HLA polymorphism accounts only for approximately one-third of the genetic predisposition to rheumatoid arthritis (RA). To investigate the role of other loci in the susceptibility to RA, we have performed an analysis of several polymorphisms in genes of immune-related function: IL-10 −1082, −819, −592 promoter single nucleotide polymorphisms (SNPs), IL-10G and IL-10R microsatellites, IL-6 −622 promoter SNP, FcγRIIIA Val/Phe-158 polymorphism, IL-1 receptor antagonist VNTR, and the IKBL+738 T/C mutation. The analysis has been performed on a case–control study and also on RA trios. IL-10G12 was found to be associated with RA in the case–control study (18% in RA patients vs 9% in controls: P=0.001; pc<0.05). This allele was also more often transmitted than not transmitted (10 vs 5). No other allele in the present study is found to be associated to RA. Our data suggest that most of the loci studied play no major role in the susceptibility to RA, the IL-10 gene being the sole exception.
Our groups have previously published a number of papers on genetic markers of rheumatoid arthritis (RA), both in family and case–control studies.1,2,3,4 Here we attempt to extend further some of those results and to investigate possible associations with a new set of markers. We have focussed on polymorphisms with presumed functional relevance or previously reported to be associated with an autoimmune disease.
IL-6 is the main mediator of the acute phase reaction, together with IL-1, mostly through its action on hepatocytes, but also on other cellular populations. The IL-6 promoter region contains a few well-characterised single nucleotide polymorphisms (SNPs), including a G/A transition at position −622 and a G/C transversion at position −174, which are in complete linkage disequilibrium.3 Allele −174C has been associated with systemic juvenile chronic arthritis, and with reduced production of this cytokine.5 A previous case–control study with Spanish RA patients has also been recently published.3
IL-10 is an anti-inflammatory cytokine and it regulates the balance of Th1–Th2 responses. The human IL-10 gene promoter contains three SNPs, at positions −1082 (A/G), −819 (T/C) and −592 (C/A).6,7,8,9 These promoter SNPs are present in any of three possible haplotypes: ACC, ATA and GCC. Two polymorphic microsatellites nearby, IL10G and IL10R, have also been described. The 1082*A allele has been associated with low and the 1082*G allele with high in vitro IL-10 production, and different IL-10G/IL-10R microsatellite haplotypes are correlated also with different levels of mRNA production.10 Some studies of IL-10 polymorphisms in RA have also been reported, mostly with negative results.11,12,13
IL-1Ra (interleukin-1 receptor antagonist) is a member of the IL-1 family, which also includes IL-1α and IL-1β. IL-1Ra binds to the IL-1 receptor but fails to transmit a signal to the target cell, thereby behaving as a natural antagonist of IL-1.14 The IL-1Ra gene contains a variable number of tandem repeats (VNTR) in its second intron. The 86 base pair repeat has three putative binding sites for transcription factors responsive to interferon and might be of functional relevance.15 The allele 2 has been found to be associated with ulcerative colitis and multiple sclerosis, but not with RA.
The IKBL gene on the MHC class III region encodes a negative regulator of NF-κB. The +738 position, usually a T, is occasionally mutated to C in certain MHC extended haplotypes.16 This mutation changes the corresponding amino acid residue from cysteine to arginine in a sequence stretch where a protein kinase C phosporylation target is located, and may therefore be of functional importance. The IKBL +738C allele has been recently reported as being a marker of severity in ulcerative colitis.17
FcγRIIIA is a member of a family of seven related genes encoding distinct isoforms of the receptor for the Fc region of human IgG.18,19 This gene has a polymorphism changing a phenylalanine for valine in the extracellular portion of the Fc receptor. The allelic variants differ in their affinity: Val158 variants bind IgG1 and IgG3 more avidly than Phe158-containing alleles. A previous case–control positive association of homozygosity for FcγRIIIA Phe158 with RA has been reported,4 but results are conflicting.20,21
The present study has been performed with three different groups of RA patients. For the transmission analysis, the 58 Spanish families of the European Consortium on RA families (ECRAF) were analysed. The clinical characteristics of the patients in this group were reported in two earlier papers where most of the families were already included.1,2 Besides, two additional RA patient cohorts were studied, both of them were already described in earlier works from our respective groups. The IL-6 and FcγRIIIA analysis was performed in samples from Granada, southern Spain. IKBL, Interleukin-1 receptor antagonist and all the IL-10 polymorphisms were studied in samples from Madrid. In each group not every sample was unambiguously genotyped, and hence the number n of samples included is indicated for each of the results. Both groups of patients were compared with controls from their respective location. DNA extraction and HLA-DRB1 typing had been done previously, and genotyping methods for each of the polymorphisms included in this study are described in Table 1 and Table 2.
Table 1 displays the frequency of the IL10R and IL10G microsatellite alleles more commonly found in our samples. All of the IL-10R alleles appeared at a similar frequency when cases and controls were compared. A significant increase could be observed in the frequency of the IL-10G12 allele, strong enough to withstand correction for the number of alleles at the IL-10G locus.
When transmission of these microsatellites was analysed, none of the alleles showed a statistically significant deviation from the 50% transmission expected. The allele IL-10G12, which was overrepresented in RA patients, was transmitted 10 times and not transmitted five times from heterozygous parents. The P-value, however, failed to reach a statistically significant level.
We also studied the IL-10 promoter. Our typing method allowed us to construct a promoter haplotype with the three SNPs studied. None of those haplotypes was differently distributed when patients were compared to controls. No difference was found when each position was separately analysed. No haplotype or SNP alone was preferentially transmitted from heterozygous parents to the affected offspring. Our typing method and the study with families were also able to assign an IL10G microsatellite to each promoter haplotype. The GCC promoter could be found with any of the different IL-10G microsatellite alleles, and it was highly associated with IL-10R3. Among the IL-10R3-positive RA patients, 88% were also GCC positive; this was the case in 45% of the IL-10R3-negative patients. The figures for controls were 87% and 50% respectively. The ATA and ACC promoters were associated with IL10-G8 through G10 and IL10-G10 through G14 microsatellite alleles, respectively. When the two IL10-G12 haplotypes, G12/ACC and G12/GCC were analysed, only the G12/ACC extended haplotype was found to be increased (14% vs 6%; P=0.0025; OR=2.43). No overt differences were apparent in the IL-10G12 GCC distribution (3.8% vs 3.4%). Regarding transmission in the RA families, both IL10-G12 haplotypes were preferentially transmitted (7 vs 5, and 3 vs 0, for IL-10G12/ACC and IL-10G12/GCC, respectively), although the differences did not reach significant P-values.
Table 2 shows the three main IL-1Ra genotypes found in our study. Alleles 3-5 are seldom found in our population, with a combined allelic frequency lower than 5%. No significant difference was found with this VNTR between RA patients and controls. When alleles rather than genotypes were analysed, differences were not found either (data not shown). Allele 2, which had been previously associated with inflammatory diseases, was not preferentially transmitted in the families under study (18 transmitted vs 31 non-transmitted; P=ns). IKBL is genetically linked to HLA-DRB1, and the results were therefore stratified according to the presence or absence of the shared epitope at the DRB1 locus. No differences were found in any of the groups. The family analysis revealed no significant differences in transmission in the haplotypes containing IKBL +738C.
Finally, Table 2 shows the phenotypic frequencies of the IL-6 promoter SNP and the FcγRIIIA 158 polymorphism, and the new results of the respective family analysis, which are presented here for the first time. No statistically significant differences are found in the phenotypic frequencies of these two genetic markers, and none of them exhibited transmission distortion, although a small trend towards increased transmission of the Phe-158 allele could be observed.
It can be concluded that none of the loci, with the exception of IL-10, is of paramount importance on its own to confer enhanced susceptibility to RA as a whole. It has been our aim to concentrate mainly on susceptibility, although we recognise that it is likely that some of the polymorphisms here studied may confer susceptibility to only a defined subset of patients, those with some specific clinical characteristics. However, without a previous understanding of the molecular consequences of the polymorphism on the disease, it is difficult to establish an a priori hypothesis as to whether an association may exist between a marker and a particular disease subset. Without an a priori hypothesis, in turn, an analysis of multiple loci trying to associate them to multiple symptoms would inevitably lead to false positive claims.
The only marker studied where a significance level was achieved was the IL-10 locus. Previous studies on IL10 microsatellite have been published, both in RA and in other diseases. Eskdale et al. find no evidence of IL-10G association with RA,13 but describe an association with IL-10R3 in both Anglo-Saxon and Afro-American populations. Unfortunately, the IL-10G data are not shown, so we could not appreciate if a similar trend to ours was also present in their population. The IL-10R3 allele is in linkage disequilibrium with the GCC promoter haplotype in our population, and neither the IL-10R3 microsatellite nor the GCC haplotype is found at an increased frequency in our study. In the results reported by Cantagrel et al, a small and clearly non-significant increase is observed in −1082G, but the −819 SNP is not investigated, and therefore they cannot discriminate ACC from ATA haplotypes.11 The IL-10G12 allele we have observed at a higher frequency is found predominantly (especially in RA patients) with the ACC haplotype, and less frequently with GCC. In contrast with GCC, the ACC haplotype is reported to be a low IL-10 in vitro producer when lymphocytes are stimulated by concanavalin-A. This genetically hard-coded lower production of IL-10 should result in a tendency to suffer from inflammatory conditions. Nevertheless, as other ACC-containing haplotypes are not associated with RA, a mechanistic explanation linking genotype with phenotype probably lies elsewhere in the vicinity of the gene, in tight linkage disequilibrium with the IL-10R2/IL-10G12/ACC extended haplotype. Some other polymorphic positions have been described in the distal region 5′ to the promoter,22,24 but to date no population data exist for most of them.
In the two cases where a case–control association has been found (IL-10G12 in the present paper and FcγRIIIA-Phe in the paper by Nieto et al4), the results could not be further confirmed by family studies; it is however important to indicate that the same trend towards RA predisposition is observed in both our case–control and TDT studies. The relatively low power of family-based studies to detect associations has been previously noted. When an OR for IL-10G12 of 2 is observed, a proportion of 10 transmitted vs 5 non-transmitted IL-10G12 alleles is to be expected. Unfortunately, it is difficult to reach a statistically significant level with these small numbers.
Besides, the association described for FcγRIIIA applies only to the homozygous status. Therefore, a TDT-positive result is only to be expected when the other progenitor has the possibility to transmit the same FcγRIIIA-Phe allele. Every time a genotype rather than an allele association exists, this kind of problem will happen, and it again makes a family study harder to perform. The conflicting results regarding association of this polymorphism with RA are extensively discussed elsewhere. Finally, as noted in Martinez et al,2 a TDT analysis is only feasible when both parents are alive, thereby biasing the results towards the less aged patients. The summation of all these factors could explain the lack of confirmation in the family study.
This work was supported by grants SAF00-213 from Plan Nacional de I+D (CICYT) FIS 01/108-03 and from the Alfonso Martin Escudero Foundation.
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Martinez, A., Pascual, M., Pascual-Salcedo, D. et al. Genetic polymorphisms in Spanish rheumatoid arthritis patients: an association and linkage study. Genes Immun 4, 117–121 (2003). https://doi.org/10.1038/sj.gene.6363931
- rheumatoid arthritis
- single nucleotide polymorphism
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