Int J Biol Sci 2013; 9(7):753-758. doi:10.7150/ijbs.6783
Association Study of Single Nucleotide Polymorphisms in XRCC1 Gene with the Risk of Gastric Cancer in Chinese Population
Department of Gastroenterology, The First Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, People's Republic of China.
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Qiao W, Wang T, Zhang L, Tang Q, Wang D, Sun H. Association Study of Single Nucleotide Polymorphisms in XRCC1 Gene with the Risk of Gastric Cancer in Chinese Population. Int J Biol Sci 2013; 9(7):753-758. doi:10.7150/ijbs.6783. Available from http://www.ijbs.com/v09p0753.htm
Gastric cancer is one of highly cancer-related deaths in the world. Previous evidence suggests that the X-ray repair cross-complementing group 1 gene (XRCC1) is one of the most important candidate genes for influencing gastric cancer risk. The objective of this study was to detect the potential association of genetic variants in XRCC1 gene with gastric cancer risk in Chinese Han population. In total, we enrolled 395 gastric cancer patients and 398 cancer-free controls in this study. The genotyping of c.910A>G and c.1804C>A genetic variants in XRCC1 gene were investigate by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and created restriction site-PCR (CRS-PCR) methods, respectively. We found the genotypes/alleles from these two genetic variants were statistically associated with the increased risk of gastric cancer (for c.910A>G, GG versus (vs.) AA: OR = 2.00, 95% CI 1.21-3.31; AG vs. AA: OR = 1.50, 95% CI 1.12-2.02; GG/AG vs. AA: OR = 1.59, 95% CI 1.20-2.10; GG vs. AG/AA: OR = 1.68, 95% CI 1.03-2.73; G vs. A: OR = 1.47, 95% CI 1.18-1.83; for c.1804C>A, AA vs. CC: OR = 2.68, 95% CI 1.46-4.94; AA vs. CA/CC: OR = 2.62, 95% CI 1.44-4.76; A vs. C: OR = 1.33, 95% CI 1.06-1.66). The allele-G of c.910A>G and allele-A of c.1804C>A genetic variants may contribute to gastric cancer susceptibility. These preliminary results indicate that these XRCC1 genetic variants are potentially related to gastric cancer susceptibility in Chinese Han population, and might be used as molecular markers.
Keywords: Gastric cancer, XRCC1 gene, Single nucleotide polymorphisms, Molecular markers, Risk factors.
Gastric cancer is a common solid tumor, and causes global health problem in the world. It is causing highly cancer-related deaths worldwide [1-5]. In the last few decades, a gradual decrease in incidence and mortality rate in gastric cancer has been observed [6-8]. However, in China, gastric cancer still remains one of the leading cause of cancer death [8-11]. Many factors could affect the development of gastric carcinogenesis. It has been reported that genetic factors may play key roles in the pathogenesis of gastric cancer [6,12,13]. Previous evidence suggests that the X-ray repair cross-complementing group 1 gene (XRCC1) is one of the most important candidate genes for influencing gastric cancer risk [5-10,12-15]. Evidence from published studies indicated that the single nucleotide polymorphisms (SNPs) in XRCC1 gene may alter the efficiency of DNA repair and influence gastric cancer susceptibility [5,8-10,12-19]. Three common genetic polymorphisms, more often found in the XRCC1 conserved sites, lead to a C to T substitution at codon 194 in exon 6, to a G to A substitution at codon 280 in exon 9, and to a G to A substitution at codon 399 in exon 10 of XRCC1 gene, leading to the amino acid alterations arginine (Arg) to tryptophan (Trp), Arg to histidine (His), and Arg to glutamine (Gln), respectively. These changes in conserved protein sites may alter the base excision repair (BER) capacity, increasing the chances of DNA damage [10,13,19-21]. Both of Arg194Trp, Arg280His and Arg399Gln genetic variants have potential affection on the susceptibility to gastric cancer [5,8-10,12-18]. However, the exact mechanism of the pathogenesis of gastric cancer still remains poorly understood. Up to now, there are no similar studies which reported the potential affection of c.910A>G and c.1804C>A genetic variants in XRCC1 on gastric cancer susceptibility. Thus, in this case-control study, the objective of this study aimed to assess whether these genetic variants influencing on gastric cancer susceptibility in Chinese Han population.
Materials and methods
A total of 793 subjects were enrolled from the First Affiliated Hospital, Medical School of Xi'an Jiaotong University in this case-control study, which consisting of 395 gastric cancer patients with a pathology-confirmed diagnosis and 398 healthy age-matched subjects who had no history of any gastric diseases as controls. All subjects were unrelated Chinese with Han nationality and lived in Xi'an city, Shaanxi province of China. Table 1 shows the demographic clinical characteristics, including sex, age, smoking, drinking, H.pyori infection, family history of gastric cancer. The protocol of this study was approved by the Ethics Committee of the First Affiliated Hospital, Medical School of Xi'an Jiaotong University. The informed consent for participation was obtained from all subjects.
Genomic DNA was extracted from peripheral venous blood of each participant using the standard extraction method and then stored at -80°C . Based on the human XRCC1 gene mRNA sequences and DNA sequences (GenBank IDs: NM_006297.2 and NC_000019.9), the specific polymerase chain reaction (PCR) primers were designed by Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA). Table 2 shows the information of primers sequences, annealing temperature, fragment sizes and region. The PCR reaction was performed in a total volume of 20 µL solution containing 50 ng template DNA, 1×buffer (Tris-HCl 100 mmol/L, pH 8.3; KCl 500 mmol/L), 0.25 µmol/L primers, 2.0 mmol/L MgCl2, 0.25 mmol/L dNTPs, and 0.5U Taq DNA polymerase (Promega, Madison, WI, USA). The PCR amplification condition was carried out in an initial 94°C for 5 minutes, then followed by 32 cycles of 94°C for 32 seconds, annealing at the corresponding temperature (given in Table 2) for 32 seconds and 72°C for 32 seconds, and a final extension at 72°C for 5 minutes. The PCR amplified products were separated by 2.0% agarose gel electrophoresis and then observed under UV light.
Clinical characteristics of gastric cancer cases and cancer-free controls.
The primers, PCR-RFLP and CRS-PCR analysis for XRCC1 genetic polymorphisms
Note: SNPs, single nucleotide polymorphisms; PCR, polymerase chain reaction; PCR-RFLP, PCR-restriction fragment length polymorphism; CRS-PCR, created restriction site PCR; Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating sequence variations.
The genotyping for c.910A>G genetic variants were detected through the PCR-restriction fragment length polymorphism (PCR-RFLP) method. The c.1804C>A genetic variants were genotyped by the created restriction site-PCR (CRS-PCR) method with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzymes for discriminating sequence variations [23-27]. According to the supplier's manual, aliquots of 5 μL PCR products were digested with 2U selected restriction enzyme (MBI Fermentas, St. Leon-Rot, Germany, given in Table 2) at 37°C for 10 hours. The digested products were separated by electrophoresis in 2.5% agarose gel and observed under UV light for analyzing the genotyping of XRCC1 genetic polymorphisms. In order to ensure the concordance of genotype results from CRS-PCR and PCR-RFLP methods, about 10% of random samples were re-analyzed by DNA sequencing method (ABI3730xl DNA Analyzer, Applied Biosystems, Foster City, CA).
The Hardy-Weinberg equilibrium (HWE) in the frequencies of allele and genotype, and general characteristics between gastric cancer patients and cancer-free controls were analyzed by chi-squared (χ2) test. The odds ratios (ORs) and 95% confidence intervals (95% CIs) from unconditional logistic regression were utilized to evaluate the potential associations between genetic variants of XRCC1 gene and the risk of gastric cancer. P value less than 0.05 was defined as statistically significant. All statistical analyses were evaluated by the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS Inc.; Chicago, IL, USA).
In this case-control study, we enrolled 395 gastric cancer patients and 398 healthy age-matched controls in Chinese Han population. The chi-squared (χ2) test indicated that there were no significant differences between gastric cancer patients and cancer-free controls in regarded to sex, age, drinking, smoking, family history of gastric cancer, and H. pyori infection (All P values > 0.05, Table 1). The demographic general characteristics of the subjects are performed in Table 1.
XRCC1 genetic variants identification
Through the PCR-RFLP and DNA sequencing methods, the c.910A>G genetic variants of XRCC1 gene were detected. DNA sequence analyses indicate that this genetic variant is a non-synonymous mutation, which causes by A to G mutations in exon9 of human XRCC1 gene and leading to the threonine (Thr) to alanine (Ala) amino acid replacement (p.Thr304Ala, Reference sequences GenBank IDs: NC_000019.9, NM_006297.2 and NP_006288.2). The PCR products were digested with HhaI restriction enzyme and divided into three genotypes, AA (238 bp), AG (238,166 and 72bp) and GG (166 and 72bp, Table 2). As for the c.1804C>A genetic variants, this genetic variants were genotyped by CRS-PCR and DNA sequencing methods. According to the DNA sequence analyses, this genetic polymorphism is a non-synonymous mutation, which causes by C to A mutations in exon17 of human XRCC1 gene and leading to the proline (Pro) to threonine (Thr) amino acid replacement (p.Pro602Thr, Reference sequences GenBank IDs: NC_000019.9, NM_006297.2 and NP_006288.2). The PCR products were digested with MaeII restriction enzyme and divided into three genotypes, CC (209 bp), CA (209,192 and 17 bp) and AA (192 and 17 bp, Table 2).
Allelic and genotypic frequencies
The allelic and genotypic frequencies of c.910A>G and c.1804C>A genetic variants are summarized in Table 3. The allele-A frequencies of c.910A>G and allele-C of c.1804C>A genetic variants were maximums in gastric cancer patients and cancer-free controls. As for c.910A>G, the allele frequencies of gastric cancer patients (A, 66.96%; G, 33.04%) were statistically significantly different from cancer-free controls (A, 74.87%; G, 25.13%, χ2 = 12.0393, P = 0.0005). The genotype frequencies in gastric cancer patients were not consistent with cancer-free controls, the differences being statistically significant (χ2 = 11.6137, P = 0.0030, Table 3). As for c.1804C>A, significantly differences were detected between the allele frequencies of gastric cancer patients (C, 71.65%; A, 28.35%) and those of cancer-free controls (C, 77.01%; A, 22.99%, χ2 = 5.9812, P = 0.0145), and the genotypic frequencies in gastric cancer patients were statistically significant different from those of cancer-free controls (χ2 = 10.6912, P = 0.0048, Table 3). The genotypic frequencies of these two genetic variants sites were corresponded to HWE in gastric cancer patients and cancer-free controls (P > 0.05, Table 3).
Association between XRCC1 genetic variants and gastric cancer risk
Table 4 presents the potential association between XRCC1 genetic variants and the risk of gastric cancer. As for c.910A>G, we detected statistically increased risk of gastric cancer in the homozygote comparison (GG versus (vs.) AA: OR = 2.00, 95% CI 1.21-3.31, χ2 = 7.43, P = 0.006), heterozygote model (AG vs. AA: OR = 1.50, 95% CI 1.12-2.02, χ2 = 7.21, P = 0.007), dominant model (GG/AG vs. AA: OR = 1.59, 95% CI 1.20-2.10, χ2 = 10.42, P = 0.001), recessive model (GG vs. AG/AA: OR = 1.68, 95% CI 1.03-2.73, χ2 = 4.39, P = 0.036) and allele comparison (G vs. A: OR = 1.47, 95% CI 1.18-1.83, χ2 = 12.03, P = 0.001). As for c.1804C>A, statistically significantly increased risk of gastric cancer were found in the homozygote comparison (AA vs. CC: OR = 2.68, 95% CI 1.46-4.94, χ2 = 10.59, P = 0.001), recessive model (AA vs. CA/CC: OR = 2.62, 95% CI 1.44-4.76, χ2 = 10.51, P = 0.001) and allele comparison (A vs. C: OR = 1.33, 95% CI 1.06-1.66, χ2 = 5.98, P = 0.014, Table 4).
The genotype and allele frequencies of XRCC1 gene polymorphisms in cases and controls
The association between gastric cancer risk and XRCC1 gene polymorphisms.
SNPs, single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval; vs., versus.
Many emergence reports indicate that gastric cancer is a common solid cancer causing by complex interactions between environmental and genetic factors. The genetic factors of candidate genes might play key roles in the susceptibility to gastric cancer. The XRCC1 gene is an important candidate gene for influencing gastric cancer susceptibility [5-10,12-15]. Earlier studies on the potential association between XRCC1 genetic variants (such as Arg194Trp, Arg280His and Arg399Gln) and gastric cancer susceptibility revealed inconsistent results that might be attributed to environmental and genetic factors [5,8-10,12-18]. ENGIN et al., suggested that the XRCC1 homozygous Gln allele at Arg399Gln was associated with 2.54 times higher risk of gastric cancer . Li et al., indicated that subjects carrying the combined XRCC1-194Arg/Trp + Trp/Trp genotype had an elevated chance of regression of gastric lesions (adjusted OR = 1.44; 95% CI 1.06-1.96), whereas subjects carrying the XRCC1-399Arg/Gln + Gln/Gln genotype had a decreased chance of regression (OR = 0.68, 95% CI 0.49-0.92) in a Chinese population . Yuan et al., reported that the XRCC1 194Trp allele significantly increased the risk of gastric cancer (OR = 2.72, 95% CI 1.04-7.24, P = 0.027), and also associated with risk of gastric cardia carcinoma and promoted distant metastasis of gastric cancer . Duarte reported that there were no evidence of a relationship between the polymorphisms XRCC1 Arg194Trp and Arg399Gln and the risk of gastric cancer in the Brazilian population . Lee et al., indicated none of these three amino acid substitution polymorphisms (Arg194Trp, Arg280His, and Arg399Gln) were associated with increased risk of gastric cancer in the Korean population . Furthermore, Lee and his colleagues found that the haplotype A (194Trp, 280Arg, and 399Arg) was associated with significant reduction in gastric cancer risk (adjusted OR = 0.65, 95% CI 0.43-0.99), whereas haplotype D (194Arg, 280Arg, and 399Arg alleles) was a risk type for gastric cancer (adjusted OR = 1.57, 95% CI 0.93-2.65) . Ratnasinghe et al., observed no association between the variant genotype in XRCC1 Arg194Trp and gastric cardia cancer, while found that the Arg399Gln was associated with reduced risk of gastric cardia cancer (relative risks (RR): 0.60, 95% CI 0.37-0.97). In the present study, the influencing of c.910A>G and c.1804C>A genetic variants in XRCC1 on the susceptibility to gastric cancer was evaluated by association analysis in 395 gastric cancer patients and 398 cancer-free control subjects. We demonstrated that these two SNPs have statistically significant impacts on the susceptibility to gastric cancer in Chinese Han population (Table 4). Our data indicated that the significant differences were shown in the frequencies of allele and genotype between gastric cancer patients and cancer-free controls for these two SNPs (All P < 0.05, Table 3). As for c.910A>G, the GG genotype was statistically associated with the increased susceptibility of developing gastric cancer compared to AA genotype and AG/AA carriers (P < 0.05). As for the c.1804C>A genetic variants, the AA genotype was statistically associated with the increased susceptibility of developing gastric cancer compared to CC genotype and CA/CC carriers (all P < 0.01). Our data indicated that the allele-G of c. 910A>G and allele-A of c.1804C>A genetic variants were increased risk factors for gastric cancer susceptibility (Table 4). To the best of our knowledge, this is the first report regarding the association between c.910A>G and c.1804C>A SNPs and the risk of gastric cancer. These findings could provide new evidence for further analysis of the biological function role of XRCC1 gene variants on gastric cancer risk. These observations suggested that XRCC1 genetic variants could be useful molecular biomarkers for assessing the susceptibility to gastric cancer. Future functional studies are necessary to verify these findings for use of c.910A>G and c.1804C>A SNPs of XRCC1 gene as molecular biomarkers in gastric cancer.
The authors have declared that no competing interest exists.
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Corresponding author: Wen Qiao, Department of Gastroenterology, The First Affiliated Hospital, Medical School of Xi'an Jiaotong University, No. 277 Yanta West Road, Xi'an710061, Shaanxi Province, People's Republic of China. Tel: +86-029-85324001; Fax: +86-029-85324001 E-mail: wenqiao_12com.