close

Вход

Забыли?

вход по аккаунту

?

Fc╨Ю╤Ц receptor gene polymorphisms in Japanese patients with systemic lupus erythematosusContribution of FCGR2B to genetic susceptibility.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 46, No. 5, May 2002, pp 1242–1254
DOI 10.1002/art.10257
© 2002, American College of Rheumatology
Fc␥ Receptor Gene Polymorphisms in Japanese Patients With
Systemic Lupus Erythematosus
Contribution of FCGR2B to Genetic Susceptibility
Chieko Kyogoku,1 Hilde M. Dijstelbloem,2 Naoyuki Tsuchiya,1 Yoko Hatta,1 Hitoshi Kato,1
Akihiro Yamaguchi,1 Toru Fukazawa,3 Marc D. Jansen,4 Hiroshi Hashimoto,3
Jan G. J. van de Winkel,4 Cees G. M. Kallenberg,5 and Katsushi Tokunaga1
Objective. Human low-affinity Fc␥ receptors
(Fc␥R) constitute a clustered gene family located on
chromosome 1q23, that consists of Fc␥RIIA, IIB, IIC,
IIIA, and IIIB genes. Fc␥RIIB is unique in its ability to
transmit inhibitory signals, and recent animal studies
demonstrated a role for Fc␥RIIB deficiency in the
development of autoimmunity. Genetic variants of
Fc␥RIIA, IIIA, and IIIB and their association with
systemic lupus erythematosus (SLE) have been extensively studied in various populations, but the results
were inconsistent. To examine the possibility that another susceptibility gene of primary significance exists
within the Fc␥R region, we screened for polymorphisms
of the human FCGR2B gene, and examined whether
these polymorphisms are associated with SLE.
Methods. Variation screening of FCGR2B was
performed by direct sequencing and polymerase chain
reaction (PCR)–single-strand conformation polymorphism methods using complementary DNA samples.
Genotyping of the detected polymorphism was done
using genomic DNA, with a specific genotyping system
based on nested PCR and hybridization probing. Association with SLE was analyzed in 193 Japanese patients
with SLE and 303 healthy individuals. In addition, the
same groups of patients and controls were genotyped for
the previously known polymorphisms of FCGR2A,
FCGR3A, and FCGR3B.
Results. We detected a single-nucleotide polymorphism in FCGR2B, (c.695T>C), coding for a nonsynonymous substitution, Ile232Thr (I232T), within the
transmembrane domain. The frequency of the 232T/T
genotype was significantly increased in SLE patients
compared with healthy individuals. When the same
patients and controls were also genotyped for FCGR2A131R/H, FCGR3A-176V/F, and FCGR3B-NA1/2 polymorphisms, FCGR3A-176F/F showed significant association. Two-locus analyses suggested that both FCGR2B
and FCGR3A may contribute to SLE susceptibility,
while the previously reported association of FCGR3B
was considered to be secondary and derived from strong
linkage disequilibrium with FCGR2B.
Conclusion. These results demonstrate the association of a new polymorphism of FCGR2B (I232T) with
susceptibility to SLE in the Japanese.
Supported by a Grant-in-Aid for Scientific Research on
Priority Areas (C) “Medical Genome Science,” a Grant-in-Aid for
Scientific Research (B) from the Ministry of Education, Science,
Sports and Culture of Japan, a Grant-in-Aid for JSPS Fellows, and by
grant C.96.1610 from the Dutch Kidney Foundation. Chieko Kyogoku
is a JSPS Research Fellow.
1
Chieko Kyogoku, MSc, Naoyuki Tsuchiya, MD, PhD, Yoko
Hatta, MSc, Hitoshi Kato, MSc, Akihiro Yamaguchi, MD, Katsushi
Tokunaga, PhD: University of Tokyo, Tokyo, Japan; 2Hilde M.
Dijstelbloem, PhD: University Hospital Groningen and University
Medical Center Utrecht, Groningen and Utrecht, The Netherlands;
3
Toru Fukazawa, MD, PhD, Hiroshi Hashimoto, MD, PhD: Juntendo
University, Tokyo, Japan; 4Marc D. Jansen, Jan G. J. van de Winkel,
PhD: University Medical Center Utrecht, Utrecht, The Netherlands;
5
Cees G. M. Kallenberg, MD, PhD: University Hospital Groningen,
Groningen, The Netherlands.
Address correspondence and reprint requests to Naoyuki
Tsuchiya, MD, PhD, Department of Human Genetics, Graduate
School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku
Tokyo 113-0033, Japan. E-mail: tsuchiya-tky@umin.ac.jp
Submitted for publication August 22, 2001; accepted in
revised form January 11, 2002.
Results of genome-wide linkage studies have
suggested that the chromosomal region 1q23 is one of
the strongest candidate regions for human systemic
lupus erythematosus (SLE) (1,2), as well as its syntenic
region in murine lupus (3). Three Fc␥ receptor type II
1242
FCGR2B GENE POLYMORPHISM IN SLE
(Fc␥RII) genes (FCGR2A, FCGR2B, and FCGR2C) and
2 Fc␥RIII genes (FCGR3A and FCGR3B) have been
physically mapped to a region of ⬃200 kb at 1q23 (4–7)
and are considered to be candidate susceptibility genes
for SLE.* The order of these genes has recently been
demonstrated, by sequencing BAC clones, to be 2A-3A2C-3B-2B (8). Association of Fc␥R polymorphisms with
SLE has been extensively studied in various populations
(9,10), and Fc␥RIIA-131R/H and Fc␥RIIIA-176V/F
polymorphisms were often shown to be associated with
SLE. In a previous study, we found an association of
FCGR3B alleles, but not of FCGR2A and FCGR3A
alleles, with SLE in the Japanese population (11). However, the results of various association studies are inconsistent among different populations with different genetic backgrounds, raising the possibility that another
gene(s) in this chromosomal region, in linkage disequiliblium with FCGR2A, FCGR3A, and FCGR3B, may
be primarily associated with SLE.
FCGR2B is the only gene among the Fc␥R family
that codes for an immunoreceptor tyrosine-based inhibitory motif (ITIM) and has the ability to transmit
inhibitory signals in B cells and myelomonocytic cells
(12). Deficiency for Fc␥RIIB in mice was recently shown
to be associated with autoimmune diseases such as
collagen-induced arthritis (13) and Goodpasture’s syndrome (14). Moreover, spontaneous development of
antinuclear antibodies and glomerulonephritis was observed when Fc␥RIIB deficiency was introduced into a
C57BL/6 background (15). Polymorphisms in the promoter region (16) and intron (17) of murine Fc␥RIIB
were recently shown to be associated with abnormal
down-regulation of Fc␥RIIB expression in germinal
center B cells, leading to abnormal up-regulation of IgG
antibody responses in (NZB ⫻ NZW)F1 mice, as well as
in other autoimmune-prone mice. In addition, 4 singlenucleotide polymorphisms (SNPs) within the coding
region, that are in complete linkage disequilibrium, were
shown to form a particular allele of the Fc␥RIIB gene in
lupus-prone mouse strains (18). These findings strongly
suggest that FCGR2B may be a susceptibility gene for
human SLE in the chromosomal region 1q23.
To date, there have been no published reports of
screening for polymorphisms of the human FCGR2B
gene. A reason for this may be the extremely high
sequence homology between human FCGR2B and
FCGR2C genes, which makes it difficult to amplify
* The designation using Roman numerals indicates the
polypeptide product, while the designation using Arabic numerals is
for the corresponding locus.
1243
FCGR2B specifically. To overcome this problem, we
carried out variation screening using complementary
DNA (cDNA), and we identified an SNP that results in
a nonconservative amino acid substitution within the
transmembrane region of Fc␥RIIB. We also genotyped
SLE patients and healthy controls for this new SNP in
FCGR2B, as well as for FCGR2A, FCGR3A, and
FCGR3B polymorphisms, in order to test whether the
SNP in FCGR2B is associated with SLE, either primarily
or in linkage disequilibrium with other FCGR genes.
PATIENTS AND METHODS
Patients and controls. One hundred ninety-three unrelated Japanese patients with SLE and 303 unrelated healthy
Japanese controls were studied. The SLE group consisted of 19
male patients and 174 female patients between the ages of 16
and 78 years (mean ⫾ SD 40.7 ⫾ 13.7). They were followed up
in the outpatient clinics of the University of Tokyo and
Juntendo University Hospitals. The patients were diagnosed
and classified for the presence or absence of clinical characteristics according to the American College of Rheumatology
criteria for SLE (19).
The control group consisted of researchers, laboratory
workers, and students (167 men, 136 women) between the ages
of 21 and 61 years (mean ⫾ SD 35.3 ⫾ 9.9). It should be noted
that the central part of Japan has been shown to be relatively
homogeneous with respect to genetic background (20), permitting the case–control approach used in this study.
To examine the frequency and linkage disequilibrium
of FCGR2B in Caucasians, 148 healthy Dutch individuals (90
males and 58 females) between the ages of 5 and 71 years
(mean ⫾ SD 37.0 ⫾ 16.2) were also genotyped. Data from
most of these subjects have been reported previously (21).
This study was reviewed and approved by the research
ethics committees of the University of Tokyo and Juntendo
University.
DNA samples. Total RNA was purified from peripheral blood mononuclear cells of SLE patients and healthy
subjects with the RNeasy Mini Kit (Qiagen, Hilden, Germany)
and was then reverse transcribed into cDNA. Genomic DNA
was purified using a QIAamp blood kit (Qiagen).
Variation screening and genotyping of the human
FCGR2B gene using cDNA. Because of size limitations with
polymerase chain reaction (PCR) amplification, FCGR2B
cDNA was divided into 2 overlapping fragments using 2 primer
sets covering the entire coding sequence from exon 1 to exon
8 of the FCGR2B gene. One primer set was placed at the
5⬘-untranslated region (5⬘-UTR) (2B 5⬘-UTR–F: 5⬘GAGAAGGCTGTGACTGCTGT-3⬘) and at the ITIM region
within exon 8 (2B ITIM-R: 5⬘-CGGGTGCATGAGAAGTGAAT-3⬘) to amplify a 944-bp cDNA fragment (fragment A)
(Figure 1A). PCR was performed in 50-␮l reaction mixtures
containing 0.2 ␮l cDNA, 0.2 ␮M of each primer, 0.4 mM
dNTPs, 2 mM MgCl2, and 2.5 units LA Taq DNA polymerase
(Takara; Otsu, Shiga, Japan), using a T-gradient thermocycler
(Biometra, Göttingen, Germany). The amplification procedure consisted of initial denaturation at 96°C for 3 minutes, 35
1244
Figure 1. A, Diagram of the cDNA structure of the FCGR2B gene
and nested polymerase chain reaction (PCR) strategy. The first PCR
amplified a 944-bp fragment (fragment A) extending from the 5⬘untranslated region (5⬘-UTR) through the immunoreceptor tyrosinebased inhibitory motif region (exon 8) and a 243-bp fragment (fragment B) extending from exon 6 through the 3⬘-UTR. Each of the
second PCR products encompassing the potential variation sites was
analyzed using single-strand conformation polymorphism (SSCP).
Positions of detected variation are indicated, and have been deposited
in the GenBank database (accession nos. AB050934 and AB051387).
SP ⫽ signal peptide; EC ⫽ extracellular domain; TM ⫽ transmembrane region; C ⫽ cytoplasmic tail. B, Representative SSCP pattern of
the FCGR2B-232I/T polymorphism. Electrophoresis was carried out
using 10% polyacrylamide gels at 10°C for 75 minutes. Separated
fragments were visualized by silver staining. C, Direct sequencing,
revealing a single-nucleotide polymorphism in exon 5, c.695T⬎C
(I232T).
cycles of denaturation at 96°C for 30 seconds, annealing at
60°C for 30 seconds, and extension at 72°C for 90 seconds,
followed by a final extension at 72°C for 5 minutes. As
previously reported (22), 2 fragments that differed in size were
observed, which were considered to be splice variants of
Fc␥RIIB, Fc␥RIIB1, and Fc␥RIIB2.
KYOGOKU ET AL
The other primer set was placed at exon 5–6 (2B exon
5.6-F: 5⬘-AAAGCGGATTTCAGCTCTCC-3⬘) and at the 3⬘UTR (2B 3⬘-UTR–R: 5⬘-TACCAGATCTTCCCTCTCTG-3⬘)
to amplify a 243-bp cDNA fragment (fragment B) (Figure 1A).
PCR was performed in 25-␮l reaction mixtures containing 0.2
␮l cDNA, 0.2 ␮M of each primer, 0.4 mM dNTPs, 3.5 mM
MgCl2, and 1.25 units LA Taq DNA polymerase. The amplification procedure consisted of initial denaturation at 96°C for
3 minutes, 40 cycles of denaturation at 96°C for 30 seconds,
annealing at 58°C for 30 seconds, and extension at 72°C for 30
seconds, followed by a final extension at 72°C for 5 minutes.
In order to genotype for potential variation sites
detected in previous experiments, the amplified fragment A
served as the template for a second PCR using 2 primer sets,
1 of which (2B exon 3-F: 5⬘-GCATCTGACTGTGCTTTCTG3⬘, 2B exon 4-R: 5⬘-CTTGGACAGTGATGGTCACA-3⬘) amplified a 275-bp fragment of the entire length of exon 4, and
the other of which (2B exon 4.5-F: 5⬘-TCCAAGCTCCCAGCTCTTCA-3⬘, 2B exon 6-R: 5⬘-TGGTTTCTCAGGGAGGGTCT-3⬘) amplified a 176-bp fragment encompassing exon
5 and exon 6 (Figure 1A). PCR was carried out in 25-␮l
reaction mixtures containing 0.5 ␮l of the first PCR product,
0.4 ␮M of each primer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 1
unit AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied
Biosystems, Norwalk, CT). The amplification procedure consisted of initial denaturation at 96°C for 10 minutes, 25 cycles
of denaturation at 96°C for 30 seconds, annealing at 60°C (for
amplification of the 275-bp fragment) or 62°C (for amplification of the 176-bp fragment) for 30 seconds, and extension at
72°C for 30 seconds, followed by a final extension at 72°C for
1 minute.
The amplified DNA fragments of 275 bp and 176 bp
were analyzed using a PCR–single-strand conformation polymorphism (PCR-SSCP) method, as previously described (23).
Electrophoresis was carried out for 90 minutes at 21°C and for
75 minutes at 10°C, using a 10% polyacrylamide gel (acrylamide:bis ⫽ 49:1). The separated fragments were visualized
with silver staining.
To exclude the presence of variation in other exons, as
well as variation that was not detected by SSCP, both fragment
A and fragment B were directly sequenced in 40 subjects.
Direct sequencing was performed using an ABI Prism 310
Genetic Analyzer (Perkin-Elmer Applied Biosystems) with a
dye-terminator method.
FCGR2B-232I/T genotyping using genomic DNA. To
increase the sample size, a method for FCGR2B-232I/T genotyping using genomic DNA was developed, based on nested
PCR and fluorescence resonance energy transfer (FRET)
technology. First, PCR primers were placed at exon 4 (2A-SF:
5⬘-AAGGACAAGCCTCTGGTCAA-3⬘) and exon 7 (2B exon
7-R: 5⬘-CCCAACTTTGTCAGCCTCAT-3⬘) to amplify a
4,323-bp fragment (Figure 2). The forward primer (2A-SF) was
originally designed for genotyping of FCGR2A (11), but can
also be used to amplify FCGR2B and FCGR2C. PCR was
carried out in 50-␮l reaction mixtures containing 0.2 ␮l cDNA,
0.4 ␮M of each primer, 0.4 mM dNTPs, 2.5 mM MgCl2, and 1
unit LA Taq DNA polymerase. The amplification procedure
consisted of initial denaturation at 96°C for 90 seconds, 40
cycles of denaturation at 96°C for 30 seconds, annealing at
60°C for 30 seconds, and extension at 72°C for 4 minutes 30
seconds, followed by a final extension at 72°C for 5 minutes.
FCGR2B GENE POLYMORPHISM IN SLE
1245
SSP) technique (25). Samples from Dutch subjects were
genotyped using PCR-SSP, as previously described (21).
Statistical analysis. Statistical analyses for association
were performed using StatView-J5.0 for Macintosh (Abacus
Concepts, Berkeley, CA). Chi-square tests were used to analyze association of the 4 Fc␥R polymorphisms with susceptibility to SLE. When sample numbers were small, Fisher’s exact
tests were used. In addition, genotype relative risk was estimated according to the method described by Lathrop (26), and
the independence of FCGR2B and FCGR3A was analyzed
using 2-locus analysis, as described by Svejgaard and Ryder
(27). Haplotype frequencies and linkage disequilibrium parameters were estimated from typing results using the EH
program (28).
Figure 2. Diagram of the genomic DNA structure of the FCGR2B
gene and the nested polymerase chain reaction (PCR) strategy for
FCGR2B-232I/T genotyping. The first PCR specifically amplified a
4,323-bp fragment of FCGR2B extending from exon 4 through exon 7.
The second PCR amplified a 863-bp product, followed by sequencespecific fluorescent hybridization probing using a LightCycler, and
melting-curve analysis. The positions of newly detected single-nucleotide polymorphisms (SNPs) within intron 4 and intron 5 are indicated
by arrows. Positions and sequences of these SNPs were deposited in
the GenBank database (accession no. AB062416). Asterisks indicate
the differences between FCGR2B and FCGR2C sequences deposited
in the database, all of which were found to be polymorphisms of the
FCGR2B gene.
A second PCR was performed based on FRET technology using a LightCycler, according to the instructions of the
manufacturer (Roche Diagnostics, Mannheim, Germany).
Primers were placed at exon 4 (2B exon 4-F: 5⬘TGTGACCATCACTGTCCAAG-3⬘) and exon 5 (2B exon
5-R: 5⬘-CTGAAATCCGCTTTTTCCTG-3⬘) to amplify an
863-bp fragment (Figure 2). Hybridization probes were fluorescein isothiocyanate–labeled 5⬘-GCTCCCAGCTCTTCACCGATGGGGATCATTGTGGCTGTG-3⬘ and LCRed 640–
labeled 5⬘-TCACTGGGATTGCTGTAGC-3⬘. PCR was
carried out in 20-␮l reaction mixtures containing 0.3 ␮l of the
first PCR product as a template, 0.5 ␮M of each primer, 0.3
␮M of the fluorescein probe, 0.3 ␮M of the LCRed 640 probe,
2 mM MgCl2, and 2 ␮l of LightCycler–DNA Master Hybridization probes (Roche Diagnostics). The amplification procedure consisted of initial denaturation at 95°C for 60 seconds, 25
cycles of denaturation at 95°C for 0 seconds, annealing at 57°C
for 15 seconds, and extension at 72°C for 40 seconds, followed
by melting-curve analysis to determine the FCGR2B-232I/T
genotype. The temperature transition rate was set at 0.1°C/
second for the melting-curve analysis.
FCGR2A, FCGR3A, and FCGR3B genotyping.
FCGR2A-131R/H genotyping of Japanese patient and control
samples was performed using PCR–restriction fragment length
polymorphism with a mismatched primer (11), FCGR3A176V/F genotyping using PCR-SSCP (11), and FCGR3BNA1/2 genotyping using a PCR–preferential homoduplex formation assay (24) and a PCR–sequence-specific primer (PCR-
RESULTS
Detection of SNPs of FCGR2B using cDNA. The
Fc␥RIIB protein consists of a signal peptide, 2 Ig-like
extracellular domains, a transmembrane region, and a
cytoplasmic tail (29) (Figure 1A). Through preliminary
variation screening of the entire coding region using
exon-specific primer sets and genomic DNA templates,
4 possible variation sites were detected in exon 4 and
exon 5 (results not shown). However, amino acid and
nucleotide sequences of Fc␥RIIB are highly homologous to Fc␥RIIA and IIC in exons 1–5 (30), raising the
possibility that some of the variations might actually
derive from FCGR2A or FCGR2C polymorphisms.
In order to amplify FCGR2B in a specific manner, nested PCR was employed using cDNA as a template, and placing the reverse primer of the first PCR in
exon 8, which is unique to FCGR2B. The second PCR
product was amplified with 2 primer sets (Figure 1A),
encompassing 2 potential variation sites, c.612G⬎A in
exon 4 (synonymous substitution) and c.772T⬎G in exon
6 (Y258D), previously identified in the human B lymphoblastoid cell line Raji (31) (designation of variation
sites is based on ref. 32), as well as possible variation
sites detected in preliminary experiments. A biallelic
polymorphism in exon 5 was detected by PCR-SSCP
analysis (Figure 1B), and direct sequencing revealed a
new SNP, c.695T⬎C (exon 5), which resulted in a
nonconservative amino acid substitution of Thr for Ile at
codon 232 (I232T) within the transmembrane region of
Fc␥RIIB (Figure 1C).
To exclude the presence of variations within
other exons, as well as variations not detected by SSCP,
variation screening of the entire coding region of
FCGR2B was performed by sequencing fragments A and
B (Figure 1A) using cDNA from 10 individuals with the
FCGR2B-232I/I genotype, 10 with the 232I/T genotype,
and 20 with the 232T/T genotype. Two synonymous
1246
KYOGOKU ET AL
Table 1. FCGR2B c.695T⬎C (I232T) polymorphism in Japanese SLE patients and healthy controls*
Genotype frequency
232 I/I
232 I/T
232 T/T
Allele positivity
I present
T present
Allele frequency
I allele
T allele
SLE
(n ⫽ 193)
Controls
(n ⫽ 303)
106 (54.9)
66 (34.2)
21 (10.9)
183 (60.4)
104 (34.3)
16 (5.3)
5.6
0.06†
172 (89.1)
87 (45.1)
287 (94.7)
120 (39.6)
5.4
1.5
0.02¶
0.23¶
278 (72.0)
108 (28.0)
470 (77.6)
136 (22.4)
3.9
0.05¶
␹2
P
OR (95%
CI)
1.0
1.1 (0.7–1.6)‡
2.3 (1.1–4.5)§
* Values are the number (%). SLE ⫽ systemic lupus erythematosus; OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval.
† By chi-square test with 3 ⫻ 2 contingency table (2 degrees of freedom [df]).
‡ ␹2 ⫽ 0.2, P ⫽ 0.65.
§ ␹2 ⫽ 5.6, P ⫽ 0.018.
¶ By chi-square test with 2 ⫻ 2 contingency table (1 df).
substitutions, c.216G⬎T (R72R) in exon 3 and
c.612G⬎A (L204L) in exon 4, were detected: the former
in an individual with the 232I/I genotype and the latter
in an individual with the 232I/T genotype. The substitution c.772T⬎G (Y258D) in exon 6, the only previously
reported variation of human FCGR2B that could possibly alter its function (33), was not detected in our
subjects, and has been shown to be rare in Caucasian
and African American populations (34). Despite a conflict at c.614T/A (F205Y) between registered sequences
(GenBank accession nos. M90731 and NM_004001, respectively), all of our samples were homozygous for
c.614A (205Y), indicating FCGR2B-205Y to be the
common allele, at least in the Japanese.
Genotyping for FCGR2B-232I/T using genomic
DNA. To increase the sample size, a method for
FCGR2B-232I/T genotyping using genomic DNA was
developed. During this process, all of the previously
reported differences between FCGR2B and FCGR2C in
intron 4 sequences (30) were found to be polymorphisms
of FCGR2B, and could not be used as specific primer
positions. Therefore, nested PCR was used to amplify a
4.3-kb FCGR2B fragment using an FCGR2B-specific
reverse primer within exon 7, followed by hybridization
probing using a LightCycler (Figure 2). Genotyping
results were confirmed to be identical to those obtained
from cDNA samples using the PCR-SSCP analysis.
Thus, further genotyping was performed using genomic
DNA samples.
Table 1 shows the frequencies of FCGR2B232I/T genotypes in the patients and healthy individuals.
Positivity was defined as the presence of 1 or 2 of the
particular alleles. Positivity for the 232I allele was significantly decreased in patients, indicating a significant
association of the 232T/T genotype with SLE. Allele
frequencies of 232I and 232T were also significantly
different between SLE patients and healthy controls,
while genotype frequencies showed a trend toward a
different distribution between patients and controls. The
odds ratios (ORs) for the development of SLE with the
T/T and I/T genotypes versus the I/I genotype were 2.3
and 1.1, respectively.
Genotype relative risk estimation by the method
of Lathrop adjusts control data for Hardy-Weinberg
equilibrium and thus reduces variance (26). Although
the genotype frequencies in healthy individuals were
compatible with Hardy-Weinberg equilibrium, the association of the specific genotype FCGR2B-232T/T was
strengthened (OR 2.35, 95% confidence interval [95%
CI] 1.4–4.0, ␹2 ⫽ 9.5, P ⫽ 0.002) when Lathrop-type
analysis was applied to our data.
FCGR2A-131R/H, FCGR3A-176V/F, and
FCGR3B-NA1/2 genotyping. To examine whether any
one of the genes in the Fc␥R family is primarily associated with SLE with other associations explained by
linkage disequilibrium, the genotypes of FCGR2A131R/H, FCGR3A-176V/F (often designated as 158V/F
by counting from the N-terminal amino acid of the
mature protein, excluding the signal peptide), and
FCGR3B-NA1/2 were compared in the same patients
and controls who were analyzed for FCGR2B. As shown
in Table 2, the distribution of genotype frequencies
differed significantly between patients and controls for
FCGR3A-176V/F, but not for FCGR2A or FCGR3B.
Positivity for the 176F allele was significantly increased
in patients, indicating a significant association of this
allele with SLE. Accordingly, the allele frequencies of
176F and 176V were significantly different between SLE
FCGR2B GENE POLYMORPHISM IN SLE
1247
Table 2. FCGR2A, FCGR3A, and FCGR3B polymorphisms in Japanese SLE patients and healthy controls*
Genotype frequency
FCGR2A
131 R/R
131 R/H
131 H/H
FCGR3A
176 F/F
176 V/F
176 V/V
FCGR3B
NA 2/2
NA 1/2
NA 1/1
Allele positivity
FCGR2A
R present
H present
FCGR3A
F present
V present
FCGR3B
NA2 present
NA1 present
Allele frequency
FCGR2A
R allele
H allele
FCGR3A
F allele
V allele
FCGR3B
NA2 allele
NA1 allele
SLE
(n ⫽ 193)
Controls
(n ⫽ 303)
8 (4.1)
72 (37.3)
113 (58.5)
␹2
P†
11 (3.6)
95 (31.4)
197 (65.0)
2.1
0.35
110 (57.0)
76 (39.4)
7 (3.6)
145 (47.8)
132 (43.6)
26 (8.6)
6.8
0.03
33 (17.1)
98 (50.8)
62 (32.1)
42 (13.9)
145 (47.8)
116 (38.3)
2.3
0.32
80 (41.5)
185 (95.9)
106 (35.0)
292 (96.4)
2.1
0.1
0.15
0.77
186 (96.4)
83 (43.0)
277 (91.4)
158 (52.1)
4.7
3.9
0.03
0.05
131 (67.9)
160 (82.9)
187 (61.7)
261 (86.1)
1.9
1.0
0.16
0.33
88 (22.8)
298 (77.2)
117 (19.3)
489 (80.7)
1.8
0.19
296 (76.7)
90 (23.3)
422 (69.6)
184 (30.4)
5.9
0.02
164 (42.5)
222 (57.5)
229 (37.8)
377 (62.2)
2.2
0.14
OR
(95% CI)
2.8 (1.2–6.5)‡
2.1 (0.9–5.1)§
1.0
* Values are the number (%). See Table 1 for definitions.
† By chi-square test with 3 ⫻ 2 contingency table (2 df) or 2 ⫻ 2 contingency table (1 df).
‡ ␹2 ⫽ 5.8, P ⫽ 0.016.
§ ␹2 ⫽ 3.0, P ⫽ 0.09.
patients and healthy controls. The ORs for the 176F/F
and 176V/F genotypes were 2.8 and 2.1, respectively.
The observed genotype frequencies in healthy
individuals were not significantly different from those
expected by Hardy-Weinberg equilibrium, but slight
deviation in the FCGR3A genotype frequency was
noted. When the Lathrop-type analysis was applied to
our data, FCGR3A-176F/F and F/V genotypes did not
show significant association, while the 176V/V genotype
had a small protective effect (OR 0.4, 95% CI 0.2–0.8,
␹2 ⫽ 5.8, P ⫽ 0.016).
Linkage disequilibrium among FCGR2A,
FCGR3A, and FCGR3B in healthy Japanese individuals.
When 2-locus linkage disequilibrium analyses were conducted among the 4 Fc␥R polymorphisms in healthy
Japanese individuals, strong linkage disequilibrium was
detected between FCGR2B and FCGR3B. Weak but
statistically significant linkage disequilibrium was also
observed between FCGR2B and FCGR2A, FCGR2B and
FCGR3A, and FCGR3A and FCGR3B, but in none of
the other combinations (Table 3). The FCGR2B-232T
allele was shown to form a haplotype with the FCGR3BNA2 allele. Strong association between FCGR2B-232T
and FCGR3B-NA2 was also observed in patients with
SLE (Table 3).
Association of FCGR genotypes with clinical
characteristics of SLE. We next analyzed the association
of FCGR2B, FCGR2A, FCGR3A, and FCGR3B polymorphisms with disease phenotypes, such as age at
onset, presence of lupus nephritis, central nervous system lupus, serositis, anti–double-stranded DNA (antidsDNA) antibodies, anti-Sm antibodies, and low complement, in patients with SLE. When genotype
frequencies were compared between patients having a
1248
KYOGOKU ET AL
Table 3. Estimated haplotype frequencies and linkage disequilibrium among FCGR2A-131R/H, FCGR2B-232I/T, FCGR3A-176V/F, and
FCGR3B-NA1/NA2 polymorphisms in Japanese subjects*
Haplotype
Controls (n ⫽ 303)
2B–2A
232I-131H
232I-131R
232T-131H
232T-131R
2B–3A
232I-176F
232I-176V
232T-176F
232T-176V
2B–3B
232I-NA1
232I-NA2
232T-NA1
232T-NA2
2A–3A
131H-176F
131H-176V
131R-176F
131R-176V
2A–3B
131H-NA1
131H-NA2
131R-NA1
131R-NA2
3A–3B
176F-NA1
176F-NA2
176V-NA1
176V-NA2
SLE patients (n ⫽ 193)
2B–3B
232I-NA1
232I-NA2
232T-NA1
232T-NA2
␹2 (1 df)
P
HF, %
LD, %
RLD
60.5
17.0
20.2
2.3
⫺2.1
2.1
2.1
⫺2.1
⫺0.48
0.48
0.48
⫺0.48
9.7
0.002
51.9
25.6
17.7
4.8
⫺2.0
2.0
2.0
⫺2.0
⫺0.30
0.30
0.30
⫺0.30
6.8
0.01
61.4
16.1
0.8
21.7
13.2
⫺13.2
⫺13.2
13.2
0.94
⫺0.94
⫺0.94
0.94
257.3
55.3
25.4
14.3
5.0
⫺0.9
0.9
0.9
⫺0.9
⫺0.15
0.15
0.15
⫺0.15
1.4
0.24
51.6
29.1
10.6
8.7
1.4
⫺1.4
⫺1.4
1.4
0.12
⫺0.12
⫺0.12
0.12
3.4
0.06
40.0
29.6
22.2
8.2
⫺3.3
3.3
3.3
⫺3.3
⫺0.29
0.29
0.29
⫺0.29
13.1
0.0003
55.4
16.6
2.1
25.9
14.0
⫺14.0
⫺14.0
14.0
0.87
⫺0.87
⫺0.87
0.87
153.1
⬍10⫺10
⬍10⫺10
* HF ⫽ estimated haplotype frequency; LD ⫽ linkage disequilibrium; RLD ⫽ relative linkage disequilibrium (see Table 1 for other definitions).
particular clinical phenotype and healthy individuals,
FCGR2B was most strongly associated with lupus nephritis (␹2 ⫽ 10.4, P ⫽ 0.01), although associations
between FCGR3A (␹2 ⫽ 7.0, P ⫽ 0.03) and FCGR3B
(␹2 ⫽ 7.1, P ⫽ 0.03) and lupus nephritis were also
detected. For age at disease onset ⬍20 years and for
anti-dsDNA positivity, only FCGR3A was significantly
associated (␹2 ⫽ 6.5, P ⫽ 0.04 and ␹2 ⫽ 7.4, P ⫽ 0.02,
respectively). No notable associations were observed for
any other clinical or immunologic characteristics.
When comparisons were made between patients
with and those without particular clinical characteristics,
the only significant difference observed was for FCGR3B
between patients with and those without nephritis (␹2 ⫽
7.4, P ⫽ 0.02). Although there was a striking difference
in the male-to-female ratio between patients and con-
trols, the results were essentially identical when sexadjusted controls were used (data not shown).
Elucidation of the contribution of each FCGR
locus. As described above, there was a weak but significant allelic association between FCGR2B-232T and
FCGR3A-176F. To dissect the contributions of the
FCGR2B and FCGR3A loci, 2-locus analysis was performed, as shown in Table 4. ORs against the combined
FCGR2B-232I/I and FCGR3A-176V/V genotype were
calculated. Elevated ORs were observed in individuals
with the combined FCGR2B-232I/T and FCGR3A176F/F genotype (OR 3.0), the combined FCGR2B232T/T and FCGR3A-176V/F genotype (OR 4.8), and
the combined FCGR2B-232T/T and FCGR3A-176F/F
genotype (OR 4.4), but not in those with the combined
FCGR2B-232I/I and FCGR3A-176V/F genotype or
FCGR2B GENE POLYMORPHISM IN SLE
1249
Table 4. Results of 2-locus analysis of FCGR2B and FCGR3A*
176V/V
232I/I
232I/T
232T/T
176V/F
176F/F
SLE
Controls
OR
SLE
Controls
OR (95% CI)
SLE
Controls
OR (95% CI)
6 (3.1)
1 (0.5)
0 (0)
21 (6.9)
5 (1.7)
0 (0)
1.0
47 (24.4)
18 (9.3)
11 (5.7)
79 (26.1)
45 (14.9)
8 (2.6)
2.1 (0.8–5.4)†
1.4 (0.5–4.0)§
4.8 (1.4–16.8)#
53 (27.5)
47 (24.4)
10 (5.2)
83 (27.4)
54 (17.8)
8 (2.6)
2.2 (0.9–5.8)‡
3.0 (1.2–7.9)¶
4.4 (1.2–15.5)**
* Values are the number (%) (n ⫽ 193 SLE patients and 303 controls). Each OR was calculated in relation to subjects with the combined
FCGR2B-232I/I and FCGR3A-176V/V genotypes. See Table 1 for definitions.
† ␹2 ⫽ 2.2, P ⫽ 0.14.
‡ ␹2 ⫽ 2.7, P ⫽ 0.10.
§ ␹2 ⫽ 0.4, P ⫽ 0.53.
¶ ␹2 ⫽ 5.2, P ⫽ 0.02.
# ␹2 ⫽ 6.1, P ⫽ 0.01.
** ␹2 ⫽ 5.2, P ⫽ 0.02.
FCGR2B-232I/I and FCGR3A-176F/F genotype. From
the comparison of ORs with the combined FCGR2B232I/T and FCGR3A-176V/F genotype (OR 1.4) and
those with the combined FCGR2B-232T/T and
FCGR3A-176V/F genotype or the combined FCGR2B232I/T and FCGR3A-176F/F genotype, it was suggested
that both FCGR2B-232T and FCGR3A-176F may contribute to susceptibility.
The contribution of each locus was further analyzed using the method of Svejgaard and Ryder (27), in
which the genotypic combination data were analyzed in
various 2 ⫻ 2 tables. For each comparison, the OR was
calculated by Haldane’s modification of Woolf’s method
and the significance was tested by Fisher’s exact test, and
then the P value was multiplied by the number of
comparisons (27). When allele positivity was analyzed
(Table 5), only modest association of FCGR3A-176F
(test 2: OR 2.4), but not of FCGR2B-232T (test 1: OR
1.3) was detected.
However, our data had shown that FCGR2B232T confers susceptibility only when it is homozygous
(Table 1); therefore, we also compared the contribution
of the FCGR2B-232T/T genotype and FCGR3A-176F
positivity (Table 6). This analysis indicated that the
association of FCGR2B-232T/T (test 1: OR 2.2) was as
significant as that of FCGR3A-176F (test 2). When P
values were multiplied by the number of comparisons,
FCGR2B showed statistical significance (corrected P
[Pcorr] ⫽ 0.04), but FCGR3A did not (Pcorr ⫽ 0.08),
indicating the stronger association of FCGR2B than
FCGR3A. The results of this analysis did not support the
notion that the association of FCGR2B and FCGR3A is
independent (tests 3, 4, 5, and 6). The OR was increased
among individuals with the FCGR2B-232T/T genotype
Table 5. Two-locus analysis of FCGR2B and FCGR3A using 2 ⫻ 2 comparisons, where ⫹⫹ ⫽ FCGR2B-232T (factor 1 [F1]) and FCGR3A-176F
(factor 2 [F2]) positive, ⫹⫺ ⫽ F1 positive and F2 negative, ⫺⫹ ⫽ F1 negative and F2 positive, and ⫺⫺ ⫽ F1 and F2 negative*
Test no.,
comparison
1,
2,
3,
4,
5,
6,
7,
8,
F1 vs. non-F1
F2 vs. non-F2
⫹⫹ vs. ⫺⫹
⫹⫺ vs. ⫺⫺
⫹⫹ vs. ⫹⫺
⫺⫹ vs. ⫺⫺
⫹⫺ vs. ⫺⫹
⫹⫹ vs. ⫺⫺
a
b
c
d
OR
␹2
P†
87
186
86
1
86
100
1
86
106
7
100
6
1
6
100
6
120
277
115
5
115
162
5
115
183
26
162
21
5
21
162
21
1.3
2.4‡
1.2
0.9
2.7
2.0
0.4
2.5
1.5
4.7
1.0
0.1
1.6
2.7
1.2
4.2
0.26
0.04§
0.34
1.00
0.40
0.14
0.41
0.06
* Of the 193 SLE patients, 86 (44.6%) were ⫹⫹, 1 (0.5%) was ⫹⫺, 100 (51.8%) were ⫺⫹, and 6 (3.1%) were ⫺⫺. Of the 303 controls, 115 (38.0%)
were ⫹⫹, 5 (1.7%) were ⫹⫺, 162 (53.5%) were ⫺⫹, and 21 (6.9%) were ⫺⫺. ORs were calculated by Haldane’s modification of Woolf’s method:
OR ⫽ [(a ⫹ 1/2)(d ⫹ 1/2)]/[(b ⫹ 1/2)(c ⫹ 1/2)]. Tests 1 and 2 investigate individual associations of F1 and F2; tests 3 and 4 investigate whether F1
is associated independently of F2; tests 5 and 6 investigate whether F2 is associated independently of F1; tests 7 and 8 investigate a combined F1
and F2 association. See ref. 26 for details; see Table 1 for definitions.
† By Fisher’s exact test.
‡ 95% CI ⫽ 1.1–5.2.
§ Corrected P ⫽ 0.08 (multiplied correction factor ⫽ 2).
1250
KYOGOKU ET AL
Table 6. Two-locus analysis of FCGR2B and FCGR3A using 2 ⫻ 2 comparisons, where ⫹⫹ ⫽ FCGR2B-232T/T (factor 1 [F1]) and FCGR3A-176F
(factor 2 [F2]) positive, ⫹⫺ ⫽ F1 positive and F2 negative, ⫺⫹ ⫽ F1 negative and F2 positive, and ⫺⫺ ⫽ F1 and F2 negative*
Test no.,
comparison
1,
2,
3,
4,
5,
6,
7,
8,
F1 vs. non-F1
F2 vs. non-F2
⫹⫹ vs. ⫺⫹
⫹⫺ vs. ⫺⫺
⫹⫹ vs. ⫹⫺
⫺⫹ vs. ⫺⫺
⫹⫺ vs. ⫺⫹
⫹⫹ vs. ⫺⫺
a
b
c
d
OR (95% CI)
␹2
21
186
21
0
21
165
0
21
172
7
165
7
0
7
165
7
16
277
16
0
16
261
0
16
287
26
261
26
0
26
261
26
2.2 (1.1–4.2)
2.4 (1.1–5.2)
2.1 (1.1–4.0)
3.5
1.3
2.2
1.6
4.6 (1.7–12.4)
5.4
4.7
4.6
–
–
4.0
–
9.2
P (Pcorr)†
0.02 (0.04) [2]
0.04 (0.08) [2]
0.04 (0.24) [6]
1.00
1.00
0.06
1.00
0.003 (0.027) [4]
* Of the 193 SLE patients, 21 (10.9%) were ⫹⫹, 0 were ⫹⫺, 165 (85.5%) were ⫺⫹, and 7 (3.6%) were ⫺⫺. Of the 303 controls, 16 (5.3%) were
⫹⫹, 0 were ⫹⫺, 261 (86.1%) were ⫺⫹, and 26 (8.6%) were ⫺⫺. See ref. 26 and Table 5 for details; see Table 1 for other definitions.
† Pcorr ⫽ corrected P, with multiplied correction factor shown in brackets.
who also carried the FCGR3A-176F allele (test 8: OR
4.6) compared with individuals with the FCGR2B232T/T genotype irrespective of the FCGR3A genotype
(test 1), suggesting the possibility that FCGR2B and
FCGR3A contribute to susceptibility in an additive
manner.
Two-locus analysis was also carried out for
FCGR2B-232T and FCGR3B-NA2, which are in strong
linkage disequilibrium. FCGR3B-NA2 was previously
shown to be associated with SLE in Japanese patients
(11). As shown in Table 7, a significant increase in the
OR was observed in individuals with the combined
FCGR2B-232T/T and FCGR3B-NA2/2 genotype (OR
2.5), but not in those with other combinations. No risk
was observed for combinations of FCGR2B-232I/T and
FCGR3B-NA1/2 genotypes (OR 1.2), or FCGR2B232I/T and FCGR3B-NA2/2 genotypes (OR 1.1), suggesting that FCGR3B-NA1/2 heterozygosity and
FCGR3B-NA2/2 homozygosity are not associated with
SLE in the absence of the FCGR2B-232T/T genotype.
Therefore, the association of FCGR3B was considered
to be secondary to FCGR2B, and derived from strong
linkage disequilibrium.
FCGR genotypes and linkage disequilibrium in
healthy Dutch individuals. To extend these observations
to other populations, we examined the FCGR2B-232I/T
polymorphism in a Dutch population. Among 148
healthy Dutch individuals, 118 (79.7%), 29 (19.6%), and
1 (0.7%) were typed to have 232I/I, I/T, and T/T,
respectively. These genotype frequencies were compatible with Hardy-Weinberg equilibrium. The allele frequency of FCGR2B-232T in the Dutch population was
10.5%, which was considerably lower than that in the
Japanese population (Table 1).
When 2-locus linkage disequilibria were analyzed
among the 4 FCGR genes, strong linkage disequilibrium
was detected between FCGR3A and FCGR3B (␹2 ⫽
48.2, P ⬍ 10⫺10). Weak, but statistically significant,
linkage disequilibrium was also observed for all other
combinations (FCGR2B and FCGR3A ␹2 ⫽ 10.6, P ⬍
0.001; FCGR2B and FCGR3B ␹2 ⫽ 10.0, P ⬍ 0.002;
FCGR2A and FCGR3A ␹2 ⫽ 15.8, P ⬍ 0.0001; FCGR2A
Table 7. Results of 2-locus analysis of FCGR2B and FCGR3B*
NA1/1
232I/I
232I/T
232T/T
NA1/2
NA2/2
SLE
Controls
OR
SLE
Controls
OR (95% CI)
SLE
Controls
OR (95% CI)
58 (30.1)
4 (2.1)
0 (0)
114 (37.6)
2 (0.7)
0 (0)
1.0
46 (23.8)
49 (25.4)
3 (1.6)
65 (21.5)
78 (25.7)
2 (0.7)
1.4 (0.9–2.3)†
1.2 (0.8–2.0)‡
2 (1.0)
13 (6.7)
18 (9.3)
4 (1.3)
24 (7.9)
14 (4.6)
1.1 (0.5–2.3)§
2.5 (1.2–5.4)¶
* Values are the number (%) (n ⫽ 193 SLE patients and 303 controls). Each OR was calculated in relation to subjects with the combined
FCGR2B-232I/I and FCGR3B-NA1/1 genotypes. See Table 1 for definitions.
† ␹2 ⫽ 1.7, P ⫽ 0.19.
‡ ␹2 ⫽ 0.8, P ⫽ 0.39.
§ ␹2 ⫽ 0.03, P ⫽ 0.87.
¶ ␹2 ⫽ 5.9, P ⫽ 0.02.
FCGR2B GENE POLYMORPHISM IN SLE
and FCGR3B ␹2 ⫽ 7.8, P ⬍ 0.01), except between
FCGR2B and FCGR2A. In the Dutch population, the
FCGR3A-176F allele was shown to form a haplotype
with the FCGR3B-NA1 allele (estimated haplotype frequencies of 176F-NA1 and 176V-NA2 30.1% and
35.9%, respectively; linkage disequilibrium 9.4, relative
linkage disequilibrium 0.67), rather than with the
FCGR3B-NA2 allele as observed in the Japanese (Table 3).
DISCUSSION
In the present study, we have identified a polymorphism in the human FCGR2B gene and demonstrated its association with SLE in the Japanese population. A number of difficulties were encountered before a
reliable method for typing FCGR2B-232I/T using
genomic DNA was established, because the FCGR2B
gene is extremely homologous to FCGR2C, even in
intron sequences flanking exons. In the process of
finding a nucleotide sequence specific for FCGR2B,
many variation sites within intron 4 were detected
(GenBank accession no. AB062416). All of the sites
previously considered to differ between FCGR2B and
FCGR2C (ref. 30 and GenBank accession nos. L08108
and L08109) were indeed found to be polymorphic, and
1 of the alleles at each site was always identical to the
FCGR2C sequence. Therefore, nested PCR with a
primer placed in exon 7, containing a sequence unique
to FCGR2B, was used for genotyping. Indeed, genotyping results obtained using genomic DNA and cDNA
were identical in 94 healthy individuals and 75 patients
whose cDNA samples were available.
In a previous study with a smaller sample size, we
found a significant association between the FCGR3BNA2 allele and SLE in the Japanese (11). In the present
study, with a larger number of patients and controls, the
association of FCGR3B-NA2 did not reach statistical
significance. Strong linkage disequilibrium between
FCGR2B-232T and FCGR3B-NA2, as well as the lack of
an independent contribution of FCGR3B-NA2 observed
in the 2-locus analysis, strongly suggest that the previously reported association of FCGR3B-NA2 was caused
by linkage disequilibrium with FCGR2B-232T. However,
functional differences between FCGR2B-232I and -232T
need to be demonstrated before final conclusions can be
drawn; functional differences involving Fc␥R-mediated
phagocytosis between FCGR3B-NA1 and -NA2 have
already been established (35).
In addition to the FCGR2B-232I/T polymorphism, the FCGR3A-176V/F polymorphism was also
1251
significantly associated with susceptibility to SLE in
Japanese patients in this study, thus replicating previous
observations in different populations by several groups
(36–40). The Lathrop-type analysis suggested that the
association of FCGR3A-176F is perhaps weaker than
that of FCGR2B, apart from a genotypic difference that
stems from slight deviations from Hardy-Weinberg equilibrium in the control population.
Previous studies demonstrating functional differences in immune complex clearance between FCGR2A131R and -131H (21,41), and between FCGR3A-176V
and -176F (42) provide strong support for FCGR2A and
FCGR3A as candidate genes for SLE susceptibility.
However, associations of these genes have conflicted in
different populations. One possible explanation for
these inconsistencies is a difference in disease phenotypes. Anti-dsDNA antibodies were shown to largely
belong to the IgG1 and IgG3 subclasses (43), suggesting
an important role for the Fc␥RIIIA-176F allele, which
has lower affinity for IgG1 and IgG3, in the clearance of
immune complexes containing anti-dsDNA antibodies.
Our results, indicating a stronger association of
FCGR3A in patients who are positive for anti-dsDNA
antibodies, are compatible with this. Likewise, several
groups have reported an association with the Fc␥RIIA131R allele, which has lower affinity for IgG2, with SLE
only in patients with lupus nephritis (44–46).
Thus, differences in physiologic roles of Fc␥
receptors in regulation of the immune response may
possibly lead to different associations with respect to
disease expression in SLE. In our study, a stronger
association with lupus nephritis was found for FCGR2B
compared with FCGR3A, suggesting that FCGR2B plays
a crucial role in renal disease, or in the overall severity
of SLE, in Japanese patients.
Based on the results of in vitro and animal
studies, which have clearly demonstrated a role for
Fc␥RIIB1 as a negative regulator of B cells (12), it is
hypothesized that the Fc␥RIIB-232I/T polymorphism
may somehow be related to decreased function of
Fc␥RIIB1, thus allowing the production of autoantibodies characteristic for SLE. Although the notion of a
functional difference associated with the FCGR2B232I/T substitution within the transmembrane region
remains speculative, a recent study did demonstrate a
role of the transmembrane domain of Fc␥RIIB1 in
inducing B cell apoptosis (47). Thus, one possibility is
that the Fc␥RIIB-232I/T polymorphism may alter apoptotic signaling, allowing survival of B cells that produce
autoantibodies, leading to autoimmune disease. Furthermore, a recent study using deletion mutants of
1252
Fc␥RIIB revealed functional contributions of multiple
tyrosine sites, including sites in the transmembrane
region (48).
An alternative possibility regarding the function
of the Fc␥RIIB2-232T allele may be that it reduces the
function of immunoreceptor tyrosine-based activation
motif (ITAM)–bearing Fc␥R in monocytes. Recently, an
in vitro study demonstrated that Fc␥RIIB2 inhibits
Fc␥RI-mediated phagocytosis when coaggregated with
Fc␥RI, a receptor that associates with the ITAMcontaining ␥ chain to transmit activation signals (49).
Furthermore, another study showed that during treatment with intravenous gamma globulin, (wellrecognized for its antiinflammatory activity), enhanced
cell surface expression of Fc␥RIIB was responsible for
the inhibition of phagocytosis mediated through activating Fc␥R (50). Thus, another possibility is that the
Fc␥RIIB-232I/T polymorphism may alter the balance
between activating and inhibitory Fc␥R and lead to the
development of autoimmune disease, either through
lowering the threshold for triggering an immune response or through down-regulating phagocytic activity.
This hypothesis may, at least in part, explain the interaction between Fc␥RIIB2-232T and IIIA-176F observed
in the 2-locus analysis.
We also found the FCGR2B-232I/T polymorphism in a Dutch population, although the allele frequency of FCGR2B-232T was substantially lower than in
the Japanese population. In addition, the haplotype
combinations between FCGR3A and FCGR3B differed
between the Japanese and the Dutch. It is highly likely
that the inconsistencies in susceptibility genes detected
in case–control studies are related to such differences in
allele frequencies and haplotype combinations among
populations. They may also explain population-related
differences in genome-wide linkage analyses, showing
that linkage of the Fc␥R gene region with SLE is more
readily detected in African American compared with
Caucasian populations (51).
There are some potential limitations in this study.
The statistical significance of the differences detected is
relatively weak. In total, 22 independent comparisons,
including 4 comparisons for each of the 4 loci (genotype
frequency, allele frequency, the positivity of each allele)
and Lathrop analysis for each genotype for FCGR2B
and FCGR3A ([4 ⫻ 4] ⫹ [3 ⫻ 2] ⫽ 22) were performed
with respect to the association of a particular allele or
genotype with SLE (see Results and Tables 1 and 2).
When the P values are corrected by multiplying the
number of comparisons, only the association of the
FCGR2B-232T/T genotype remains significant (Pcorr ⫽
KYOGOKU ET AL
0.044). However, as mentioned above, FCGR3A-176F
has been shown to be associated with SLE in several
previous studies in various populations, and it is unlikely
that our current data represent a Type I error.
On the other hand, the association of FCGR2B232T is a new finding which needs to be confirmed in the
future in independent studies in Japanese as well as
other populations. In some populations in which population admixture is a significant problem, the advantage
of family-based association studies over case–control
association studies, to avoid detection of spurious associations, has been established. However, the Japanese
have been shown to be relatively homogeneous with
respect to genetic background (20). In fact, in our
previous association studies of a number of genes on
various chromosomes, using Japanese SLE and control
cohorts substantially overlapping those used in this
study, significant association was not detected, suggesting that these 2 cohorts, at least, do not differ considerably with respect to the genetic background.
In conclusion, our present findings strongly suggest that both FCGR2B and FCGR3A contribute to the
genetic susceptibility to SLE in the Japanese. The
association of the FCGR2B-232T/T genotype seems to
be stronger than that of FCGR3A-176F. The role of
Fc␥R genes in the genetic background of SLE appears
to be highly complicated, with multiple genes associated
with different aspects of disease in different populations.
To confirm a contribution of FCGR2B, further genetic
studies of the FCGR2B polymorphism in various populations, as well as functional analyses of the 2B-232T
allele, are necessary. Polymorphism screening in the
promoter region of FCGR2B and FCGR2C should also
be performed, when reliable genomic sequences of these
loci become available. In addition, our observations
indicate that all FCGR genotypes should be determined
in every study population, in order to fully evaluate
which gene contributes primarily to SLE susceptibility in
each population.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Jun Ohashi (University of Tokyo) for statistical analysis, to Michiko Shiota for
technical assistance, and to Dr. Sachiko Hirose (Juntendo
University) for stimulating discussions.
REFERENCES
1. Moser KL, Neas BR, Salmon JE, Yu H, Gray-McGuire C, Asundi
N, et al. Genome scan of human systemic lupus erythematosus:
FCGR2B GENE POLYMORPHISM IN SLE
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
evidence for linkage on chromosome 1q in African-American
pedigrees. Proc Natl Acad Sci U S A 1998;95:14869–74.
Shai R, Quismorio FP Jr, Li L, Kwon O-J, Morrison J, Wallace DJ,
et al. Genome-wide screen for systemic lupus erythematosus
susceptibility genes in multiplex families. Hum Mol Genet 1999;
8:639–44.
Wakeland EK, Wandstrat AE, Liu K, Morel L. Genetic dissection
of systemic lupus erythematosus. Curr Opin Immunol 1999;11:
701–7.
Peltz GA, Grundy HO, Lebo RV, Yssel H, Barsh GS, Moore KW.
Human Fc␥RIII: cloning, expression, and identification of the
chromosomal locus of two Fc receptors for IgG. Proc Natl Acad
Sci U S A 1989;86:1013–7.
Qiu WQ, de Bruin D, Brownstein BH, Pearse R, Ravetch JV.
Organization of the human and mouse low-affinity Fc␥R genes:
duplication and recombination. Science 1990;248:732–5.
Su Y, Brooks DG, Li L, Lepercq J, Trofatter JA, Ravetch JV, et
al. Myelin protein zero gene mutated in Charcot-Marie-Tooth
type 1B patients. Proc Natl Acad Sci U S A 1993;90:10856–60.
Callanan MB, Le Baccon P, Mossuz P, Duley S, Bastard C,
Hamoudi R, et al. The IgG Fc receptor, Fc␥RIIB, is a target for
deregulation by chromosomal translocation in malignant lymphoma. Proc Natl Acad Sci U S A 2000;97:309–14.
Su K, Edberg JC, Wu J, McKenzie SE, Kimberly RP. Single
nucleotide polymorphisms in the FcgRIIB gene promoter which
alter receptor expression and associate with systemic lupus erythematosus in African Americans [abstract]. Arthritis Rheum
2001;44(Suppl 9):S248.
Van der Pol W-L, van de Winkel JGJ. IgG receptor polymorphisms: risk factors for disease. Immunogenetics 1998;48:222–32.
Salmon JE, Pricop L. Human receptors for immunoglobulin G:
elements in the pathogenesis of rheumatic disease. Arthritis
Rheum 2001;44:739–50.
Hatta Y, Tsuchiya N, Ohashi J, Matsushita M, Fujiwara K,
Hagiwara K, et al. Association of Fc␥ receptor IIIB, but not of Fc␥
receptor IIA and IIIA, polymorphisms with systemic lupus erythematosus in Japanese. Genes Immun 1999;1:53–60.
Ravetch JV, Lanier LL. Immune inhibitory receptors. Science
2000;290:84–9.
Yuasa T, Kubo S, Yoshino T, Ujike A, Matsumura K, Ono M, et
al. Deletion of Fc␥ receptor IIB renders H-2b mice susceptible to
collagen-induced arthritis. J Exp Med 1999;189:187–94.
Nakamura A, Yuasa T, Ujike A, Ono M, Nukiwa T, Ravetch JV,
et al. Fc␥ receptor IIB-deficient mice develop Goodpasture’s
syndrome upon immunization with type IV collagen: a novel
murine model for autoimmune glomerular basement membrane
disease. J Exp Med 2000;191:899–905.
Bolland S, Ravetch JV. Spontaneous autoimmune disease in
Fc␥RIIB-deficient mice results from strain-specific epistasis. Immunity 2000;13:277–85.
Jiang Y, Hirose S, Sanokawa-Akakura R, Abe M, Mi X, Li N, et
al. Genetically determined aberrant down-regulation of Fc␥RIIB1
in germinal center B cells associated with hyper-IgG and IgG
autoantibodies in murine systemic lupus erythematosus. Int Immunol 1999;11:1685–91.
Jiang Y, Hirose S, Abe M, Sanokawa-Akakura R, Ohtsuji M, Mi
X, et al. Polymorphisms in IgG Fc receptor IIB regulatory regions
associated with autoimmune susceptibility. Immunogenetics 2000;
51:429–35.
Slingsby JH, Hogarth MB, Walport MJ, Morley BJ. Polymorphism
in the Ly-17 alloantigenic system of the mouse Fc␥RII gene.
Immunogenetics 1997;46:361–2.
Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield
NF, et al. The 1982 revised criteria for the classification of systemic
lupus erythematosus. Arthritis Rheum 1982;25:1271–7.
Tokunaga K, Imanishi T, Takahashi K, Juji T. On the origin and
dispersal of East Asian populations as viewed from HLA haplo-
1253
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
types. In: Akazawa T, Szathmary EJ, editors. Prehistoric Mongoloid dispersals. Oxford: Oxford University Press; 1996. p. 187–97.
Dijstelbloem HM, Bijl M, Fijnheer R, Scheepers RHM, Oost WW,
Jansen MD, et al. Fc␥ receptor polymorphisms in systemic lupus
erythematosus: association with disease and in vivo clearance of
immune complexes. Arthritis Rheum 2000;43:2793–800.
Brooks DG, Qiu WQ, Luster AD, Ravetch JV. Structure and
expression of human IgG FcRII (CD32). J Exp Med 1989;170:
1369–85.
Komata T, Tsuchiya N, Matsushita M, Hagiwara K, Tokunaga K.
Association of tumor necrosis factor receptor 2 (TNFR2) polymorphism with susceptibility to systemic lupus erythematosus.
Tissue Antigens 1999;53:527–33.
Fujiwara K, Watanabe Y, Mitsunaga S, Oka T, Yamane A, Akaza
T, et al. Determination of granulocyte-specific antigens on neutrophil Fc␥ receptor IIIb by PCR-PHFA (preferential homoduplex
formation assay) and gene frequencies in the Japanese population.
Vox Sang 1999;77:218–22.
Bux J, Stein E-L, Bierling P, Fromont P, Clay M, Stroncek D, et al.
Characterization of a new alloantigen (SH) on the human neutrophil Fc␥ receptor IIIb. Blood 1997;89:1027–34.
Lathrop GM. Estimating genotype relative risks. Tissue Antigens
1983;22:160–6.
Svejgaard A, Ryder LP. HLA and disease associations: detecting
the strongest association. Tissue Antigens 1994;43:18–27.
Xie X, Ott J. Testing linkage disequilibrium between a disease
gene and marker loci [abstract]. Am J Hum Genet 1993;53(Suppl):
1107.
Van de Winkel JGJ, Capel PJA. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol
Today 1993;14:215–21.
Warmerdam PAM, Nabben NMJM, van de Graaf SAR, van de
Winkel JGJ, Capel PJA. The human low affinity immunoglobulin
G Fc receptor IIC gene is a result of an unequal crossover event.
J Biol Chem 1993;268:7346–9.
Warmerdam PAM, van den Herik-Oudijk IE, Parren PWHI,
Westerdaal NAC, van de Winkel JGJ, Capel PJA. Interaction of a
human Fc␥RIIb1 (CD32) isoform with murine and human IgG
subclasses. Int Immunol 1993;5:239–47.
Den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion.
Hum Mutat 2000;15:7–12.
Van den Herik-Oudijk IE, Westerdaal NAC, Henriquez NV,
Capel PJA, van de Winkel JGJ. Functional analysis of human
Fc␥RII (CD32) isoforms expressed in B lymphocytes. J Immunol
1994;152:574–85.
Lehrnbecher T, Foster CB, Zhu S, Leitman SF, Goldin LR, Huppi
K, et al. Variant genotypes of the low-affinity Fc␥ receptors in two
control populations and a review of low-affinity Fc␥ receptor
polymorphisms in control and disease populations. Blood 1999;94:
4220–32.
Salmon JE, Edberg JC, Kimberly RP. Fc gamma receptor III on
human neutrophils: allelic variants have functionally distinct capacities. J Clin Invest 1990;85:1287–95.
Wu J, Edberg JC, Redecha PB, Bansal V, Guyre PM, Coleman K,
et al. A novel polymorphism of Fc␥RIIIa (CD16) alters receptor
function and predisposes to autoimmune disease. J Clin Invest
1997;100:1059–70.
Koene HR, Kleijer M, Swaak AJG, Sullivan KE, Bijl M, Petri MA,
et al. The Fc␥RIIIA-158F allele is a risk factor for systemic lupus
erythematosus. Arthritis Rheum 1998;41:1813–8.
Salmon JE, Ng S, Yoo D-H, Kim T-H, Kim SY, Song GG. Altered
distribution of Fc␥ receptor IIIA alleles in a cohort of Korean
patients with lupus nephritis. Arthritis Rheum 1999;42:818–9.
Zuñiga R, Ng S, Peterson MGE, Reveille JD, Baethge BA,
Alarcón GS, et al. Low-binding alleles of Fc␥ receptor types IIA
and IIIA are inherited independently and are associated with
1254
40.
41.
42.
43.
44.
45.
systemic lupus erythematosus in Hispanic patients. Arthritis
Rheum 2001;44:361–7.
Seligman VA, Suarez C, Lum R, Inda SE, Lin D, Li H, et al. The
Fc␥ receptor IIIA–158F allele is a major risk factor for the
development of lupus nephritis among Caucasians but not nonCaucasians. Arthritis Rheum 2001;44:618–25.
Warmerdam PAM, van de Winkel JGJ, Vlug A, Westerdaal NAC,
Capel PJA. A single amino acid in the second Ig-like domain of the
human Fc␥ receptor II is critical for human IgG2 binding.
J Immunol 1991;147:1338–43.
Koene HR, Kleijer M, Algra J, Roos D, von dem Borne AEGK, de
Haas M. Fc␥RIIIa-158V/F polymorphism influences the binding
of IgG by natural killer cell Fc␥RIIIa, independently of the
Fc␥RIIIa-48L/R/H phenotype. Blood 1997;90:1109–14.
Amoura Z, Koutouzov S, Chabre H, Cacoub P, Amoura I, Musset
L, et al. Presence of antinucleosome autoantibodies in a restricted
set of connective tissue diseases: antinucleosome antibodies of the
IgG3 subclass are markers of renal pathogenicity in systemic lupus
erythematosus. Arthritis Rheum 2000;43:76–84.
Duits AJ, Bootsma H, Derksen RHWM, Spronk PE, Kater L,
Kallenberg CGM, et al. Skewed distribution of IgG Fc receptor IIa
(CD32) polymorphism is associated with renal disease in systemic
lupus erythematosus patients. Arthritis Rheum 1995;38:1832–6.
Salmon JE, Millard S, Schachter LA, Arnett FC, Ginzler EM,
Gourley MF, et al. Fc␥RIIA alleles are heritable risk factors for
KYOGOKU ET AL
46.
47.
48.
49.
50.
51.
lupus nephritis in African Americans. J Clin Invest 1996;97:
1348–54.
Song YW, Han C-W, Kang S-W, Baek H-J, Lee E-B, Shin C-H, et
al. Abnormal distribution of Fc␥ receptor type IIa polymorphisms
in Korean patients with systemic lupus erythematosus. Arthritis
Rheum 1998;41:421–6.
Pearse RN, Kawabe T, Bolland S, Guinamard R, Kurosaki T,
Ravetch JV. SHIP recruitment attenuates Fc␥RIIB-induced B cell
apoptosis. Immunity 1999;10:753–60.
Fong DC, Brauweiler A, Minskoff SA, Bruhns P, Tamir I,
Mellman I, et al. Mutational analysis reveals multiple distinct sites
within Fc␥ receptor IIB that function in inhibitory signaling.
J Immunol 2000;165:4453–62.
Pricop L, Redecha P, Teillaud J-L, Frey J, Fridman WH, SautèsFridman C, et al. Differential modulation of stimulatory and
inhibitory Fc␥ receptors on human monocytes by Th1 and Th2
cytokines. J Immunol 2001;166:531–7.
Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor. Science
2001;291:484–6.
Gray-McGuire C, Moser KL, Gaffney PM, Kelly J, Yu H, Olson
JM, et al. Genome scan of human systemic lupus erythematosus by
regression modeling: evidence of linkage and epistasis at 4p1615.2. Am J Hum Genet 2000;67:1460–9.
Документ
Категория
Без категории
Просмотров
0
Размер файла
250 Кб
Теги
lupus, japanese, patients, polymorphism, fcgr2b, erythematosuscontribution, systemic, genes, receptov, genetics, susceptibility
1/--страниц
Пожаловаться на содержимое документа