A regulatory variant in FCRL3 gene is associated with susceptibility for multiple autoimmune diseases Yuta Kochi Laboratory for Rheumatic Diseases SNP.

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 diseaseGenetic predisposition in autoimmune disease

Wandstrat et al, Nat Immunol 2001

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

HLA and non-HLA genes 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 How to discover genetic predisposition of human autoimmune diseases ?human autoimmune diseases ?

Linkage analysis

- using family members of patients

Case-control association study

- whole genome approach

- candidate gene approach

Whole genome survey using SNPsWhole genome survey using SNPs by linkage disequilibrium mapping by linkage disequilibrium mapping

～ ～ SNP center, RIKEN 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 1q21-23Candidate region 1q21-23

FcγRI

FCRL1~5

CD1

FcγRII/III

CD3Z

1q21

mCh3

1q23

mCh1

human

Ps （ PSORS4 ）

MS

RA

SLE

mouse

Lupus models(sle1, swrl1)

CIA （ Mcia2)

EAE,TMEVD(Eae3, Tmevd2)

NOD(Idd10, Idd17)

FCRL ：　 Fc receptor-like

Analysis of 1q21-23 region using SNPsAnalysis 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 regionAssociation 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 FCRL3Association test in FCRL3 genegene

fcrl3_3 　 　 fcrl3_4 　　　　 fcrl3_5 　　　　　　　 　　　 fcrl3_6

-169 　　　 -110 　　　 exon1 　　 exon2 　　 　　　 　 exon3

ATG

SNPsa Allele Allele1 frequency Recessive trait comparison

ID Location 1/2RA

patientsControls OR (95% CI) 2 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 0.42 0.35 2.05 (1.51-2.78) 21.6 0.0000033

fcrl3_6 Intron3 A/G 0.42 0.34 2.02 (1.49-2.75) 20.8 0.0000052

aSNPs with P<0.0001 in allele frequency comparison test

Functional analysis of FCRL3 variantsFunctional 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 activityFCRL3 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 NFFCRL3 variant alters NFB binding (1)B binding (1) in silicoin silico prediction by TRANSFAC prediction by TRANSFAC

allele core match matrix match

-169C 1.000 0.957

-169T 0.760 0.824

Similarity with NFB consensus motif

CGGGAAGTCC [C/T] TCGGGAAGTCC [C/T] T

-169C/T-169C/T

FCRL3 variant alters NFkB binding (2)FCRL3 variant alters NFkB binding (2)EMSA (gel shift assay)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 expressionFCRL3 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 QuantificationAllele-Specific Transcript QuantificationASTQASTQ

–169 C +358 C

Exon1 Exon2 EagI site

FCRL3 transcripts from B-cells with -169C/T genotype were quantified by RFLP.

From susceptibility allele122 bp

　 85 bp

+358C/G 　　 CC 　 GG 　　　　　　　　　 　　C/G

122 bp

　 85 bp

Genomic DNA control

Transcripts

C/G ratio

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

T G

PCR amplification of cDNA

122 bp C

85 bp G Digestion by EagI

FCRL3FCRL3 variant affects gene expressionvariant affects gene expression

High affinity to -169C

SNP-169C/T

exon1 exon2 exon3

High expression in -169C

c-Relp50

NFB

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

FCRL3 expression in organsFCRL3 expression in organs

Quantified by TaqMan-PCR

FCRL expression in tonsilFCRL expression in tonsilin situin situ hybridization hybridization 　　（（ Miller et al, Miller et al, Blood Blood 20022002 ））

　 FCRL1 　　 　 FCRL2 　　 　 FCRL3 　　 　　 FCRL4　　　　 FCRL5 　　　

Light zone Mantle zoneMarginal zone Light zoneMantle zone

FCRL3 is strongly expressed in centrocytes of GC light zone.

FCRL3 expression in RA synoviumFCRL3 expression in RA synoviumin situin situ hybridization hybridization

T-cells

Anti-CD3

100x

B-cells

Anti-CD20

100x

FCRL3

ISH

100x 400x

Does FCRL3 variant influence the disease outcome?Does FCRL3 variant influence the disease outcome?

Genotype and autoantibodies in RA patientsGenotype 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 Is FCRL3 variant a common genetic predisposition in autoimmune diseases? in autoimmune diseases?

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

Disease

Number

of

subjects

Allele C

frequency

Recessive-trait comparison

OR (95% CI) 2 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.

FCRL3FCRL3 　　 Fc receptor-like 3Fc receptor-like 3

Type I membrane protein

Extra-cellular domain - six Ig-like domains

- high homology with FcR

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 autoimmunityRole of FCRL3 in autoimmunity

BCR

FcγR bⅡ

FAS

FCRL3

CD40

FCRL3 expresision

　 with -169C allele

?

B　cell

Clonal selection in GC

Self-reactive clonesOrgan damage

CD40 CD40L

B　cell T　cell

Antigen presentation

AgMHC TCR

Autoantibody

RFa-CCP

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

Recent discovery of non-HLA genes Recent discovery of non-HLA genes associated with human autoimmunitiesassociated with human autoimmunities

Genetic predisposition of RAGenetic predisposition of RA

PADI4 　(RA)

PTPN22 　(RA,SLE,T1D)

FCRL3 (RA, SLE,AITD)

SLC22A4/5 　（ RA, Crohn)

HLA (most autoimmunities)

CTLA4 　 (RA, SLE ， AITD, T1D)

① Antigen production

② Antigen presentation

③ Lymphocyte signaling

④ inflammation

MIF 　（ RA, UC)

Conclusion

A SNP in the promoter region of FCRL3 was associated with susceptibility for multiple autoimmune diseases.

FCRL3 variant alters the binding affinity of NFB 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.

AcknowledgmentLaboratory 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