Division of Rheumatology, The Hospital for Rheumatic Diseases, Hanyang University, Seoul, 1Department of Microbiology, Hanyang University Medical College, Seoul and 2Department of Microbiology and Immunology, Catholic University College of Medicine, Seoul, Korea.
Correspondence to:
D. H. Yoo, The Hospital for Rheumatic Diseases, Hanyang University, Seoul, 133792, Korea. E-mail: dhyoo{at}hanyang.ac.kr
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Abstract |
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Methods. A total of 299 SLE patients meeting 1982 ACR criteria and 144 Korean disease-free controls were enrolled. Genotyping for the FcRIIa 131 R/H and Fc
RIIIa 176 F/V was performed by polymerase chain reaction (PCR) of genomic DNA using allele-specific primers. HLA-DRB1 typing was performed by the PCR-SSOP method.
Results. There was significant skewing in the distribution of the three FcRIIa genotypes between the SLE patients and the controls [P = 0.002 for R/R131 vs R/H131 and H/H131, relative risk (RR) 2.6 (95% CI 1.35.2)], but not in Fc
RIIIa genotypes. HLA-DRB1*15 allele was significantly more prevalent among SLE patients than the control population [P < 0.02, RR = 1.7 (1.12.6)]. HLA-DRB1 genotypes or allele frequencies of the SLE patients with nephritis did not differ significantly from those of the SLE patients without nephritis. We analysed the combined effects of the two candidate genes on SLE susceptibility. HLA-DRB1*15 allele was a significant predictor of SLE in individuals who were not homozygous for Fc
RIIa-R/R131 [RR = 2.1 (1.23.7), P < 0.008], and the Fc
RIIa-R/R131 genotype vice versa [RR = 5.3 (1.915.4), P < 0.001]. However, an additive or synergistic effect of both susceptible genes on relative risk for SLE was not evident.
Conclusions. Our results suggest that FcRIIa-R/R131 homozygote and HLA-DRB1*15 allele are independent risk factors in Korean SLE patients without additive or synergistic effects.
KEY WORDS: SLE, HLA-DR type, FcRIIa and IIIa polymorphism.
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Introduction |
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Several genetic strategies have been used to define susceptibility genes of genetically complex diseases like SLE. These involve the systemic genome-wide scan approach, animal models and the candidate gene approach. To date, there are six published genome-wide screens in SLE families and these screens have identified six regions meeting criteria for significant linkage (LOD score >3.3) [49]. Using several animal models of SLE and genetic dissection, similar candidate genes or gene families responsible for susceptibility to SLE in both mouse and humans were suggested [1014]. Candidate gene approaches in both population association and family linkage studies have been widely used and thus there are hundreds of published studies. Despite several potential difficulties such as population admixture and genetic drift, this association study has the significant advantage over the other strategies of having greater power to detect small effects [3, 15, 16]. Among these candidate genes, the HLA region, complement components and low-affinity receptors for IgG showed significant association with SLE susceptibility [3, 15].
HLA genes have received significant attention in human SLE, and there is evidence supporting a role for specific extended HLA haplotypes as genetic risk factors for disease expression in several populations [1721]. HLA-DR2 and -DR3 have shown consistent associations with SLE in European Caucasian populations [22]. In Korea, significant increased frequency of HLA-DRB1*1501 to DRB5*0101, and DR9 was reported in SLE patients [23, 24]. These HLA regions also showed stronger association with autoantibody production in SLE than with disease expression itself [22]. Although the precise genes attributed to these associations are not known, genome-wide scan studies have indicated that this MHC region is a very important gene location for susceptibility to SLE [4, 5, 9]. Recent studies have shown that Fc receptors (Fc
R) could be one of the non-MHC susceptibility genes for SLE [4, 2528]. Fc
RIIa and IIIa are located within the same cluster in chromosome 1q21-23 and they have allelic variants which differ substantially in their functional capacities of immune complex clearance. Fc
RIIa alleles, R131 and H131, differ at amino acid position 131 in the extracellular domain and also differ in their ability to bind human IgG2: R131 is the low-binding allele, H131 is high binding and heterozygotes have intermediate function. Fc
RIIIa alleles, F176 and V176, which differ in one amino acid at position 176 in the extracellular domain and differ in their ability to bind human IgG1 and IgG3: F176 is the low-binding allele, V176 is high binding. Although there are a few reports about linkage disequilibrium (LD) between Fc
RIIa and IIIa in other ethnic groups, there have been no reports to date in Korean populations.
We have previously reported that FcRIIa-R131 homozygote and R131 allele were predisposing factors for SLE development [29]. Recently, the importance of epistasis and epistatic interaction between two susceptibility alleles in multigenic diseases such as SLE has been emphasized in genetic pathogenesis based on various genetic studies. Thus we studied whether the epistatic effect between HLA-DR and Fc
RIIa/IIIa among the many important candidate genes in SLE was observed in Korean patients with SLE.
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Patients and methods |
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FcRIIa/IIIa genotyping and Fc
RIIIa genomic DNA sequencing
For genotyping of FcRIIa and Fc
RIIIa, DNA was isolated from peripheral blood (Puregene kit, Gentra systems, Minneapolis, MN) and polymerase chain reaction (PCR) was performed as previously described [29, 31, 32]. To confirm the Fc
RIIIa genomic sequence of 17 individuals whose PCR results were inadequate, PCR was done with primers to amplify a portion of exon 4 of Fc
RIIIa which corresponds to EC2 [29, 32]. PCR products were purified by Qiagen Column (Qiagen, Germany) and genomic DNA sequencing was performed on an ABI PRISM 310 (Perkin Elmer, USA).
HLA-DRB1 typing
HLA-DRB1 typing was performed by the PCR-SSOP method, which was essentially the same as that described at the 12th International Workshop, with minor modifications [33]. Specific HLA-DRB1 primers were used to amplify DRB1 products, which were then denatured and immobilized on a nylon membrane and probed with a series of digoxigenin-labelled oligonucleotides specific for the known hypervariable sequences. Stringent washing was performed in the presence of tetramethyl ammonium chloride (TMAC, Sigma chemical Co., St. Louis, USA). The hybridized probe was detected according to the manufacturers instructions with the anti-digoxigenin antibody conjugated with alkaline phosphatase, followed by addition of chemiluminescent substrate CSPD (Boehringer Mannheim, GmbH, Germany). Chemiluminescence was detected by exposing to radiographic film.
Statistical analysis
The distribution of the allele and genotype of FcRIIa, Fc
RIIIa and HLA-DRB1 between two groups (SLE patients vs controls, SLE with nephritis vs SLE without nephritis, SLE with nephritis vs controls) was compared with 3 x 2 or 2 x 2 contingency tables using the
2-test.
We used the EH program for haplotype analysis [34, 35]. The frequency of each haplotype of FcRIIa 131Fc
RIIIa 176 (RF, RV, HF and HV) can be calculated from this linkage utility program. The odds ratio (OR) for patients with the low-binding haplotype Fc
RIIa R131Fc
RIIIa F176 (RF) and the high-binding haplotype Fc
RIIa H131Fc
RIIIa V176 (HV) for disease susceptibility and prevention from disease development was calculated, respectively.
Linkage disequilibrium between FcRIIa and Fc
RIIIa was assessed using the D value for non-random assortment of alleles [36]. To observe the additive effect of Fc
RIIa/IIIa and HLA-DRB1 on the development of SLE or lupus nephritis, groups with both risk alleles were compared with reference groups without both risk alleles using
2. We applied corrected P values to this multiple testing [37]. In brief, when an initial 2 x C contingency table is partitioned into non-independent 2 x 2 tables, the value of corrected a' is obtained as follows: a' = a/2(C - 1). The value of C in our multiple testing is 4.
A probability of 0.05 (2-tailed) was used to reject the null hypothesis that there was no difference in the distribution of allele and genotype between the groups. Odds ratios with 95% confidence intervals (95% CI) were calculated from 2 x 2 contingency tables by 2-test. The mean age at onset and age of clinical and ACR diagnosis were compared using independent sample t-test or ANOVA, and the amount of proteinuria using the MannWhitney U-test or KruskalWallis test. Here we used SPSS 10.0 for Windows for statistical analysis.
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Results |
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Distribution of HLA-DR type between SLE patients and controls
HLA-DRB1*15 allele was significantly more prevalent among SLE patients than the control population [P = 0.015 for HLA-DRB1*15 allele vs non-HLA-DRB1*15 allele, RR 1.7 (95% CI 1.12.6)] (Table 3). No HLA-DR genotype or allele of the SLE patients with nephritis differed significantly from those of the SLE patients without nephritis. However, comparing the lupus nephritis patients with controls, DRB1*07 was more prevalent among the lupus nephritis group [RR = 2.2 (1.24.3), P < 0.002] (Table 3). The frequency of HLA-DRB1*01 and *04 allele was lower in SLE patients than controls. With respect to clinical manifestations and autoantibody profiles of SLE, the frequencies of neurological disorder and anti-RNP antibody in SLE patients with HLA-DRB1*15 allele were significantly increased compared with controls (Table 4). Besides HLA-DRB1*15, the other HLA-DR types were also associated with some clinical manifestations or laboratory findings. Increased frequency of arthritis and bacterial infection in patients with HLA-DRB1*04, increased serum creatinine level in patients with HLA-DRB1*01, haemolytic anaemia in patients with HLA-DRB1*13 or DRB1*07, anti-RNP antibody in patients with HLA-DRB1*11, anti-La antibody in patients with HLA-DRB1*07 or DRB1*08, lupus anticoagulant in patients with HLA-DRB1*12, and Raynauds phenomenon and low C4 in patients with HLA-DRB1*09 were observed (Table 4).
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Discussion |
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FcR genes in chromosome 1q23 and MHC class II genes in chromosome 6p are among candidate genes of SLE. There are various proposals for the mechanism underlying the higher association of these polymorphisms in SLE patients. HLA class II molecules elicit the T-helper cell response after presentation of specific antigens by antigen-presenting cells. Therefore, autoantibody production mediated by T-helper cells specific to autoantigens, a characteristic feature in SLE, can be attributed to HLA class II molecules [3, 40]. The role of Fc
R polymorphisms in the pathogenesis of SLE has been focused on their dysregulation of immune complex handling and enhanced immune complex-mediated organ injury [25, 38, 39]. Despite this reasonable theoretical background and many positive results, a significant debate in interpreting these association data is posed by the significant LD across the region. In fact, reported associations of SLE with several MHC class III genes such as TNF-
, TAP genes, HSP70 and complement genes as well as DQ alleles may reflect LD with HLA-DR allele or the epistatic effects of several genes on extended haplotypes [4145]. Also, the Fc
receptors are very tightly clustered at 1q23, and the interpretation of the association result of one locus is potentially complicated by LD in the region [25, 38, 46].
This study showed similar results to previous reports on Korean populations: FcRIIa-R/R131 homozygote and HLA-DRB1*15 allele were significantly prevalent in SLE patients, but not Fc
RIIIa polymorphism or HLA-DR3 [23, 24, 29]. In addition, several non-HLA-DRB1*15 alleles were associated with some clinical manifestations and some autoantibodies as shown in Table 4. Among these associations, increased frequency of arthritis in patients with HLA-DR4which has been reported as a susceptible allele in rheumatoid arthritiswas interesting and suggested that HLA-DR4 allele participates in the development of arthritis in SLE as in RA. We further analysed the effects of Fc
RIIa polymorphism on arthritis in SLE patients. There was no association between the two variables (data not shown). Although we showed there are many associations between various HLA-DRB1 alleles and clinical manifestations or laboratory findings, interpretations about their protective or predictive effects in SLE are obscure and further study is needed to clarify their associations.
We analysed the combined effects of both candidate genes on SLE, HLA-DRB1 and FcRIIa-R/R131. HLA-DRB1*15 allele was a significant predictor of SLE in Fc
RIIa-R/R131 homozygote-negative individuals, and Fc
RIIa-R/R131 homozygote vice versa. Also, ORs of HLA-DRB1*15 and Fc
RIIa-R/R131 homozygote for SLE in Fc
RIIa-R/R131 homozygote-negative and HLA-DRB1*15-negative individuals, respectively, are more increased than initial ORs in total individuals. These results suggest that the two susceptible genes may not have an additive or synergistic effect on OR for SLE, but rather each gene has independent association with SLE. Also one candidate gene may have a more pathogenic role, especially within the individuals without the other susceptible gene, suggesting HLA-DRB1 and Fc
RIIa are probably involved in different pathogenic pathways. There are a few reports that have analysed the association of these two genes in SLE development. Manger et al. [25] analysed the distribution of Fc
RIIa polymorphism and HLA-DR3 in Caucasian SLE patients. Although they did not present the data in detail, they suggested that HLA-DR3 haplotypes did not seem to represent additional risk factors for the association of the Fc
RIIa-R131 allele polymorphism with SLE. We also obtained the results about LD between Fc
RIIa and Fc
RIIIa in Korean populations, which showed no significant associations. These results were consistent with other ethnic groups [28].
In addition, we analysed the combination effect between HLA-DRB1*07 and FcRIIa polymorphism, and HLA-DRB1*15 and Fc
RIIIa polymorphism, and obtained no significant combination effects (data not shown).
Our study had some limitations to interpretation. Control groups were ethnically matched with SLE patients, but not sex and age matched. Also the size of the control group was too small to conclude the exact role of the combination of two candidate genes in SLE susceptibility.
In conclusion, our results suggest that FcRIIa-R/R131 homozygote and HLA-DRB1*15 allele are independent risk factors in Korean SLE patients without additive and synergistic effects.
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Acknowledgments |
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References |
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