ARTICLE
Auteur(s) : Xian-Dun Zhai1, Yao-Nan Mo1, Xiao-Qi
Xue1, Gui-Sen Zhao1, Lin-Bo Gao2,
Hong-Wei Ai1, Yi Ye2
1Institute of Forensic Medicine, Hennan University of
Science and Technoogy, Luoyang 471003, Henan, China
2Department of Forensic Biology, West China School of
Preclinical and Forensic Medicine, Sichuan University, Chengdu
610041, Sichuan, China
Article reçu le 31 Mai 2009, accepté le 22 Juillet 2009
Introduction
Esophageal squamous cell carcinoma (ESCC) is a malignancy arising
from the epithelial cells lining the esophagus. Moreover, it is
characterized by poor prognosis and a wide incidence variation in
different geographical regions [1, 2]. In china, ESCC occurs mainly
in a few sharply demarcated areas of the country located on the
south side of the Taihang Mountains on the borders of three
provinces (Henan, Shanxi and Hebei), where the incidence (about
250,000 cases diagnosed every year) and mortality rates rise to a
considerable height [3].
The mechanism of ESCC pathogenesis, however, is not yet well
known. Many factors contribute to the development of ESCC,
including nitrosamine carcinogens; cigarette smoking, alcohol
drinking, and genetic variation may be major risk factors of ESCC
[4-7].
Recent genetic association studies on cancer risk have focused
on assessing effects of single nucleotide polymorphisms (SNPs) in
candidate genes, among which DNA repair genes are increasingly
studied because of their pivotal role in maintaining genome
integrity. Sequence variants in DNA repair genes are thought to
modulate DNA repair capacity and consequently are suggested to be
associated with altered cancer risk [8].
Among these, X-ray repair cross-complementing groups 1 (XRCC1)
and excision repair cross-complementing group 2 (ERCC2, also known
as XPD) are DNA-repairing enzymes, located in chromosome 19. The
XRCC1 gene product, a scaffolding protein, is thought to play a
critical role in the DNA single-strand break repair and base
excision repair pathway. The ERCC2 protein is absolutely necessary
in nucleotide excision repair pathway that is responsible for bulky
adducts and repair of UV-induced DNA damage [9].
So far, only relatively small study is reported about the
association between XRCC1 codon 280 polymorphisms and ESCC [10].
Some valuable studies on the association of ERCC2 codon 751 and
ESCC have been reported. Of these, however, ERCC2 codon 751 was
researched among different populations or races, but the conclusion
was various [11-13]. So with the help of these studies, we examined
the association between these two polymorphisms and ESCC risk in a
hospital-based, case-control study of 200 ESCC cases and 200
controls of a population of Luoyang, the high-incidence regions of
esophageal cancer in the West part of Henan Province of China.
Materials and methods
Study population
The case group consisted of 200 diagnosed patients with
histologically confirmed ESCC in Luoyang, Henan Province, China,
between 2006 and 2008. All study subjects were ethnically
homogeneous Han nationality and permanent residents of Luoyang.
Patients with secondary and recurrent tumors were excluded. The
control group comprised 200 healthy volunteers who visited the
general health check-up during the same period and were frequently
matched to the cases by age, gender, alcohol intake and smoking
status. Selection criteria for controls were no evidence of any
personal or family history of cancer. These cancer-free controls
were genetically unrelated to the cases and all subjects were of
the Han ethnic group. Data on age, gender, alcohol intake and
smoking status and amount were derived from questionnaires by two
trained investigators. Among the cases and controls, the average
age was 61.4 ± 9.7 and 61.3 ± 9.5, respectively. After adjustment,
there were no difference of age, sex, alcohol intake and cigarette
smoking use between the case group and control group. Written
informed consent was obtained from all the subjects, and the study
was performed with the approval of the ethics committee of Chinese
Human Genome.
Genotyping
Genomic DNA was extracted from peripheral blood by proteinase K
digestion and phenol/chloroform extraction. The XRCC1 codon
Arg280His and ERCC2 codon Lys751Gln polymorphisms were determined
by polymerase chain reaction (PCR) restriction fragment length
polymorphism (PCR-RFLP) analysis. Primer sequences and reaction
conditions are shown in table 1.
Table 1 Primer sequences and reaction conditions for
XRCC1 and ERCC2 polymorphisms.
|
Gene locus
|
Primer sequence
|
Annealing temperature (°C)
|
Restriction enzyme
|
|
Arg280His
|
F:5′-CCAGTGGTGCTAACCTAATC-3′
|
59
|
RsaI
|
|
R:5′-CCTACATGAGGTGCGTGCTGT-3′
|
|
|
|
Lys751Gln
|
F:5′-TCAAACATCCTGTCCCTACT-3′
|
62
|
PstI
|
|
R:5′-CTGCGATTAAAGGCTGTGGA-3′
|
|
|
Statistical analysis
Genotype and allele frequencies of XRCC1 gene and ERCC2 gene were
compared between ESCC cases and controls using the χ2
test and Fisher’s exact test when appropriate, and odds ratios (OR)
and 95% confidence intervals (CIs) were calculated to assess the
relative risk conferred by a particular allele and genotype.
Hardy-Weinberg equilibrium was tested for with a goodness of fit
χ2 test with one degree of freedom to compare the
observed genotype frequencies among the subjects with the expected
genotype frequencies. Statistical significance was assumed at the P
< 0.05 level. The SPSS statistical software package version 11.5
was used for all of the statistical analysis.
Results
The study was performed on a series of 200 ESCC patients and 200
controls. To find potential associations, we tested each single
variant as well as each pair of variants against cases and
controls. The genotype and allele frequencies of XRCC1 codon 280
and ERCC2 codon 751 polymorphisms between the controls and the
cases are shown in table 2. The genotype
distributions were in Hardy-Weinberg equilibrium in each group
studied. The frequencies of the Arg/Arg, Arg/His, and His/His
genotypes of XRCC1 codon 280 were 78.0, 21.0, and 1.0% in controls,
and 76.0, 22.5, and 1.5% in cases, respectively. The frequencies of
Arg and His alleles of XRCC1 codon 280 were 88.5 and 11.5% in
controls, and 87.3 and 12.7% in cases, respectively. The
frequencies of the Lys/Lys, Lys/Gln, and Gln/Gln genotypes of ERCC2
codon 751 were 74.0, 25.5, and 0.5% in controls, and 83.5, 15.5,
and 1.0% in cases, respectively. The frequencies of Lys and Gln
alleles of ERCC2 codon 751 were 86.8 and 13.2% in controls, and
91.3 and 8.7% in cases, respectively.
Table 2 showed that individuals with
the heterozygous Lys/Gln of ERCC2 Lys751Gln were more prevalent in
controls (25.5%) than in patients (15.5%), but the Gln/Gln genotype
frequency was rare in this investigation. The Gln allele was
associated with a borderline decrease in the risk of ESCC ([OR] =
0.628; 95% [CI] = 0.400-0.986). The genotype frequency of XRCC1
Arg280His polymorphisms was similar between cases and controls in
this investigation. No significant association of Arg280His
polymorphisms with ESCC risk was found (P > 0.05, OR = 1.125;
95% CI = 0.735-1.720).
In addition, we analysed the genotype distribution about the two
groups’ characteristics and found the difference was not
significant (data not shown).
Table 2 The genotype and allele frequencies of two
polymorphisms of XRCC1 and ERCC2 gene between ESCC patients and
controls.
|
Genotype
|
Controls (N = 200) (%)
|
Cases (N = 200) (%)
|
OR (95% CI)
|
|
XRCC1 Arg280His
|
|
Arg/Arg
|
156 (78.0)
|
152 (76.0)
|
1.00
|
|
Arg/His
|
42 (21.0)
|
45 (22.5)
|
1.10 (0.68-1.77)
|
|
His/His
|
2 (1.0)
|
3 (1.5)
|
|
|
Arg allele frequency
|
354 (88.5)
|
349 (87.3)
|
1.00
|
|
His allele frequency
|
46 (11.5)
|
51 (12.7)
|
1.125 (0.735-1.720)
|
|
ERCC2 Lys751Gln
|
|
Lys/Lys
|
148 (74.0)
|
167 (83.5)
|
1.00
|
|
Lys/Gln
|
51 (25.5)
|
31 (15.5)
|
0.539 (0.327-0.887)
|
|
Gln/Gln
|
1 (0.5)
|
2 (1.0)
|
|
|
Lys allele frequency
|
347 (86.8)
|
365 (91.3)
|
1.00
|
|
Gln allele frequency
|
53 (13.2)
|
35 (8.7)
|
0.628 (0.400-0.986)
|
Discussion
Maintenance of the genomic integrity by DNA repair genes is an
essential component of normal cellular growth and differentiation
[14, 15]. The ability to monitor and repair carcinogen-induced DNA
damage is an important determinant of susceptibility to
carcinogenesis [16]. Considerable evidence showed that reduced DNA
repair capacity might play a role in cancer development.
Hundreds of research articles worldwide have reported the
relationship of DNA repair gene (such as XRCC1, XRCC2, and ERCC2,
etc.) polymorphisms and various cancer risks. Among them, XRCC1
Arg280His and ERCC2 Lys751Gln were frequently analysed in different
cancer types. In our study, the two polymorphisms chosen were
analysed in the search for association with ESCC.
Our results tested the hypothesis that polymorphisms in XRCC1
and XPD are involved in the susceptibility to ESCC in a Chinese
population, and the study for the XPD Lys751Gln polymorphism showed
an association with ESCC and the presence of the XPD 751Gln allelic
decreased the risk for ESCC (P < 0.05, OR = 0.628; 95% CI =
0.400-0.986). We found that no statistical differences were
observed for XRCC1 280His. (P > 0.05, OR = 1.125; 95% CI =
0.735-1.720).
Previous molecular epidemiological studies have found that the
XPD 751Gln allele is associated with increased risks for head and
neck cancer [17], melanoma skin cancer [18] and lung cancer [19,
20]. Also, a number of studies have investigated the role of the
ERCC2 Lys751Gln polymorphism in the etiology of esophageal cancer.
Ye et al., Liu et al. and Tse et al. [21-23]
reported that variant genotypes of Lys751Gln polymorphism were
associated with a higher risk of esophageal adenocarcinoma. Yu
et al. [12] reported that genotype Gln/Gln was associated with
an increased risk of ESCC in a Chinese population (Hubei, China).
In Wang’s analysis, small associations of the XPD Lys 751 Gln
polymorphism with cancer risk for esophageal cancer (for Lys/Gln
versus Lys/Lys: OR = 1.34; 95% CI = 1.10-1.64; for Gln/Gln versus
Lys/Lys: OR = 1.61; 95% CI = 1.16-2.25) are revealed [24]. However,
Ferguson et al. and Doecke et al. reported that there
were no statistically significant associations between these
polymorphisms and risk of esophageal adenocarcinoma [25, 26].
Ranbir reported the ERCC2 (Lys/Gln-Gln/Gln) genotype was not
associated with risk of ESCC in a North Indian population [11].
Xing et al. [27] reported that no significant association
between Lys751Gln polymorphism and the risk of esophageal cancer
was found in a Chinese population (Beijing, China). While a
significant protective effect was observed for the XPD Lys751Gln
homozygous variant (C/C) genotype in patients with esophageal
adenocarcinoma (OR = 0.24; 95% CI = 0.07-0.88) in Canada, and their
results were the same as ours [28]. We found that the Lys/Gln
genotype was associated with a borderline decrease in the risk of
ESCC (OR = 0.539; 95% CI = 0.327-0.887); in addition, we observed a
marginally reduced risk of developing ESCC in individuals with the
allele Gln (OR = 0.628; 95% CI = 0.400-0.986).
As far as we know, for the XRCC1 Arg280His, Moullan et al.
[29] reported that Arg280His polymorphism was significantly
associated with breast cancer risk; Skjelbred et al. [30]
suggest the XRCC1 280His allele was associated with an increased
risk of colorectal adenomas, and Sak et al. [31] found
Arg280His was marginally associated with increased bladder cancer
risk, but our results showed that there was no significant
association between Arg280His polymorphism and ESCC risk,
consistent with Lee et al. [10].
From the above statements, we realized that the results were
different even if the targets belonged to the same race. The
obviously conflicting results from previous molecular
epidemiological studies of the association between DNA repair gene
polymorphisms and cancer risk warrant cautious interpretation of
our results. Reasons for the previous inconsistent findings may
include small sample sizes, inappropriate study design and may be
different LD with other SNPs in different populations. So, we
initially concluded that the distribution of the XRCC1 Arg280His
and ERCC2 lys751Gln polymorphisms in the ESCC cases was more
dependent on the environmental, geographical regions or habitual
factors of life than on the race of people.
This study has some limitations. One is that detailed
information on the survival of ESCC was not available, which
restricted our further analysis on the role of the XRCC1 and ERCC2
in cancer prognosis. Another is that the association between XRCC1
and ERCC2 gene polymorphisms and ERCC should be tested in groups of
different ethnically disparate populations. The third is that the
study sample size is relatively small; thus chance findings cannot
be excluded. Further, we did not have enough power to detect a
modest excess risk.
In summary, in this case-control study, we found that ERCC2
lys751Gln polymorphisms was linked to the risk of developing ESCC,
and the XRCC1 Arg280His polymorphism was not significantly
associated with it. Future studies should be based on larger
samples and needed to better understand the different pathways and
factors (the environmental factors especially) contributing to
these associations. Further studies are warranted to confirm this
finding.
Acknowledgments
We thank all patients and controls who agreed to join our study.
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