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
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
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
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…
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
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