u Autoimmune diseases afflict some 50 million people in the US.

u Systemic autoimmunity (SLE, RA)

uOrgan-specific autoimmunity• Single organ (IDDM, Thyroid disease)• Multiple organs (APS)

Inherited and Acquired Risk for Autoimmunity

Inherited and Acquired Risk for Autoimmunity

uMonogenic disorders– APS1 (APECED)

– Genetically homogeneous, Phenotypically homogenous

u Acquired disease– Chemical- or virus -induced

u Multifactorial diseases• SLE• Multiple sclerosis, etc.• Type 1 diabetes

• Genetically heterogeneous, phenotypically homogeneous

Autoimmune PolyglandularSyndromes (APS)

uAPS1 (APECED): > 2 of 3 disorders• Hypoparathyroidism (HPT)• Addison’s disease (AD)• Mucocutaneous candidiasis (MC)

uAPS2: AD plus IDDM or autoimmune thyroid diseases (ATD)

uAPS3: ATD and others but no AD

Component Diseases and their Frequencies in Autoimmune Polyglandular Syndromes

Component Diseases

Endocrine components:Hypoparathyroidism

Addison Disease

Hypothyroidism

Hyperthyroidism

IDDM

Ovarian failure

Testicular failure

Pernicious Anemia

Hypopituitarism

APS1 APS2 APS3

80%

70% (early)

5%

0%

2-5%

60%

14%

15% (early)

+-

0%

100% (late)

70%

++

52%

4%

2%

1% (late)

+

0%

0%

70%

+-

+-

0%

0%

++

--

Component Diseases and their Frequencies in Autoimmune Polyglandular Syndromes

Component Diseases APS1 APS2 APS3

Non-endocrine components:Mucocutaneous Candidiasis

Alopecia

Vitiligo

Chronic active hepatitis

Keratopathy

Squamous cell carcinoma, Septicemia

Intestinal Malabsorption

Enamel hypoplasia

90%

26%

10%

11%

35%

++

18%

77%

0%

1%

5%

0%

0%

+

0%

0%

0%

+-

>10%

0%

0%

--

0%

0%

Distribution of Numbers of Disease Components Among APS1 Patients

0

5

10

15

20

25

30

% o

f C

ases

1 2 3 4 5 6 7 8

No. of Disease Components

APS 1

uRare: <1/100,000

uRecessively inherited

uSingle gene on chromosome 21q

Hypothetical structure of AIRE

Gel Shift Assay with AIRE Consensus sequence

ChIP mop ChIP

Fig. 3: ChIP microarray analysis with mouse thymus. Green spots indicate higher intensities for the ChIP-enriched probes than the unenriched probes and most likely contain sequence sites bound by the Aire protein. Yellow (equal intensities for both probes) or red (lower intensities for the ChIP-enriched probe) represent non-specific immunoprecipitated DNA (noise).

Search for target genes regulated by AIRE- Chromatin Immunoprecipitation Microarray

Fig3. Expression differences measured by semiquantitative RT-PCR of 11 genes from Aireknock-out mice, B6 mice was used as control.cDNAs were normalized to B-actin. Experiment has been done in 2 independently isolated MECsamples from both Aire knock-out and B6 mice.

Mechanism of Disease

Regulation of autoantigen expression in the thymus. Deficiencies in expression of peripheral autoantigens in thymus results in defective elimination of autoreactive T cells.

Regulation of key cytokines and growth factors involved in thymocyte development.

AIRE gene

1 2 3 4 5 6 7 8 9 10 11 12 13 14

AIRE protein

1 545L L L LPHD1 PHD2PRR

Mutations

R15L

L28P T90C

K83E

IVS3+1T/C

R203X

R257X13bpdel

ins CCTG

1249 del C1264 del C

1313 del C

1422 ins AC

X546C

1291 ins A

R139X318-353del

Q173X

IVS9-1G/A

1284 ins A

1320 del C1391 del C

The Autoimmune Regulator (AIRE) is Mutated in APS1 Patients

Population Frequencies of Major Mutations

82%

13%0%

5%

73%

27%

0%

0%

56%28%

16%

0%

95%

0%5%0%

0%

14%

64%

22% Mutations:

1) R257X

2) 13bpdel

3) R139X

4) Others

Finland Italy Sardinia

UK US

Type 1 Diabetes (T1D or IDDM)Type 1 Diabetes (T1D or IDDM)

u Due to destruction of pancreatic beta cells by one’s own immune system

♦ Autoantibodies: IAA, ICA, GAD & IA2♦ Autoreactive T cells

u Affects 0.4% of the population (1 million Americans)

u Age of onset for most patients: before 17 yearsuMultifactorial disease

♦ ~50% concordance in MZ twins♦ Multiple genetic factors♦ Environmental triggers

Type 1 DiabetesType 1 Diabetes

Environment Immune Response

Genetics

Natural History of Type 1 DiabetesNatural History of Type 1 Diabetes

Genetic Predisposition

Prediabetes/Autoimmunity(Autoantibodies,Insulitis)

Clinical Disease

- virus- diet- toxins

- virus- diet - toxins

Initiators Promoters

IDDM Susceptibility Intervals in Humans (2004)IDDM Susceptibility Intervals in Humans (2004)

Loci Region Genes/Status

IDDM 1IDDM 2IDDM 3IDDM 4IDDM 5IDDM 6IDDM 7IDDM 8IDDM 9IDDM 10IDDM 11IDDM 12IDDM 13IDDM 15D14S70-D14S276D16S515-D16S520D19S247-D19S226

D19S225D1S1644-AGT

6p2111p1515q2611q136q2518q2q336q273q10p13-q1114q24-q312q332q336q2114q12-q2116q22-q2419p1319q131q

HLA-DR, DQ and othersInsulin??

??????CTLA4???

???

IDDM18 - IL12p40

Controls NFkB

?

HLA Genotypes % in % in IDDM Absolute RiskDR-DQB1/DR-DQB1 controls (Sensitivity) (Specificity)

03-0201/04-0302 (DR3/4) 1.8 35.0 1/15

04-0302/04-0302 (DR4/4) 0.7 10.0 1/20

03-0201/03-0201 (DR3/3) 1.5 9.0 1/40

04-0501/04-0301 (DR1/4) 0.6 2.0 1/60

15-0602/ 50.7 20.0 1/15,000

Predictability for IDDM Risk by HLAPredictability for IDDM Risk by HLA

Family History Increases Risk for T1DFamily History Increases Risk for T1D

1st Degree Relative

1/4-5

1/8

1/10

1/15

1/125

1/15,000

GeneralPopulation

1/15

1/30

1/40

1/60

1/600

1/15,000

DR/DQ

DR 3/4, DQ 0201/0302

DR 4/4, DQ 0300/0302

DR 3/3, DQ 0201/0201

DR 4/X, DQ 0302/X

X/X, 3/X

DR 0403 or DQ 0602

Islet Cell Autoantibodies increase Islet Cell Autoantibodies increase specificity of the screeningspecificity of the screening

uMeasure 4 autoantibodies ♦ GADA: Glutamic acid decarboxylase autoantibody

♦ IA2A: Insulinoma-associated antigen 2 autoantibody

♦ IAA: Insulin autoantibody

♦ ICA Islet cell autoantibody

Timing and Sequence of Ab AppearanceTiming and Sequence of Ab Appearance

0

1

2

3

4

5

18m 20m 22m 24m 30m 33m 39mIDD

Age (months)

Ab

Tite

r

GAD IA-2A Cut off

1

10

100

1000

18m 20m 22m 24m 30m 33m 39mIDD

Age (months)

Ab

Tite

r

ICA ICA Cutoff IAA IAA Cutoff

0

1

2

3

4

5

12m 15m 18m 21m 24m 27m 33m 36mIDD

39m 42m

Age (months)

Ab

Tite

r

GAD IA-2A Cutoff

1

10

100

1000

12m 15m 18m 21m 24m 27m 33m 36mIDD

39m 42m

Age (months)

Ab

Tite

r

ICA ICA Cutoff IAA IAA cutoff

Specificity and Sensitivity of AbScreening in Relatives

00.10.20.30.40.50.60.70.80.9

0 0.01 0.02 0.03 0.04

1-Specificity

Sens

itivi

tyiY

ICA&IA2ß

GAD

ICA&GAD ICA&IAA

IA2ß

GAD or IAA GAD or ICA

IAA

GAD or IA2ß

ICAGAD or IA2

ICA&IA2

???

MicroarrayMicroarray--based prediction markersbased prediction markers

AbN T1D AbPAbN T1D AbP

118 genes

AbP/IDD vs AnN

18 genes

AbP vs IDD

MultiMulti--gene Modelsgene Models

Model 11276, 65 clones

Canonical Variate 2

Can

onic

al V

aria

te 1

Ab-Ab+Idd

Normal Kernel Model

1 of 5 models

4-9% error

Model 16562, 35 Clones

Canonical Variate 2

Can

onic

al V

aria

te 1

Ab-Idd

Discovery of two subsets of AbP SubjectsDiscovery of two subsets of AbP Subjects

Model 16562, 35 Clones

Canonical Variate 2

Can

on

ical

Var

iate

1

Ab-IddAb+

Progression to Diabetes among AbP SubjectsProgression to Diabetes among AbP Subjects

0

0.25

0.5

0.75

1

0 1 2 3 4

Time of Follow-Up (years)

Pro

bab

ility

of S

urv

ival

AbN- like

T1D- like

n=6

n=13

TEDDY Centers(preceding study, year started)

• Colorado (DAISY, 1993) • Finland (DIPP, 1994)• Georgia/Florida (PANDA, 1997)• Germany (BABY-DIAB, 1989)• Sweden (DiPiS, 2000)• Washington (DEW-IT, 2000)• Data Coordinating Center (Tampa, FL)

The Environmental Determinants of Diabetes in the Young (TEDDY)

TEDDY Screening and Enrollment

Pregnant Women

Newborns

Screening for HLA-DR,DQ genotypes

Low-RiskExcluded from Study

95% of general population70% of first-degree relatives

High/Moderate RiskFollow up

5% of general population30% of first-degree relatives

Europe:FinlandSwedenGermany

N. America:ColoradoGeorgia/FloridaWashington

Sibling/offspring cohort

6 centers

General population cohort

6 centers

Screened = 216,000 = 4,800

Eligible (high risk) = 11,880 (5.5%) = 1,440 (30%)

Enrolled = 5,940 (50%) = 1,152 (80%)

Autoimmunity cases= 238-500 = 152-300 by age 15

T1DM cases = 238 (4%) = 152 (13.3%) by age 15

TEDDY Proposed Study Population

Major Goals of TEDDY

Identify environmental triggers for T1DInfectious agentsDietary factorsPsycho-social factors

Understand gene/environment interactions

Refine screening strategies

Summary

Individuals predisposed to monogenic autoimmune diseases are readily detectable by newborn screening.

Most common autoimmune diseases are multifactorial.

Multifactorial diseases can be predicted by a combination of genetic and other risk factors.

Screening for autoimmunity may soon become an important issue for PHL.

AcknowledgementsAcknowledgementsMouse microarray

Sarah Eckenrode, Ph.DQingguo Ruan

Ping Yang

Weipeng ZhengQuanZhen Li, Ph.DHuman microarray

HongShan Zhao, Ph.D

Christin Collins

PANDAAndy Muir, M.D.Diane HopkinsAna CaroDesmond Schatz, MDMark Atkinson, PhD

University of Colorado, Denver

Marian Rewers, MD

George Eisenbarth, MD, Ph.D

& DAISY team

Proteomics

Sharad Purohit, Ph.D,

Dan Eisenman

BioinformaticsRichard McIndoe, Ph.DYan Zhang, Ph.D Yihua Huang, Ph.DJohn Hopkins

BiostatisticsRobert Podosky, Ph.DMark Yang, Ph.D, Prof.James Yang, Ph.D

uGet Get TEDDY slides

Day 3 Thymectomized Mice

uDevelop organ-specific autoimmunity• Prostatitis• Orchitis• Epididymovasitis• Thyroiditis• Gastritis• Oophoritis

uDeficiency in CD4+ CD5high regulatory T-cells

Induction of Autoimmune Diseases in Mice by Eliminating CD4+ and CD25+ (IL-2Rα+) T-cells

uOrgan-specific autoimmune diseasesuGraft-versus-host disease (GVHD)-like

wasting disease

• Gastritis

• Oophoritis

• Thyroiditis

• Insulitis

• Adrenalitis

• Glomerulonephritidis

• Sialoadenitis

• Arthritis

Mater(Maternal effect gene)

B6 KO

B-actin 1

B-actin 2

2nd PCR

B-actin 3

B-actin 4

B-actin 5

B6 KO

B-actin

1

2Mgat5 (32X)

No difference found: ATP4a, ATP4b

No amplification in TEC:After nest PCR: EAPA1, EAPA2

1st PCR: BMP1, BMP5, BMP8a, INF_gamma, GAD65

BMP8bBMP10

B-actin

ICA69

Rbp3 (nest PCR)

Hypothetical KRAB box containing protein

B-actin

KRAB

Ag H Ki Li LN Lu Ov Pa Mu Sp Te Th BMDC Pr

B-actin

HPRT

4F9R

B6 Aire-/-

Aire expression in Bone-Marrow DC

N Thymus1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

S1 S2 S3 T1 T2 T3 T4 T5 C1 C2 P MS1 S2 S3 T1 T2 T3 T4 T5 C1 C2 P M

Bone morphogenetic protein

S1 S2 T1 T2 T3 T4 T5 C1 C2 P M

Mannoside acetylglucosaminyltransferase V (Mgat5)

Fig. 4: PCR confirmation of Aire/DNA complexes from ChIP. ChIP-enriched DNA was amplified using primers specific for the flanking region of the Aire binding site. Two genes are shown here as examples. S1-S3: NOD spleen pools. T1-T5: Thymus pools 1-5. C1 & C2: Mock ChIP control. P: positive control using regular mouse genomic DNA. M: marker.

0

2040

60

80

100

120

140160

1 2

Tgn + control

Tgn + AIRE

0

2040

60

80

100

120

140160

1 2

Fabp2 + control

Fabp2 + AIRE

Thyroglobulin Fatty acid binding protein 2

Fig. 6: Luciferase co-transfection assay for promoter activity. Data from two replicates were shown for each promoter.

AIRE binds to the promoters with specific sequence motifs

AIRE Contains Protein Domains of Transcription Factors

u4 LXLL motifs: binds nuclear receptorsu1 SAND domain: mediates DNA

interaction of nuclear proteinsu2 PHD (CYS4-His-CYS3) zinc-finger

domains found in transcription factorsu1 proline rich region

+/+ -/-

B-actin

Bmp 9

Fig. 5: PCR analysis of expression of the bone morphorgentic protein 9 (Bmp 9) in whole thymus tissue from Aire(+/+) and knockout Aire(-/-) mice. mRNA was reversed transcribed into cDNA. Serial dilutions (2 fold) of the RT products were used as template for RT-PCR for β actin (26 cycles) and Bmp 9 (30 cycles).

1 1/2 1/4 1/8 1/16 1 1/2 1/4 1/8 1/16 Dilution

Expression difference in KO mice