Association of ALDH3B2 gene polymorphism and risk factors with susceptibility of esophageal squamous cell carcinoma in a Chinese population: a case-control study involving 2,358 subjects

Background Esophageal cancer (EC) is the sixth leading cause of cancer-associated death worldwide. The interaction of environmental risk factors and genetic factors might contribute to the carcinogenesis of EC synergistically. Results All seven single locus polymorphisms of ALDH3B2 were not associated with risk of ESCC as evaluated by allelic, dominant, co-dominant, recessive and Cochran-Armitage trend tests. Stratified analyses showed these SNPs were not correlated with the susceptibility of ESCC according to different age, gender, cigarette smoking and alcohol drinking status. None of the major haplotypes were related with ESCC susceptibility. Materials and Methods We conducted a hospital-based case–control study to evaluate the combined effects of environmental risk factors and the single nucleotide polymorphisms (SNPs) of ALDH3B2 gene on the development of esophageal squamous carcinoma (ESCC). A total of 1043 ESCC cases and 1315 controls were recruited for this study. Seven ALDH3B2 SNPs and four environmental factors were selected as independent variables. ALDH3B2 SNPs were determined by ligation detection reaction method. Conclusions Our study suggested that ALDH3B2 rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907 and rs9787887 polymorphisms were not implicated with altered susceptibility of ESCC according to different age, gender, cigarette smoking and alcohol drinking status. Yet this conclusion needs to be verified in larger studies among different ethnic populations with validation design, the biological function of these SNPs in carcinogenesis are subject to further investigation.


INTRODUCTION
Esophageal cancer ranks the ninth most common cancer and the sixth most common cause of cancer death worldwide [1]. Despite remarkable advances in the therapeutic strategy, extensive treatment may be associated with a noticeable decline in health-related quality of life and yet a poor prognosis [2]. Approximately 70% of global esophageal cancer cases occur in China, with esophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (> 90%) [3]. Alcohol consumption [4,5], tobacco use [4,6], poor oral hygiene, low socioeconomic status and nutritional deficiencies have been reported risk factors for esophageal cancer [7][8][9][10]. The fact that only a subset of cohort that are exposed to the risk factors eventually develop esophageal cancer suggested a critical role of genetic factors, including single nucleotide polymorphisms (SNPs), in the esophageal carcinogenesis.
Among all risk factors, others and we have repeatedly verified the indisputable role of alcohol consumption in the ESCC carcinogenesis [11,12]. Extensive evaluations and reviews of alcohol-related cancers have been published in the Monographs of the International Agency of Research on Cancer (IARC) [13,14], with most convincing evidence (Group I) for alcohol-drinking-related cancers is targeted on esophagus and some other organs [15]. Specific enzymes, whose activity and expression are influenced by genetic polymorphisms, regulate the metabolism of alcohol [11]. In humans, alcohol is primarily metabolized by two major groups of enzymes termed alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) [16]. In the cytosol of hepatocytes, ADHs catalyse the oxidation of ethanol to acetaldehyde, which is further oxidized to acetate by ALDHs in the mitochondria [11]. Although alcohol is not a carcinogen per se, its metabolite acetaldehyde is a toxin and carcinogen that rapidly binds to protein and DNA. It has profound effects on carcinogenesis by forming with DNA carcinogenic DNA adducts, by inhibiting DNA repair and by regulating DNA methylation. Acetaldehyde is degradated by ALDHs, which renders ALDHs a pivotal role in the carcinogenesis. Indeed, high ALDH1 expression predicts unfavorable outcomes in patients with ESCC [17]. Individuals with ALDH2 Lys allele possess a higher risk of esophageal cancer, in correlation with a higher concentration of blood acetaldehyde after drinking alcohol [18]. There is a strong association between ALDH2 Glu487Lys polymorphism and the risk of esophageal cancer [19]. ALDH2 rs671 [20] and rs886205 [21] polymorphisms have also been demonstrated to correlate with ESCC, respectively.
Similarly, the aldehyde dehydrogenase 3 family member B2 (ALDH3B2) is also a key member of ALDH family. Originally identified as ALDH8, ALDH3B2 encodes a member of the aldehyde dehydrogenase family, a group of isozymes that may play a major role in the detoxification of aldehydes generated by alcohol metabolism and lipid peroxidation. As compared with ALDH1/2, little is known on ALDH3 family (including ALDH3B2) with respect to their roles in carcinogenesis. The association between ALDH3B2 polymorphisms and ESCC has not been investigated. Hence, in this hospitalbased case-control study, we performed genotyping analyses of the seven SNPs in 1043 ESCC cases and 1315 controls in a Chinese population.

Characteristics of the study subjects
The characteristics of the study subjects, including the demographics and environmental risk factors, are presented in Table 1. The controls and cases were well matched in age and gender (χ 2 test, p = 0.121 and 0.880, respectively). However, the cigarette-smoking rate (43.53% vs.26.70%, p < 0.001) and alcohol drinking rate (31.54% vs.7.07%, p < 0.001) were both significantly higher in the ESCC cases.
As shown in Table 2, the genotyping successful rates were all beyond 98.81%. In the control subjects, the genotype frequencies for the seven polymorphisms reached Hardy-Weinberg equilibrium (p-value for HWE, all p > 0.05, Table 2).

Associations between risk of ESCC and seven polymorphisms
As shown in Table 3, the single locus analyses showed no statistically significant difference in genotype frequencies of seven SNPs between the cases and controls (p > 0.05). As assessed by the allelic, dominant, co-dominant, recessive and Cochran-Armitage trend tests, there are no correlations between these seven polymorphism sites with the risk of ESCC (Table 3).

Stratification analyses on seven polymorphisms and risk of ESCC
To further evaluate the effects of ALDH3B2 rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907 and rs9787887 on ESCC risk with different gender, age, smoking and alcohol drinking status, stratification analyses were performed as demonstrated in the Tables 4-10.
Our analyses demonstrated that neither gender, age, cigarette smoking nor alcohol drinking has detectable impacts on the susceptibility of ESCC after stratified analyses. www.impactjournals.com/oncotarget

Linkage disequilibrium analyses and association test
Linkage disequilibrium analyses in both controls and cases were performed as shown in Tables 11, 12 and Figures 1, 2. There were strong correlations between these seven loci. Association test was conducted using Haploview software (version 4.2), strong associations were detected between these seven loci.

Haplotype analyses of ALDH3B2 polymorphisms and susceptibility of ESCC
As demonstrated in Table  13, haplotype analyses showed that ALDH3B2 G rs34589365 G rs3741172 T rs4646823 G rs78402723 C rs7947978 G rs866907 A rs9787887 was most common haplotype in both groups (66.3% in controls, 64.6% in cases). As compared with G rs34589365 G rs3741172 T rs4646823 G rs78402723 C rs7947978 G rs866907 A rs9787887 , none of the haplotypes was associated with the susceptibility of ESCC.

Power calculation
The power calculation was performed using the "Power and Sample Size Calculation" software (http:// biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize). Based on the assumption that the type I error probability for a two sided test α equals 0.05, the probability of exposure in controls P 0 is 0.126 in control. In the current study, using ligation detection reaction method, the successful rate of genotyping exceeded 98%. There were in total 1315 controls and 1043 cases successfully genotyped. The ratio of control/case (m) equals 1.261, and the correlation coefficient for exposure between matched case and controls (f) is 0.619. The power value is 0.936 as calculated by the "Power and Sample Size Calculation" software.

DISCUSSION
In this hospital-based case-control epidemiological study, we investigated the association between tagging SNPs of ALDH3B2 and the risk of developing esophageal squamous cell carcinoma in a Chinese population. We found ALDH3B2 rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907 and rs9787887 polymorphisms were not implicated with altered susceptibility of ESCC according to age, gender, cigarette smoking and alcohol drinking stratification analyses.
Despite a suspected association between alcohol drinking and death to cancer reported in an epidemiological study as early as 1903, it took until 1988 for the research community to agree on the potential risk through the International Agency for Research on Cancer (IARC). Clear Patterns have emerged between alcohol consumption and esophageal cancer. Essentially, alcohol and its metabolite acetaldehyde were both designated as type 1A carcinogen [13]. The cytotoxic properties, the ability to form DNA-acetaldehyde adducts and to generate additional mutagenic species at concentrations attainable in vivo may underlie the carcinogenic effects [22]. Most of the acetaldehyde generated during alcohol metabolism in vivo is rapidly eliminated by aldehyde dehydrogenase (ALDH), which renders ALDH an important role in carcinogen balancing and therefore carcinogenesis. In fact, there is ample evidence showed that subjects with an inactive form of ALDH2 (heterozygous for ALDH2 mutation) have an increased risk of developing various types of head and neck cancers as a consequent of intense exposure to acetaldehyde. Case-control studies of various Japanese drinking populations [23][24][25][26][27][28][29] and Chinese  alcoholics [30] have consistently reported that the inactive ALDH2 encoded by the ALDH2*1/2*2 genotype is a strong risk factor for esophageal cancer. In resemblance with ALDH2, ALDH3 also plays a pivotal role in the alcohol metabolism, we thus hypothesize that ALDH3 family may be of potential relevance to carcinogenesis.  In line with our speculation, overexpression of ALDH3 protects cells from 4-hydroxynonenal induced apoptosis, suggesting a functional relevance of ALDH3 in the carcinogenesis. On the other hand, many of the sphere cells and stem cells reported in different organs have recently been found to be associated with elevated ALDH1A1 enzyme activity [31][32][33]. ALDH1A1 expression or activity may be used with other cell surface markers to identify tumor-initiating cells in hepatocellular, prostate and breast solid carcinomas [34][35][36]. ALDH1A1 has also been detected to be associated with early metastasis and poor clinical outcome [33]. In addition, the ALDH enzymes also play a pivotal role in epithelial homeostasis. Deregulation of these enzymes is associated with multiple cancers [37-  rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907 and rs9787887 were not associated with the susceptibility of ESCC in the current study, the cigarette smoking rate and alcohol drinking rate were significantly higher in the ESCC cases, exemplifying the significance of interaction between the environmental and genetic risk factors in causing esophageal squamous carcinoma.
Our finding that there are more male than female subjects in the case group was in consistent with the comprehensive data recently published by the National Office for Cancer Prevention and Control as well as the National Cancer Center of China. The fact that smoking and alcohol drinking are far more prevalent in male subjects implicated the importance of these risk factors in carcinogenesis of ESCC.
One of the limitations of our previous studies investigating the association between SNPs and risk of  Haplotypes were composited by ALDH3B2 rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907, rs9787887. All those frequency < 3% were ignored in analysis, most common haplotype ALDH3B2G rs34589365 G rs3741172 T rs4646823 G rs78402723 C rs7947978 G rs866907 A rs9787887 was selected as reference. www.impactjournals.com/oncotarget ESCC was the sample size [12]. To overcome that, we have recruited a total of 2358 subjects including 1043 ESCC cases and 1315 controls in the current study. Yet, we do acknowledge there are still some limitations in this study. First, this study is limited by the sample sources, future studies designed and conduced in multiple ethnical populations and various geographic locations would be more convincing. Second, the lack of a validation cohort compromised the power of our study. Third, we are refrained by the lack of technical support to establish single nucleotide mutation cell/animal model, the actual biological function of these SNPs in esophageal carcinoma remains obscure, the underlying mechanisms are yet to be further dissected. Last but not least, the detailed information with regard to cancer metastasis and survival were not provided, the correlations between SNPs and outcomes have not been analyzed as this follow-up is still ongoing.

Ethics statement
This hospital-based case-control study was approved by the Review Board of Jiangsu University (Zhenjiang, China). All subjects provided written informed consents. This study has complied with the World Medical Association Declaration of Helsinki with regard to ethical

Study populations
A total of subjects consisting of 1043 ESCC cases and 1315 non-cancer controls frequency-matched to the cases regarding age and gender (p = 0.121 and 0.880, respectively) were enrolled in this study (Table 1). All patients and controls were consecutively recruited from the Affiliated People's Hospital of Jiangsu University (Zhenjiang, China) from October 2008 to January 2017. All cases of esophageal cancer were diagnosed as ESCC histologically. The exclusion criteria included cancer history, metastasized cancer or chemotherapy/radiotherapy history.
Each subject was individually questioned by experienced interviewers with a questionnaire to obtain information on demographic information and related risk factors (including alcohol consumption and cigarette smoking). After written informed consent was provided, two milliliters of venous blood were collected from each subject. The "Smokers" cohort included individuals who smoked one cigarette per day for more than one year. Subjects who had more than three alcoholic drinks a week for more than six months were included in the "Alcohol drinkers" cohort.

Genomic DNA extraction, SNP selection and genotyping
Genomic DNA was isolated from peripheral blood using QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany) as reported [12]. Sample DNA were amplified by PCR according to the manufacturer's protocol. Gene polymorphisms were analyzed by the ligation detection reaction (LDR) method with technical support from Genesky Biotechnology Inc. (Shanghai, China). 10% of the total samples were randomly selected for repeated analyses for quality control. Pilot linkage disequilibrium analyses were performed in the Chinese Han population to choose the SNP loci with moderate correlation, and tag SNPs were selected for further analyses.

Statistical analyses
Statistical analyses were conducted using SPSS 23.0 statistical package (SPCC Inc., Chicago, IL). Hardy-Weinberg equilibrium for genotypes was tested by goodness-of-fit χ 2 in control group. Variations of demographic characteristics and genotypes of the ALDH3B2 rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907 and rs9787887 between the controls and cases were evaluated using the chi-square (χ 2 ) test to examine the statistical differences. The associations between these seven SNPs and risk of ESCC were analyzed by PLINK software (v1.07, available at http://zzz.bwh.harvard.edu/plink/download. shtml). Crude ORs and adjusted ORs when adjusting for age, sex, smoking and alcohol drinking status were also computed using logistic regression analyses. Bilateral probability tests were taken, p value < 0.05 was considered statistically significant.

CONCLUSIONS
The esophageal squamous carcinoma is associated with various factors including gene, environment and life-style. Our findings that ALDH3B2 rs34589365, rs3741172, rs4646823, rs78402723, rs7947978, rs866907 and rs9787887 polymorphisms were not implicated with altered susceptibility of ESCC in different age, gender, cigarette smoking and alcohol drinking status, when interpreted with caution, could be helpful in evaluating the susceptibility to ESCC.