0

2

4

6

8

10

12

Parent Sibling

Hemminki et al Diabetologia (2009)52:1820: Swedish Multigenerational Register

PROBAND

Standardized Incidence Ratios

No Association MS

INS

PT

PN

22

IL2

RA

SH

2B

3

ER

BB

3

PT

PN

2

CL

EC

16

A

CT

LA

4

IL1

8R

AP

PT

PN

2

CC

R5

IFIH

1

CT

SH

CD

22

6

IL2

RA

PR

KC

Q

IL2

BA

CH

2

UB

AS

H3

A

RG

S1

IL7

RA

CIQ

TN

F6

TN

FA

IP3

TN

FA

IP3

TA

GA

P

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

Insulin productionand metabolism

Unknownfunction

Immunity cell apoptosisprotection

Locus

Od

ds r

ati

o

6.50-

HLA

Od

ds R

ati

oGenome-wide Associations in Type 1 Diabetes

Modified from Concannon, Rich, Nepom NEJM 360:1646 2009

GENERAL POPULATION NEWBORN COHORT (NECs) Followed to the Development of Anti-islet Autoantibodies and then Diabetes

HIGH RISK=DR3/4:DQ2/DQ8 lacking protective DP and DR4 alleles

Baschal et al Diabetes 56:2405, 2007

Father Mother Father FatherMother Mother

Haplotype SharingHaplotype Sharing

Extreme Risk for Diabetic Autoimmunity inDR3-DQB1*0201/DR4-DQB1*0302 Siblings

Share 2 MHC haplotypes: 29 (16 cases)

Share 0 or 1: 19 (3 cases)

p=0.03

HR=3.4, 95% CI (1.1, 7.0)

Share 2 MHC haplotypes: 29 (11 cases)

Share 0 or 1: 19 (1 case)

p=0.04

HR=6.1, 95% CI (1.04, 11.81)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.50

102030405060708090

100 Siblings at high risk (Share 2)

Siblings at low risk (Share 0 or 1)

% Autoantibody Positive

Age (y)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.50

102030405060708090

100

% With Diabetes

Age (y)

N = 29

N = 19

Aly TA. Extreme genetic risk for type 1A diabetes. PNAS. September 2006.

Human Type 1 diabetes susceptibility regionshttp://www.t1dbase.org/cgi-bin/dispatcher.cgi/Welcome/display

PTPN22

(IFIh1)

(CTLA4)

(IL2)

HLA

IL2RA INS

ERBB3

(PTPN11)

KIAA0350 PTPN2

8/4/08

1

2

3

4

5

6

7ODDS RATIO

Modified from Todd et al. Robust Associations of four new chromosome regions from genome-wide anlayses of type 1 diabetes Nature Genetics June 6 2007

0

10

20

30

40

50

60

Control Case

E-41 6 7 8 7 13 14 10 6 7 6

Expanded Reference Group for Type 1 Diabetes: 2,000 cases and 7,670 controls

Genome-wide association study of 14,000 cases and seven common diseases and 3,000 shared controls: WTCCC Nature June 2007 661-677

Modified from Genome-Wide Analysis: Todd et al Nature Genetics June 2007

Chrom “Gene” rs OR Pvalue

6p21 DR-DQ 3129934 6.9 (<<-100)

11p15 INS 689 2.3 (<-7)

1p13 PTPN22 2476601 1.9 (<-80)

10p15 IL2R/CD25 52580101 1.7 (<-5)

12q24 C12orf30 3184504 1.3 -16

12q13 ERBB3e 2292239 1.3 -20

18p11 PTPN2 1893217 1.2 -14

16p13 KIAA0350 12708716 1.2 -18

2q33 CTLA4 3087243 1.1 (<-5)

10p15 IL2R/CD25 11594656 1.1 (<-5)_

Type 1A Diabetes( ) Odds Ratios

• MHC(6.9) DQ>DR>DP>>HLA-A,B

• Insulin(2.3)

• PTPN22(1.89): Lymphocyte Tyrosine Phosphatase

• CTLA-4(.85),PTPN2(1.30),KIAA0350(.81),RBM17-CD25(.75)• ERBB3e(1.22),12q13(1.28)• IFIH1(.82)?, CD226(1.17)?• Other

? + MHC LINKED GENE(S) X

Extreme Risk for Type 1A DiabetesExtreme Risk for Type 1A Diabetes

High risk cohort: DR3/4-DQ8 siblings that share both MHC haplotypes identical-by-descent with their proband, N=29 Low risk cohort: DR3/4-DQ8 siblings that do not share both MHC haplotypes identical-by-descent with their proband, N=19

Aly T et al. PNAS 2006

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.50

102030405060708090

100 Siblings at high risk

Siblings at low risk

% Autoantibody Positive

Age (y)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.50

102030405060708090

100

% With Diabetes

Age (y)

Updated 5/7/07

Genetic Mapping at 3-Kilobase Resolution Revelas Inositol 1,4,5-Trisphosphate Receptor 3 as risk Factor for type 1

Diabetes in Sweden• 2,360 Illumina snp analysis MHC and centromere

• ITPR3 Intronic snp (rs2296336) identified as associated DM in survey and confirmatory Swedish population

• DR/DQ linkage present but does not obviate association

• Patient: 107/643 snp C/C, Control: 35/481 C/C

Roach et al, Am J Human Genetics, Volume 79, Oct 2006

Aly et al. Diabetes 55:1265-1269, 2006Analysis of 656 SNPs of case (N=17) and control (N=15) HLA-DR3-B8-haplotypes with Illumina™

technology• The HLA-DR3-B8-A1 haplotype is a common (carried in 7% of

Caucasians), conserved (>99.9% by SNP assay comparison), and extended (3 Mb) haplotype.

• HLA-DR3-B8-Al haplotype, genotyped at 656 SNPs in the MHC• Control and Case (islet autoantibody positive and/or diabetic)

had remarkable conservation approximately 3 million base pairs.

• Provides excellent genomic segment to analyze relation to diabetes centromeric and telomeric to region for DR3 haplotypes.

HLA-A

HLA-BMICA

DRB1DQB1

DPB1

221,21211,1111,2111,1,22122111111,1,1,1,111,11,1,1211,11211,21,1,1,1,1,21211,21,2HLA A211,1,11121,1,2211,1,21,21,211,22111222121,1,11,1,1,1,1,1,11,1,1,11,21,221,221,22121,12221,21,1,1211,1,121,221,2211,rs1266250122211,1HLA C1,1,11,11111,1111,1,21HLA B1,1,11,121,11,111MIC-A21,22211,21,1,21,21212221212221112111,11,121,1,21,1,1,21,1,121,1,11221,1,21,11,1,1,1,2121,1,1,21,1,11,1,1,1,11,1221221212221,1,21211,2221,1,1,1,221,11,1,11,1,1,211,1,21,111,21,11,1121,1,21,221,1,1,1,1,1,1122111,21,1,1,1221,21221111,211,1211,1,21,11,2221,1,1,1,1,1,1,1,1,1,1,121,21,1,1,1,1,1,1,111,11,21221,1,1,1,1,21,1,21,11,1,1,1,2DRB1211,1,2211,1,DQA11,21,21,DQB111,21,1,1,1,11,1,11,211222221111121,1,2211,1,11121,2111,11221221,2221,1211,1,21121211,1,1,21,1,1,221121,221,1,1111,21,21,1,11,222221,1,2211,222212112212122111221,11DPA212DPB111,2122212222111,21221DPB2221,1,1,21,1212222222121,1,21?11?212222121222122112212222211112111112221,21,1,21,1,1,221,21,1,111,2

Columns = Haplotypes: HLA-DR3-B8-A1 HLA-DR3-non-B8

Rows represent SNPs in the MHC region; Figure is Xcel spreadsheet with 656 miniturized rows or SNPs;Major allele = grey;Minor allele = yellow;Unknown allele = blue;(phase not determined)

Aly et al, Diabetes 55: 1265, 2006

Smyth et al: Nature Genetics 38:617-619, 2006 Genome wide assocaition study of

nonsynonymous SNPS: Interferon-induced helicase (IFIH1) region

• O.R.= .82 and .87 G allele A946T of IFIH1cases: 2,029+2,471; controls: 1,755+4,593

• TDT =46.8% transmission of G alleleTransmitted 912 versus 1,037 not (expected with null hypothesis 975)

• Genes in region “not distinguishable” fibroblast activation protein (FAP), IFIH1, granacalcin (GCA), potassium channel KCNH7

Interferon Induced Helicase SNP (IFIH1 A946T) association type 1 DM

Smyth et al, Nature Genetics 2006 rs1990760

Case N (%) Controls N (%) OR (95% c .i) P value

Alleles

A 5,526 (65) 7,117 (60.9)G 2,980 (35) 4,567 (39.1) 0.85 9.3x10(-8)Genotypes

A/A 1,810 (42.6) 2,183 (37.4)A/G 1,906 (44.8) 2,751 (47.1) 0.841.3X10(-4)G/G 537 (12.6) 908 (15.5) 0.73 1.1x10(-6)

Natural peptides selected by diabetogenic DQ8 and murine I-Ag7 molecules show common sequence homology

Suri et al JCI 115:2268, 2005Structure of Human insulin peptide DQ8, Lee et al Nature

Immunology 6:501, 2001

Crystal DQ8;B:9-23: S H L V E A L Y L V C G E R G

P1 P4 P9

I-Ag7 v,e,q I,L A,s D,E

12% 20% 30,11% 45%

P6

DQ8 E,d A,S A,V,s E,D

27,17% 19% 20% 60,25%

Preferred AA in Bound Peptides

Wiley Nat Immunol

% amino acid at position

Type 1A DiabetesType 1A Diabetes

• Monogenic:Monogenic: Single gene defect.Single gene defect. APS-I: AIRE autosomal recessive APS-I: AIRE autosomal recessive XPID: Scurfy Gene X-linked XPID: Scurfy Gene X-linked

• Polygenic:Polygenic: Summation of small effects of Summation of small effects of multiple genes creating diabetes multiple genes creating diabetes susceptibility (e.g. NOD mouse)susceptibility (e.g. NOD mouse)

• Oligogenic:Oligogenic: MHC+few major genesMHC+few major genes Genetic heterogeneity with Genetic heterogeneity with different major non-MHC genes different major non-MHC genes

for different families (e.g. BB rat) for different families (e.g. BB rat)

BDC-July01

Type 1A Diabetes

• MonogenicAPS-I (AIRE)XPID (Scurfin:Foxp3)

• OligogenicBB rat (Ian4/5+MHC)LETL Rat (Cblb+MHC)IDDM17

• Polygenic

NOD Mouse

?APS-II

?Type 1A

?=Polygenic/HeterogeneousBDC

Spontaneous Animal ModelsSpontaneous Animal Models

• BB ratBB ratHomozygosity Lymphopenia (Ch4;Ian)Homozygosity Lymphopenia (Ch4;Ian)RT1-U class II (Ch 20)RT1-U class II (Ch 20)Additional Loci (Ch2,18,X)Additional Loci (Ch2,18,X)

• NOD mouseNOD mousePolygenic: class II + class I loci + IL-2 Polygenic: class II + class I loci + IL-2

linked polymorphism + >12 iddm locilinked polymorphism + >12 iddm loci• Long-EvansTokushima RatLong-EvansTokushima Rat

RT1-U MHCRT1-U MHCHomozygosity Chr 11-Cblb geneHomozygosity Chr 11-Cblb gene

BDC

APS-I

• Autoimmune Polyendocrine Syndrome Type 1

• Autosomal Recessive mutations AIRE (Autoimmune Regulator) gene

• Mucocutaneous Candidiasis/Addison’s Disease/Hypoparathyroidism

• 18% Type 1 Diabetes

• “Transcription Factor” in Thymus

BDC

XPID: X-linked polyendocrinopathy, immune dysfunction and diarrhea

• XLAAD: “Autoimmunity Allergic Dysreg”

• Scurfin gene (Foxp3/JM2)

• Immunopathogenesis: Th2 Cytokines, abnormal activation (Il-4,5,13)-Scurfy+Nude: No Autoimmunity-CD4+ into Nude: Disease-Bone Marrow into irradiated: No Disease-Require Antigen Stimulation for Disease-Mixed Chimera: No Disease

BDC

Foxp3/JM2 GeneFork Head HomologyZn Zip

X

X

Scurfy

D

ORF

XLAAD-100

XLAAD-200

Zn = Zinc-finger domain, Zip = Zip Motif

ORF = Predicted Open Reading FrameBDC

Other Genes

• Insulin Gene VNTR Type 1A DiabetesProtection with greater thymic messenger RNA

• AIRE gene APS-I syndromeAutosomal recessive: 18% Diabetes

• Scurfy gene of XPID SyndromeNeonatal death overwhelming autoimmunity

• Ian 4/5 recessive lymphopenia gene BB rat• Cblb recessive autoimmune gene LETL rat• LYP inhibitor T cell activation and CTLA-4 gene in man

Human Leukocyte Antigen

human MHC

cell-surface proteins

important in self vs. nonself distinction

present peptide antigens to T cells

CLASS I: A,B,C CLASS II: DR,DQ,DP

HLAJ. Noble

The Major Histocompatibility Complex

Human

Mouse

DP DQ DR B C A

K I-A I-E D L

Chromosome 6

Chromosome 17

Class II Class III Class I

Class II Class III Class IClass I

Complement Proteins

Cytokines Class I-like genesand pseduogenes

Antigen Processing Genes

The Major Histocompatibility Complex

0 base pairs 1 million

1 million 4 million

DPB1DPA1

LMP2TAP1

LMP7

TAP2DQB1

DQA1DRB1

DRA

CYP 21BC4A HSP70

TNFB C E A

MICA

Class I Region

MHC Class II Region

Class III Region

DQB1*0402

Asp57

Leu56

-chain

-chain

BDC

HLA-Peptide: TCR

CDR1

CDR2CDR3

CDR3CDR2

CDR1

NH3+

COO-

TCR

alpha

TCR

beta

2 Helix α1 Helix

BDC

Format: Gene locus*Serologic specificity = 2 digits

Allele = 2 digits

Silent polymorphism (if present) = 1 digit

examples: DRB1*0405

DQB1*0302

A*68012

B*2701

HLA nomenclatureJ. Noble

TERMINOLOGY

DRB1*02

DQB1*0302DRB1*0401

DRB1*0401

DRB1*0301

DQB1*0302

DRB1*0401

DQB1*02

Allele:

Haplotype:

Genotype

J. Noble

DP DQ DR B C A

++++ +++ ? ? +

WHICH HLA LOCI ARE INVOLVED?

J. Noble

LOCUS NUMBER OFALLELES*

NUMBER OFPROBES

DRB1 241 40DRB3 26 2DRB4 9 1DRB5 14 2DQA1 20 12DQB1 44 26DPA1 19 17DPB1 86 48

A 165 57B 328 83C 88 34

*as of January, 2000

HLA POLYMORPHISMJ. Noble

HLA Class I and II Alleles (January 2001)

0

50

100

150

200

250

300

350

400

450

Class II Alleles

NU

MB

ER

OF

AL

LE

LE

S

A B1 A1 B1 A1 B1 A B C

Class I Alleles DR DQ DP

207

412

100

2

271

2045

19

93

Locus/Loci Unique Alleles/HaplotypesDRB1 34

DQB1 16

DPB1 23

A 33

B 52

DRB1-DQB1 57

DRB1-DQB1-DPB1 232

DRB1-DQB1-B 313

DPB1-DRB1-DQB1-B 558

DPB1-DRB1-DQB1-B-A 779

Alleles and Haplotypes in HBDI Type 1 Diabetes Families

J. Noble

HLA type Risk

DR3

DR4

DR3/DR4

DR2

Overall incidence = 1/300

Incidence for DR3/DR4 (DQB1*0302) = 1/15

TYPE 1 DIABETESJ. Noble

Heirarchy of IDDM risk: 0405>0402>0401>0404>0403

observed DR4 allele distribution in CaucasianAFBAC controls

0401 50.0%0404 20.3%0402 12.5%0407 9.4%0405 4.7%0408 1.6%0403 1.6%

DR4 SUBTYPESJ. Noble

Common HLA Haplotypes

• High RiskDR3: DQB1*0201, DQA1*0501, DRB1*0301 DR4: DQA1*0301, DQB1*0302, DRB1*0401

• ProtectiveDR2: DQB1*0602, DQA1*0102,, DRB1*1501

DQB1

DQA1DRB1

DRA6p

BDC

Diabetes Risk by HLADQ and DR Haplotypes

RISK DRB1 DQA1 DQB1

HIGH 0401,0405,0402 (DR4) 0301 0302

0301 (DR3) 0501 0201

0801 0401 0402

MODERATE 0401 0301 0301

0401 0301 0303

0403 0301 0302

0101 0101 0501

1601 0102 0502

LOW 1101 0501 0301

PROTECTIVE 1501 (DR2) 0102 0602

0701 0201 0303

1401 0101 0503

BDC

HBDI Series: Transmission from Parents with second haplotype not DQ2 or DQ8

0

10

20

30

40

50

60

70

80

90DQ8DQ20401/04020102/05020101/05010102/060403/030303/03010501/03010201/020103/06030102/06020101/0503(1401)

N= 406/333/33/ 79/ 72/ 55/ 55/ 44/ 42/ 38/ 29/ 37/ 4

0

1

2

3

4

5

Od

ds r

ati

o

0

20

40

60

80

Tra

ns

mis

sio

n f

req

ue

ncy (

%)

******

**

* *

*p< 0.05 vs. control haplotype

High risk

ProtectiveModerate risk

461 389 40 51 182 82 99 20 121 55 124 27 135 34

HBDI Families: Odds Ratio

HBDI Families: Transmission from Heterozygous Parents

BDC

00.5

1

1.52

2.53

3.54

4.55

Percent Diabetic

DRB 0401/0401DQB0303/0302

DRB0404/0404DQB0302/0302

DRB 0401/0401-DQB0301/0301

Absolute Risk of Childhood Diabetes by DRB1 genotype for DQB1*0302 or DQB1*0301 homozygous

Individuals

0-4 yr

0-14yr

Paul et al. Absolute Risk of childhood DM by HLA class II Genotype;

Lambert et al J. Clin Endocr Metab 89:4037-4043, 2004

Myth of Protection by DQB 57 AspEight Highest Risk Genotypes DQbeta

0

1

2

3

4

5

6

Paul et al. Absolute Risk of Childhood-Onset Type 1 Diabetes;

J Clin Endocr Metab: 89:4037-4043, 2004

DRB1*1401 and DQB1*0602Dominant Protection

05

101520253035404550

TDT: % Transmission to Diabetic

N=37

N=4

N=307 N=6

N=11

JCEM:85:3793-3797,2000

IDDM risk by age 20 HLA-DR DQB1 Frequency %

High 1:15 3/4 0201/0302 2.4

Moderate 4/x 0302/ 12.71:60-1:200 4/4 0302/ 3.0

3/3 0201/0201 1.4

Average 1:300 3/x 0201/ 12.53/4 0201/not 0302 1.0

Lower than 1:300 4/x, 4/4 /not 0302 6.6others 60.4

DAISY 7/96

HLA-Defined IDDM Risk GroupsHLA-Defined IDDM Risk GroupsDenver Population, n=9,338Denver Population, n=9,338

BDC

0 10 20 30 40 50

0.0

0.2

0.4

0.6

0.8

1.0

6 and younger n= 387-10 n= 3311-14 n= 4215-24 n= 3725 and older n= 37

Difference significant (log-rank and gen'ld wilcoxon p= 0.001 , 0.001 )

ProportionProportion of Twins Without Diagnosis of DM of Twins Without Diagnosis of DM

Years Since DM Diagnosis in Index TwinRedondo, Diabetologia

Incidence or Hazard of IDDM Incidence or Hazard of IDDM Developing in TwinDeveloping in Twin

Discordance TimeDiscordance Time

Inci

den

ceIn

cid

ence

00 1010 2020 3030 4040

0.0

0.0

0.01

0.01

0.02

0.02

0.03

0.03

0.04

0.04

0.05

0.05

6 and younger n= 386 and younger n= 387-10 n= 337-10 n= 3311-14 n= 4211-14 n= 4215-24 n= 3715-24 n= 3725 and older n= 3725 and older n= 37

Redondo, Diabetologia

a,b c,d

a,d

a,b c,da,b c,d a,b c,d

share 1 share 0share 2

a,d a,d a,d b,ca,ca,d

AFBAC: Affected Family-Based Controls

J. Noble

Insulin Gene (INS)

Class I VNTR26-63 repeats

21 alleles

Predisposing

IDDM2

Insulin Gene (INS)

Class III VNTR140-200 repeats

15 alleles

IDDM2

Protective

The IDDM2 Locus

VNTR = Variable Number of Tandem Repeats

IDDM2 Genotypes in U.S. Caucasians

I/III III/III0

20

40

60

80

100

I/I

IDDM Controls

VNTR Class

%

Pugliese et al., J. Autoimm 7: 687- 694, 1994

Pugliese et al., J. Autoimm 7: 687- 694, 1994

Transmission of VNTR Alleles to the Affected Offspring

0

10

20

30

40

50

60

Class I VNTR Class III VNTR

Transmitted

Non-Transmitted

0

5

10

15

20

25

30

35

40

45

50

Class I VNTR Class III VNTR

Transmitted

Non-Transmitted

Father Mother

• Parent-of-origin effects influence the transmission of IDDM2 alleles

• Parent-of origin effects may be mediated by imprinting (repression of expression of one of the parental alleles, usually by methylation)

VNTR Main ClassesClass I (Predisposing) Class III (Protective)

VNTR SUBTYPES

by refined sizing & flanking polymorphisms at HUMTH01 locus

Class III alleles (n= 15)301

302

303

304-Z8 VPH

305-Z8 VPH

306-Z8 VPH

306-Z PH

307-Z PH

308-Z PH

309-Z PH

310-Z PH

311

312

313

314

315

Class I alleles (n= 21)

598 (27 repeats)

613 (28 repeats)

626 (29 repeats)

641 (30 repeats) Z-4

655 (31 repeats) Z-4

669 (32 repeats) Z-4

683 (33 repeats) Z-4

698 (34 repeats) Z-4

714 (35 repeats) Z-4/Z-16

728 (36 repeats) Z-4

742 (37 repeats) Z-4

756 (38 repeats) Z-12

771 (39 repeats) Z-12

786 (40 repeats) Z-12

800 (41 repeats) Z-6

814 (42 repeats) Z-16

828 (43 repeats) Z

843 (44 repeats) Z

858 (45 repeats)

Class I alleles 598-858 vary in size by the number of repeats, which each allele representing a single increment in the number of repeats ranging between 27 and 45, top to bottom.

Most alleles are predisposing, although allele 814, the most common allele reported, is protective when transmitted from heterozygous 814/class III fathers.

Z, Z-4, Z8, Z-12, and Z-16 are alleles at the HUMTH01 locus. The most commonly observed Z alleles are shown for each allele.

For class I alleles, Z alleles tend to cluster with number of repeats around 31 (IS or small sub-class, 40 (IM or medium subclass) and 42 (IL or large subclass) repeats. There appears to be strong correlation between the IS and IM subclasses and the Z-4 and Z-12 alleles, respectively, suggesting that these patterns mark ancestral lineages.

Class III VNTR alleles in linkage disequilibrium with Z8 were more protective than those in linkage disequilibrium with Z. Such haplotypes are known as VPH and PH (very protective and protective haplotypes).

VNTR LINEAGESbased on analysis of repeat sequence by MVR-PCR & flanking polymorphisms

IC+ (Predisposing

IC- (Predisposing)

ID- (including 814, protective when transmitted by heterozygous ID-/III fathers)

IIIA (Protective, corresponding to PH)

IIIB (Protective, corresponding to VPH)

Fig. 2. Insulin gene VNTR variants and classification.

HUMTHO1 INS IGF2

Hph I Dra III Pst IVNTR

Exon 1 Intron 2 UTRExon 2 Exon 3Intron 1Promoter

• VNTR stimulates INS steady-state transcription in ß-cells

• VNTR length inversely correlates with INS mRNA steady-state levels in ß-cells in vivo

VNTR Effectson Pancreatic INS Transcription

PredisposingClass I VNTR

ProtectiveClass III VNTR

Pancreas INS Transcription

• Protective class III VNTR alleles are associated with LOWER (~30%) INS transcription than diabetes-predisposing class I VNTR alleles

INS is transcribed in Human Thymus

-No

RN

A

-Ski

n

-Lun

g

-In

test

inal

Mu

cosa

-Isl

et C

ells

(u

ndilu

ted

)

-Isl

et C

ell

(1:2

50

0)

-Th

ymus

-Th

ymus

-No

RN

A

-Ski

n

-Lun

g

-In

test

inal

Mu

cosa

-Isl

et C

ells

(u

ndilu

ted

)

-Th

ymus

-Th

ymus

ß-actin mRNA INS mRNA

Pugliese et al. Nature Genetics 15:293-297, 1997

PredisposingClass I VNTR

ProtectiveClass III VNTR

Thymus INS Transcription VNTR alleles affect INS

transcription in thymus

PredisposingClass I VNTR

ProtectiveClass III VNTR

Pancreas INS Transcription

Class I VNTR

cDN

A

DN

A

cDN

A

DN

A

Class III VNTR

Pugliese et al. Nature Genetics

15:293-297, 1997

Class I VNTR

Class III VNTR

• INS Monoallelic expression observed in ~15-20% of heterozygous thymus specimens

• Class III VNTR always the non-expressed allele(5/5 thymi; 2 fetal & 3 post-natal cases 5 & 8 months, 3 yrs old)

Pugliese et al. & Vafiadis et al. Nature Genetics 15:293-297 & 15: 289-292, 1997

Parental Effects at the IDDM2 Locus“Imprinting” (INS Monoallelic Expression)

in Human Thymus

Genomic cDNA

IDDM2 Effects in the Thymus

• Allelic Variation Effects: in the thymus, protective class III VNTR alleles are associated with higher INS transcription (2-3 fold) than predisposing alleles

• Parental Effects: may prevent the expression of class III VNTR alleles and cause loss of protection

• Hypothesis: INS expression in the thymus and its regulation affect diabetes risk by modulating thymic selection processes and in turn tolerance to insulin

A. Pugliese

Percent BabyDiab (Offspring) Autoantibody Positive at age 5

yearsHLA and Insulin Gene VNTR

0

5

10

15

20

25

30

DR3-4 DQ8 4-4 DQ8 Other HLA

INS I/I

INS I/III III/III

Walter et al, Diabetologia (2003) 46:712-720

“Minimal” Influence CTLA-4 Polymorphisms Human Type 1 DM in Contrast to Graves

DiseaseUeda et al, Nature 2003: 423:506• CTLA-4 “susceptible” G allele transmitted 53.3%

to affected offspring in 3,671 Diabetic families (RR=1.14)

• G/G A/G A/AGraves Disease: 41% 46% 23%Control : 29% 48% 14%

• G/G Genotype Associated with ½ soluble CTLA-4 Splice Variant

• Idd5.1 Of NOD Mouse Possibly CTLA-4 variant at Position 77 with G allele increasing exon 2 deleted splice variant, “ligand independent form”

0

10

20

30

40

50

60

70

80

90

100

C/C C/T T/T C/C C/T T/T

Diabetic

Control

Denver Sardinia

% with Genotype

Bottini et al. Nature Genetics; 36: 337-338

P<.001 P<.05

LYP Gene (PTPN22) Polymorphism

0

10

20

30

40

50

60

70

Britain Ireland U.S. Romania

T (Trp) Allele C (Arg) Allele

% Transmitted LYP(PTPN22) Alleles Diabetes 2004, 53:3020

Replication of an Association Between the LYP Locus with Type 1 DM…

Modified from Mustelin T and Tasken K, Biochem.J.Bottini

LYP:PTPN22-PEP in Mouse

Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant

Vang,..Bottini, Nature Genetics Nov 2005

• Tryptophan Replacing Arginine: R620W PTPN22 gene, increases risk diabetes, rheumatoid arthritis, etc.

• LYP-Trp620 inhibits T cells greater than LYP-Arg620-Less IL-2 secretion from genotype Trp/Arg vs Arg/Arg-Transfection T lymphocytes LYP-R gene greater inhibition IL-2 secretion

• Early TCR signaling inhibited more by disease associated LYP-Trp620 variant

• Gain of function variant associated with autoimmunity suggests possibility of pharmacologic inhibition of PTPN22 as therapeutic

LYP polymorphism:Autoimmunit

y

A C to T transition in position 1861 of Lyp cDNA leads to an Arg-Trp polymorphism in position 620, within the P1 domain of the final protein

Lyp1 NQESAVLATAPRIDDEIPPPLPVRTPESFIVVEEAGEFSPNVPKSLSSAVKVKIGTSLEW 656PEP NQETAVEAPSRRTDDEIPPPLPERTPESFIVVEEAGEPSPRVTESLP--LVVTFGASPEC 653 ***:** *.: * ********* ************** **.*.:**. : *.:*:* *

Lyp1 GGTSEPKKFDDSVILRPSKSVKLRSPKSELHQDRSSPPPPLPERTLESFFLADEDCMQAQ 716PEP SGTSEMKS-HDSVGFTPSKNVKLRSPKSDRHQD-GSPPPPLPERTLESFFLADEDCIQAQ 711 .**** *. .*** : ***.********: *** .*********************:***

Lyp1 SIETYSTSYPDTMENSTSSKQTLKTSGKSFTRSKSLKILRNMKKSICNSCPPNKPAESVQ 776PEP AVQTSSTSYPETTENSTSSKQTLRTPGKSFTRSKSLKIFRNMKKSVCNSSSPSKPTERVQ 771 :::* *****:* **********:*.************:******:***..*.**:* **

Lyp1 SNNSSSFLNFGFANRFSKPKGPRNPPPTWNI 807PEP PKNSSSFLNFGFGNRFSKPKGPRNPPSAWNM 802 .:**********.*************.:**:

P1

P2

P3

P4

Bottini

T cell activation

LypW620 LypR620

TCR engagementCsk

Csk

Lck

Bottini

Hypothesis: LYP “mutation”

PIP2

IP3 + DAG

PLC1

Ca++

NFAT activation

calcineurin

TCR

CD4 CD45CD28

MAPK

Shc

Ras

SOS

Grb2

PKC

NFB

Functional Variant LYP Functional Variant LYP associated with Type 1 associated with Type 1

Diabetes Diabetes Bottini, Nature Gen:36:337Bottini, Nature Gen:36:337

(ION)(PMA)

PTK

Zap70 LckLck

TcFynLYP-Csk Inhibition

0

5

10

15

20

25

30

35

40

45

50

G/G G/A A/A

Diabetes

Control

SUMO4 SNP: M55V: IDDM5

Guo et al, Nature Genetics 2004, 36:837-841

Functional Variant of SUMO4, new IkBalpha modifier

Not Confirmed:

Kosoy et al, Genes Immun 2005 6:231

Smth et al Nat Genet 2005, 37:110

Confirmed Japan: See Ikegami slide set

0

10

20

30

40

50

60

70

80

C/C A/C A/A

Swiss DM

Swiss Ctrl

German DM

German Ctrl

Pax4 Transcription Factor: A (residue 1168) or C (Proline/Histidine P321H)

Biason-Lauber et al, Diabetologia 2005, 48:900-905Association of childhood type 1 DM with a variant of PAX4: possible link to beta cell regenerative capacity

RR=3.75!, p<.0001 Not Confirmed

Maier et al Diabetologia 48:2180 2005

Gylvin et al Diabetologia 48:2183 2005