Correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma: A meta-analysis and trial sequential analysis

Background This study aims to uncover the potential correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma. Methods Literatures reporting the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma published before 1st June, 2019 were searched in PubMed, Embase, Cochrane, Wanfang and CNKI. Eligible literatures were enrolled and their data were extracted. OR and its 95% CI were calculated for assessing the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma. The included data were weighted by an inverse variance and then analyzed by a fixed or random effects model. Heterogeneity test and sensitivity analysis were performed on the enrolled reports. STATA12.1 and TSA (trial sequential analysis) were utilized for analyses.


Introduction
Bronchial asthma is a chronic allergic condition of the respiratory tract.The pathogenesis of asthma is complex, involving diverse inflammatory cells and structural cells (1)(2)(3).Asthma-induced chronic inflammation and structural change result in the high reactivity of the airways and limitation of the generally reversible expiratory flow (4,5).It is estimated that there are approximately 300 million people suffering from asthma and 180,000 people die of asthma globally (6,7).The etiology and pathogenesis of asthma have not been comprehensively explored yet (8,9).Generally, environmental and genetic factors both contribute to the occurrence of asthma (10).Exposure to allergic substances, pollutants, tobacco and smog have been recognized as intrinsic and extrinsic risk factors for asthma (11).Individuals with different genetic backgrounds exhibit different levels of susceptibility to asthma, highlighting the role of genetic components in the occurrence and progression of asthma (12,13).
LTC4S is a key enzyme for the production of cysteinyl leukotrienes (CysLTs).Polymorphism A-444C of Cytidine (C) instead of Adenosine (A) is present in promoter region -444 of LTC4S gene.The frequency of C (-444) variant allele is 22.6% in normal controls and 43.6% in aspirin asthma patients (14,15).Relevant studies have uncovered that LTC4S is upregulated in eosinophils of individuals carrying C-444 allele, thus contributing to the enhanced intracellular synthesis capacity of CysLTs (16,17).Current researches focus on genetic understanding of asthma pathogenesis (17).It is reported that LTC4S -444 A>C polymorphism in Han population is closely linked to disease severity and pulmonary dysfunctional level in adult non-acute asthma (18).This polymorphism is identified to be closely related to high reactivity of the respiratory tract, chronic inflammation, respiratory remodeling, and decreased lung function in patients with bronchial asthma (15)(16)(17)(18).
So far, several researches on underlying the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma have been published (15)(16)(17)(18).However, the conclusion was controversial.This study searched for relevant studies and analyzed their potential correlation.

Literature search
Literatures reporting the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma published before 1 st June, 2019 were searched in PubMed, Embase, Cochrane, Wanfang and CNKI.Key words searched were as follows: »Leukotrienes C4 synthase« or »LTC4S -444 A>C« or »single nucleotide polymorphism« or »variants«, or »polymorphism«, and »asthma«, and »risk« or »susceptibility«.No limitations were set on publication regions.Enrolled studies and their citations were manually examined by two researchers independently.Studies with larger sample size or latest published were selected if data overlapping.

Inclusion and exclusion criteria
Inclusion criteria were applied as follows: (1) Case-control or cohort studies; (2) Studies that analyzed the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma; (3) OR and 95% CI or relative data that could be used to calculate them were provided.
Exclusion criteria were applied as follows: (1) Cross-sectional studies, case reports, abstracts and reviews; (2) Studies that only analyzed asthma; (3) Inadequate data that could not calculate OR and their 95% CI; (4) Low-quality and repeated studies.

Data extraction
Baseline data extraction: First author, year of research, ethnicity, control resource, genotyping method, OR and its 95% CI.Data acquisition was independently carried out by two reviewers, and a third reviewer was responsible for re-evaluating disagreements.

Statistical analysis
The heterogeneity in enrolled studies was tested using the c 2 test at a test level of a= 0.10, and represented as I 2 value.Gene polymorphisms included in this analysis were studied in at least three case-control 1.24; Allele model (A): 1.07, 0.98-1.18).Subgroup analyses carried out in Asian and Caucasian population, as well as in population-based and hospital-based controls obtained the same conclusions.Conclusion: No significant correlation is identified between the LTC4S -444 A>C polymorphism and susceptibility to asthma.Researches with high-quality and large sample size are required for further validation in multi-center hospital.
Klju~ne re~i: LTC4S -444 A>C polimorfizam, osjetljivost, astma, Meta-analiza studies.P<0.10 or I 2 >50% was considered to be statistically heterogeneous and a random effects model was applied; Otherwise, a fixed effects model was adopted.OR and its 95% CI in each model were calculated and analyzed by Z test: D (AC + CC vs. AA); R (CC vs. AC + AA); (C) Homo (CC vs. AA); (D) Hetero (AC vs. AA); (E) A (C allele vs.A allele).Genotype in control group was calculated by c 2 test.P<0.05 considered that genotype in control group was not consistent with Hardy-Weinberg equilibrium (HWE).At last, Begg's test and Egger's test were utilized for evaluating publication bias.Statistical analysis was performed using Stata 12.1 and TSA.

Heterogeneity
Heterogeneity was observed in all genetic models.Interestingly, subgroup analysis can reduce heterogeneity.In this analysis, neither the ethnicity nor control source can lead to heterogeneity.Galbraith radial plots in five genetic models showed no significant heterogeneity (Figure 5).

Sensitivity analysis
Individual influence on OR was assessed by sensitivity analysis.Pooled OR in our analysis was not influenced by removal of any single research each time, verifying the robust conclusion (Figure 6).

Publication bias
Publication bias in this study was assessed using Begg's test and Egger's test.The systematic shape of funnel diagram indicated no significant publication

TSA results
The cumulative z-curve did not cross the test sequence monitoring boundary.Meanwhile, case numbers did not exceed the required amount of information, indicating that our conclusion are required for further conclusive evidence (Figure 8).

Discussion
Bronchial asthma is a common chronic respiratory disease.Its morbidity and mortality throughout the world have been risen sharply (1)(2)(3).It is estimated that there are over 25 million asthma patients in China, most of whom are children (4,5).Prevention and intervention of asthma are insufficient in our country (5)(6)(7).In recent years, genetic mutations are considered to be important risk factors for asthma.A great number of susceptible genes to asthma have been identified (9)(10)(11).Researches on candidate genes associated with susceptibility to bronchial asthma have become a hot topic in etiology (12,13).
LTC4S is an important enzyme in the cysteine leukotriene synthesis pathway.As a strong inflamma-tory mediator, cysteinyl leukotriene is widely involved in many inflammatory pathological processes (14,15).Cysteinyl leukotrienes is able to alter endothelial cell permeability and vascular endothelial cell migration by activating their receptors CysTL1 and CysTL2, thus influencing smooth muscle spasm and microvascular leakage.LTC4S is located on the chromosome 5q35 (16)(17)(18).Current researches on the correlation between LTC4S -444 A>C and asthma are controversial (17,18).Meta-analysis is a powerful tool that yields a more credible conclusion than that of an individual study, especially in controversial conclusions obtained from one common research (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31).In this paper, 15 independent case-control studies involving 3,791 asthma patients and 2,185 controls were analyzed (32,33).Our findings showed no significant relationship between the CC genotype of LTC4S -444 A>C polymorphism and susceptibility to asthma.Such a conclusion may be explained by differences of sample size, genotyping method, research design and statistical approach.Subgroup analyses were conducted based on ethnic and control group sources.Identically, no significant relationship was observed no matter in Asian or Caucasian population, nor population-based or hospital-based control group.Notably, subjects in control group could be healthy or accompanied with other diseases except for asthma, which may influence the research quality.TSA reduces random errors caused by repeated measurements of inadequate data and provides a reliable conclusion through combining multiple relevant researches.Here, TSA was conducted to control the risk of type I errors and estimate the necessarily for further experiments.In our analysis, the cumulative zcurve did not cross the monitoring boundary, and the sample size was insufficient.Therefore, we strongly considered that the conclusion obtained from this analysis required for solid validation.In addition, asthma is a multifactorial disease.The pathogenesis of asthma is closely linked to the interaction of various genes and environmental factors, not a single gene.Therefore, the interaction between environmental factors and genetic variations is of significance in assessing genetic polymorphism.In the future research, more data are needed to take into consideration of gene polymorphism in influencing asthma.

Conclusions
No significant correlation between the LTC4S -444 A>C polymorphism and susceptibility to asthma.Researches with high-quality and large sample size are required for further validation in multi-center hospital.

Figure 1
Figure 1 Flow diagram of literature search and selection process.

Figure 2
Figure 2 Forest plots of the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma model.

Figure 3
Figure 3 Forest plots of subgroup analysis by ethnicity of the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma in fixed-effects model.(A) Dominant model; (B) Recessive model; (C) Homozygous model; (D) Heterozygous model; (E) Allele model.

Figure 4
Figure 4 Forest plots of subgroup analysis by source of controls of the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma in fixed-effects model.(A) Dominant model; (B) Recessive model; (C) Homozygous model; (D) Heterozygous model; (E) Allele model.

Figure 5
Figure 5 Galbraith plot of the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma.(A) Dominant model; (B) Recessive model; (C) Homozygous model; (D) Heterozygous model; (E) Allele model.

Figure 8
Figure 8  Trial sequential analysis of the correlation between LTC4S -444 A>C polymorphism and susceptibility to asthma.The required information size was calculated based on a two side α= 5%, β= 15% (power 85%), and a relative risk reduction of 20%.

Table I
Characteristics of studies that investigated the association between LTC4S -444 A>C polymorphism and susceptibility to asthma.

Table II
Meta-analysis results for the included studies of the association between LTC4S -444 A>C polymorphism and susceptibility to asthma.