1. Genetic factors in autoimmune myasthenia gravis Henri-Jean Garchon Inserm U567 - Institut Cochin Team « Chronic Inflammation and Immune System » December 2009 Euromyasthenia

2. 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

3. Seronegative Follicular hyperplasia Early Onset ThymomasLate-onset MG Anti-MuSK antibodies F >> M Onset age < 40 High anti-AChR M = F Onset age : 30-60 anti-AChR + M > F Onset age > 60 anti-AChR + Normal thymus Early Onset F > M Onset age < 40 Thymic alteration Anti-titin Ab Anti-AChR antibodies Heterogeneity of MG

4. Seronegative DR3↗ Follicular hyperplasia Early Onset ThymomasLate-onset MG Anti-MuSK DQ5↗ Normal thymus Early Onset HLA heterogeneity A25↗ A2↘ DR3↗ DR7↘ DR3↗ DR7↗ DR3↘

5. Non-MHC genes associated with MG Locus symbol, gene productVariant or markerMechanism 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 22Coding (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 4Coding (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 microsatelliteunknown ADRB2,  2-adrenergic receptor Coding SNP (Gly16Arg)unknown IL1B, interleukin-1  SNP in exon 5Disease-associated allele associated with ‘high- secretor’ phenotype IL10, interleukin-10Microsatellite, SNPs in upstream regionPotentially 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 polymorphismsAntigenic peptide recognition IGH, immunoglobulin heavy chainGm allotypesunknown IGK, immunoglobulin  -chain Coding SNP, conservative changeunknown

6. Three genetic factors in MG The PTPN22 R620W coding variant The CHRNA1 promoter variant The MHC

7. The Arg620Trp (R620W) variant of the PTPN22 gene Tyrosine Phosphatase Associated 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 ~1.5-2 Souris ptpn22-KO, LD analysis and functional studies indicate that R620W is causal

8. R620W: gain of fonction PTPN22 T-cell Receptor Csk PTK inactive PTK active P Activation Csk PTPN22*620W PTK active PTK inactive P Inactivation  IL2 production

9. The Arg620Trp (R620W) variant of the PTPN22 gene 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

10. 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 C192 C193 ACh binding site NH 2 COOH M4 M3 M2 M1  MIR

11. Thymic expression of auto-antigens Central 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

12. CHRNA1 polymorphism CHRNA1: chr 2, 10 exons, 17 kb 16 re-sequenced individuals 70 bi-allelic polymorphisms –43 common ( MAF > 5% ) 1 2 3P3A 456789 1000 pb -3295 pb+1 pb +16549 pb (Matthieu Giraud, Nature 2007)

13. Association of group B polymorphisms with early onset of MG with thymus hyperplasia 1 2 3P3A 456789 -3295 pb+1 pb +16549 pb P=0.00015 OR=2.66 (1.6-4.4) French Oxford (N.Willcox & D.Beeson) Group B TagSNP SNP29 SNP11SNP9 IRF8

14. 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 level P=0.0062 AIRE and CHRNA1 levels (Richard Taubert and Bruno Kyewski)

15. Transcriptional activation in a TEC line Luciferase Relative Activity AIRE (ng) SNP11 Constructs

16. A IRF8 CHRNA1 G IRF8 CHRNA1 mTEC Autoimmunity thresholds

17. F G A E C B TNF DR C4B Bf DQ DP The Major Histocompatibility Complex (MHC)

18. The issue of linkage disequilibrium Genetic markers Disease locus LD = non random association of alleles at neighbour loci At the basis of association mapping A strong LD confounds fine mapping

19. Recombinants 8.1 haplotypes (microsatellite-based) Reconstruction in trios Assisted with Simwalk Transmission tested with TDT C. Vandiedonck (PNAS 2004)

20. FGAECBTNFDRC4BBfDQDP MYAS1 auto-Ab titers tel cen 0 0.5 1 1.5 2 2.5 log 10 (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 PatientsControls (n=130) (n=105) Multiple effects of the MHC in EOMG

21. 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

22. 1040 SNPs; ~5 Mbases; Corrected P values - Log P MHC SNP mapping in MG Ivo Gut, CNG

23. 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)

24. p = 2.76x10 -4 DRB1 TNFBCEA 9N-IMHC125 Protective microsatellites alleles in MG patients Family-based association study TNFd OR=0.52 (0.29-0.94) D6S265 OR=0.30 (0.13-0.65) MYAS1

25. FGAECBTNFDRC4BBfDQDP 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 Cormier Nick Willcox Alex Marx, Ph. Ströbel

26. 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

27. Acknowledgements Inserm Jean-Grégory Cormier Matthieu Giraud Claire Vandiedonck Centre National de Génotypage Ivo Gut German Cancer Centre Bruno Kyewski Richard Taubert Mannheim Institute of Pathology Alex Marx Philipp Ströbel Oxford Neurosciences group Angela Vincent David Beeson Nick Willcox Manchester University Xiayi Ke ICGEB, Trieste Franco Pagani Francisco Baralle French Neurologists Philippe Gajdos Bruno Eymard Christine Tranchant

30. Comparing two DR3 haplotypes: DR3- B8 and DR3-B18 DR3-DQ2BA

31. Conservation of the core 8.1 haplotype

32. 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 variant Disease Subphenotype Cellular events Gene expression

33. 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)

34. Acknowledgements Inserm: Myasthenia Gravis Jean-Grégory Cormier Claire Vandiedonck Matthieu Giraud Inserm: SpA Maxime Breban Gilles Chiocchia Roula Saïd-Nahal Félicie Costantino Emma Walton Centre National de Génotypage Ivo Gut German Cancer Centre Bruno Kyewski Richard Taubert Mannheim Institute of Pathology Alex Marx Philipp Ströbel Oxford Neurosciences group Angela Vincent David Beeson Nick Willcox Manchester University Xiayi Ke ICGEB, Trieste Franco Pagani Francisco Baralle French Neurologists Philippe Gajdos Bruno Eymard Christine Tranchant

35. Non-MHC genes not associated with MG Locus symbol, gene productMarker tested CHRNB1,  -subunit of muscle nicotinic acetylcholine receptor Microsatellite CHRNE,  -subunit of muscle nicotinic acetylcholine receptor Intronic insertion-deletion polymorphism IL1RN, IL1 receptor antagonistVariable number tandem repeat in intron 2 IL4, interleukin 4Variable number tandem repeats IL6, interleukin 6Promoter SNP (-174C/G), alters an estrogen response element CCR2, chemokine receptor 2SNP (Val64Ile) CCR5, chemokine receptor 532bp deletion in open reading frame FCGR3B, type 2B low affinity receptor for IgG Coding (F158V, NA1/NA2) IL12B, interleukin 12 p40 subunit3’untranslated region SNP B7H3, B7 homolog 3SNPs

36. The multifactorial nature of autoimmune diseases Polygenicity, heterogeneity Nongenetic factors Often modest effects Complex mechanisms: –multiple pathways involved

37. 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

38. 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 10 6 bp  associated with numerous immune phenotypes and autoimmune diseases  and with MG with early onset/ thymus hyperplasia  suspected role of the central region DR3 A1 DRX A1 DR3 AZ B8 locus DR3B8 A1 DRx Ay Mapping the locus  strategy of recombinant haplotypes  combining family-based and case- control design

39. MG patients without the 8.1 haplotype Additive modelDominant model AlleleZ scoreP p TNFb*73.2810.0010363.1870.001437 FBAT 8.1 patientsnon 8.1 patients

40. 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 2 Between 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

41. F G A E C B TNF DR C4B Bf DQ DP MYAS1 60% 5% 0 50 100 - / -+ / -+ / + % OR = 6.52 (3.37-13-5) OR = 42.6 (11.4-182) P = 7.1 x 10 -11 PatientsControls (n=130) (n=105) Auto-Ab expression 0 0.5 1 1.5 2 2.5 log 10 (Ab titers) non 8.1 8.1 w/o DR3 8.1 P = 8 x 10 - 6 Multiple effects of the MHC in MG

42. MHC and heterogeneity of MG in French Caucasian patients ATA+ Thymoma Normal thymus Thymus hyperplasia MuSK+ DQ5 DR3 DR7 titin + titin - DR3 DR7 A2 (B2 thymomas) A25

43. CHRNA1 allelic associations CHRNA1 polymorphisms in clusters Patients ABCDE Common homo Rare homo Hetero Undefined Group: :TagSNPs

44. Transcriptional control by SNP11 in a TEC line 1 2 3P3A 456789 -3295 pb+1 pb +16549 pb SNP11SNP9 * *

45. SNP11 disrupts the binding of IRF8 (interferon-response factor 8) 1 2 3P3A 456789 -3295 pb+1 pb +16549 pb SNP11 Early onset G A IRF-8 A G Anti- IRF-8 A G Anti- IRF-1 Super-shift A EMSA in RAW cells:

46. CHRNA1 promoter activity is IRF8-dependent (RNA interference) Construct: IRF-8 siRNA Non-targeting RISC-free IRF8 Firefly *

47. CHRNA1 promoter activation in response to IFN  depends on SNP11 SNP11 Luciferase Relative Activity Construct IFN  - + - + IRF-8 *

48. IRF8 binds to the endogenous CHRNA1 promoter by chromatin immunoprecipitation

49. 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

50. 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