Association between Glutathione S-Transferase T1 Null Genotype and Gastric Cancer Risk: A Meta-Analysis of 48 Studies

Background Glutathione S-transferases (GSTs) have proved to be involved in the detoxifying several carcinogens and may play an important role in carcinogenesis of cancer. Previous studies on the association between Glutathione S-transferase T1 (GSTT1) polymorphism and gastric cancer risk reported inconclusive results. To clarify the possible association, we conducted a meta-analysis of eligible studies. Methods We searched in the Pubmed, Embase, and Wangfang Medicine databases for studies assessing the association between GSTT1 null genotype and gastric cancer risk. The pooled odds ratio (OR) and its 95% confidence interval (95%CI) was calculated to assess the strength of the association. A total of 48 studies with a total of 24,440 individuals were ultimately eligible for meta-analysis. Results Overall, GSTT1 null genotype was significantly associated with increased risk of gastric cancer (Random-effect OR = 1.23, 95%CI 1.13–1.35, P OR <0.001, I2 = 45.5%). Significant association was also found in Caucasians, East Asians, and Indians (P Caucasians = 0.010; P East Asians = 0.003; P Indians = 0.017). After adjusting for other confounding variables, GSTT1 null genotype was also significantly associated with increased risk of gastric cancer (Random-effect OR = 1.43, 95%CI 1.20–1.71, P OR <0.001, I2 = 48.1%). Conclusion The meta-analysis provides strong evidence for the significant association between GSTT1 null genotype and increased risk of gastric cancer.


Introduction
Gastric cancer is the second most frequent cause of cancer death worldwide, and the global burden of gastric cancer continues to increase largely in economically developing countries [1,2]. Though there are many achievements in the treatment of gastric cancer in terms of the combined therapy, novel anti-tumor agents and personalized treatments the, the survival of gastric cancer patients is still poor [3,4]. Currently, the prevention intervention is regarded as the best option to reduce the high rated of gastric cancer mortality. Effective prevention strategies should be based on specific risk profiles of gastric cancer, including Helicobacter pylori, environmental factors, and the host genetic polymorphisms [2]. In addition, genetic susceptibility to gastric cancer has been a research focus, and identifications of risk factors for gastric cancer are important for us to understand the biology of gastric carcinogenesis and develop some effective interventions. Glutathione S-transferases (GSTs) have proved to be involved in the detoxifying several carcinogens and may play an important role in carcinogenesis of cancer [5][6][7]. The theta class of GSTs is encoded by the Glutathione S-transferase T1 (GSTT1) gene located on the long arm of chromosome 22 (22q11.23), and the homozygous deletion (null genotype) of GSTT1 gene causes complete absence of GST enzymes activity [8]. Previous studies on the association between Glutathione S-transferase T1 (GSTT1) polymorphism and gastric cancer risk reported inconclusive results . To clarify the possible association, we conducted a meta-analysis of eligible studies by searching three electronic databases.

Identification of Eligible Studies
We searched in the Pubmed, Embase, and Wangfang Medicine databases for studies assessing the association between GSTT1 null genotype and gastric cancer risk. The literature strategy used the following keywords: (''Glutathione S-transferase T1'', ''GSTT1'' or ''GSTT'') and (''gastric cancer'', ''gastric carcinoma'', ''stomach cancer'' or ''stomach carcinoma''). The references of the retrieved articles were also hand searched at the same time to identify additional published articles. The references of eligible studies and relevant reviews were also checked for other literature not indexed into common databases. There was no language restriction applied in this meta-analysis. The inclusion criteria of eligible studies were as following: (1) Case-control study; (2) The cases were patients with histopathologicaly proved gastric cancer; (3) The controls were gastric cancer-free individuals; (4) Reported the frequencies of GSTT1 polymorphism in both cases and controls or the odds ratio (OR) and its 95% confidence interval (95%CI) of the association between GSTT1 null genotype and gastric cancer risk. Family-based studies and studies containing overlapping data were all excluded.

Data Extraction
Relevant data were extracted from all the eligible studies independently by two reviewers, and disagreements were settled by discussion and the consensus among all reviewers. The main data extracted from the eligible studies were as following: the first author, year of publication, country, ethnicity, characteristics of cases, characteristics of controls, total numbers of cases and controls, the genotype frequency of GSTT1 polymorphism, adjusted variables, and adjusted ORs and corresponding 95%CIs. Different ethnicities were mainly categorized as Caucasians, East Asians, Indians, Africans, and Mixed. If a study did not specify the ethnicity or if it was not possible to separate participants according to such phenotype, the group was termed ''mixed''. For studies including subjects of different ethnic populations, data were collected separately whenever possible and recognized as an independent study.

Quality Assessment
Quality of eligible studies in present meta-analysis was assessed using the Newcastle Ottawa scale (NOS) as recommended by the Cochrane Non-Randomized Studies Methods Working Group. This instrument was developed to assess the quality of nonrandomized studies, specifically cohort and case-control studies [49]. This scale awards a maximum of nine stars to each study: four stars for the adequate selection of cases and controls, two stars for comparability of cases and controls on the basis of the design and analysis, and three stars for the adequate ascertainment of the exposure in both the case and control groups. Given the variability in quality of eligible studies found on our initial literature search, we considered studies that met 5 or more of the NOS criteria as high quality.

Statistical Methods
The strength of the association between GSTT1 null genotype and gastric cancer risk was assessed by calculating the pooled OR with its corresponding 95%CI, and the significance of the pooled OR was determined by the Z-test. To assess the heterogeneity among the included studies more precisely, both the chi-square based Q statistic test (Cochran's Q statistic) to test for heterogeneity and the I 2 statistic to quantify the proportion of the total variation due to heterogeneity were calculated [50,51]. If obvious heterogeneity existed among those included studies (P Q statistic ,0.05), the random-effect model (DerSimonian and Laird method) was used to pool the results [52]. When there was no obvious heterogeneity existed among those included studies (P Q statistic .0.05), the fixed-effect model (Mantel-Haenszel's method) was used to pool the results [53]. Subgroup analyses were performed by ethnicity, the adjusted status of the estimates, and the quality of studies. The kinds of ethnicity were mainly defined as Caucasians, East Asians, and Indians. Publication bias was investigated with the funnel plot and its asymmetry suggested risk of publication bias. The asymmetry of funnel plots was further assessed by both the Begg's test and the Egger's linear regression test [54,55]. All statistical tests for this meta-analysis were performed with STATA (version 11.0; Stata Corporation, College Station, TX). A P value less than 0.05 was considered statistically significant, and all the P values were two sided.

Publication Bias
In the meta-analysis of total 48 studies, the shape of the funnel plot did not reveal any evidence of obvious asymmetry (Figure 3). In addition, both the Begg's test and Egger's test provided statistical evidence for the symmetry of the funnel plot (P Begg = 0.333; P Egger = 0.145). Therefore, there was no obvious risk of publication bias in the present meta-analysis.

Discussion
Previous studies on the association between GSTT1 polymorphism and gastric cancer risk reported inconclusive results. To clarify the possible association, we conducted a meta-analysis of a total of 48 studies with 24,440 individuals [56][57][58][59][60][61][62][63]. Overall, GSTT1 null genotype was significantly associated with increased risk of gastric cancer (Random-effect OR = 1.23, 95%CI 1.13-1.35, P OR ,0.001, I 2 = 45.5%). Significant association was also found in Caucasians, East Asians, and Indians (P Caucasians = 0.010; P East Asians = 0.003; P Indians = 0.017). After adjusting for other confounding variables, GSTT1 null genotype was also significantly associated with increased risk of gastric cancer (Random-effect OR = 1.43, 95%CI 1.20-1.71, P OR ,0.001, I 2 = 48.1%). Therefore, the meta-analysis provides strong evidence for the significant association between GSTT1 null genotype and increased risk of gastric cancer.
Endogenous products and environmental factors could result in the production of reactive oxygen species (ROS) and nitrogen metabolites causing cell injury and genetic instability [64,65]. GSTs are the most important family of phase II isoenzymes known to detoxify a variety of electrophilic compounds, including carcinogens, chemotherapeutic drugs, environmental toxins, and DNA products generated by reactive oxygen species damage to intracellular molecules, chiefly by conjugating them with glutathione [66]. GSTs play a major role in cellular antimutagen and antioxidant defense mechanisms, and these enzymes may regulate pathways that prevent damage from several carcinogens. GSTs have proved to be involved in the detoxifying several carcinogens and may play an important role in carcinogenesis of cancer [66]. These enzymes also play a crucial role in protection of DNA from oxidative damage by ROS [66]. Therefore, the polymorphisms in GSTT1 gene can causes the dysfunction of GSTs and result in less protection of DNA from damages caused by ROS [8]. The null genotype of GSTT1 gene can cause the complete absence of GST enzymes activity, which may increase the host's susceptibility to DNA damage and some cancers. Thus, there is obvious biochemical evidence for the relationship of GSTT1 polymorphism with cancer risk [8].
Nowadays, a great number of studies have been published to assess the association between GSTT1 null genotype and risks of some cancers. Currently, GSTT1 null genotype has been proven to be associated with risks of some cancers, such as lung cancer and hepatocellular carcinoma [67,68]. The significant associations further suggest that GSTT1 null genotype can affect the individual susceptibility to common malignancies, and has important roles the carcinogenesis of some cancers.
A meta-analysis in 2010 was performed to assess the association between GSTT1 null genotype and risk of gastric cancer by including thirty-six studies with 4,357 gastric cancer cases and 9,796 controls [69]. The previous meta-analysis concluded that GSTT1 gene polymorphism may be not associated with increased gastric cancer risk among Europeans, Americans, and East Asians, and more large-scale studies based on the same racial group were needed [69]. In the present meta-analysis, we performed a updated literature search and included 12 new studies, and the total sample size (24, 440 individuals) was nearly two times of that from the previous meta-analysis. To the best our knowledge, our meta-analysis is the largest meta-analysis of the association between GSTT1 null genotype and gastric cancer risk. Therefore, compared with the previous meta-analysis, the present metaanalysis has greater statistical power and can provide a more precise assessment on the association between GSTT1 null genotype and gastric cancer risk.
Some limitations of this study should be acknowledged. Firstly, there was some heterogeneity in both the meta-analysis of total 48 studies and the subgroup analyses by ethnicity. The differences from the selection criteria of cases or controls, the adjusted confounding variables, and the ethnicity result in the heterogeneity. Secondly, most studies in the meta-analysis were retrospective design which could suffer more risk of bias owing to the methodological deficiency of retrospective studies. Those there was no obvious risk of publication bias in the present metaanalysis, the risks of other potential bias were unable to be excluded. Some misclassification bias was possible because most studies could not exclude latent gastric cancer cases in the control group. Therefore, more studies with prospective design and low risk of other bias are needed to provide a more precise estimate of the association between GSTT1 null genotype and gastric cancer risk. Finally, we could not address gene-gene and gene-environmental interactions in the association between GSTT1 null genotype and gastric cancer risk. The latter may be important for genes that code proteins with detoxifying function, but would require detailed information on exposures to various potential carcinogens and individual-level data and would be most meaningful only for common exposures that are found to be strong risk factors for the disease. Thus, more studies analyses on the gene-gene and gene-environmental interactions are needed.
In conclusion, the meta-analysis provides strong evidence for the significant association between GSTT1 null genotype and increased risk of gastric cancer. In addition, more studies with well design are needed to further assess the possible gene-gene and gene-environmental interactions in the association between GSTT1 null genotype and gastric cancer risk. Figure S1 Flow diagram in the meta-analysis of the association between GSTT1 null genotype and gastric cancer risk. (TIF)