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Yuta Kochi Laboratory for Rheumatic Diseases SNP Research Center, RIKEN

A regulatory variant in FCRL3 gene is associated with susceptibility for multiple autoimmune diseases. Yuta Kochi Laboratory for Rheumatic Diseases SNP Research Center, RIKEN. Genetic predisposition in autoimmune disease. Much higher concordance rate for disease

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Yuta Kochi Laboratory for Rheumatic Diseases SNP Research Center, RIKEN

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  1. A regulatory variant in FCRL3 gene is associated with susceptibility for multiple autoimmune diseases Yuta Kochi Laboratoryfor Rheumatic Diseases SNP Research Center, RIKEN

  2. Genetic predisposition in autoimmune disease • Much higher concordance rate for disease in monozygotic twins than dizygotic twins supports; - genetic predisposition in autoimmune diseases - presence of multiple genes with disease risk Wandstrat et al, Nat Immunol 2001

  3. HLA and non-HLA genes • HLA haplotypes comprise the major genetic predisposition to most autoimmune diseases. • Multiple non-HLA genes are involved, with • relatively low contribution to the disease risk.

  4. How to discover genetic predisposition of human autoimmune diseases ? • Linkage analysis - using family members of patients • Case-control association study - whole genome approach - candidate gene approach

  5. Whole genome survey using SNPs by linkage disequilibrium mapping～ SNP center, RIKEN ～ • Hypothesis-free whole-genome approach -comprehensive analysis to discover disease related genes -identification of novel pathologic mechanisms of disease • Large-scale case-control screening using SNPs - 100,926 SNPs in gene-containing region - comparison of the allele frequency between 830 RA patients and 658 controls - localization of candidate regions by LD-mapping

  6. mouse human CIA（Mcia2) EAE,TMEVD (Eae3, Tmevd2) Ps （PSORS4） NOD (Idd10, Idd17) MS SLE Lupus models (sle1, swrl1) RA Candidate region 1q21-23 FcγRI FCRL1~5 CD1 FcγRII/III CD3Z 1q21 mCh3 1q23 mCh1 FCRL ：　Fc receptor-like

  7. Analysis of 1q21-23 region using SNPs • LD mapping • - 491 SNPs • - genotyped for 658 controls • 　　 → 110 LD blocks （Δ＞0.5） • Association study • <1st screeening> • - 491 SNPs • - genotyped for 94 RA patients • - Allele frequency comparison test • → associations in 9 SNPs （P<0.01) • <2nd screening> • - 9 SNPs • - genotyped for 736 RA patients • → strong association • in an intronic SNP of FCRL3 gene • 　　　　　　（OR 1.39, P=0.000035） • ↓ • Further analysis of FCRL complex FCRLs

  8. Association study in FCRL region • Case-control allele-frequency comparison tests • 41 SNPs　（newly identified 16 SNPs) • 830 RA patients vs 658 controls • →　peak of association in FCRL3 promoter region

  9. -169-110exon1 exon2exon3 ATG fcrl3_3fcrl3_4fcrl3_5fcrl3_6 Association test in FCRL3gene

  10. -169-110exon1 exon2exon3 ATG fcrl3_3fcrl3_4fcrl3_5fcrl3_6 Functional analysis of FCRL3 variants • How do the FCRL3 variants cause the disease? • None of variants with disease risk alter the amino acid sequence of the protein. • Do the variants affect FCRL3 expression?

  11. FCRL3 variant affects promoter activity Evaluation of FCRL3 promoter activity by Luciferase assay • Promoter activity in three promoter haplotypes （nt -523 ～ +203） • Enhancing activity of sequence around SNP -169 C/T （nt -189 ～ -160）

  12. FCRL3 variant alters NFkB binding (1)in silico prediction by TRANSFAC -169C/T CGGGAAGTCC [C/T] T Similarity with NFkB consensus motif

  13. FCRL3 variant alters NFkB binding (2)EMSA (gel shift assay) • EMSA • 30 bp oligo around -169C/T • Nuclear proteins from Raji cells • 　　　→ higher binding affinity in C allele • Super-shift assay • anti-NFκB antibodies • 　　　→ shifted by anti-p50，p65，c-Rel Abs

  14. FCRL3 genotypes and expression FCRL3 expression in B-cells from healthy donors quantified by TaqMan-PCR FCRL3 expression was regressed by the number of disease risk allele（n = 0,1,2) 　　　　　　　　　　　　　　　　　　 （R2 = 0.49，P = 0.0076）

  15. PCR amplification of cDNA 122 bp C Digestion by EagI 85 bp G +358C/G CC GGC/G 122 bp 85 bp From susceptibility allele Transcripts C/G ratio 1.67 1.44 1.60 1.76 1.70 mean 1.68 C/G Genomic DNA control 122 bp 85 bp C/G ratio 1.11 1.05 1.02 1.03 1.11 mean 1.06 Allele-Specific Transcript QuantificationASTQ –169 C +358 C T G Exon1 Exon2 EagI site FCRL3 transcripts from B-cells with -169C/T genotype were quantified by RFLP.

  16. NFkB p50 c-Rel High affinity to -169C FCRL3variant affects gene expression High expression in -169C exon1 exon2 exon3 SNP -169C/T

  17. Where is FCRL3 expressed ?In what cells does FCRL3 function?

  18. FCRL3 expression in organs Quantified by TaqMan-PCR

  19. FCRL expression in tonsilin situ hybridization（Miller et al, Blood 2002） FCRL1FCRL2FCRL3FCRL4FCRL5 Marginal zone Mantle zone Light zone Light zone Mantle zone FCRL3 is strongly expressed in centrocytes of GC light zone.

  20. FCRL3 expression in RA synoviumin situ hybridization B-cells Anti-CD20 100x T-cells Anti-CD3 100x FCRL3 ISH 100x 400x

  21. Does FCRL3 variant influence the disease outcome?

  22. Genotype and autoantibodies in RA patients

  23. Is FCRL3 variant a common genetic predisposition in autoimmune diseases?

  24. Association of SNP -169C/T with AITD and SLE

  25. FCRL3Fc receptor-like 3 • Type I membrane protein • Extra-cellular domain - six Ig-like domains - high homology with FcgR • Intra-cellular domain - four tyrosine motifs - binding of Syk to ITAM binding of SHP1/SHP2 to ITIM (Xu MJ et al, BBRC 2002) 734 a.a. ITAM; immunorecepter tyrosine-based activating motif ITIM; immunorecepter tyrosine-based inhibitory motif

  26. Autoantibody RF a-CCP FCRL3 expresision with -169C allele Self-reactive clones Organ damage ? Antigen presentation Ag MHC TCR CD40 CD40L B cell T cell Role of FCRL3 in autoimmunity BCR FCRL3 FcγRⅡb B cell FAS CD40 Clonal selection in GC

  27. Recent discovery of non-HLA genes associated with human autoimmunities • CTLA4 - T-cell inhibitory receptor - T1D, AITD, RA, SLE, MS - Expression of soluble CTLA4 is decreased with the disease risk haplotype • PTPN22 - Tyrosine phosphatase - T1D, RA, SLE, AITD - TCR signaling is decreased in cells with the disease risk allele • PADI4 - Protein citrullinating enzyme - RA - mRNA is more stable with the disease risk haplotype

  28. Genetic predisposition of RA PADI4(RA) ① Antigen production MIF　（RA, UC) PTPN22(RA,SLE,T1D) HLA(most autoimmunities) ② Antigen presentation FCRL3 (RA, SLE,AITD) SLC22A4/5　（RA, Crohn) ③ Lymphocyte signaling ④ inflammation CTLA4(RA, SLE，AITD, T1D)

  29. Conclusion • A SNP in the promoter region of FCRL3 was associated with susceptibility for multiple autoimmune diseases. • FCRL3 variant alters the binding affinity of NFkB and regulates gene expression. • High FCRL3 expression and augmented autoantibody production were observed in individuals with the disease-risk genotype. • FCRL3 may play an important role in the breakdown of peripheral tolerance and B-cell driven autoimmunity.

  30. Acknowledgment RA sample collection Masao Yukioka Shigeyuki Wakitani Shigeto Tohma Tsukasa Matsubara Ryota Teshima Yuichi Nishioka Shinichi Yoshino Masakazu Nagashima SLE and AITD sample collection Takehiko Sasazuki Senji Shirasawa Akio Mimori Takao Koike Wako Yumura Shigeru Otsubo -- and many other collaborators Laboratory For Rheumatic Diseases Kazuhiko Yamamoto Ryo Yamada Akari Suzuki Shinya Tokuhiro Xiaotian Chang Kyoko Kobayashi Emi Kanno Miyako Yamanaka Keiko Myozen Keiko Komakine SNP Reseach Center, RIKEN Yusuke Nakamura Hiroto Kawakami Atsushi Takahashi Tatsuhiko Tsunoda Akihiro Sekine Yozo Ohnishi University of Tokyo Tetsuji Sawada

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