12
Hemminki et al Diabetologia (2009)52:1820: Swedish
Multigenerational Register
10
Standardized Incidence Ratios
8
6
No Association MS
4
2
0
Ce
Ad
lia
dis
c
on
's
PROBAND
Ty
pe
Gr
av
es
1D
M
M
S
Pe
rn
ic
Bi
iou
sA
lia
ry
Ci
rr
ne
mi
a
Parent
Rh
eu
m
Sa
rc
oid
Ar
th
Sibling
SL
E
U
Co
l
itis
W
eg
gr
an
ul
As
th
m
om
a
0.00
0.00
Insulin
production
and metabolism
Modified from Concannon, Rich,Locus
Nepom
NEJM 360:1646 2009
Locus
INS
PTPN22
INS
IL2RA
PTPN22
SH2B3
IL2RA
ERBB3
SH2B3
PTPN2
ERBB3
CLEC16A
PTPN2
CTLA4
CLEC16A
IL18RAP
CTLA4
PTPN2
IL18RAP
CCR5
PTPN2
IFIH1
CCR5
CTSH
IFIH1
CD226
CTSH
IL2RA
CD226
PRKCQ
IL2RA
IL2
PRKCQ
BACH2
IL2
UBASH3A
BACH2
RGS1
UBASH3A
IL7RA
RGS1
CIQTNF6
IL7RA
TNFAIP3
CIQTNF6
TNFAIP3
TAGAP
TNFAIP3
TAGAP
0.00
1.00
1.00
0.75
0.75
0.50
0.50
INS
PTPN22
IL2RA
SH2B3
ERBB3
PTPN2
CLEC16A
CTLA4
IL18RAP
PTPN2
CCR5
IFIH1
CTSH
CD226
IL2RA
PRKCQ
IL2
BACH2
UBASH3A
RGS1
IL7RA
CIQTNF6
TNFAIP3
TNFAIP3
TAGAP
HLA
Odds ratio Odds Ratio
Odds ratio
Odds ratio
Genome-wide Associations in Type 1 Diabetes
2.50
2.25
2.00
6.501.75
1.50
2.50
2.50
1.25
2.25
2.25
1.00
2.00
2.00
0.75
1.75
1.75
0.50
1.50
1.50
0.25
1.25
1.25
0.25
0.25
Locus
Immunity
 cell apoptosis
protection
Unknown
function
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
Haplotype Sharing
Siblings Share
Both Haplotypes
Siblings Share
One Haplotype
Siblings Share
No Haplotypes
Family B
Family C
Family A
Haplotypes
A
B
C
D
A
B
C
D
A
B
C
D
HLA-A
1
2
29
30
1
2
29
30
1
2
29
30
HLA-DRB1
3
3
4
4
3
3
4
4
3
3
4
4
Father
Mother
Father
Mother
Father
Mother
Diabetic
Proband
DAISY
Sibling
Diabetic
Proband
DAISY
Sibling
Diabetic
Proband
DAISY
Sibling
HLA-A
1
29
1
29
1
29
1
30
1
29
2
30
HLA-DRB1
3
4
3
4
3
4
3
4
3
4
3
4
A
C
A
C
A
C
A
D
A
C
B
D
Extreme Risk for Diabetic Autoimmunity in
DR3-DQB1*0201/DR4-DQB1*0302 Siblings
% Autoantibody Positive
100
90
80
70
60
50
40
30
20
10
0
0.0
% With Diabetes
Siblings at high risk (Share 2)
100
90
Siblings at low risk (Share 0 or 1)
80
N = 29
70
60
50
40
30
20
N = 19
10
0
2.5 5.0 7.5 10.0 12.5 15.0 17.5
0.0
Age (y)
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)
2.5
5.0
7.5 10.0 12.5 15.0 17.5
Age (y)
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)
Aly TA. Extreme genetic risk for type 1A diabetes. PNAS. September 2006.
8/4/08
Human Type 1 diabetes susceptibility regions
http://www.t1dbase.org/cgi-bin/dispatcher.cgi/Welcome/display
IL2RA INS
HLA
(IFIh1)
PTPN22
(IL2)
(PTPN11)
(CTLA4)
KIAA0350
ERBB3
PTPN2
ODDS RATIO
7
6
5
4
3
2
IN
S
P
TP
N
22
C
D
25
12
q2
4
E
R
B
B
3e
P
TP
N
K
2
IA
A
03
50
C
TL
A
4
C
D
25
H
LA
cl
as
s
II
1
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
Expanded Reference Group for Type 1 Diabetes: 2,000 cases and 7,670 controls
60
E-41 6
7
8
7
13
14
10
6
7
6
50
40
30
20
10
PT
PN
22
C
TL
A
4
3p
21
IL
2R
A
12
p1
3
12
q1
3
12
q2
4
16
p1
3
17
q2
1
18
p1
1
22
q1
3
0
Control
Case
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
6p21
11p15
1p13
10p15
12q24
12q13
18p11
16p13
2q33
“Gene”
DR-DQ
INS
PTPN22
IL2R/CD25
C12orf30
ERBB3e
PTPN2
KIAA0350
CTLA4
rs
3129934
689
2476601
52580101
3184504
2292239
1893217
12708716
3087243
OR
6.9
2.3
1.9
1.7
1.3
1.3
1.2
1.2
1.1
Pvalue
(<<-100)
(<-7)
(<-80)
(<-5)
-16
-20
-14
-18
(<-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 Diabetes
% Autoantibody Positive
100
90
80
70
60
50
40
30
20
10
0
0.0
Siblings at high risk
Siblings at low risk
2.5
5.0
7.5 10.0 12.5 15.0 17.5
Age (y)
% With Diabetes
100
90
80
70
60
50
40
30
20
10
0
0.0
2.5
5.0
7.5 10.0 12.5 15.0 17.5
Age (y)
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
Updated 5/7/07
Aly T et al. PNAS 2006
Genetic Mapping at 3-Kilobase
Resolution Revelas Inositol 1,4,5Trisphosphate 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, 2006
Analysis 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.
Columns = Haplotypes: HLA-DR3-B8-A1
Key:
Case haplotype
Negative control haplotype
2
1,
2
1
1,
1
1,
2
1
1,
2
1
2
1
1
1,
1,
1
1,
1
1,
1
2
1,
1
2
1
1,
2
1,
1,
2
1
2
1,
HLA A
2
1
1,
1
2
1,
2
1
1,
2
1,
2
1
1,
2
1
2
1
2
1,
1
1,
1,
1
1,
1
1,
2
1,
2
1,
2
1
2
1,
1
2
1,
2
1,
1
2
1,
1
2
1,
2
1,
2
1
1,
rs12662501
2
1
HLA C
1,
1
1,
1
1,
1
1,
2
1
HLA B
1,
1
2
1,
1
MIC-A
2
1,
2
1
2
1,
2
1,
1
2
2
1
2
2
1
2
1
1,
1
2
1,
2
1,
2
1,
1
2
1,
1
2
1,
2
1,
1
1,
2
1
1,
2
1,
1
1,
1
1,
2
1
2
1
2
2
1,
2
1
1,
2
1,
2
1,
1
1
1,
2
1
1,
2
1
1,
2
1,
1
2
1,
2
1,
2
1,
1,
1
2
1
1,
2
1,
1
2
1,
2
1
2
1
1,
2
1,
1
2
1,
2
1,
1
2
1,
1,
1,
1,
1
1,
2
1,
1,
1
1,
1
2
1
2
1,
1,
2
1,
2
1,
1
1,
DRB11,
2
1
2
1
DQA1
1,
2
1,
DQB1
1
2
1,
1
1,
1
1,
2
1
2
2
1
1
2
1,
2
1
1,
1
2
1,
1
1,
1
2
1
2
1,
2
1,
1
2
1,
2
1
1
2
1,
2
1,
2
1
2
1,
2
1,
1
1,
2
1,
1
1,
2
2
1,
2
1
1,
2
2
1
1
2
1
2
2
1
2
1,
1
DPA
2
DPB11,
1
2
1
2
1
2
2
1
1,
2
1
2
1
DPB21,
2
2
1,
1
2
2
2
1
2
1,
2
1
?
1
?
2
1
2
1
2
2
1
2
1
2
1
2
2
1
1
2
1
1
2
1,
2
2
1,
2
1,
1,
1
2
1,
HLA-A
HLA-B
MICA
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)
DRB1
DQB1
DPB1
Aly et al, Diabetes 55:
1265, 2006
HLA-DR3-non-B8
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 IFIH1
cases: 2,029+2,471; controls: 1,755+4,593
• TDT =46.8% transmission of G allele
Transmitted 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 (%)
Alleles
A
5,526 (65)
G
2,980 (35)
Genotypes
A/A 1,810 (42.6)
A/G 1,906 (44.8)
G/G
537 (12.6)
Controls N (%)
OR (95% c.i) P value
7,117 (60.9)
4,567 (39.1)
0.85 9.3x10(-8)
2,183 (37.4)
2,751 (47.1)
908 (15.5)
0.84 1.3X10(-4)
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, 2005
Structure 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
Wiley Nat Immunol
Preferred AA in Bound Peptides
I-Ag7
DQ8
% amino acid at position
P1
P4
P6
v,e,q
I,L
12%
20% 30,11%
E,d
A,S
A,V,s
E,D
27,17%
19%
20%
60,25%
A,s
P9
D,E
45%
Type 1A Diabetes
• Monogenic:
• Polygenic:
• Oligogenic:
BDC-July01
Single gene defect.
APS-I: AIRE autosomal recessive
XPID: Scurfy Gene X-linked
Summation of small effects of
multiple genes creating diabetes
susceptibility (e.g. NOD mouse)
MHC+few major genes
Genetic heterogeneity with
different major non-MHC genes
for different families (e.g. BB rat)
Type 1A Diabetes
• Monogenic
APS-I (AIRE)
XPID (Scurfin:Foxp3)
• Polygenic
NOD Mouse
?APS-II
• Oligogenic
BB rat (Ian4/5+MHC)
LETL Rat (Cblb+MHC)
IDDM17
?Type 1A
?=Polygenic/Heterogeneous
BDC
Spontaneous Animal Models
• BB rat
Homozygosity Lymphopenia (Ch4;Ian)
RT1-U class II (Ch 20)
Additional Loci (Ch2,18,X)
• NOD mouse
Polygenic: class II + class I loci + IL-2
linked polymorphism + >12 iddm loci
• Long-EvansTokushima Rat
RT1-U MHC
Homozygosity 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 Gene
Zn
Fork Head
Homology
Zip
ORF
X
D
XLAAD-100
XLAAD-200
X
Scurfy
Zn = Zinc-finger domain, Zip = Zip Motif
ORF = Predicted Open Reading Frame
BDC
Other Genes
• Insulin Gene VNTR Type 1A Diabetes
Protection with greater thymic messenger RNA
• AIRE gene APS-I syndrome
Autosomal recessive: 18% Diabetes
• Scurfy gene of XPID Syndrome
Neonatal 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
J. Noble
HLA
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
The Major Histocompatibility Complex
Class III
Class II
Human
Chromosome 6
DP
DQ
Antigen Processing
Genes
Mouse
Chromosome 17
Class I
Class II
K
I-A
DR
B
Complement
Proteins
Cytokines
Class III
I-E
Class I
C
A
Class I-like genes
and pseduogenes
Class I
D
L
The Major Histocompatibility Complex
LMP7
DQA1
DPA1
DPB1
LMP2
TAP2
DQB1
DRB1
DRA
MHC Class II Region
0 base pairs
1 million
MICA
CYP 21B
C4A
1 million
TAP1
HSP70
B
C
E
A
TNF
Class III Region
Class I Region
4 million
DQB1*0402
 -chain
Leu56
-chain
Asp57
BDC
HLA-Peptide: TCR
2 Helix
NH3+
α1 Helix
TCR
alpha
TCR
beta
BDC
COO-
J. Noble
HLA nomenclature
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
J. Noble
TERMINOLOGY
Allele:
Haplotype:
Genotype
DRB1*0401
DRB1*0401
DQB1*0302
DRB1*0401
DQB1*0302
DRB1*0301
DRB1*02
DQB1*02
J. Noble
WHICH HLA LOCI ARE
INVOLVED?
DP
DQ
DR
B
C
A
+
+++
+++
?
?
+
HLA POLYMORPHISM
LOCUS
DRB1
DRB3
DRB4
DRB5
DQA1
DQB1
DPA1
DPB1
NUMBER OF
ALLELES*
241
26
9
14
20
44
19
86
A
165
B
328
C
88
*as of January, 2000
NUMBER OF
PROBES
40
2
1
2
12
26
17
48
57
83
34
J. Noble
HLA Class I and II Alleles
(January 2001)
412
NUMBER OF ALLELES
450
400
350
271
300
207
250
200
150
100
50
100
93
45
2
20
19
0
A
DR
B1 A1 B1 A1
DQ
B1
A
B
C
DP
Class II Alleles
Class I Alleles
J. Noble
Alleles and Haplotypes
in HBDI Type 1 Diabetes Families
Locus/Loci
Unique Alleles/ Haplotypes
DRB1
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
J. Noble
TYPE 1 DIABETES
Overall incidence = 1/300
HLA type
Risk
D R3

D R4

D R3/D R 4
 
D R2

Incidence for DR3/DR4 (DQB1*0302) = 1/15
DR4 SUBTYPES
J. Noble
Heirarchy of IDDM risk: 0405>0402>0401>0404>0403
observed DR4 allele distribution in Caucasian
AFBAC controls
0401
0404
0402
0407
0405
0408
0403
50.0%
20.3%
12.5%
9.4%
4.7%
1.6%
1.6%
Common HLA Haplotypes
6p
DQB1 DRB1
DQA1
DRA
• High Risk
DR3: DQB1*0201, DQA1*0501, DRB1*0301
DR4: DQA1*0301, DQB1*0302, DRB1*0401
• Protective
DR2: DQB1*0602, DQA1*0102,, DRB1*1501
BDC
Diabetes Risk by HLA
DQ and DR Haplotypes
RISK
DRB1
DQA1
DQB1
HIGH
0401,0405,0402 (DR4)
0301
(DR3)
0801
0401
0401
0403
0101
1601
1101
1501
(DR2)
0701
1401
0301
0501
0401
0301
0301
0301
0101
0102
0501
0102
0201
0101
0302
0201
0402
0301
0303
0302
0501
0502
0301
0602
0303
0503
MODERATE
LOW
PROTECTIVE
BDC
HBDI Series: Transmission from Parents with
second haplotype not DQ2 or DQ8
N= 406/333/33/ 79/ 72/ 55/ 55/ 44/ 42/ 38/ 29/ 37/ 4
90
80
70
60
50
40
30
20
10
0
DQ8
DQ2
0401/0402
0102/0502
0101/0501
0102/0604
03/0303
03/0301
0501/0301
0201/02
0103/0603
0102/0602
0101/0503(1401)
HBDI Families: Odds Ratio
5
Odds ratio
4
*
*
*p< 0.05 vs. control
haplotype
3
2
1
*
0
*
*
*
*
*
Transmission frequency (%)
HBDI Families: Transmission from Heterozygous Parents
80
60
40
461 389 40
51 182
82 99 20 121
55
*
*
124 27 135 34
High risk
Moderate risk
Protective
20
03
/0
30
05
2
01
04 /0
01 2
/0
01 40
02 2
/0
01 50
01 2
/0
01 50
02 1
/0
60
03 4
/0
30
1
03
/0
30
02 3
01
01 /0
03 2
/0
05 60
01 3
/0
01 30
01 1
/0
01 503
1
02
/0
60
02
2
01
/0
30
3
0
BDC
Absolute Risk of Childhood Diabetes by DRB1
genotype for DQB1*0302 or DQB1*0301 homozygous
Individuals
5
4.5
4
3.5
3
Percent
2.5
Diabetic
2
1.5
1
0.5
0
0-4 yr
0-14yr
DRB 0401/0401
DQB0303/0302
DRB0404/0404
DQB0302/0302
DRB 0401/0401DQB0301/0301
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 Asp
Eight Highest Risk Genotypes DQbeta
6
5
4
3
2
1
0
*
*
*
2
2
2
2
2
4
3
3
0
0
0
0
0
3
3
/0
30
30
03
03
03
0
0
2
/
/
/
/
/
0
0
/
/
0
1
2
1
1
1
2
1
3
0
0
0
0
0
0
0
0
02
03
02
02
02
03
02
Paul et al. Absolute Risk of Childhood-Onset Type 1 Diabetes;
J Clin Endocr Metab: 89:4037-4043, 2004
DRB1*1401 and DQB1*0602
Dominant Protection
TDT: % Transmission to Diabetic
50
45
40
35
30
25
20
15
10
5
0
N=4
N=11
N=307
N=6
N=37
02
06
B
02
06
B
Q
tD
no
02
01
06
B
15
Q
R
D
D
01
15
R
tD
no
Q
D
;
01
3
03
05
B
0
05
Q
D
1;
Q
D
;
01
40
t1
15
R
D
no
14
R
D
JCEM:85:3793-3797,2000
HLA-Defined IDDM Risk Groups
Denver Population, n=9,338
IDDM risk by age 20
HLA-DR
DQB1
High 1:15
3/4
0201/0302
2.4
Moderate
1:60-1:200
4/x
4/4
3/3
0302/
0302/
0201/0201
12.7
3.0
1.4
Average 1:300
3/x
3/4
0201/
0201/not 0302
12.5
1.0
Lower than 1:300
4/x, 4/4
others
/not 0302
6.6
60.4
DAISY 7/96
BDC
Frequency %
0.4
0.6
0.8
1.0
Proportion of Twins Without Diagnosis of DM
0.2
6 and younger n= 38
7-10 n= 33
11-14 n= 42
15-24 n= 37
25 and older n= 37
0.0
Difference significant (log-rank and gen'ld wilcoxon p= 0.001 , 0.001 )
0
10
Redondo, Diabetologia
20
30
40
50
Years Since DM Diagnosis in Index Twin
Developing in Twin
Incidence
0.02 0.03 0.04
0.05
Incidence or Hazard of IDDM
0.0
0.01
6 and younger n= 38
7-10 n= 33
11-14 n= 42
15-24 n= 37
25 and older n= 37
0
Redondo, Diabetologia
10
20
Discordance Time
30
40
AFBAC: Affected Family-Based Controls
J. Noble
a,b
c,d
a,d
a,b
a,d
c,d
a,d
share 2
a,b
a,d
c,d
a,b
a,c
a,d
share 1
c,d
b,c
share 0
The IDDM2 Locus
IDDM2
Insulin Gene (INS)
Predisposing
Class I VNTR
26-63 repeats
21 alleles
IDDM2
Insulin Gene (INS)
Protective
Class III VNTR
140-200 repeats
15 alleles
VNTR = Variable Number of Tandem Repeats
IDDM2 Genotypes in U.S. Caucasians
IDDM
Controls
100
80
60
%
40
20
0
I/I
I/III
III/III
VNTR Class
Pugliese et al., J. Autoimm 7: 687- 694, 1994
Transmission of VNTR Alleles to the Affected Offspring
Father
Mother
60
50
45
50
40
35
40
30
Transmitted
Non-Transmitted
30
20
25
20
15
10
10
5
0
0
Class I VNTR
Class III VNTR
Class I VNTR
Class III VNTR
• 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)
Pugliese et al., J. Autoimm 7: 687- 694, 1994
Fig. 2.
Insulin gene VNTR variants and classification.
HUMTHO1
VNTR
INS
IGF2
Hph I
Promoter
Exon 1
Intron 1
Dra III
Exon 2
Intron 2
Pst I
Exon 3
UTR
VNTR Main Classes
Class I (Predisposing)
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.
Class III (Protective)
VNTR SUBTYPES
by refined sizing & flanking polymorphisms at HUMTH01
locus
Class I alleles (n= 21)
Class III alleles (n= 15)
598 (27 repeats)
301
613 (28 repeats)
302
626 (29 repeats)
303
641 (30 repeats) Z-4
304-Z8 VPH
655 (31 repeats) Z-4
305-Z8 VPH
669 (32 repeats) Z-4
306-Z8 VPH
683 (33 repeats) Z-4
698 (34 repeats) Z-4
306-Z
PH
714 (35 repeats) Z-4/Z-16
307-Z
PH
728 (36 repeats) Z-4
308-Z
PH
742 (37 repeats) Z-4
309-Z
PH
756 (38 repeats) Z-12
310-Z
PH
771 (39 repeats) Z-12
311
786 (40 repeats) Z-12
312
800 (41 repeats) Z-6
313
814 (42 repeats) Z-16
828 (43 repeats) Z
314
843 (44 repeats) Z
315
VNTR LINEAGES
858 (45 repeats)
based on analysis of repeat sequence by MVR-PCR & flanking polymorphisms
IC+ (Predisposing
IIIA (Protective, corresponding to PH)
IC- (Predisposing)
IIIB (Protective, corresponding to VPH)
ID- (including 814, protective when transmitted by
heterozygous ID-/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 Z12 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 Effects
on Pancreatic INS Transcription
• VNTR stimulates INS steady-state transcription in ß-cells
• VNTR length inversely correlates with INS mRNA steady-state
levels in ß-cells in vivo
• Protective class III VNTR
alleles are associated with
LOWER (~30%) INS
transcription than diabetespredisposing class I VNTR
alleles
Pancreas INS Transcription
Predisposing
Class I VNTR
Protective
Class III VNTR
ß-actin mRNA
-No RNA
-Skin
-Lung
-Intestinal Mucosa
-Islet Cells (undiluted)
-Islet Cell (1:2500)
-Thymus
-Thymus
-No RNA
-Skin
-Lung
-Intestinal Mucosa
-Islet Cells (undiluted)
-Thymus
-Thymus
INS is transcribed in Human Thymus
INS mRNA
Pugliese et al. Nature Genetics 15:293-297, 1997
VNTR alleles affect INS
transcription in thymus
Thymus INS Transcription
Predisposing
Class I VNTR
Class I
VNTR
Protective
Class III VNTR
Pancreas INS Transcription
Class III
VNTR
Pugliese et al. Nature Genetics
15:293-297, 1997
Predisposing
Class I VNTR
Protective
Class III VNTR
Parental Effects at the IDDM2 Locus
“Imprinting” (INS Monoallelic Expression)
in Human Thymus
Genomic
cDNA
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
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 years
HLA and Insulin Gene VNTR
30
25
20
INS I/I
INS I/III III/III
15
10
5
0
DR3-4 DQ8
4-4 DQ8
Other HLA
Walter et al, Diabetologia (2003) 46:712-720
“Minimal” Influence CTLA-4 Polymorphisms Human
Type 1 DM in Contrast to Graves Disease
Ueda 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/A
Graves 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”
% with Genotype
LYP Gene (PTPN22) Polymorphism
100
90
80
70
60
Diabetic
Control
50
40
P<.001
P<.05
30
20
10
0
C/C
C/T
Denver
T/T
C/C
C/T
T/T
Sardinia
Bottini et al. Nature Genetics; 36: 337-338
% Transmitted LYP(PTPN22) Alleles Diabetes 2004, 53:3020
Replication of an Association Between the LYP Locus with Type 1 DM…
70
60
50
40
30
20
10
0
Britain
Ireland
U.S.
T (Trp) Allele
C (Arg) Allele
Romania
LYP:PTPN22-PEP in Mouse
Bottini
Modified from Mustelin T and Tasken K, Biochem.J.
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 LYPTrp620 variant
• Gain of function variant associated with autoimmunity suggests
possibility of pharmacologic inhibition of PTPN22 as therapeutic
LYP polymorphism:Autoimmunity
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
P1
Lyp1
PEP
NQESAVLATAPRIDDEIPPPLPVRTPESFIVVEEAGEFSPNVPKSLSSAVKVKIGTSLEW 656
NQETAVEAPSRRTDDEIPPPLPERTPESFIVVEEAGEPSPRVTESLP--LVVTFGASPEC 653
***:** *.: * ********* ************** **.*.:**. : *.:*:* *
P2
Lyp1
PEP
GGTSEPKKFDDSVILRPSKSVKLRSPKSELHQDRSSPPPPLPERTLESFFLADEDCMQAQ 716
SGTSEMKS-HDSVGFTPSKNVKLRSPKSDRHQD-GSPPPPLPERTLESFFLADEDCIQAQ 711
.**** *. .*** : ***.********: *** .*********************:***
P3
Lyp1
PEP
SIETYSTSYPDTMENSTSSKQTLKTSGKSFTRSKSLKILRNMKKSICNSCPPNKPAESVQ 776
AVQTSSTSYPETTENSTSSKQTLRTPGKSFTRSKSLKIFRNMKKSVCNSSSPSKPTERVQ 771
:::* *****:* **********:*.************:******:***..*.**:* **
P4
Lyp1
PEP
SNNSSSFLNFGFANRFSKPKGPRNPPPTWNI 807
PKNSSSFLNFGFGNRFSKPKGPRNPPSAWNM 802
.:**********.*************.:**:
Bottini
Hypothesis: LYP “mutation”
TCR engagement
Csk
Csk
Lck
LypW620
LypR620
T cell activation
Bottini
Functional Variant LYP
associated with Type 1
Diabetes
Bottini, Nature Gen:36:337
Zap70
TCR
LYP-Csk Inhibition
Fyn
PLCg1
PIP2
(ION)
CD4
CD28
Lck
Lck
Tc
Shc
Grb2
SOS
(PMA)
Ras
IP3 + DAG
Ca++
PKC
MAPK
calcineurin
NFkB
NFAT activation
CD45
PTK
Not Confirmed:
SUMO4 SNP: M55V: IDDM5
50
Kosoy et al, Genes Immun 2005 6:231
45
Smth et al Nat Genet 2005, 37:110
Confirmed Japan: See Ikegami slide set
40
35
30
Diabetes
Control
25
20
15
10
5
0
G/G
G/A
Guo et al, Nature Genetics 2004, 36:837-841
Functional Variant of SUMO4, new IkBalpha modifier
A/A
Pax4 Transcription Factor: A (residue 1168) or C (Proline/Histidine P321H)
80
RR=3.75!, p<.0001
70
Not Confirmed
Maier et al Diabetologia 48:2180 2005
Gylvin et al Diabetologia 48:2183 2005
60
50
Swiss DM
Swiss Ctrl
German DM
German Ctrl
40
30
20
10
0
C/C
A/C
A/A
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