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The Association between GSTM1, GSTT1 Genetic Variants and Gastric Carcinoma Susceptibility in Chinese: A Systematic Review Article

Background: Glutathione S-transferases (GSTs) have been investigated as potential carcinoma susceptible genes. However, the relationship between GSTs (GSTM1, GSTT1) variants and gastric carcinoma (GC) risk has been controversial in Chinese population. Methods: A comprehensive literature search strategy (PubMed, Chinese Biomedical Database, Chinese National Knowledge Infrastructure, Wan fang Database, etc.) was launched. Crude odds ratios (ORs) and confidence intervals (95% CI) were applied to estimate the strength of the association. Results: Significant associations between GSTs genetic polymorphisms and GC were evidenced under random-effects model (ORGSTM1=1.56, 95% CI: 1.39 to 1.76, I2=50.7%, P<0.0001; ORGSTT1=1.24, 95% CI: 1.10 to 1.39, I2=43.6%, P=0.014; ORGSTM1-GSTT1=1.51, 95% CI: 1.26 to 1.81, I2=59.7%, P=0.004). The pooled ORs were not qualitatively changed when any single study was omitted by sensitivity analysis. Conclusion: Our results indicated an increased GC risk in Chinese population with GSTM1 and GSTT1 null genotype and GSTM1-GSTT1 dual null genotype. Further multi-center studies are needed to investigate the gene-gene and gene-environment interactions on the susceptibility of GC.


Introduction
Gastric carcinoma (GC) is one of the most common malignant tumors and is the second leading cause of cancer-related death across the worldwide (1). GC is a major health issue in China (2); its incidence is high, accounts for over 40% of all new GC cases (3). Studies involved in twins, familial clustering, and different ethnicities have identified that genetic factors contributed to GC susceptibility (4). Glutathione S-transferases (GSTs) family, known as phase II isoenzymes, has proved to be involved in detoxifying several carcinogens and plays a critical role in the deactivation of toxic and carcinogenic electrophile (5)(6)(7). The GST family included four gene subfamilies (GSTA, GSTM, GSTT, and GSTP), GSTM1 and GSTT1 arelocated in 1p13.3 and 22q11.23 in the human chromosome, and has been studied widely (8)(9)(10)(11). Polymorphisms within GSTM1 and GSTT1 genes either decrease or abolish their enzyme activities (12). The most common variant of GSTM1 and GSTT1 genes is homozygous deletion (null geno-type), which can detoxify several xenobiotics and lower the defense against oxidative stress (8,(13)(14). A meta-analysis involved in 46 studies observed evidence for GSTT1 null polymorphism and GC risk in East Asians and Indians, but not in Caucasian, and Middle Eastern and African populations (15). Another meta-analysis with 8,203 GC cases and 13,866 controls showed that GSTT1 null allele was associated with increased risk of GC in Europeans and Asians (16). Whereas, no statistical significance was observed for the GSTT1, GSTM1 genotypes and GC risk in Taiwanese (17). The above indicate that these associations vary in different populations. Substantial studies have investigated the associations between GSTM1 and GSTT1 genetic polymorphisms and GC risk in Chinese population. However, the results have been controversial. Therefore, we performed a meta-analysis to explore the above association with increased sample size and statistical power.

Literature review
Two reviewers independently conducted a comprehensive literature search in PubMed, EM-BASE, Web of science, Chinese Biomedical Database, Chinese National Knowledge Infrastructure and Wan fang Data, up to Apr 2016 without language restriction. Besides, we also searched two websites (http://www.baidu.com and http://scholar.google.com). The reference lists of available articles were also retrieved simultaneously. The following search strategies were used: ("glutathione s-transferase" or "GST" or "GSTM1" or "GSTT1") AND ("gastric" or "stomach ") AND ("cancer" or "carcinoma" or "tumo(u) r" or "neoplasm") AND ("China" or "Chinese" or "Taiwan"). When there was more than one article published, only the latest and /or the most comprehensive one would be adopted. Inclusion and exclusion criteria All inclusive studies should comply with the following criteria: 1) case-control or cohort studies; 2) the articles provided raw data or sufficient information to calculate odds ratios (ORs) with 95% confidence intervals (CIs); 3) if studies contained overlapping data, only the one with the largest sample size was included. Exclusion criteria were: 1) not related case-control or cohort studies; 2) abstract, case report, review article, and other meta-analysis; and 3) studies that contained overlapping data. Data extraction and synthesis According to the inclusion criteria, relevant data were extracted from the included studies by two independent reviewers. Discrepancy was resolved by discussion among all reviewers. The following data were extracted: first author, years of publication, geographical location, study time, criteria of pathologic diagnosis, source of control, characteristic of cases and controls, genotype frequencies of null GSTM1, null GSTT1 and dual null GSTM1-GSTT1 in cases and controls (Table  1). Meanwhile, sub-group analyses based on geographical location, number of cases, source of control and test material were also performed.

Statistical analysis
1) ORs and 95% CIs were applied to evaluate the strength of associations between the GSTs and gastric carcinoma risk; 2) statistical heterogeneity was calculated by Q and I 2 statistics (18). The Q test and I 2 were used to evaluate the proportion of the total variation from heterogeneity (19), When P value of heterogeneity tests was (P≤0.1), a random-effect model was performed. Otherwise, a fixed-effect model was used (20). Heterogeneity was divided into high heterogeneity (I 2 ≥50%) and low heterogeneity (I 2 <50%); 3) in order to explore the potential heterogeneity, subgroup analysis were also performed by geographical location (Northeast China, North China, East China, Central China, South China, Southwest China, Northwest China, and Taiwan), number of cases (<100 vs. ≥100), and sources of control (population-based, hospital-based, mixed); 4) Sensitivity analysis was used to determine the stability of the results after removing one study at a time. Galbraith plot was also performed to identify the potential heterogeneity; 5) The potential publication bias was assessed using Begg's funnel plot (21) and Egger's linear regression test (22), and P<0.05 was regarded as representative of statistically significant; and 6) all analyses were performed by STATA version 12.0 (Stata Corporation, College station, TX, USA), and all P values were two-sided.

The selection and characteristics of studies
After a comprehensive search of the above databases, a total of 142 articles were identified, 46 irrelevant articles were excluded by reviewing their abstracts, 16 articles were excluded for overlapping data, 36 articles were excluded for metaanalysis, review, only cases and other pop-ulations, and other 7 articles were excluded due to unavailable information. Finally, the remaining 37 full-text publications  were used to evaluate the associations of GSTM1 and GSTT1 genetic polymorphisms with gastric carcinoma susceptibility (Fig. 1). The characteristics of the included studies were shown in Table 1 (Fig. 2c).

Results of Sub-group analysis
We did not detect significant increased risk for GC in either North or Taiwan in GSTM1 metaanalysis or in the East or Taiwan in GSTT1 metaanalysis. Cases number <100 had a higher risk than cases number ≥100 in both GSTM1 and GSTT1 meta-analysis. In addition, populationbased studies had a higher risk than hospitalbased studies in GSTM1 meta-analysis. The heterogeneity test demonstrated that studies from Taiwan were major sources of heterogeneity for GSTM1 meta-analysis (I 2 =71.2%).
In the analysis of the relationship between GSTM1-GSTT1 genetic polymorphisms and GC risk, significant associations were found in South China, Northwest of China and hospital-based studies, however, we observed high heterogeneities in South China (I 2 =71.4% ).

Galbraith plot and sensitivity analysis
In this meta-analysis, Galbraith plot was used to identify the possible sources of heterogeneity. Three articles, two articles and two articles were identified as outliers by Galbraith plot in GSTM1, GSTT1 and GSTM1-GSTT1 meta-analysis, respectively. (Data not shown  (Fig. 3).
The fail-safe number (N fs0.05 ) was 1000 and 248 in GSTM1 and GSTT1 meta-analysis respectively, which indicated that if we want to turn the results, at least 1000 and 248 non-statistically significant studies should be further included in relevant meta-analysis. Therefore, our results were robust and reliable.

Discussion
The pooled and sub-group analysis identified a positive association between GSTM1, GSTT1 and GSTM1-GSTT1 genetic polymorphisms and GC susceptibility in Chinese population. This is consistent with previous studies. A meta-analysis showed homozygous deletion in GSTM1 increased risk of GC in different ethnics (including Japanese, Chinese, Indians, Caucasians and Africans) (60). However, significant heterogeneity was noticed. Studies from East China and Taiwan were the main heterogeneity for GSTM1 meta-analysis. The eastern region is rich in seafood, which is typically high in salt for longer storage. Fujian, an eastern coastal region, is a representative highrisk area for GC. Inhabitants' diet includes dried shrimp sauce and pickled fish (48,61). As well known, a high salt diet is a significant risk factor for the development of GC. The high osmotic pressure caused by dietary salt can damage the gastric mucosa, which will lead to extensive diffuse hyperemia, necrosis, hemorrhage etc. (62) and then accelerate the potential carcinogenicity of carcinogenic compounds. Meanwhile, studies in Chinese have confirmed that pickled food is rich in amine, which can synthesize a hard carcinogenic substance (N-nitroso compound) in the stomach. Thus, traditional Asian pickled vegetables have been classified as possible human carcinogen by the International Agency for Research on Cancer (IARC) (63,64). Furthermore, the population from East China, such as Fujian, Shanghai, and Southern Jiangsu, favors of sweet food. Available nutrition epidemiological studies have considered sugar as a vital risk factor for GC. Increasing daily sugar intake was responsible for the susceptibility of stomach cancer in both male and female in island residents (65). Diet with high sugar can damage the gastric mucosa, thus accelerate the absorption of carcinogenic substances (66). To further explore the potential heterogeneity, we performed Galbraith plot analysis. In the GSTM1 meta-analysis, three studies were identi-fied as potential heterogeneous sources (27,34,49). These three studies with small sample size might contribute to potential bias. While in the GSTT1 meta-analysis, two studies were spotted as outliers (29,43), no statistical significant heterogeneity was observed after omitted those two studies (I 2 =30.6%). Due to the heterogeneity and publication bias, the following limitations should be claimed: 1) studies included in our meta-analysis were mainly hospital-based studies, which were not as representative as population-based studies; 2) our meta-analysis included few studies with relatively small sample size, which might contribute to potential publication bias; 3) the sample size included in our meta-analysis is not very large, which may not have sufficient statistical power to evaluate the relevant associations; 4) we did not assess the gene-gene and gene-environment interactions due to unavailable data; 5) we spotted publication bias, but the fail-safe number illustrated the impact of publication bias was negligible, and the conclusion was reliable.

Conclusion
The findings indicate that GSTs genetic polymorphisms are associated with the increased GC risk in Chinese. However, larger sample size and multi-center studies are needed to confirm our findings, and gene-gene and gene-environment interactions should be explored further in the future.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.