Identification of rheumatoid arthritis-associated PADI4, SLC22A4/A5, FCRL3 & PTPN22 International Symposium on Inflammatory Diseases of Barrier Organs 3-4 June 2005, Kiel, Germany Ryo Yamada Center for Genomic Medicine Kyoto University Kyoto, Japan

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

Major findings from SNP-based studies 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. PTPN22 : T1DM, SLE, RA & AITD –Lymphoid-specific intracellular phophatase FCRL3 : RA, SLR & AITD –Fc receptor homolog on B-cell membrane

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

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

この部分だけ！ PADI4 Citrullination Antigenicity

Human and Mouse PADI Gene cluster

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

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 –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)

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

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

SNP < § 0.064* < § 0.12* SNP < § 0.066* < § 0.12* SNP < § 0.24* < § < § 0.19* SNP < § 0.35* < § 0.40 * < § 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

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

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

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

NFkB regulates FCRL3 expression Binds C > T

Lymphoid organs and B cell- specific expression

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

RA における FCRL3 の働き ( 仮 説）

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

PTPN22 10kb Genes Exons SNPs PTPN22 Chromosome 1p13 Arg→Trp J C A P value F ST P value F ST P value F ST SNP < § § J vs. C J vs. A C vs. A §P 0.05 Mori, et al. J Hum Genet 2005 May 10

Connection of multiple autoimmune diseases by susceptible genes/loci

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

MΦ Anti-oxydant transporter

PADI4 4SNPs シトルリン化酵素 SLC22A4 3SNPs 有機カチオントランスポーター 代表基質： Ergothioneine SLC22A5 1SNP 有機カチオントランスポーター 代表基質： carnitine PDCD1 1SNP 免疫グロブリンスーパーファミ リー 分子 PTPN22 1SNP TCR シグナル抑制性制御酵素 locus gene SNP 数・機能 Crohn RA SLE T1D PADI4 PTPN22 PDCD1 SLC22A4/A5 対象とする自己免疫疾患関連遺伝子 Crohn ：クローン病 SLE ：全身性エリテマトーデ ス T1D ：Ⅰ型糖尿病

疾患感受性 SNP アレル頻度 人種による比較 J C A PADI4 SLC22A4/5 PDCD1 PTPN22 疾患 既報での関連 あり なし RA J ・ C C RA J C Crohn Crohn C － (J: 多型なし ) RA, SLE C － T1D 人種差 なし あり

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

Lab. Rheumatic Diseases –Dr. K. Yamamoto –Dr. A. Suzuki –Dr. X. Chang –Dr. Y. Kochi –Dr. S. Tokuhiro –Ms. R. Kawaida –Ms. M. Mori –Ms. K. Kobayashi –Ms. M. Ohtake –Ms. E. Kanno –Ms. K. Komakine SRC, RIKEN –Dr. A. Sekine –Dr. T. Tsunoda –Dr. Y. Nakamura –Mr. H. Kawakami –Mr. T. Kawaguchi Clinical Institutes –Dr. T. Sawada And many other collaborators Thank you all !