Genetic factors in autoimmune myasthenia

gravis

Henri-Jean Garchon

Inserm U567 - Institut CochinTeam « Chronic Inflammation and Immune System »

December 2009

Euromyasthenia

MG as a genetic disease• Issues

– Very few multicase families– Rare disease– Several levels of heterogeneity– Multifactorial

• However– Well-defined phenotype– One well-established genetic factor:

• The Major Histocompatibility Complex

Seronegative

Follicular hyperplasiaEarly Onset

Thymomas Late-onset MG

Anti-MuSK antibodies

F >> MOnset age < 40High anti-AChR

M = FOnset age : 30-60

anti-AChR +

M > FOnset age > 60

anti-AChR +

Normal thymusEarly Onset

F > MOnset age < 40

Heterogeneity of MG

SeronegativeDR3↗

Follicular hyperplasiaEarly Onset

Thymomas Late-onset MG

Anti-MuSKDQ5↗

Normal thymusEarly Onset

HLA heterogeneity

A25↗

A2↘

DR3↗DR7↘

DR3↗

DR7↗DR3↘

Non-MHC genes associated with MG

Locus symbol, gene product Variant or marker Mechanism

CHRNA1, -subunit of muscle nicotinic acetylcholine receptor

promoter SNP (-478A/G), G allele alters binding of IRF8 transcription factor and response to interferon-

Causal, affects the gene promiscuous expression in thymus and tolerization

PTPN22, cellular tyrosine phosphatase 22 Coding (Arg620Trp); Trp allele impairs binding to Csk kinase

Causal, immunoregulatory, increases phosphatase activity and impairs T cell activation, including IL2 production

FCGR2A, type 2A low affinity receptor for IgG

coding (R131H), H variant increases receptor affinity for IgG2

Potentially modifies regulation of B cell activation

CTLA4, cytotoxic T cell late antigen 4 Coding (Thr17Ala), alteration of glycosylation pattern by the signal peptide variant; 3’UTR microsatellite

Molecular and immunological mechanisms debated

CHRNG, CHRND; - and -subunits of muscle nicotinic acetylcholine receptor

Intronic microsatellite unknown

ADRB2, 2-adrenergic receptor Coding SNP (Gly16Arg) unknown

IL1B, interleukin-1 SNP in exon 5 Disease-associated allele associated with ‘high-secretor’ phenotype

IL10, interleukin-10 Microsatellite, SNPs in upstream region Potentially upregulates IL10 expression, promoting B-cell growth

IFNG, interferon- Non coding SNP (+874A/T), maps to a putative NF-B binding site

Potentially alters IFN- production

TCRA, T-cell receptor locus Restriction fragment length polymorphisms Antigenic peptide recognition

IGH, immunoglobulin heavy chain Gm allotypes unknown

IGK, immunoglobulin -chain Coding SNP, conservative change unknown

Three genetic factors in MG

The PTPN22 R620W coding variant

The CHRNA1 promoter variant

The MHC

The Arg620Trp (R620W) variant

of the PTPN22 gene

Tyrosine PhosphataseAssociated with several

autoimmune diseases in various populations:– Type 1 diabetes, – Autoimmune thyroiditis – Rheumatoid arthritis– Addison’s disease– But NOT: multiple sclerosis,

sjögren’s syndrome, inflammatory bowel disease

Relative risks ~1.5-2Souris ptpn22-KO, LD analysis

and functional studies indicate that R620W is causal

R620W: gain of fonction

PTPN22

T-cell Receptor

Csk

PTKinactivePTK

active

P

Activation

CskPTPN22*620W

PTKactive PTK

inactive

P

Inactivation IL2 production

The Arg620Trp (R620W) variant

of the PTPN22 gene

7,4

13,7

9,8 9,4

0

2

4

6

8

10

12

14

16

T allele

T a

llel

e fr

eq.

(%)

Controls (296)

EOMG (293)

LOMG (97)

Thymomas (80)

• The risk T allele is associated with nonthymoma MG without anti-titin antibodies.

• No effect on AChR autoantibody titers, gender, HLA type, association with other autoimmune diseases

• Smoking?

OR=1.97 (1,32-2,97); P=0.0006

• Vandiedonck, Annals of Neurology 2006• Lefvert, J. Neuroimmunology 2008• Greve, Human Immunology 2009• Chuang, Genes Immunity 2009

The CHRNA1 gene

• Role of -subunit in synthesis and structure of AChR

• Target of autoantibodies (main immunogenic region)

• Mutation of CHRNA1 causes CMG

Membrane

unfolded

Cytoplasmic

Extracellular

C192C193

ACh binding

site

NH2

COOH

M4M3

M2M1

MIR

Thymic expression of auto-antigensCentral tolerance

Promiscuous expression of auto-antigens in medullary Thymic Epithelial Cells (mTECs)

Central in immune tolerance Mechanism: negative selection of T cells, positive selection of

Tregs

Type I diabetes VNTR of the INS gene -> decreased thymic expression of INS Accelerated diabetes of NOD mice with proinsulin2-ko locus

APS1 ou APECED (Autoimmune Polyendocrinopathy Candidiasis-Ectodermal Dystrophy)

Mutations in the AIRE gene (Auto Immune REgulator) Expressed in medullary Thymic Epithelial Cells (mTECs) AIRE-ko mice: Defective expression of auto-antigens in mTECs

CHRNA1 polymorphism

• CHRNA1: chr 2, 10 exons, 17 kb• 16 re-sequenced individuals• 70 bi-allelic polymorphisms

– 43 common ( MAF > 5% )

1 2 3 P3A 4 5 6 7 8 9

1000 pb

-3295 pb +1 pb+16549 pb

(Matthieu Giraud, Nature 2007)

Association of group B polymorphisms with early onset of MG with thymus

hyperplasia

1 2 3 P3A 4 5 6 7 8 9

-3295 pb +1 pb +16549 pb

0,23

0,11 0,11

0

0,1

0,2

0,3

50 130 151

Perc

ent

0,21

0,11 0,11

0

0,1

0,2

0,3

46 104 109

Perc

ent

Early onset

Late onset

Controls P=0.00015OR=2.66 (1.6-4.4)

French Oxford(N.Willcox & D.Beeson)

Group B TagSNPSNP29SNP11SNP9

IRF8

Association of SNP rs16862847 (B11) with a decrease of CHRNA1 expression in mTECs

ex-vivo

Effect of SNP B11 (rs16862847)1.8-fold decrease of CHRNA1

levelP=0.0062

AIRE and CHRNA1 levels

(Richard Taubert and Bruno Kyewski)

Transcriptional activation in a TEC line

Lu

cife

rase

Rel

ativ

e A

ctiv

ity

AIRE (ng)

SNP11

SNP11

Constructs

A

IRF8

CHRNA1

G

IRF8CHRNA1

mTEC mTEC

Autoimmunity thresholds

FGA

E

CBTNF

DR

C4BBf

DQ

DP

The Major Histocompatibility Complex (MHC)

The issue of linkage disequilibrium

Geneticmarkers

Diseaselocus

LD = non random association of alleles at neighbour loci

At the basis of association mapping

A strong LD confounds fine mapping

Recombinants

8.1 haplotypes(microsatellite-based)

Reconstruction in trios

Assisted with Simwalk

Transmission tested with TDT

C. Vandiedonck (PNAS 2004)

F G A E CB TNF DRC4B

Bf DQ DP

MYAS1

auto-Ab

titers

tel cen

0

0.5

1

1.5

2

2.5log10 (Ab titers)

non 8.1

8.1 w/o DR3

8.1

P = 8 x 10- 6

60% 5%

0

50

100

- / - + / - + / +

% OR = 6.52 (3.37-13-5)

OR = 42.6 (11.4-182)

P = 7.1 x 10-11

Patients Controls(n=130) (n=105)

Multiple effects of the MHC in EOMG

MHC SNP genotyping

• 438 subjects:

– 178 patients + 172 relatives

– 88 controls

– MG patients with thymic follicular hyperplasia

• « Gene-centric » Illumina bead arrays (CNG)

• 1040 SNPs genotyped:

– Call rate > 95%

– In Hardy-Weinberg equilibirium in both patients and

controls

– Minor allele frequency > 1%

– Average density = 1 SNP/5 kb

1040 SNPs; ~5 Mbases; Corrected P values

- L

og

P

MHC SNP mapping in MG

Ivo Gut, CNG

Reconstruction of SNP haplotypes

MHC position (between 28.9 and 33.9 Mbases)

Each row is a haplotype; each column is a SNP Yellow matches the 8.1 haplotype allele; blue indicates the alternative allele.

Patient haplotypes (n=210) Control haplotypes (n=170)

- lo

g1

0 (

p v

alu

e)

p = 2.76x10-4

DRB1 TNF B C E A

9N-I MH C125

Protective microsatellites alleles in MG patients

Family-based association study

TNFdOR=0.52 (0.29-0.94)

D6S265OR=0.30 (0.13-0.65)

MYAS1MYAS1

F G A E CB TNF DRC4B

Bf DQ DP

MYAS1

auto-Ab

titers

tel cen

Complex but significant effects

Protection

OR = 0.36 (0.23-0.55)P = 4.2 x10-7

Jean-Grégory CormierNick WillcoxAlex Marx, Ph. Ströbel

Myasthenia Gravis Genetics Consortium

(MGGC)• Genome-wide association study

– MG samples from Sweden, France, UK, Norway …– Genotyping by Peter Gregersen– Data analysis by Michael Seldin

• Goals– To extend the recruitment for GWAS– To pursue the genes underlying association

signals– To seek funding

• Open group

Acknowledgements

InsermJean-Grégory CormierMatthieu GiraudClaire Vandiedonck

Centre National de GénotypageIvo Gut

German Cancer CentreBruno KyewskiRichard Taubert

Mannheim Institute of PathologyAlex MarxPhilipp Ströbel

Oxford Neurosciences groupAngela VincentDavid BeesonNick Willcox

Manchester UniversityXiayi Ke

ICGEB, TriesteFranco PaganiFrancisco Baralle

French NeurologistsPhilippe GajdosBruno EymardChristine Tranchant

Comparing two DR3 haplotypes: DR3-B8 and DR3-B18

DR3-DQ2BA

Conservation of the core 8.1 haplotype

Conclusion

• Current genomic and statistical tools allow to identify functional (causal) variants

• The MHC has a special status in MG:– Large effects– Multiple sub-phenotypes– Protective alleles

Genetic varia

nt

Disease

Subphenotype

Cellular e

vents

Gene express

ion

Genetic factors in autoimmune MG

• The HLA antigens, in the major histocompatibility complex (MHC)– HLA-DR3/B8/A1 (8.1) haplotype (Compston, Dawkins,

Janer)– Patients with early disease-onset and thymus follicular

hyperplasia– Observed in all Caucasian populations

• Non MHC genes– Regulatory: B2AR, IL10 (IL6, IL4,CCR2, CCR5)– Effector: CTLA4, IL1, IL1RA, IgKappa, (B7H3)– Target: CHRNA1, CHRNG, CHRND (CHRNB)

Acknowledgements

Inserm: Myasthenia GravisJean-Grégory CormierClaire VandiedonckMatthieu Giraud

Inserm: SpAMaxime BrebanGilles ChiocchiaRoula Saïd-NahalFélicie CostantinoEmma Walton

Centre National de GénotypageIvo Gut

German Cancer CentreBruno KyewskiRichard Taubert

Mannheim Institute of PathologyAlex MarxPhilipp Ströbel

Oxford Neurosciences groupAngela VincentDavid BeesonNick Willcox

Manchester UniversityXiayi Ke

ICGEB, TriesteFranco PaganiFrancisco Baralle

French NeurologistsPhilippe GajdosBruno EymardChristine Tranchant

Non-MHC genes not associated with MG

Locus symbol, gene product Marker tested

CHRNB1, -subunit of muscle nicotinic acetylcholine receptor

Microsatellite

CHRNE,-subunit of muscle nicotinic acetylcholine receptor

Intronic insertion-deletion polymorphism

IL1RN, IL1 receptor antagonist Variable number tandem repeat in intron 2

IL4, interleukin 4 Variable number tandem repeats

IL6, interleukin 6 Promoter SNP (-174C/G), alters an estrogen response element

CCR2, chemokine receptor 2 SNP (Val64Ile)

CCR5, chemokine receptor 5 32bp deletion in open reading frame

FCGR3B, type 2B low affinity receptor for IgG

Coding (F158V, NA1/NA2)

IL12B, interleukin 12 p40 subunit 3’untranslated region SNP

B7H3, B7 homolog 3 SNPs

The multifactorial nature of autoimmune diseases

Polygenicity, heterogeneityNongenetic factorsOften modest effectsComplex mechanisms:

– multiple pathways involved

Autoimmune myasthenia gravis (MG)a model of antibody-mediated

autoimmunity Prevalence : 1x10-4

Clinical features Weakness of skeletal muscles

Target of autoimmune response is known: muscle acetylcholine receptor (AChR)

Pathogenic anti-AChR autoantibodies 90% cases Passive transfer Highly specific (diagnosis) Quantitative trait

Heterogeneous disease Thymus anomaly in > 50% patients Follicular hyperplasia

=> women with onset < 40 yrs with high titers of anti-AChR antibodies

Thymic tumor

AChR

The ancestral 8.1 MHC haplotype

Associates HLA-A1-B8-DR3(A1 Cw7 B8 C4AQO C4B1 C2C Bfs DR3 DQ2) highly conserved in Caucasians extends over >3 x 106 bp associated with numerous immune

phenotypes and autoimmune diseases

and with MG with early onset/ thymus hyperplasia

suspected role of the central regionDR3 A1

DRX A1

DR3 AZ

B8

B8

B8

locus

DR3

B8 A1

DRx

Ay

Mapping the locus strategy of recombinant haplotypes combining family-based and case-

control design

MG patients without the 8.1 haplotypeAdditive model Dominant model

Allele Z score P Z score p

TNFb*7 3.281 0.001036 3.187 0.001437

FBAT

8.1 patients non 8.1 patients

Conserved SNP sequence of the 8.1 haplotype

1 #105_1604_AIMG

7 +++++++++++++++++++++++++++++++++++++

29 ........................+++++++++++++

1 #20_64_AIMG

7 +++++++++++++++++++++++++++++++++++++

9 ++++++++++++++++++++++++++........+..

1 #47_365_AIMG

15 +++++++++++++++++++++++++++++++++++++

91 ...+.....................++++++++++++

Reconstruction with PHASE 2Between HLA-B and AIF1

Alignment with the 8.1 haplotypes defined with microsatellites

Patients: 64 /210

Controls: 18 /170

OR = 3.7 (2.04-6.95)P = 2.8 x 10-6

10 SNP-wide sliding widow over 600 kb

+ indicates identity of 10/10 SNPs. indicates ≥ 1 mismatch/10 SNPs

FGA

E

CB

TNF

DR

C4BBf

DQ

DP

MY

AS

1

60% 5%

0

50

100

- / - + / - + / +

% OR = 6.52 (3.37-13-5)

OR = 42.6 (11.4-182)

P = 7.1 x 10-11

Patients Controls(n=130) (n=105)

Auto-Abexpressi

on

0

0.5

1

1.5

2

2.5log10 (Ab titers)

non 8.1

8.1 w/o DR3

8.1

P = 8 x 10- 6

Multiple effects of the MHC in MG

MHC and heterogeneity of MG in French Caucasian patients

ATA+

ThymomaNormalthymus

Thymus hyperplasia

MuSK+ DQ5

DR3 DR7

titin +

titin -

DR3

DR3

DR7 A2(B2 thymomas)

A25

CHRNA1 allelic associations

CHRNA1 polymorphisms in clusters

Pat

ient

s

A B C D E

Common homo

Rare homo

HeteroUndefined

Group::TagSNPs

Transcriptional control by SNP11 in a TEC line

Luciferase Relative Activity

SNP11SNP9

SNP11SNP9

SNP11SNP9

SNP11SNP9

1 2 3 P3A 4 5 6 7 8 9

-3295 pb +1 pb +16549 pb

SNP11SNP9

**

SNP11 disrupts the binding of IRF8

(interferon-response factor 8)1 2 3 P3A 4 5 6 7 8 9

-3295 pb +1 pb +16549 pb

SNP11

Early onsetG

A

IRF-8IRF-8

A G

Anti-

IRF-8

A G A G

Anti-

IRF-1

Super-shift

A

EMSA in RAW cells:

CHRNA1 promoter activity is IRF8-dependent

(RNA interference)

Construct:

IRF-8

siRNA

Non-targeting

RISC-free

IRF8

Firefly

*

CHRNA1 promoter activation in response to IFN depends on SNP11

SNP11

SNP11

Luciferase Relative Activity

Construct IFN

-

+

-

+

IRF-8

*

IRF8 binds to the endogenous CHRNA1 promoter by chromatin immunoprecipitation

Role of the self-antigen expression

A regulatory polymorphism of the self-antigen gene modifies the autoimmune phenotype in myasthenia gravis

It modulates its level of expression in mTECs, in conjunction with AIRE

It alters binding of a transcription factor, IRF-8, and controls its upregulation in response to IFN

Molecular mechanism of transcriptional control of a self-antigen gene in thymic epithelium

Importance of thymic expression of a self-antigen in central tolerance and in pathogenesis of human autoimmune diseases

Three genomic levels of genetic investigation

• Whole genome– Identify the candidate regions or genes

• Chromosomal region– Identify the candidate gene(s)

• Candidate gene– Identify the causative variant– Understand the biological mechanism