of the fragmented human chromosome are joined to the hamster
chromosomes in a process of repair resulting in human-hamster hybrid
chromosomes.
Human chromosomal segments are therefore present as insertions or trans-
locations in the hamster chromosomes.
Markers that are closer together in a human chromosome will be more
likely to be on the same fragment after irradiation, while those that are far
apart will be in separate fragments.
5.4.3.3 Other Disorders Associated with ABCA4 Mutations

Mutations in the ABCA4 gene are associated with a range of phenotypes that include
dystrophies involving the central and peripheral retina. The involvement of ABCA4
gene mutations in the pathogenesis of Stargardt’s disease, a juvenile form of macu-
lar dystrophy, gave rise to the idea that this gene may also be involved in the patho-
genesis of age-related macular degeneration (ARMD), a late-onset disease with a
complex etiology that resembles Stargardt’s disease, since it also affects the central
retina. Mutations in ABCA4 are found in about 16% of ARMD patients, who were
heterozygous for these sequence changes (Allikmets et al. 1997a).
Clinical heterogeneity associated with specific ABCA4 mutations can also occur
within the same family. In such cases, different members of a family may manifest
phenotypes such as RP, CRD, or Stargardt’s disease in association with different
genotypes of ABCA4 mutations. This is illustrated by the patterns of both genotype
and phenotype obtained from families that have some individuals with CRD and
others affected with RP. In these cases, individuals having RP are found to be homo-
zygous for mutations predicted to have severe effects (such as null mutations). On
the other hand, members of the same family with a phenotype of CRD have at least
one allele that is predicted as having a relatively “mild” effect, i.e., it is expected to
be only partially defective in its function and may thus retain some degree of activ-
ity. An example of this is a family with RP-affected individuals being homozygous
for a splice site mutation of IVS30+1G->T at the conserved splice donor dinucleo-
tide of intron 30 of the ABCR gene, while those with cone-rod dystrophy had two
mutations IVS30+1G->T and IVS40+5G->A and were thus compound heterozy-
gotes (Cremers et al. 1998). In these patients, the more severe phenotype of RP is
associated with two “severely defective” alleles produced by mutation of the con-
served splice junction sequence at +1 position of the intron. Since the base at this
splice donor position is absolutely conserved, a mutation of this base is almost cer-
tain to lead to a complete splicing error and produce a mutant mRNA. In other
words, we do not expect any of the mRNA molecules to be correctly spliced. The
consequence of this is a complete loss of function of this gene due to absence of any
normal mRNA (and protein). The relatively more “mild” phenotype of CRD in the
same family is associated with one mutant allele involving the conserved +1
position of the splice donor site and a second allele with a mutation affecting the +5
position of the intronic splice site. The mutant at +5 would be expected to retain
partial (residual) function since it may lead to some degree of correct splicing, even
if at a low level, and is therefore classified as a “mild” mutation. The overall effect
of the two mutations, one mild and one severe, is presumably to give rise to at least
a low level of functional mRNA and protein, thereby retaining a fraction of the nor-
mal activity in patients who are compound heterozygotes. Similar correlations have
been made of patients’ genotypes, i.e., mild versus severe types of mutations, in
association with Stargardt’s disease (milder phenotype) and RP (severe phenotype),
respectively, within a family (Rozet et al. 1999). These observations have thus led
to a model of genotype-phenotype correlations with ABCA4 mutations in which
mild and severe phenotypes are associated with mild and severe types of mutations,
respectively. The various retinal dystrophies associated with mutations in ABCA4
are considered to be a continuum of phenotypes ranging in severity from low to
high—from ARMD, Stargardt’s disease, cone-rod dystrophy, to RP. There is exten-
sive mutational heterogeneity in the ABCA4 gene, and thousands of mutations have
been documented. A very frequent mutant allele among patients with Stargardt’s
disease from Western Europe is the substitution mutation 2588G>C, occurring in
the first base of exon 17 of ABCA4 and leading to two mutant transcripts as demon-
strated by analysis of the mRNA from leukocytes of patients and controls—one
transcript carries a deletion of a glycine residue at position 863 (Gly863del) corre-
sponding to a 3 bp deletion as a result of missplicing; and a second transcript is
normally spliced but has a missense change of glycine-863 to alanine (Gly863Ala).
The 2588G>C mutation has a heterozygote frequency of about 30% in Europeans
with Stargardt’s disease and is found in about 3% of the control population (Maugeri
et al. 1999).
5.4.4 Crumbs Homolog 1 (CRB1)
Associated disorders—autosomal recessive LCA, autosomal recessive RP, RP with

PPRPE, RP with Coats-like reaction.
5.4.4.1 Function of CRB1

The Crumbs protein plays a critical role in the apicobasal polarity of epithelial cells
and in maintaining cell-cell contacts. The protein is found localized on the apical
membranes of epithelial cells, and mutants of crumbs in drosophila have a disrup-
tion in the organization of epithelial cell layers (Tepass et al. 1990). It was identified
as a key component of the protein complex that is required for maintaining cell
polarity. An important structure required for cell polarity is the junctional complex,
which is present around the circumference of the apical part of the cell. In verte-
brates, the junctional complex is comprised of the zonula adherens or adherens
junction, flanked by tight junctions and desmosomes. The adherens junction is made
up by the apposed plasma membranes with a gap of 10–20 nm between them. The
plasma membranes of adjacent cells are bridged by rod-shaped structures made of
cadherin molecules extracellularly, connected through alpha- and beta-catenins to

actin filaments extending on their intracellular side. In polarized epithelial cells, the
adherens junctions extend around the circumference of the cell along with the actin
belt (Meng and Takeichi 2009). The major function of adherens junctions is to
maintain cell-cell contact, and loosening of the junctions causes disorganization and
disruption of tissues.
In Drosophila, mutation of crb produces an embryonic lethal phenotype, and
embryos die during gastrulation, due to loss of epithelial integrity in the ectodermal
layer. Homozygous loss of function Crumbs mutants in Drosophila show a com-
plete absence of cuticle, with only a few grains remaining giving the appearance of
crumbs (thus giving the protein its name). Studies on various mutants suggest that
Crb together with other proteins defines the localization of the zonula adherens. In
Crb mutants or in the presence of its overexpression, the zonula adherens is dis-
rupted (Tepass 1996; Rashbass and Skaer 2000).
The Drosophila Crumbs protein is a transmembrane protein with a small extra-
cellular domain and a large intracellular domain-containing 30 EGF-like repeats
and 4 laminin AG-like repeats. The intracellular domain is involved in the formation
of the Crumbs protein complexes.
5.4.4.2 Mapping and Isolation of the Human CRB1 Gene

The locus for the CRB1 gene on chromosome 1q31-q32, designated as the RP12
locus, was initially mapped in a large family from the Netherlands having autosomal
recessive RP with clinical heterogeneity within the family. A subset of members,
apart from an early-onset RP, presented with an added feature known as para-arteri-
olar preservation of the RPE (PPRPE). In PPRPE, one finds that the RPE adjoining
the retinal blood vessels is spared from degeneration. The presence of PPRPE was
not uniform in the family studied; it had 20 affected members with RP and PPRPE
and 11 members with RP but no PPRPE (van Soest et al. 1994). Linkage analysis in
the family showed evidence of linkage heterogeneity in the entire family; the RP
with PPRPE phenotype was found to be completely linked to a marker on 1q31.
The phenotype of autosomal recessive RP with PPRPE was earlier reported in a
series of five patients with retinitis pigmentosa that includes night blindness in the
early stages of disease with onset in the first to second decades of life and one or
more of the patients having optic nerve head drusen and clumped pigmentary depos-
its. The characteristic feature in this series of patients was that of para-arteriolar
preservation of the RPE (PPRPE) involving the presence of preserved RPE adjoin-
ing and beneath the retinal arterioles, detectable by fluorescein angiography. Severe
loss of vision is reported in this form of retinitis pigmentosa by the second decade
(Heckenlively 1982).
The identification of the RP12 gene was made by a subsequent study involving
the isolation of genes that are specific to or enriched in the retina and RPE in their
expression pattern. Sequencing of several cDNA clones from such a library and
selecting for novel genes that were not represented in any of the databases led to the
identification of the cDNA for CRB1. The complete human cDNA was found to be
about 4 kilobases long, encoding a protein of 1376 amino acids. The cDNA was
mapped to the RP12 locus on chromosome 1q31-32 (den Hollander et al. 1999).
The CRB1 gene was found to be specifically expressed in the human brain and neu-
ral retina but not in several other tissues examined. Thus, its map position and its
retina-specific expression pattern made it a good candidate gene for the RP12 locus.
Screening of the gene in a series of 15 patients with RP and PPRPE revealed patho-
genic mutations in 10 patients. These included missense, splice, and frameshift
mutations. The predicted CRB1 protein showed the most similarity—about 55%
similarity and 35% identity to Drosophila Crumbs (Crb) protein. Its sequence con-
tains 19 EGF-like domains, 3 laminin AG-like domains, a C-type lectin domain, and
a signal peptide. Although the mammalian Crb homolog does not have a transmem-
brane domain, the similarity in the arrangement of its other domains in both dro-
sophila and humans led to its designation as Crumbs homolog 1 (CRB1). It is
expressed in all epithelial tissues of ectodermal origin in Drosophila. However, in
mice, its expression is evident from day 11 of the embryo, being found in the prolif-
erating retinoblasts. In the postnatal and adult stages, it is expressed in the photore-
ceptors and in some cells in the inner nuclear layer (den Hollander et al. 2002).
Mutations in CRB1 are associated with multiple forms of retinal dystrophy. In
addition to the RP12 phenotype mentioned above, CRB1 mutations are also patho-
genic in a subset of patients with LCA. The first demonstration of the association of
CRB1 mutations to LCA came from a study on European probands, in which seven
out of fifty-two patients tested were positive for CRB1 mutations. Again, various
types of changes such as missense, splice, and nonsense mutations were detected.
Screening of a series of patients with RP revealed one proband with CRB1 muta-
tions consisting of a missense and stop mutation on one allele and a second mis-
sense mutation on the other allele. Interestingly, the proband with these mutations
and his brother had a variant of RP associated with a condition known as Coats-like
exudative vasculopathy, resulting in additional loss of vision. Coats-like exudative
vasculopathy is a relatively rare complication of RP, occurring in 1–3% of patients
with RP, characterized by vascular abnormalities, yellow extravascular lipid deposi-
tions, and retinal detachment (den Hollander et al. 2001). The association of CRB1
mutations with the phenotype of Coats-like exudative vasculopathy was further sub-
stantiated in a further series of eight patients, thus providing confirmation that CRB1
mutations are a risk factor for this condition. The same study also showed that
Coats-like vasculopathy may be present as a complication of RP to a variable extent
even in the same family. In other words, it was present in some members with CRB1
mutations but not in others, since there were RP-affected members within a family
that had this vasculopathy, and also some that did not. In addition, the Coats-like
reaction may occur unilaterally in certain affected individuals. There is evidently no
firm correlation of genotype with phenotype in these CRB1-associated phenotypes
since the same mutations have been associated with RP and PPRPE, as well as with
RP and Coats-like reaction in different individuals. From the foregoing, it appears
that the CRB1 allele is necessary but not sufficient for the presence of the Coats-like
vasculopathy. These observations also imply that there may be other genetic modi-
fiers in addition to CRB1 mutations that influence the development of vasculopathy
in these patients.
5.5 Genes Encoding Splicing Factors 111
CRB1 mutations may occur in up to about 10% of cases of LCA as determined

from studies in a series of patients with LCA and other early-onset retinal diseases.
CRB1-associated LCA patients may present with certain peculiar features such as
nummular (coin-shaped) pigmentation at the level of the RPE and an increased reti-
nal thickness as assessed on optical coherence tomography. A rod-cone type of
dystrophy was observed as predominant in these patients, and only a few had a
cone-rod type of pattern on clinical and electrophysiologic evaluation (Henderson
et al. 2011).
5.5 Genes Encoding Splicing Factors
Mutations in a number of genes encoding mRNA splicing factors are associated

with dominant forms of RP. Although these splicing factors are ubiquitous in
expression and function, and are required for pre-mRNA splicing in general, muta-
tions in several of these genes give rise specifically to retinal diseases. These include
PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200.
5.5.1 Precursor RNA Processing Factor 31 (PRPF31; RP11)
Associated disorders—autosomal dominant RP.
5.5.1.1 Mapping and Identification of the RP11 Locus

The first locus for a splicing factor to be associated with ADRP was designated as
RP11. The RP11 locus was mapped to chromosome 19q in families from the UK
and, independently, from Japan (Al-Maghtheh et al. 1994, 1996; Xu et al. 1995). A
notable feature of the phenotype in all the families mapped to this locus was incom-
plete expressivity of the disease. The members of the families mapped to RP11 were
reported to show a “bimodal” expressivity, with some of the obligate carriers of the
disease gene being asymptomatic throughout life. In other words, although half of
the individuals in the affected family were considered at risk by virtue of having
inherited the specific haplotype linked to the disease, only 31% were actually symp-
tomatic and showed signs of disease. Manifestations in the symptomatic members
included characteristic features of RP such as night blindness beginning in the first
decade, retinal bony spicule pigment deposits, macular atrophy, and pigment epithe-
lial atrophy. Symptomatic individuals in the family, however, had children who
were either symptomatic or asymptomatic. In addition, there was no significant dif-
ference in the numbers of symptomatic versus asymptomatic children born to
asymptomatic disease haplotype carriers (Evans et al. 1995).
Haplotype data from the RP11-linked families enabled the mapping of the criti-
cal interval on chromosome 19q to a 3 cM interval on chromosome 19. Physical
mapping of this interval onto BAC clones yielded a region of a few hundred kilo-
bases harboring the RP11 gene. Sequencing of all genes in the critical interval
showed mutations in the PRPF31 gene (precursor RNA processing factor 31), with
homology to the yeast pre-mRNA splicing factor (Prp31p) gene. PRPF31 is widely
expressed in several tissues including the retina. Pathogenic mutations were identi-
fied in the families linked to the RP11 locus, as well as in some sporadic cases with
RP (Vithana et al. 2001). With several independent families being mapped to the
RP11 locus, it appears to be a relatively frequent cause of RP.
A significant feature of families with PRPF31 mutations is the incomplete pen-
etrance of the phenotype, such that there are mutation carriers who do not develop
the disease. The analysis of PRPF31 mRNA in a large family with a deletion in the
PRPF31 gene indicated that the asymptomatic individuals had a higher expression
of the mRNA from the wild type PRPF31 allele on the homologous chromosome,
as compared with symptomatic individuals, thus contributing to an overall higher
level of the normal PRPF31 mRNA in the former. The same difference in PRPF31
protein levels was also observed between symptomatic and asymptomatic mutation
carriers. In other words, asymptomatic carriers and non-carriers of the mutation had
similar levels of normal protein, which was higher than that of symptomatic carri-
ers. Thus, the increased expression of the normal copy of the gene appears to com-
pensate for the defect in the mutant copy, thereby preventing the manifestation of
the disease in a subset of mutation carriers. This phenomenon provided a molecular
basis for the incomplete penetrance of PRPF31 mutations (Vithana et al. 2003).
Interestingly, well before the discovery of the RP11 gene, the segregation of
markers at this locus was associated with reduced penetrance in RP11-linked fami-
lies. From a study of markers and their haplotypes at the RP11 locus in such fami-
lies, it was proposed that the penetrance of mutations at this locus is influenced by
wild type alleles at the RP11 locus on the homologous chromosome or by a closely
linked gene inherited from non-carrier parents (McGee et al. 1997). The frequency
of PRPF31 mutations is about 5–10% in populations with ADRP from Europe
including the UK, France, and Belgium (Waseem et al. 2007; Audo et al. 2010; Van
Cauwenbergh et al. 2017).
5.5.2 Precursor RNA Processing Factor 6 (PRPF6)

Yet another splicing factor apart from PRPF31 that is central to the trinuclear
snRNP complex of U4/U6/U5 is PRPF6. The prior knowledge that mutations of
various splicing factor genes are pathogenic for RP suggested that the PRPF6 gene
is also a reasonable candidate gene for RP especially in view of its involvement in
the formation of the abovementioned splicing complex. Screening of the PRPF6
gene in over 188 families led to the detection of a missense mutation in one family
with ADRP, suggesting that mutations in PRPF6 are a very rare cause of RP. The
residue affected was highly conserved in the protein sequence, and functional
impairment of the mutant was demonstrated by the detection of misspliced tran-
scripts for various endogenous genes from patients’ lymphoblastoid cell lines. In
addition, staining of the protein with specific antibodies showed mislocalizaton of
the mutant protein as compared with the wild type, in lymphoblastoid cell lines
from patients and normal controls, respectively (Tanackovic et al. 2011).
5.5 Genes Encoding Splicing Factors 113
5.5.3 Precursor RNA Processing Factor 4 (PRPF4)
5.5.3.1 PRPF4 and Retinal Disease

The gene for PRPF4, another splicing factor that is part of the complex with tri-
snRNP proteins, was again considered as a good candidate gene for retinitis pig-
mentosa. Direct evidence that a mutation in PRPF4 is associated with retinal
disease came from a zebrafish model. A knockdown of the Prpf4 gene in zebrafish
using morpholino oligonucleotides led to a phenotype resembling RP (Linder
et al. 2011). The involvement of PRPF4 mutations in retinal disease in humans
was demonstrated upon screening of a series of 85 probands from families with
ADRP, which led to the identification of a missense mutation of Arg192His in one
family. The pathogenicity of this missense mutation was further established in a
zebrafish model of RP. This consisted of the zebrafish strain in which endogenous
Prpf4 expression was knocked down by the use of morpholinos against Prpf4
mRNA. The Prpf4 morpholino, which was designed to abolish the expression of
zebrafish Prpf4 mRNA, was co-injected with either wild type or mutant tran-
scripts (coding for Arg192His mutant) of Prpf4. It was found that only the wild
type transcripts could rescue the retinal degeneration phenotype of the morpho-
lino-treated zebrafish. The mutant transcripts failed to rescue the retinal defects in
the zebrafish, although the amount of protein expressed from the mutant was
comparable with that of the wild type transcript (Linder et al. 2014). Apart from
changes in the cellular layering of the retina, this model system provided an
opportunity to investigate changes occurring at the molecular level due to the
mutant Prpf4 transcript. It was observed that the mutant Prpf4 protein was unable
to form complexes with the other splicing factors, in contrast to the wild type
protein.
The effects of various mutant Prpf4 transcripts having missense and deletion
mutations on zebrafish embryos by overexpression of these further demonstrated
the impact of a deficiency of normal Prpf4; it was found that the mutant transcripts
led to widespread defects with increased mortality of the embryos in which the
endogenous prpf4 gene was silenced (Chen et al. 2014).
5.5.4 Precursor RNA Processing Factor 8 (PRPF8; RP13)

The RP13 locus was mapped in a large family of British ancestry from South
Africa, with onset of night blindness within the first decade. Extensive retinal
degeneration was observed by middle age, with characteristic changes in the fun-
dus, including pigmentary deposits in the mid-peripheral retina. Based on an analy-
sis of the family of 46 members, the disease was mapped to chromosome 17p13
(Greenberg et al. 1994). The same RP13 locus was also mapped in additional fami-
lies with RP from the UK, the Netherlands, and the USA, with a total of five fami-
lies having ADRP mapped to this locus. Further refinement of the locus by haplotype
analysis in the same families subsequently placed the disease locus within an inter-
val of about 3 centimorgans. Physical mapping of this region led to the identifica-
tion of the PRPF8 gene in the mapped region (McKie et al. 2001). PRPF8 encodes
a large protein of 2335 amino acids coded by 42 exons. Screening of the gene for
mutations in the mapped families revealed pathogenic missense changes, all within
exon 42, close to the 3′ end of the transcript.
An extended series of over 300 patients with ADRP were also tested for patho-
genic changes in this gene, and this process identified four more missense mutations
in the same region of the gene. Mutations in PRPF8 are reported in a few more
families with ADRP from other populations as well, also located near the 3′ end of
the gene (Kondo et al. 2003).
5.5.5 Pre-mRNA Processing Factor 3 (PRPF3; HPRP3, RP18)
5.5.5.1 Mapping and Identification of the RP18 Gene

A locus for autosomal dominant RP (RP18) was mapped in two large families of
English and Danish origins in separate studies, to an interval of 1 cM on chromo-
some 1q21. In one study, the locus for ADRP in a seven-generation Danish fam-
ily with characteristic features of RP was mapped close to the centromere region
of chromosome 1 at 1p13-q23, to an interval of about 4 cM (Xu et al. 1996b).
Another study mapped the same locus on chromosome 1 in an English family of
four generations with autosomal dominant RP, characterized by loss of visual
fields and reduced visual acuity in the third-fourth decades. The appearance of
the fundus was characteristic of RP with RPE atrophy and pigmentary deposits in
the retina (Inglehearn et al. 1998). The gene encoding a splicing factor HPRP3,
present within this chromosomal region, was considered a suitable candidate for
the disease in these families, due to the previous associations of the genes for
splicing factors PRPF31 and PRPC8 with ADRP. Screening of the HPRP3 gene
showed a single mutation of Thr494Met in both families. The same mutation was
also found in three additional patients upon screening a larger series of 150 unre-
lated patients with simplex and dominant RP, suggesting that it is highly recur-
rent in this population (Chakarova et al. 2002). The threonine residue at position
494 is a site of phosphorylation in the protein, and substitution of methionine in
place of threonine in the mutant protein led to a lack of phosphorylation; this was
observed to interfere with its interactions with other splicing factors and with
snRNPs, thereby possibly leading to loss of activity of the protein (Gonzalez-
Santos et al. 2008). A second mutation of Pro493Ser was also found in a single
patient.
5.5.6 Pim1-Associated Protein Gene (PAP1; RP9)

5.6 Genes Encoding Ciliary/Centrosomal Proteins 115

The RP9 locus was mapped to chromosome 7p13-p15 and localized to an interval of
within four centimorgans in a large pedigree of autosomal dominant RP from England
with over 40 affected individuals (Inglehearn et al. 1993, 1994). This interval was
subsequently refined using additional microsatellite markers, to a region of <2 Mb.
Within the critical interval, one of the genes selected as a candidate for the retinal
disease in the family was that encoding the Pim1 kinase-interacting protein (also
known as PAP1, Pim1-associated protein). EST data for this transcript suggested a
widespread expression profile, showing it to be expressed in most tissues. Evaluation
of the Pim1 kinase-interacting gene revealed missense mutations in the original RP9
family as well as in a second simplex RP patient out of a series of 300 cases of differ-
ent forms of RP (Keen et al. 2002). The PAP1 protein localizes to nuclear compart-
ments containing “splicing speckles,” which are reported to contain active transcription
and splice proteins. This pattern of distribution of the protein was therefore suggestive
of a role in splicing. An effect of the mutant PAP1 protein on splicing was indeed
demonstrated in vitro using splicing assays in cell lines (Maita et al. 2004).
5.5.7 Small Nuclear Ribonuclear Protein 200 (SNRNP200)

Another gene associated with autosomal dominant RP, which functions in mRNA
splicing, is the SNRNP200 gene, encoding the U5 SNRNP200 KDa helicase (MIM
601664). This gene is located on chromosome 2cen-q12.1 at the RP33 locus. RP33
was mapped in a large four-generation family with ADRP by linkage analysis, and
fine mapping, with the critical region being localized to about 9 Mb (Zhao et al. 2009).
Five genes in this interval were considered as possible candidates for RP, including the
SNRNP200 gene, which was selected on the basis of the knowledge of other splicing
factor genes being associated with ADRP. The involvement of SNRNP200 in the
pathogenesis of ADRP in the family was indeed confirmed by the detection of a mis-
sense mutation of serine-1087 to leucine (Ser1087Leu). The mutation was observed to
be completely penetrant—i.e., all individuals carrying the mutation developed the dis-
ease. As with the other splicing factors, the SNRNP200 transcript is widely expressed
in a number of tissues including the retina. The SNRNP200 protein, known as hBrr2 in
humans, is 2136 amino acids in length and plays a central role in pre-mRNA splicing.
The yeast ortholog of hBrr2, known as Brr2p, is required for unwinding of the U4/U6
RNAs and for disassembly of the spliceosome (Raghunathan and Guthrie 1998).
5.6 Genes Encoding Ciliary/Centrosomal Proteins
5.6.1 Retinitis Pigmentosa 1 (RP1)
Associated disorders—autosomal dominant RP, autosomal recessive RP.

The RP1 locus was one of the earliest loci to be mapped for ADRP. The gene was
mapped and identified simultaneously by two groups. The RP1 locus was first
mapped in a seven-generation family from the USA to the pericentromeric region of
chromosome 8 (Blanton et al. 1991). Linkage mapping of two other families with
ADRP from Australia and the UK also placed the gene within the same locus on
chromosome 8q11-q12 and further refined this region to about 4 cM (Xu et al.
1996a). The RP1 locus, spanning about 4 Mb in length, showed partial synteny with
mouse chromosome 4. Analysis of expressed sequence tags (ESTs) mapping to the
human and the orthologous mouse loci led to the cloning of the RP1 cDNA and the
gene (Sullivan et al. 1999). All families which mapped to the RP1 locus had similar
clinical features such as late onset of symptoms and slow progression of the disease.
Another feature is the wide variation in the severity and onset of the disease, with
mutation carriers being without any signs and symptoms even after the fifth decade
of life. Interestingly, among the three families that were originally mapped to this
locus, two families (one each from the USA and Australia) had the same mutation
of Arg677Ter (a stop mutation at Arg677). The third family also had a truncating
mutation in the RP1 gene.
A second study on the RP1 gene isolated the mouse gene through another
approach and independently mapped and identified the gene in families in which the
disease was mapped to the RP1 locus. In this case, the Rp1 cDNA was obtained
using a mouse model of oxygen-induced retinopathy, based on changes in its expres-
sion in response to retinal hypoxia. A differential display analysis of genes whose
expression was significantly altered by changes in oxygen levels led to the isolation
of the Rp1 gene in mouse, among several others that also showed a similar response.
The human RP1 cDNA was cloned from a human retinal EST library, based on its
homology to the mouse Rp1 cDNA, and mapped onto human chromosome 8, within
the previously mapped RP1 locus (Pierce et al. 1999). Its expression was clearly
dependent on oxygen levels, since it was stimulated by hypoxia and suppressed by
hyperoxia. The RP1 gene was thus a plausible candidate gene in families with RP,
in which the disease was previously mapped to the same interval on chromosome
8q. Screening of RP1 indeed showed the truncating mutation arginine-677 to Stop
(Arg677Ter) in 9 out of 241 index cases screened, indicating a frequency of about
3–4% in North America. Incidentally, this study as well as the one by Sullivan and
coworkers identified the Arg677Ter mutation in the original family mapped to the
chromosome 8q locus by Blanton et al. (1991). It appears that the Arg677Ter muta-
tion in the RP1 gene is the most frequent mutation in ADRP after the rhodopsin
mutations at residues 23 and 347, which are found in about 10% and 4% of cases,
respectively. The overall frequency of all mutations in RP1 is about 7% in patients
with ADRP in this population. The majority of mutations involve truncations of the
protein (Bowne et al. 1999). These studies clearly established the role of RP1 in reti-
nal dystrophies, although its function in the retina was characterized in subsequent
studies.
Mutations in the RP1 gene are also found in some families with autosomal reces-
sive RP. A study of three consanguineous Pakistani families having ARRP with
multiple affected individuals in each obtained evidence of homozygosity to markers
on chromosome 8 in all affected members recruited, by testing for various known
loci for RP (Khaliq et al. 2005). Analysis of the RP1 gene, present at this locus,
revealed mutations in all three families. Interestingly, the same missense mutation
of threonine-373 to isoleucine (Thr373Ile) occurred in two of the families tested.
The third family had an insertion mutation. The disease in these families was severe
in nature with an early onset, in contrast to the families with ADRP having muta-
tions in the same gene. Patients are reported to have an onset of disease since early
childhood and were essentially blind by the second decade of life. Characteristic
features of RP were noted in the retina in these individuals such as the presence of
attenuated blood vessels, pigment deposits in the retina, and a pale optic disc.
The RP1 gene has four exons and has a transcribed sequence of almost 7000 bp,
coding for a protein of 2095 amino acids. The protein was at first localized to the
connecting cilia of rods and cones in human and mouse retinas, as detected by
immunofluorescence with specific antibodies (Liu et al. 2002). Subsequent studies,
however, demonstrated that it is co-localized with acetylated alpha-tubulin in the
photoreceptor axoneme, which extends throughout the length of the photoreceptor,
rather than in the connecting cilium (Liu et al. 2004). The N-terminus of RP1 shares
homology with doublecortin, a microtubule protein. It is expressed in photoreceptor
cells, specifically within the connecting cilium and axoneme of the photoreceptors.
The pathologic effects of the disruption of the RP1 gene on the retina were assessed
by the generation of Rp1 knockout mice. The disruption of the Rp1 gene in a mouse
model led to rapid retinal degeneration, with rod outer segments (OS) becoming
shortened and disorganized. A key feature of the retinas of Rp1 knockout mice is the
misalignment of the OS discs. Homozygous knockout mice showed reductions in
the number of photoreceptors with a decrease in thickness of the outer nuclear layer
after the first few weeks, and the defect was marked after 3 months of age. The outer
segments of the photoreceptors progressively shortened after birth and showed a
disorganized structure. In vitro experiments suggest that RP1 is a microtubule-
associated protein and appears to have a role in regulating the length of the axoneme
of the photoreceptors (Gao et al. 2002).
5.6.2 Leber Congenital Amaurosis 5 (LCA5)
Associated disorders—autosomal recessive LCA.

The LCA5 locus was the fifth to be mapped for LCA. The gene was mapped in a
family containing three affected members in two branches of the family linked by a
common ancestor. The family belonged to a genetically isolated population from
Pennsylvania, known as the Old River Order Brethren, who were migrants there
during the eighteenth century. The patients presented with poor vision, high hypero-
pia, defective pupillary reflexes, and a normal-looking retina.
5.6.2.1 Mapping of the LCA5 Gene

Mapping of the LCA gene in the Pennsylvania family of which 27 individuals were
included led to the localization of the LCA5 gene to chromosome 6q11-16. Various
candidate genes in this interval, such as the GABA receptor genes, which are
expressed in the retina, were tested and found to be negative for pathogenic muta-
tions in the family (Dharmaraj et al. 2000a). The LCA5 locus was also mapped
subsequently in another consanguineous family from Pakistan, of which thirteen
members (five of them affected) were analyzed. This enabled the narrowing of the
LCA5 locus on chromosome 6. In contrast to the LCA5-linked family from
Pennsylvania, in which the fundus was essentially unremarkable, the affected mem-
bers of the Pakistani family had optic disc pallor, narrowing of the retinal vessels,
and atrophic changes around the fovea (Mohamed et al. 2003).
5.6.2.2 Identification of the LCA5 Gene

The LCA5 gene was identified in a study of LCA families of various ethnicities
including Pakistani. Mapping of the disease locus by homozygosity mapping effec-
tively narrowed the critical interval for the disease to approximately 700–800 kb, on
the basis of shared haplotypes in homozygous affected individuals in all the families
(den Hollander et al. 2007a). Evaluation of the genes in this region, including an
uncharacterized ORF designated as C6OR152 (renamed later as LCA5), revealed
mutations involving frameshifts or terminations in all the families screened.
However the frequency of mutations in LCA5 in LCA patients appears to be rather
low, as suggested by a subsequent study, which estimated about 2% of patients to
have LCA5 mutations out of a large series of over 1000 probands with LCA and
recessive RP tested (Mackay et al. 2013).
5.6.2.3 Function of LCA5/Lebercilin

Characterization of the protein encoded by the LCA5 gene, named as lebercilin,
showed that it was expressed in a wide range of tissues, including the eye, nervous
system, gut, and respiratory organs. The protein is localized to the ciliary axoneme
located at the base of the primary cilia in all ciliated cells. The function of lebercilin
is connected to the intraflagellar transport of proteins, as evident from its presence
as part of a large complex of proteins involved in intraflagellar transport (IFT).
Furthermore, LCA-associated mutants of the lebercilin protein failed to interact
with the intraflagellar transport proteins. The interaction of lebercilin and IFT pro-
teins possibly occurs in vivo as well since it co-localizes with the latter proteins in
the connecting cilia of photoreceptors (Boldt et al. 2011). Knockdown of Lca5 in
mice led to abnormalities in retinal structure and function and an altered localiza-
tion of various proteins in the photoreceptors.
5.6.3 Topoisomerase 1 Binding RS-Like Protein Gene (TOPORS)

The topoisomerase 1 binding RS-like protein was initially discovered and char-
acterized as an interacting protein for the human topoisomerase1 enzyme, using the
yeast two-hybrid screen. Based on the abundance of Arg-Ser (RS) dipeptides in the
predicted sequence of the protein, it was named as topoisomerase 1 binding RS
protein (Haluska Jr et al. 1999). The unique features of the protein are the presence
of the RS peptides and a RING-type zinc finger domain, thus having similarity to
RING finger proteins, which are a class of transcription factors and to RS proteins.
The latter are a group of proteins involved in RNA processing. Despite these fea-
tures, TOPORS functions in the protein degradation pathway, as an ubiquitin ligase
with an E3 type of ubiquitin ligase activity conferred by the RING domain. As an
E3 enzyme, it is involved in the final step of ubiquitination. It is found to interact
with a range of E2 ubiquitin-conjugating enzymes suggesting that it acts on a wider
range of substrates and hence plays a critical role in the ubiquitin-proteasome sys-
tem (Rajendra et al. 2004). Notably, it interacts with a key component of the 26S
proteasome, an organelle responsible for protein degradation, and localizes to the
centrosome, as well as to the junctions of the outer segments of photoreceptors with
the RPE (Czub et al. 2016).
The TOPORS gene is associated to a form of ADRP that was originally mapped
to chromosome 9p21 (known as the RP31 locus). The RP31 locus was mapped in a
French Canadian family of twenty-six individuals from three generations, of which
fourteen were affected (Papaioannou et al. 2005; Chakarova et al. 2007). The phe-
notype in the family was characterized by a perivascular cuff of retinal pigment
epithelial atrophy surrounding the vascular arcades, early rod dysfunction, and a
high degree of variability in visual functions between members of the family as
assessed by visual acuities, fields, and electroretinography. Three of the affected
individuals were asymptomatic, and the onset of symptoms ranged from the first to
the fifth decades. The disease was mapped to an interval of 14 Mb, with over 50
genes within the mapped locus. Screening of the TOPORS gene in the family
revealed a pathogenic mutation consisting of a heterozygous one base insertion.
Further screening of a series of German patients with ADRP showed a deletion in
one patient out of sixty-four patients screened. Both the mutations predicted a
frameshift in the protein.
5.6.4 FAM161A
5.6.4.1 Mapping and Identification

The FAM161A gene maps to chromosome 2p14-15, a chromosomal region that
overlaps with a locus for ARRP, known as the RP28 locus. RP28 was originally
mapped in consanguineous Indian families by Gu and coworkers (1999).
Subsequently, multiple families of Israeli and Palestinian origins were studied by
homozygosity mapping using high-density SNP arrays; 11 families had affected
individuals that were homozygous for markers within a common interval on chro-
mosome 2p, and the region of homozygosity was shared by all affected individuals.
The common haplotype in these families, spanning a region of about 5 Mb, over-
lapped with the RP28 locus (Bandah-Rozenfeld et al. 2010). Evaluation of several
genes in the critical region for the disease in these families identified mutations in
the FAM161A gene. Three null mutations leading to premature stop codons were
detected in multiple families, amounting to an overall frequency of 11% (corre-
sponding to 20 out of 172 families tested). Mutations in FAM161A are thus very
frequent in these populations, which comprised different ethnicities including the
North African Jews, Ashkenazi Jews, Syrian Jews, and Arab Muslims. Founder
effects in these families were evident from common haplotypes shared over several
megabases flanking the FAM161A gene. Phenotypic features in the FAM161-linked
families are reported to be very variable, with a range of severity and manifestations.
Common features reported in the abovementioned families are pallor of the optic
disc and attenuation of the retinal blood vessels, present in all patients, whereas bone
spicule-like pigmentary deposits were not evident. Thinning of the outer nuclear
layer was seen on OCT imaging, with relative sparing in the region of the fovea.
Another simultaneous study identified the FAM161A gene in the original family
that was mapped to the RP28 locus, using a different approach. Patients’ DNA was
subjected to targeted capture of all exons within the RP28 genomic interval. High
throughput sequencing of the genes in the captured DNA was used to detect
sequence changes (Langmann et al. 2010). In parallel, the authors used chromatin
immunoprecipitation and sequencing (ChIP-Seq) of genomic regions in mouse,
which were bound by the transcription factor CRX (see Box 5.3). CRX is the cone-
rod homeobox protein, functioning as a retina-specific transcription factor that regu-
lates several genes with retinal expression. This strategy was based on the observation
that the majority of retina-specific genes contained CRX-binding sites. Among
these are several genes that are RP-associated. Alignment of the mouse genomic
CRX-binding sequences with the human orthologs present in the RP28 locus identi-
fied the FAM161A gene as a strong candidate. The FAM161A gene has a strong
CRX-binding site in its promoter and was thus detectable in the ChIP-Seq analysis,
thereby emerging as a good candidate for the RP28 gene. Its role in the pathogenesis
of the RP28 form of RP was confirmed by the detection of homozygous affected
individuals in the family having a mutation leading to a stop codon, Arg229Ter;
unaffected members of the family were either heterozygous carriers or had two
normal alleles. In this study as well, it was noted that the patients had no specific or
unique clinical features associated with the FAM161A mutations. In addition,
another nonsense mutation was found in three German families out of over 100
probands tested, and the affected individuals also shared a common haplotype of
markers in this genomic region. Mutation of the FAM161A gene was thus estab-
lished as a pathogenic basis in a small subset of ARRP families.
Box 5.3 ChIP-Seq

Chromatin immunoprecipitation (ChiP) sequencing is a method to identify
DNA sequences bound by a protein of interest. It is carried out by first cross-
linking the DNA and DNA-bound proteins.
The protein along with its bound DNA is then subjected to immunoprecipi-
tation using antibodies specific to the protein.
The bound DNA is released by reversing the cross-links with protein, and
the protein is digested. The DNA fragments are then ligated to adaptors and
cloned to generate libraries.
The cloned libraries are then subjected to high-throughput sequencing
using next-generation sequencing (NGS) systems, to generate massively par-
allel, short reads that can be aligned to the genome. Any protein of interest,
including transcription factors, chromatin-associated proteins, etc., can be
investigated by this technique for determining their binding sites to genomic
DNA.
5.6.4.2 Expression and Function of FAM161A

The FAM161A transcript is expressed at relatively higher levels in the human retina
and testes, as compared with other tissues. The protein is found in the inner segments
of both types of photoreceptors and in the plexiform layers, but not detected in the
outer segments (Langmann et al. 2010). During development, it is found to be
expressed in the retinal progenitor cells. In the postnatal retina, it is specific to the
photoreceptor precursors and the mature photoreceptors (Bandah-Rozenfeld et al.
2010). Studies of Fam161a−/− mice having knockouts of both copies of the Fam161a
gene by insertional mutagenesis confirmed the pathological effects of the lack of func-
tional Fam161a protein in the retina. The knockout mice showed severe degeneration
of the photoreceptors that was evident by 1 month of age and progressed to an absence
of the outer nuclear layer by 5 months (Karlstetter et al. 2014). Thinning of the outer
retina in these mice was accompanied by disorganization of the cellular layers and
activation of glial cells, with abnormal appearance and migrations of microglia. In
addition, the connecting cilium was markedly shorter and distorted in the mutants,
there was a misalignment of outer segment discs in the photoreceptors, and the micro-
tubule doublets in the cilium were spread in the proximal half of the cilium, with the
consequent dilation of the base of the outer segments. The FAM161A protein has a
wide network of interactions, and these have been mapped using the yeast two-hybrid
system. The proteins interacting with FAM161A include components of the cilia-cen-
trosome and microtubules and members of the Golgi network (Di Gioia et al. 2015).
5.6.5 R
etinitis Pigmentosa 3 (RP3)/Retinitis Pigmentosa GTPase
Regulator (RPGR)
Associated disorders—X-linked RP.
5.6.5.1 Mapping of the RP3 Locus

The RP3 locus for XLRP possibly represents the major locus among XLRP patients
in Europe and North America. A clue to a locus for XLRP came through the evalu-
ation of a patient with a syndrome that included four different disorders—RP,
Duchenne muscular dystrophy (DMD), chronic granulomatous disease (CGD), and
McLeod phenotype. The location of the genes for these disorders, DMD, CGD, and
McLeod phenotype, all on the X chromosome, strongly suggested that the syn-
drome in this patient represented a contiguous gene deletion syndrome involving
deletions of all three genes. The presence of RP in this patient therefore pointed to
the existence of a locus for RP also in the region of the Xp21 deletion in this patient.
Subsequently, another study mapped XLRP to the same locus, i.e., the RP3 locus,
on chromosome Xp21 through a linkage mapping study in a large Caucasian family.
However, the analysis of crossovers in this family indicated that the RP3 gene was
located outside the boundary of the contiguous gene deletion mentioned above; this
study effectively narrowed down the disease interval for RP3 to less than one mega-
base. Thus RP3 was mapped distal to the RP2 locus on the short arm of the X chro-
mosome (Fujita et al. 1996). As is characteristic of XLRP, males in the affected
family were severely affected; they manifested initial symptoms of the disease dur-
ing their teens and had severe visual impairment by their fourth decade. Females
that were heterozygote carriers also exhibited milder signs and symptoms of the
disease. The symptoms that were reported were sensitivity to bright light and night
blindness. Partial signs of the disease in females included a reduction in ERG
responses and presence of bony spicule pigment deposits in certain localized areas
of the retina. Some females, however, were noted to be severely affected.
5.6.5.2 Identification of the RP3 Gene

The mapping of the RP3 gene led to its cloning and isolation, as reported in two
parallel studies. The RP3 gene was also isolated by the method of YAC representa-
tional hybridization (YRH) (Roepman et al. 1996a). To isolate the gene, the authors
generated probes from a YAC clone of interest, which contained the RP3 genomic
region. The idea was to use these fragments derived from the RP3 locus as probes
to detect the corresponding regions in genomic DNA of RP patients. Restriction
enzyme fragments of the YAC DNA, after being amplified by PCR, were used as
probes to screen the genomic DNA from a series of RP patients by means of
Southern blots. The investigation of the genomic DNAs from several unrelated
patients with RP by this method revealed an anomalous pattern of DNA fragments
in one patient among a series of patients that were tested. The DNA in this patient
revealed a microdeletion of a few kilobases in length. Based on the position of the
deletion, the complete RP3 gene was isolated from neighboring genomic DNA
clones in cosmid vectors. The genomic DNA clone harboring the RP3 gene was in
turn used as a probe to isolate cDNA clones for this gene from a retinal cDNA
library. Sequence analysis and alignment of both the genomic and cDNAs for RP3
facilitated the identification of the exons of the gene (Roepman et al. 1996b).
Identification of the RP3 locus was also approached by mapping various deletion
syndromes similar to that of the patient mentioned above; however, there were differ-
ences in the presence of one or more of the phenotypes in the patients, as well as in the
extent of the deletions present (Meindl et al. 1996). One patient with classical XLRP
was found to have a relatively small deletion of 75 kb in the RP3 region. Screening of
the genes present in the deletion region mapped in this patient led to the identification
of the RP3 gene. The gene was designated as RPGR (retinitis pigmentosa GTPase
regulator). The gene thus isolated contained 19 exons, about 60 kb in length and ubiq-
uitously expressed. The RPGR cDNA, of about 2 kb in length, was isolated from a
human retinal cDNA library using probes designed from available EST sequences and
based on predicted exonic sequences of the gene. The complete cDNA product corre-
sponded to an encoded protein of 815 amino acids. The RPGR peptide has six tandem
repeat motifs with homology to the highly conserved repeats of a protein known as
regulator of chromatin condensation 1 (RCC1). It is a guanine nucleotide exchange
factor (GEF). GEFs are proteins which bind to GTP-hydrolyzing enzymes (GTPases)
and facilitate the release of GDP formed as a result of GTP hydrolysis.
5.6.5.3 Mutations in the RP3/RPGR Gene

Various studies including the ones mentioned above identified mutations in a frac-
tion of RP3-linked families. For example, in the study by Roepman and coworkers
(mentioned above), screening the RPGR gene coding sequences in 28 families with
XLRP led to the detection of additional mutations in families with RP, amounting to
about 20% of families with mutations at this locus. In the second mapping effort
mentioned above by Meindl and coworkers, mutations in RPGR were found at a
frequency of about 10% upon testing of several families of British and German
origins. A noticeable discrepancy in these families was that although the RP3 locus
represented the mapped disease locus in the majority (two-thirds) of XLRP fami-
lies, as indicated by linkage studies, not more than 20% of families appeared to have
detectable mutations in the gene. There was thus a considerable gap in the number
of families mapped to the RP3 locus and the number of families that were in fact
found to be positive for mutations in the RP3/RPGR gene.
In an attempt to explain the apparently “mutation-negative” families mapped to
the RP3 locus, Vervoort et al. used exon prediction programs to detect putative
exons and performed RT-PCR analysis of RPGR transcripts to detect unidentified
coding regions of RPGR and to characterize transcripts of RPGR in various adult
tissues. These experiments showed that new exons that were unknown until then
were indeed expressed as part of the RPGR transcript. These were designated as
open reading frames 14 and 15 (ORF14, ORF15). The ORF14 was generated from
the transcription of exon 14 and intron 14. ORF15 consisted of exon 15 and a part
of intron 15 and was expressed in all tissues examined including the retina and a
retinoblastoma cell line. The ORF15 contained a polyadenylation signal suggesting
that it is an alternative terminal exon. Mutations were found in ORF15 in almost
70% of 47 XLRP families tested. These consisted of deletions, nonsense mutations,
and missense changes. Thus, the “missing” mutations in RPGR were found within
ORF15, and overall, the study found 80% of mutations in RP3 to be located in
ORF15 (Vervoort et al. 2000). A peculiar feature of ORF15 is its very high purine
content, making up a repetitive sequence, with the encoded amino acids being
mostly the acidic amino acids glutamic and aspartic acid, as well as glycine. The
high rate of mutation in the RPGR-ORF15 is ascribed to the repetitive purine-rich
sequence of the gene, which may have a higher tendency to form unusual DNA
structures and cause errors during replication. Taking the RPGR exons 1–14 and
exon ORF15 together, mutations in this gene occur in about 70% of XLRP patients.
5.6.6 Centrosomal Protein 290 KDa (CEP290)
The CEP290 gene encodes a protein that is a critical component of the cilia. It was
first discovered through its association with the syndromic retinal dystrophy Joubert
syndrome (JBTS), which is comprised of the combination of cystic kidney disease
(or nephronophthisis), retinal dystrophy, cerebellar vermis aplasia (a malformation
of the cerebellum), and mental retardation. A related disorder to JBTS is Senior-
Loken syndrome (SLSN) which involves nephronophthisis and retinal dystrophy. In
a genome-wide linkage scan on a series of consanguineous families with SLSN,
JBTS, or nephronophthisis from various parts of the world (Sayer et al. 2006), link-
age was established to an interval of about 1 megabase on chromosome 12q21.
Screening of the various genes in the interval led to the identification of truncating
mutations in the CEP290 gene in families with SLSN and JBTS thus establishing
the CEP290 as the gene for these diseases. The CEP290 gene is made up of 55
exons encoding a transcript of about 8 kb, and a protein of 2479 amino acids,
expressed in the brain and placenta. Immunostaining of kidney cells with a specific
monoclonal antibody showed the localization of CEP290 protein within the centro-
somes. Further, analysis of mouse photoreceptor cells by immunogold labeling
showed that it was highly expressed in the connecting cilia. The discovery of
CEP290 as the JBTS gene was also made in a study of several consanguineous
families of Italian and Asian origins, with neurological, retinal, and renal manifesta-
tions. Genome-wide linkage analyses mapped the locus in these families to chromo-
some 12, to a region containing the CEP290 gene. This gene was considered an
appropriate candidate gene in these families since it encodes a centrosomal-ciliary
protein and members of this group of ciliary protein genes were known to be
involved in the pathogenesis of other related disorders (Valente et al. 2006).
Screening of the CEP290 gene revealed mutations comprising deletions and frame-
shifts in several families. The CEP290 protein is found to be expressed in the cen-
trioles in non-ciliated cells and in the cilia of ciliated cells and across multiple
tissues examined in the mouse, with high levels of expression in the cerebellum.
The association of CEP290 mutations with a non-syndromic retinal disease,
LCA, was discovered in a linkage study of a French Canadian family, in which sig-
nificant linkage was found to chromosome 12q21, coinciding with the CEP290
locus. Though the CEP290 was a strong candidate gene for the disease in the family,
screening of all the exons and splice junctions of CEP290 did not reveal any muta-
tions. The search for the mutation was then pursued by screening of the entire tran-
script for CEP290 by RT-PCR. This analysis revealed an aberrantly spliced
transcript, which involved insertion of a cryptic exon between exons 26 and 27 of
the gene, in the affected individuals. In addition, the authors also noted a small
amount of normal CEP290 transcript was observed, in the affected members.
Sequencing the genomic sequence surrounding the cryptic exon identified the
change in CEP290. A deep intronic change was found in intron 26, c.2991+1655A>G,
located about 1.5 kb downstream of the exon, which segregated with the disease in
the family (den Hollander et al. 2006). Screening of a series of over 70 additional
patients with LCA showed a few more homozygous individuals carrying this
5.7 Genes Involved in the Metabolism of Retinoids 125
mutation and several heterozygous carriers. The heterozygotes for the

c.2991+1655A>G intronic mutation were found to have a different mutation in the
second allele of CEP290 and were thus compound heterozygotes. Overall, accord-
ing to the available data, mutations in CEP290 account to one-fifth of the total
patients screened especially from Europe and North America, implying that it is one
of the most frequent causes of LCA in these populations. An interesting association
of the CEP290 genotype with phenotype is that the splice mutant associated with
LCA retains partial activity (since some amount of normal mRNA is produced)
which is presumably sufficient to maintain the functioning of the kidney and cere-
bellum. In the syndromic disorders on the other hand, mutations of CEP290 lead to
complete loss of function of the protein. All of these abovementioned diseases are
autosomal recessive in inheritance. Over 100 mutations are reported in the CEP290
gene with the majority of them being truncating mutations and one-fifth involving
splice site changes. Very few amino acid substitutions are reported. Apart from the
intronic mutation c.2991+1655A>G, another recurrent mutation is Lys1575Stop,
which is the second most common mutation in LCA patients, reported mainly in
families from France and Belgium (Coppieters et al. 2010).
5.7 Genes Involved in the Metabolism of Retinoids
5.7.1 C
ellular Retinaldehyde Binding Protein Gene (CRALBP,
RLBP1)
Associated disorders—autosomal recessive RP, retinitis punctata albescens,

Newfoundland rod-cone dystrophy, Bothnia dystrophy.
Various genes which function in the generation or transport of retinoids are asso-
ciated with RP and other retinal disease phenotypes. One gene in this pathway is
that for cellular retinaldehyde binding protein (CRALBP, RLBP1; OMIM 180090),
mutations in which are associated with various forms of retinal dystrophies. The
RLBP1 gene is expressed in the RPE and Muller cells of the retina and in the pineal
gland (Crabb et al. 1988). It binds to 11-cis retinol and thereby facilitates the con-
version of 11-cis retinol to 11-cis retinal by the enzyme 11-cis retinol dehydroge-
nase. 11-cis retinal then enters the photoreceptors to combine with opsin and form
a visual pigment, thus completing the cycle.
The cDNA for CRALBP was cloned from a bovine retinal cDNA expression
library using both mono- and polyclonal antibodies against the CRALBP protein.
The bovine cDNA sequences were used to isolate the human retinal cDNA clone for
CRALBP. Bovine and human sequences share about 90% identity at the cDNA and
protein levels. Association of a mutation in the RLBP1 gene with retinal disease was
first demonstrated in an Indian family with autosomal recessive RP by Maw and
coworkers (1997). Both affected sibs in the family were homozygous for a sequence
variation leading to change of Arg150Gln in CRALBP. The mutant protein was
impaired in its solubility and in binding to 11-cis retinal. A subsequent study associ-
ated mutations in the RLBP1 gene with a phenotype known as retinitis punctata
albescens (RPA). RPA essentially manifests with small yellow, punctate deposits at
the level of the RPE. It is often associated with features of RP such as night blind-
ness in the early stages, attenuated vessels, and reduced ERG responses (Morimura
et al. 1999). Three unrelated families, among over 300 probands with different reti-
nal dystrophies, were found to have mutations in RLBP1, suggesting it to be a rare
cause of retinal dystrophy. Significantly, affected members from all three families
with the RLBP1 mutations had the phenotype of RPA. The family described by
Maw and coworkers was originally categorized as having RP, but a feature worth
noting in this context is that they also reported the presence of intraretinal yellow
deposits in the affected members, thus raising the possibility that the phenotype in
this family was also RPA in association with RP. These studies together suggest that
RLBP1 mutations give rise to retinal dystrophy with RPA, a clinically separate
entity from retinitis pigmentosa.
Other phenotypes related to RPA are also associated with RLBP1 gene muta-
tions—a disorder known as Bothnia dystrophy belongs to this category (Burstedt
et al. 1999). It was so called as it was predominantly seen in families from Bothnia,
a province in Northern Sweden, adjacent to the Gulf of Bothnia. This disease was
mapped within the vicinity of the RLBP1 gene on chromosome 15, in seven families
from the same area of Sweden. Screening of the coding regions of the RLBP1 gene
revealed the Arg234Trp mutation in all families. Bothnia dystrophy manifests with
night blindness in early childhood; macular atrophy; clinical characteristics of RPA,
particularly the formation of deposits within the retina appearing as white dots; and
decreasing visual acuity with age, with legal blindness in the fourth decade (Burstedt
et al. 2001). Due to this variant form of RP occurring in a population from a geo-
graphically restricted region, several families with this phenotype have been identi-
fied with the same mutation of Arg234Trp in CRALBP, inherited in an autosomal
recessive mode. A striking feature of Bothnia dystrophy is therefore the genetic
homogeneity in the affected population from Sweden. They constitute the largest
number of families with retinal dystrophy carrying the same mutation, with over 65
unrelated individuals being homozygous for the mutation Arg234Trp. This is due to
a founder effect, in which the population in a specific region has descended from a
few ancestors (founders) who have migrated there after having branched off from a
larger population. This phenomenon limits genetic diversity within such popula-
tions, and an ancestral mutation could reach a fairly high frequency in the descen-
dants. The frequency of the RLBP1 mutation is estimated to be up to 1–2% in the
population in Bothnia district, and the prevalence of the dystrophy is 1 in 4500, both
being significantly higher than elsewhere in the world. In fact, specific pathogenic
variants in any given gene in Mendelian disorders generally have a very low fre-
quency (≪1%) in most populations in the absence of founder effects.
Yet another variation of the same clinical condition, discovered in a geographi-
cally isolated population, is the Newfoundland rod-cone dystrophy (NFRCD).
Inhabitants of Newfoundland, a province in eastern Canada, are migrants to this
region from different parts of Europe, settled there from about the seventeenth cen-
tury onward. Over 50% of the population is of English and Irish ancestry. Studies
on this population identified families with a form of retinal dystrophy, similar to
Bothnia dystrophy, but with some differences (Eichers et al. 2002). The affected
members showed an early onset of night blindness, loss of rod responses earlier than
cones, visual field loss with a small central island of vision, and blindness by the
fourth decade. Differences from RP include the attenuation of retinal vessels rela-
tively late in the disease course and lack of other features of RP such as pallor of
optic discs and pigmentary deposits in the retina. Two mutations predicted to disrupt
splicing were discovered in the RLBP1 gene in six different families with the same
phenotype, occurring in combinations of either the same mutation in both alleles or
a different mutation in each of the alleles, in either homo- or compound heterozy-
gous individuals, respectively. These mutations involved the last base of the third
exon and the second base in the intron, both at the exon-intron junction and thus
affecting the splice donor site.
Mutations in RLBP1 also occur in patients with yet another retinal disorder
known as fundus albipunctatus (FA; OMIM 136880). This entity is a form of sta-
tionary night blindness with round white dots in the mid-peripheral retina. Analysis
of four pedigrees with FA from Saudi Arabia revealed one family with a missense
change consisting of Arg150Gln (Katsanis et al. 2001).
However, apart from RLBP1, mutations in other genes are associated with the
phenotype of fundus albipunctatus as well. Two studies published at around the
same time reported mutations in the 11-cis retinol dehydrogenase (RDH5) gene in
association with FA. In a study from North America, mutations were detected in two
probands with FA out of a series of patients with retinal diseases (Yamamoto et al.
1999). The second study identified mutations in RDH5 in two families of European
descent with a diagnosis of FA; patients were homozygous or compound heterozy-
gous, respectively, for various missense changes in RDH5 (Gonzalez-Fernandez
et al. 1999).
Knockout of the Rlbp1 gene in mice helped to understand the role of CRALBP
in the retina. Mice with a knockout of both copies of the Rlbp1 gene (Rlbp1−/−) are
severely impaired in the regeneration of 11-cis retinal. The consequences of this
biochemical defect are a considerable delay in the recovery of the full reserve of
rhodopsin and a resulting delay in dark adaptation as compared with wild type mice.
Despite this, the retinas of knockout mice were normal in their morphology, and the
number of photoreceptor nuclei and the organization of the cellular layers showed
no significant changes, even after several months (Saari et al. 2001). These defects
suggest that the CRALBP affects the isomerization of all-trans to 11-cis retinol.
One way in which CRALBP might influence this reaction is by stimulating the con-
version of the all-trans to the 11-cis isomer by binding to the product of the reaction,
11-cis retinol. The binding is then coupled with oxidation of 11-cis retinol to 11-cis
retinal. Thus, sequestration and removal of the product might possibly stimulate the
kinetics of the reaction toward generating more 11-cis retinol.
5.7.2 Retinal Pigment Epithelial 65 KDa Protein (RPE65) Gene
Associated disorders—autosomal recessive RP, LCA.

5.7.2.1 Characterization of the RPE65 Gene

The RPE65 protein is a retinoid isomerase, mediating the conversion of all-trans to
11-cis retinol. It was isolated and cloned from bovine RPE and was suspected to be
an important protein in the metabolism of rod outer segments due to its abundant
expression in the RPE and the timing of its expression, which correlated with the
development of the rod outer segments (Hamel et al. 1993). The protein was iso-
lated by the use of a monoclonal antibody raised against human RPE cells and
identified as a novel 65 kDa protein, conserved in mammals, birds, and frogs. This
RPE-specific protein was most effectively solubilized in the presence of detergent
suggesting that it is associated with the RPE cell membranes. Cell fractionation by
differential solubilization and centrifugation demonstrated that the protein was pref-
erentially associated with the microsomal membrane fraction, where it is the major
protein. Morphologically well-differentiated RPE cells showed detectable expres-
sion of the RPE65 protein from postnatal day 4, about 1–2 days before the photore-
ceptors develop their outer segments, suggesting that the expression of this protein
may be coordinated with other developmental events in the retina.
The protein is associated with the microsomal membranes though it does not
have a transmembrane domain, and has an expression pattern which is highly spe-
cific for the RPE (Båvik et al. 1993). The sequencing of the protein together with the
availability of specific antibodies against RPE65 paved the way for cloning of the
cDNA. The protein from RPE extracts was first purified by immunological methods
including Western blot and immunoaffinity chromatography. Partial sequencing of
the protein thus obtained made it feasible to obtain the sequences of short peptides;
such peptide sequences were then used to design degenerate oligonucleotide probes
corresponding to the RPE65 cDNA. Therefore, one approach employed to obtain-
ing the cDNA was by screening of bovine RPE cDNA libraries with oligonucleotide
probes specific to RPE65 (Hamel et al. 1993). The RPE65 cDNA thus isolated con-
sisted of approximately 3 kilobases, encoding a protein of 533 amino acids. In
another approach, a cDNA expression library from bovine RPE cells made in the
bacteriophage lambda vector was screened with polyclonal affinity-purified anti-
bodies against the RPE65 protein in order to isolate its complete cDNA (Båvik et al.
1993). The RPE65 cDNA contains an open reading frame of 533 amino acids, and
the protein has an apparent molecular weight of 65 KDa on denaturing polyacryl-
amide gel electrophoresis. Shortly thereafter, the human RPE65 gene was mapped
to chromosome 1 and the transcript of about 3.0 kB in length isolated from the
human RPE, predicting a protein that was >98% identical to the bovine cDNA
sequence (Nicoletti et al. 1995). A peculiar feature of the RPE65 transcripts is that
they were not stable and appeared to be lost upon culture of the RPE cells.
RPE65 was suspected to have a role in the metabolism and/or transport of reti-
noids on the basis of its association with retinol-binding protein (RBP). It was iden-
tified in bovine RPE extracts as a component of a high molecular weight membrane
receptor complex for RBP (Båvik et al. 1993). A clue to its function came from the
observation that it was associated with 11-cis retinol dehydrogenase in RPE micro-
somes. The retinol dehydrogenase is a stereospecific enzyme belonging to the fam-
ily of short-chain alcohol dehydrogenases, and it converts 11-cis retinol to 11-cis
retinaldehyde (Simon et al. 1995). The function of RPE65 was further elucidated by
means of a mouse knockout model of the Rpe65 locus. These Rpe65−/− mice slowly
developed degeneration of the photoreceptors by about 15 weeks of age, with loss
of cell layers in the outer nuclear layer of the retina, corresponding to photoreceptor
cell loss, shortening of the rod outer segments, and disorganization of outer segment
discs (Redmond et al. 1998). These changes were accompanied by an attenuation of
rod electrical responses as assessed by electroretinography. Rhodopsin was found to
be completely absent in the Rpe65−/− mice, and the retinas accumulated all-trans
retinyl esters. Lipid droplets, possibly containing retinyl esters, were seen on elec-
tron microscopy of sections of the retinas from knockout mice. These observations
implied that the conversion of all-trans retinyl esters to 11-cis retinoids was blocked
in the Rpe65 knockout animals. The conclusion from these studies was that there
was an absence of isomerohydrolase activity in the Rpe65-deficient mice, thus
pointing to Rpe65 as the protein responsible for this activity.
5.7.2.2 Mutations of the RPE65 Gene

Since the RPE has an essential role in maintaining the photoreceptors in a viable and
functional state, it was apparent that a gene which is abundantly and specifically
expressed in the RPE is a probable candidate for retinal dystrophies. Hence, the
involvement of the RPE65 gene in the pathogenesis of retinal diseases was sus-
pected, even before its function was elucidated. The association of RPE65 mutations
with retinal diseases was established in three independent studies which reported
mutations in patients with LCA as well as RP. In one study, Gu and coworkers
screened Indian and German patients with ARRP for RPE65 mutations, by a combi-
nation of approaches including linkage mapping and direct screening of RPE65 (Gu
et al. 1997). They mapped the disease gene to chromosome 1p31, to a region which
harbored the RPE65 gene, in an Indian family. A pathogenic mutation in RPE65,
involving a non-conservative missense substitution, was found in the affected indi-
viduals in this family. Further analysis of 20 consanguineous Indian families identi-
fied a second family that mapped to the same locus, found to have a disease-associated
mutation of a splice junction in RPE65. Direct screening of the RPE65 gene in 86
probands of German origin revealed three families with splice site and deletion
mutations that segregated with the disease. Together, these mutations amount to a
frequency of about 5% among probands with childhood-onset severe retinal dystro-
phy in this study. A second study involved the screening of 12 probands with LCA
for mutations, and two affected individuals of one family were found to be com-
pound heterozygotes for two truncating mutations in the RPE65 gene (Marlhens
et al. 1997). A third study by Morimura and coworkers screened almost 150 cases
with various retinal dystrophies and found about 2% of cases with recessive RP and
16% of cases with LCA to have pathogenic changes in RPE65. Most of the changes
detected were missense substitutions (ten out of fourteen mutations), one splice site
mutation, and three termination mutations caused either by insertions or stop codons
(Morimura et al. 1998). Together, these studies established the role of RPE65 muta-
tions in the pathogenesis of early-onset RP and LCA. The RPE65 locus is designated
as LCA2, which is the second gene that was found to be associated with LCA.
Genetic testing of larger populations of LCA patients of various ethnicities

shows that mutations in RPE65 are associated with LCA in approximately 2–7% of
patients (Lotery et al. 2000; Dharmaraj et al. 2000b; Hanein et al. 2004; den
Hollander et al. 2007b). The methods used for detection of mutations, however,
varied in different studies, and the frequency of detected mutations is dependent on
the efficiency of the technique. The methods generally employed include screening
of the gene with single-strand conformation polymorphism (SSCP) and sequencing,
linkage analysis using microsatellite or SNP markers at the gene locus followed by
sequencing of the gene, or direct sequencing.
5.7.2.3 Animal Models of RPE65 Knockout

An important large animal model of retinal dystrophy is the Briard dog, which is a
naturally occurring breed of dogs with retinal blindness segregating through mem-
bers of this breed. The Briard dog is an ancient breed which is found across Europe
and the USA and is a highly inbred strain. Many dogs of this breed are affected with
retinal diseases ranging from congenital stationary night blindness (CSNB) to
severe daytime blindness. The affected dogs show diminished ERG responses;
changes in the retina and RPE, including the presence of a vacuolated RPE with
cytoplasmic inclusions; a shortening of photoreceptor outer segments; and eventual
loss of photoreceptors. The presence of extensive RPE changes in the blind Briard
dog led to an investigation of the RPE65 gene in these dogs, due to the RPE-specific
expression of this gene. The genetic defect in the Briard dogs was indeed found to
be a 4 bp deletion at positions 487–490 in the canine RPE65 cDNA, thus leading to
a frameshift in the protein. The deletion was reported in the Swedish Briard dog and
found to be the same in Briards originating in the USA and Canada as well, indicat-
ing a founder effect (Aguirre et al. 1998; Veske et al. 1999). The discovery of the
genetic basis of the blindness in the Briard dog as the RPE65 gene spurred efforts
toward development of gene therapy for patients with blindness due to RPE65
mutations. Thus, the Briard dog represents a naturally occurring canine model for
human LCA. Its advantage over a small-animal model such as rodent or guinea pig
is that being a large animal, it has an eye size that is more similar to humans than the
rodent models for RP. It was therefore regarded as a suitable model system to test
for the efficacy of gene replacement therapy with RPE65, with the results of the
gene therapy in the Briard dog being presumably more comparable to the conse-
quences of gene therapy in human patients, as compared with small mammals such
as rodents.
Gene delivery in the Briard dog was carried out by introducing the RPE65 gene
in an expression cassette consisting of the adeno-associated virus (AAV) vector with
the canine RPE65 cDNA. The effectiveness of this gene therapy system was first
tested in vitro, by introduction of the construct into cultured RPE cells explanted
from Rpe65−/− animals. Infection of the RPE65-negative cells with AAV-RPE65
demonstrated the expression of RPE65 protein in these cells. These experiments thus
established that the AAV vector system used could in fact transduce RPE cells and
also direct the expression of the RPE65 gene from the cassette. The AAV-RPE65
gene therapy construct was therefore suitable for in vivo replacement of the RPE65
gene in the Briard dog. Subretinal injection of the AAV-RPE65 gene therapy con-
struct into blind Briard dogs showed evidence of visual recovery, though to a partial
degree in treated animals as compared with untreated ones. ERG responses were
detectable in treated dogs, albeit to a fraction (16%) of the normal response. Pupillary
responses were similarly restored partially in treated dogs, with the magnitude of
response being intermediate to those of the normal and untreated animals. The
recovery of vision-guided behavior was also evident in these dogs, as assessed by
their responses to visual obstacles placed in their path (Acland et al. 2001). Further
studies showed that the recovery of vision in the Briard dogs treated with the RPE65
gene construct was durable and that treatment with a single injection sustained the
visual responses for several years. Thus, the gene replacement therapy in the blind
Briard dogs provided proof of concept for this mode of treatment in humans.
5.7.2.4 Human Gene Therapy Trials with RPE65 for LCA Patients

Human clinical trials for RPE65 gene replacement therapy for LCA were initiated
at various centers, using an adeno-associated virus 2 (AAV2) vector packaged with
the RPE65 gene. The initial trials were designed to test for safety and efficacy of the
gene delivery system in patients. One of the human gene therapy trials for LCA was
conducted by Maguire and coworkers and involved investigators at multiple centers.
They employed adeno-associated virus 2 (AAV2), a replication-defective AAV vec-
tor, having the human RPE65 cDNA under the control of the chicken β-actin pro-
moter in a clinical trial of (Maguire et al. 2008). The gene therapy vector was
administered by injection into the subretinal space of three patients with
LCA. Efficacy was estimated by various visual parameters assessed from preopera-
tive and postoperative visits at 1 month after treatment, of each patient. Objective
measures of vision included pupillary light reflexes and nystagmus. Subjective mea-
sures that were evaluated were visual acuity, visual fields, and mobility testing. The
investigators found no evidence of significant immune responses against the vector
or its dissemination into other parts of the body. The average of multiple measure-
ments of pupillary reflexes in treated and untreated eye of the patients indicated an
increased response in the treated eyes. Sensitivity to light was noted to be about
three times greater in the treated eyes compared to baseline. In addition, the
responses in these eyes surpassed those of the untreated eyes, even though the worse
eye of each patient was chosen for gene therapy. Importantly, all patients treated
showed improved visual acuity, which was perception of hand motions pretreat-
ment. After the gene therapy, the subjects were able to read letters on the first three-
four lines of the Snellen visual acuity chart. These observations thus showed the
safety of the gene therapy vector and also resulted in improvements in visual func-
tion that were noticeable within a month of treatment.
A second clinical trial of RPE65 gene replacement in LCA patients was carried out
by Bainbridge and coworkers; they also used the AAV2-RPE65 system for gene ther-
apy. The expression of the RPE65 gene in this case was driven by the human RPE65
promoter (Bainbridge et al. 2008). Subretinal injection of the gene therapy vector into
three patients aged 17–23 years again did not result in significant adverse events in
these cases. Visual acuity was observed to decrease below baseline values with
injection but returned to baseline values within 6 months after injection. No changes
were observed in any of the visual parameters including acuity, visual fields, or elec-
troretinographic responses. One of the three patients treated showed improvement of
retinal function by microperimetry and in vision-guided mobility in dim light.
The third trial for RPE65 gene delivery to LCA patients carried out contempora-
neously with the two clinical trials mentioned above also involved three young adult
subjects with LCA due to RPE65 mutations (Hauswirth et al. 2008). The construct
used was an AAV2 vector with the cytomegalovirus (CMV) immediate early
enhancer-chicken beta-actin promoter driving the expression of the human RPE65
cDNA. For each subject, data were collected on multiple visits for 6 months before
and 3 months after surgery. Visual parameters including full-field sensitivity and
visual acuity were tested, and retinal structure was assessed by optical coherence
tomography (OCT). In addition, ocular inflammation, presence of vector in body
fluids, and immune responses to AAV2 were monitored. There was no significant
inflammatory response in any of the subjects, and vector was not detectable in blood
even within a few days after treatment. Visual acuities fell below baseline (pretreat-
ment) levels immediately after surgery in all three patients, but recovered to base-
line within 3 months after. Full-field sensitivity increased to variable degrees in all
patients and showed a significant improvement above baseline for all eyes as a
group, which was most evident in low light conditions.
Overall, the results of the three trials for RPE65 gene therapy showed safety of
the AAV gene delivery system, but differences in the gene expression cassettes that
were used and variations in injection volumes in the three studies complicate any
correlations of vector dosage with visual outcome between the studies.
Further evaluation of the three cohorts of patients included in these initial trials
as well as cohorts recruited later provided a long follow-up of 3 years posttreatment
in the earlier sets of subjects, as well as a short follow-up in subsequently recruited
patients with LCA (Jacobson et al. 2012; Bainbridge et al. 2015). The observations
in these follow-up studies suggested significant increases in sensitivity of the full
visual field in response to both blue and red light, as well as in pupillary light
reflexes and mobility tasks, although there was no consistent improvement in visual
acuity as a result of treatment, in most cases. Any improvements in visual acuity that
were observed were below the magnitude that can be regarded as clinically signifi-
cant. The increase in visual sensitivity was dose-dependent, found in a larger num-
ber of patients given a high dose of the vector than those that received a low dose.
The sensitivity declined after a few months of treatment, though it remained better
than pretreatment (baseline) levels.
From the foregoing, the gene replacement therapy was found to be safe, and no
serious adverse events were detected in any of the trials. Visual outcomes were vari-
able, and there was evidence of improved visual function in some patients as
reported by different groups of investigators, though there were differences in the
treatment outcomes between patients within each study as well as between studies.
RPE65 gene therapy in human trials thus provided important leads for future thera-
pies in such patients and also paved the way for future trials in retinal blinding dis-
eases such as LCA.
5.8 Genes Encoding Transcription Factors 133
5.8 Genes Encoding Transcription Factors
5.8.1 N
uclear Receptor Subfamily 2 Group E Member 3
(NR2E3, PNR)
Associated disorders—autosomal dominant RP, autosomal recessive RP, enhanced

S-cone syndrome (ESCS), Goldmann-Favre syndrome (GFS).
5.8.1.1 Characterization of the NR2E3 Gene

The NR2E3 gene (also known as PNR—photoreceptor nuclear receptor) encodes a
photoreceptor-specific transcription factor, and its cDNA was isolated during a
search for human nuclear receptors related to an orphan nuclear receptor, known as
TLX. The TLX protein is the vertebrate homolog of the receptor encoded by Tailless
gene (Tll) in Drosophila. The TLX gene, also known as nuclear receptor subfamily
2 group E member 1 (NR2E1), is expressed in the retina, brain, and olfactory epithe-
lium and is important in eye and brain development as indicated by knockout mod-
els in mouse and Xenopus. Using degenerate PCR primers to screen Drosophila
embryonic mRNA sequences related to Tll/TLX, the cDNA clone for a closely
related nuclear receptor was isolated (Kobayashi et al. 1999). A search for ESTs
similar to the PNR sequence identified a closely related sequence in humans. The
EST sequence was used to isolate the cognate cDNA from human cell lines includ-
ing a retinoblastoma cell line. This led to the identification of a cDNA of about
1.9 kb with a sequence suggestive of a nuclear receptor with a ligand-binding
domain. Based on its photoreceptor-specific expression pattern, it was named as
photoreceptor nuclear factor (PNR). The PNR protein was found to bind to some of
the same target sequences as TLX, suggesting that the two receptors have an over-
lapping function.
5.8.1.2 Animal Model of PNR/NR2E3 Knockout

A naturally occurring mouse mutant known as the rd7 mouse, bearing a recessive
mutation, represents an animal model for knockdown of the NR2E3 locus. The rd7
mouse manifests with late-onset retinal degeneration, white spots all over the retina
at birth, and waves of disorganized photoreceptor layers with the appearance of
whorls and rosette-like structures visible upon histology of the retina. The wave-like
appearance disappeared at about 5 months of age. Photoreceptor degeneration was
progressive, and the thickness of the outer nuclear layer reduced to half by about
16 months. These changes were mirrored by changes in ERG responses. Both cone
and rod responses were almost normal at 1 month but severely diminished by
16 months of age. The rd7 locus was mapped to mouse chromosome 9, and the Pnr
cDNA was identified as one of a pool of transcripts that was expressed specifically
in the photoreceptors; it was mapped to the rd7 locus by linkage mapping (Akhmedov
et al. 2000). Expression of the Pnr transcript was found in the photoreceptors of the
mouse retina, but not in the brain, kidney, or other organs. The mouse Pnr sequence
was thus an ortholog of the human PNR cDNA, which was reported earlier by
Kobayashi and coworkers.
The evaluation of a postmortem human retina from an individual with ESCS

associated with a missense mutation in the NR2E3 gene, by histopathology and
immunofluorescence, provided an insight into the structural changes in the diseased
retina. The features of the ESCS retina paralleled those of the rd7 mouse in the
absence of rods, an excess of cones with a distortion in the ratio of S to L/M cones,
and an abnormal layering of the retina (Milam et al. 2002).
5.8.1.3 NR2E3 Mutations in Retinal Diseases

The pathogenic role of the NR2E3 gene in human retinal diseases was investigated in
a study of about 400 patients with various types of retinal dystrophy including a sub-
set of patients with enhanced S-cone syndrome (ESCS) (Haider et al. 2000). ESCS is
a form of autosomal recessive retinal dystrophy in which the sensitivities of rods as
well as long and medium wavelength cones are abnormally low, while the sensitivity
of short wavelength (S-cones) is considerably enhanced. Patients with ESCS tend to
have supernormal ERG responses to short wavelength (blue) light and subnormal
responses to medium and long wavelengths of (green and red) light. The extent of
retinal degeneration may vary from mild to severe, and abnormalities generally pres-
ent may include cystic changes in the fovea and vitreous degeneration. Other variable
features that have been noted are retinoschisis, pigmentary retinopathy, yellow flecks
in the retina, and macular scarring. The ratio of S-cone to L/M cone sensitivity is
severalfold higher than normal. A histological evaluation made on the eyeball of a
patient with ESCS, obtained for postmortem analysis, shed light on the changes in the
different layers of the retina due to this disease. There were disruptions in the photo-
receptor layers, and relative excess of S-cones as compared with other types was also
observed. In addition, the layering of the retina was abnormal, and cones and inner
retinal neurons were seen to be intermixed (Haider et al. 2000; Milam et al. 2002).
Mutations in the NR2E3 gene occur in a significant proportion of all probands
with ESCS as determined in the abovementioned study. While the mutations
detected included splice site mutations, deletion, and missense changes, the major-
ity of mutations were missense changes located in the DNA-binding and ligand-
binding domains of the protein.
NR2E3 mutations are also associated with recessive RP, as discovered in an endog-
amous population of Crypto-Jews1 from Portugal. A consanguineous family of
Crypto-Jews comprising three generations with nine affected individuals was evalu-
ated by linkage and homozygosity mapping to identify the disease locus. The shortest
region of homozygosity mapped in the affected individuals was a region of about
5 cM on chromosome 15. Physical mapping using YAC clones within the region of
homozygosity narrowed down the mapped region and refined the locus for the PNR
gene to the same interval. Screening of the coding regions of this gene identified a
missense mutation of Arg311Gln in the ligand-binding domain in NR2E3, present in
all affected members and absent in the control population (Gerber et al. 2000). The
Crypto-Jews is a term which refers to Jews in Spain and Portugal during the fifteenth to seven-
1@
teenth centuries when the Spanish Inquisition was at its peak. Persecution of Jews led to their
forming a group that practiced Judaism in secret and hence referred to as the “Crypto-Jews.”
impact of the mutant on the function of NR2E3 was determined by a two-hybrid assay
in mammalian cells. The assay employs two fusion constructs to detect interaction
between hybrid molecules made with two different proteins (A and B) or between
two-hybrid molecules of the same protein (A and A) of interest. The proteins to be
tested for interaction are made as fusion proteins. One protein (A) is fused with the
Gal4 DNA-binding domain, and the other (B) is fused with the transactivation domain
of VP16. VP16 is a powerful transcriptional activator that is normally involved in the
activation of genes in herpes simplex virus 1 (HSV1). It can activate genes when its
transactivation domain is fused to heterologous DNA-binding domains such as that of
the GAL4 protein. If the two proteins A and B interact with each other within the cell,
the use of the fusion proteins, i.e., GAL4 protein A and VP16 protein B, leads to the
DNA binding and transactivation domains of the GAL4 and VP16 proteins, respec-
tively, to be brought close together. One can thereby detect an increase in transcription
from promoter-reporter constructs bearing a Gal4-binding site. In the case of homo-
meric interactions, the same protein is present in the two fusion constructs, i.e., with
the Gal4 DNA-binding domain and the VP16 activation domain. Such an assay with
NR2E3 mutants, using two fusion constructs of the NR2E3 mutant, showed that
homodimerization is affected in the case of the missense mutant Arg311Gln.
The NR2E3 gene is also involved in the pathogenesis of other types of retinal
diseases including RP and pigmentary retinopathies. One of the phenotypes associ-
ated with NR2E3 mutations is autosomal dominant RP. A study of a Belgian family
of four generations with ADRP in 25 affected individuals mapped the disease to
chromosome 15q22-25, over a region of 7 cM (Coppieters et al. 2007). This region
included the NR2E3 gene, and a substitution of Gly56Arg in NR2E3 was identified
as the pathogenic change. Evaluation of a series of probands with various retinal
dystrophies revealed two more families with the same mutation in NR2E3. The
families with this mutation were of Belgian and French origin, and the affected
individuals across the three families shared a common haplotype of SNPs extending
over several kilobases on either side of the NR2E3 gene, suggesting a common ori-
gin for this mutation. The mutation appears to have a relatively high frequency in
the European population since it was found in three out of forty-seven families
tested, amounting to a frequency of about 6%. The clinical features of ADRP in the
family with the NR2E3 mutation showed some typical features of ADRP, but there
were differences as well. These included a relatively late onset of decline in cone
function, concentric rings of hyperautofluorescence in the retina, and limited intra-
retinal pigment deposits. The Gly56Arg mutation was also identified in another
ADRP family of four generations from Switzerland, in which the disease was
mapped to chromosome 15q (Escher et al. 2009). Interestingly, NR2E3 mutations
have been associated with two phenotypes, ESCS and RP, in different members of
the same family, an American family, in which individuals with ESCS were com-
pound heterozygotes for two mutations—Gly56Arg and Arg311Gln—and those
with RP were heterozygotes for Gly56Arg, with the other allele being wild type.
The inheritances of the two disorders were autosomal recessive for ESCS-affected
members (the proband and her sibling) and autosomal dominant in the case of
RP-affected individuals (son and grandchildren of the proband).
Mutations in NR2E3 are also found in patients with Goldmann-Favre syndrome

(GFS), a disease that again involves enhanced sensitivity to short wavelength (blue)
light, similar to ESCS, and in another form of recessive RP that manifests with
clumped pattern of pigmentation in the retina, known as clumped pigmentary retinal
degeneration (CPRD). Patients with CPRD have features of RP such as night blind-
ness and loss of ERG responses. There is an apparent overlap between these condi-
tions, as clumped pigments are also reported in ESCS and GFS. The same mutation
has been found in association with more than one phenotype, and certain mutations
such as an intronic change in the intron 1 splice acceptor, together with a missense
change of Arg311Gln, are recurrent, being identified in multiple families (Sharon
et al. 2003). Thus the different phenotypic entities that are associated with NR2E3
mutations may in fact overlap with each other, and on the other hand, the same
mutation in NR2E3 shows phenotypic heterogeneity.
5.8.2 Neural Retina Leucine Zipper (NRL)
5.8.2.1 Characterization of the NRL Gene

The neural retina leucine zipper (NRL) is a transcription factor that regulates the
expression of several photoreceptor-specific genes. The cDNA for NRL was iso-
lated and characterized by Swaroop and coworkers, from a subtracted cDNA library
made from the adult human retina. This process involved the hybridization of
single-stranded DNA inserts from a retinal cDNA library with transcripts made
from a cDNA library from lymphoblastoid cells. This led to elimination of constitu-
tively (ubiquitously) expressed mRNAs from the retinal library. The excess unbound
cDNA after hybridization essentially represented transcripts that were absent from
the lymphoblastoid cells and were therefore specific to the retina. The retina-specific
transcripts were of interest due to their potential role in maintaining or regulating
processes that are important in this tissue. The clone for the NRL cDNA was isolated
from this pool of retinal-enriched cDNAs and found to be highly specific to the
retina based on its expression pattern assessed by Northern blot analysis. It is
expressed in the retina and in retinoblastoma cell lines and absent in several other
tissues examined (Swaroop et al. 1992).
Analysis of the major NRL cDNA clone thus isolated predicted an open reading
frame of 237 amino acids, and in vitro translation of the cDNA yielded a polypep-
tide of about 30 kDa. The polypeptide sequence comprises a basic DNA-binding
motif and a periodic repeating sequence of leucine residues. The protein sequence
has homology with the fos/jun proteins although the protein itself did not show any
dimerization with fos or jun proteins when tested by in vitro experiments. The pro-
tein was hence named as neural retina leucine zipper (NRL) based on the high level
of expression of its transcript in the neural retina and the presence of leucine zipper
motifs in the peptide sequence. In situ hybridization of NRL cDNA probe to the
primate retina suggested that NRL is specifically expressed in the neural retina,
particularly in the photoreceptor layer. The NRL gene and its transcript are evolu-
tionarily conserved and are found in various mammals and other species tested. The
murine Nrl protein shows a high degree of sequence similarity with human NRL,
especially in the amino-terminal region and encodes a polypeptide of the same size
as the human, of 237 amino acids (Farjo et al. 1993).
5.8.2.2 Function and Interactions of NRL

The NRL gene has a key function in the retina, being a transcriptional regulator for
several photoreceptor-specific genes. The first photoreceptor-specific gene that
was shown to be regulated by NRL is the rhodopsin gene. This was demonstrated
by a study in cell lines involving transient expression assays. In addition, the NRL
protein binds to the RHO promoter as demonstrated by gel shift experiments from
retinal cell lines that expressed NRL. Specific binding to the NRL-response ele-
ment, defined as the consensus of TGC(N)6-7GCA, occurred with NRL protein
from retinal nuclear extract, thereby substantiating the role of NRL in the regula-
tion of transcription of retinal genes (Rehemtulla et al. 1996). Further, the regula-
tion of the Rhodopsin promoter by NRL of the rhodopsin promoter through its
binding site in the proximal part of the promoter is synergistic to its regulation by
CRX. This synergy in activation results from a direct interaction between the leu-
cine zipper of the NRL protein and the homeodomain of the CRX protein. A direct
interaction between the two proteins was demonstrated by a yeast two-hybrid assay
with an NRL-leucine zipper fusion protein with a heterologous DNA-binding
domain (i.e., that of the bacterial LexA protein). This was used as a “bait” to screen
a bovine retinal cDNA library to identify interacting proteins. This search yielded
the CRX cDNA as a major interacting partner for NRL (Mitton et al. 2000).
Moreover, the interaction with CRX requires the latter’s homeodomain, as demon-
strated in vitro by interaction assays using fusion proteins of glutathione
S-transferase (GST), i.e., GST-NRL or CRX fusion proteins, deletion of the CRX
homeodomain abolished binding to NRL, and mutations in CRX that are associ-
ated with retinal disease interfered with its binding to NRL.
5.8.2.3 Animal Model of Nrl Knockout

A mouse knockout model of the Nrl locus was created by homologous recombina-
tion to generate both homozygous (Nrl−/−) and heterozygous (Nr+/−) knockout mice.
This model helped elucidate the functions of Nrl in the retina and also underscore
the specific effects of its absence. The heterozygous knockout mice, Nr+/−, displayed
ERG responses similar to wild type mice, whereas the homozygous Nrl knockout
mice, Nrl−/−, showed complete loss of rod function with cone responses being unaf-
fected. In fact, the amplitude of cone responses was higher in the Nrl−/− than in
normal (wild type) mice, and the increase was found to be particularly mediated by
the S-cones. This pattern mimicked the phenotype of patients with ESCS, associ-
ated with mutations in the NR2E3 gene. Notably, despite the heightened cone
response, no Nr2e3 transcripts were detectable in these retinas suggesting that Nrl
was upstream of Nr2e3 in the timing of its expression and in the developmental
sequence (Mears et al. 2001). Thus knockout of Nrl is likely to lead to the loss of
expression of Nr2e3. Changes observed in the histology of the retinal outer nuclear
layer of the knockout mice were the presence of whorls and rosettes and eventual
thinning of the ONL. There was an absence of rhodopsin expression in the homozy-
gous knockout mice, whereas the heterozygous knockout animals were comparable
to wild type in this regard.
5.8.2.4 NRL Mutations in Retinal Diseases

The involvement of mutations in the NRL gene with human retinal disease was first
demonstrated in a large family with ADRP, in which the disease locus was mapped
to chromosome 14q11. The affected members of the family had a mutation leading
to a missense change of serine-50 to threonine in the NRL protein that cosegregated
completely with the disease in the pedigree and was absent in a normal control
population (Bessant et al. 1999). The NRL mutant protein was also functionally
abnormal, as demonstrated by its defective activation of the rhodopsin promoter in
cell lines, in comparison with the wild type protein. Few mutations have been iden-
tified in NRL, and they appear to mostly involve the amino acid residues 50 and 51,
suggesting that these are functionally critical. A screen of over 180 patients with
ADRP revealed mutations of Ser50Pro, Ser50Leu, and Pro51Thr in 4 unrelated
patients (DeAngelis et al. 2002).
The mouse models for Nrl and Nr2e3 knockout are similar to each other in that
they have enhanced number of S-cones and a reduction in the number of rods. Based
on the premise that analogous to these mouse models, humans with two mutant
alleles of the NRL gene would have similar phenotypes to those having two mutant
alleles of the NR2E3 gene, a large series of over 700 patients were investigated for
recessive NRL mutations. The patients included subgroups that had different retinal
diseases such as RP, cone-rod dystrophy, and LCA, although the majority of those
included had autosomal recessive RP or isolate RP. Through this screen, NRL muta-
tions were discovered in a family of two affected individuals with clumped pigmen-
tary retinal degeneration (CPRD). CPRD is a phenotype that is a form of autosomal
recessive RP, but distinct from it. It involves the presence of clumped pigmentary
deposits at the level of the RPE. Two mutations involving frameshift and truncation
of the protein at amino acid 75 (L75fs), and a second allele with Leu160Pro substi-
tution, were identified in a family of two affected siblings who were compound
heterozygotes for the two alleles (Nishiguchi et al. 2004). One of the affected mem-
bers in this family that was evaluated had night blindness from early childhood and
a slowly progressive loss of the peripheral visual field. The retina showed pigment
clumps at the periphery in the RPE layer, distinct from the bone spicule type of pig-
ments seen in RP, and rod and cone ERG responses were severely reduced. Through
an analysis of visual fields under light stimuli of different colors, a relative preserva-
tion of S-cone function was inferred in these patients. A similar disorder of enhanced
S-cone function and CPRD, in three Moroccan families with biallelic missense and
frameshift mutations in NRL, further substantiates the association of recessive NRL
mutations with this phenotype. The patients in these families presented with an
early-onset retinal dystrophy with nystagmus, hyperopia, and constriction of visual
fields (Littink et al. 2018).
5.9 Genes Involved in Various Other Pathways 139
5.9 Genes Involved in Various Other Pathways
5.9.1 RP25/Eyes Shut (EYS)
5.9.1.1 Mapping and Identification

The RP25 locus, situated at chromosome 6q14, was identified by a combination of
homozygosity screening and linkage mapping of Spanish families with ARRP using
microsatellite markers (Ruiz et al. 1998). The loci that were selected for evaluation
were those of the genes encoding receptors for gamma-aminobutyric acid (GABA), a
major inhibitory neurotransmitter in the central nervous system. The GABA receptors
are also widely expressed in the retina and therefore considered as candidate genes
for RP. Screening of 17 probands from inbred families for homozygosity at various
loci for GABA receptor genes across several chromosomes revealed that 2 probands
were homozygous for the selected markers at the GABA receptor gene cluster on
chromosome 6. Ruiz and coworkers extended their analyses to 12 non-consanguineous
families with ARRP, which were tested for linkage to markers in the same region on
chromosome 6q. Data from two additional families in this set were suggestive of link-
age of the disease to this locus. This was further confirmed by a combined multipoint
analysis of the four families (two consanguineous and two non-consanguineous).
Thus the disease locus was mapped to chromosome 6q. Screening of the GABA
receptor genes in this region, however, failed to identify pathogenic mutations.
The RP25 locus was independently mapped in other families with ARRP. A
study on a three-generation Pakistani family mapped the disease to the RP25 locus.
The family, consisting of 20 members in three generations, 12 of whom were
affected with RP, showed significant linkage to the RP25 locus on chromosome 6q.
The presence of recombinants at the locus enabled the refinement of the critical
interval for the disease to 2.4 cM from the previously mapped region of 16 cM. This
data effectively led to the exclusion of the GABA receptor genes as candidate genes
for the disease (Khaliq et al. 1999).
Yet another study that mapped the RP25 locus involved a homozygosity screen
using SNP arrays on a large number of Dutch families. One of the families had a
contiguous segment of homozygosity on chromosome 6q12-q11, shared by two
affected members (Collin et al. 2008). The region of homozygosity fully overlapped
with the already mapped RP25 locus. A search for genes within the mapped interval
found that there were five genes that were previously known, including the EGFL11
(EGF-like-domain, multiple 11) gene. This gene was found to be expressed in the
retina at high levels, and the encoded protein had several predicted EGF-like
domains. Screening of the annotated 11 exons of the EGFL11 gene in the family,
however, failed to detect any mutations. The authors went on to analyze adjacent
genomic regions for gene sequences. More sequences that were part of the same
gene were identified by exon prediction methods, which encoded putative proteins
with EGF-like domains, suggesting that these sequences may code for a longer
isoform of the EGFL11 transcript. By means of long-range RT-PCR on mRNA from
the human retina, followed by mapping of the cDNA thus obtained, Collins et al.
isolated a longer transcript of about 10.5 kilobases, representing a length of 2 Mb of
genomic DNA and comprising 44 exons. The predicted protein from the isolated
transcript has 3165 amino acids, with a signal peptide for secretion through a mem-
brane, several EGF-like domains, and five laminin AG-like domains. It was desig-
nated as the human eyes shut homolog (EYS) based on its similarity to the Drosophila
“eyes shut” or spacemaker protein. Apart from being related in sequence to the
predicted protein product of the RP25 gene, the Drosophila eyes shut protein is also
known to be important for photoreceptor development in insects (Box 5.4).
Box 5.4 The Drosophila Eyes Shut Gene

• The compound eye of Drosophila is made up of individual units known as
ommatidia. Each ommatidium has a cornea on its anterior side, a conical
crystalline structure through which the light rays converge onto the photo-
receptive structure known as the rhabdomere.
• Seven rhabdomeres make up an ommatidium. Each rhabdomere is sepa-
rated from the other by the inter-rhabdomeral space (IRS), which is recog-
nized to be critical for vision.
• The eyes shut gene in Drosophila was identified from mutant flies that
lacked an inter-rhabdomeral space. Notably, the rest of the structure of the
eye was normal in these flies, including the external morphology, retina
cell fates, and patterning. The defect was specific for the IRS.
• The eyes shut transcript in Drosophila is about 12 kb in length and encodes
a protein of 2176 amino acids.
• The Eys protein was localized exclusively in the IRS of adult fly retinas
and synthesized concomitantly with the formation of the IRS.
• Similar to the human EYS protein, the Drosophila Eys contains 14 EGF
(epidermal growth factor-like) domains, 4 LamG (laminin G-like) domains,
and a threonine/serine-rich region that is predicted to be highly
glycosylated.
Additional families with ARRP were tested for mutations in this gene, and the
families were selected for screening of the RP25 gene based on the affected mem-
bers being homozygous at consecutive SNPs in the same locus. Overall, three fami-
lies in this study were found to have pathogenic, truncating mutations in EYS, with
two of these families sharing the same mutation.
One of the further developments in this field was the mapping of more families
to the RP25 locus by Barragán and coworkers. Linkage of 3 families to the RP25
locus was followed by evaluation of 18 more families of Spanish origin, and 5 fami-
lies in this group were suggestive of linkage. Altogether, these studies suggested
that RP25 accounts for over 27% of ARRP in Spanish families. Through an analysis
of recombinants in these families, the RP25 locus was localized to 2.6 cM (Barragán
et al. 2008).
Another study that identified the RP25 gene involved the systematic screening of
the RP25 genomic interval on chromosome 6q, which included over 110 genes. The
detection of the RP25 gene was facilitated by findings that made it possible to nar-
row down the critical interval—one was the identification of a large deletion in one
family with ARRP that was originally linked to this locus, by the technique of com-
parative genomic hybridization (CGH). The deletion defined a smaller region of 100
kilobases based on the position and length of the deletion clone (Abd El-Aziz et al.
2008). The genomic region of the deletion contained several predicted genes, which
were analyzed for mutations in the RP25-linked Spanish families. The full RP25
gene was characterized by RT-PCR of RNA from various cell lines through isola-
tion and mapping of the complete cDNA, and the 43 exons were thus identified.
Based on the presence of multiple EGF-like domains and laminin domains, and the
similarity of the RP25 protein to the Drosophila spacemaker (Spam) protein
encoded by the eyes shut (eys) gene, the human gene was named as EYS, encoding
the protein SPAM. The Drosophila eyes shut gene was identified from the study of
mutant flies that had disorganized rhabdomeres (see box item). Mutations were
detectable in the EYS gene in multiple families with ARRP (see below).
5.9.1.2 EYS Mutations

Mutations in EYS are apparently among the most frequent among the various ARRP
loci, being detected in three families from the Netherlands as well as five Spanish
families as reported in the initial studies that mapped the EYS gene (Collin et al.
2008; Abd El-Aziz et al. 2008). All mutations reported were nonsense or frameshift
mutations. In fact, EYS probably represents a major gene for RP in the Spanish
population since it accounts for about 15% of cases as deduced from a study of 94
families (Barragán et al. 2010). The prevalence of EYS mutations in families with
ARRP is also found to be relatively high in various other populations of European
and Asian descent.
The EYS gene also appears to be a frequent cause of RP in Indian populations. A
report on families from Southern India detected mutations in 7–8% of patients with
RP based on whole exome sequencing of 14 families with ARRP and 100 sporadic
cases. Mutations were identified in ten families, and the affected individuals had
missense changes, stop mutations, and frameshifts. However, the majority of
patients in this study had missense mutations and were either homo- or heterozy-
gous for these alleles (Di et al. 2016).
5.9.1.3 EYS: Animal Models

Animal models of EYS knockdown are mainly confined to the zebrafish mutant
since rodents do not possess the eyes shut gene. A zebrafish model of EYS knock-
down was made by targeting the gene in single cell embryos through gene editing
using TALENS (transcription activator-like effector nucleases). The EYS−/− fish
displayed abnormalities in their retinal structure such as a decreased thickness of
the outer nuclear layer (ONL) with time and a concomitant increase in the numbers
of apoptotic cells in the retina. The outer segments of the photoreceptors shortened
progressively, and there was a decrease in the numbers of photoreceptor cells,
particularly the cones. A significant change noted was the mislocalization of outer
segment proteins, which began in very early stages of the embryo, when there were
no structural changes evident as yet (Lu et al. 2017).
5.9.2 Retinitis Pigmentosa 2 (RP2)
Associated disorders—X-linked RP.
5.9.2.1 Mapping and Isolation

The first locus to be mapped for X-linked RP was designated as RP2 and mapped by
linkage analysis to chromosome Xp11.3 in a set of 24 families with XLRP, onto the
proximal short arm of the X chromosome, using X-chromosome specific probes for
RFLPs (Bhattacharya et al. 1984). X-linked RP is among the most severe forms of
RP, with onset in the first decade in affected males, progressing to blindness in about
the third decade. Females are variably affected, and heterozygous carriers of muta-
tions may show the signs and symptoms of the disease.
However, various other studies mapped XLRP to essentially the same locus
using large affected families; the results of these different studies, though suggestive
of linkage of the disease with the same genetic marker (a microsatellite marker on
the X-chromosome DXS7), estimated the disease locus at somewhat different posi-
tions relative to DXS7 in different families, raising the idea that there might be two
different loci in this region that were close together. Thus while XLRP in some
families was mapped by linkage analysis as telomeric to DXS7 at Xp21 (Nussbaum
et al. 1985; Denton et al. 1988), in other families, the locus was mapped as centro-
meric to DXS7, at Xp11 (Friedrich et al. 1985). In order to clarify the position of the
XLRP gene, multipoint analysis with seven markers was carried out on a large num-
ber of British kindreds with XLRP by Wright and coworkers (1987); this data
mapped the XLRP locus to the proximal region of the short arm of the X chromo-
some, placing it outside the Xp21 region. These various linkage results on XLRP
were attributed to the existence of two distinct loci, thus implying genetic heteroge-
neity in XLRP. The possibility of two XLRP loci was again apparent from results of
multipoint linkage analysis and by the use of tests to determine linkage heterogene-
ity in a set of nine Australian families of British descent. In these families, the dis-
ease locus in some mapped proximal and, in others, distal to DXS7 (Chen et al.
1989). Taking data from various studies together, RP2 locus was mapped, broadly
within a 13 cM interval on chromosome Xp11. A subsequent analysis of two large
families by genotyping additional markers to refine this region led to the positioning
of the RP2 gene within a reduced interval of 5 cM on chromosome Xp11.3 to 11.23,
based on the presence of recombinants at these loci (Thiselton et al. 1996).
The mapped genomic region for RP2 was then isolated from cloned human
genomic DNA in yeast artificial chromosomes (YACs; Box 5.5) (Schwahn et al.
1998). YAC clones that contained the RP2 region were digested with restriction
enzymes, and the DNA fragments thus generated were ligated to short linkers.
These digested fragments were then amplified by PCR, to generate a set of ampli-
cons that represented the genomic region containing the RP2 locus in the YAC
clone. The amplicons could thus, in turn, be used to interrogate the genomic DNA
of RP patients to look for alterations, by subjecting the DNA to Southern blotting.
Any structural anomaly (such as an insertion, duplication, or deletion) in this region
of the patient’s genome would be detectable as an alteration in the pattern of
genomic DNA displayed in the blot. Such an experiment is known as YAC represen-
tational hybridization (YRH).
Box 5.5 Yeast Artificial Chromosome

The yeast artificial chromosome (YAC) is an artificial vector for cloning large
fragments of DNA.
DNA fragments ranging in size from 100 to 1000 kilobases can be cloned
into YAC vectors.
YACs contain a centromere, an autonomous replicating sequence (ARS,
which is the yeast the origin of replication), a pair of telomeres (normal ends
of eukaryotic chromosomes), selectable marker genes, and a restriction
enzyme site for cloning.
The YRH method was carried out in the manner outlined above, using genomic
DNA from a series of patients with RP. Southern blots of the genomic DNA digests
of patients were probed with amplicons from the YAC insert containing the RP2
genomic region; this process led to the detection of an aberration in the pattern of
genomic DNA fragments in one patient. The fragments containing the aberration
were cloned from the patient’s DNA and sequenced in order to characterize it.
Comparison of the pattern of patient DNA with normal control DNA suggested that
a genomic insertion of over 6 kilobases had occurred at this locus in the patient. The
insertion involved a LINE 1 (long interspersed nuclear elements), or L1 sequence.
The insertion of the L1 element in the RP2 region in the patient’s genome provided
a clue about the location of the RP2 gene; thus it was apparent that the L1 insertion
event was possibly disrupting the sequence of the RP2 gene and leading to retinitis
pigmentosa in this patient. This suggested that in order to identify the RP2 gene, one
had to study the genomic regions adjacent to the inserted L1 sequences. These
sequences were analyzed for the presence of exons by a method known as “exon
trapping” (see Box 5.6; Schwahn et al. 1998). Such an experiment indeed showed
the presence of exonic sequences with high homology to a mouse EST from a blas-
tocyst cDNA library. The identification of portions of exons facilitated the isolation
of the cDNA for RP2, by making specific probes complementary to the coding
sequences. The RP2 cDNA thus isolated was about 4 Kb in size, encoding a poly-
peptide of 350 amino acids. Comparison of the cDNA sequence with the genomic
sequence of the RP2 region revealed five exons and identified the position of the L1
insertion as the first intron of the gene. The RP2 transcript is widely expressed in
various fetal and adult tissues and in the retina. The encoded peptide shows homol-
ogy to a protein involved in beta-tubulin folding and represented a novel peptide,
whose identity and function were unknown before.
Box 5.6 Exon Trapping

This is a method used to identify exons in any piece of genomic DNA and
thereby isolate the gene contained therein.
The genomic DNA fragment of interest is cloned into a specific expression
plasmid, known as the “exon trapping vector,” which has splicing signals
incorporated in it. The vector contains a portion of a gene made up of two
exons flanking an intron in the middle. In addition, it contains promoter
sequences necessary for expression of mRNA in mammalian cells.
The DNA fragment to be tested is cloned into the intron in the exon trap-
ping vector, which contains a polylinker site for cloning. The recombinant
vector is then transfected into a eukaryotic cell line.
The mRNA generated from the gene sequences in the exon trapping vector
is a spliced transcript formed by exons in the vector and the insert after
removal of the introns. mRNA is isolated, reverse transcribed into cDNA
using a primer complementary to the bordering exon coded in the vector,
amplified, and sequenced.
Any sequences from the test genomic DNA that are present in the final
amplified cDNA represent “trapped” exons in the insert that were spliced to
the exons present in the vector.
5.9.2.2 Mutations in RP2

An evaluation of the sequence of the RP2 gene in a series of patients with X-linked
RP revealed mutations in several patients, and 10 out of 38 patients tested (about
18%) had mutations in RP2, thus providing more evidence for its involvement in the
disease. The frequency of RP2 mutations has been estimated at about 10–30%
among patients with XLRP, by different studies, and the mutations are mostly
frameshift or stop mutations. A screen for mutations in a subset of 33 XLRP fami-
lies with disease linked to the RP2 locus found mutations in 6 families (Hardcastle
et al. 1999). The only missense change found in this study was an arginine-118 to
histidine (Arg118His), incidentally also reported in an earlier study by Schwann
and coworkers. A similar mutational pattern involving a predominance of truncating
mutations and a frequency of about 10% of XLRP patients was also evident in other
studies—five out of fifty-one XLRP patients excluded for involvement of the RPGR
gene had mutations in the RP2 gene (Mears et al. 1999). Relative mutation frequen-
cies of RP2 and RPGR (RP3) were also established in a larger study of 127 families
with XLRP, as about 12% and 51%, respectively. In the case of RPGR, almost two-
thirds of mutations detected were located in the ORF15 region of the gene (Pelletier
et al. 2007). RP2 mutations account for isolate RP among males in about one-third
of cases. The disease is generally more severe in affected males, having an onset in
the first decade of life, with rapid progression of the disease.
5.9.2.3 Function of RP2

The RP2 protein is localized to the plasma membranes and is detectable in all layers
of the retina. By immunohistochemistry, it is found in the photoreceptor outer
5.10 Genetics of Usher Syndrome: A Form of Syndromic Retinitis Pigmentosa 145
segments, inner segments, outer nuclear and plexiform layers, inner nuclear layer,
inner plexiform layer, and the retinal ganglion cell layers. Staining with rod and
cone-specific markers along with anti-RP2 antibody showed that RP2 is specifically
detected in the plasma membrane of the photoreceptors, particularly on its intracel-
lular aspect, and not within the outer segments discs or in the nucleus or cytoplasm
of the rods and cones (Grayson et al. 2002). The localization of RP2 appears fairly
ubiquitous, being detected throughout the retina, by immunofluorescence studies. It
is expressed in the outer and inner nuclear layers, the inner plexiform layer, and
ganglion cell layer. Fractionation of retinal cells shows that RP2 is found to be in the
plasma membrane fraction (Holopainen et al. 2010). It is also detected in the con-
necting cilia of photoreceptors in sections of the mouse retina and in the ARPE19
cell line. RP2 appears to mediate vesicular transport of proteins. It is observed in
vesicles that move from the Golgi and toward the plasma membrane by live cell
imaging, suggesting a role for the protein in Golgi vesicle trafficking. Knockdown
of RP2 by siRNA demonstrated a reduction in the number of vesicles formed for
transport, as well as vesicle-mediated protein trafficking away from the Golgi
(Evans et al. 2010). High-resolution imaging and immunoelectron microscopy
revealed that it localized in the centriole and basal body of the connecting cilium.
RP2 may function as a GTPase activator for another important ciliary protein,
named as Arl3 (ADP-ribosylation factor (ARF)-like 3). The RP2 protein interacts
with the GTP-bound form of Arl3 and is thus possibly an effector protein for Arl3.
The latter is a microtubule-associated protein and is a member of the Ras superfam-
ily of small G proteins, localized in the connecting cilium of the photoreceptors.
RP2 in concert with other cofactors is found to stimulate the GTPase activity of
native tubulin and is thus proposed to be required for assembly of tubulin heterodi-
mers (Bartolini et al. 2002).
5.10 G
enetics of Usher Syndrome: A Form of Syndromic
Retinitis Pigmentosa
The Usher syndrome is a disorder involving retinitis pigmentosa (RP) and sensori-
neural deafness (resulting from abnormalities in the inner ear). Vision loss in Usher
syndrome is progressive with onset from childhood. Usher syndrome is classified
into several types most of which show autosomal recessive inheritance. It is classi-
fied on the basis of the severity of the deafness and vestibular dysfunction. Usher
type 1 is more severe, and patients experience profound hearing loss and absence of
vestibular function, while those affected with Usher syndrome type 2 have congeni-
tal deafness which is moderate to severe and normal vestibular function. Usher syn-
drome type 3 is more variable in its onset, and manifestations and vestibular
functions can range from normal to absent. The age at diagnosis for Usher type 3 is
in the second-third decades. There is, however, a large overlap in the clinical signs
and symptoms between the different subtypes, and these characterizations may not
be useful to diagnose individual cases. Usher types 1 and 2 are generally more com-
mon, accounting for about 30–40% and 50–65% of cases, respectively, among
patients from Europe and North America. Usher syndrome type 3 is reported to be
more common in Finland, occurring in about 40% of patients, due to a founder

effect (Petit 2001), and among Ashkenazi Jews.
Genetic heterogeneity of each of the two types further subdivides Usher syn-
drome type 1 into types 1A, 1B, and 1C and type 2 into 2A, 2C, and 2D. The sub-
types of Usher syndromes 1 and 2 are not clinically distinguishable within
themselves.
5.10.1 Usher Syndrome 1
The genetic locus for Usher syndrome 1 was mapped in families from Western
France to chromosome 14q. Some of the families with Usher type 1 did not map to
the same locus suggesting genetic heterogeneity for this disorder (Kaplan et al.
1992). Linkage to two more loci was established at chromosomes 11q (Kimberling
et al. 1992) and 11p (corresponding to USH1B and USH1C, respectively; Smith
et al. 1992). Mutations at the USH1B locus are the most common among these. A
re-evaluation of families mapped to the USH1A locus on chromosome 14 failed to
confirm the validity of this locus, thus negating earlier conclusions on this (Gerber
et al. 2006). It is now held that an USH1A locus does not exist. Till date, seven
genetic loci for Usher syndrome 1 (USH1B–H) have been mapped on chromosomes
(see Table 5.2). The various Usher proteins encoded by these genes make up an
interactome, forming an extensive network of interactions between themselves and
with other proteins that have been associated with retinal ciliopathies. Among these
proteins, the USH1C protein harmonin and the USH1G protein known as SANS
(short for scaffold protein containing ankyrin repeats and SAM domain) are regarded
as “scaffold” proteins since they interact directly with all other proteins. Details of
various USH1 genes are depicted in the table.
Table 5.2 Usher Syndrome 1 (USH1) loci and genes

Locus Chromosome Gene Protein Function
USH1B 11q13 MYO7A Myosin 7A Actin-based motor protein
USH1C 11p15 PDZ73 PDZ domain- Scaffold protein for network
containing of various Usher proteins
protein, harmonin
USH1D 10q22 CDH23 Cadherin 23 Part of protein complex that
connects the stereocilia in
auditory hair cells
USH1E 21q21 Not known – –
USH1F 10q21 PCDH15 Protocadherin 23 Interacts with CDH23 to
form filaments that connect
stereocilia
USH1G 17q25 SANS Scaffold protein
USH1H 15q22-23 Not known – –
Shown above are details of loci, genes, and proteins associated with various subtypes of Usher
syndrome type 1
5.10.1.1 Usher 1B
The USH1B locus on chromosome 11 was mapped through a genome-wide linkage
analysis of microsatellite markers in 27 families with Usher syndrome 1. All affected
individuals were noted to have profound hearing loss, absent vestibular reflexes, and
retinitis pigmentosa. Significant linkage was obtained in a region of chromosome
11q in all families tested with no evidence of genetic heterogeneity (Kimberling
et al. 1992, Genomics 14: 968–94).
Mutations in the myosin 7A (MYO7A) gene, at the USH1B locus, are the most
common cause of Usher syndrome. The clue to the identification of MYO7A as the
USH1B gene came from a mouse mutant known as shaker 1 (sh1). Shaker 1 is a
deafness mutant, and the phenotype in these mice was mapped to chromosome 7, a
region that is orthologous to the human Usher 1B genomic locus on chromosome
11. Shaker 1 deafness mutant mice display a similar pathology to Usher syndrome
in humans, though without any apparent retinopathy. The gene responsible for the
phenotype in this mutant mouse strain was identified as one which encodes an
unconventional myosin. This discovery suggested that the human ortholog of the
mouse myosin gene is a candidate for Usher syndrome 1B in humans (Weil et al.
1995, Nature 374: 60–61).
Cloning and Identification of MYO7A

The USH1B locus was mapped by linkage analysis and homozygosity mapping of a
large family to a 6 cM interval of chromosome 11q13 (Guilford et al. 1994). The
genomic fragment containing this mapped USH1B interval was isolated in a yeast
artificial chromosome (YAC) clone and found to have sequences that are comple-
mentary to the mouse myosin gene (Weil et al. 1995). Sequencing of the same
region and the adjacent genomic regions in the YAC clone, and its cDNA from a
retinal cDNA library, led to identification of exons of the human myosin 7A
(MYO7A) gene, thus providing the complete coding sequence, which consists of 50
exons. Various mutations were discovered in a few families with Usher syndrome in
which the disease was mapped to the USH1B locus.
The mRNA for MYO7A is expressed in the ear, testes, and retina. The inner ear
contains inner and outer hair cells, equipped with mechanosensory organelles called
hair bundles that are required for audition. They are made up of stereocilia, which
are connected together by links. Expression of MYO7A protein is found in the hair
cells of the cochlea, particularly within the stereocilia of these cells (Hasson et al.
1995). The MYO7A and other Usher proteins are present as part of a network of
proteins associated with the links, required for the organization of the hair bundles
in the inner ear, anchoring them to the actin filaments in the bundles (Bonnet and
El-Amraoui 2012). Essentially MYO7A functions as an actin-based molecular
motor, required for the transport of organelles such as phagosomes and melano-
somes in the RPE; it also functions in translocating proteins including transducin
and opsin proteins between outer and inner segments of the photoreceptors. In the
retina, MYO7A protein is present in the apical part of the RPE cells but not in the
inner retinal neurons. Its absence results in a mislocalization of the melanosomes in
the RPE, a slowed degradation of phagosomes, and a reduced amount of RPE65
protein, possibly as a result of impaired translocation (Lopes and Williams 2015).

Apart from the RPE, it was shown by immunoelectron microscopy to be expressed
in the connecting cilia of photoreceptor cells (Liu et al. 1997a). These expression
patterns together with the pathogenic mutations found in Usher syndrome patients
suggest that myosin 7A is essential for the normal structure and/or functioning of
the photoreceptors as well as the sensory cells of the inner ear.
Mutations in MYO7A
The involvement of MYO7A and its mutational spectrum in Usher syndrome 1 have
been substantiated by several studies, although the frequency of MYO7A mutations
cannot be deduced from these studies due to the limitations of the screening meth-
ods used. The MYO7A gene exhibits a great deal of mutational heterogeneity, and
mutations encompassing different types of changes including missense, nonsense,
deletion, and splice mutations are found to be distributed throughout the coding
region. A partial screening of the first 14 exons of the MYO7A gene in 189 probands
with Usher syndrome type 1, including familial and simplex cases with a diagnosis
of Usher syndrome type I, revealed mutations in MYO7A in 20 probands by means
of heteroduplex analysis and sequencing (Weston et al. 1996). Many of the patients
were heterozygotes for the mutations, with missense mutations being the most fre-
quent type. Another screen for mutations using the method of single-strand confor-
mation polymorphism (SSCP) from Israel including several families of mixed
ethnicity revealed mutations in 15 families out of 28 screened (Adato et al. 1997).
Mutations in MYO7A were also reported in patients with recessive non-syndromic
deafness, including two Chinese families with non-syndromic deafness (Liu et al.
1997b) and a family with hearing loss and vestibular dysfunction (phenotype
DFNB2, MIM no. 600060) that was mapped to the same locus on chromosome 11
(Weil et al. 1997).
Animal Models of Myo7a Knockout

Mouse models that are defective at the Ush1b locus have been useful in studying the
effects of the mutations on the retina and on the subcellular localization of the
Myo7A protein. The study of three shaker 1 (sh1) mouse lines having missense and
stop mutant alleles of Myo7a showed that there was no change in photoreceptor cell
number at 6 months of age as compared with normal controls and the ultrastructure
of the photoreceptors was normal in appearance by electron microscopy (Liu et al.
1999). In addition, the localization of mutant Myo7a protein in the shaker 1 mice
was found to be similar to the wild type protein in normal mice, being located in the
connecting cilium of the photoreceptor, suggesting that Myo7a does not depend on
its own motor functions for its targeting. However, homozygous mutant mice
showed mislocalizaton of opsin with abnormal amounts of the protein being detected
in the connecting cilia of photoreceptors. This suggests that proper trafficking of
opsin to the outer segments depends on Myo7a. Exposure to normal light conditions
did not appear to cause photoreceptor degeneration in shaker1 mice even at
15 months of age. Despite the absence of gross structural defects in these mice, the
photoreceptors of mutant mice were more sensitive to exposures of light and
susceptible to light-induced damage as compared with wild type mice, since they
had functional defects. Subnormal functioning of photoreceptors in these Myo7a
mutant mice is reflected in the decreased amplitudes of the electroretinographic
(ERG) responses, as compared with the controls. Diminished ERG responses were
observed from postnatal day 20 up to several months, essentially showing no change
with age (Libby and Steel 2001). The relative preservation of retinal structure in
Myo7a mutant mice, as well as the functional decline measured by ERG, was also
confirmed in a subsequent study (Colella et al. 2013).
Gene replacement was effective in overcoming the defect in transport in the
shaker 1 mice caused by a mutant Myo7A gene. Subretinal delivery of an adeno-
associated viral vector system (AAV2) carrying a human MYO7A cDNA under the
control of the CMV promoter in the shaker1 (sh1) mice led to the correction of the
defect in transport of opsin as compared with the control untreated sh1 mutant mice.
Similarly, the transport of melanosomes into the RPE microvilli was restored after
gene replacement with the MYO7A construct.
Clinical trials of gene therapy with the human MYO7A gene in a lentivirus vector
have been initiated for patients with Usher syndrome (this trial is registered on the
Clinical Trials Database maintained by the NIH with Clinical Trial identifier
NCT01505062).
5.10.1.2 Usher 1C
Mapping and Identification of the USH1C Locus

Studies on a group of families with Usher syndrome 1 led to the mapping of the
locus for Usher syndrome type 1C to chromosome 11p. Analysis of 8 French-
Acadian families and 11 British families with Usher syndrome type 1 suggested that
all families mapped to the locus on chromosome 11, without any genetic heteroge-
neity among the families tested (Smith et al. 1992). This locus was refined further
to an interval of 2–3 cM at 11p15-p14, by genotyping of an extended set of 27 fami-
lies from the French-Acadian population (Keats et al. 1994). The Acadians are
French settlers in the Canadian provinces in Eastern Canada, who migrated there
during the seventeenth century and formed a genetically isolated population that
essentially consisted of the descendants of a small group of founders (ancestors).
The USH1C gene was also mapped using a different route, based on the study of
families with a contiguous gene deletion syndrome involving the phenotypes of
hyperinsulinism, profound congenital deafness, enteropathy, and renal dysfunction
(Bitner-Glindzicz et al. 2000). Among these disorders, hyperinsulinism has been
associated with loss of function of potassium channels, and mutations in two genes,
ABCC8 and KCNJ11, both encoding potassium channels, occur in patients with
hyperinsulinism. Therefore, analysis of the ABCC8 gene was undertaken in these
families. PCR amplification of the various exons showed that a large part of the
gene, toward its 5′ end, was indeed deleted. Further mapping of the deletion region
on chromosome 11 by fluorescence in situ hybridization (FISH) of metaphase chro-
mosomes from the affected families, followed by amplification using primers for
sequence-tagged sites in the region, defined the deletion as one that covered a
genomic sequence of 120 kilobases on chromosome 11p. Characterization of the

ORFs located in the deleted region by Blast analysis led to the identification of a
novel gene, which was named as the Usher 1C (USH1C) gene. This gene was in fact
known from earlier studies to be associated with diseases of the gut and found to be
highly expressed in the gut epithelium. The abovementioned families with the syn-
drome had a deletion covering most of the gene. Further, point mutations inactivat-
ing the USH1C gene were detected in other families with Usher syndrome 1. The
mutations consisted of insertions or deletions in various families. In an Acadian
family with Usher 1C, a substitution of c.216G-to-A was found to result in a dele-
tion in the cDNA, possibly due to the creation of a new splice site (Bitner-Glindzicz
et al. 2000).
The USH1C gene was also identified in another study, and its cDNA isolated
from a subtracted cDNA library that was enriched for transcripts expressed in the
sensory cells of the cochlea (Verpy et al. 2000). The transcript thus obtained showed
a high degree of similarity to human cDNAs encoding PDZ-domain proteins. Since
these domains are known to organize multiprotein complexes, especially in synaptic
junctions and microvilli, the isolated gene was considered as a potential candidate
gene for Usher syndrome and named as harmonin (based on its presumed ability to
organize multiprotein complexes). It was subsequently mapped to the USH1C locus
on chromosome 11p14. Determination of the coding sequences and exon-intron
structure of the human harmonin gene was achieved by comparison of mouse and
human harmonin cDNA sequences with the genomic sequence from the same region
on human chromosome 11.
Screening of the gene in patients with Usher type 1 showed pathogenic muta-
tions leading to splice site changes, in separate families from Lebanon. A remark-
able finding was that 11 Acadian families were evaluated and all found to have an
expansion of a repeat sequence of 45 bp (a VNTR sequence) in intron 5 of the
USH1C gene. While normal control individuals had five to six repeats, patients with
Usher syndrome 1 showed nine repeats of the sequence (Verpy et al. 2000). The
predominance of this variant the Acadian population has been confirmed in various
studies. The VNTR expansion is in linkage disequilibrium with another point muta-
tion involving a silent change c.216G>A (Bitner-Glindzicz et al. 2000; Savas et al.
2002). The c.216G>A change is shown to result in aberrant splicing of the USH1C
gene, leading to a deletion of coding sequences.
The c.216G>A allele also appears highly prevalent in French Canadian families
from Quebec affected with Usher syndrome 1, amounting to a frequency of about
40%, and is in complete linkage disequilibrium with an expansion of the VNTR
sequence referred to above. All patients from Quebec had an increased number of
repeats as compared with normal individuals (Ebermann et al. 2007a).
Expression of the USH1C Gene

The transcript and protein for USH1C are highly expressed in fetal tissues including
eye, ear, gut, kidney, and brain. Alternative splicing of the transcript made up of 28
exons generates several different transcribed products of harmonin; 8 transcripts are
detectable in the inner ear, predicting separate isoforms of the protein. The protein
contains three major domains—PDZ domains, coiled-coil domains, and proline,

serine, threonine-rich (PST) domain. The various isoforms differ in the domain
structure, that is, the presence of one or more PDZ domains and coiled-coil domains
and a PST domain (Verpy et al. 2000). These domains participate in a variety of
protein-protein interactions and together confer the harmonin protein the properties
of a scaffold.
The harmonin mRNA consists of multiple alternately spliced transcripts. Of
these, some transcripts include exons specific to the inner ear and are comprised of
spliced products that are different from the constitutively expressed transcripts that
are found in the rest of the tissues. In the ear, harmonin is present in the sensory hair
cells of the organ of Corti and in the vestibular organ. The distribution of mutations
likewise shows the same correlation with phenotype, since mutations associated
with Usher syndrome 1C are all located in the constitutively expressed exons, while
the mutations which are associated with non-syndromic deafness occur in alterna-
tively spliced transcripts specific to the inner ear (Reiners et al. 2006). Harmonin
expression in the retina occurs prominently in the inner and outer segments of pho-
toreceptors, as well as in the ribbon synapses of the outer plexiform layer. It has
been shown by immunoelectron microscopy that it is expressed in the synapses of
photoreceptors as well as of horizontal and bipolar cells (Williams et al. 2009).
Animal Models of Usher Syndrome 1C

Two spontaneously occurring mouse mutants of the Ush1c locus have been charac-
terized. The underlying gene alterations in the two mutant strains consist of a large
intragenic deletion of over 12 kb that eliminates most of the gene (dfcr) and a single
base deletion leading to frameshift and premature termination (dfcr-2J) (Johnson
et al. 2003). The two strains showed congenital deafness and vestibular dysfunction
which manifested with circling and head tossing behavior. The retina showed no
abnormalities in its structure in either of the strains, except for a slight degree of
retinal degeneration in the peripheral retina in some of the mice. The ERG responses
of these mice were normal. Thus, in contrast to the disease in human Usher syn-
drome type 1C, deletion of the constitutively expressed exons in the dfcr mutant
mice does not lead to an appreciable retinal disease phenotype.
The clinical entity comprising Usher syndrome 2 is genetically heterogeneous, and

various loci that are mapped in different families with a diagnosis of Usher syn-
drome type 2 are designated as USH2A, USH2B, USH2C, and USH2D. These sub-
types as mentioned earlier are based on genetic heterogeneity, though they are not
clinically distinct (Table 5.3).
Among these, the USH2B locus, which was originally mapped to chromosome
3p23-24 in a Tunisian family, was subsequently eliminated in the list of Usher syn-
drome loci based on a further analysis of the same family after more members were
included. A genome-wide linkage analysis of the family, which consisted of some
Table 5.3 Loci for Usher syndrome types 2 and 3

Locus Chromosome Gene Protein Function
USH2A 1q41 USH2A Usherin Component of the
extracellular basement
membrane
USH2C 5q14-21 USH2C VLGR1 (very large G Part of the periciliary
protein-coupled membrane complex of
receptor 1)/ADGRV1 photoreceptors
(adhesion G protein-
coupled receptor VI)/
GPR98
USH2D 9q32 USH2D Whirlin Part of a multiprotein
complex of three USH2
proteins in the
photoreceptors. Exact
function not clear
USH3A 3q25 USH3A Clarin Not known. Thought to
participate in sensory
synapses
USH3B 20q – – –
Details of loci for Usher syndrome types 2 and 3, and the corresponding proteins if known, are
listed above. Note: The USH2B locus has been withdrawn
members with non-syndromic RP and others with Usher syndrome phenotypes,

failed to confirm linkage to the USH2B locus on chromosome 3. The re-analysis
took into account the affection status of only members having Usher syndrome
(leaving out the RP-affected individuals) in the family. This strategy led to the map-
ping of Usher syndrome in this family to the (already known) USH2C locus on
chromosome 5. Thus the USH2B locus has been withdrawn. The family members
with non-syndromic RP were found to have a mutation in a different gene alto-
gether, which segregated with the RP phenotype (Hmani-Aifa et al. 2009).
5.10.2.1 Usher Syndrome 2A Gene (USH2A)

Associated phenotypes—autosomal recessive RP, Usher syndrome type 2.
Mapping and Identification of the USH2A Gene

Mapping of the gene for Usher syndrome type 2 by linkage analysis of 22 families
showed significant linkage at chromosome 1q thus identifying the USH2A locus
(Lewis et al. 1990). The absence of linkage with the same markers in families with
Usher syndrome type 1 confirmed that Usher syndrome 2 is a distinct genetic entity.
The USH2A locus was also mapped to the same locus by a larger study of over 70
families with Usher type 2 predominantly of Caucasian origin and was refined to a
region of about 2 centimorgans on chromosome 1q41. Out of all families, it was
found that about six families did not show linkage to this region, thereby indicating
that there is further genetic heterogeneity in Usher syndrome type 2 (Kimberling
et al. 1995). The analysis of the mapped region in more families with Usher syn-
drome type 2 enabled the disease interval to be narrowed down to about 300 kilo-
bases. The 300 kb region of the genome that was defined as the critical interval for
Usher syndrome 2A was cloned into a bacterial artificial chromosome (BAC) vec-
tor. Analysis of the cloned sequences in the BAC clone showed that it contained an
ORF having similarity to epidermal growth factor motifs in the laminin family of
proteins. Using probes based on the sequences of the ORF, a cDNA for USH2A of
about 4.7 kilobases was isolated from a retinal cDNA library. The USH2A cDNA
thus obtained encoded a putative protein of 1551 amino acids with a predicted
molecular mass of 171 kilodaltons (Eudy et al. 1998). The exon-intron structure of
the gene as determined in this initial study showed the presence of 21 exons. A par-
tial screening of the coding regions of the USH2A gene thus obtained resulted in the
identification of three deletion mutations—c.2314delG, c.2913delG, and
4353-54delCT—making up about 15% of alleles among 96 patients screened. Of
these, the mutation of c.2314delG was found to be most frequent, with 10% of cases
being homozygotes and 13% being heterozygotes for this allele.
Mutations in USH2A
The high frequency of the same c.2314delG deletion mutation mentioned above
was confirmed in subsequent analyses of Usher syndrome 2 patients, mostly of
American and European origin. It constitutes about 16% of all alleles in the patients
screened. However, a more comprehensive screen for mutations in all the 21 known
exons of the USH2A gene in 57 probands with Usher syndrome 2 detected about
50% of possible mutant alleles in these patients (Weston et al. 2000). Other studies
that tested the USH2A gene also detected mutations in a similar proportion of
patients, and only one mutant allele was identified in a significant number of cases.
Thus, the analyses of 21 exons of the USH2A gene failed to explain the genetic
basis for Usher syndrome 2 in about half of all the patients with Usher syndrome 2.
The apparent absence of mutations in these patients suggested that there might be
additional coding regions in the USH2A gene apart from the 21 exons already known
and may be the sites of the “missing mutations.” With the aid of exon prediction
programs, van Wijk and coworkers identified putative exonic sequences in the
USH2A gene downstream of the 21st exon. The predicted exonic sequences were
indeed present in the USH2A cDNA isolated by reverse transcription and PCR from
a retinoblastoma cell line, as well as human cochlea and retina (van Wijk et al. 2004).
The amplified cDNA fragments thus isolated were sequenced and mapped to get the
complete sequence of USH2A transcript, which consisted of 51 additional exons.
The length of the protein encoded by the longest open reading frame (ORF) is 5303
residues with a predicted mass of 576 kDa. The “missing mutations” were detected
upon screening of the newly found exons in several additional families with Usher
syndrome type 2. Together with the mutations in the previously known 21 exons, the
ones in the “novel” exons explained the pathogenic basis of the disease in the major-
ity of families. About 75% of families of over 100 families tested were found to have
mutations in the novel exons 22–73. Among the families positive for mutations, both
mutant alleles of the USH2A gene were found in almost 90% of patients (Dreyer
et al. 2008). Analysis of the entire gene thus filled in the gaps in the genetic basis of
Usher syndrome type 2 in the families that were mapped to the same locus and led
to a complete characterization of the genetic basis for Usher syndrome type 2A.
In addition to Usher syndrome, mutations in the USH2A gene are also associated
to non-syndromic RP, as discovered in a survey of 224 patients with autosomal
recessive RP. A particular missense mutation of cysteine-594 to phenylalanine
(Cys594Phe) in the gene was found in several families, accounting to 4.5% of the
patient population screened (Rivolta et al. 2000). The segregation of this allele with
RP in all these families and its absence in patients with Usher syndrome suggest that
it is specific to non-syndromic RP. Taken together, mutations in the USH2A gene
may be a relatively frequent cause of ARRP, particularly in populations from North
America.
Functions of the USH2A Gene

As mentioned in the previous section, the identification of several additional exons
in the USH2A gene predicted a much larger open reading frame than the coding
sequence that was known at the time it was first discovered. Thus, the gene has
two transcript variants. The short USH2A transcript that was originally isolated by
Eudy et al. (1998) is about 5 kilobases encoding a protein of molecular mass 171
kilodaltons, which is entirely extracellular in location. The long transcript variant,
consisting of 51 additional exons, is about 15 kilobases long, encoding a putative
protein of about 600 kilodaltons. The sequence of USH2A contains an open read-
ing frame (ORF) that has similarity to the epidermal growth factor (EGF) motifs
present in the laminin family of proteins. The USH2A protein has two isoforms
termed “a” and “b.” The isoform “a” has motifs that are common in extracellular
matrix proteins and to extracellular domains of transmembrane proteins, particu-
larly the laminin and EGF-like domains. Biochemical studies have corroborated
these predictions since USH2A is found to be a component of the extracellular
basement membrane, interacting with laminins and collagens (Reiners et al.
2006).
The long murine Ush2a transcript is >15 kb in length and encodes the protein,
usherin, with an ORF of 5193 aa, which is 82% identical in amino acid sequence to
the longest human usherin protein. The domains of the protein consist of various
laminin-type domains, a fibronectin-type domain, and a transmembrane region. The
usherin protein in the retina is about 600 kDa and is localized in the connecting cilia
of the photoreceptors (Liu et al. 2007). Generation of a targeted knockout of the
Ush2a gene in mice recapitulates the phenotype of Usher syndrome 2A in humans—
it is associated with retinal and auditory defects. The photoreceptor defects are
reportedly evident only from about 10 months of age, with the retinas being without
fairly normal in the knockout mice, as are the ERG responses. From 10 months of
age onward, a progressive loss of the outer nuclear layer, with shortening of the
outer and inner segments of the photoreceptors, has been observed. By 20 months
of age, the mice display a loss of over half of the photoreceptors, and ERG responses
are substantially reduced. The changes in the retina are accompanied by the loss of
the hair cells in sections of the cochlea in the inner ear. Thus, the Ush2a mouse
model is unique among various Usher syndrome murine models in that it closely
resembles the human phenotype of Usher syndrome type 2A, manifesting with both
visual and auditory defects.
5.10.2.2 Usher Syndrome 2C
Mapping and Identification of USH2C

Various linkage studies of families with Usher syndrome type 2 detected linkage
heterogeneity in the families—i.e., some of the families did not show linkage to the
USH2A locus on chromosome 1—as outlined in the preceding section. Analysis of
several such families identified yet another locus on chromosome 5q14-21, and it
was designated as USH2C (Pieke-Dahl et al. 2000).
A search for reference transcripts in the mapped genomic interval from expressed
sequence tag (EST) databases yielded a candidate gene known as very large G
protein-coupled receptor gene 1 (VLGR1), which was taken as a probable candidate
gene for Usher syndrome based on its expression in both the retina and cochlea. The
VLGR1 gene is alternatively known as adhesion G protein-coupled receptor V1
(ADGR VI) gene or G protein-coupled receptor 98 (GPR98). In addition to the
expression pattern of the mRNA for GPR98, another factor in favor of its being a
gene for Usher syndrome was the similarity of the protein to other Usher syndrome
proteins with respect to the presence of certain conserved sequence motifs—it has
domains similar to the cadherin superfamily of membrane proteins (cadherin/proto-
cadherin genes are associated with other types of Usher syndrome as well) and also
to the USH2A protein.
Mutations in USH2C/VLGR1
Screening of ten separate probands with Usher syndrome that were mapped to the
Usher syndrome 1C (USH1C) locus showed truncating mutations in five probands
and their affected siblings, thus confirming the role of the VLGR1 gene in the patho-
genesis of Usher syndrome 1C. Screening over 150 patients with Usher syndrome
type 2 that were negative for mutations in the USH2A gene, for these same VLGR1
mutations thus identified, showed that two probands in this series had a truncating
mutation in VLGR1 (Weston et al. 2004).
5.10.2.3 Usher 2D (USH2D, Whirlin)

The whirlin gene was identified in a study that used a mouse model of deafness to
map the gene responsible for the phenotype. The whirler mouse is a strain of mouse
having a form of recessive deafness and displays a head shaking or tossing behavior.
Whirler mice have abnormalities in the inner ear—the stereocilia of the hair cells
are shortened in length in the inner hair cells and arranged in an abnormal shape in
the outer hair cells. Genetic mapping in this strain of mice to localize the Whirler
gene mapped the gene onto mouse chromosome 4. Construction of a BAC clone of
this region of the mouse chromosome 4 and sequencing of genes within the BAC
clone identified seven genes in the clone—these included one novel gene with
twelve exons and two isoforms, a short C-terminal isoform and a longer isoform.
The predicted protein sequence of both the isoforms consisted of PDZ domains and
a proline-rich domain, with the longer isoform encoding a protein of about 900
amino acids and the shorter isoform encoding a 465-amino acid protein (Mburu
et al. 2003). In order to determine which of the genes in the BAC clone represents
the Whirler gene, the entire BAC clone containing the same locus from wild type
mice was used to create transgenic mice. These transgenic mice were crossed with
the mutant wi/wi mice (whirler homozygotes, having a knockout of the gene).
Rescue of the Whirler phenotype was achieved in the offspring that inherited the
BAC transgene. All progeny that received the transgene had normal hearing and
showed no head tossing behavior. Thus, these results indicated that the BAC clone
indeed contained the gene responsible for the phenotype of Wi/Wi mutant mice—
that is, the gene that was defective or missing in the Wi/Wi mutants was being
replaced by one of the genes in the BAC clone. Analysis of the corresponding genes
in the wi/wi mutant identified a mutation in a novel gene named as the Whirlin gene
that led to the Whirler phenotype. Whirlin (Whrn) was confirmed as the gene associ-
ated with the phenotype of the whirler mouse as it had a deletion of 592 bp in these
mice, with frameshift and truncation of the protein. This mutation was absent in the
normal inbred strain of mice.
Common structural motifs such as the PDZ domains are shared by different pro-
teins involved with deafness-related phenotypes—these include the mouse Whirlin
protein as well as the harmonin protein encoded by the USH1C gene. The human
whirlin gene was identified and mapped by analysis of human cDNA sequences
homologous to the Whrn gene and to the human genome draft sequence. Sequence
analysis of the 12 exons that were identified in the human gene showed a truncating
mutation in a family with non-syndromic deafness mapped to the DFNB31 locus on
chromosome 9q32, thus establishing its role in the auditory cells in humans (Mburu
et al. 2003). The whirlin gene is expressed in the cochlea and vestibule of the inner
ear, particularly in the stereocilia in these organs. A search for similar proteins
showed that the USH1C protein harmonin is closely related to whirlin, since it also
has PDZ domains, the two proteins having 65% similarity in their PDZ domains.
The gene for Usher syndrome 2D was suspected to be the same as the DFNB31
gene, on the basis of interactions of its encoded protein with other Usher syndrome
proteins such as usherin (the Usher 2A protein) and VLGR1b (Usher 2C), thus
forming a part of the Usher protein network.
The USDH2D locus was mapped in a German family with Usher syndrome type
2, consisting of seven individuals. Of these, two siblings were affected with Usher
syndrome type 2 (congenital hearing and vision loss), and a parent of the affected
siblings had progressive non-syndromic hearing loss between the third and sixth
decades. The presence of a shared haplotype of markers at the DFNB31 locus on
chromosome 9 among the two affected individuals suggested that it could be the
potential disease locus. Analysis of the whirlin/DFNB31 gene revealed mutations
that were pathogenic, and the affected members were compound heterozygotes for
a splice site and a nonsense mutation (Ebermann et al. 2007b). Of note, both the
mutations are located in the 5′ portion of the gene, in the first and second exons that
are a part of the long isoform of the transcript. The gene encodes a PDZ domain-
containing scaffold protein that is expressed in both stereocilia and in photoreceptor
cells and also co-localizes with the USH2A and VLRG1b proteins in both these
cells. The long isoform of whirlin includes exons 1–12 and therefore has a longer
N-terminal segment than the short isoform, which is encoded by exons 6–12. In
contrast, the mutations associated with non-syndromic deafness are all found to be
in the region of the gene coding for the C-terminal half of the protein. The Usher
syndrome mutations therefore affect both the short and the long isoforms. This
study thus established the DFNB31 or whirlin is the same as the USH2D gene.
Usher syndrome type 3 is defined by the progressive nature of the hearing loss pres-
ent along with progressive pigmentary retinopathy. It is thus distinguished from
Usher syndrome types 1 and 2, in which the hearing loss is congenital. It is noted to
be relatively common in Finland, making up about 40% of the patients with Usher
syndrome in that population. The USH3 locus was mapped in Finnish families with
a diagnosis of Usher syndrome type 3. The patients were characterized by bilateral
progressive hearing loss with retinitis pigmentosa. A set of 11 families from Finland
was tested. Disease in these families was excluded for linkage to any of the previ-
ously known USH loci. Mapping by genome-wide linkage analysis located the dis-
ease gene to a 5 centimorgan region of chromosome 3q21-25 (Sankila et al. 1995).
Further analysis of the haplotypes in these families refined the disease interval to
about 200 kilobases. The families showed linkage disequilibrium at this locus, with
one major founder haplotype present in more than half of the families. Exclusion of
genes by sequencing and a recombination within this region led to a further reduced
interval of about 60 kilobases. Analysis of ESTs within this region identified a
retina-specific gene that contained 4 exons spread over about 18 kilobases of DNA.
The sequence of the USH3 gene thus identified was predicted to encode a trans-
membrane protein of 120 amino acids having two transmembrane segments and a
cytoplasmic amino terminus. The USH3 gene is widely expressed in several tissues
as well as the retina. Mutations detected in the Usher syndrome type 3 families in
the Finnish families included a more frequent truncating mutation Tyr100Ter
(Y100X) and a less frequent missense change of Met44Lys (M44K). A third muta-
tion was found in an Italian patient (Joensuu et al. 2001). However, a search for
mutations in additional families in a subsequent study involving Jewish and Finnish
families showed that several patients in whom both the clinical diagnosis and link-
age data were compatible with Usher syndrome type 3 did not have detectable
mutations.
The apparent absence of mutations in many of the Usher type 3 families led to a
re-analysis of the transcript and gene sequence of USH3, and additional exons were
discovered (Fields et al. 2002). USH3 cDNA was again isolated from human retina
and Y79 cell lines, and the sequence of the complete cDNA thus assembled by the
technique of RACE (rapid amplification of cDNA ends) showed novel sequences at
the 5′ and 3′ ends of the transcript that was described previously. The revised gene
sequence had additional bases in the exons 1, 3, and 4, and only the second exon
remained the same as what was characterized earlier. The cDNA of the revised
USH3 gene was found to be about 1.6 kilobases in length and has 198 base pairs of
additional sequence. The new USH3 sequence thus obtained was screened for
mutations in patients with Usher syndrome 3, and about 40% of patients tested had
detectable mutations, a few of which were in the newly identified first exon.
Mutations reported by Joensuu et al. (previous para) were found to be recurrent
among families studied, though the positions at which they occur are revised based
on the new exonic sequences characterized—e.g., the Y100X is re-designated as
Y176X (Tyr176Ter). The Y176X mutation is recurrent in the Finnish population.
The missense change of N48K (Asn48Lys) shows recurrence in the Jewish popula-
tion, with a high carrier frequency for this allele in the Ashkenazi Jews (Fields et al.
2002; Ness et al. 2003); these observations suggest a common ancestral origin for
the mutations that are highly prevalent in each of the populations.
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Genetics of Eye Diseases

ISBN 978-981-13-7145-5 ISBN 978-981-13-7146-2 (eBook)

© Springer Nature Singapore Pte Ltd. 2019

Hyderabad, India Chitra Kannabiran

such rare variants are tagged by more common single-nucleotide polymorphisms

1 Genetics in Corneal Diseases�������������������������������������������������������������������� 1

4.3 Primary Angle Closure Glaucoma (PACG)���������������������������������������� 75

5.8.1 Nuclear Receptor Subfamily 2 Group E Member 3

1.1 Corneal Dystrophies

1.1.1 Introduction and Classification

1. There were discrepancies between the conventional text book definition of a

© Springer Nature Singapore Pte Ltd. 2019 1

the TGFBI-induced corneal dystrophies such as Reis-Bücklers corneal dystro-

In order to address the shortcomings of the conventional classification, a newer

Box 1.1 Classification of Corneal Dystrophies

1.1.2 Epithelial and Subepithelial Corneal Dystrophies

1.1.2.1 Meesmann Corneal Dystrophy (MECD)

Manifestations and Clinical Features

Manifestations and Clinical Features

Table 1.1 Keratin gene mutations in MECD

characteristic of amyloid. Electron microscopy (EM) shows the amyloid deposits

1.1.2.3 Lisch Epithelial Corneal Dystrophy (MIM 300778)

1.1.3 Corneal Stromal Dystrophies

1.1.3.1 Macular Corneal Dystrophy (MCD, MCDC1 [OMIM 217800])

specific expression in the corneal epithelium and keratocytes, it was considered as a

1.1.4.1 Transforming Growth Factor-Induced (TGFBI) Gene

Manifestations and Clinical Features

1.1.4.3 T  hiel-Behnke Corneal Dystrophy [(CDTB, CDB2); Curly Fiber

in the deposits. The cornea is characterized by a layer of fibrous tissue described as

Box 1.2 Epithelial Recurrent Erosion Dystrophy (ERED)

The corneal manifestations include early recurrent erosions, with diffuse

Manifestations and Clinical Features

1.1.4.5 Lattice Corneal Dystrophy (LCD)

autosomal dominant mode of inheritance and associated with mutations in TGFBI.

1.1.5 Corneal Endothelial Dystrophies

1.1.5.1 Congenital Hereditary Endothelial Dystrophy (CHED)

1.1.5.2 Posterior Polymorphous Corneal Dystrophy (PPCD)

Table 1.2 Mutations identified in PPCD3

1.1.5.3 Fuchs Endothelial Corneal Dystrophy (FECD)

Table 1.3 Types of FECD and their genetic loci

1.1.5.4 G  enetics of Corneal Endothelial Dystrophies in Indian

Keratoconus involves corneal thinning (ectasia) leading to conical protrusion of the

1.2.1 Mapped Loci for Keratoconus

Keratoconus is genetically heterogeneous, and a large number of loci have been

Table 1.5 Mapped loci for keratoconus

1.2.2 Genes Associated with Keratoconus

2.1 Congenital Cataracts

Congenital cataracts can be hereditary in nature or secondary to various insults in

© Springer Nature Singapore Pte Ltd. 2019 31

2.1.1 Genetics of Congenital Cataract

Table 2.1 Mutations in crystallin genes associated with congenital cataracta, b

2.1.2 Mutations in Alpha-Crystallins

2.1.2.1 Alpha A-Crystallin (CRYAA)

subjects (Zhang et al. 2009b). The phenotype of congenital or developmental cata-

2.1.3 Mutations in Beta-Crystallins

2.1.3.1 Acidic Beta-Crystallins

1. A Brazilian family with the c.215+1G>A mutation had pulverulent nuclear

Yet another mutation in bA3/A1-crystallin that is also highly recurrent consists

Beta A2 and Beta A4-Crystallins

four-­generation family included in a study involving mostly Caucasian patients. The

2.1.3.2 Basic Beta-Crystallins

Several missense mutations in CRYBB1 are reported in families with dominant

to a highly similar “acceptor.” In the gene conversion involving CRYBB2 and

Hyderabad, India Chitra Kannabiran

1 Genetics in Corneal Diseases�� 1

4.3 Primary Angle Closure Glaucoma (PACG)�� 75

5.8.1 Nuclear Receptor Subfamily 2 Group E Member 3

1.1 Corneal Dystrophies

1.1.1 Introduction and Classification

1.1.2 Epithelial and Subepithelial Corneal Dystrophies

1.1.2.1 Meesmann Corneal Dystrophy (MECD)

1.1.2.3 Lisch Epithelial Corneal Dystrophy (MIM 300778)

1.1.3 Corneal Stromal Dystrophies

1.1.3.1 Macular Corneal Dystrophy (MCD, MCDC1 [OMIM 217800])

1.1.4.1 Transforming Growth Factor-Induced (TGFBI) Gene

1.1.4.3 T hiel-Behnke Corneal Dystrophy [(CDTB, CDB2); Curly Fiber

1.1.4.5 Lattice Corneal Dystrophy (LCD)

1.1.5 Corneal Endothelial Dystrophies

1.1.5.1 Congenital Hereditary Endothelial Dystrophy (CHED)

1.1.5.2 Posterior Polymorphous Corneal Dystrophy (PPCD)

1.1.5.3 Fuchs Endothelial Corneal Dystrophy (FECD)

1.1.5.4 G enetics of Corneal Endothelial Dystrophies in Indian

1.2.1 Mapped Loci for Keratoconus

1.2.2 Genes Associated with Keratoconus

2.1 Congenital Cataracts

2.1.1 Genetics of Congenital Cataract

2.1.2 Mutations in Alpha-Crystallins

2.1.2.1 Alpha A-Crystallin (CRYAA)

2.1.3 Mutations in Beta-Crystallins

2.1.3.1 Acidic Beta-Crystallins

four-generation family included in a study involving mostly Caucasian patients. The

2.1.3.2 Basic Beta-Crystallins

2.1.4 Mutations in Gamma-Crystallins

2.1.4.1 Mutations in CRYGC

2.1.4.2 Mutations in CRYGD

2.1.4.3 Mutations in CRYGS

2.1.5.1 Connexin Genes

2.1.5.2 Lens Intrinsic Membrane Protein 2 (LIM2, MP19, MP20)

2.1.6 Mutations in Genes Encoding Transcription Factors

3.1 Genetics of EL

3.2 Genetics of Isolated EL

4.1 Primary Congenital Glaucoma

4.1.1 Genetics of PCG

4.1.1.1 GLC3A/Cytochrome P4501B1 (CYP1B1) Gene

4.1.1.2 The GLC3C/LTBP2 (Latent Transforming Growth Factor Beta

4.2 Primary Open-Angle Glaucoma (POAG)

4.2.1 Genetics of POAG

4.2.1.2 Optineurin Mutations in POAG

4.3 Primary Angle Closure Glaucoma (PACG)

4.3.1 Genetics of PACG

4.3.1.1 Genetics of PACG in Indian Patients

5.1 eneral Features of Major Forms of Non-syndromic

5.1.1 Retinitis Pigmentosa

5.1.2 Leber Congenital Amaurosis

5.2 Homozygosity Mapping in Retinal Disorders

5.3 Genes Involved in Phototransduction

5.3.1.1 Cloning and Isolation of the RHO Gene