| BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most common human malignancies which caused the sixth highest mortality rate in the world. Global epidemiology results suggest that the distribution of ESCC has obvious regional and national differences. Higher prevalence can be presented in such as China, South Africa, Saudi Arabia. There is a 500-fold difference in higher and lower incidence areas. In the north of China, the incidence of ESCC was significantly higher than other areas, such as Henan province, the junction area of Shandong and Hebei province, especially Anyang of Henan. Approximately,32.22 per 100,000 people died because of ESCC in Anyang, which is 30 times more than Yunnan, one typically lower risk area.Multi-environmental factors, multiple gene variants and multi-stages involved in the pathogenesis of ESCC. Drinking, smoking, inadequate nutritional intake, environmental and genetic factors were associated with the occurrence of ESCC. The relationship between the incidence of ESCC and genetic variation is one of the focus in studies recently.Under long time of certain environment, individuals can form corresponding point mutation due to long-term options. This is one of important manifestations of individual genetic variation. Single nucleotide polymorphism (SNP) is exactly such genetic variation which reflects the level of one single base mutation. The regional and national distribution of SNP differences may explain the reason why human beings suffer from different susceptibility, severity and treatment effect to the same disease. In recent years, significant progress has been achieved in functional SNP of complex diseases and drug effect associated SNP. Furthermore, the important role of SNP in functional disorders has also gradually been revealed.DNA double strand breaks (DSB) including loss of coding genes, chromosome deletions, rearrangements, restructuring and so on is a serious injury form of DNA,which could lead to serious consequence of the cells, induce cell dysfunction, even cell death or turn to tumor. Non-homologous end joining (NHEJ) and homologous recombination (HR) are two mainly ways to repair the DSB damage. PRKDC and XRCC4 are two important genes involved in NHEJ pathway, In this tudy, we hypothesized that those two gene polymorphisms may be associated with the occurrence of ESCC.ObjectiveIn the present study, environmental and genetic backgrounds were matched in those two groups. In order to discuss whether the occurrence of ESCC was related to XRCC4 and PRKDC polymorphisms, based on candidate gene approach, we used high-throughput DNA sequencing and PCR-RFLP technique to detect the SNP genotypes of XRCC4 and PRKDC. Besides, we aimed to provide important theoretical basis to screen high risk individuals, implement targeted interventions and reduce the incidence of ESCC ultimately.MethodsA case-control study was conducted in Anyang Tumor Hospital of Henan province. A total of 189 ESCC patients with mean age 60.23±7.91 were recruited. All cases were confirmed by endoscopy or surgical excision biopsy histopathology diagnosed with ESCC. All samples were collected preoperatively and without radiotherapy or chemotherapy.189 cases of healthy control subjects were collected with mean age 59.40±7.86. They were long-term residents of Han population with no history of autoimmune disease, cancer and other diseases in Anyang. All subjects were agreed to participate in the study voluntary.Demographic data, smoking history, alcohol consumption and family history of all the subjects were queried and noted by specialized researchers.5ml of venous blood samples were collected into anticoagulant tubes at the following recruited next morning. Samples were placed in -20℃ refrigerator waiting to extract DNA which following the experimental procedure strictly. By browsing NCBI (http://www.ncbi.nhn.nih.gov/snp), we got the complete genome sequence of PRKDC and XRCC4 gene in NHEJ repair pathway. According to related papers and Haploview software, we selected rs7003908 in PRKDC gene and rs1805377 in XRCC4 gene to study. Minor allele frequencies of both SNPs we selected reached greater than 10%. RFLP and DNA sequencing were used to genotype two SNP of all the subjects.Selecting 10 samples recognized genotypes using RFLP to perform DNA sequencing. If the results confirm the RFLP results, we could accept the reliability of RFLP. Data were expressed as mean±standard deviation, frequency, relative frequency and percentage. Statistical software STATA.11 was used to conduct chi-square test to verify whether the genotype frequencies met the Hardy-Weinberg equilibrium. If the frequencies met the equilibrium, the data would be included in the analysis. Statistical software SPSS 19.0 was used to perform independent samples t-test to analyze the age homogeneity of variance of the two groups. Univariate conditional logistic regression analysis was used to detect the distribution of rs1805377 (XRCC4), rs7003908 (PRKDC) on smoking history, drinking history, family history of esophageal in the case and control groups matched with gender and age. When one variable showed statistically significant, then the variable would be brought in multivariate logistic regression analysis. The odds ratio (OR) was the calculation symbol of regression analysis and generally taken its 95%confidence interval (CI). The larger values in this interval, the stronger correlation between SNP and ESCC risk. Related experiments performing statistical tests were carried out on both sides probability test. P<0.05 indicated statistical significance, P<0.01 indicated a significant difference.Results1. The univariate conditional logistic regression suggested that 87 smokers in the cases group accounted for 46.0% while the number was 72 (38.1%) in the control group (P<0.05). After the correction by age and gender, OR=1.882,95%CI= (1.045,3.390).2.54 patients of ESCC had drinking habit (28.6%), which was significantly higher than 39 (20.6%) in control group (P<0.05). After correction by age and gender, OR= 1.75,95%CI=(1.010,3.031).3.68 patients of ESCC had family history of esophageal cancer (36.0%), which was significantly higher than 41 (21.7%) in control group (P<0.05). After correction by age and gender, OR= 1.929,95%CI= (1.222,3.044).4. Distributions of rs1805377 in XRCC4 and rs7003908 in PRKDC gene both met the Hardy-Weinberg equilibrium (P> 0.05).5. Rs1805377 in XRCC4 gene could be genotyped into wild-type genotype AA, wild-type genotype AG and GG. Compared with A A, AG genotype and G allele carriers (AG and GG) did not affect the risk of ESCC. After correction by smoking, drinking, family history and other confounding factors, ORsmoking=0.933 (95% CI= 0.620,1.406), ORdrinking=0.818 (95%CI=0.392,1.707) and ORfamily history=0.878 (95% CI=0.585,1.319). These results showed no significant difference in genotypes frequency between two groups.6. Rs7003908 in PRKDC gene could be genotyped into wild-type genotype AA, wild-type genotype AC and CC. Frequency of CC genotype in the case group was significantly higher than control (P=0.013). After correction by smoking, drinking, family history and other confounding factors, OR=3.637,95% CI= (1.313,10.072). There was no interaction (P>0.05) among smoking, alcohol consumption and genetic polymorphisms.Conclusions1. Respectively, smoking, drinking and family history of esophageal cancer could act as independent risk factors that significantly increased the possibility risk of ESCC.2. Rs1805377 in XRCC4 and rs7003908 in PRKDC were found in Han population of Anyang.3. The genotypes frequency of rs1805377 in XRCC4 and rs7003908 in PRKDC were in line with Hardy-Weinberg equilibrium law (P> 0.05).4. XRCC4 gene polymorphism rs1805377 had no significant correlation with ESCC.5. CC genotype of rs7003908 in PRKDC gene might cause 3.64 times higher risk for ESCC. |
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