Replication Study of ESCC Susceptibility Genetic Polymorphisms Locating in the ADH1B-ADH1C-ADH7 Cluster Identified by GWAS

China was one of the countries with highest esophageal squamous cell carcinoma (ESCC) incidence and mortality worldwide. Alcohol drinking has been identified as a major environmental risk-factor related to ESCC. The alcohol dehydrogenase (ADH) family are major enzymes involved in the alcohol-metabolizing pathways, including alcohol dehydrogenase 1B (ADH1B) and ADH1C. Interestingly, ADH1B and ADH1C genes locate tandemly with ADH7 in a genomic segment as a gene cluster, and are all polymorphic. Several ESCC susceptibility single nucleotide polymorphisms (SNPs) of the ADH1B-ADH1C-ADH7 cluster have been identified previously through a genome-wide association study (GWAS). In the study, we examined the association between five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) and risk of developing ESCC. Genotypes were determined in two independent case-control sets from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Our data demonstrated that these ADH1B-ADH1C-ADH7 cluster SNPs confer susceptibility to ESCC in these two case-control sets, which were consistent to results of the previous GWAS.

Alcohol drinking has been identified as a major environmental risk-factor related to ESCC [2,3]. Ethanol is metabolized in vivo by alcohol dehydrogenase (ADH) family and aldehyde dehydrogenase (ALDH), which are all polymorphic in human beings [18][19]. The total activity of ADH is significantly higher in cancer tissue than in healthy mucosa [18]. The ALDH and ADH polymorphisms influence individual diversity in alcohol-oxidizing capability and drinking behavior [19]. Among the ADH family, the major enzymes involved in the alcohol-metabolizing pathways are alcohol dehydrogenase 1B (ADH1B) and ADH1C. ADH1B and ADH1C exist as several homo-and heterodimers of ADH1A subunits, exhibit high activity for ethanol oxidation and play an essential role in ethanol catabolism. ADH7 is also a member of the ADH family. Although less efficient in ethanol oxidation compared to ADH1B or ADH1C, ADH7 is the most active as a retinol dehydrogenase. Therefore, ADH7 may take part in the synthesis of retinoic acid, a hormone important for cellular differentiation. Interestingly, the aforementioned three genes locate tandemly in a genomic segment as a gene cluster. Wu et al. identified several new ESCC susceptible SNPs, including ADH1B rs1042026 and rs17033, ADH1C rs1614972 and rs1789903 as well as ADH7 rs17028973 through a GWAS based on analyses of in 2031 ESCC cases and 2044 controls with independent validation in 8092 ESCC cases and 8620 controls [4]. Considering the importance of ADH1B-ADH1C-ADH7 cluster in ESCC, we conducted this replication case-control study to validate the association between ADH1B rs1042026 and rs17033, ADH1C rs1614972 and rs1789903 as well as ADH7 rs17028973 SNPs and ESCC risk.

Study subjects
This study consisted of two case-control sets: (a) Hangzhou set: 617 patients with ESCC from Cancer Research Institute, Zhejiang Cancer Hospital (Hangzhou, Zhejiang Province, China) and sexand age-matched (65 years) 537 controls. Patients were recruited between January 2012 and March 2013 at Zhejiang Cancer Hospital. Control subjects were individuals who underwent a physical examination in the same hospital during the same time period as the patients were collected. (b) Jinan study: 540 patients with ESCC from Shandong Cancer Hospital, Shandong Academy of Medical Sciences (Jinan, Shandong Province, China) and sexand age-matched (65 years) 550 controls. Patients were recruited between June 2009 and April 2012 at Shandong Cancer Hospital. Control subjects were randomly selected from a pool of 4500 individuals from a community cancer-screening program for early detection of cancer conducted in Jinan city during the same time period as the patients were collected. The diagnosis of all patients was histologically confirmed. Individuals who smoked one cigarette per day for over 1 year were considered as smokers. Subjects were considered as alcohol drinkers, if they drank at least once per week. All subjects were ethnic Han Chinese. At recruitment, the written informed consent was obtained from each subject and each participant was then interviewed to collect detailed information on demographic characteristics, such as sex and age, and related risk factors, such as cigarette smoking, and alcohol drinking. This study was approved by the Review Board of Zhejiang Cancer Hospital and the Review Board of Shandong Cancer Hospital.

SNP selection and genotyping
A total of five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) were included in the current study. These SNPs are ones identified by a previous ESCC GWAS in Chinese Han population [4]. All ADH1B-ADH1C-ADH7 cluster SNPs were analyzed by the MassArray system (Sequenom Inc., San Diego, California, USA). A 15% blind, random sample of study subjects was genotyped in duplicates and the reproducibility was 100%.

Statistical analyses
Pearson's x 2 test was used to examine the differences in demographic variables and genotype distributions of five ADH1B-ADH1C-ADH7 cluster SNPs between patients and controls. The associations between genotypes of these SNPs and ESCC risk were estimated by ORs and their 95% CIs computed by logistic regression models. All ORs were adjusted for age, sex, smoking or drinking status, where it was appropriate. We tested the null hypotheses of multiplicative gene-environment interaction and evaluated departures from multiplicative interaction models by including main effect variables and their product terms in the logistic regression model [4,[20][21][22][23]. A P value of less than 0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All analyses were performed using Statistical Analysis System (version 9.0; SAS Institute) and SPSS 16.0 (SPSS Inc.).
The ESCC risk associated with the ADH1B-ADH1C-ADH7 cluster SNPs was further examined by stratifying for smoking  status and alcohol drinking history due to the key role of these enzymes in metabolism of ethanol and other toxics in Jinan casecontrol set (Table 4 and 5). Interestingly, we found higher odds of those five polymorphisms for developing ESCC among smokers than those among non-smokers (Table 4 and 5). Similar results were also observed among alcohol drinkers except ADH1B rs17033 genetic variant (Table 4 and 5). However, no evident gene-smoking interaction or gene-drinking interaction exists in Jinan case-control set (Table 4 and 5). We also examined whether there are gene-environment interaction between five ADH1B-ADH1C-ADH7 cluster genetic variants and age and sex, but the results were negative (data not shown).

Discussion
In the current study, we examined the association between five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) and risk of developing ESCC in a two-stage case-control design. In this replication study, we successfully validated results of a previous GWAS that these five SNPs confer susceptibility to ESCC [4]. However, no significant gene-smoking interaction or gene-drinking interaction between these ADH1B-ADH1C-ADH7 polymorphisms and ESCC was observed in this Chinese Han population.
Several molecular epidemiological studies using a candidate gene approach indicated a set of SNPs associated with ESCC susceptibility, primarily variations that are related to alcohol metabolism [24][25][26][27][28][29][30]. As a powerful and successful tool to identify common disease alleles, GWAS could interrogate a large amount of tagging SNPs that serve as surrogates for untested common SNPs across the genome. In published GWAS of cancers of the upper aerodigestive tract, including ESCC in individuals of European [28,30], Japanese [9] and Chinese [4], have shown that SNPs in the ADH genes contribute to susceptibility of ESCC. Our results in this study are consistent to these reports and highlight the importance of genetic variants of the ADH genes in ESCC development.
There might be several limitations in the current case-control study. First, because it was a hospital-based study and the cases were from the hospital, inherent selection bias may exist. Thus, it is important to validate these findings in a population-based prospective study from the same geographic regions. Second, the statistical power of our study may be limited by the sample size, especially for statistical analyses of gene-covariate interaction. Third, future studies will need to address the biological function of these polymorphisms in the genesis of ESCC.