B-cell Lymphoma 2 rs 17757541 C > G Polymorphism was Associated with an Increased Risk of Gastric Cardiac Adenocarcinoma in a Chinese Population

Apoptosis, also called programmed cell death, is a major control mechanism by which cells die if DNA damage is not repaired (Lowe et al., 2000). Apoptosis has been considered as a fundamental component in cancer pathogenesis (Alenzi et al., 2010), key genes related to apoptosis include B-cell lymphoma 2 (BCL2) (Kroemer et al., 1997), FAS, FAS ligand (FASL)/FASLG (Nagata et al., 1999), erythroblastic leukemia viral oncogene homolog 2 (ERBB2) (Yu et al., 1998), vascular endothelial growth factor receptor 2 (VEGFR2)/KDR (Kasahara et al., 2000) and other genes (Nagata et al., 1997; Elmore et al., 2007). BCL2 is initially identified as an antiapoptotic regulatory protein (Cory et al., 2002). However, it also serves as an inhibitor of proliferation (Zinkel et al., 2006). The FASL-FAS system mainly forms the death-inducing signaling complex (Holler et al., 2003). ERBB2 (HER2/ Neu) is a member of the ERBB family of transmembrane receptor tyrosine kinases associated with apoptosis,

Considering the biological and pathologic significance of BCL2, FAS, FASL/FASLG, ERBB2 and VEGFR2/KDR, it is possible that functional genetic variations in these genes may contribute to the development of cancers such as the gastric cardiac adenocarcinoma (GCA).The objective of this investigation was to evaluate the association between functional polymorphisms BCL2 rs17757541 C>G, BCL2 rs12454712 T>C, FAS rs2234767 G>A, FASL/FASLG rs763110 C>T, ERBB2 rs1136201 A>G and VEGFR2/KDR rs11941492 C>T variants and GCA susceptibility in a hospital-based case-control study.For this purpose we here performed genotyping analyses for these six single nucleotide polymorphisms (SNPs) using a smaple of 243 GCA cases and 476 controls in a Chinese population.

Ethical approval of the study protocol
This hospital-based case-control study was approved by the Review Board of Jiangsu University (Zhenjiang, China).We have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals.All subjects provided written informed consent to be included in the study.

Study subjects
Two-hundred and forty three subjects with gastric cardiac cancer were consecutively recruited from the Affiliated People's Hospital of Jiangsu University and Affiliated Hospital of Jiangsu University (Zhenjiang, China) between October 2008 and July 2010.All cases of gastric cardiac cancer were diagnosed as GCA by pathologic means.The exclusion criteria were patients who previously had: cancer; any metastasized cancer; radiotherapy or chemotherapy.Among 476 controls, 380 controls were patients without cancer frequency-matched to the cases with regard to age (±5 years) and sex recruited from the two hospitals mentioned above during the same time period.Another 96 controls were recruited from hospitals in Changzhou city (which is a neighbouring city of Zhenjiang) as previous described (Cheng et al., 2012).

Most of the controls were admitted to the hospitals for the treatment of trauma.
Each subject was personally questioned by trained interviewers using a pre-tested questionnaire to obtain information on demographic data (e.g., age, sex) and related risk factors (including tobacco smoking and alcohol consumption).After the interview, 2-mL samples of venous blood were collected from each subject.
Individuals who smoked one cigarette per day for >1 year were defined as "smokers".Subjects who consumed ≥3 alcoholic drinks a week for >6 months were considered to be "alcohol drinkers".

Isolation of DNA and genotyping by a custom-by-design 48-Plex SNPscanTM Kit
Blood samples were collected from patients using Vacutainers and transferred to tubes lined with ethylenediamine tetra-acetic acid (EDTA).Genomic DNA was isolated from whole blood with the QIAamp DNA Blood Mini Kit (Qiagen, Germany).Sample DNA (10 ng) were amplified by PCR according to the manufacturer's recommendations.The SNP genotyping work was performed using a custom-by-design 48-Plex SNPscanTM Kit (Genesky Biotechnologies Inc., Shanghai, China) as previously described (Sun et al., 2013;Wei et al., 2013;Yin et al., 2013;Zheng et al., 2013) (Figure 1).This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies Inc., which was based on double ligation and multiplex fluorescence PCR.For quality control, repeated analyses were done for 4% of randomly selected samples with high DNA quality.

Statistical Analyses
Differences in the distributions of demographic characteristics, selected variables, and genotypes of the BCL2 rs17757541 C>G, BCL2 rs12454712 T>C, FAS rs2234767 G>A, FASL/FASLG rs763110 C>T, ERBB2 rs1136201 A>G and VEGFR2/KDR rs11941492 C>T variants between the cases and controls were evaluated using the χ 2 test.
The associations between BCL2 rs17757541 C>G, BCL2 rs12454712 T>C, FAS rs2234767 G>A, FASL/ FASLG rs763110 C>T, ERBB2 rs1136201 A>G and VEGFR2/KDR rs11941492 C>T genotypes and risk of GCA were estimated by computing the ORs and their 95% CIs using logistic regression analyses for crude ORs and adjusted ORs when adjusting for age, sex, smoking

Characteristics of the study population
Characteristics of cases and controls included in the study are summarized in Table 1.The cases and controls appeared to be adequately matched on age and sex as suggested by the χ 2 tests (P = 0.923 and P = 0.197, respectively).As shown in Table 1, no significant difference was detected on drinking status between the cases and the controls (P = 0.217), but smoking rate was higher in GCA patients than in control subjects (P = 0.004).The primary information for six genotyped SNPs was in Table 2.For the six SNPs, the genotyping was successful ranging from 96.94% to 97.50% in all 719 samples.The concordance rates of repeated analyses were 100%.Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database for all six SNPs (Table 2).The observed genotype frequencies for these six polymorphisms in the controls were all in HWE (Table 2).

Associations between six polymorphisms and risk of GCA
The genotype distributions of BCL2 rs17757541 C>G, BCL2 rs12454712 T>C, FAS rs2234767 G>A, FASL/FASLG rs763110 C>T, ERBB2 rs1136201 A>G and VEGFR2/KDR rs11941492 C>T in the cases and the controls are shown in Table 3.In the single locus analyses, the genotype frequencies of BCL2 rs17757541 C>G were 65.11% (CC), 32.34% (CG), and 2.55% (GG) in the case patients and 72.69% (CC), 25.38% (CG), and 1.94% (GG) in the control subjects, and the difference was not statistically significant (P = 0.117).When the BCL2 rs17757541 CC homozygote genotype was used as the reference group, the CG genotype was associated with a significantly increased risk for GCA (CG vs. CC: OR 1.42, 95% CI 1.01-2.01,P = 0.046).When the BCL2 rs17757541 CC homozygote genotype was used as the reference group, the GG genotype was not associated with the risk for GCA (GG vs. CC: OR 1.47, 95% CI 0.52-4.21,P = 0.470).In the recessive model, when the BCL2 rs17757541 CC/CG genotypes were used as the reference group, the GG homozygote genotype was not associated with the risk for GCA (OR 1.33, 95% CI 0.47-3.78,P = 0.595).In the dominant model, the BCL2 rs17757541 CG/ GG variants were associated with a significantly increased risk of GCA, compared with the BCL2 rs17757541 CC genotype (CG/GG vs. CC: OR 1.43, 95% CI 1.02-2.00,P = 0.039) (Table 3).After adjusting for age, sex, smoking and drinking, a borderline statistically increased risk of GCA was observed in the heterozygote comparing model (CG vs. CC: adjusted OR 1.39, 95% CI 0.98-1.98,P = 0.063) and dominant model (CG/GG vs. CC: OR 1.40, 95% CI 0.99-1.96,P = 0.054) (Table 3).
None of the BCL2 rs12454712 T>C, FAS rs2234767 G>A, FASL/FASLG rs763110 C>T, ERBB2 rs1136201 A>G and VEGFR2/KDR rs11941492 C>T polymorphisms achieved a significant difference in the genotype distributions between cases and controls (Table 3).Logistic regression analyses revealed that the five polymorphisms were not associated with the risk of GCA (Table 3).

Stratification analyses of BCL2 rs17757541 C>G polymorphisms and risk of GCA
To evaluate the effects of BCL2 rs17757541 C>G genotypes on GCA risk according to different age, sex, smoking and alcohol drinking status; we performed the stratification analyses.A significantly increased risk of GCA associated with the BCL2 rs17757541 C>G polymorphism was evident among male patients, older patients and patients who smoking or drinking (Table 4).

Table 1 . Distribution of Selected Demographic Variables and Risk Factors in Gastric Cardiac Adenocarcinoma (GCA) Cases and Controls
a Two-sided χ 2 test and student t test; Bold values are statistically significant (P <0.05) Figure 1.

Table 2 . Primary Information for Six Genotyped SNPs
c MAF: minor allele frequency; d HWE: Hardy-Weinberg equilibrium