1. Large-scale Linkage Disequilibrium Mapping of Rheumatoid Arthritis-associated Genes in Japan ～ Results and Perspectives ～ December 9, 2005 Hanyang University Seoul, Korea Ryo Yamada Unit of Human Disease Genomics, CGM, Kyoto University, Kyoto Japan Lab. For Rheumatic Diseases, SRC, RIKEN, Yokohama, Japan

3. DNA-variations and their Effects on Transcripts, Peptides, Molecules … Phenotypes http://www.microbe.org/espanol/news/human_genome.asp

4. Missense SNPs in PADI4 gene RA-susceptible haplotype and non-susceptible haplotype with three missense SNPs and one silent SNP in coding region Allele specific mRNA stability and enzymatic activity and risk to RA Allele-specific molecular difference No allele-specific difference in molecular structure

5. Central Dogma & DNA Variations ＤＮ Ａ mRNA Peptide Transcription Translation Transcription initiation point Transcription termination point Splicing and mRNA maturation Translation initiation point Codon triplets Translation termination point Variations Post-translational peptide modifications Molecules

6. Not one way, but Multiple Bifurcations & Quantitative Variations ＤＮＡ ｍＲＮＡ１ ｍＲＮＡ２ Ｐｅｐｔｉｄｅ１ Ｐｅｐｔｉｄｅ２ Ｐｅｐｔｉｄｅ３ Ｍｏｌｅｃｕｌｅ３ Ｍｏｌｅｃｕｌｅ２Ｍｏｌｅｃｕｌｅ１ Ｍｏｌｅｃｕｌｅ４ Transcript variations Peptide variations Molecular variations

7. ＤＮＡ ｍＲＮＡ１ ｍＲＮＡ２ Ｐｅｐｔｉｄｅ１ Ｐｅｐｔｉｄｅ２ Ｐｅｐｔｉｄｅ３ Ｍｏｌｅｃｕｌｅ３ Ｍｏｌｅｃｕｌｅ２Ｍｏｌｅｃｕｌｅ１ Ｍｏｌｅｃｕｌｅ４ Phenotype1 Phenotype2 Phenotype3 Phenotype4

8. ＤＮＡ ｍＲＮＡ１ ｍＲＮＡ２ Ｐｅｐｔｉｄｅ１ Ｐｅｐｔｉｄｅ２ Ｐｅｐｔｉｄｅ３ Ｍｏｌｅｃｕｌｅ３ Ｍｏｌｅｃｕｌｅ２ Ｍｏｌｅｃｕｌｅ１ Ｍｏｌｅｃｕｌｅ４ Phenotype1 Phenotype2 Phenotype3 Phenotype4

9. Monogenic determination ～ Recessive trait ～ ＤＮＡ ｍＲＮＡ１ Ｐｅｐｔｉｄｅ１ Ｍｏｌｅｃｕｌｅ１ Disease mutation and mal-functional molecule Disease phenotype Non-disease phenotype

10. Monogenic Trait Complex Trait Missense Silent Non-coding Depth of transmission of allele-specific molecular difference depends on type of polymorphism Missense mutation and significant change of molecular function

11. Susceptible Non-susceptible Association study of Complex Traits with DNA-markers DNA RNA Peptides

12. Susceptible Non-susceptible Association study with DNA-markers Simplified Architecture Bypasses Elements between DNA and Phenotypes … Pros and Cons

13. Time course Triggering Event Disease Manifest ations Diag nosi s Clinical F/U Birth No observation In pre-clinical phase RNA, proteins and others DNA More dynamic and more direct information from Non-DNA analyses; Amount of Information is more but might be difficult to define their representatives among them. DNA-analyses Data is Simple and Fixed throughout the Life. Time course Birt RNA, proteins and othersDNA

14. Another big world of RNA genes Non-coding RNA x 23,000 in mammals

15. Coding-gene World and Variations Coding DNA Coding mRNA Non-coding-gene World and Variations DNA Functional RNA Effects on transcription Effects on translation ?? Effects on phenotypes??

16. Large-scale LD mapping and Identification of RA-Susceptible PADI4 Polymorphisms and Follow-up Replication Studies

17. Genetics and Genetic Analysis of Rheumatoid Arthritis Twin and family studies –Relative risk to monozygotic twin ( λ MZ ) 12~62 –Relative risk to siblings (λ sib ) 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

18. Two Ways of Whole Genome LD Mapping 9 8 7 6 5 4 1 2 3 1 23 7 6 98 4 5 Coding Gene-based Approach Map-based Approach Gene A Gene B Gene A Gene B Gene D Gene C Gene D

19. SNP distribution of RIKEN study

20. SNPs Samples Replication-based analysis SNPs Samples Stage 1 Stage 2 One-Stage Design Joint analysis SNPs Samples Stage 1 Stage 2 Two-Stage Design Michael Boehnke : Design Considerations in Large Scale Genetic Association Studies Design Considerations in Large Scale Genetic Association Studies HapMap Tutorials 836 vs. 658 two-stage joint screening

21. 12,890 / 21,153 genes 12,890 （ 60.9% ） genes were evaluated with block/SNPs No. SNPs per gene and density of SNPs 5.0±6.4 /gene 0.2±0.3 /kb No. coding genes in autosomal chromosomes ： 21,153 Covered with SNPs not in block Covered with block Not covered 4,509 8,381 8,263 12,890 Gene 200520002001200220032004 10k 20k 40k 30k 50k RIEN project started 27,283Genes

22. Major findings from SNP-based studies Japanese study (RIKEN) –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. –FCRL3 : RA, SLR & AITD Fc receptor homolog on B-cell membrane US study (A.Begovich et al.) –PTPN22 : T1DM, SLE, RA & AITD Lymphoid-specific intracellular phophatase

23. MΦ Anti-oxydant transporter

24. PADI4 Missense SNPs Stability of transcript SLC22A4 Intronic SNPs Transcriptional regulation FCRL3 Transcriptional regulation PTPN22 Missense SNP Molecular function? RR ～ ２ Promoter SNP RR ～ ２

25. Multiple Genes and Multiple Diseases

27. Rheumatoid Arthritis and PADI4 Citrulline and a-CCP Antibody Antiperinuclear Anti-keratin Anti-Sa Very many and heterogeneous autoantibodies are detectable in RA sera. Sensitivity and specificity vary. Some of RA-autoantibodies are extremely specific but their relatively low sensitivity limited their clinical utility. Their epitope target turned out to be common and citrulline residue in the molecules. Anti-CCP antibody has established as a reliable clinical marker of RA and they could predict development of RA several years before clinical onset. Citrulline is a non-coding native amino-acid and they are in proteins only after enzymatic conversion from arginine by PADI.

28. Anti-citrullinated peptide anyibody ~Most reliable autoantibody for RA

29. ＰＡＤＩ４ Ca 2+ -dependent post-translational modification Loss of ionic NH2+ of Arg residue Effects on intra- and inter- molecular interactions C=NH 2 + NH 2 CH 2 HCNH 3 + COO - NH C=O NH 2 CH 2 HCNH 3 + COO - NH ArginineCitrulline PADIs

31. この部分だけ！ PADI4 Citrullination Antigenicity

32. Association Plots in the PADI Cluster -log 10 (P) ＝５ PADI4

33. Enzyme substrate Missenes SNPs, but no allelic difference in enzyme activity.

34. RA-susceptible variant transcript is more stable.

35. Hypothetical mechanism of RA-susceptible variant

36. K Arita M Sato et al. Nat Str & Mol Biol 2004 ＰＡＤＩ４ Ca 2+ -dependent post- translational modification

38. Multiple studies on PADI4 padi_92 or padi_94 Asian Caucasian 3 “Positive” reports & 3 “Negative” reports

39. FCRL3 Fc 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

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

41. NFkB regulates FCRL3 expression Binds C > T

42. Lymphoid organs and B cell- specific expression

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

44. Hypothetical mechanism Clonal selection depends on reactivity to autoantigens Arthritis FCRL3 expression

45. Perspectives Genetic determinants of prognosis and clinical responsiveness Coding genes and non-coding genes Ethnic diversity and genetic factors Combination of multiple genetic factors with or without environment factors

46. Network of genes with susceptible polymorphisms and genes without susceptible variations but functionally important A network of protein–protein interactions in yeast by Benno Schwikowski, Peter Uetz3 & Stanley Nature Biotechnology 18 :1257 - 1261 (2000)

47. Caucasians Japanese PDCD1 MIF1CTLA4SLC22A4/A5 PTPN22TNFRSF1PADI4 Susceptible allele Preliminary calculation with random-effect model

48. Ethnic variations

49. Sugar transporter signature sequence motif N C 12 3 4 5 6 7 89 10 11 Nucleotide binding motif * * * * + + + + + SLC22A4(/A5)

50. Recessive association Relative Risk = 2 Chromosome 5q31 IL3, IL4, IL5, IL9, IL13, CSF2, IRF1 and TCF7 are in the region

51. The SNPs associated with RA or Crohn disease RILSLC22A4SLC22A5IRF1 10kb Genes Exons SNPs RUNX1 binding site RA & Crohn Crohn L503F Heat shock element- binding site Psoriatic Arthritis Tokuhiro, S. et al. Peltekova, V. D. et al. Barton, A. et al. Japanese Caucasian

52. SLC22A4 Ergothioneine transporter SLC22A5 Carnitine transporter

53. SNP1 0.29 0.09 0.04 <0.00001 § 0.064* <0.00001 § 0.12* 0.14 0.012 SNP2 0.29 0.09 0.04 <0.00001 § 0.066* <0.00001 § 0.12* 0.14 0.012 SNP3 0.001 0.39 0.04 <0.00001 § 0.24* <0.00001 § 0.018 <0.00001 § 0.19* SNP4 0.001 0.52 0.58 <0.00001 § 0.35* <0.00001 § 0.40 * <0.00001 § 0.0038 J vs. C J vs. A C vs. A J C A P value F ST P value F ST P value F ST §P 0.05 Ethnic difference in allele frequency of disease-related SNPs IRF1 RIL SLC22A4SLC22A5 10kb Genes Exons SNPs intron1 intron2 Leu→Phe promoter (slc2F2 ) (slc2F1) Mori, et al. J Hum Genet 2005 May 10

54. Japanese Caucasian 0W0 1M0 0W0 1M0 0M1 1M1 Assumption of common responsible variant Segregation

55. SNP Research Center, RIKEN, Yokohama, Japan –Lab. for Rheumatic DIseases Kazuhiko Yamamoto Akari Suzuki Yuta Kochi Mikako Mori –Akihiro Sekine –Tatsuhiko Tsunoda –Yusuke Nakamura Clinical Institutes of Collaboration –University of Tokyo Hospitals Tetsuji Sawada –National Sagamihara Hospital Shigeto Tohma Toshihiro Matsui Center for Genomic Medicine, Kyoto University, Kyoto, Japan –Fumihiko Matsuda –Shohei Chida –Alexandre Vasilescu –Hitomi Hiratani –Victor Renault –Masao Yamaguchi –Katsura Hirosawa –Kenei Ohigashi