Association Between Single Nucleotide Polymorphisms in the XRCC 1 Gene and Susceptibility to Prostate Cancer in Chinese Men

Prostate cancer (Pca) is the most commonly diagnosed cancer among men, accounting for 10% of male cancerrelated mortality (Jemal et al., 2008). The aetiology of PCa remains poorly understood and the genetic and environmental factors have been proved with the susceptibility to PCa (Pienta et al., 1993; Lichtenstein et al., 2000; Schaid, 2004). A number of studies suggested that X-ray repair complementing group 1 (XRCC1) is an important candidate gene for mediating the genetic influence on Pca (Hirata et al., 2007; Geng et al., 2009; Agalliu et al., 2010; Mandal et al., 2010; Dhillon et al., 2011; Kuasne et al., 2011; Langsenlehner et al., 2011; Wei et al., 2011; Wei et al., 2011; Berhane et al., 2012; Mittal et al., 2012). It has been reported that several SNPs, such as C26304 (Arg194Trp), G27466A (Arg280His), and G28152A (Arg399Gln) et al., were associated with Pca (Hirata et al., 2007; Geng et al., 2009; Mandal et al., 2010; Dhillon et al., 2011; Langsenlehner et al., 2011; Wei et al., 2011; Wei et al., 2011; Berhane et al., 2012; Mittal et al., 2012). However, up to date, the association analysis between the c.910A>G variant of XRCC1


Subjects
This present case-control study was performed on a total of 381 subjects (Han Chinese), including 193 prostate patients and 188 cancer-free volunteers.The diagnosis of prostate cancer was confirmed by the doctor according to the clinical, pathological and laboratory examinations.The general characteristics for these subjects, including age, smoking status, drinking status, body mass index (BMI), family history of prostate cancer, Gleason glade and PSA level (ng/ml), were summarized (Table 1).This study was approved by the local ethics committee and all subjects signed the study informed consent forms.

Genotyping
Genomic DNA was extracted from peripheral blood samples by standard phenol/chloroform/isoamyl alcohol extraction protocol.One pair of primers was designed using Primer Premier 5.0 software.Primers sequences, annealing temperature, region and fragment sizes were shown in Table 2. Polymerase chain reaction (PCR) amplifications were performed on 20 μl reaction mixture containing 50 ng mixed DNA template, 10 pM of each primer, 0.20 mM dNTP, 2.5 mM MgCl 2 and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China).The PCR protocol was 95°C for 5 min followed by 32 cycles of 94°C for 30 s, 60.9°C annealing for 30 s, and72°C for 30 s, and a final extension at 72°C for 10 min.The PCR-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze the c.910A>G genotype.Aliquots of 5 μl PCR products were digested with 2U restriction enzyme at 37°C for 10 h.The digested products were separated by electrophoresis in 2.5% agarose gel.The PCR amplified products were sequenced by ABI 3730 sequencer (Bioasia Biotechnology Co., Ltd.Shanghai, China).

Statistical analysis
The Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS Inc.; Chicago, IL, USA) was used to analyze the data.Allele and genotype frequencies were calculated by counting.The Chi-squared (χ 2 ) test was utilized to evaluate the significant departure from the Hardy-Weinberg equilibrium.Odds ratios (OR) and 95% confidence intervals (95% CI) adjusted for age, smoking status, drinking status, body mass index (BMI), family history of prostate cancer were then estimated by multiple logistic regression analysis to investigate the association between the XRCC1 polymorphism and susceptibility of prostate cancer.P value<0.05 was considered statistically significant.

General characteristics
A total of 381 subjects were recruited in this study, including 193 prostate cancer patients and 188 healthy controls.The general characteristics for these subjects were performed in Table 1.No significant difference between prostate cancer patients and healthy controls were detected in terms of age (P=0.5356).Additionally, no significant differences were detected in BMI, smoking and drinking status between the prostate cancer patients and healthy controls (P=0.7732,P=0.2626 and P=0.2952, respectively).

Genotyping of XRCC1 polymorphism
In this work, a novel allelic variant corresponding to the AgG mutations and Threonine (Thr) to Alanine (Ala) amino acid replacement in the exon9 of XRCC1 gene could be detected by PCR-RFLP.The PCR product of c.910A>G was digested with HhaI enzyme.Three possible genotypes were defined by three distinct banding patterns: AA (225 bp), AG (225, 138 and 87 bp), and GG (138 and 87 bp).The allelic and genotypic frequencies of the c.910A>G polymorphism were presented in Table 3.The polymorphism site was fitted with Hardy-Weinberg equilibrium (P>0.05).Allele frequencies in Pca patients and healthy controls were 0.5570 and 0.6915 for A allele, and 0.4430 and 0.3085 for G allele, respectively.Frequencies of the AA, AG, and GG genotypes were 0.3471, 0.4197, and 0.2332 in prostate cancer patients, while the frequencies of those genotypes in healthy subjects were performed to be 0.5000, 0.3830, and 0.1170.The allele and genotype frequencies of prostate cancer  patients were significantly different from those of the healthy controls (χ 2 =14.6745, p=0.0001 and χ 2 =12.8895, p=0.0016, respectively, Table 3).

Discussion
Pca is a complex disease and the genetic factors play a key role in contributing to the susceptibility.Common genetic SNPs in the cancer-related gene play a major role in tumorigenesis.The present study firstly indicated that the XRCC1 c.910A>G polymorphism was associated with susceptibility to PCa.The G allele might be a risk allele for PCa in Chinese men (G versus A: OR=1.89, 95% CI 1.56-2.42,χ 2 =14.67,P<0.000, Table 4).As shown in Table 3, the allelic and genotypic frequencies were statistically associated with the risk of PCa between patients and controls (P=0.0001and P=0.0016, respectively).Our findings suggested that the GG genotype was strongly associated with increased susceptibility to Pca compared to AA genotype and AG/AA carriers (OR=2.95,95% CI 1.46-5.42,P=0.001 and OR=2.17, 95% CI 1.33-4.06,P=0.003, Table 4).Previous studies have been evaluated the relationship between the susceptibility of Pca and SNPs in XRCC1 gene, such as the (Hirata et al., 2007;Geng et al., 2009;Mandal et al., 2010;Dhillon et al., 2011;Langsenlehner et al., 2011;Wei et al., 2011;Wei et al., 2011;Berhane et al., 2012;Mittal et al., 2012), but not including the c.910A>G variant.To our knowledge, this is the first report demonstration the susceptibility of the XRCC1 c.910A>G polymorphism with Pca.Results from this study provided more information to help understand and determine the role of XRCC1 gene in Pca.However, the results from these studies still remain inconsistent.Therefore, further investigation will be necessary to clarify the role of the c.910A>G and other polymorphisms of XRCC1 gene in susceptibility to Pca on larger different ethnic populations.