Cytotoxic T-Lymphocyte Associated Antigen 4 Polymorphisms and Asthma Risk: A Meta-Analysis

Background A number of studies assessed the association of cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) gene polymorphisms with asthma in different populations. However, the results were contradictory. We performed a meta-analysis to examine the association between CTLA-4 polymorphisms and asthma susceptibility. Methods Pubmed, EMBASE, HuGE Navigator, and Wanfang Database were searched. Data were extracted independently by two reviewers. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Results Seventeen studies involving 6378 cases and 8674 controls were included. Significant association between +49 A/G polymorphism and asthma was observed for AA vs. AG+GG (OR = 1.18, 95% CI 1.01–1.37, P = 0.04). There were no significant associations between −318 C/T, −1147 C/T, CT60 A/G, −1722 C/T, or rs926169 polymorphisms and asthma risk. Conclusions This meta-analysis suggested that the +49 A/G polymorphism in CTLA-4 was a risk factor for asthma.


Introduction
Asthma is a major public health problem worldwide. The disease affects over 300 million people [1]. In developed countries, the prevalence of asthma has increased considerably over the past three decades [2]. Asthma is a complex inflammatory disorder that results from interactions between more than 100 susceptibility genes and multiple environmental factors [3,4]. It is, therefore, important to identify the gene variants contributing to asthma pathogenesis. Numerous studies have focused on this field, and the cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) gene has been extensively studied.
CTLA-4, a B7-binding protein, was initially described as a classical type I glycoprotein on the surface of activated T cells [5]. Cumulative evidence suggested that CTLA-4 may play an important role in the pathogenesis of asthma. CTLA-4 is a powerful negative regulator of T cell activation and is associated with Th cell differentiation. Oosterwegel et al. [6] demonstrated that CTLA-4 was a potent and critical inhibitor of Th2 cell differentiation. Expression of CTLA-4 in Th2 cells was much higher than in Th1 cells [7]. CTLA-4 was also demonstrated to suppress the production of cytokines produced by Th2 cells [7]. A number of studies showed that administration of CTLA-4-Ig significantly ameliorated airway hyperresponsiveness (AHR), reduced the level of eosinophils in average bronchoalveolar lavage fluid and serum IgE, as well as cytokine production in murine asthma model [8][9][10][11]. Recently, Choi et al. [12] reported that intranasal administration of Hph-1-ctCTLA-4 could significantly reduce infiltration of inflammatory cells, secretion of Th2 cytokines, serum IgE levels and AHR in a mouse model of allergic airway inflammation. Lin and co-workers demonstrated that decreased allergic inflammation by surfactant protein D was mediated by an increased expression of CTLA-4 in T cells [13].
The human CTLA-4 gene is located on chromosome 2q33.2 [14]. Several single nucleotide polymorphisms (SNPs) of the CTLA-4 gene have been identified. Some of these studies have demonstrated a significant association of CTLA-4 polymorphisms with atopy or asthma [15][16][17]. However, the results were not consistent in other studies [18,19].
Considering a single study may lack the power of providing a reliable conclusion, we performed a meta-analysis to investigate the relationship between CTLA-4 gene variants and asthma. To our knowledge, this is the first meta-analysis of the association between CTLA-4 polymorphisms and asthma susceptibility.

Publication search
Pubmed, EMBASE, HuGE Navigator, and Wanfang Database were searched (Last search was updated on March, 2012). The following MeSH terms were used in Pubmed: ''asthma'' and ''polymorphism, genetic'' and ''CTLA 4 antigen''. The search terms used in EMBASE and Wanfang Database were as follows: (asthma or asthmatic) and (cytotoxic T-lymphocyte associated antigen 4 or CTLA4 or CTLA-4 or CD152) and (polymorphism or mutation or variant). We also searched the reference list of original reports and review articles related to CTLA-4 polymorphism and asthma risk to identify studies not included in the computerized databases.

Inclusion and exclusion criteria
Studies fulfilled the following criteria were included in this metaanalysis: (1) asthma diagnosed by a physician or according to asthma guidelines, (2) evaluation of the polymorphisms in CTLA-4 gene and asthma risk performed, (3) using a case-control design, (4) genotype distributions in both asthma cases and controls should be available for estimating an odds ratio (OR) with 95% confidence interval (CI). Studies were excluded if one of the following existed: (1) not clinical studies, and (2) reviews and abstracts. For overlapping studies, only the one with the largest sample size was included. There was no language restriction.

Qualitative assessment
Two authors independently assessed the quality of each study. Any disagreement was resolved by consensus. Quality assessment scores of molecular association studies of asthma were used to assess the quality of selected articles [20]. This quality scoring system was based on both traditional epidemiologic considerations and genetic issues. Total scores ranged from 0 (worst) to 15 (best). Studies with quality scores #4 were defined as low quality studies [21].

Data extraction
Two investigators (Nie and Chen) independently reviewed full manuscripts of eligible studies, and the relevant data were extracted into predesigned data collection forms. We verified accuracy of data by comparing collection forms from each investigator. Any discrepancy was resolved by discussion or a third author (Xiu) would assess these articles. The following data were collected from each study: first author's name, year of publication, original country, ethnicity, age, atopic status,sample size, asthma and atopy definition, genotyping method, the polymorphisms in CTLA-4 gene, and genotype number in cases and controls. Authors of the included studies were contacted via Email when additional study data were needed.

Statistical analysis
When the data from at least 2 similar studies were available, meta-analysis was performed. ORs and 95% CIs were employed to assess the strength of association between SNPs in +49 A/G, 2318 C/T, 21147 C/T, CT60 A/G, 21722 C/T, rs926169 and asthma risk. OR1, OR2, and OR3 were calculated for the genotypes 1) AA vs. GG (OR1), AG vs. GG (OR2), and AA vs. AG (OR3) for the +49 A/G and CT60 A/G polymorphisms, 2) CC vs. TT (OR1), CT vs. TT (OR2), and CC vs. CT (OR3) for the 2318 C/T, 21147 C/T, and 21722 C/T polymorphisms, and 3) AA vs. CC (OR1), AC vs. CC (OR2), and AA vs. AC (OR3) for the rs926169, respectively. These pairwise differences were used to indicate the most appropriate genetic model as following: if OR1 = OR3?1 and OR2 = 1, then a recessive model was suggested; if OR1 = OR2?1 and OR3 = 1, then a dominant model was suggested; if OR2 = 1/OR3?1 and OR1 = 1, then a complete overdominant model was suggested; if OR1.OR2.1 and OR1.OR3.1 (or OR1,OR2,1 and OR1,OR3,1), then a codominant model was suggested [22]. Once the best genetic model was identified, this model was used to collapse the three genotypes into two groups (except in the case of a codominant model) and to pool the results again. A random-effects model, using the Mantel-Haenszel method, was used to calculate the   Departure from Hardy-Weinberg equilibrium (HWE) in controls was tested by the chi-square test. The Q statistic and the I 2 statistic were used to test for heterogeneity among the studies included in the meta-analysis. Sensitivity analyses were performed by including studies not in HWE. In addition, sensitivity analyses were also done by ethnicity and atopic status. Graphic exploration with funnel plots was used to evaluate the publication bias visually. The Begg's test and the Egger's test were used to assess publication bias statistically [23,24].
All statistical tests were performed by using the Revman 5.1 software (Nordic Cochrane Center, Copenhagen, Denmark), STATA 11.0 software (Stata Corporation, College Station, TX), and SPSS 18.0 software (Chicago, IL, USA). A P value,0.05 was considered statistically significant, except for tests of heterogeneity where a level of 0.10 was used.
The 21147 C/T, CT60 A/G, 21722 C/T, and rs926169 polymorphisms. Six studies studied the association between 21147 C/T polymorphism and asthma risk [16,28,29,33,35,38]. Total sample sizes for asthma and control groups were 1866 and 1677, respectively. The estimated OR1, OR2 and OR3 were 1.29, 1.16 and 1.04, respectively ( Table 3). These estimates suggested a codominant genetic model. The pooled OR was 1.29 (95% CI 0.87-1.91, P = 0.21) and 1.16 (95% CI 0.77-1.73, P = 0.48). Only 5 studies and 3 studies were eligible for meta-analysis on CT60 A/ G, 21722 C/T, and rs926169 polymorphisms. Dominant genetic models were chosen based on the estimated OR1, OR2 and OR3 of these three polymorphisms. Results from our meta-analysis demonstrated that CT60 A/G, 21722 C/T, and rs926169 polymorphisms were not risk factors for asthma. Summary of comparisons are listed in Table 3.

Discussion
This meta-analysis of 17 case-control studies including 6378 cases and 8674 controls systematically evaluated the association between +49 A/G, 2318 C/T, 21147 C/T, CT60 A/G, 21722 C/T, and rs926169 polymorphisms in the CTLA-4 gene and asthma risk. We found that +49 A/G polymorphism was a modest risk factor for developing asthma in the overall study population. The results revealed that carriers of the AA homozygote had 18% increased asthma risk compared to those individuals with the G allele carriers (AG+GG). In the sensitivity analysis, we found that individuals carrying AA homozygote had increased asthma risk in Asians, but not in Caucasians. These results suggested that interactions between different ethnicities and genetic variants may contribute to asthma risk. However, there were only 4 studies on Caucasians for this polymorphism [16,28,31,33]. It is therefore possible that the observed ethnic difference was due to chance. More studies with Caucasian population are required to validate the effect of ethnic differences on asthma risk through the +49 A/ G polymorphism. In addition, significant heterogeneity was observed in the Caucasians subgroup (I 2 = 56%) but not in the Asians subgroup (I 2 = 7%). Furthermore, asthma is a complex disease. Both genetic and environmental factors affect the risk of asthma in different populations. It is possible that different asthma risks in Asians and Caucasians were due to exposure to various environmental factors. However, no reported article was performed to assess the effect of environment-CTLA-4 interactions in different ethnicities. In the future, more studies should be designed to analyze these associations. We also carried out sensitivity analysis for atopic status. We found that atopic patients had increased asthma risk, suggesting a possibility of atopic status differences in asthma pathogenesis.
Ligers and co-workers showed that CTLA-4 cell-surface expression was significantly increased in individuals carrying the AA genotype, compared to levels in carriers of the AG and GG genotypes [39]. CTLA-4 +49 G.A caused 17 Ala. 17 Thr substitution in the leading peptide of CTLA-4 [40]. 17 Thr substitution increased binding of CTLA-4 to B7.1, causing stronger inhibition on T cell activation then CTLA-4 17 Ala [41]. In addition, T cells with +49 GG genotype had higher activation and proliferation rates compared to those with +49 AA genotype [41]. Recently, the G allele of the +49 A/G polymorphism was reported to have a strong association with autoimmune diseases [42][43][44]. Considering the inverse relationship between allergic diseases (Th2 dominant) and autoimmune diseases (Th1 dominant), and the role of CTLA-4 polymorphisms in determining the Th1/Th2 balance [45], it is biologically plausible that the A allele of the +49 A/G polymorphism could increase the susceptibility of asthma. Our findings and a previous study by Yang et al. [46] supported this speculation. Furthermore, Jones et al. [47] indicated +49 A allele was associated with infant atopic dermatitis. However, how A allele of the +49 A/G polymorphism influences asthma risk is unclear. Park et al. [48] reported significantly lower serum sCTLA-4 levels in Behcet's disease patients with the CTLA-4 +49 G allele than those in healthy controls. Serum sCTLA-4 concentrations also increased in patients with allergic asthma and after allergen inhalation in sensitized asthmatic subjects [49][50][51][52]. These data suggested that +49 A/G polymorphism could influence asthma susceptibility through affecting serum sCTLA-4 level.
Results from our meta-analysis showed the lack of associations between the 2318 C/T, 21147 C/T, CT60 A/G, 21722 C/T, or rs926169 polymorphisms and asthma risk. However, these results should be interpreted with caution. Because 2318 C/T was shown to be associated with asthma severity and may serve as a clinically useful marker of severe asthma [17]. More studies are required to assess the associations between 21147 C/T, CT60 A/ G, 21722 C/T, or rs926169 polymorphisms and asthma risk,  since less than 6 case-control studies were included in this metaanalysis. A positive association between these polymorphisms and asthma could not be ruled out because studies with small sample size may have insufficient statistical power to detect a slight effect.
Publication bias and heterogeneity may influence the results of meta-analyses. In our meta-analysis, only studies indexed by the selected databases were included. Negative studies were less likely to be published in journals and be available in computerized database [53], resulting in potential overestimation of effect sizes. In this meta-analysis, Begg's test and Egger's test showed significant publication bias, thus the current results should be interpreted cautiously. In addition, there was no significant heterogeneity in most of the overall comparisons for all 4 polymorphisms. Therefore, heterogeneity did not seem to have influenced the results, suggesting the reliability of our results.
Some limitations of this meta-analysis should be considered. First, the number of available studies that could be included was moderate. Therefore, the results could be influenced by factors like random error. Second, only 7 of the 17 studies were conducted in non-Asian population. Third, the overall outcomes were based on individual unadjusted ORs, while a more precise evaluation should be adjusted by other potentially suspected factors including age, sex and lifestyle. Finally, this study could not address genegene and gene-environment interactions, due to insufficient information from the primary publication.
In conclusion, our meta-analysis suggested that the +49 A/G polymorphism in CTLA-4, but not the 2318 C/T, 21147 C/T, CT60 A/G, 21722 C/T, or rs926169 polymorphisms, represented a risk factor for asthma. Future large-scale studies are still needed to validate our findings. Moreover, gene-gene and geneenvironment interactions should also be considered in future studies.