1. 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

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. 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

6. 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

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= ）
　　　　　　　　　　↓
　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. 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 ( )
24.3
fcrl3_4
-110
A/G
0.25
0.18
3.01 ( )
16.1
fcrl3_5
Exon2
C/G
2.05 ( )
21.6
fcrl3_6
Intron3
0.34
2.02 ( )
20.8
aSNPs with P< in allele frequency comparison test
fcrl3_3　 　fcrl3_4　　　　 fcrl3_5　　　　　　　 　　　fcrl3_6
-169　　　-110　　　 exon1 　　exon2　　 　　　 　exon3
ATG

10. 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

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
CGGGAAGTCC [C/T] T
Similarity with NFkB consensus motif
allele
core match
matrix match
-169C
1.000
0.957
-169T
0.760
0.824

13. 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

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 = ）

15. Allele-Specific Transcript Quantification ASTQ
–169 C 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
mean 1.06
mean 1.68
C/G
FCRL3 transcripts from B-cells with -169C/T genotype were quantified by RFLP.

16. FCRL3 variant affects gene expression
c-Rel
p50
NFkB
High affinity to -169C
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 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.

20. FCRL3 expression in RA synovium in 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
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= by regression analysis.
bP=0.029 by Fisher's exact test.

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

24. 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 ( )
15.7 HT
158
0.42
1.62 ( )
5.2
0.022
AITD total
509
0.45
1.74 ( )
18.5
SLE
564
0.41
1.49 ( )
9.8
0.0017
RAa+AITD+SLE
2437
1.52 ( )
24.2
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.

25. 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

26. 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

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. 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)

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 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