Elemente de Genetică

în
Diabetul Zaharat

Dr. Cristian Guja MD, PhD

Institutul de Diabet, Nutriţie şi Boli Metabolice

“Prof. NC Paulescu”, Bucureşti, România
“An expert is someone called in at the last
minute to share the blame”

Sam Ewing,
Former baseball champion
Informaţia genetică

Genom = Totalitatea cromozomilor

Genom uman
Structura unei gene
Dogma biologiei moleculare

Duplicarea ADN
ADN polimerază

Transcrierea
ARN polimerază
Translaţia – Sinteza proteică

mRNA matur

secvenţe pentru
fixarea AA
anticodon pentru
secvenţa ARNm
Sinteza proteică –
modificări post-
translaţionale
Polimorfisme simple - SNP

C/C T/T T/C

Homozygous C/C Homozygous T/T Heterozygous T/C

Mutaţii simple (punctiforme)
Transmitere genetică simplă (Mendeliană)
 Transmiterea genetică în DZ
Forma comună (frecventă în populaţie) a DZ nu se transmite
după un model Mendelian clasic

Din punct de vedere genetic, DZ este complex şi poligenic (există

mai mulţi loci implicaţi, fiecare cu mai multe alele de
susceptibilitate/protecţie care interacţionează unele cu celelalte dar
şi cu diverşi factori de mediu în generarea riscului global de boală)

Pentru a fi exprimate fenotipic, genele formei comune a DZ tip 2

sunt dependente de interacţiunea cu factorii de mediu (alimentaţie,
sedentarism, obezitate, etc.).

DZ tip 2 este de asemenea multigenic (mai multe combinaţii

diferite ale unor variante genice pot duce la un tablou fenotipic
similar de diabet zaharat).
Încărcătura
genetică în
DZ
comparativ
cu alte boli
genetice
Metoda PCR - Clasică
 Metode moderne de
genotipare de mare viteză

Affymetrix
PCR clasic + hibridizare
Identificare produşi prin fluorescenţă
Până la 1.000.000 SNP simultan pe o probă ADN

Illumina
PCR clasic + oligo-ligare
Identificare produşi prin fluorescenţă
Până la 1.000.000 SNP simultan pe o probă ADN
 Metode de Studiu

Studiul Genelor Candidate

- Foloseşte cunoştinţele fiziopatologice şi ţinteşte gene a
căror funcţie le-ar putea implica direct în etiopatogenia bolii
 Metode de Studiu

Screening-ul Întregului Genom – Genome Wide Scans

GENOME WIDE LINKAGE STUDIES
- Cu ajutorul markerilor (microsateliţilor)
- Cel puţin 250 markeri la distanţă de aproximativ
20 cM
GENOME WIDE ASSOCIATION STUDIES
- Foloseşte mii de polimorfisme SNP (cu localizare
cunoscută) repartizate uniform în tot genomul
GW Linkage Studies -
Microsateliţi
 GW Linkage Studies

Genome Wide Linkage Studies

- Cu ajutorul markerilor (microsateliţilor)
- Cel puţin 250 markeri la distanţă de aproximativ 20 cM
- Actual cel puţin 400 de markeri, la 10 cM

Demonstrarea linkage-ului unor alele ale unui anumit

microsatelit indică că în regiunea respectivă se află un
locus/genă de asociere cu boala.
Regiunea va fi ulterior restrânsă prin introducerea în analiză
a mai multor microsateliţi la intervale mai mici
Ulterior se trece la studiul genelor candidate din zona
respectivă.
GWS Linkage
 GW Association Studies

Genome Wide Association Studies

Cu ajutorul SNP
Harta SNP disponibila prin Human Genome Project
GeneChip 500K-700K Mapping Array Set (Affymetrix,
Illumina)
Loturi de cazuri/control – mii, zeci de mii cazuri
GWS Association
Factori Genetici în DZ tip 1
 Significant evidence of linkage to type 1 diabetes at HLA
region on chromosome 6p21.3.
Suggestive evidence of linkage near CTLA4 on chromosome
2q32.3 and near INS on chromosome 11p15.5.
Five non – HLA chromosome regions showed some evidence of
linkage to type 1 diabetes.

Diabetes 58:1018, 2009

 Gene DZ tip 1 identificate prin metoda
de studiu a genelor candidate

IDDM1 – Genele HLA de pe cromozomul 6p21

IDDM2 – Gena insulinei de pe cromozomul 11p15

IDDM12 – Gena CTLA4 de pe cromozomul 2q33

Gena Lyp/PTPN de pe cromozomul 1p13

Regiunea IL2RA/CD25 de pe cromozomul 10p15

Gena IFIH1 de pe cromozomul 2q24

 Dinamica identificării gene DZ1 după GWA

Mai mult de 60 gene

DZ1 în prezent

Bakay et al. Genes 2013;4:499-521

Mecanism gene DZ1

Concannon, NEJM, 2009

 DZ1 – Boală autoimună

Bakay et al. Genes 2013;4:499-521

Factori Genetici în DZ tip 2
 Loci T2DM identificaţi prin GWS Linkage Scans
Chromosome Reference
1q21-q23 Mori et al Diabetes 2002; Hsueh et al Diabetes 2003
2q34, 2q37 (NIDDM1) Horikawa et al Nat Genet 2000
3q27 Vionnet et al AJHG 2000, Kissebah et al. PNAS 2000
6q24-q27 Sale et al. Diabetes 2004
7p22-p21 Watanabe et al Am J Hum Genet 2000
9p21, 9q13-21 Luo et al Diabetologia 2001
10q Duggirala et al. Am J Hum Genet 1999
11p13-p12 Mori et al Diabetes 2002
12q24 (NIDDM2) Mahtani et al Nat Genet 1996, Rotimi et al Diabetes 2004
14q11-q13 Mori et al Diabetes 2002
15q13-q21 Mori et al Diabetes 2002
18p11 Parker et al. Diabetes 2001, Aulchenko Diabetes 2003
20q12-q13 Ghosh et al. Am J Hum Genet 2000
 Gene Candidate DZ2 Confirmate

Gena KCJN11 de pe cromozomul 11

Adiponectina pe cromozomul 3q27

PPAR pe cromozomul 3p24-p25

Gena HNF4α de pe cromozomul 20q12

Gena HNF1β (TCF2) de pe cromozomul 17q21

Gena Wolframin pe cromozomul 4p

Dinamica descoperirilor în Genetica DZ

Diabetologia Nov 2007

 Dinamica descoperirilor în Genetica DZ2
De la 2 loci în 2002 la > 150 actual

Prasad et al. Genes (Basel). 2015 Mar 12;6(1):87-123. doi: 10.3390/genes6010087.

 DZ2 – Deficiență beta celulară

Prasad et al. Genes (Basel). 2015 Mar 12;6(1):87-123. doi: 10.3390/genes6010087.

 Factorii genetici în DZ tip 2
In special proteine beta celulare
Proteine implicate în patogenia DZ tip 2

TCF1
TCF2
CDKAL1 HNF4A
CDKN2A/B HHEX
TCF7L2

WFS1

IGF2BP2

SLC30A8

KCJN11
ABCC8
Preluat după NEJM
 Asocierea a fost replicată în multiple populaţii (Zeggini et al.
Diabetologia 2007) şi este cea mai puternică raportată pînă în prezent
(OR < 1.7) (Sladek et al. Nature 2007)

“In UK samples, the variants in PPARγ, KCNJ11 and TCF7L2

are jointly capable of generating a discriminative value of
approximately 58%”
 Defecte genetice ale functiei beta
celulare / actiunii insulinei

ADA Diagnosis and Classification of Diabetes Mellitus 2014

Mecasnism insulinosecretie mediata de glucoza

1
5
6
2
3
4 7
Proteins associated with monogenic diabetes
 Fenotype features of MODY

Feature MODY T2DM

Transmission Monogenic, Autosomal dominant Polygenic
Heredity Usually a minimum of 3 Rarely multiple generations
generations
Penetrance 80-95% Variable, 10-40%
Pathophysiology  cell dysfunction  cell dysfunction/Insulin-resistance
Age at onset Childhood, adolescence sor young Adult, usually >40-60 yrs;
adult, usually before 25 yrs increasingly in children (obese)
Obesity Very rare Frequent (80% cases)
Metabolic Synd Absent Most often present
MODY types and involved genes
MODY types and involved genes
 MODY types and involved genes
Glucokinase and Transcription Factor
Diabetes
 MODY types and involved genes
Glucokinase and Transcription Factor
Diabetes
 MODY Probability Calculator
http://www.diabetesgenes.org/content/mody-probability-calculator
Probability >25% - genetic testing may be offered.
 Genetic defects in insulin action
Severe insulin-resistance syndromes

Primary disorders of insulin action

Hereditary
Donohue Syndrome (Leprechaunism)
Rabson-Mendenhall Syndrome
Type A insulin-resistance Syndrome

Lipodistrophy disorders
Generalized Congenital Lipodistrophy (Berardinelli-Seip)
Partial Familial Lipodistrophy (Dunnigan-Köberling)

George S, Savage D and O’Rahilly S in Williams and Pickup Textbook of Diabetes

 Donohue Syndrome (Leprechaunism)

Most severe, 1/106 births

Severe uterine and psotnatal growth delay, large

ears, ocular protrusion, micrognatia, acanthosis
nigricans, hirsutism, ginecomasty, abdominal distension.

Insulinemia 10000 pmol/l

Life expectancy < 2 yrs

Mutations in both copies of the IR gene (chromosome

19p13.2), with severe alteration of its function.
Donohue Syndrome (Leprechaunism)
 Rabson-Mendenhall Syndrome

Intermediate Severity.

Acanthosis nigricans, hirsutism, growth delay,

precocious pseudo-puberty precoce, genital hipertrophy.
Overt diabetes, very difficult to control.

Poor prognosis, survival beyond 20 yrs quite rare

Tipically mutations in both copies of the IR gene

Mutant receptors preserve some degree of

functionality
Rabson-Mendenhall Syndrome
Type A Insulin Resistance Syndrome
Females with hiperandrogenism and severe PCOS

Oligo/amenohrea, hisutism, acne, virilization,

acanthosis nigricans

Mutations in IR gene in 20-25% of patients.

Typically only one copy of the gene is mutated

According to the definition it is not encountered in

man.

Male carriers of the mutation are hyperinsulinemic

and frequently associate impaired glucose tolerance and
acanthosis nigricans
 Congenital generalized lipodystrophy
(Berardinelli-Seip)

Complete absence of the adipose tissue

Hyperinsulinemia, secondary diabetes, hepatomegaly
(NASH), PCOS
Very low leptin levels
Autosomal-recessive transmission; at least three different
defects
BSCL1 on chromosome 9q34

BSCL2 on chromosome 11q13.

 Congenital
generalized
lipodystrophy
 Neonatal Diabetes

Neonatal diabetes is defined as a form of diabetes

that is clinically diagnosed during the first 6 months of
life.

Neonatal hyperglycemia can be transient (remission

later in life) – Transient Neonatal Diabetes (TNDM) or
permanent –Permanent Neonatal Diabetes (PNDM).
 Transient Neonatal Diabetes

TNDM accounts for approximately for 50% cases of

NDM, with an incidence of ~1/100,000 live births.

Main clinical feature - it may either spontaneously remit

or be so mild as not to require treatment.

Usually diabetes will relapse, most often during

adolescence.

The majority (about 80%) of cases of TNDM are caused

by abnormalities of an imprinted locus on chromosome 6q24

The main cause seems to be a defect in maternal

methylation, most often due to recessive mutations in the
ZPF57 (Zinc Finger Protein) gene
 Permanent Neonatal Diabetes

Mean age at diagnosis - 6 weeks.

80% diagnosed < 3 months, almost all before the age
of 6 months.
Age of 6 Mos – border between PNDM and autoimmune
T1D.
Majority small for date babies – failure of in utero
insulin mediated weight gain
Usually symptomatic hyperglycaemia, usually
accompanied by DKA and blood glucose higher than 500
mg/dL, classiccally leading to insulin treatment from
onset.
 Permanent Neonatal Diabetes
Genetic basis

Activating mutations of KCJN11 gene on

chromosome 11 that encodes Kir6.2 component of the
beta cell KATP channel.
(Gloyn et al. NEJM 2004, Hattersley et al. Diabetes 2005)

Activating mutations of ABCC8 on chromosome 11

that encodes component of the beta cell KATP channel.
(Babenko et al. NEJM 2006)

Mutations of INS gene on chromosome 11p15

encoding pre-proinsulin, with defective folding of the
proinsulin molecule.
(Stoy et al. PNAS 2006)
 KATP function - Physiology

Both Kir6.2 and SUR1 are needed for the correct functioning of KATP
ATP closes KATP binding to Kir6.2,
SUs bind to SUR1 leading to KATP closure and insulin secretion stimulation
 Kir6.2 Mutations – Functional Significance

Loss of function mutations of KCJN11 gene are

associated with blockage of Kir6.2 in “closed position”,
followed by unrestricted insulin secretion - Sindromul
Familial neonatal hyperinsulinemic hypoglycemic
syndrome.
(Thomas et al. Hum Mol Genet 1996)

It was hypothesized that activatory mutations (“gain

of function”) of KCNJ11 could lead to the blockage in
an “open position” of Kir6.2, with consequent loss of
insulin secretion ability and a form of neonatal
monogenic diabetes
(Gloyn et al. NEJM 2004)
Up to 60% of PNDM patients exhibit gain of function mutations
of KCJN11

Most frequent SNPs V59M, R201H, R201C şi Y330C

Hatterslay et al. Diabetes, 2005

 7 mutations of ABCC8 in 9 of a series of 34 patietns
with PNDM

Most frequent SNPs L213R şi I1424V

Babenko et al. Diabetes, 2006

Stoy et al. PNAS, 2007
 INS – Functional significance

Mutations are located in key positions of pre-

proinsulin molecule

They alter the space structure affecting mainly the

correct folding/packaging of proinsulin, blocking the
progression through the ER.

A reaction known as Unfolded Protein Response,

leads to the local breakdown of proinsulin and
generates a functional stress inside the ER.
Increased ER stress leads to beta cell apoptosis.

The same process explains the murine models of T2D

– Akita and Munchen mice
Stoy et al. PNAS, 2007
 KATP associated PNDM
Possible therapeutic consequences

Mutant KATP channels do not respond to the

physiological stimulus (glucose), and remain opened.
However KATP can close following binding of SU’s to
SUR1.
SU’s “bypass” the physiological pathway but finally lead
to the same chain of events associated with insulin
secretion.
SUs are largely used in T2D treatment.
It was hypothesized that SU’s could be used in the
treatment of KATP associated PNDM.
(Gloyn et al. NEJM 2004)
SU’s effect in PNDM

Gloyn NEJM 2004

Tonini et al. Diabetologia 49: 2210, 2006.
 PNDM treatment with SU

The most commonly used drug for the treatment of

PNDM is glibenclamide (glyburide).

The usual dose used is 0.4-1 mg/kg/day which is

higher than the current dose used for the treatment of
T2DM.

Incidence of hypo’s - very low

No cases of severe hypo’s reported even in patients

receiving very high doses ( > 2 mg/kg) of glibenclamide.

Pearson et al. NEJM 355: 467-477, 2006.

 Largest series of PNDM treated with SU (49 patients)

90% of patients can be successfully switched to SU

treatment

Mean HbA1c improved from 8.1% to 6.4%after 12

weeks

Pearson et al. NEJM 355: 467-477, 2006.

Iafusco et al. Diabetologia 54: 2736, 2011.
Rafiq et al. Diabetes 31: 204, 2008
OMIM http://www.ncbi.nlm.nih.gov/Omim
Human Genome Database http://www.gdb.org
T1DBase http://www.t1dbase.org
Perlegen http://www.genome.perlegene.com
HapMap http://www.hapmap.org
ORPHANET http://orphanet.com
UCSC Genome browser http://www.genome.ucsc.edu
db SNP http//www.ncbi.nlm.nih.gov/SNP
GEO http//www.ncbi.nlm.gov/geo/
GWA Studies http://www.genome.gov/GWAstudies.
Genetic association db http//www. geneticassocitiondb.nih.gov/
Whole genome resources http//www. ncbi.nlm.nih.gov/WGA/
Wellcome Trust http//www.wtccc.org.uk/
Consortium
ARLEQUIN http://lgb.unige.ch/arlequin/
STRUCTURE http://pritch.bsd.uchicago.edu/structure.html
HaploBlockFinder http://cgi.uc.edu/cgi-
bin/kzhang/haploBlockFinder.cgi
Primer design http://www.autoprimer.com/
Beckman Coulter
 Crazy Medical Student Sane

Hard Working ?
Attention Span

Significant Non Existent Very Not so much

Attitude
Psychiatry ER

Nice Neutral Mean

Hates adults Hates children Patient asleep Patient dead Surgery

Paediatrics Medicine Anesthesia Pathology

NO ! Afraid of the light Afraid of the dark

Think big Think small

Dentistry Radiology Dermatology Oftalmology