Interleukin 4 gene polymorphism (−589C/T) and the risk of asthma: a meta-analysis and met-regression based on 55 studies

Numerous investigations have previously evaluated the association of interleukin (IL) 4 gene polymorphisms and the risk of asthma, conferring inconsistent results. To resolve the incongruent outcomes yielded from different single studies, we conducted the most up-to-date meta-analysis of IL4 gene −589C/T (rs2243250) polymorphism and susceptibility to asthma. A systematic literature search was performed in ISI web of science, Scopus, Medline/PubMed databases prior to September 2020, and the pooled odds ratio (OR) and their corresponding 95% CI were calculated to determine the association strength. Literature search led to retrieving of 49 publications (55 case-control studies) containing 9572 cases and 9881 controls. It was revealed that IL4 gene −589C/T polymorphism increased the risk of asthma across all genetic models, including dominant model (OR = 1.22), recessive model (OR = 1.17), allelic model (OR = 1.21), and TT vs. CC model (OR = 1.34), but not the CT vs. TT model. The subgroup analysis by age indicated that IL4 gene -589C/T polymorphism was significantly associated with asthma risk in both pediatrics and adults. Additionally, the subgroup analysis by ethnicity revealed significant association in Asian, American, and Europeans. Finally, subgroup analysis by East Asian and non-East Asian populations indicated significant associations. The current meta-analysis revealed that IL4 gene -589C/T polymorphism was a susceptibility risk in both pediatrics and adults in the whole and different ethnic groups.


Background
Asthma is one of the most common atopic disorders of the respiratory tract, which results in wheezing, coughing, breathlessness, and bronchial obstruction [1]. The prevalence and incidence of asthma raised regularly and it estimated more than 300 million persons of the world are affected by this disease [2]. The main causes of asthma are not completely clear. However, is has been postulated that asthma is mediated by interactions between specific external stimulating factors, including pollutants, viral and bacterial infections, allergens, tobacco smokes, etc., and genetics of the patients. Additionally, increasing number of studies recommend that genetic factors play a critical role in the etiology of asthma by their interactions with the environmental elements [3,4]. The heritability of asthma is estimated to be 35 to 95% [5]. Numerous studies have examined the correlation between genetic variations of pro and antiinflammatory genes and susceptibility to asthma [6,7]. In recent decades, single nucleotide polymorphisms (SNP) have become one of the frequently studied models of DNA variation in analyses of the association between genetics and susceptibility to disease [8,9].
The role of immunological factors especially cytokines in modulating and controlling the inflammatory response of the respiratory tracts is essential in the evolution, progression, and exacerbations of asthma [10]. Interleukin (IL)-4 is a key ingredient of the immune system required in the regulation of response to an allergen through controlling the isotype switching of antibody in B lymphocytes to IgG and IgE class [11]. Elevated serum levels of IgE are suggestive of allergic reactions and resemble a high level of IL-4 mRNA assembly [12]. Moreover, it acts as a growth factor to facilitate the differentiation of T helper (Th) 2 cells and mast cells. These characteristics of IL-4 accentuate on the crucial roles of cytokines in the pathogenesis asthma [13,14]. Additionally, IL4 gene polymorphisms, like promoter region (C + 33 T) SNP [15], and 3017 G/T SNP in intron 2 [16], have been associated with IgE levels, which might be involved in the pathogenesis of asthma.
The IL4 gene is located on chromosome 5q31 [17]. The -589C/T (rs2243250) polymorphism has been recognized on upstream of the transcription initiation site [18]. It has been demonstrated that the binding of a transcription factor is enhanced by the appearance of the polymorphic T allele that may result in an overexpression of the IL4 gene and, thus, raising the power of any immunological response that dependents on IL-4 [19]. To date, many studies have examined the association between IL4 gene -589 C/T polymorphisms and the risk of asthma, but their outcomes have not been consistent. Therefore, we performed this meta-analysis to analyze the relationship between the -589C/T polymorphisms and susceptibility to asthma.

Methods
This study conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement including; literature review, study selection, inclusion and exclusion criteria, data extraction and quality assessment, and statistical analysis [20]. No ethics committee confirmation was necessary for this meta-analysis, which does not contain any studies with human participants or animals performed by any of the authors.

Literature review
A comprehensive search was performed in the ISI web of science, Scopus, Medline/PubMed databases to retrieve published articles prior to September 2020. The following main key words and Medical Subject Headings (Mesh) were searched: ("asthma" [Mesh] OR "asthmatic") AND ("interleukin-4" OR "IL-4" OR "rs2243250") AND ("single nucleotide polymorphism" OR "SNP" OR "polymorphisms" OR "mutation" OR "variation"). No restrictions were placed on language, sample size, population or publication date.

Study selection
The retrieved publications by primary literature search were imported into Endnote X8 software. The duplicate studies were removed and title and abstract of remain studies were reviewed by two investigators. Full-text verification was performed if we could not categorize studies based on title and abstract. Any disagreements during study selection was discussed and resolved by consensus.

Inclusion and exclusion criteria
The following inclusion criteria were used to distinguish eligible studies: i) studies with distinct case and control group evaluating the association between IL-4 C589T polymorphism and susceptibility to asthma; ii) studies with calculable or extractable data for odds ratio (OR) and 95% confidence intervals (CIs); iii) studies with sufficient data for alleles and genotypes in case and control group. The duplicates, reviews, book chapters, and meta-analysis were excluded. The application of these criteria results in 49 qualified studies for the metaanalysis.

Data extraction and quality assessment
Two of our authors independently and according to an extraction checklist extracted the following data: the first author, journal and year of publication, country of origin, ethnicity, number of subjects in the case and the control groups for each gender, mean or range of age, genotyping method, genotype counts in the case and the control group. The quality of each study was assessed using the Newcastle-Ottawa Scale (NOS) criteria [21]. Studies with scores 0-3, 4-6 or 7-9 were low, moderate or high-quality, respectively.

Statistical analysis
Statistical analyses were carried out using STATA (version 14.0; Stata Corporation, College Station, TX) and SPSS (version 23.0; SPSS, Inc. Chicago, IL). The strength of association between polymorphism and asthma susceptibility was estimated by odd ratios (ORs) and 95% confidence intervals (CIs) for the dominant model, recessive model, allele contrasts, and additive comparison. Heterogeneity among included studies was measured via Q statistics (P value< 0.1 considered statistically significant) and I 2 -test (I 2 values of 25, 50 and 75% were described as low, moderate, and high heterogeneity, respectively). In the presence of heterogeneity random effect model (REM) was used, however fixed effect model (FEM) was applied in homogeneous condition [22,23]. In order to assessed the predefined sources of heterogeneity among included studies, subgroup analysis and meta-regression analysis based on year of population, the continent of the study population, and genotyping method were performed. The genotypic frequency distribution in the controls was checked for consistency of the Hardy-Weinberg equilibrium (HWE). Furthermore, publication bias was computed by the Begg's and Egger's test and visual examination of the funnel plot (P value< 0.05 considered statistically significant) [24,25]. Additionally, to calculate overall effect size in absence of each study, a sensitivity analysis was conducted.

Search results and characteristics of the selected studies
Our primary search retrieved 2121 potential articles. After removing of duplicate articles (n = 301), 1820 articles remain for abstract and full-text screening. Of 1820 articles, 1612 were excluded base on title and abstract and 159 articles based on full-text reading. Ultimately 49 publications with 9579 cases and 9881 controls met the inclusion criteria and their data were extracted for meta-analysis. Among these 49 publications, four of them, including Basehore et al. [16], Donfack et al. [26], Zhang et al. [27], and Baye et al. [28] examined two or three different populations with separate case and control; therefore, we assumed them as 9 case-control studies collectively (55 studies). The detailed information on study selection process is illustrated in Fig. 1, Tables 1, and 2.

Subgroup analysis by age
We stratified eligible articles into three groups including: pediatrics (16 articles), adults (21 articles) and mixed (cover both range;18 articles). The results highlighted a predisposing role of IL4 gene -589C/T polymorphism for the asthma risk in pediatrics and adults under all genotype models. However, no significant association was detected in mixed group ( Table 3, Fig. 3).

Subgroup analysis by ethnicity 1 (continent)
In this subgroup we categorized studies by their continent: including Asia (32 articles), Europe (11 articles), America (10 articles), Africa (1 article), and Oceania (1 article). Since there was only one study for each one of the African and Australian population, these studies were excluded from the analysis. The results indicated that presence of

Subgroup analysis by ethnicity 3
Finally, subgroup analysis of eligible articles according ethnicity including Caucasians (41 articles), African-Americans (9 articles), and Arabs (5 articles) showed that there was no significant association between IL4 gene -589C/T SNP and asthma risk in  (Table 3, Fig. 4).

Meta-regression analyses
Meta-regression analyses were performed to explore potential sources of heterogeneity among included studies ( Table 4). The findings indicated that none of the expected heterogeneity parameter were the source of heterogeneity (Fig. 5).

Publication bias
To check existence of publication, Egger's linear regression and Begg's funnel plot test were used. The shape of   6).

Sensitivity analysis
The impact of individual study on pooled OR was evaluated by sequential omission of each studies. The result showed that no individual study significantly affected the pooled ORs under all genotype models of the IL4 gene -589C/T polymorphism (Fig. 7).

Discussion
To date, several individual case-control replication studies have attempted to divulge the association of IL4 gene -589C/T polymorphism and risk of asthma. Due to some differences, however, these disperse investigation demonstrated incongruous reports. The differences in the race of study subjects, diversity in the diagnostic criteria of the patients, limited sample sizes may be the cause of such inconsistent results [74]. On the other hand, meta-analysis is a tool that has the potential to solve the problem of  inconsistency by removing the confining issues of insufficient statistical power in the individual studies. Therefore, to resolve the mentioned confining factors about the IL4 gene -589C/T polymorphism, the present most up-to-date meta-analysis was conducted to determine a bona fide estimation of the association between IL4 gene -589C/T polymorphism and susceptibility to asthma. Our analysis indicated that this SNP was associated with increased risk of asthma in the overall population as well as during subgroup analysis by age groups and ethnicity/continent. Asthma is a complicated pulmonary disease, characterized by airway hyperresponsiveness, airway inflammation, and airway remodeling [75,76]. During asthma, there is a hyperactivity of Th2 responses, in which the cytokines of the type 2 immunity, such as IL-4, IL-5, and      IL-13 promote the harmful inflammatory events in the airways. Studies have reported that local administration of IL-4 gene plasmids prior to antigen challenge could stimulate the airway hyperresponsiveness and accumulation of eosinophils in mice [77]. This phenotype of asthma is commonly referred to "eosinophilic" asthma. On the other side, "noneosinophilic" asthma is characterized by low frequency of eosinophils in the involved sites, but other inflammatory cells are dominant in the effector phase, such as neutrophils, mixed granulocyte inflammatory cells, or even little number of inflammatory cells, called paucigranulocytic inflammation. Th17 mediated IL-17 axis and lack of significant Th2/Th17 inflammation have been attributed to the noneosinophilic asthma [78]. Among the SNPs in the IL4 gene, the -589C/T (rs2243250) polymorphism has been widely investigated in susceptibility to asthma. It has been shown that the T allele of this SNP leads to increased affinity of the binding of transcription factors in comparison to the C allele, leading to overexpression of IL4 mRNA [79,80]. As a consequence, it is a biological justification that IL4 gene −589C/T SNP impresses the IL-4 expression and, hence, could affect the asthma susceptibility. Previously, three meta-analysis studies have attempted to disclose the association of IL4 gene −589C/T SNP with the risk of asthma. Wang et al. in 2012 indicated that the T allele of IL4 gene −589C/T SNP increased the risk of asthma (OR = 1.12). Basically, individuals carrying the T allele had a 24% increased risk of asthma in comparison to the CC homozygote model. Subgroup analysis revealed the association of this polymorphism in the Caucasians [81]. In addition, Nie et al. in 2013 included 40 studies involving 7345 cases and 7819 controls in their meta-analysis [18]. This meta-analysis indicated that TT vs. CC (OR = 1.40) and CT vs. CC (OR = 1.22) models were significantly associated with increased risk of asthma. In the subgroup analysis by ethnicity, significant associations were found among Asians and Caucasians, but not in the African-Americans. In addition, the subgroup analysis by atopic status revealed no significant association among atopic asthma patients and nonatopic asthma patients. On the other side, Zhang et al.  [75] by evaluating pediatric asthma risk by evolving 17 case-control studies (15 publications) containing 3427 cases and 4247 controls revealed that IL4 -589C/T polymorphism was associated with increased risk of asthma in pediatrics. Furthermore, the subgroup analyses by ethnicity, indicated significant association in Caucasians and Asians. Our analysis was performed on 55 case-control studies containing 9572 cases and 9881 controls. It was observed that IL4 gene -589C/T polymorphism increased the risk of asthma across all genetic models, including dominant model (OR = 1.22), recessive model (OR = 1.17), allelic model (OR = 1.21), and TT vs. CC model (OR = 1.34), but not the CT vs. TT model. Furthermore, subgroup analysis by age indicated that IL4 gene -589C/ T polymorphism was significantly associated with asthma risk in both pediatrics and adults. The subgroup analysis by ethnicity revealed significant association in Asian, American, and Europeans. Finally, subgroup analysis by East Asian and non-East Asian populations indicated significant associations.
This meta-analysis bears some limitations and caveats. First, the analysis was according to crude estimation of IL4 gene -589C/T polymorphism association with asthma susceptibility, regardless of the effect of confounding factors, like age, sex, environmental factors, and contribution of other genes in LD with IL4 gene. Second, we did not analyze other genes that could be contributing in understanding of cytokine involvement in the susceptibility to asthma.

Conclusion
All in all, here we carried out the most up-to-date analysis of the IL4 gene 589C/T polymorphism and asthma risk prior to September 2020. Our meta-analysis further confirmed some results of the previously performed meta-analysis, while rejected some of them. In a whole, IL4 gene -589C/T polymorphism increased the risk of asthma across all genetic models. Moreover, the subgroup analysis by age indicated that IL4 gene -589C/T polymorphism was significantly associated with asthma risk in both pediatrics and adults. Also, the subgroup analysis by ethnicity revealed significant association in Asian, American, and Europeans. Ultimately, subgroup analysis by East Asian and non-East Asian populations indicated significant associations.