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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