ARC Epidemiology Unit, University of Manchester, UK and
1 Rheumatology Division, Hospital Xeral-Calde, Lugo, Spain
Correspondence to:
A. Barton.
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Abstract |
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Objective. To investigate the association of CTLA-4 with RA in Spanish and UK subjects.
Methods. Caucasoid UK RA patients (n=192), UK controls (n=96), Spanish RA patients (n=136) and Spanish controls (n=144) were typed for an A/G bi-allelic polymorphism in exon 1 of CTLA-4 using polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) (enzyme).
Results. No significant differences in the frequency of the G allele or the GG genotype were found in either the UK or Spanish RA patients compared with controls.
Conclusion. No significant evidence was found of an association between RA and CTLA-4.
KEY WORDS: CTLA-4, SNP, Rheumatoid arthritis, Susceptibility locus, autoimmunity, genetic
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Introduction |
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Only one RA susceptibility locus, the major histocompatibility complex (MHC), has been identified to date, although a single-locus model for inheritance of RA has been rejected. HLA has been estimated as accounting for approximately 40% of the genetic component of susceptibility to RA [3]. It is likely that a number of other genes are involved, each contributing a small amount to the total genetic component.
T cells play a major role in the pathogenesis of RA. Based on animal models of inflammatory disease, the success of anti-T-cell therapy in RA and the association with HLA DR4, it has been proposed that RA is a T-cell-mediated autoimmune disease. Regulators of T-cell activity are, therefore, candidates for influencing disease susceptibility. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is a negative regulator of T-cell activation. Mice deficient in CTLA-4 develop a rampant lymphoproliferative disorder resulting in premature death [4]. The CTLA-4 gene in humans lies on chromosome 2q33 and a microsatellite polymorphism has been identified in the 3' untranslated region of the gene. This polymorphism has been previously linked to type I insulin-dependent diabetes mellitus (IDDM) in an Italian population but not in a UK population [5]. A bi-allelic G/A polymorphism has also been identified at position 49, exon 1 of the gene [6]. This encodes a Ala/Thr substitution and, although unlikely to be functionally important, may be in linkage disequilibrium with a nearby disease susceptibility gene. The G allele has been associated with IDDM in Spanish, Belgian and Italian populations [5], and with Graves' disease in German [7], US [8] and Hong Kong Chinese [6] patients. Recently, an association with the G allele has been described in a subset of patients with RA in a German population. RA patients carrying the HLA DRB1*0401 subtype were significantly more likely to be homozygous for the GG genotype, although no differences in G allele frequencies were seen in the data set as a whole [9]. Furthermore, an association of the G allele with early-onset persistently pauciarticular juvenile chronic arthritis with chronic iridocyclitis has been reported [10].
CTLA-4 is a potential candidate for other T-cell-mediated autoimmune diseases. In this study, we investigated the association of CTLA-4 with RA in UK and Spanish populations.
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Materials and methods |
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Normal individuals (n=96), with no history of inflammatory joint disease, were recruited from general practice. Fifty-seven per cent were female and the mean age was 48 yr with a range of 1971 yr.
Spanish samples.
DNA was obtained from 136 patients with RA from the Galicia region of northern Spain. These patients were all referred to the Xeral-Calde Hospital by general practitioners or by self-referral to the Accident and Emergency Department. Seventy-one per cent of patients were female. The mean age at disease onset was 49 yr (range 1678 yr) and the average disease duration was 11.6 yr (056 yr). All were seropositive, 81% were erosive and 18% nodular. Only four cases had never received DMARD therapy and the others on average had received 2.7 DMARDs.
Control samples were obtained from the same population (n=144). Forty-nine per cent of controls were female and age ranged from 16 to 90 yr.
Polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP)
A G/A polymorphism exists at position 49 in exon 1 of the CTLA-4 gene [6]. A recognition site for the restriction enzyme BbvI (New England Biolabs, Hertfordshire, UK) is created when the G allele is present but not when the A allele is present. An additional BbvI recognition site upstream of the polymorphism acts as an internal control.
Primers were synthesized with sequences complementary to DNA at position 635 (forward: 5'-GTCAAGGGACCATTGAAG-3') and 1301 (reverse: 5'-CTTTGCAGAAGACAGGGATGA-3'). DNA containing the polymorphic site was amplified by PCR under the following conditions: 1 min denaturation at 95°C, 1 min annealing at 55°C and 2 min elongation at 72°C for 35 cycles. Restriction enzyme digestion was performed at 37°C overnight and digested samples subjected to electrophoresis in a 2% agarose gel for 30 min at 200 V in Trisborate electrophoresis buffer. The gels were viewed under a transilluminator.
DNA from 192 Caucasian UK RA patients, 96 healthy UK controls, 136 Spanish RA patients and 144 healthy Spanish controls was analysed for the bi-allelic G/A polymorphism.
Statistical analysis
Allele frequencies in patient and control groups were calculated by direct counting. The association of the G allele with RA was assessed by calculating the odds ratio (OR) and 95% confidence intervals (95% CI) for the G phenotype and GG genotype. The level of significance was determined by 2 and Fisher's exact tests. No correction was made for multiple testing. Assuming a dominant mode of inheritance, the study had 80% power to detect a genotypic relative risk (RR) of 2.5 in the UK group and 2.1 in the Spanish group at the 5% significance level.
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Results |
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RA is a heterogeneous disease and, therefore, associations may be found in subgroups of patients. No association of the G allele with subgroups stratified for sex, seropositivity and the presence of nodules was demonstrated. A previous study in a German population found that RA patients who were HLA-DRB1*04 positive, particularly those carrying the HLA-DRB1*0401 subtype, were significantly more likely to be homozygous for the GG genotype [9]. HLA data were available for 113 UK RA patients, 77 UK controls, 120 Spanish RA patients and 132 Spanish controls. In the UK individuals for whom HLA data were available, the GG genotype did not occur more frequently in HLA-DRB1*04-positive RA patients (2=1.66, P=0.44) or in those carrying the HLA-DRB1*0401 subtype (
2=0.27, P=0.87) than in controls (Table 2
). No association with the GG genotype was demonstrated in the Spanish cohort stratified for HLA.
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Discussion |
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The aim of this study was to determine whether a polymorphism in the CTLA-4 gene is associated with RA. The results show no significant difference in the frequency of the G allele in UK or Spanish patients with RA.
One other study has investigated the association of the CTLA-4 dimorphism with RA in a German population. In the data set as a whole, no significant differences in the distribution of genotypes were seen in RA patients compared with controls. However, further analysis showed that RA patients carrying the HLA-DRB1*0401 subtype were significantly more likely to be homozygous for the GG genotype [9]. We have not replicated this result in either the Spanish or UK groups.
Nistico et al. [5] demonstrated linkage and association of the CTLA-4 gene with IDDM in Spanish and Italian patients, but failed to show evidence of linkage in UK, US or Sardinian patients with IDDM. The lack of association in the UK RA group (OR for G phenotype=0.68; 95% CI=0.401.16; P=0.23) could be due to an ethnic specificity of the polymorphism such that, in this population, the G allele may not be in as strong linkage disequilibrium with the predisposing allele of the aetiological mutation. Spanish patients with RA were, therefore, compared with controls for the presence of the G allele. Again, however, the frequency of G allele-positive individuals was equal in both groups (OR for G phenotype=0.83; 95% CI=0.511.36; P=0.42). It should be noted that the Spanish RA patients studied came from north-west Spain and had similar HLA associations as UK RA patients. This is in contrast to other Spanish RA populations where patients are more likely to carry HLA DR1, DR10 or *0405 subtypes [14].
We have ruled out a significant role for CTLA-4 as the study had the power to detect a gene with a RR of 2.5 and 2.1 (80% power, 5% significance level) in UK and Spanish RA groups, respectively. It is possible that the RR of CTLA-4 in RA is small and that an association has, therefore, been missed.
The results presented herein have not demonstrated an association between a polymorphism of CTLA-4 and RA.
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References |
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