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    Asian J Androl 2007; 9 (3): 331-338

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- Original Article -

Relationship between XRCC1 polymorphisms and susceptibility to prostate cancer in men from Han, Southern China

heng Xu1, Li-Xin Hua1, Li-Xin Qian1, Jie Yang1, Xin-Ru Wang2, Wei Zhang1, Hong-Fei Wu1

1Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
2School of Public Health, Nanjing Medical University, Nanjing 210029, China

Abstract

Aim: To investigate the association among XRCC1 polymorphisms, smoking, drinking and the risk of prostate cancer (PCa) in men from Han, Southern China. Methods: In a case-control study of 207 patients with PCa and 235 cancer-free controls, frequency-matched by age, we genotyped three XRCC1 polymorphisms (codons 194, 280 and 399) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP) method. Results: Among the three polymorphisms, we found that the XRCC1 Arg399Gln variant allele was associated with increased PCa risk (adjusted odd ratio [OR]: 1.67, 95% confident interval [CI]: 1.11_2.51), but the XRCC1 Arg194Trp variant allele had a 38% reduction in risk of PCa (adjusted OR: 0.62, 95% CI: 0.41_0.93). However, there was no significant risk of PCa associated with Arg280His polymorphism. When we evaluated the three polymorphisms together, we found that the individuals with 194Arg/Arg wild-type genotype, Arg280His and Arg399Gln variant genotypes had a significantly higher risk of PCa (adjusted OR: 4.31; 95% CI: 1.24_14.99) than those with three wild-type genotypes. In addition, we found that Arg399Gln variant genotypes had a significant risk of PCa among heavy smokers (adjusted OR: 2.04; 95% CI: 1.03_4.05). Conclusion: These results suggest that polymorphisms of XRCC1 appear to influence the risk of PCa and may modify risks attributable to environmental exposure. (Asian J Androl 2007 May; 9: 331_338)

Keywords: XRCC1; polymorphism; prostate cancer; genetic susceptibility; molecular epidemiology

Correspondence to: Dr Hong-Fei Wu, Department of Urology, First Affiliated Hospital of Nanjing Medical University, 300 Guang-zhou Rd., Nanjing 210029, China.

Tel: +86-25-8371-8836 ext. 6603 Fax: +86-25-8378-0079
E-mail: wu-hongfei@hotmail.com
Received 2006-08-29 Accepted 2006-11-08

DOI: 10.1111/j.1745-7262.2007.00263.x


1 Introduction

Prostate cancer (PCa) is one of the most common malignancies in men in the Western world. Prostate is the leading site for cancer incidences, accounting for 31% of new cancer cases in men [1]. The incidence of PCa varies greatly with race and geography. The incidence in black Americans is 60 times higher than that of the Han population in China, so the research on pathogenesis of PCa from a genetic aspect is of important significance [2]. It is well known that in the carcinogenic process multiple points at which genetically-determined host characteristics and/or environmental factors might influence individual susceptibility through affecting DNA-repair capacity and other cellular processes [3]. The mechanism of PCa development is similar to that of other major tumors, which are dependent on the interactions of genetic factors and environmental agents. Living organisms suffer continuous damage from diverse environmental agents and normal cellular metabolism products. DNA repair is essential in protecting the genome of cells from environmental hazards, such as tobacco smoking. Reduced DNA repair capacity (DRC) might result in a higher risk for many people in developing cancer [4]. Therefore, the ability to monitor and repair carcinogen-induced DNA damage is the determinant factor of host susceptibility to carcinogenesis.

A complex system of DNA repair enzymes has a vital role in protecting the genome of cells from all kinds of carcinogenic exposure. The DNA repair enzyme XRCC1 is thought to be involved in base excision repair (BER) of oxidative DNA and single-strand breaks repair [5]. Human XRCC1 is mapped at human chromosome 19q13.2_13.3, spans a genomic distance 33 kb, contains 17 exons, and transcripts a protein of 633 amino acids (69.5 kDa). Although XRCC1 has no known enzymatic activity, it can interact with several important repair proteins through its different domains, such as DNA ligase III at its breast cancer susceptibility gene C terminus II (BRCT-II) domain, DNA polymerase b at its NH2 terminus, poly (ADP-ribose) polymerase (PARP) 1 and 2 at its BRCT-I domain, human AP endonuclease, polynucleotide kinase (PNK), human 8-oxoguanine DNA glycosylase (OGG1) and proliferating cell nuclear antigen (PCNA) at the central section of XRCC1 protein [6]. Three common polymorphisms that lead to amino acid substitutions in XRCC1 gene have been described in codon 194 (exon 6, base C to T, amino acid Arg to Trp), codon 280 (exon 9, base G to A, amino acid Arg to His) and codon 399 (exon 10, base G to A, amino acid Arg to Gln) [7].

Although a large number of molecular epidemiological studies have been conducted to evaluate the role of the three polymorphisms on cancer risk, the results are conflicting rather than conclusive. In the present study, we genotyped the three polymorphisms and evaluated the associations between the polymorphisms and their haplotypes with PCa risk in a hospital-based case-control study.

2 Material and methods

2.1 Study subjects

The study subjects consisted of 207 cases with newly diagnosed PCa and 235 cancer-free male controls recruited from the First Affiliated Hospital of Nanjing Medical University (Nanjing, China) between September 2003 and April 2006. All cases were patients diagnosed with PCa through biopsy of puncture or operation. Serological (prostate specific antigen [PSA], prostatic acid phosphatase), physical and other auxiliary examinations were conducted on all controls to exclude the possibility of PCa, and any control was excluded from the study if he ever had an abnormal PSA test (i.e., ¡Ý 4ng/dL), or he ever had an abnormal digital rectal examination, or he had any previous cancer diagnosis. The mean age of the cases was 71.2 vs. 69.7 years in the controls. All subjects were ethnic Han Chinese, permanently residing in Jiangsu or Anhui Province, China. Each subject was informed about the aims and requirements of the study, and informed consent for participation was obtained in accordance with institutional guidance at Nanjing Medical University. A structured questionnaire was administered by interviewers to collect information on demographic data and lifestyle characteristics. In our research, smoking more than five cigarettes per day for more than 5 years was defined as a `smoking habit'. Taking smoke into the lung when smoking was defined as "deep smoking", while only taking smoke into the mouth was defined as "superficial smoking". Pack-years of smoking (cigarettes per day/20) × (years with smoked) were calculated to indicate the cumulative smoking dose. "Drinking habit" was defined as drinking at least three times per week for more than 10 years. "Family history of cancer" was defined as any cancer in first-degree relatives (parents, siblings or children). After interview, approximately 5 mL of venous blood sample was collected from each subject.

2.2 Genotyping

Genomic DNA was isolated and purified from anticoagulated blood by the traditional phenol/chloroform extraction and ethanol precipitation, dissolved in TE buffer (pH = 7.4) and stored at _20ºC for genotyping.

The XRCC1 Arg194Trp, Arg280His and Arg399Gln polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Three separate PCR assays were used to detect the polymorphisms in exon 6, exon 9 and exon 10 of XRCC1 using primers of (a) XRCC1 194F, 5'-GCCCCGTCCCAGGTA-3'; and XRCC1 194R 5'-AGCCCCAAGACCCTTTCACT-3' and (b) XRCC1 280F, 5'-CCAGTGGTG CTAACCTAATC-3'; and XRCC1 280R 5'-CACTCAGCACCAGTACCACA-3' and (c) XRCC1 399F, 5'-TTGTGCTTTCTCTGTGTCCA-3'; and XRCC1 399R 5'-TCCTCCAGCCTTTACTGATA-3'. PCR conditions were 95ºC for 5 min, followed by 37 cycles of 95ºC for 40 s, 60ºC (for codon 194) or 55ºC (for codon 280 and codon 399) for 40 s, 72ºC for 60 s and a final elongation step at 72ºC for 10 min. Following PCR, 10 µL aliquot were removed and subjected to restriction digestion separately with Pvu II (for codon 194), Rsa I (for codon 280) and Msp I (for codon 399) (New England Biolabs, Ipswich, MA, USA). The Pvu II restricted products of XRCC1 codon194 Arg/Arg, Arg/Trp and Trp/Trp genotypes had band sizes of 491, 491/294/197 and 294/197 bp, respectively. The Rsa I restricted products of XRCC1 codon 280 Arg/Arg, Arg/His and His/His genotypes had band sizes of 145/56, 201/145/56 and 201 bp, respectively. The Msp I restricted products of XRCC1 codon 399 Arg/Arg, Arg/Gln and Gln/Gln genotypes had band sizes of 375/240, 615/375/240 and 615 bp, respectively.

2.3 Statistical analysis

All differences in select demographic variables, pack-years of smoking, smoking method, alcohol use, family history of cancer, frequencies of the XRCC1 genotypes between the case and control groups were evaluated by using χ2-test. Unconditional univariate and multivariate logistic regression analyses were performed to obtain the crude and adjusted odds ratios (OR) for the risk of PCa and their 95% confidence intervals (CI). When we selected variables for the multivariate analysis, any variable whose univariate test has a P-value < 0.2 was a candidate for the multivariable model along with all variables of known clinical importance. Once the variables had been identified, we began with a model containing all of the selected variables. The multivariate adjustment included age, pack-years of smoking, alcohol use and family history of cancer. Considering the potential interaction among the three polymorphisms on the risk for PCa, the associations between the combined genotypes of the three polymorphisms risk of PCa was evaluated. The genotypes were further stratified by subgroups of age, pack-years of smoking, smoking method, alcohol use and family history of cancer. All tests of statistical significance were two-sided. All statistical analyses were performed with Statistical Analysis System software (version 9.1.3e; SAS Institute, Cary, NC, USA).

3 Results

3.1 Characteristics of the study population

Table 1 shows the demographic characteristics of the case and control groups. The cases and controls appeared to be well-matched in age (grouping by 70 years old, P = 0.092) and gender (all male). However, there were more heavy smokers (56.0%) and deep smokers (42.5%) among the cases than the controls (28.5% and 23.4%, respectively), and these differences were statistically significant (P < 0.001). In addition, cases had more often been alcohol user (26.1% vs. 17.9%, P = 0.037), and more often had a first-degree relative with cancer (23.2% vs. 8.1%, P < 0.001).

3.2 Genotype distributions of XRCC1 polymorphisms among cases and controls

The distribution of XRCC1 genotypes in case patients and control subjects and their associations with risk of PCa are presented in Table 2. The genotype frequencies of these three polymorphisms among the controls were all in agreement with the Hardy-Weinberg equilibrium (χ2-test: P = 0.214 for Arg194Trp, 0.524 for Arg280His and 0.680 for Arg399Gln). The frequency of XRCC1 194Arg/Arg genotype was 49.8% in cases and 39.1% in controls, suggesting that 194Arg/Arg genotype might be a risk genotype. Logistic regression analysis revealed that the 194Arg/Trp heterozygote had a significantly decreased risk (adjusted OR: 0.62; 95% CI: 0.40_0.95) and the 194Trp/Trp homozygote had a non-significantly decreased risk (adjusted OR: 0.62; 95% CI: 0.31_1.25), compared with 194Arg/Arg homozygous wild-type. When we combined the variant genotypes (Arg/Trp+ Trp/Trp) assuming a co-dominant allele effect, the combined variant genotypes had a 38% reduction in risk of PCa (adjusted OR: 0.62; 95% CI: 0.41_0.93). However, the Arg399Gln variant allele was associated with increased PCa risk. The data indicated that individuals with 399Gln allele (Arg/Gln+Gln/Gln) were more prevalent in the cases (47.9%) compared to the controls (34.9%). The combined variant genotypes were at a 1.67-fold greater risk of PCa than homozygous wild-type genotype, and the adjusted ORs for 399Arg/Gln and 399Gln/Gln genotypes were 1.61 (95% CI: 1.05_2.47) and 2.09 (95% CI: 0.85_5.14), respectively, as shown in Table 2. However, the XRCC1 Arg280His polymorphism was not associated with a significantly increased risk of PCa.

3.3 Association between the combined genotypes of XRCC1 polymorphisms and risk of PCa

Combined genotype analyses of the three XRCC1 polymorphisms are shown in Table 3. Assessment of intra-gene interactions in the logistic regression models of the three XRCC1 polymorphisms revealed statistically significant interactions (P < 0.001). Compared with wild-type for all three genotypes, Arg399Gln variant alone was associated with a statistically significant increased risk of PCa (adjusted OR: 3.19; 95% CI: 1.48_6.88). Moreover, we found that the individuals with 194Arg/Arg wild-type genotype, Arg280His and Arg399Gln variant genotypes had a significantly higher risk of PCa (adjusted OR: 4.31; 95% CI: 1.24_14.99) than those with three wild-type genotypes.

3.4 Stratified analyses of the association between XRCC1 polymorphisms and risk of PCa

The dichotomized genotypes (the XRCC1 194Arg/Trp+Trp/Trp versus Arg/Arg genotypes, the XRCC1 280Arg/His+His/His vs. Arg/Arg genotypes and the XRCC1 399Arg/Gln+Gln/Gln versus Arg/Arg genotypes) were further examined for subgroups of the variables listed in Table 1. The stratified, adjusted ORs are presented in Table 4. Among individuals no more than 70 years old, the OR for XRCC1 Arg194Trp combined variant genotypes was 0.55 (95% CI: 0.30_1.00). Among non-smokers, the OR for XRCC1 Arg194Trp combined variant genotypes was 0.48 (95% CI: 0.24_0.95). Among heavy-smokers (Pack-years of smoking ¡Ý 20), the OR for XRCC1 Arg399Gln combined variant genotypes was 2.04 (95% CI: 1.03_4.05). Among non-drinkers and subjects without a family history of cancer, the ORs for XRCC1 Arg399Gln combined variant genotypes were 1.74 (95% CI: 1.07_2.83) and 2.02 (95% CI: 1.26_3.25), respectively.

4 Discussion

The XRCC1 protein has no known catalytic activity but serves to orchestrate BER through its role as a central scaffolding protein for DNA ligase III, DNA polymerase β, and PARP, and also through its function in recognizing and binding to single-strand breaks [8]. The Arg399Gln polymorphism is located in the region of the BRCT-I interaction domain of XRCC1 with poly(ADP-ribose) polymerase, the presence of the variant 399Gln allele has been shown to be associated with measurable reduced DRC as assessed by the persistence of DNA adducts [9,10], tumor-suppressor gene P53 mutations [11], increased red blood cell glycophorin A [9], elevated levels of sister chromatid exchanges [10] and prolonged cell-cycle delay [12]. Both the XRCC1 Arg194Trp and Arg280His variants occur in the newly identified PCNA binding region [13], but few studies have examined the influence of the 194Trp allele on the function of the XRCC1 protein, and there are relatively fewer studies conducted to examine the association between Arg280His variant and cancer risk. To the best of our knowledge, it is the first study that has investigated the association of these three polymorphisms and the risk of PCa in a Chinese population.

In this study, we investigated the associations of three polymorphisms of the DNA repair gene XRCC1 with the risk of PCa in a southern Chinese population. When we evaluated each polymorphism separately, we found that the Arg194Trp variant genotypes had a lower risk of PCa than the wild-type genotype, but the Arg399Gln variant genotypes were associated with a significantly increased risk of PCa compared with the wild-type genotypes. However, the Arg280His polymorphism had no main effect on PCa risk. When we analyzed these three polymorphisms together, we found that individuals with Arg399Gln variant genotype, Arg194Trp and Arg280His wild-type genotypes were associated with a statistically significant increased risk of PCa compared with wild-type for all three genotypes (adjusted OR: 3.19; 95% CI: 1.48_6.88). Moreover, we found that the individuals with 194Arg/Arg wild-type genotype, Arg280His and Arg399Gln variant genotypes had a much higher risk of PCa (adjusted OR: 4.31; 95% CI: 1.24_14.99). Such findings suggest that polymorphisms in XRCC1 may contribute to the risk and therefore play a role in the etiology of PCa.

A lot of researchers have studied the associations between polymorphisms of the DNA repair gene XRCC1 and cancer risk. Stern et al. [14] reported a case-control study of the association between XRCC1 genotypes and bladder cancer in a white US population. They found some evidence of a protective effect for subjects that carried at least one copy of the codon 194 variant allele relative to those homozygous for the common allele (adjusted OR: 0.59; 95% CI: 0.3_1.0). Ritchey et al. [15] reported that XRCC1 399Gln/Gln genotype was associated with an increased risk of PCa (OR: 2.18; 95% CI; 0.99_4.81). Chen et al. [16] also reported that a significantly increased risk of PCa was observed in white men with the XRCC1 399Gln allele (OR: 1.6, 95% CI; 1.1_2.4). Our results from the Chinese population reported here are consistent with previous findings that the XRCC1 194Arg/Arg, 399Arg/Gln and Gln/Gln genotypes are the at-risk genotypes [14_16]. There are few studies investigating the Arg280His polymorphism and cancer risk to date, and most reported a non-significant risk of cancer association with 280His allele [14, 17]. Therefore, these data strongly support our molecular epidemiologic findings that functional polymorphisms influencing the activity of XRCC1 are associated with an increased risk of cancer.

Genetic polymorphisms often vary between ethnic groups. For example, the frequencies of XRCC1 194Arg/Arg, Arg/Trp and Trp/Trp in our 235 southern China control group were 39.1%, 49.8% and 11.1%, respectively, compared with 83%, 17%, and 0%, respectively, of 197 cancer-free white men controls in the study by Stern et al. [14]. Similarly, the frequencies of the XRCC1 280Arg/Arg, Arg/His, and His/His in our controls were 82.1%, 16.6%, and 1.3%, respectively, compared with 90.7%, 9.3%, and 0%, respectively, of 312 white men in a study by Moullan et al. [18]. For the XRCC1 Arg399Gln polymorphisms, we found the frequencies of Arg/Arg, Arg/Gln, and Gln/Gln (65.1%, 30.6% and 4.3%, respectively) among controls were also different to the study by Olshan et al. [19] (38.5%, 50.9% and 10.6%, respectively). This variation needs to be investigated further.

Tobacco smoking might be a risk factor for PCa. We found that the OR for XRCC1 Arg399Gln combined variant genotypes was 2.04 (95% CI: 1.03_4.05) among heavy-smokers (pack-years of smoking ¡Ý 20). But we did not find any evidence for interactions between the polymorphisms of the XRCC1 gene and other risk factors, such as alcohol drinking and family history of cancer. There was a significantly increased risk associated with the Arg399Gln variant genotypes among non-drinkers or subjects without a family history of cancer. This observed association might suggest that the cancer patients without the established risk factors may have some other unknown exposures, or have other genetic factors that are linked to the putative XRCC1 risk genotypes. Another possibility is that the results are purely chance as the numbers in the subgroups compared were relatively small. Again, larger studies are needed to verify the findings.

In conclusion, the XRCC1 Arg194Trp and Arg399Gln polymorphisms have a major effect on the risk of PCa. The variant genotypes may modulate the risk of PCa associated with smoking. However, these results may be biased by the relatively small number of subjects in the various subgroups analyzed and therefore need to be validated by larger studies. Future molecular epidemiological studies of the three and other sequence variants of the XRCC1 gene and their association with DNA repair phenotypes will help us understanding the role of the XRCC1 gene in the etiology of PCa.

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