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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
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
CIA（Mcia2)
1q21
FcγRI
mCh3
Ps （PSORS4）
EAE,TMEVD
(Eae3, Tmevd2)
FCRL1~5
NOD
(Idd10, Idd17)
MS
CD1
SLE
Lupus models
(sle1, swrl1)
1q23
mCh1
FcγRII/III
CD3Z
FCRL ： Fc receptor-like
RA
Analysis of 1q21-23 region using SNPs
FCRLs
 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
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
RA
Recessive trait comparison
Controls
OR (95% CI)
c2
0.42
0.35
2.15 (1.58-2.93)
24.3
0.00000085
A/G
0.25
0.18
3.01 (1.71-5.29)
16.1
0.000060
Exon2
C/G
0.42
0.35
2.05 (1.51-2.78)
21.6
0.0000033
Intron3
A/G
0.42
0.34
2.02 (1.49-2.75)
20.8
0.0000052
ID
Location
1/2
fcrl3_3
-169
C/T
fcrl3_4
-110
fcrl3_5
fcrl3_6
aSNPs
Allele1 frequency
patients
with P<0.0001 in allele frequency comparison test
-169
-110
exon1
exon2
exon3
ATG
fcrl3_3
fcrl3_4
fcrl3_5
fcrl3_6
P
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?
-169
-110
exon1
exon2
exon3
ATG
fcrl3_3
fcrl3_4
fcrl3_5
fcrl3_6
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
-169C/T
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)
 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
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
Digestion by EagI
85 bp G
+358C/G
CC
GG
C/G
122 bp
Transcripts
From susceptibility allele
85 bp
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
FCRL3 transcripts from B-cells with -169C/T genotype were quantified by RFLP.
FCRL3 variant affects gene expression
NFkB
p50 c-Rel
High affinity to -169C
High expression in -169C
exon1
SNP
-169C/T
exon2
exon3
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
FCRL1
FCRL2
Marginal zone
Mantle zone
（Miller et al, Blood 2002）
FCRL3
FCRL4
Light zone
Light zone
FCRL5
Mantle zone
FCRL3 is strongly expressed in centrocytes of GC light zone.
FCRL3 expression in RA synovium
in situ hybridization
T-cells
B-cells
Anti-CD3
Anti-CD20
100x
100x
FCRL3
ISH
100x
400x
Does FCRL3 variant influence the disease outcome?
Genotype and autoantibodies in RA patients
Rheumatoid factor
Anti-CCP antibody
n
Serum level
n
(N=148)
±SEM (IU/ml)
(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
Genotype
CCP; cyclic citrullinated peptide
aR2=0.049,
bP=0.029
P=0.0065 by regression analysis.
by Fisher's exact test.
Is FCRL3 variant a common genetic predisposition
in autoimmune diseases?
Association of SNP -169C/T with AITD and SLE
Number
Disease
of
subjects
Allele C
Recessive-trait comparison
frequency
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
0.42
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
BCR
RF
a-CCP
FCRL3
FcγRⅡb
FCRL3 expresision
with -169C allele
?
FAS
Self-reactive clones
Organ damage
B cell
Antigen presentation
Ag
CD40
Clonal selection in GC
MHC
TCR
CD40
CD40L
B cell
T cell
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
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)
③ Lymphocyte signaling
SLC22A4/5 （RA, Crohn)
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 diseaserisk 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
RA sample collection
Masao Yukioka
Shigeyuki Wakitani
Shigeto Tohma
Tsukasa Matsubara
Ryota Teshima
Yuichi Nishioka
Shinichi Yoshino
Masakazu Nagashima
SNP Reseach Center, RIKEN
Yusuke Nakamura
Hiroto Kawakami
Atsushi Takahashi
Tatsuhiko Tsunoda
Akihiro Sekine
Yozo Ohnishi
SLE and AITD sample collection
Takehiko Sasazuki
Senji Shirasawa
Akio Mimori
Takao Koike
Wako Yumura
Shigeru Otsubo
University of Tokyo
Tetsuji Sawada
-- and many other collaborators