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 Gene-based Approach Map-based Approach Gene A Gene B Gene A Gene B Gene D Gene C Gene D 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, (2003)

Public gene database Build (NCBI)No. mRNANo. genes When completion of human genome sequence was announced

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

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

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

The SNP changes affinity of RUNX1 RILSLC22A4SLC22A5IRF1 10kb Genes Exons SNPs Exons SNPs RA-associated SNP ……CAGGTTATGTGG C/T GAAGGATAAG…… RUNX1 binding site

The SNPs associated with RA or Crohn disease RILSLC22A4SLC22A5IRF1 10kb Genes Exons SNPs RUNX1 binding site RACrohn L503FHeat shock element- binding site

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, (2003)

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

3 genes with RUNX1 binding sequenceSLE RA Psoriasis

Table DiseaseGeneCytobandFunction of geneRUNX1 binding motifLocation Length evaluated with reporter assay SLEPDCD1 2q37.3 An immunoreceptor with tyrosine-based inhibitory motif TGCg/aGT Intron 4Not Done Psoriasis SLC9A3R1 17q24-q25 Regulatory molecule of functional membrane TGTg/aGT Intergene102 bp NAT9 N-acetyltransferase Rheumatoid arthritis SLC22A4 5q31 Organic cation transporter TGTGGt/c Intron124 bp

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 )

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 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

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 F van Gaalen R de Vries et al. Arthritis & Rheum 2004

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

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

Results of Affected Sib-pair Linkage Analyses

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

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 haplotype25 ％ in population RA-non-susceptible haplotype60 ％ in population ● RR of Individuals with two copies of susceptible types is 2.

RA patients with 2 copies of susceptible type were more frequently to be positive for anti-citrullinated filaggrin antibody copies1 copy0 copy Number of copies of RA-susceptible allele Fraction Positive Negative P-0.04

Following UK study on PADI variants(1)

UK study on PADI variants(2)

Hypothetical mechanism of RA-susceptible variant

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.

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