Meta-Analysis of Microsomal Epoxide Hydrolase Gene Polymorphism and Risk of Hepatocellular Carcinoma

Background Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH). Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent. Methods PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Results Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.35, 95% CI = 1.04–1.75, p = 0.02), homozygote comparison (OR = 1.65, 95% CI = 1.07–2.54, p = 0.02) and a recessive genetic model (OR = 1.54, 95% CI = 1.21–1.96, p<0.001), while individuals carrying the Arg139Arg mEH genotype had no association with increased or decreased risk of HCC. Conclusion The 113His- allele polymorphism in mEH may be a risk factor for hepatocarcinogenesis, while the mEH 139Arg- allele may not be a risk or protective factor. There is substantial evidence that mEH polymorphisms interact synergistically with other genes and the environment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions.


Introduction
As the most frequent primary cancer of the liver, hepatocellular carcinoma (HCC) is a significant cause of cancer morbidity and mortality worldwide, and treatment options are limited. The estimated incidence of new HCC cases each year is more than 0.5 million [1]. Sub-Saharan Africa and Southeast Asia are the regions with highest incidence of HCC (.20 per 100,000 people) [2]. Epidemiologically, HCC is strongly associated with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcohol consumption, and aflatoxin B1 (AFB) contamination of foodstuffs, among other factors [3][4]. In developing countries, AFB together with chronic HBV infection contributes to the high incidence of HCC. However, not all individuals with these factors appear to have the same risk of developing HCC. HCC exhibits a high degree of genetic heterogeneity: multiple molecular pathways may give rise to subsets of hepatocellular neoplasms.
HCC pathogenesis remains incompletely understood. It is known to involve chronic inflammation, hepatocyte hyperplasia and ultimately malignant transformation [5]. Current thinking is that HCC is a multifactorial disease, the etiology of which involves various host and environmental factors. Moreover, host and environmental factors may interact synergistically in HCC pathogenesis and progression.
The liver has a complex detoxification system. In this complex system, several enzymes participate in the metabolism of both exogenous and endogenous metabolites produced by oxidative stress in chronic infections, such as HBV and HCV infections. The genes encoding these enzymes are polymorphic, and the variant sequences lead to enzymes with altered activity. Any genetic defect in these xenobiotic-metabolizing enzymes may lead to accumulation of viral integration products and xenobiotics. This should increase the risk of liver diseases, such as cirrhosis and HCC.
Microsomal epoxide hydrolase (mEH) is one of these enzymes. As a phase II metabolic enzyme, mEH is involved in the hydrolysis of various epoxides and deleterious chemical epoxide intermediates generated by phase I oxidation reactions [6][7]. Since epoxides are highly reactive oxidative metabolites, mEH is considered to act on the most toxicologically active forms of drugs and environmental chemicals. Despite its important protective function, its net effects on the body can be complex, since it plays a dual role of procarcinogen detoxifier and activator [8].
The gene encoding mEH consists of nine exons and eight introns. Two relatively frequent single nucleotide polymorphisms occur in the coding region: Tyr113His in exon 3 and His139Arg in exon 4. These polymorphisms modify protein stability: Tyr113His decreases enzyme activity, while His139Arg increases it [9].
In 1995, Mcglynn and coworkers [10] explored the association between mEH polymorphism and risk of HCC. They found that the Tyr113His mEH gene polymorphism is associated with risk of developing HCC. The study also found a synergistic increase in the risk of HCC when HBV infection and susceptible genotype occurred together [10]. More recently, epidemiological studies have evaluated the association between the Tyr113His and His139Arg mEH gene polymorphisms and risk of HCC in diverse ethnicities, with inconsistent results [11][12][13][14][15][16][17][18][19][20]. Some of these studies also revealed synergistic gene-gene [13,[17][18][19] and gene-environment [10,16,20] interactions. A single case-control study may fail to completely demonstrate these complicated genetic relationships because of small sample size. In order to provide stronger evidence of the effects of these two mEH polymorphisms on HCC risk, we carried out a meta-analysis by combining data from numerous published studies.

Search Strategy
All case-control studies of mEH polymorphism and HCC risk published up to August 31, 2012 were identified through systematic searches in PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure (CNKI) databases using English and Chinese. The search terms used were: mEH; HYL1; EPHX; microsomal epoxide hydrolase; these four terms in combination with polymorphism, variation, genotype, genetic and mutation; and all of the above terms in combination with hepatocellular carcinoma, HCC, liver cancer, liver tumor, liver neoplasms and hepatic tumor. The references of each article identified were also manually searched to identify additional relevant publications.

Inclusion Criteria
A study was included in the meta-analysis if it satisfied the following criteria: (a) it assessed the association between HCC and the Tyr113His and His139Arg mEH gene polymorphisms, (b) it used a case-control design, and (c) it provided sufficient published data for estimating an odds ratio (OR) with a 95% confidence interval (95% CI). In the case of multiple studies based on the same population, we selected the study with the largest number of participants.

Data Extraction
Literature searches and identification of eligible articles based on the inclusion criteria were carried out independently by two authors (JHZ and BDX). These authors independently extracted the following data: first author's name, year of publication, ethnicity or country, source of controls (hospital-or populationbased), exon number, numbers and genotypes of cases and controls, and Hardy-Weinberg equilibrium (HWE) of controls. Discrepancies were resolved by consensus.

Statistical Methods
The unadjusted OR with 95% CI was used to assess the strength of the association between the Tyr113His and His139Arg mEH polymorphisms and HCC risk based on the genotype frequencies in cases and controls. Subgroup analysis stratified by ethnicity was performed. Ethnicity was categorized as Chinese and mixed. The meta-analysis examined the association of different genotypes at Tyr113His mEH with HCC risk by comparing the 113 Tyr allele (113T-allele) with the 113 His allele (113H-allele), the homozygous comparison (His113His vs. Tyr113Tyr), and recessive and dominant genetic model. The same approach was used to examine the association of His139Arg mEH and HCC risk.
All statistical tests for this meta-analysis were performed using RevMan 5.0 and Stata 11.0 softwares. Fixed-effect and randomeffect models were used to calculate a pooled OR. The statistical significance of the pooled OR was determined using the Z-test, and P,0.05 was considered statistically significant. The assumption of heterogeneity was evaluated by applying a chi square-based Q-test among the studies. P.0.10 for the Q-test indicates a lack of statistical heterogeneity, suggesting the variability in effect sizes is larger than that expected from chance alone [21]. In these cases, a pooled OR was calculated for each study using the fixed-effects model. Otherwise, the random-effect model was used. To assess the reliability of the outcomes in the meta-analysis, a sensitivity analysis was performed by excluding one study at a time. Smallstudy bias was assessed by Harbord's modified test [22]. HWE in the control group was assessed using the asymptotic test, with P,0.05 considered significant.

Description of Studies
A total of 223 potentially relevant publications up to August 31, 2012 were systematically identified through PubMed, EMBASE, Google Scholar and CNKI. After screening the title and/or abstracts, 174 (78%) were excluded because they did not satisfy the inclusion criteria. An additional 36 publications were excluded because they did not examine Tyr113His and His139Arg mEH polymorphisms or they were review articles. Three publications [19,23,24] were based on the same participants with HCC, so they were considered as one study. In the end, 11 studies [10][11][12][13][14][15][16][17][18][19][20] were included in this meta-analysis based on our search strategy and inclusion criteria (Fig. 1).
We established a database according to the information extracted from each article. Detailed characteristics of the 11 studies are listed in Tables 1 and 2. Overall, 1,696 HCC cases and 3,600 controls were retrieved. Six of the studies involved Chinese subjects [10][11][15][16]18,20]. Of these, three studies involving 232 cases and 322 controls were published in Chinese, while the other three involving 540 cases and 1021 controls were published in English. The populations of the remaining five studies came from the UK [12], Sudan [13], Italy [14], Gambia [17], and India [19]. All studies had a case-control design. Three studies [12,14,19] included a control population with liver disease (hospital-based control). These three studies [12,14,19] involved 202 HCC cases and 536 controls. The number of populationbased controls was 3,064. However, some of the population-based controls (health population) had liver disease. Therefore, subgroup analysis by source of controls was not conducted. Of the total number of 5,296 subjects considered in the meta-analysis, at least 2,000 (37.8%) had one or more of the following: alcoholic liver disease, HBV or HCV infection, and cirrhosis. Most studies did not report detailed data on the percentages of their subjects with these background conditions. Therefore we could not define subgroups for meta-analysis based on liver disease, HBV/HCV infection or cirrhosis status.
All 11 studies described the Tyr113His polymorphism; however, only 6 studies [12][13][14][15][16]19] described the His139Arg polymorphism. The distribution of genotypes among controls did not show HWE in several studies [10][11][13][14][15][17][18]20] ( Tables 1 and 2). Table 3 shows the relationship between the Tyr113His mEH polymorphism and HCC risk. The statistical heterogeneity of Tyr113His mEH allelic contrast, homozygote comparison, and dominant and recessive genetic models was analyzed for all 11 studies. Statistically significant heterogeneity was observed in all studies except for the recessive genetic model for the Chinese subgroup. Therefore, random-effect models were used to analyze the OR.

Test of Heterogeneity
For the heterogeneity of His139Arg mEH, statistically significant heterogeneity was observed for allelic contrast (P = 0.003, I 2 = 72%) and the dominant genetic model comparison (P,0.001, I 2 = 76%). However, no heterogeneity was found between studies for the homozygote comparison and recessive genetic model comparison (I 2 = 0%) (Table S1). Table 3 shows the summary ORs for the Tyr113His mEH polymorphism and HCC risk on the basis of 1,696 HCC cases and 3,600 controls. We observed an association between Tyr113His mEH genotype and HCC risk in the total population based on all 11 studies. Given the ethnic differences in the allele frequency of this sequence variant, we evaluated the effect of the Tyr113His mEH polymorphism separately in Chinese and in other populations. Given that not only some of the healthy population in the control arm had liver disease, but also most studies did not report detailed data on the percentages of their subjects with these background conditions, we did not compute overall ORs stratified by source of control.

Sensitivity Analysis
In the allele contrast, the result was altered after excluding the study by Mcglynn and coworkers [10], with OR of 1.24 (95% CI 0.98 to 1.58, P = 0.08). In the recessive genetic model contrast, the results were not altered after excluding studies one by one. However, in the dominant genetic model contrast, the result was statistically significant after excluding the study by Kiran et al. [19], with OR of 0.66 (95% CI 0.46 to 0.94, P = 0.02). Therefore, the results should be interpreted with caution. For the His139Arg mEH polymorphism and HCC risk, the results were not altered based on allelic contrast, dominant or recessive genetic model comparison.

Discussion
HCC involves complex, multistep and heterogeneous malignant tumorigenesis. The pathogenesis of HCC involves host and environmental factors, as well as modulation of molecular signaling pathways implicated in malignant transformation of hepatocytes and tumor progression [25]. Polymorphism in the mEH gene has been associated with risk of various malignancies [26][27][28], including HCC [14]. Two relatively common genetic polymorphisms occur in the mEH gene: Tyr113His in exon 3 and His139Arg in exon 4. The C to T transition in the Tyr113His polymorphism eliminates an EcoRV restriction site, while an A to G transition in the His139Arg polymorphism creates an Rsa1 cleavage site [9]. The Tyr113His polymorphism decreases mEH activity by 40-50%, while the His139Arg polymorphism increases it by 25% [9].
Some studies reported an association between the Tyr113His polymorphism and HCC risk, while others found no such association. The most likely reason for the inconsistencies among these studies is that they are single case-control studies with small sample sizes. To help resolve these conflicting results using a larger sample size, we conducted meta-analysis of published studies. Our results for the total population suggest an increased HCC risk for subjects carrying the Tyr113His mEH genotype. Our approach also allowed us to look for potential ethnic differences in the association. Analysis of ethnic subgroups showed that in the Chinese population, the 113His-allele was strongly associated with increased risk of HCC based on allelic contrast, homozygote comparison and the recessive genetic model. However, no association was observed for other populations (UK, Sudan, Italy, Gambia, and India). Six studies [12][13][14][15][16]19] reported the association of His139Arg mEH polymorphism and HCC risk. The results of the present meta-analysis revealed that the 139Argallele is not associated with increased or decreased risk of HCC.
The clinical significance of mEH polymorphisms extends beyond HCC. Polymorphisms in this gene have been reported to be susceptibility factors for several human diseases, such as type 2 diabetes mellitus [29], alcohol dependence [30], and chronic obstructive pulmonary disease [31]. In fact, two earlier metaanalyses [32][33] suggested a potential protective effect of the Tyr113His polymorphism and a potential harmful effect of the His139Arg polymorphism on lung cancer. While some studies have reported that mEH polymorphisms do not affect susceptibility to esophageal carcinoma [34] or colorectal cancer [35][36], other studies [27,37] have shown that the mEH 113His allele is associated with increased risk of ovarian cancer. Several studies in our meta-analysis indicate that mEH polymorphisms can interact with environmental factors to module HCC risk (Table 4) [10,16,18,20]. Two such synergistic geneenvironment interactions are that smoking populations with mEH polymorphism are at increased risk of HCC [16,20], while a synergistic effect of smoking, drinking, and mEH polymorphism was observed in HBV-infected individuals [20]. Moreover, the association between mEH polymorphism and HCC depended on HBV status, suggesting a synergistic increase in risk of HCC when HBV infection and susceptible genotype occur together [10,18].
Our meta-analysis also revealed gene-gene interactions between the mEH polymorphisms and genotypes associated with elevated risk of AFB toxicity. Individuals with a greater number of suspected AFB-related high-risk genotypes (GSTM1-null, mEH-YH/HH, XRCC1-AG/GG) faced a greater risk of HCC than those without any high-risk genotypes, especially among individuals with higher AFB exposure [13,[17][18][19]. These results demonstrate that the etiology of HCC is complex and involves host and environmental factors that may interact synergistically.
The 113His codon variant is relatively common in the mEH gene. However, the allele frequency differs significantly among   [28] also observed that the rate of homozygous His113His is greatest in the Asian population (18-42%) and intermediate in the European population (about 10%). These findings are consistent with our metaanalysis. In our analysis, the Chinese population had the highest frequency of His113His: 35-56% among HCC cases and 26-44% among healthy individuals and individuals with other liver diseases. The frequencies of His113His in populations from the UK, Sudan, Italy, and India were 11-28% among HCC cases and 7-29% among healthy individuals and individuals with other liver diseases. The frequency in the Gambian population was lowest at 5% (Table 1). Our data revealed that His113His mEH is a risk factor for hepatocarcinogenesis, but the complete picture is more complex. Both China and Gambia have among the highest incidences of HCC in the world, as well as a high prevalence of HBV infection and dietary exposure to AFB, which are the two main risk factors for HCC [39][40][41]. Nevertheless, Chinese had the highest frequency of His113His in our meta-analysis, while Gambians had the lowest. This discrepancy indicates that hepatocarcinogenesis has a complex, multifactorial etiology. It may also reflect the possibility that mEH mitigates the carcinogenic effects of AFB [42]. Polymorphisms in numerous other genes, such as those encoding epidermal growth factor [43] and tumor necrosis factoralpha [44] are also associated with the risk of HCC. It may be that any single nucleotide polymorphism such as His113His is insufficient on its own to cause HCC, though it does increase the risk of the disease.
Of the total number of 5,296 subjects considered in the metaanalysis, at least 2,000 (37.8%) had one or more of the following: alcoholic liver disease, HBV or HCV infection, and cirrhosis. Since the studies in our meta-analysis often did not report detailed statistics on the proportion of HCC or control subjects with these background conditions, we could not perform subgroup analysis to separate the contribution of mEH polymorphism from that of possible confounders like HBV or HCV infection. Some other limitations of this meta-analysis also should be considered when interpreting the results. Bias may result from our exclusion of unpublished data, as well as papers published in languages other than English and Chinese [45]. Second, the results may be affected by additional confounding factors, such as tumor status, age or gender, but most studies either did not report these baseline data or aggregated them in different ways, making it impossible to include them in the meta-analysis. Third, there was significant heterogeneity among the studies. Fourth, five studies were based on non-overlapping countries and ethnicities and relied on quite small samples. Fifth, the result in the allelic contrast was altered after excluding one study. Therefore, the result should be explained with caution. Moreover, the distribution of genotypes among controls did not show HWE in several studies.
In conclusion, this meta-analysis suggests that the His113His mEH polymorphism may be associated with increased risk of HCC, especially in the Chinese population. Moreover, the mEH gene is involved in synergistic gene-gene and gene-environment interactions. These results suggest that sequence variation in exon 3 of the mEH gene may play an important role in the occurrence of HCC. In contrast, no association was observed between mEH gene variation in exon 4 and the risk of HCC. However, since this meta-analysis included few studies from non-Asian populations, large, well-designed studies in Caucasian and African populations are warranted to re-evaluate these associations.
This meta-analysis is guided by the PRISMA statement (Checklist S1).

Supporting Information
Table S1 Overall and stratified meta-analyses of the association between mEH polymorphism His139Arg and risk of hepatocellular carcinoma. (DOC)