Asian J Androl 2007; 9 (3): 331–338

DOI: 10.1111/j.1745-7262.2007.00263.x www.asiaandro.com

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Original Article .

Relationship between XRCC1 polymorphisms and susceptibility to prostate cancer in men from Han, Southern China Zheng Xu1, Li-Xin Hua1, Li-Xin Qian1, Jie Yang1, Xin-Ru Wang2, Wei Zhang1, Hong-Fei Wu1 1 2

Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China School 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 cancerfree 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

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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%

Correspondence to: Dr Hong-Fei Wu, Department of Urology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd., Nanjing 210029, China. Tel: +86-25-8371-8836 ext. 6603 Fax: +86-25-8378-0079 E-mail: [email protected] Received 2006-08-29 Accepted 2006-11-08

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

© 2007, Asian Journal of Andrology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All rights reserved. Tel: +86-21-5492-2824; Fax: +86-21-5492-2825; Shanghai, China

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XRCC1 polymorphisms and risk of prostate cancer

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 β 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

Materials 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

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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., ≥ 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'; a nd XRCC1 399R 5'-TCCTCCAGCCTTTACTGATA-3'. PCR conditions were 95ºC for 5 min, followed by 37 cycles of http://www.asiaandro.com; [email protected]

Asian J Androl 2007; 9 (3): 331–338

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, packyears 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

Table 1. Distribution of selected variables and XRCC1 alleles in prostate cancer (PCa) cases and controls. a Two-sided χ2-test. Case (n = 207) Control (n = 235) Variables Pa n % n % Age (years) ≤70 97 46.9 129 54.9 0.092 >70 Pack-years of smoking 0 70

1

0.89 (0.46–1.70)

1

1.40 (0.62–3.12)

1

1.75 (0.91–3.36)

57/50

53/56

86/87

24/19

55/66

55/40

1 37/46

0.48 (0.24–0.95) 29/76

1 52/99

0.99 (0.42–2.33) 14/23

1 36/80

1.24 (0.61–2.49) 30/42

1

0.54 (0.16–1.78)

1

0.45 (0.08–2.69)

1

1.18 (0.35–3.93)

12/18

13/28

22/40

3/6

16/29

9/17

1

1.12 (0.56–2.26)

1

1.22 (0.53–2.80)

1

2.04 (1.03–4.05)

54/28

62/39

91/54

25/13

56/44

60/23

1 37/46 1 23/20 1 43/26

0.48 (0.24–0.95) 29/76 0.84 (0.35–2.03) 30/38 1.06 (0.46–2.44) 45/29

1 52/99 1 45/48 1 68/46

0.99 (0.42–2.33) 14/23 0.97 (0.31–3.05) 8/10 1.22 (0.45–3.36) 20/9

1 36/80 1 29/42 1 43/31

1.24 (0.61–2.49) 30/42 2.28 (0.91–5.70) 24/16 1.33 (0.61–2.91) 45/24

1 81/78 Ever 1 22/14 Family of history cancer No 1 80/86 Yes 1 23/6

0.69 (0.42–1.13) 72/115 0.76 (0.28–2.06) 32/28

1 122/160 1 43/33

1.15 (0.61–2.17) 31/33 0.76 (0.24–2.35) 11/9

1 75/124 1 33/29

1.74 (1.07–2.83) 78/69 0.93 (0.34–2.54) 21/13

0.79(0.49–1.27) 79/130 0.32(0.01–1.26) 25/13

1 127/178 1 38/15

1.26 (0.69–2.28) 32/38 0.57 (0.13–2.64) 10/4

1 78/145 1 30/8

2.02 (1.26–3.25) 81/71 0.32 (0.09–1.08) 18/11

Pack-years of smoking 0