Effects of Polymorphisms -1112C/T and +2044A/G in Interleukin-13 Gene on Asthma Risk: A Meta-Analysis

Background Associations between interleukin-13 (IL-13) polymorphisms and asthma risk remained controversial and ambiguous. Therefore, we performed a meta-analysis to assess the associations between IL-13 polymorphisms and asthma susceptibility. Methods Pubmed, EMBASE, Chinese National Knowledge Infrastructure (CNKI) and Wangfang databases were searched. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to calculate the strength of association in the random-effects model. Results Thirty-four studies were included in this meta-analysis. The results indicated that IL13 -1112C/T polymorphism was significantly associated with asthma risk (OR = 1.20, 95% CI 1.08–1.34, P = 0.0009) in a dominant genetic model. When stratifying for race, IL13 -1112C/T polymorphism exhibited increased asthma risk in Caucasians (OR = 1.30, 95% CI 1.09–1.55, P = 0.003), while no significant association was found in Asians and African Americans. In the subgroup analysis based on atopic status, significant association was observed in atopic patients (OR = 1.25, 95% CI 1.07–1.45, P = 0.004) but not in the non-atopic patients. In addition, a significant association between IL13+2044A/G polymorphism and asthma risk was observed (OR = 1.18, 95% CI 1.08–1.28, P = 0.0002). In the subgroup analysis by ethnicity, there were significant associations between IL13+2044A/G polymorphism and asthma risk in Asians (OR = 1.19, 95% CI 1.04–1.36, P = 0.01) and Caucasians (OR = 1.22, 95% CI 1.06–1.40, P = 0.005) but not in African Americans. In the subgroup analysis stratified by atopic status, a marginal significant association was found in atopic patients (OR = 1.12, 95% CI 1.00–1.26, P = 0.05). Conclusions This meta-analysis suggested that the IL13 -1112C/T and +2044A/G polymorphisms were risk factors for asthma.


Introduction
Asthma is one of the most common chronic respiratory diseases, characterized by wheezing, cough, and bronchial hyperresponsiveness. It is believed to be a multifactorial disorder with a strong genetic component [1,2]. Interleukin-13 (IL-13) is a central effector cytokine of allergic inflammation. Huang et al. [3] found that a significant enhancement of both IL-13 transcripts and secreted proteins in the allergen-challenged bronchoalveolar lavage (BAL) compared with the saline-challenged control sites of asthmatic and rhinitic patients. Furthermore, in human subjects with asthma, the IL-13 concentration in peripheral blood was increased across disease severity in a stable state and was upregulated at exacerbations [4,5]. Recently, Corren et al. [6] reported that lebrikizumab treatment in 219 adults who had inadequately controlled asthma was associated with improved lung function. These results strongly suggested that IL-13 had an important role in the pathogenesis of asthma and the IL-13 gene may be a susceptibility gene of asthma.
So far, a lot of studies investigated the association between the IL-13 gene polymorphisms and susceptibility of asthma . Most of them focused on two polymorphisms: -1112C/T and +2044A/G. However, the results from these studies were inconsistent. Although two meta-analyses on these polymorphisms have be published [41,42], some inconsistent results still existed. For example, Yang et al. [41] reported that IL-13+2044A allele was associated with an increased risk of asthma among Asians but not among Caucasians. However, Cui et al. [42] found that IL-13+2044A/G polymorphism was associated Caucasians but not Asians. In addition, these two meta-analyses did not evaluate the association between IL-13 polymorphisms and atopic asthma. Hence, we performed a meta-analysis of all eligible studies to derive more precise estimation of the associations of IL-13 21112C/T and +2044A/G polymorphisms with asthma risks. This was, to our knowledge, the most comprehensive metaanalysis of the association between IL-13 polymorphisms and asthma susceptibility.

Publication Search
Published studies were identified through a computerized search of Pubmed, EMBASE, Chinese National Knowledge Infrastructure (CNKI) and Wangfang databases (Last search was updated on October, 2012). The search terms were used as follows: (asthma or asthmatic) and (interleukin-13 or IL-13) and (polymorphism or mutation or variant). We also perused the reference lists of all retrieved articles and relevant reviews. There was no language restriction.

Inclusion and Exclusion Criteria
Studies included in the current meta-analysis should meet the following criteria: (1) evaluation of the polymorphisms in IL-13 gene and asthma risk, (2) using a case-control design, (3) genotype distributions in both 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 relevant to IL-13 or asthma risk, (2) not designed as case-control studies, (3) genotype frequencies or number not offered, (4) nonclinical studies, (5) editorials, reviews and abstracts, and (6) not consistent with Hardy-Weinberg equilibrium (HWE). In the case of overlapping studies, only the one with the largest sample numbers was included.

Data Extraction
Data were extracted from all eligible studies independently by two of the authors (Nie and Liu). The relevant data were extracted into predesigned data collection forms. The following information was collected from each study: first author's name, year of publication, original country, ethnicity, age group, atopic status, sample size, genotyping method, and genotype number in cases and controls. We verified accuracy of data by comparing collection forms from each investigator. If a decision could not be made regarding inclusion, the full text of the article was examined.

Qualitative Assessment
Two authors (Nie and Liu) assessed the quality of each study independently. The quality scoring system was based on traditional epidemiologic considerations and asthma genetic issues [43]. Scores ranged from the lowest zero to the highest fifteen. Studies with quality scores #4 were defined as low quality studies [44].

Statistical Analysis
When the data from at least 3 similar studies were available, meta-analysis was performed. The strength of the association between the IL-13 polymorphisms and asthma risk was measured by ORs and 95% CIs. The statistical significance of summary OR was determined with Z test. OR1, OR2, and OR3 were calculated for the genotypes: 1). TT vs. CC (OR1), TC vs. CC (OR2), and TT vs. TC (OR3) for the -1112C/T polymorphism, 2). AA vs. GG (OR1), AG vs. GG (OR2), and AA vs. AG (OR3) for the +2044A/ G polymorphism. These pairwise differences were used to indicate the most appropriate genetic model [45][46][47]. 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. We used a randomeffects model to calculate the pooled ORs. Heterogeneity among studies was examined with I 2 statistic. I 2 takes a value of 0-100% (I 2 = 0-25%, no heterogeneity; I 2 = 25-50%, moderate heterogeneity; I 2 = 50-75%, large heterogeneity; I 2 = 75-100%, extreme heterogeneity). A chi-square based Q-test was also performed to check the betweenstudy heterogeneity, which was considered to be significant for P,0.10. To explore the source of the heterogeneity and evaluate the ethnic-specific, atopic-specific effects, subgroup analyses were performed by ethnicity and atopic status. To access the stability of the metaanalysis, one-way sensitivity analyses were carried out. We did cumulative meta-analysis by undertaking sequential randomeffects pooling, starting with the earliest studies. Results were presented as a series of mini meta-analyses, which were ordered chronologically in a forest plot to show the consequence of adding studies on the effect size. Departure from HWE in controls was tested by the chi-square test. Publication bias was assessed by visual inspection of funnel plots, in which the standard error of log (OR) of each study was plotted against its log (OR). Funnel plot asymmetry was assessed by Egger's linear regression test [48].
All statistical tests were performed by using the Revman 5.1 software (Nordic Cochrane Center, Copenhagen, Denmark) and STATA 11.0 software (Stata Corporation, College Station, TX). A P value ,0.05 was considered statistically significant.

Study Characteristics
The flow chart in Figure 1 summarizes this literature review process. In this current study, a total of 34 eligible studies met the inclusion criteria . Four articles reported two cohorts [12,14,33,38], and each cohort was considered as a case-control study. There were 22 studies on -1112C/T polymorphism and 31 studies on +2044A/G polymorphism. There were 18 studies performed using Asians, 13 studies using Caucasians, and 4 studies using African Americans. Ten studies were performed in adults and seventeen in children. Seven studies included only atopic asthma patients, six studies included both of atopic and non-atopic asthma patients but data for these patients could be separately extracted, and 19 studies did not report detailed information. Quality scores for the individual studies ranged from 5 to 12. The characteristics of each study included in this meta-analysis are         Table 1. Genotype frequencies and HWE examination results are listed in Table 2.
We conducted one-way sensitivity analysis to evaluate the stability of the meta-analysis. As shown in Figure 3, the statistical significance of the results was not altered when any single study was omitted. Cumulative meta-analyses of IL-13 21112C/T polymorphism association were also conducted. The inclination toward significant association with asthma risk was found ( Figure 4). The funnel plot was seemed symmetrical ( Figure 5). However, Egger's test indicated significant publication bias (P = 0.021).
In the one-way sensitivity analysis, there was little modification of the estimates after exclusion of individual study (Figure 7). Cumulative meta-analysis showed that the evidence was consistent over time ( Figure 8). The shape of the funnel plots seemed symmetrical in the dominant genetic model (Figure 9). Egger's test was used to provide statistical evidence of funnel plot symmetry. The result did not show any evidence of publication bias (P = 0.684).

Discussion
Hallmarks of asthma include airway inflammation predominated by eosinophils, mucus hyperproduction, and airway hyperre- sponsiveness (AHR) [49]. A considerable weight of evidence supporting a role for IL-13 in asthma was derived from animal models. For instance, previous studies showed that acute administration of IL-13 itself was sufficient to recapitulate eosinophilic inflammation in nonimmunized mice or recombination-activating gene-deficient mice [50,51]. In addition, blockade of IL-13 alone in vivo through IL-13 gene targeting in mice prevented and reversed established mucus cell changes, suggesting a key role of IL-13 in mucus hyperproduction [52,53] Furthermore, AHR can be induced by IL-13 overexpression and blockade of IL-13 by the soluble receptor-Fc fusion protein abrogated allergen-induced AHR [54]. Taken together, these results suggested that IL-13 was a critical cytokine in the development of asthma. IL-13 was one of the most studied of the candidate genes for asthma. IL-13 21112C/T polymorphism led to increased IL-13 transcription in polarized TH2 cells and enhanced IL-13 secretion by mitogen-stimulated mononuclear cells [55]. Moreover, Arima et al. [56] indicated that the IL-13+2044A/G polymorphism may be a functional variant. Studies demonstrated that the AA genotype resulted in decreased affinity of IL-13 for IL-13Ra2 and increased expression of IL-13 [56]. Thus, it is biologically plausible that these two polymorphisms could influence the susceptibility to asthma.
In the present meta-analysis, we explored the association between the IL-13 21112C/T and +2044A/G polymorphisms and asthma risk, including 34 eligible case-control studies. For IL-13 21112C/T polymorphism, 5834 cases and 8110 controls were included. We found that individuals with the 21112T allele (TT or TC) showed an increased risk of asthma in the overall population. The results from meta-analysis showed that carriers of the TT or TC genotype had 20% increased asthma risk compared to those individuals with the CC carriers. In the stratified analysis by ethnicity, the significant association was observed in Caucasians, but not in Asians and African Americans. It is possible that different genetic backgrounds may account for these differences. However, there were only four studies using African Americans. Thus, the positive association between African Americans and asthma could not be ruled out because studies with small sample size may have insufficient statistical power to detect a slight effect. In addition, significant heterogeneity (I 2 = 52%, P = 0.10) may also distort the result. In the subgroup analysis by atopic status, we found IL-13 21112C/T polymorphism exhibited increased atopic asthma risk. For IL-13+2044A/G polymorphism, 8118 cases and 11147 controls were included. There was a significant association between this polymorphism and asthma risk. When subgroup analysis was performed according to ethnicity, significant associations were showed in Asians and Caucasians, but not in African Americans. Only three studies were performed with African Americans, thus the positive association still can not be excluded. The subgroup analysis based on atopic status found that IL-13+2044A/G polymorphism was marginally associated with allergic asthma risk. Taken together, these results suggested that IL-13 polymorphisms may play a role in the etiology of allergic asthma.
A recent meta-analysis performed by Yang et al. [41] found +2044A/G polymorphism was associated asthma risk in Asians but not in Caucasians. Another meta-analysis conducted by Cui et al. [42] showed this polymorphism was more pronounced among Caucasians but not among Asians. Results from our study were inconsistent with these meta-analyses. We found significant associations in both Asians and Caucasians. There are several potential explanations for the different results. First, different inclusion and exclusion criteria were used in these two metaanalyses [41,42]. For example, Cui et al. [42] only included English papers. However, Yang et al. [41] included articles published in English and Chinese. Thus, although these two meta-analyses were published in the same year, it was possible that different results may be observed. Second, different numbers of subjects were included in the two meta-analyses [41,42]. For +2044A/G polymorphism, Cui et al. [42] included 8439 subjects in their study, while Yang et al. [41] only included 5695 subjects in their meta-analysis. Third, we noted that three studies (n = 806) performed using Caucasians and nine studies (n = 4241) performed using Asians were included in Yang's study [41]. Moreover, six studies (n = 4202) conducted in Caucasians and five studies (n = 3673) conducted in Asians were included in Cui's study [42]. Therefore, different statistical power might be another reason for the discrepant results. For +2044A/G polymorphism, our meta-analysis included eleven case-control studies (n = 7096) in Caucasians and sixteen case-control studies (n = 10373) in Asians, thus our study was more conclusive and more powerful. Additionally, our study had some advantages. First, we attempted to find as many publications as we could by means of various searching approaches. Second, it is the first time studying the atopic specificity and IL-13 polymorphisms interactions. Third, the methodological issues for meta-analysis, such as, one-way sensitivity analysis and cumulative meta-analysis were well investigated.
Results from one-way sensitivity analysis and cumulative metaanalysis suggested high stability and reliability of our results. Besides, we had to mention the importance of heterogeneity and publication bias, which might influence the results of metaanalysis. In our study, moderate heterogeneity was observed for the IL-13 21112C/T and +2044A/G polymorphisms. We used subgroup analysis to explore the sources of heterogeneity. After subgroup analysis by atopic status, the heterogeneity was effectively decreased and disappeared. Therefore, the main source of heterogeneity was from atopic status. Moreover, funnel plots and Egger's tests were used to find potential publication bias. The results indicated that there was significant publication bias for IL-13 21112C/T polymorphism. Thus, our results should be interpreted with caution and more studies are still needed to evaluate the effect of IL-13 21112C/T polymorphism on asthma risk.
Several limitations need to be addressed. First, the numbers of published studies were not sufficient for a comprehensive analysis, particularly for African Americans. Second, our results were based on unadjusted estimates. Lacking of the original data of the eligible studies limited the evaluation of the effects of the gene-gene and gene-environment interactions in asthma. Third, Vercelli [57] suggested that IL-13 21112C/T and +2044A/G were in high linkage disequilibrium. However, we did not carry out haplotype analysis due to limited data.
In conclusion, this meta-analysis suggested that IL-13 21112C/ T and +2044A/G polymorphisms may be associated with the risk of asthma. Well-designed studies with larger sample size and more ethnic groups should be considered to further confirm these associations, especially in African Americans.