Identification of rheumatoid arthritis-associated PADI4, SLC22A4/A5, FCRL3 & PTPN22

1. Identification of rheumatoid arthritis-associated PADI4, SLC22A4/A5, FCRL3 & PTPN22 International Symposium on Inflammatory Diseases of Barrier Organs 3-4 June 2005, Kiel, Germany Ryo Yamada Center for Genomic Medicine Kyoto University Kyoto, Japan

2. Genetics and Genetic Analysis of Rheumatoid Arthritis • Twin and family studies • Relative risk to monozygotic twin ( λMZ ) • 12~62 • Relative risk to siblings (λsib) • 2~17 • HLA locus explains 1/3-1/2 of total genetic components. • There are multiple non-HLA genes. • Multiple linkage studies • Many candidate-approach studies

4. Major findings from SNP-based studies • PADI4 : RA • Post-translational enzyme to produce targets of the most RA-specific autoantibodies. • SLC22A4 /A5 : RA & Crohn • Ergothioneine or carnitine transporter expressed in hematologic lineages. • PTPN22 : T1DM, SLE, RA & AITD • Lymphoid-specific intracellular phophatase • FCRL3 : RA, SLR & AITD • Fc receptor homolog on B-cell membrane

5. ＰＡＤＩ４Ca2+-dependent post-translational modification Arginine Citrulline NH2 NH2 C=NH2 + C=O NH NH CH2 CH2 CH2 CH2 CH2 CH2 PADIs HCNH3+ HCNH3+ COO- COO- Loss of ionic NH2+ of Arg residue Effects on intra- and inter- molecular interactions

6. ＰＡＤＩ４Ca2+-dependent post-translational modification K Arita M Sato et al. Nat Str & Mol Biol 2004

8. PADI4CitrullinationAntigenicity この部分だけ！

9. Human and Mouse PADI Gene cluster

11. Association Plots in the PADI Cluster PADI4 -log10(P)＝５

12. Enzyme substrate

13. Hypothetical mechanism of RA-susceptible variant

14. Following studies • Case-control association studies • UK (Barton, A. J., Worthington et al. 2004 Arthritis & Rheum): • Similar allele frequency • No statistical significance • Japanese (Kuwahara, M., Kamatani, N et al. 2004 ACR Poster) • Allelic effect on expression • Higher concentration of PADI4 in PBMC from individuals with more copies of “RA-susceptible haplotype”(Harney, S.M., Brown, M.A. et al. 2004 ACR Poster) • Allelic effect on autoantibody-production • Combinatorial effect on anti-CCP Ab production between HLA and PADI4 polymorphisms”(Constantin, A., Cantagrel, A., et al. 2004 ACR Poster)

15. SLC22A4(/A5) * Nucleotide binding motif * C * + + * + + 5 3 1 2 4 6 10 7 8 9 11 + N Sugar transporter signature sequence motif

17. The SNPs associated with RA or Crohn disease 10kb RIL SLC22A4 SLC22A5 IRF1 Genes Caucasian Japanese Exons Crohn Psoriatic Arthritis RA & Crohn SNPs Heat shock element-binding site RUNX1 binding site L503F Tokuhiro, S. et al. Barton, A. et al. Peltekova, V. D. et al.

18. Ethnic difference in allele frequency of disease-related SNPs 10kb SLC22A4 SLC22A5 IRF1 RIL Genes Exons SNPs intron1 intron2 Leu→Phe promoter(slc2F2 ) (slc2F1) J vs. C J vs. AC vs.A J CAP value FST P value FST P value FST SNP10.290.090.04 <0.00001§0.064*<0.00001§0.12* 0.14 0.012 SNP20.290.090.04 <0.00001§0.066*<0.00001§0.12* 0.14 0.012 SNP30.0010.390.04 <0.00001§0.24*<0.00001§0.018 <0.00001§0.19* SNP40.001 0.520.58 <0.00001§0.35* <0.00001§ 0.40*<0.00001§0.0038 §P<0.01, ＊FST> 0.05 Mori, et al. J Hum Genet 2005 May 10

19. Assumption of common responsible variant Japanese Caucasian 0W01M0 0W01M0 0M1 1M1 Segregation

20. FCRL3Fc Receptor-like 3 Transmembranous protein （734aa) FcγR homolog ６ Ig-like domains outside Multiple tyrosin motifs (ITAM, ITIM, hemi-ITAM) Expressed by B cells in germinal centers

21. FCRL3 and others in 1q21-23 マウス ヒト FcγRI FCRL1~5 CD1 FcγRII/III CD3Z CIA（Mcia2) 1q21 mCh3 1q23 mCh1 EAE,TMEVD (Eae3, Tmevd2) Psoriasis Multiple sclerosis NOD (Idd10, Idd17) SLE Lupus models (sle1, swrl1) RA

22. NFkB regulates FCRL3 expression Binds C > T

23. Lymphoid organs and B cell- specific expression

24. Expression of FCRL3 in lymphocyte aggregates in RA synovial tissue B-cell CD20 100x T-cell CD3 100x FCRL3 Antisense 100x 400x

25. RAにおけるFCRL3の働き(仮説）

26. PTPN22 • 1p13 • 110kDa intracellular tyrosine phosphatase • Functional SNP （1858 C->T, R620W) • Positive association • IDDM (Caucasian, Italian)Bottini N et al. • RA (Caucasian)Begovich AB et al. • SLE (Caucasian)Kyogoku, C. et al. • No Tryptophan (W) allele in Japanese

27. PTPN22 10kb GenesExonsSNPs PTPN22 Chromosome1p13 Arg→Trp J vs. C J vs. AC vs.A J CAP value FST P value FST P value FST SNP1 0.00 0.08 0.02 <0.00001§ 0.040 0.00032§ 0.011 0.16 0.016 §P<0.01　 ＊FST >0.05 Mori, et al. J Hum Genet 2005 May 10

28. Connection of multiple autoimmune diseases by susceptible genes/loci

29. Environmental Entry of Antigen Production of Antigen PADI4 Genetic Body PTPN22 Response to Antigen Presentation of Antigen Recognition of Presented Antigen RUNX1 SLC22A4/A5 Immune System Transient Autoreaction Manifestation of phenotype Maintained Autoreaction From the Atlas of Renal PathologyAmerican Journal of Kidney Diseases, Volume 32, Issue 1 Medical Encyclopedia

30. MΦ Anti-oxydant transporter

31. 対象とする自己免疫疾患関連遺伝子 Crohn PDCD1 SLE RA T1D PADI4 PTPN22 locusgeneSNP数・機能　 PADI44SNPs シトルリン化酵素 SLC22A43SNPs 　　　　　　　 有機カチオントランスポーター 代表基質：Ergothioneine SLC22A51SNP 有機カチオントランスポーター 代表基質：carnitine PDCD11SNP 　　　　　　　 免疫グロブリンスーパーファミリー 分子 PTPN22 1SNP TCRシグナル抑制性制御酵素 1 2 3 4 SLC22A4/A5 Crohn：クローン病SLE：全身性エリテマトーデスT1D：Ⅰ型糖尿病

32. 疾患感受性SNP アレル頻度　人種による比較 JCA 人種差 　なし 　あり 　あり 　あり 　あり 疾患　　　　 既報での関連　あり　　　なし　　 RAJ・CC RA JCCrohn Crohn C　　－(J:多型なし) RA, SLE C－T1D RA, SLEC　　 －　T1D PADI4 SLC22A4/5 SLC22A4/5 PDCD1 PTPN22

33. Caucasian PDCD1 MIF1 CTLA4 SLC22A4/A5 PTPN22 TNFRSF1 PADI4 Preliminary calculation with random-effect model Susceptible allele Japanese

34. Lab. Rheumatic Diseases Dr. K. Yamamoto Dr. A. Suzuki Dr. X. Chang Dr. Y. Kochi Dr. S. Tokuhiro Ms. R. Kawaida Ms. M. Mori Ms. K. Kobayashi Ms. M. Ohtake Ms. E. Kanno Ms. K. Komakine SRC, RIKEN Dr. A. Sekine Dr. T. Tsunoda Dr. Y. Nakamura Mr. H. Kawakami Mr. T. Kawaguchi Clinical Institutes Dr. T. Sawada And many other collaborators Thank you all !