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: firstname.lastname@example.org 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)  0.04 (0.08)  0.04 (0.24)  1.00 1.00 0.06 1.00 0.003 (0.027)  * 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. 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