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 Today’s contents 1. 自己免疫疾患関連遺伝子の同定 2. SNPによる大規模LDマッピング 3. 関節リウマチとPADI、抗シトルリン化ペプ チド抗体 4. PADI4多型の同定 Genetic predisposition in autoimmunities Wandstrat A. and Wakeland E. Nat Immunol 2(9) 802,2001 HLA and autoimmunities Overlaps of associated genes 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 Genetic analysis of common diseases • Hypothesis-free whole-genome approach – In order to identify novel pathologic mechanisms Large-scale case-control screening with SNPs Two Ways of Whole Genome Approach Map-based Approach 2 1 6 3 7 5 4 Gene D Gene A Gene B Gene C Gene-based Approach 12 3 8 910 567 4 Gene D Gene A Gene B Gene C 8 9 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) Public gene database Build (NCBI) No. mRNA No. genes 27 37823 37486 28 51689 51258 29 46438 45753 30 35273 33809 31 27634 25336 32 34839 32208 33 When completion of human genome sequence was announced. 37498 34731 34 25334 22193 Adopted SNPs from JSNP db ~Gene-based approach~ • 105,123 SNPs – In 16,676 genes • 5.6 SNPs per a gene in average Gene structure 5flank Exon Intron 3flank Inter-gene Undetermined 4022 18305 68542 2076 10024 2154 4% 17% 65% 2% 10% 2% 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 Identification of SLC22A4 and RUNX1 as RA-associated genes RA or Crohn disease-associated SNPs in Ch5q31 Cytokine cluster Chromosome 5q31 IL3, IL4, IL5, IL9, IL13, CSF2, IRF1 and TCF7 are in the region • Recessive association • Relative Risk = 2 The SNP changes affinity of RUNX1 10kb Genes RIL SLC22A4 SLC22A5 IRF1 Exons SNPs RA-associated SNP 1 2 3 4 Exons SNPs ……CAGGTTATGTGG C/T GAAGGATAAG…… RUNX1 binding site 5 6 78 9 10 The SNPs associated with RA or Crohn disease 10kb Genes RIL SLC22A4 SLC22A5 Exons RA Crohn SNPs RUNX1 binding site L503F Heat shock elementbinding site IRF1 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) RA, SLE and Psoriasis-associated SNPs disrupt RUNX1 binding motif 3 genes with RUNX1 binding sequence SLE Psoriasis RA Table Disease Gene Cytoband Function of gene RUNX1 binding motif Location Length evaluated with reporter assay SLE PDCD1 2q37.3 An immunoreceptor with tyrosine-based inhibitory motif TGCg/aGT Intron 4 Not Done TGTg/aGT Intergene 102 bp TGTGGt/c Intron1 24 bp SLC9A3R1 Psoriasis 17q24-q25 NAT9 Rheumatoid arthritis SLC22A4 Regulatory molecule of functional membrane N-acetyltransferase 5q31 Organic cation transporter Identification of RA-susceptible variant in PADI4 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) 感度 特異度 PPV リ ウマ トイド因 子陽性 54% 89% 85.70% 抗シ トルリ ン化 ペプチド抗体陽 性 38% 98.70% 97.40% リ ウマ トイド因 子&抗シ トルリ ン化ペプチド抗 体陽性 30% 100% 100% 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 Peptidyl citrullination Arginine Citrulline NH2 C=NH2 + NH CH2 CH2 CH2 HCNH3+ COO- NH2 C=O NH CH2 PADIs CH2 CH2 HCNH3+ COO- Loss of ionic NH2+ of Arg residue Effects on intra- and intermolecular interactions Detection of citrullinated filaggrin by SDS-PAGE and Western blotting Augmentation of T-cell response by citrullinated peptides J.Hill E Cairns et al. J Immunol 2003 HLA class II genes and aCCP • Carrier of SE DRB1 was associated with aCCP positivity SE status OR(95% CI) for aCCP positivity SE/SE 13.3 (4.6-40.4) SE/x 3.3 (1.8-6.0) x/x 1 SE: Shared epitope P <0.001 <0.001 F van Gaalen R de Vries et al. Arthritis & Rheum 2004 Molecular modification as peptidyl-citrullination alters tertially structure of selfpeptides. Production of anti-citrullinated antibodies under the influence of HLA molecules ? Chromosome 1 PADI cluster Result of 1st step screening by case-control association Results of Affected Sib-pair Linkage Analyses Association Plots in the PADI Cluster PADI4 Exons -log10(P)＝５ 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. Fraction RA patients with 2 copies of susceptible type were more frequently to be positive for anti-citrullinated filaggrin antibody 100 90 80 70 60 50 40 30 20 10 0 P-0.04 Positive Negative 2 copies 1 copy 0 copy Number of copies of RA-susceptible allele Following UK study on PADI variants(1) Haplotype frequency Haplotype frequency in British and Japanese 100% Others 80% 60% haplotype 2 40% RA-susceptible haplotype haplotype 1 20% 0% Japanese British Ethnic groups UK study on PADI variants(2) Japan UK Case Control Case Control N 830 736 839 481 Susceptible haplotype frequency 0.32 0.25 0.32 0.30 Protective haplotype frequency 0.52 0.60 0.56 0.59 Hypothetical mechanism of RA-susceptible variant Summary for PADI4 • Hypothesis-free approach identified an RAassociated 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. Acknowledgment • 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 http://134.160.84.101/ra/
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