AIM: To investigate the role of metabolic enzyme and DNA repair genes in susceptibility of esophageal squamous cell carcinoma (ESCC).

METHODS: A case-control study was designed with 454 samples from 128 ESCC patients and 326 gender, age and ethnicity-matched control subjects. Genotypes of 69 single nucleotide polymorphisms (SNPs) of metabolic enzyme (aldehyde dehydrogenase-2, ALDH2; alcohol dehydrogenase-1 B, ADHB1; Cytochrome P450 2A6, CYP2A6) and DNA repair capacity genes (excision repair cross complementing group 1, ERCC1; O⁶-methylguanine DNA methyltransferase, MGMT; xeroderma pigmentosum group A, XPA; xeroderma pigmentosum group A, XPD) were determined by the Sequenom MassARRAY system, and results were analyzed using unconditional logistic regression adjusted for age, gender.

RESULTS: There was no association between the variation in the ERCC1, XPA, ADHB1 genes and ESCC risk. Increased risk of ESCC was suggested in ALDH2 for frequency of presence C allele of SNP [Rs886205: 1.626 (1.158-2.284)], XPD for C allele [Rs50872: 1.482 (1.058-2.074)], and MGMT for A allele [Rs11016897: 1.666 (1.245-2.228)]. Five variants of MGMT were associated with a protective effect on ESCC carcinogenesis, including C allele [Rs7069143: 0.698 (0.518-0.939)], C allele [Rs3793909: 0.653 (0.429-0.995)], A allele [Rs12771882: 0.719 (0.524-0.986)], C allele [Rs551491: 0.707 (0.529-0.945)], and A allele [Rs7071825: 0.618 (0.506-0.910)]. At the genotype level, increased risk of ESCC carcinogenesis was found in homozygous carriers of the ALDH2 Rs886205 [CC vs TT, odds ratios (OR): 3.116, 95% CI: 1.179-8.234], MGMT Rs11016879 (AA vs GG, OR: 3.112, 95% CI: 1.565-6.181), Rs12771882 (AA vs GG, OR: 2.442, 95% CI: 1.204-4.595), and heterozygotes carriers of the ALDH2 Rs886205 (CT vs TT, OR: 3.930, 95% CI: 1.470-10.504), MGMT Rs11016879 (AG vs GG, OR: 3.933, 95% CI: 2.216-6.982) and Rs7075748 (CT vs CC, OR: 1.949, 95% CI: 1.134-3.350), respectively. Three variants were associated with a protective effect on ESCC carcinogenesis, carriers of the MGMT Rs11016878 (AG vs AA, OR: 0.388, 95% CI: 0.180-0.836), Rs7069143(CT vs CC, OR: 0.478, 95% CI: 0.303-0.754) and Rs7071825 (GG vs AA, OR: 0.493, 95% CI: 0.266-0.915). Increased risk of ESCC metastasis was indicated in MGMT for frequency of presence C allele [Rs7068306: 2.204 (1.244-3.906)], A allele [Rs10734088: 1.968 (1.111-3.484)] and C allele [Rs4751115: 2.178 (1.251-3.791)]. Two variants in frequency of presence C allele of CYP2A6 [Rs8192720: 0.290 (0.099-0.855)] and A allele of MGMT [Rs2053139: 0.511 (0.289-0.903)] were associated with a protective effect on ESCC progression. Increased risk of ESCC metastasis was found in heterozygote carriers of the MGMT Rs7068306 (CG vs CC, OR: 4.706, 95% CI: 1.872-11.833).

CONCLUSION: Polymorphic variation in ALDH2, XPD and MGMT genes may be of importance for ESCC susceptibility. Polymorphic variation in CYP2A6 and MGMT are associated with ESCC metastasis.

Esophageal cancer (EC) is one of the most common malignancies throughout the world, ranking the eighth in incidence and the sixth in mortality among tumors of all sites[1]. However, the incidence of EC varies extremely among different regions[1,2]. The high-risk areas for esophageal squamous cell carcinoma (ESCC) (with an incidence ranging from 15 to 150/100 000) included distinct areas of South America, and the so-called “Asian Esophageal Cancer Belt” from eastern Turkey, through Iraq, Iran and the southern former Soviet Union (Kazakhstan, Turkmenistan, Uzbekistan, Tajikistan) to Mongolia and Western/Northern China[3]. Residents in the Asian EC Belt have a 10-100 fold greater chance suffering from EC than those living in the neighboring areas. ESCC is the most frequent type of EC in Asia. More than half of all ESCC cases in the world occurred in China, in the southern parts of the Taihang mountains at the borders of Henan, Shanxi and Hebei Provinces (Lin xian/Linzhou and Anyang County in Henan, and Cixian in Hebei, which will be designated below as “Linxian area”), in northern Jiangsu (Huai’an county) and in northern Xinjiang (with age standardized rates of 90-150/100 000)[4,5].

Epidemiological studies revealed that the incidence of ESCC is associated with environmental factors, such as tobacco smoking, alcohol consumption, exposure to Nitrosamines, and nutritional deficiencies[6-8]. However, since only a fraction of individuals exposed to these risk factors actually develop EC, the role played by genetic determinants in response to environmental exposures needs to be addressed.

Data have also identified chronic alcohol consumption as a significant risk factor for alimentary tract cancer[9]. Ethanol is almost totally broken down by oxidative metabolism in vivo. Individuals who accumulate acetaldehyde due to polymorphic differences in the genes encoding the enzymes responsible for acetaldehyde generation and detoxification have been thought to show an ethanol associated carcinogenesis.

Ethanol is first metabolized into acetaldehyde through several enzymatic and nonenzymatic mechanisms, the main enzymatic pathways being alcohol dehydrogenase, cytochrome P450 (CYP) and catalase. Most of the acetaldehydes generated during alcohol metabolism in vivo are promptly eliminated by aldehyde dehydrogenase-2 (ALDH2), and alcohol dehydrogenase-1B (ADH1B, previously called ADH2)[10,11].

Cytochrome P450 is a Phase I enzyme responsible for activating most environmental pre-carcinogens, whereas glutathione S-transferases (GSTs) are Phase II enzymes capable of detoxifying the electrophile carcinogens that result from the action of CYP enzymes. CYP2A6, the main enzymes capable of activating nitrosamines and other carcinogens in humans, are expressed in esophageal mucosa of Brazilian patients, with a high degree of variation in expression[12]. The CYP2A6 gene is located on chromosome 19, and 30 different alleles have been described[13].

DNA repairing capacity (DRC) is an essential component in EC progression. Sufficient DNA repair activity ensures the stability and fidelity of the genome when exposed to carcinogens in the process of cell growth and differentiation[14], whereas instability in the genome in cancer patients may indicate a possible involvement of defective DRC[15]. DNA repair processes generally consist of direct reversal (DR), base excision repair, nucleotide excision repair (NER), or mismatch repair pathways[16]. Each pathway is specific for the repair of one or more types of DNA damage.

Key proteins in the transcription-coupled NER pathway (involved in correcting UV-induced lesions, chemical adducts, and crosslinks) include xeroderma pigmentosum group A and D (XPA and XPD), and excision repair cross complementing group 1 (ERCC1)[17,18].

O⁶-methylguanine DNA methyltransferase (MGMT) is a single protein responsible for the DR pathway. The major defense against alkylating mutations is from MGMT, a 207 amino acid DNA repair protein that transfers potentially carcinogenic O⁶ alkylation adducts from the DNA to a cysteine residue of MGMT[19,20]. For each adduct removed, an MGMT molecule is inactivated, hence the capacity for each cell to repair DNA depends upon the total number of MGMT molecules in the cell.

When genes essential for a variety of DNA repair pathways display aberrant activities, this may influence DRC with consequent carcinogenesis and progression. Several studies suggested that reduced expression of certain DNA repair genes were associated with the risk of environment-related esophageal adenocarcinoma[21-23]. There are few studies that examined the roles of gene polymorphism, metabolic enzyme and several DNA repair genes in risk of ESCC.

In the present study, Sequenom MassARRAY system was utilized to determine the relationship between ESCC and seven genes (ADHB1, ALDH2, ERCC1, MGMT, XPA, XPD, and CYP2A6) involved in two different DNA damage and repair pathways. The relationship between the polymorphism of candidate DNA repair genes and ESCC was determined in peripheral blood mononuclear cells from 128 patients with newly diagnosed, untreated ESCC and 326 healthy controls from the northern Xinjiang.

ESCC is one of the major health issues in China because of its high incidence and poor survival. Although the exact mechanism on EC is unclear, several possible mechanistic pathways have been proposed, including metabolic enzyme and DNA repair factors.

Alcohol consumption and tobacco smoking are established major risk factors for ESCC in Western populations[25,26]. However, studies in high-incidence regions are scarce and their results have been inconsistent[27-29].

In the present study, we first conducted a case-control study to evaluate the associations between metabolic enzyme polymorphisms of ADHB1, ALDH2 and CYP2A6 and ESCC risk in population from northern Xinjiang, China. Consistent with previous studies[30-32], the present study demonstrated that alcohol consumption and cigarette smoking are strongly associated with the incidence of ESCC. Moreover, individuals with ALDH2 for frequency of presence C allele of SNP in Rs886205 and heterozygote carriers of the ALDH2 in Rs886205 (CT vs TT) had significantly increased risk for ESCC carcinogenesis while individuals with frequency of presence C allele in CYP2A6 of Rs8192720 were inversely associated with ESCC metastasis.

Genomic fidelity and genetic stability usually depend on the efficiency of DRC when an organism is exposed to environmental and endogenous carcinogens. Peltomäki[33] showed that aberrant DRC might be involved in the pathogenesis of some cancers.

Previous molecular epidemiological studies have found that the XPD polymorphism is associated with increased risks for head and neck cancers[34]. However, inconsistent findings were also reported, including inverse associations with esophageal adenocarcinoma[35]. Our results suggested that XPD for frequency of presence C allele of SNP in Rs50872 increased the risk of ESCC carcinogenesis, which is consistent with the reported findings[36,37].

MGMT is a major gene in the pathway of DNA repair and frequently found to be silenced by CpG island hypermethylation in many cancers, such as gastric cancer[38] and esophageal adenocarcinoma[39]. However, the studies on the ESCC are still rare. Fang et al[40] reported that 29% (5 of 17) of normal esophageal tissues, 50% (10 of 20) of basal cell hyperplasia, 67% (8 of 12) of dysplasia, and 72% (13 of 18) of ESCC samples obtained from Linzhou City of Henan Province in northern China had DNA hypermethylation in MGMT promoter region. Until now, no investigation has been carried out about the associations between MGMT polymorphism and ESCC in a population of high incidence region of Northern Xinjiang. Our findings displayed that MGMT for frequency of presence A allele of SNP in Rs11016897, carriers of MGMT Rs11016879 (AA vs GG, AG vs GG), Rs12771882 (AA vs GG), and Rs7075748 (CT vs CC) significantly increased the risk of ESCC carcinogenesis. Our results are in agreement with the previous studies on EC[41-43].

Furthermore, increased risk of ESCC metastasis was indicated in MGMT for frequency of presence C allele in Rs7068306, A allele in Rs10734088 and C allele in Rs4751115, and carriers of MGMT Rs7068306 (CG vs CC).

In addition, variants of MGMT were associated with a protective effect on ESCC carcinogenesis, including C allele in Rs7069143, C allele in Rs3793909, A allele in Rs12771882, C allele in Rs551491, and A allele in Rs7071825, and the carriers of the MGMT in Rs11016878 (AG vs AA), Rs7069143 (CT vs CC) and Rs7071825 (GG vs AA). Frequency of C allele in Rs8192720 and A allele in Rs2053139 were associated with a protective effect on ESCC metastasis which was inconsistent with previous studies[44,45].

Difference in study population may be one of the reasons leading to the different results. However, since this is only a retrospective study and our findings could not be stratified because of the relatively small number in the groups, further epidemiological studies with enlarged samples are worth doing to warrant the results.

A limitation of our study may be missing covariate data. There have been many studies of individual environmental risk factors for EC[46-48]. We did not collect information on dietary factors and occupational history and so we were unable to study gene-environmental interactions in the etiology of ESCC. Gene-smoking and gene-alcohol interactions cannot be accurately measured due to sample size considerations. However, an improved understanding of the main separate effects of genes and environment will allow detailed gene-environmental interactions to be examined in larger-scale studies in the future. Our study population size is too small to avoid spurious results although they are suggestive, and further larger validation studies are certainly needed. The biological meaning and function of interesting SNPs need to be confirmed especially in advanced researches.

However, the strong points in our study are that both cases and healthy control subjects were recruited from the same area and matched on age, gender, ethnicity and residence, and all our control subjects were under Hardy-Weinberg equilibrium. Moreover, all our cases were pathologically confirmed, and followed by a strict quality control from genotyping.

To conclude, this study provides no evidence on a role of common variation of genes ERCC1, XPA and ADHB1 in ESCC. However, our data indicated that variation in the ALDH2, XPD and MGMT genes may be related to the risk of ESCC carcinogenesis. The polymorphic variation in CYP2A6 and MGMT are associated with ESCC metastasis.