| Gastric cancer is one of the most common malignant tumors of the word. The age-standardized morbidity and mortality of gastric cancer in China occupy the second and third position respectively in all malignant tumors. It threatens the human’s life and health seriously. The tumorigenesis of gastric cancer is a complex, multistep and multifactorial process with interaction between the external environment factors and inherent genetic factors. Single Nucleotide Polymorphisms(SNPs) is the main form of genetic differences between individuals. Therefore, exploring the association between SNPs in the genome and gastric cancer susceptibility, clarifing the mechanisms of genetic susceptibility to gastric cancer, screening high-risk populations and taking corresponding preventive measures is one of the effective strategies to control the high incidence of gastric cancer.Genomic instability is one of the important characteristics of the tumor. Our genetic material DNA is continually threatened by exogenous damage factors like chemicals,ionizing radiation as well as endogenous damage factors including metabolic products and reactive oxygen species.The destroyed chemical structure of DNA may frequently change the high fidelity transmission of genetic information transfer process, destroy the stability of the genome, and eventually lead to the occurrence of cancer or precancerous lesions.Within all kinds of DNA damage, DNA double strand breaks(DSBs) is the most serious consequence as it can trigger cell permanently stunted growth or cell death. DNA repair is a series cellular responses to recover normal DNA sequence structure and maintain a relatively stable genetic information. If DSBs can be well repaired, cell will get through the cell cycle checkpoint and restart normal activities, on the contrary, the efficient DSBs repair will result in apoptosis, genetic diseases and even tumorigenesis. Many of the gene products are involved in DSBs repair. As a key encoding protein gene of MRN(MRE11-RAD50-NBN) complex, NBN plays a crucial role in maintaining genomic stability and preventing cell apoptosis, inflammation and tumorgenesis. Genetic variation of the NBNgene may predispose to Nijmegen Breakage Syndrome(NBS). NBS is an autosomal recessive disorder characterized by microcephaly, immunodeficiency, spontaneous chromosomal instability, sensitivity to ionizing radiations and a high incidence of malignancy. Beside classical miscoding mutations associated with either clinical signs of the NBS disorder or a functionally altered NBN protein, a certain number of common genetic variants in NBN may also bring to subtle structural alterations of proteins and consequently modulation of cancer susceptibility.In this study, we hypothesized that the genetic variants in NBN may play an important role in the development of gastric cancer.Objective To investigate the association between the potentially functional single nucleotide polymorphisms(SNPs) in the 3’-untranslated region of NBN gene with gastric cancer risk.Methods 1. We conducted a case-control study with 1,140 gastric cancer cases and1,547 cancer-free controls. Cases were recruited from hospitals in Jiangsu province, and controls were randomly selected from those who participated in a community-based screening program of non-infectious diseases, and were frequency-matched to cases based on age and sex.2. The SNPs were selected from the NCBI database(http://www.ncbi.nlm.nih.gov/projects/SNP) and the International Hap Map Project database(http://hapmap.ncbi.nlm.nih.gov/) based on the following two criteria: 1) located in the exons, 3’ or5’untranslated region of NBN gene, and 2) with a minor allele frequency(MAF) of at least10% in CHB population. According to above criteria, a total of 10 SNPs were selected.Linkage disequilibrium(LD) analysis with an r2 threshold of 0.80 was further applied to filter these SNPs. As a result, 4 SNPs were included in our following analysis, including rs10464867(G>A), rs14448(A>G), rs1063053(G>A) and rs1063045(G>A).3. We genotyped NBN gene SNPs rs10464867, rs14448, rs1063053 and rs1063045 using Taq Man allelic discrimination assay and analyzed the association between the SNPs and gastric cancer risk. The chi-square test was used to evaluate distribution differences of demographic characteristics, selected variables and genotypes between cases and controls.Logistic regression analyses were employed to test the association between genetic variants and gastric cancer risk, to evaluate odds ratios(ORs) and 95% confidence intervals(CIs) with an adjustment for age, sex, smoking and drinking status. To examine the differences between subgroups, we calculated Cochran’s Q statistic to test the heterogeneity of effect sizes(ORs and 95% CIs) derived from corresponding subgroups.The interaction analysis between genotypes and environment were also performed. We further conducted a haplotype analysis to assess the associations between the haplotype created by these four SNPs and gastric cancer susceptibility. Haplotypes were created by a Confidence Intervals approach, which is the default algorithm in Haplo View 4.2 software,95% confidence bounds on D prime were generated and each comparison was called "strong LD", "inconclusive" or "strong recombination". A block was created if 95% of informative comparisons were "strong LD". As a result, one block was created and analyzed in current study. Haplotypes were obtained for each sample using the PHASE computer program, and the haplotype frequencies were estimated via permutation methods.4. The potentially biological function was predicted and analysed with Regulome DB,University of California Santa Cruz(UCSC), Haplo Reg and The Genotype Tissue Expression(GTEx) database. We also performed differential expressed analysis of NBN m RNA expression level with RNA-Seq data of paired gastric adenocarcinoma tissues from The Cancer Genome Atlas(TCGA).Results 1. Logistic regression analysis revealed that the A allele of rs10464867 were strongly associated with a decreased risk of gastric cancer(per-allele OR = 0.81, 95% CI:0.71–0.94, P = 5.00×10-3). No significant correlations between other three polymorphism variants(rs14448, rs1063045, rs1063053) and gastric cancer risk were found.2. In the stratification analysis, we further evaluated the associations of the four SNPs on gastric cancer risk in subgroups based on age, gender, smoking, drinking status and tumor sites. A significant difference between subgroups of gender were observed for the association of rs10464867 and rs14448 with gastric cancer risk(P for heterogeneity =0.041 for rs10464867 and rs14448). The association between A allele of rs10464867 and decreased risk of gastric cancer was more significantly in elder individuals(per-allele OR= 0.72[0.59-0.88], P = 1.00×10-3), and male individuals(per-allele OR = 0.73[0.62-0.87],P = 3.70×10-4). As well, the G allele of rs14448 shown the same results with rs10464867.The association between G allele of rs14448 and decreased risk of gastric cancer was more significant in elder individuals(per-allele OR = 0.84[0.71-1.00], P = 0.049), and male individuals(per-allele OR = 0.81[0.70-0.94], P = 6.00×10-3).Besides, A significant difference between subgroups of smoking status were observed for the association of rs1063053 and rs1063045 with gastric cancer risk(P for heterogeneity = 0.033 for rs1063053; P=0.022 for rs1063045).3. In interaction analysis, we detected a significant interaction between genotypes of rs10464867 and rs14448 and gender, respectively(P=0.033 for rs10464867; P=0.031 for rs14448), Similarly, we also observed a significant interaction between rs1063053 and rs1063045 genotypes and somking status(P=0.033 for rs1063053; P=0.017 for rs1063045)on gastric cancer risk.4. In haplotype analysis, haplotypes were created for rs10464867, rs14448 and rs1063053 in one block. We then performed association analysis and observed that haplotype GAA and AGG were significantly associated with a decreased risk of gastric cancer risk as compared to haplotype GAG, OR were 0.88(0.77-0.99) and 0.76(0.65-0.89),P value were 0.034 and 6.39×10-4, respectively.5. Our further functional annotation analysis based on GTEx revealed that rs2284135,which is in perfect Linkage Disequilibrium(LD)(r2=0.98) with rs10464867, is an e QTL loci of NBN. rs2284135 A allele can significantly increase the expression of NBN gene in whole blood(beta=0.31, P = 0.038). It suggested that rs10464867 may exert important roles in the progression of gastric cancer, and regulate the expression of NBN gene by rs2284135.6. Differential expressed analysis of NBN gene based on TCGA RNA-Seq data further revealed that the expression level of NBN m RNA was significantly higher in gastric adenocarcinoma tissues than adjacent normal tissues, suggesting a potential vital role of NBN played in gastric carcinogenesis.Conclusions In this current study, we found that rs10464867, located in the 3’TUR of NBN gene was a new independent susceptibility variant of gastric cancer in Chinese,providing important clues that genetic variants in NBN might also involve in thesusceptibility of gastric cancer and further highlight the potentially pleiotropic effect of NBN SNPs on multiple cancers. It also provids clues to clarify the gastric cancer pathogenic mechanisms of the functional loci at NBN gene. |
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