Gene-based Large Scale LD-mapping of Rheumatoid Arthritis-associated Genes and Variation in Associated Polymorphisms Among Ethnic Groups PADI4, SLC22A4/A5 & Others Ryo Yamada Laboratory for Rheumatic Diseases SNP Research Center RIKEN Yokohama Japan

Overview 1.Hypothesis-free gene-based LD mapping screening 2.Three major findings through the screening –RA-specific susceptible gene: PADI4 –Multiple-autoimmunity crossing: SLC22A4(/A5) & RUNX1 3.Ethnic variability

1.Hypothesis-free gene-based LD mapping screening 2.Three major findings through the screening –RA-specific susceptible gene: PADI4 –Multiple-autoimmunity crossing: SLC22A4(/A5) & RUNX1 3.Ethnic variability

Gene-based LD-mapping Gene-based Approach Map-based Approach Gene A Gene B Gene A Gene B Gene D Gene C Gene D 10

PADIs cluster in Chr1p HAPMAPSRC RIKEN 5Mb PADI2 PADI1 PADI3 PADI4 PADI6 Map-BasedGene-Based

Number of genes in 1p with or without genotyped SNPs by status of curation status curated predicted ■ genes with more than one SNPs genotyped ■ genes without SNPs genotyped number of genes Gene-based allocation Genes

70,422 SNPs 6,126 blocks 43,170 common haplotypes 22,612 htSNPs SNPs … Blocks … Haplotypes … Tagging SNPs Phase I Pick up of associated SNPs Phase II Pick up of associated blocks Under data-mining PADI4 SLC22A4 RUNX1 and more

Phase I Identification of associated SNPs with single SNP comparison Determination of extension of LD from the associated SNPs Discovery of SNPs in the extension Identification of causative variants and genes of association and susceptibility

-log(P) plots from small-scale comparison Chromosome 1 PADI cluster

Association Plots in the PADI Cluster with Denser and Larger Scale Comparison -log 10 (P) ＝５ PADI4

SNPs … Blocks … Haplotypes … Tagging SNPs Phase I Pick up of associated SNPs Phase II Pick up of associated blocks Under data-mining PADI4 SLC22A4 RUNX1 and more 70,422 SNPs 6,126 blocks 43,170 common haplotypes 22,612 htSNPs

Phase II Identification of LD blocks Identification of associated blocks Identification of variants and genes as origin of association and susceptibility Now under data-mining

Genes SNP Block Inter-block relation Haplotypes Tagging SNPs Linkage Disequilibrium Mapping Chromosome 1p Genes with SNPs

Tagging SNPs

Number of SNPs and htSNPs in block Number of blocks min max med mean±SD -- SNP ± htSNP ± 2.5 Number of SNPs and htSNPs per block Number of haplotypes in block Number of blocks Distribution of number of haplotypes per block Median = 6 ← Mean ±SD = 8.2±6.5 ← Maximum = 44 ← Blocks, haplotypes and tagging SNPs

70,422 SNPs 6,126 blocks 43,170 common haplotypes 22,612 htSNPs Phase I Pick up of associated SNPs Phase II Pick up of associated blocks Under data-mining PADI4 SLC22A4 RUNX1 and more ?? Phase III ?? Pick up of associated SNPs/segments by combining physically unlinked loci

1.Hypothesis-free gene-based LD mapping screening 2.Three major findings through the screening –RA-specific susceptible gene: PADI4 –Multiple-autoimmunity crossing: SLC22A4(/A5) & RUNX1 3.Ethnic variability

Three major findings through the screening RA-specific susceptible gene: –PADI4 –Post-translational enzyme to produce targets of the most RA-specific autoantibodies. Multiple-autoimmunity crossing: –SLC22A4(/A5) Function-unknown transporter expressed in hematologic lineages. Three SNPs in 500 kb are associated with; –RA, Crohn ’ s disease, Lupus and/or Psoriatic arthritis –RUNX1 Leukemia-responsible gene SNPs in three loci disrupt RUNX1 binding sequence and are associated with SLE, psoriasis or RA.

Identification of RA-susceptible variant in a gene with RA-specific function

Anti-citrullinated peptide antibody most specific and reliable clinical test for RA

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

この部分だけ！ PADI4 Citrullination Antigenicity

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

Enzyme substrate

Hypothetical mechanism of RA-susceptible variant

Following studies Case-control association studies –UK (Barton, A. J., Worthington et al Arthritis & Rheum) : Similar allele frequency No statistical significance –Replicated in Japanese (Kuwahara, M., Kamatani, N et al 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 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 ACR Poster)

Identification of multiple autoimmunity-associated gene(s) through RA-gene screening

Sugar transporter signature sequence motif N C Nucleotide binding motif * * * * SLC22A4(/A5)

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

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

3 genes with a SNP that disrupts RUNX1 binding sequence SLE RA Psoriasis

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

Genes associated with multiple autoimmune diseases

1.Hypothesis-free gene-based LD mapping screening 2.Three major findings through the screening –RA-specific susceptible gene: PADI4 –Multiple-autoimmunity crossing: SLC22A4(/A5) & RUNX1 3.Ethnic variability

SNPs associated with RA, Crohn disease or Psoriatic arthritis RILSLC22A4SLC22A5IRF1 10kb Genes Exons SNPs RUNX1 binding site RA Crohn L503F Heat shock element- binding site Psoriatic Arthritis Tokuhiro, S. et al. Peltekova, V. D. et al. Barton, A. et al. Yamazaki, K et al. JAPANESE CAUCASIANS NOT Polymorphic in Japanese

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

Caucasian Japanese PDCD1 MIF1CTLA4SLC22A4/A5 PTPN22TNFRSF1PADI4 Susceptible allele Preliminary calculation with random-effect model

Lab. Rheumatic Diseases –Prof. K. Yamamoto –Dr. A. Suzuki (PADI4) –Dr. X. Chang (RUNX1) –Dr. Y. Kochi –Dr. S. Tokuhiro (SLC22A4) –Ms. R. Kawaida –Ms. M. Mori (Ethnicity) –Ms. K. Kobayashi (Ethnicity) –Ms. M. Ohtake –Ms. E. Kanno –Ms. K. Komakine Sankyo Co. SRC, RIKEN –Prof. Y. Nakamura –Dr. A. Sekine (HT Genotyping) –Dr. A. Iida (SNP Identification) –Dr. T. S Saito (SNP Identification) –Dr. T. Tsunoda –Mr. H. Kawakami (Data Handling) –Mr. T. Kawaguchi (WG statistics) Clinical Institutes –Dr. T. Sawada –Dr. S. Yoshino –And many others And many other collaborators Thank you all !