In the current latest meta-analysis, we first investigated simultaneously and comprehensively the most-studied SNPs of CTLA-4 on RA susceptibility and provided more reliable and robust evidence. From the data integration of 66 studies in 21681 cases and 23457 controls, we found that the rs3087243 SNP was associated with RA risk in Caucasians and Asians, the rs231775 SNP of CTLA-4 was associated with RA risk in Asians but not in Caucasians and Africans, and the rs5742909 SNP was not associated with RA risk in both Caucasians and Africans.
The CTLA-4 gene, located on chromosome 2q33, encodes a 223 amino acid receptor protein on T cell surface which is responsible for T cell immune regulation. As an antagonist of the costimulatory receptor CD28 which bind the same ligand B7 as CTLA-4, CTLA-4 with higher affinity transmits an inhibitory signal and subsequently plays a suppressive role in regulating T-cell activation , which suggest it is involved in the pathological processes of many autoimmune disorders[12–15]. It is widely believed that RA is a T cell-mediated autoimmune disease , of which the chronic inflammation and damage of the joints are typical . Therefore, the effect of CTLA-4 on RA pathogenesis has get growing attentions.
Previous research had found that serum levels of soluble CTLA-4 were increased in RA patients and had a positive correlation with Disease Activity Score in RA patients and even proposed that serum levels of CTLA-4 could serve as a new marker of RA disease activity [64, 65]. Besides, function experiments in vivo indicated that gene delivery of CTLA4 by intra-articular injection could alleviate experimental arthritis . Furthermore, CTLA-4Ig administration on RA synovial macrophages and T helper cells downregulated the production of proinflammatory cytokines, and these evidences suggested that CTLA-4 constituted a treatment target for RA [67, 68]. In fact, blockade of CTLA-4 by CTLA-4Ig had been successfully applied to treatment for RA .
As we all know, the protein level, structure and function in large part are determined by gene. Apart from these function research, numerous studies on correlation between CTLA-4 and RA risk from gene level also had been conduct to investigate genetic factors [8, 9, 13–15, 17–20, 24–27, 32–61]. However, the results were inconsistent or contrary likely due to the ethnic background, geographic environment, clinical heterogeneity, limited sample size, insufficient data and so on. Thus, it was urgently necessary to perform a comprehensive up-date Meta-analysis as an effective methodology to draw an overall objective appraisal on the association between CTLA-4 polymorphism and RA susceptibility.
In the present meta-analysis, we extracted 66 studies with 21681 cases and 23457 controls to inspect the correlation between three most-often SNPs in the CTLA-4 gene and the risk of RA. There were 22 studies with 16394 cases and 17453 controls for rs3087243 SNP, 34 studies with 11452 cases and 12444 controls for rs231775 SNP, and 10 studies with 2477 cases and 2941 controls for rs5742909 SNP. For rs3087243 polymorphism, our findings demonstrated a decreased susceptibility of RA both in total and in Caucasians in any gene mode. In total, carriers with allele A reduced an approximate 13% risk of RA than ones with allele G and genotype AA reduced 20% or so than genotype GG. Moreover, a decreased susceptibility of RA was respectively also found among Asians in the allele and homozygote comparison and among Latin Americans in the heterozygote and dominant comparison. However, only one study was included in Latin Americans and so it needed to enlarge sample size to further research. For rs231775 polymorphism, significant association did exist among the whole population in all genetic models except recessive model: compared with allele A and genotype AA, allele G and genotype GG and GA respectively was associated with an increased risk of RA. The same association was observed in Asians and Latin Americans in the subgroup analysis. On the contrary, no significant association between rs231775 SNP and RA risk could be detected in Caucasians and Africans using any gene model after excluding the Elshazli R’s study  with the apparent heterogeneity. Here, it should be noted that only one case–control study was included in Africans and Latin Americans, so the conclusions should be cautiously explained. For rs5742909 polymorphism, no significant association between this locus polymorphism and RA risk was observed among any population in any model. Although the heterogeneity existed in some genetic model, but no obvious change had happened in heterogeneity and P value for the pooled ORs when each study was removed by sensitivity analysis.
With regard to the diverse results of the same SNP on different populations, it maybe be attributed to clinical and genetic real heterogeneity of RA, interaction of genetic background and region environment, and even lack of vigorous statistical power.
It should be pointed out that previous several meta-analyses have summarized the effect of CTLA-4 polymorphism on RA risk [18–20]. But a few points need be taken notice. On one hand, the conclusions they achieved were discordant as the following: the conclusion of Li’s (2014) study  on the association of rs231775 SNP of CTLA-4 with RA was different from the others; the genetic models which indicated significant association were diverse in these analyses. These differences were mainly originated from divergent diagnostic criteria, limited number of studies and sample sizes in previous meta-analyses. On the other hand, all these meta-analyses only focused one of the three well-studied loci except Li’s study  on two. As we all know, the expression and function of the protein are determined by the whole gene. Therefore, it is of great necessity to investigate simultaneously the effect of all the 3 SNPs on RA risk to obtain an overall evaluation. Besides, the number of included studies in previous meta-analyses was small. Some original association studies [9, 15, 24–27] have emerged in the past few years and they can be incorporated. Taking these points into considerations, we updated the meta-analysis to achieve a more valid and comprehensive estimation on the association of CTLA-4 gene and RA susceptibility.
To our knowledge, this is a latest meta-analysis with the largest number of included studies, the largest sample size and the most-wide studied SNPs, so the conclusion draw is relatively most reliable and authentic. However, some limitations of our study should be acknowledged. Firstly, the sample sizes in some specific population such as African or Latin American and in some individual studies with less than thousands of participants were not sufficient enough to detect the genetic association between CTLA-4 SNPs and RA susceptibility. Especially, the results of certain populations including only one study should be interpreted with the caution. Secondly, we only investigated the role of three locus polymorphisms. As CTLA-4 gene had various SNPs, the function of protein CTLA-4 depended on the whole gene and RA was a multigene susceptibility disease, more SNPs of CTLA-4 should be included. Thirdly, certain degree of heterogeneity still existed in rs5742909 polymorphism and some genetic models. Although the elimination of each single study did not distinctly alter the P value, the results must still be treated cautiously. Fourthly, inadequate raw data in individual studies result in the inability to calculate the number of the genotypes in certain studies and perform stratification analysis by gender or clinical variables such as RF etc.