大規模連鎖不平衡マッピングによる 関節リウマチ関連遺伝子解析

1. 大規模連鎖不平衡マッピングによる関節リウマチ関連遺伝子解析大規模連鎖不平衡マッピングによる関節リウマチ関連遺伝子解析 山田　亮 理化学研究所 遺伝子多型研究センター 関節リウマチ関連遺伝子研究チーム 平成16年9月18日

2. Today’s contents • 自己免疫疾患関連遺伝子の同定 • SNPによる大規模LDマッピング • 関節リウマチとPADI、抗シトルリン化ペプチド抗体 • PADI4多型の同定

3. Genetic predisposition in autoimmunities Wandstrat A. and Wakeland E. Nat Immunol 2(9) 802,2001

4. HLA and autoimmunities

6. Overlaps of associated genes

7. 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

8. Genetic analysis of common diseases • Hypothesis-free whole-genome approach • In order to identify novel pathologic mechanisms Large-scale case-control screening with SNPs

9. Two Ways of Whole Genome Approach Map-based Approach 7 6 2 8 1 3 5 9 10 4 Gene D Gene A Gene B Gene C Gene-based Approach 8 9 10 2 3 1 7 4 6 5 Gene D Gene A Gene B Gene C

10. Whole Genome Survey with SNPs by Linkage Disequilibrium Mapping • Prospects • Map-based approach • Markers are evenly distributed throughout the genome • No. of SNPs to cover the whole genome: 500,000 – 1,000,000 • Gene-based approach • Markers are distributed in gene-containing regions • No. of SNPs to cover the whole genome: 50,000 – 100,000 D. Botstein & N. Risch Nat Genet 33 Suppl,228-237(2003)

11. Public gene database

12. Adopted SNPs from JSNP db~Gene-based approach~ • 105,123 SNPs • In 16,676 genes • 5.6 SNPs per a gene in average

14. Case-control association tests for screening the whole genome • Two steps • (1) 94 cases vs. 658 controls • (2) 846 cases vs. 658 controls • 1,165 /105,123 SNPs passed an initial step (1) • 50 / 1,165 SNPs passed the second step (2) • in progress • 50 SNPs are located in 25 LD blocks

15. Identification of SLC22A4 and RUNX1 as RA-associated genes

16. RA or Crohn disease-associated SNPs in Ch5q31 Cytokine cluster

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

18. The SNP changes affinity of RUNX1 10kb RIL SLC22A4 SLC22A5 IRF1 Genes Exons SNPs RA-associated SNP 8 1 2 3 4 5 6 7 9 10 Exons SNPs ……CAGGTTATGTGGC/TGAAGGATAAG…… RUNX1 binding site

19. 10kb RIL SLC22A4 SLC22A5 IRF1 Genes Exons RA Crohn SNPs L503F Heat shock element-binding site RUNX1 binding site The SNPs associated with RA or Crohn disease

20. SLC22A4 and RUNX1 • SLC22A4 • Organic cation transporter • Unknown physiologic function • In 5q31 cytokine cluster linked with Crohn disease and asthma/atopy • RUNX1 • Hematologic transcriptional factor • Responsible for leukemia • Links with autoimmune diseases (SLE, psoriasis) Tokuhiro S et al. Nat Genet 35, 341-348 (2003)

21. RA, SLE and Psoriasis-associated SNPs disrupt RUNX1 binding motif

22. 3 genes with RUNX1 binding sequence SLE Psoriasis RA

24. Identification of RA-susceptible variant in PADI4

25. 関節リウマチ診断における、最も信頼できる自己抗体関節リウマチ診断における、最も信頼できる自己抗体 抗シトルリン化ペプチド抗体（抗CCP抗体)

26. Citrulline • Citrulline： • One of native amino acids, but not among 20 coding amino acids • Free citrulline and peptidyl citrulline • Hereditary citrullinemia (a metabolic disorder) • Unclear physiologic function of peptidyl citrullination

27. Arginine Citrulline NH2 NH2 C=NH2 + C=O NH NH CH2 CH2 CH2 CH2 CH2 CH2 PADIs HCNH3+ HCNH3+ COO- COO- Peptidyl citrullination Loss of ionic NH2+ of Arg residue Effects on intra- and inter- molecular interactions

28. Detection of citrullinated filaggrin by SDS-PAGE and Western blotting

30. Augmentation of T-cell response by citrullinated peptides J.Hill E Cairns et al. J Immunol 2003

31. HLA class II genes and aCCP • Carrier of SE DRB1 was associated with aCCP positivity F van Gaalen R de Vries et al. Arthritis & Rheum 2004

32. Molecular modification as peptidyl-citrullination alters tertially structure of self-peptides. Production of anti-citrullinated antibodies under the influence of HLA molecules ?

33. Chromosome 1 PADI cluster Result of 1st step screening by case-control association

34. Results of Affected Sib-pair Linkage Analyses

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

37. Two main transcript variants in PADI4 663 amino acids, 4 SNPs with 3 amino acid substitutions Gly 55 Ser, Val 82 Ala, Gly 112 Ala RA-susceptible haplotype 25％ in population RA-non-susceptible haplotype 60％ in population ●RR of Individuals with two copies of susceptible types is 2.

38. 100 90 P-0.04 80 70 60 Positive Fraction 50 Negative 40 30 20 10 0 2 copies 1 copy 0 copy Number of copies of RA-susceptible allele RA patients with 2 copies of susceptible type were more frequently to be positive for anti-citrullinated filaggrin antibody

39. Following UK study on PADI variants(1)

40. UK study on PADI variants(2)

41. Hypothetical mechanism of RA-susceptible variant

42. Summary for PADI4 • Hypothesis-free approach identified an RA-associated gene that was functionally strongly relevant to RA. • Functional variants of PADI4 were consisted of SNPs. • Many issues are still present to understand PADI and citrullination in RA pathogenesis.

43. Lab. Rheumatic Diseases Dr. K. Yamamoto Dr. A. Suzuki Dr. X.Chang Dr. Y. Kochi Mr. S. Tokuhiro Ms. R. Kawaida Ms. K. Kobayashi Ms. M. Ohtake Ms. E. Kannno Ms. K. Komakine SRC, RIKEN Dr. A. Sekine Dr. T. Tsunoda Dr. Y. Nakamura Mr. H. Kawakami Clinical Institutes Dr. T. Sawada And many other collaborators Acknowledgment

44. http://134.160.84.101/ra/