Endometrial cancer is one of the most frequent gynecological malignancies in the world.1 Although the incidence in Asian nations, including Japan, is lower than that in industrialized Western nations,2 the most recent epidemiological data show that there has been a marked increase in the age-standardized incidence rate in Japan from 3.9 in 1990, 5.1 in 2000, to 8.1 in 2007.3 Major risk factors of endometrial cancer are as follows: obesity, nulliparity, late menopause, exogenous unopposed estrogen, tamoxifen, diabetes, hypertension, high dietary fat consumption, radiation therapy, and hereditary nonpolyposis colorectal cancer.4
Much of the risk for endometrioid carcinoma can be attributed to exposure to unopposed estrogen resulting primarily from unopposed postmenopausal estrogen therapy and obesity4,5; however, a large proportion of endometrial cancer cases cannot be completely explained by these factors. Recent technological advances in genotyping enable researchers to uncover the association between single nucleotide polymorphisms (SNPs) and susceptibility to diseases, including cancer. An important aspect of the application of these techniques has been the discovery of genetic variation in DNA repair genes, which results in defects in DNA repair and is related to the risk for certain cancers.6,7 Evidence exists to indicate that polymorphisms in DNA repair genes associate with endometrial cancer.8–11 In contrast, the association of polymorphisms in DNA repair genes with endometrial cancer in the Japanese population remains to be investigated in detail.
Here, we conducted a hospital-based case-control study to examine the association between various polymorphisms in base excision repair (BER) DNA pathway genes (OGG1, MUTYH, XRCC1, APEX1, and PARP1) among Japanese postmenopausal women with and without endometrial cancer. We also determined whether these associations were influenced by obesity and gene-gene interactions.
MATERIALS AND METHODS
The subjects included 91 postmenopausal patients newly and histologically diagnosed with endometrial carcinoma between January 2001 and November 2005 at the Aichi Cancer Center Hospital in Japan. The distribution of histological subtypes among cases was 59 with endometrioid adenocarcinoma (64.8%), 26 with other types of carcinoma (28.6%), and 6 with tumors that we were unable to identify (6.6%). Controls (n = 261) were randomly selected from 11,814 women who were diagnosed as cancer-free at the Aichi Cancer Center Hospital between January 2001 and November 2005 and who were also postmenopausal. All subjects were recruited within the framework of the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), as described elsewhere.12–14 In brief, we asked all first-visit outpatients aged 20 to 79 years to provide information on lifestyle factors using a self-administered questionnaire and a sample of blood. The patients were also asked about their lifestyle when healthy or before the current symptoms developed. A trained interviewer checked responses. Our previous study showed that the lifestyle patterns of first-visit outpatients were comparable with a randomly selected sample of the population of Nagoya City.15 All participants provided written informed consent, and the Institutional Ethical Committee of Aichi Cancer Center approved this study.
Smoking and drinking habits were categorized as never, former, and current. Former smokers and drinkers (designated “ever-smokers” and “ever-drinkers”) were included with current smokers and drinkers for analysis. Body mass index (BMI) at enrollment was calculated from self-reported height and weight as BMI (kg/m2) = weight (kg)/height (m2). In the present study, regular exercise was defined as at least 1 session each month.
Genotyping of DNA Repair Genes
DNA was extracted from the buffy coat fraction of blood samples using a Blood Mini Kit (Qiagen KK, Tokyo, Japan) and analyzed using the polymerase chain reaction (PCR) TaqMan method with 7500 Real-Time PCR System (Applied Biosystems).16 The probes used were designed to detect rs1052133 (OGG1), rs36053993 (MUTYH), rs1799782 (XRCC1), rs25487 (XRCC1), rs1130409 (APEX1), and rs1136410 (PARP1). Genotyping was conducted in duplicate in cases that deviated from the Hardy-Weinberg equilibrium (HWE). Linkage disequilibrium (LD) was evaluated by means of LD coefficients (D’).
To assess the strength of associations between the polymorphisms and risk for endometrial cancer, odds ratios (ORs) with 95% confidence intervals (CIs) per allele were estimated using unconditional logistic models adjusted for potential confounders. Potential confounders considered in the multivariate analyses were age, smoking (never or ever), consumption of alcohol (never or ever), current BMI (<25 or ≥25 kg/m2), regular exercise (yes or no), age at menarche (≤12, 13–14, or ≥15 years), age at menopause (≤39, 40–54, or ≥55 years), parity (0, 1–2, or ≥ 3), history of diabetes (yes or no), history of hypertension (yes or no), use of contraceptives (yes or no), and hormone replacement therapy (yes or no). Differences in categorized demographic variables between the cases and the controls were determined using the χ2 test. Age, BMI, age at menarche, age at menopause, and parity between the cases and the controls were compared using the Mann-Whitney U test. To explore the interaction between current BMI and each locus, we conducted further analysis stratified by BMI (<25 or ≥25 kg/m2) based on our previous study.12 Furthermore, we evaluated the interactions between XRCC1 haplotype and current BMI and rs1136410 (PARP1). These were assessed by logistic regression models including score for current BMI (<25, 0; ≥25, 1), score for each locus (major homozygote, 0; heterozygote, 1; minor homozygote, 2), score for XRCC1 haplotype (haplotype CG, 0; CA, 1; TG, 2), and their interaction terms.
Genotypes were designated as scores. We applied a retrospective profile-likelihood method in logistic regression to estimate haplotype effects using the “haplologit” command.17 P values less than 0.05 were considered statistically significant. All analyses were conducted using STATA version 10.1 (Stata Corp, College Station, TX). Linkage disequilibrium estimates were calculated using STATA.
Baseline characteristics of the 91 endometrial cancer patients and the 261 controls are shown in Table 1. The median age for the cases was lower than that for the controls (P = 0.003). In contrast, current BMI was significantly higher than in the controls (P = 0.008). Smoking was more common among the cases. Alcohol consumption did not differ significantly between the groups. With respect to the subjects’ reproductive history, low parity was more prevalent (P = 0.016) and age at menopause was higher among the cases than the controls (P = 0.002). A history of hormone replacement therapy was more common in the cases. There were no significant differences between the groups in their histories of diabetes, hypertension, and contraceptive use.
The distributions of genotypes of 6 DNA repair gene polymorphisms and their impacts on endometrial cancer risk are shown in Table 2. Genotype distributions of each locus among the controls were in accordance with the HWE, without taking into account the data for OGG1 and MUTYH. In the per-allele model, the significant associations between endometrial cancer risk and XRCC1 are as follows: rs1799782 (C > T, Arg194Trp) (CT: OR, 1.79; 95% CI, 1.01–3.18; TT: OR, 2.87; 95% CI, 1.25–6.64; trend P = 0.004) and XRCC1 rs25487 (G > A, Arg399Gln) (GA: OR, 0.64; 95% CI, 0.37–1.11; AA: OR, 0.14; 95% CI, 0.02–1.12; trend P = 0.008).
Table 3 shows the interaction between current BMI (≤25 or >25) and each locus. A significant interaction between BMI and XRCC1 rs25487 was observed (interaction P = 0.048).
Table 4 shows the impacts of XRCC1 haplotype rs1799782 and rs25487 on the risk for endometrial cancer, which were in complete LD (D′ = 1.00), and specifically in relation to BMI (<25 or >25) and PARP1 rs1136410 polymorphism (TT or TC/CC). The XRCC1 TG haplotype was significantly associated with the increased risk for endometrial cancer (OR, 1.49; 95% CI, 1.02–2.18). In contrast, only a marginally significant interaction between the XRCC1 CA haplotype and current BMI was observed (interaction P = 0.087). The postmenopausal women with XRCC1 TG haplotype and PARP1 rs1136410 C allele had a significantly increased risk (OR, 1.63; 95% CI, 1.01–2.62). However, a significant interaction between XRCC1 haplotype and PARP1 rs1136410 polymorphism was not detected.
Here, among the selected polymorphisms in genes involved in the BER pathway, our analyses revealed a significant association between polymorphisms in XRCC1 and endometrial cancer risk in postmenopausal Japanese women. The XRCC1 polymorphisms rs1799782 and rs25487 in XRCC1 were in complete LD, and the TG haplotype was significantly associated with endometrial cancer. Only a marginally significant interaction between the CA haplotype and current BMI was observed, whereas an interaction between XRCC1 haplotype and PARP1 rs1136410 was not significant.
This is the first study, to our knowledge, to explore the associations of genetic variation in BER genes and endometrial cancer in a Japanese population. Evidence indicates that estrogens and their metabolites damage DNA by the formation of bulky DNA adducts,18 which are repaired by the nucleotide excision repair pathway. Therefore, earlier studies examined the association between the genetic variations in nucleotide excision repair pathway and endometrial cancer.8
The BER pathway functions to repair small lesions in DNA, such as oxidized or reduced bases and fragmented or nonbulky adducts.6 There is extensive evidence indicating that the accumulation of oxidative damage to DNA leads to genomic instability, resulting in cancer.19 However, there are relatively few biological and epidemiological studies that focus on the role of BER in endometrial carcinogenesis. For example, Samulak et al9 concluded that the XRCC1 Arg399Gln polymorphism may be associated with the incidence of sporadic endometrial cancer in Polish women, and Cincin et al10 reported that the XRCC1 Gln399Gln and HOGG1 Ser326Cys genotypes increase the risk for type I endometrial cancer in Turkish women. In another study of Polish women, Sobczuk et al11 found that gene-gene interaction between the ERCC2 751Gln and XRCC1 194Trp variants increased the risk for endometrial cancer.
To investigate the association between endometrial cancer and polymorphisms in the BER pathway (OGG1, MUTYH, XRCC1, APEX1, and PARP1) in Japanese women, we selected 6 coding SNPs. Our findings suggest that XRCC1 polymorphisms play an important role in endometrial cancer among postmenopausal women. XRCC1 encodes a scaffolding protein that plays a pivotal role in the BER pathway by forming a complex with PARP, DNA ligase 3, and DNA polymerase β.20 Functional studies on XRCC1 polymorphisms are limited; however, XRCC1 399Gln (rs25487, Arg399Gln) in human cells might be associated with higher sensitivity of DNA damage after exposure to various mutagens, and XRCC1 194Trp (rs1799782, Trp194Arg) seems to have lower mutagen sensitivity in vitro.7 Although these common nonsynonymous SNPs in XRCC1, including rs25487,21,22 rs25489,21 and rs1799782,21,22 have been studied extensively in various cancers, the evidence is inconsistent regarding whether these are etiologically associated. Meta-analyses of these XRCC1 polymorphisms and cancer risk demonstrate that an A allele carrier of rs25487 is likely to have an increased risk for bladder,6 breast,6,23 lung,23 and colorectal cancer.24 In contrast, individuals carrying the T allele of rs1799782 are at decreased risk for skin7 and upper digestive tract cancer23 as well as all types of cancers.24,25 Because these findings are inconsistent with those reported here, further investigation of larger populations and functional studies are warranted. Alternatively, this apparent discrepancy could be explained by our selection of Japanese subjects.
The interaction between rs25487 in XRCC1 and current BMI was significant in our study. Obesity is an established risk factor of endometrial cancer, and it affects the synthesis and the bioavailability of endogenous sex steroid hormones, mainly estrogens.5 It is known that the levels of markers of oxidative stress correlated significantly with BMI and that excess estrogen exposure leads to the elevated generation of reactive oxygen species.26–28 Moreover, Dai et al29 demonstrated a significant interaction of oxidative stress markers and BMI on breast cancer risk among Chinese women, although endometrial cancer was not addressed in this study.
In contrast, a gene-gene interaction between XRCC1 haplotype and PARP1 polymorphism was not significant in our present study. However, on the basis of their mechanisms of action19,20 and studies of genetic associations,30 an interaction between XRCC1 and PARP1 is biologically plausible.20 Polymorphisms in BER genes and their interactions might represent a target for further biological and epidemiological research on the carcinogenesis of endometrial cancer.
Potential limitations of our study warrant consideration. First, we selected 6 coding SNPs in 5 BER genes on the basis of previous studies. Work is now in progress in our laboratory to search for other BER gene polymorphisms that might be relevant to the etiology of endometrial carcinogenesis. Second, our study design represents a hospital-based case-control study. However, we previously examined the external validity of the HERPACC study.15 Further, the HERPACC system is less prone to intrinsic information bias because we collected data before the diagnosis of the subjects. Finally, the sample size of our study was relatively small, and our study was conducted within central Japan. Therefore the HWE of OGG1 rs1052133 could be disturbed. We plan to conduct studies of larger populations in Japan. Consequently, we could consider the difference between histological subtype. In our preliminary stratified analysis by histological subtype, we observed the same trend (data not shown) despite the small sample size. A larger sample is required to clarify the difference between histological subtype.
In conclusion, our research reveals a significant association between XRCC1 rs1799782 and rs25487 and endometrial cancer risk in postmenopausal Japanese women. XRCC1 rs1799782 and rs25487 were in complete LD. The TG haplotype was significantly associated with endometrial cancer. In contrast, a marginally significant interaction between the CA haplotype and current BMI was observed, but we did not detect a significant interaction between XRCC1 and PARP1 polymorphisms. Our study adds to the evidence supporting a role for polymorphisms in DNA repair genes in the etiology of endometrial cancer.
The authors thank the administrative staff at the Division of Epidemiology and Prevention at Aichi Cancer Center Research Institute for their support of this study.
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