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 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

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.

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

Hypothesis-free whole-genome approach 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

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

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

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

Association test in FCRL3 gene SNPsa Allele Allele1 frequency Recessive trait comparison ID Location 1/2 RA patients Controls OR (95% CI) c2 P fcrl3_3 -169 C/T 0.42 0.35 2.15 (1.58-2.93) 24.3 0.00000085 fcrl3_4 -110 A/G 0.25 0.18 3.01 (1.71-5.29) 16.1 0.000060 fcrl3_5 Exon2 C/G 2.05 (1.51-2.78) 21.6 0.0000033 fcrl3_6 Intron3 0.34 2.02 (1.49-2.75) 20.8 0.0000052 aSNPs with P<0.0001 in allele frequency comparison test fcrl3_3　 　fcrl3_4　　　　 fcrl3_5　　　　　　　 　　　fcrl3_6 -169　　　-110　　　 exon1 　　exon2　　 　　　 　exon3 ATG

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? fcrl3_3　 　fcrl3_4　　　　 fcrl3_5　　　　　　　 　　　fcrl3_6 -169　　　-110　　　 exon1 　　exon2　　 　　　 　exon3 ATG

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）

FCRL3 variant alters NFkB binding (1) in silico prediction by TRANSFAC CGGGAAGTCC [C/T] T Similarity with NFkB consensus motif allele core match matrix match -169C 1.000 0.957 -169T 0.760 0.824

FCRL3 variant alters NFkB binding (2) EMSA (gel shift assay) 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

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）

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

FCRL3 variant affects gene expression c-Rel p50 NFkB High affinity to -169C High expression in -169C exon1 exon2 exon3 SNP -169C/T

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

FCRL3 expression in organs Quantified by TaqMan-PCR

FCRL expression in tonsil in situ hybridization　（Miller et al, Blood 2002） 　FCRL1　　 　 FCRL2　　 　 FCRL3　　 　　FCRL4　　　　FCRL5　　　 Marginal zone Mantle zone Light zone Light zone Mantle zone FCRL3 is strongly expressed in centrocytes of GC light zone.

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

Does FCRL3 variant influence the disease outcome?

Genotype and autoantibodies in RA patients Rheumatoid factor Anti-CCP antibody Genotype n (N=148) Serum level ±SEM (IU/ml) n (N=71) Positivity (%) -169 C/C 29 479.9 ±91.3a 17 100.0b -169 C/T 75 323.7 ±47.3a 35 94.3b -169 T/T 44 216.4 ±44.0a 19 73.7b CCP; cyclic citrullinated peptide aR2=0.049, P=0.0065 by regression analysis. bP=0.029 by Fisher's exact test.

Is FCRL3 variant a common genetic predisposition in autoimmune diseases?

Recessive-trait comparison Association of SNP -169C/T with AITD and SLE Disease Number of subjects Allele C frequency Recessive-trait comparison OR (95% CI) c2 P GD 351 0.46 1.79 (1.34-2.39) 15.7 0.000074 HT 158 0.42 1.62 (1.07-2.47) 5.2 0.022 AITD total 509 0.45 1.74 (1.35-2.24) 18.5 0.000017 SLE 564 0.41 1.49 (1.16-1.92) 9.8 0.0017 RAa+AITD+SLE 2437 1.52 (1.29-1.79) 24.2 0.00000084 Controls 2037 0.37 aRA represents sum of three sets (n=1364). GD = Graves’ disease; HT = Hashimoto’s thyroiditis; AITD = Autoimmune thyroid disease; SLE = Systemic lupus erythematosus.

FCRL3 Fc 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

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

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

③ Lymphocyte signaling 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)

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.

Acknowledgment 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 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