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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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