¹Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

²Matsuta Clinic, Tokyo, Japan

Correspondence to: Dr N Tsuchiya, Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: tsuchiya-tky@umin.ac.jp

Supported by the Grant-in-Aid for Scientific Research on Priority Areas (C) 'Medical Genome Science', the Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan, Health Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan, and the Grant-in-Aid for JSPS Fellows.

We recently detected a new single nucleotide polymorphism of FcRIIB gene, which alters an amino acid within the transmembrane domain from Ile to Thr (I232T), and its association with SLE in the Japanese. This study was performed to examine whether FCGR2B-I232T was associated with susceptibility to rheumatoid arthritis in the Japanese. At the same time, FCGR2A, 3A and 3B polymorphisms were also examined. Genotyping of FCGR2B-I232T, FCGR2A-H131R, FCGR3A-F176V and FCGR3B-NA1/2 polymorphisms were performed using genomic DNA. Association with RA was analyzed in 382 Japanese patients with RA and 303 healthy individuals using a case-control approach. In addition, the same groups of patients and controls were genotyped for HLA-DRB1 to examine possible interaction with FCGR genes. Significantly different distribution of genotype, allele carrier and allele frequencies was not observed between patients with RA and healthy individuals in any of the four polymorphisms. When the subjects were stratified according to the carriage of HLA-DRB1 shared epitope (SE), significant increase of FCGR3A-176F/F genotype was observed in SE positive patients compared with SE positive healthy individuals (P=0.009, P_corr=0.07). In conclusion, FCGR3A-176F/F genotype was considered to confer risk through genetic interaction with HLA-DRB1 SE.

Genes and Immunity (2002) 3,488-493. doi:10.1038/sj.gene.6363921

Fc receptor; FCGR3A polymorphism; rheumatoid arthritis; HLA-DRB1 shared epitope

Introduction

Fc receptors (FcRs) are members of the immunoglobulin superfamily that bind to the Fc portion of IgG. Human low-affinity FcRs consist of three FcRII (IIA, IIB, IIC) and two FcRIII (IIIA, IIIB), which vary in their cellular distribution and affinity for different IgG isotypes. FcRs are expressed in immune effector cells and are thought to play an important role in the pathogenesis of autoimmune diseases.¹

Among the five FcRs genes (FCGR the designation using Roman numerals indicate the polypeptide product, while the designation using Arabic numerals refers to the corresponding locus), FCGR2A-H131R, FCGR3A-F176V (often designated as F158V by counting from the N-terminal amino acid of the mature protein, excluding the signal peptide) and FCGR3B-NA1/2 polymorphisms had previously been known.¹ Association of FCGR polymorphisms has been extensively studied in a variety of autoimmune and/or inflammatory diseases. Recently, we identified a single nucleotide polymorphism (SNP) in FcRIIB gene coding for a nonsynonymous substitution within the transmembrane domain, Ile232Thr (I232T), and demonstrated that FCGR2B-232T/T genotype was significantly increased in Japanese patients with systemic lupus erythematosus (SLE).²

Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by chronic inflammation of the synovial as well as extra-articular tissues. Currently, the only established susceptibility gene to RA is HLA class II. The association of certain HLA-DRB1 alleles encoding the 'shared epitope' (SE) with susceptibility to RA is widely accepted.³ In Japanese, unlike in Caucasians, the most common allele of SE is DRB1*0405; nevertheless, significant association with SE has been confirmed.⁴

Functional importance of FcRs in the pathogenesis of RA has been shown by animal studies. DBA/1 mice lacking FcR chain, and thus lacking FcRI and FcRIII signaling, were protected from collagen-induced arthritis (CIA), while DBA/1 mice lacking FcRIIB developed arthritis.⁵ 129/SvJ (H-2^b) and C57BL/6 (H-2^b) hybrid background mice, usually resistant to CIA, became susceptible to CIA and developed cartilage destruction when FcRIIB gene deficiency was introduced.⁶ In humans, only a limited number of studies have been reported on the association of FCGR polymorphisms with RA. In Caucasians living in southern Spain,⁷ in the United Kingdom (UK),⁸ and in North Indians/Pakistanis,⁸ FCGR3A-176F/V polymorphism was reported to be associated with RA. However, the allele showing higher genetic risk for RA was opposite in these two studies. In the previous study, we reported lack of significant association of FCGR2A, 3A and 3B polymorphisms with RA in 115 Japanese patients and 217 healthy individuals.⁹ However, FCGR2B polymorphism, which was considered to be most relevant to pathogenesis, has not been examined for association with RA.

The purpose of this study was to investigate the association of FCGR2B-I232T with RA. At the same time, we evaluated the association of FCGR2A, 3A and 3B in a larger sample size. In addition, possible genetic interaction between FCGRs and HLA-DRB1 was examined.

Results

Genotyping of FCGR2B, 2A, 3A and 3B gene polymorphisms

Table 1 shows the frequencies of FCGR2B-I232T, FCGR2A-H131R, FCGR3A-F176V and FCGR3B-NA1/2 genotypes in 382 patients and 303 healthy individuals. Significant difference in genotype frequencies was not observed between patients and healthy individuals in all FCGR gene polymorphisms. Accordingly, allele carrier frequencies and allele frequencies did not show any difference between patients and healthy individuals.

Genotype relative risk estimation by the method of Lathrop adjusts control data for Hardy-Weinberg equilibrium and thus reduces variance.¹⁰ This type of analysis did not reveal association of any of the FCGR genes with RA either. Since a tendency of increase in the genotype frequency of FCGR3A-176F/F was observed, association of FCGR3A was also analyzed using Armitage's test for trend.¹¹ This analysis resulted in P-value of 0.15, but the difference still did not reach statistical significance.

Analyses on the association of FCGR2B, 2A, 3A and 3B polymorphisms with disease phenotypes in patients with RA, such as age of onset under 40 and presence of rheumatoid factor (RF), did not reveal significant association either (data not shown).

We next examined whether any of the haplotypes formed by four FCGR alleles is more frequently found in RA compared with controls. Since the genotypes of the family of the subjects were not available, haplotype frequencies were estimated using EH program.¹² As shown in Table 2, the estimated haplotype frequencies were quite similar in RA and healthy individuals. Haplotypes formed by FCGR2A-131H, 3A-176F, 3B-NA1 and 2B-232I was the most common haplotype in the Japanese.

Genetic interaction between FCGR3A and HLA-DRB1 shared epitope

We next examined whether FCGR polymorphisms may confer susceptibility to RA in concert with the already established susceptibility gene, namely, HLA-DRB1. As previously described, striking association of HLA-DRB1 SE with susceptibility to RA was present in our subjects (Table 3).⁴ Genotype frequency of each FCGR was compared between patients and controls after stratification according to the presence of SE. Significant difference in the genotype frequency was only detected in FCGR3A-F176V polymorphism when compared between patients with SE and healthy individuals with SE (P=0.03, P_corr=0.24 by ² test, P=0.017, P_corr=0.14 by Armitage's test for trend), but not in other comparisons. This association derived from the increase of FCGR3A-176F/F homozygotes in patients carrying SE compared with controls carrying SE (²=6.8, P=0.009, P_corr=0.072, F/F vs F/V and V/V combined).

As shown in Table 4, odds ratio for the development of RA was 4.84 in individuals with SE and FCGR3A-F/F genotype, which was significantly higher when compared with 2.71 in those with SE, but with FCGR3A-F/V or V/V genotypes.

Discussion

In the present study, we failed to detect significant overall association of FCGR2A, 2B, 3A and 3B polymorphisms with RA in the Japanese population. However, a slight trend for the increase of FCGR3A-176F/F was observed in RA, which reached statistical significance when this genotype was compared between patients and controls carrying HLA-DRB1 SE, while such a trend was not observed in patients and controls without SE. These results indicated that genetic interaction may be present between HLA-DRB1 and FCGR3A genes.

Two recent papers also demonstrated association of FCGR3A polymorphisms with RA. Nieto et al⁷ showed that FCGR3A-176F/F is associated with RA in the southern Spanish population. In contrast, Morgan et al⁸ demonstrated association of FCGR3A-176V allele in UK Caucasian and North Indians/Pakistanis. Interestingly, both groups provided evidence for a genetic interaction between FCGR3A and HLA-DRB1. Therefore, although P_corr did not reach statistical significance, our present data showing the association of FCGR3A-176F/F with RA in individuals carrying SE are considered to confirm the observation by Nieto et al⁷ in a different population. It is difficult to understand why a discrepancy in the associated allele is present among studies; it could be related to genetic heterogeneity, ethnic difference or technical difference.

Significant linkage disequilibria were observed between the alleles of FCGR gene family, which are closely located on 1q23 in the order of 2A-3A-2C-3B-2B.¹³ However, the status of linkage disequilibrium has been shown to be different in different populations, and does not simply accord with the gene order.² Thus, one conceivable possibility was that the association of FCGR3A with RA might be caused by the linkage disequilibrium with another primarily associated gene, and because of the difference in the allele frequency and linkage disequilibrium, each of the FCGR3A alleles was detected to be associated in each population. However, our present data indicate that other FCGR genes are not likely to be such genes of primary significance, including FCGR2B, which was most intriguing in terms of functions. However, the possibility that other polymorphisms in FCGR2B, including recently reported promoter SNP,¹³ might be associated with RA cannot be excluded at this stage. In addition, another recently reported gene within this chromosomal region encod-ing Fc receptor-related molecule might be another candidate.¹⁴

RA is characterized by the production of RF, which reacts with the Fc portion of the IgG molecule. Immune complexes containing RF can interact with FcRs, which may contribute to the inflammation in RA. The expression of FcRIIIA on macrophage in the synovium and dermis was suggested to be involved in the immune complex-induced tissue damage in RA.¹⁵ A major role for macrophage FcRIIIA in the induction of tumor necrosis factor following receptor ligation by small immune complex was shown in RA.¹⁶ At this point, it is not clear how FcRIIIA-176F protein, previously shown to be associated with lower affinity to IgG1, IgG3 and reduced signaling in NK cells,¹ is associated with the pathogenesis of RA.

Interestingly, genetic interaction between MHC and FcR has also been shown in the mouse model of SLE.¹⁷ The molecular mechanism underlying such an interaction remains an important subject for future research. One of the likely speculations may be an effect of FCGR3A polymorphisms on phagocytosis and/or activation of antigen presenting cells, which express HLA-DRB1 and present putative 'arthritogenic' antigens.

There are some limitations in this study; namely, the sex ratio and age distribution are different between the patients and controls. However, significant difference of FCGR genotype frequencies was not observed between male and female controls; therefore, adjustment of the male-to-female ratio between patients and controls did not affect the results (data not shown). Since the controls are younger compared with the patients, it is possible that up to 2 or 3 individuals out of 303 of controls might develop RA in the future (the estimated prevalence of RA is 1% or less in Japan). In that sense, our current study design is considered to be a conservative one, and the significant difference observed in this study would remain unchanged even if age-matched controls were employed.

In conclusion, among the FCGR2A, 2B, 3A and 3B polymorphisms, only FCGR3A-176F/F genotype was associated with RA in HLA-DRB1 SE positive subjects. These results indicated that FCGR3A-176F, or other allele(s) in linkage disequilibrium with it (but not FCGR2A, 2B and 3B alleles), may confer genetic risk to RA in concert with HLA-DRB1 SE, in the Japanese.

Patients and methods

Subjects

A total of 382 unrelated, Japanese patients with RA and 303 unrelated, Japanese controls were examined. The group of patients with RA consisted of 40 males and 342 females between the ages of 21 and 84 (mean±sd 58.8±11.6). They were followed in the outpatient clinics of the University of Tokyo and Matsuta Clinic (Tokyo, Japan). The patients were diagnosed according to the American College of Rheumatology criteria for RA.¹⁸ Eighty three percent of the patients were positive for RF, and the majority had erosive disease.

The group of healthy individuals, 167 males and 136 females between the ages of 21 and 61 (mean±sd 35.3±9.9) consisted of researchers, laboratory workers and students. It should be noted that the central part of Japan has been shown to be relatively homogeneous with respect to genetic background, permitting the case-control approach employed in this study.¹⁹

This study was reviewed and approved by the research ethics committee of the University of Tokyo.

Genotyping of FCGR3A-F176V polymorphism

The genomic DNA used for genotyping was purified from the peripheral blood cells of patients and healthy individuals using a QIAamp blood kit (Qiagen, Hilden, Germany).

FCGR3A-F176V genotyping was performed using PCR-single strand conformation polymorphism (SSCP) method. 152 bp fragment was amplified using primer set of 3A-F: 5'-TATTTACAGAATGGCAAAGG-3' and 3A-R: 5'-GTGATGGTGATGTTCACAGT-3'. PCR reaction was carried out in 25 l reaction mixtures containing 1 l of genomic DNA, 0.4 M of each primer, 0.2 mM dNTPs, 2.5 mM MgCl₂ and 1 U AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems, Norwalk, CT, USA). The amplification procedure consisted of initial denaturation at 96°C for 10 min, 35 cycles of denaturation at 96°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 60 s, followed by a last extension at 72°C for 5 min. The amplified DNA fragment was applied to 10% polyacrylamide gel (acrylamide:bis=49:1) containing 5% glycerol. Electrophoresis was carried out for 90 min at 4°C. The separated fragments were visualized with silver staining.

Genotyping of FCGR2B, 2A and 3B polymorphisms

FCGR2B-I232T genotyping was carried out by nested PCR and fluorescence resonance energy transfer (FRET) technology using LightCyclerÔ (Roche Diagnostics, Mannheim, Germany), as described in Kyogohu et al² FCGR2A-H131R genotyping was performed using PCR-restriction fragment length polymorphism method with a mismatched primer, and FCGR3B-NA1/2 genotyping using a PCR-preferential homoduplex formation assay and PCR-sequence specific primers method.^2,9

Genotyping of HLA-DRB1 alleles

HLA-DRB1 alleles were determined at the sequence level using PCR microtiter plate hybridization assay.⁴

Statistical analysis

Statistical analyses for association were performed using StatView for Windows, version 5.0. (SAS Institute Inc., Cary, NC, USA). ² tests were used to analyze association of the FCGR polymorphisms with susceptibility to RA. In addition, genotype relative risk of FCGRs was estimated according to Lathrop,¹⁰ and Armitage's test for trend in proportions was performed according to Sasieni.¹¹ In the analysis of association of FCGR genotypes in the subjects stratified with respect to HLA-DRB1, the P-values were corrected for multiple comparisons (P_corr) by multiplying the P-values by 8 (comparisons of four FCGR loci in each of SE positive and SE negative groups). Haplotype frequencies were estimated using EH program.¹²

Acknowledgements

The authors thank Michiko Shiota and Aya Kawasaki (Department of Human Genetics, The University of Tokyo) for HLA-DRB1 genotyping, and Dr Jun Ohashi (Department of Human Genetics, The University of Tokyo) for statistical suggestions. Chieko Kyogoku is a JSPS Research Fellow.

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Table 1 FCGR2B, FCGR2A, FCGR3A and FCGR3B polymorphisms in Japanese patients with RA and healthy individuals

Table 2 Estimated haplotype frequencies formed by four FCGRs alleles in the Japanese patients with RA and healthy individuals

Table 3 FCGR genotype frequencies in the patients and healthy individuals stratified according to HLA-DRB1 SE

Table 4 Genetic interaction between HLA-DRB1 SE and FCGR3A-176F/F