Single Nucleotide Polymorphisms of DNA Base-excision Repair Genes ( APE 1 , OGG 1 and XRCC 1 ) Associated with Breast Cancer Risk in a Chinese Population

Breast cancer is the most frequently diagnosed cancer in women and the leading cause of cancer death in females worldwide (Jemal et al., 2011). Numerous factors have been associated with the increased risk of breast cancer, including ionizing radiation (IR), heterocyclic aromatic amines, alcohol, reactive oxygen radicals, bulky DNA adducts, oxidized DNA bases, and DNA strand breaks (Hu et al., 2002; Smith et al., 2003; Dumitrescu 2005). Mammalian cells utilize different pathways to repair diverse types of DNA damage, and thus conserve genome stability and integrity. Faulty DNA repair machinery might contribute to insufficient repair of damaged DNA and subsequent genomic instability, which may lead to deletions, amplifications, and/or mutations of crucial genes, leading to breast carcinogenesis (Parshad et al., 1996). However, data from previous studies suggest that proteins encoded by various polymorphic variants of DNA repair genes might vary in their properties and activities, and thus possibly link to individual differences in cancer


Introduction
Breast cancer is the most frequently diagnosed cancer in women and the leading cause of cancer death in females worldwide (Jemal et al., 2011). Numerous factors have been associated with the increased risk of breast cancer, including ionizing radiation (IR), heterocyclic aromatic amines, alcohol, reactive oxygen radicals, bulky DNA adducts, oxidized DNA bases, and DNA strand breaks (Hu et al., 2002;Dumitrescu 2005). Mammalian cells utilize different pathways to repair diverse types of DNA damage, and thus conserve genome stability and integrity. Faulty DNA repair machinery might contribute to insufficient repair of damaged DNA and subsequent genomic instability, which may lead to deletions, amplifications, and/or mutations of crucial genes, leading to breast carcinogenesis (Parshad et al., 1996). However, data from previous studies suggest that proteins encoded by various polymorphic variants of DNA repair genes might vary in their properties and activities, and thus possibly link to individual differences in cancer Cancer Center,Daping Hospital and Research Institute of Surgery,Third Military Medical University,Chongqing,China *For correspondence: dongwang64@hotmail.com Hao Luo, Zheng Li, Yi Qing, Shi-Heng Zhang, Yu Peng, Qing Li, Dong Wang* susceptibility (Goode et al., 2002;Misra et al., 2003).
Identifying an association between single nucleotide polymorphisms (SNPs) in candidate genes and the risk of human cancers has been a popular area of research in molecular cancer epidemiology, and genes involved in DNA repair have been increasingly studied because of their critical roles in maintaining genomic integrity (Hoeijmakers, 2001;Wood et al., 2005). The efficient and potent base excision repair (BER) pathway plays a major role in maintaining integrity of the genome by correcting somatic mutations induced by endogenous free radicals produced during cellular metabolism or by exogenous exposure to chemicals and ionizing radiation (Nock et al., 2006).
Human BER pathway consists of at least 11 DNA damage specific glycosylases and additional core proteins, each with a specialized function. Among these proteins, 8-oxoguanine DNA glycosylase (OGG1), apurinic/ apyrimidinic endonuclease 1 (APE1), and X-ray repair cross-complementing group 1 protein (XRCC1) are three key enzymes in this repair pathway (Hung et al., 2005;Robertson et al., 2009). BER pathway activity begins with recognition and excision of a damaged base by the specific DNA glycosylase. For example, OGG1 recognizes and removes an oxidized 8-oxoguanine base by releasing the modified base, and thus creating an apurinic/apyrimidinic site. This abasic site is then excised by APE1, leaving behind a 5'-deoxiribose phosphate residue. Subsequently, this residue is removed by the AP-lyase activity of DNA polymerase β, and a correct nucleotide is inserted. Finally, DNA ligase DNA III seals the nick in the DNA strand to restore the integrity of DNA double strand. The XRCC1 gene encodes a protein that acts as a scaffolding protein by interacting with a complex of DNA repair proteins, including poly (ADP-ribose ADP) polymerase, DNA ligase III, and DNA polymerase β, and coordinates the gap-sealing process in the short-batch BER (Petermann et al., 2006;Maynard et al., 2009).
Polymorphic variants of OGG1, XRCC1, and APE1 genes have been reported to be responsible for functional changes at the protein level which may be related to cancer risk (Vodicka et al., 2007;Baute and Depicker, 2008), including nasopharyngeal carcinoma (Li et al., 2013), bladder cancer (Mittal et al., 2012), lung cancer (Osawa et al., 2010), esophageal cancer (Zhai et al., 2009), and cervical cancer (Li et al., 2012). Similarly, epidemiologic studies have examined the association between these inheritable BER pathway variations and the susceptibility to breast cancer; however, the results have been inconclusive. Although the genetic variants OGG1 Ser326Cys, APE1 Asp148Glu, and XRCC1 Arg399Gln in the BER pathway play critical roles in repairing base damage, few studies have investigated the association between breast cancer risk and these genetic polymorphisms in Chinese women. Furthermore, no studies have been conducted to examine the combined and synergistic effects of these 3 genes in Chinese women. To investigate potential differences in relative breast cancer risk by race, and the association between the genetic variants of these three BER genes and breast cancer risk, we conducted a hospital-based study of 194 patients with incident breast cancer and 245 cancer-free control subjects who were frequency-matched by age, age at menarche, age at menopause, menopausal status, pregnancy, body mass index, and family history of cancer.

Study Subjects
This case-controlled study included 194 new incident breast cancer patients and 245 cancer-free control subjects. All participants were of Chinese Han nationality. The cancer patients had been histologically diagnosed at the Daping Hospital, Third Military Medical University (Chongqing, China) during January 2007 to December 2010, and did not have a reported previous history of cancer. Control subjects were randomly selected among individuals receiving health examinations at the Health Examination Center of the same hospital during the same period; any of these subjects who had a history of cancer were excluded from the study. The control subjects were frequency-matched with the cancer patients based on age (mean age, age at menarche, age at menopause), family history of cancer, residential area, and nationality. The protocol for this study was approved by the ethics committee of Daping Hospital, and written informed consent was obtained from each participant. A structured questionnaire was administered by well-trained interviewers to collect information on demographic and anthropometric characteristics of the enrolled subjects. For purposes of the questionnaire, a family history of cancer was defined as any self-reported cancer in a first-degree relative, including parents, siblings and children.

Blood Sample Processing
Venous blood samples were collected from the antecubital vein of 194 cancer patients and 245 control subjects, and immediately centrifuged at 2000 rpm (10 min, 4 ℃) to remove the serum. Genomic DNA was isolated from peripheral blood leukocytes using a standard phenol-chloroform extraction method (Saxena et al., 2006).

SNPs Selection and Genotyping
Based on the current literature, four common nonsynonymous SNPs of BER genes: OGG1 (rs1052133; Ser326Cys; C/G; in exon 7), XRCC1 (rs25487; Arg399Gln; G/A; in exon 10), APE1 (rs1130409; Asp148Glu; T/G; in exon 5) and the promoter polymorphism of APE1: APE1 (rs1760944; -141T/G; in the promoter region) were chosen    for genotyping. Genetic polymorphisms were detected by PCR-CTPP (PCR with confronting two-pair primers) as previously described (Hamajima, 2001). Primer pairs and product lengths were designed for each allele and the correct allele was identified based on product length. PCR primers were designed based on GenBank reference sequences, and are shown in Table 1. PCR amplification was performed in a 25-µL mixture volume in glass capillaries. The PCR mixtures contained 2 µL of genomic DNA, 0.5 U of TaqMan SNP Genotyping Assay Mix (40x) (Life Technologies, Grand Island, NY, USA), 12.5 pmol of each primer, 1.5 mM MgCl2, 5 µL of PCR buffer (5x), and 0.30 mM of dNTPs. Reaction conditions included an initial denaturation at 95℃ for 10 min, then 30 cycles of denaturation at 95℃ for 1 min, annealing at 64℃ (OGG1 Ser326Cys) or 66℃ (XRCC1 Arg399Gln) or 58 (APE1_141T/G) or 60℃ (APE1 Asp148Glu) for 1 min, and elongation at 72℃ for 1 min. PCR products were analyzed by agarose gel electrophoresis. Genotype results were regularly confirmed by randomly selecting 5% of the samples for direct measurement of DNA sequencing. The results were reproducible and showed no discrepancies in genotyping.

Statistical Analysis
Breast cancer patients were compared with control subjects for basic demographic variables and genotypes. All statistical analyses were performed with SPSS software (v.16.0 for Windows). Differences in categorical variables among demographic variables (including pregnancy, menopausal status, body mass index (BMI), and family history of cancer) between cancer patients and control subjects were compared using Pearson's chisquared (χ 2 ) test. Continuous variables, including age at menarche and age at menopausal were examined using the t-test. Unconditional logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (95% CI) with adjustments for age, age at menarche, pregnancy, menopausal status, body mass index, and family history of cancer. Homozygous common alleles were used as a reference category. The statistical significance of interactions was determined using the likelihood ratio test, which compared logistic models with and without interaction terms. Statistical analyses of polymorphisms of OGG1, XRCC1, and APE1 genes as exposure variables and breast cancer as the dependent variable were performed using multivariate logistic regression analysis. A P value < 0.05 was used to indicate statistical significance in all studies. Deviations in the frequency of each SNP genotype in control subjects, as determined by the Hardy-Weinberg equilibrium (HWE) formulas, were assessed using the χ 2 test. The number of homozygous adverse genotypes per individual was calculated to evaluate the potential combined effects of OGG1 Ser326Cys, XRCC1 Arg399Gln, and APE1 Asp148Glu on breast cancer risk by unconditional logistic regression analysis.

Study Subjects
A total of 439 subjects (194 breast cancer patients and 245 cancer-free control subjects) were enrolled in this study. Characteristics of the study population are shown in Table 2. The cancer patients did not significantly differ from control subjects in terms of age, age at menarche, age at menopause, menopausal status, pregnancy, body mass index, and family history of cancer. Table 3 shows the distributions of OGG1 (Ser326Cys), XRCC1 (Arg399Gln), and APE1 (Asp148Glu;-141T/G) genotypes and allele frequencies among control subjects. The frequency of each genotype in the control population was accordant with the Hardy-Weinberg equilibrium (P > 0.05).

Single Genotype Distribution and Breast Cancer Risk
The genotype distributions and allele frequencies for DNA repair gene polymorphisms in breast cancer patients and control subjects are shown in Table 4. We observed that subjects harboring either heterozygous or homozygous XRCC1 Gln allele showed a significantly increased risk of breast cancer [ (OR = 1.529; 95% CI: 1.012~2.310; P = 0.044); (OR = 2.189; 95% CI: 1.063~4.507; P = 0.033), respectively], when compared to subjects harboring homozygous Arg allele, which was used as a reference. The data were adjusted for age, age at menarche, pregnancy, menopausal status, body mass index, and family history of cancer. A further analysis using the chi-square test demonstrated that when compared with the Arg allele, a significant susceptibility to breast cancer risk was associated with the XRCC1 Gln allele, (OR = 1.403; 95% CI: 1.044~1.886; P = 0.025). The OGG1 Cys/ Cys genotype and APE1 Glu/Glu genotype both showed protective effects against developing breast cancer (OR = 0.693 and OR = 0.78, respectively), though the effects were not statistically significant. Also, there was no statistically significant difference in distribution of the -141T/G polymorphism of the APE1 gene between patients and control subjects.
A subgroup analysis stratified by body mass index (BMI) and menopausal status (Table 5) showed no statistically significant interaction between body mass index or menopausal status and the four BER gene loci polymorphisms. The OGG1 Ser/Cys and OGG1 Cys/Cys genotypes showed a significant protective effect against developing breast cancer for women with a low BMI (< 24 kg/m 2 ), (OR = 0.426, 95% CI 0.200~0.905, P = 0.027, and OR = 0.323, 95% CI 0.146~0.715, P = 0.005, respectively). Moreover, we found that among postmenopausal women, those with the XRCC1 Arg/Gln+ Gln/Gln allele were at a significantly increased risk of breast cancer (OR = 2.432; 95% IC,1.304~4.538;P = 0.005). The increased risk also was observed in women with a high BMI (≥ 24 kg/m 2 ) and allele XRCC1 Arg/Gln+ Gln/ Gln (OR = 1.948; 95% IC 1.169~3.246; P = 0.011). The APE1 -141T/G polymorphism TG+GG showed a protective effect (OR = 0.569) in women with a low BMI (< 24 kg/m 2 ). However, the effect was not statistically significant. Similarly, no statistically significant differences were found in genotype or allele distributions of APE1 Asp148Glu between cancer patients and control subjects in a subgroup analysis.
When analyzing the incidence of different polymorphisms in the study population, we found more than one gene variant in a large number of individuals. Several studies have verified a higher susceptibility to cancer and  :http://dx.doi.org/10.7314/APJCP.2014.15.3.1133 DNA Base-excision Repair Genes andBreast Cancer Risk cancer recurrence with increasing numbers of putative risk alleles Smith et al., 2008;Li et al., 2011). Therefore, we also considered whether gene-gene interactions may increase an individual's susceptibility to cancer. The incidence of the three genotypes in which a single polymorphism may increase the risk of cancer (XRCC1 399Gln, APE1 148Asp, and OGG1 326Ser) is shown in Table 6. Because individuals rarely contain all three of these risk genotypes, individuals with two or three risk alleles were combined into a single group. In our study, we found that breast cancer was associated with pair-wise combinations of these three alleles (XRCC1 399Gln, APE1 148Asp, and OGG1 326Ser), with an OR of 2.183 (95% CI = 1.095~4.353).

Discussion
Breast cancer is one of the most commonly diagnosed cancer and the leading cause of cancer death in females globally. Increasing evidence suggests that insufficient or faulty repair of DNA damage plays an important role in the carcinogenesis of breast cancer (Parshad et al., 1996;Jyothish et al., 1998). Several well established risk factors, including UV light, smoking, dietary factors, exposure to reactive oxygen species and endogenous or exogenous estrogens are thought to be associated with DNA damage. The BER pathway consists of a series of coordinated sequential reactions to recognize and dispose of damage resulting from reactive oxygen species, hydroxylation reactions, and other cellular processes (Krokan et al., 2000;Hung et al., 2005;Hoeijmakers, 2007). Accordingly, SNPs in key repair genes of the BER pathway may impair DNA repair capacity, which may have a subsequent impact on cancer susceptibility and occurrence. In this study we investigated the possible relation between sequence variants in four BER gene loci (OGG1 Ser326Cys, XRCC1 Arg399Gln, APE1 Asp148Glu, and APE1 -141T/ G) and breast cancer risk. We found that Chinese women with the XRCC1 399Gln allele have an increased risk of breast cancer, while the OGG1 326Cys allele confers a significant protective effect against breast cancer in women with a low BMI (< 24 kg/m 2 ). When analyzing the effect of the gene-gene interactions on breast cancer susceptibility, we found that breast cancer susceptibility was associated with at least two risk genotypes (XRCC1 399Gln, APE1 148Asp, and OGG1 326Ser). To the best of our knowledge, this is the first case-controlled study to examine the association between the three BER SNPs and the risk of breast cancer in Chinese women.  The human 8-oxoguanine DNA glycosylase (OGG1) gene is located on chromosome 3p26, in a region that frequently shows a loss of heterozygosity in several human cancers. The product of this gene (8-oxoguanine DNA glycosylase) is a key enzyme involved in removal of 8-oxodeoxyguanosine, which is one of the most common substances produced by oxidative stress, and is highly mutagenic (Rossner et al., 2006;Tudek, 2007). A meta-analysis of 4,963 breast cancer cases and 4,776 control subjects on the role of OGG1 326Cys in breast cancer showed a significant protective effect against breast cancer in European women. However, no significant association between the OGG1 326Cys allele and breast cancer was found among Asian women (Yuan et al., 2010). Additionally, another meta-analysis of OGG1 Ser326Cys, which included data from 11 studies, also failed to observe an association between OGG1 Ser326Cys and breast cancer risk in the in European or Asian subjects or in an analysis stratified by ethnicity, source of control subjects, and menopausal status (Gu et al., 2010). We found that OGG1 Ser326Cys conferred a significant protective effect against breast cancer among women having a low BMI (< 24 kg/m 2 ) and the OGG1 Ser/Cys genotype (P = 0.027) or OGG1 Cys/Cys genotype (P = 0.005). The different findings in these studies may be related to different genetic backgrounds of the subjects and the heterogeneous nature of exposure to breast carcinogens within the study populations. Due to the relatively small sample size and limited demographic characteristics of our study population, the possibility that our results may be related to internal and external exposure to risk factors requires further verification.
The gene for X-ray repair cross-complementing group 1 (XRCC1) has 17 exons which span ~31.9 kb, and is located at chromosome 19q13.2. While the protein encoded by XRCC1 has no known enzymatic activity, it is thought to act as a scaffold protein which coordinates the activities of other proteins such as DNA polymerase β, DNA ligase III, and poly (ADP-ribose) polymerase (PARP), which function at the site of DNA damage (Campalans et al., 2005) by recognizing and binding to single-strand breaks (Dalhus et al., 2009). The XRCC1 Arg399Gln polymorphism is located within the XRCC1 BRCA1 carboxyl-terminal domain (BRCT I) and is suggested to affect protein structure and function. In the population of Chinese subjects, we found that individuals who were either the heterozygous or homozygous 399 codon of XRCC1Gln allele were at a significantly increased risk of breast cancer compared with those harboring the XRCC1-399 Arg allele (P = 0.044; P = 0.033). Furthermore, an analysis using the chi-square test showed a significant susceptibility to breast cancer was associated with the XRCC1 Gln allele, compared to the Arg allele (P = 0.025). A woman's breast cancer risk was also shown to be associated with postmenopausal status and high BMI (≥ 24 kg/m 2 ). In agreement with our findings, a meta-analysis of XRCC1 Arg399Gln conducted using 31 studies, which included 10,465 breast cancer cases and 10,888 control subjects, suggested an increased breast cancer risk with a recessive effect for the Arg399Gln variant in an Asian population (Gln/Gln vs. Arg/Arg+Arg/ Gln: OR = 1.59) (Li et al., 2009). Similarly, another metaanalysis of XRCC1 polymorphisms and their association with breast cancer risk found that the 399Gln allele might act as a recessive allele in its association with breast cancer risk (Saadat and Ansari-Lari, 2009). In addition, this polymorphism has been associated with an increased risk of breast cancer in Caucasian (Roberts et al., 2011), Iranian (Saadat et al., 2008), and Portuguese (Silva et al., 2007) postmenopausal women.
The APE1 gene consists of five exons and four introns, has a 2.21-kb span, is located at chromosome 14q11.2-q12, and encodes a 317 amino acids protein. This protein is the rate-limiting enzyme in the BER pathway (Hoeijmakers, 2001). In addition to its role in DNA repair, APE1/Ref-1 also functions as a redox activator of numerous cellular transcription factors that are thought to be important in carcinogenesis, including AP-1, NF-kB, Myb, HIF-1a, HLF, PAX, and p53 (Ando et al., 2008). Polymorphisms in a promoter region can influence interactions between transcriptional factors and their ability to recognize DNA sequences in a promoter region, and thus affect gene expression. To our knowledge, no studies have yet been conducted to examine a possible link between the APE1-141T/G promoter polymorphism and the risk of breast cancer. In current study, we failed to find an association between the APE1-141T/G promoter polymorphism and breast cancer risk in Chinese Han women. However, other recent studies have investigated the role of this polymorphism in other cancers. Li et al (Li et al., 2011) reported that individuals homozygous for the variant APE-141GG were somewhat protected against lung cancer overall (OR = 0.62), and were particularly protected against lung adenocarcinoma (OR = 0.65). Additionally, a study in glioblastoma showed that individuals homozygous for the 141GG genotype exhibited a 46% reduced risk of glioblastoma compared to individuals who were 141TT homozygous (Zhou et al., 2011). APE1 Asp148Glu variants are the most common APE1 polymorphisms identified in the general population that result in a single amino acid substitution (Hung et al., 2005). Zhang et al. (2006) found no association between the APE1 Asp148Glu variant and breast cancer risk among non-Hispanic white Americans, whereas Suleeporn et al. (2008) reported a significant protective effect of the APE1 148Glu allele in Thai women. Subsequently, a meta-analysis of the APE1 Asp148Glu polymorphism , which included 5 studies with a total of 2,539 breast cancer patients and 2,572 control subjects, showed no obvious association between APE1 148Glu and breast cancer (Wu et al., 2012). Consistent with this result, we also found no significant association between APE1 Asp148Glu polymorphisms and breast cancer in our study.
When more than one genetic variant exist in an individual's BER related genes, it is possible that the total effect of these variants have a significant impact on DNA repair activity and breast cancer risk (Mohrenweiser et al., 2003;. In our study, we analyzed the impact of gene-gene interactions on breast cancer susceptibility, and found that a high risk of breast cancer was associated with at least two risk genotypes (XRCC1 399Gln, APE1 148Asp, and OGG1 326Ser) with OR values of 2.183 (95% CI = 1.095~4.353). This finding suggests that the interaction between these two genotypes in the BER pathway might contribute to a higher susceptibility to cancer. It should be noted that we currently lack a full understanding of how polymorphisms affect gene function, and a comprehensive analysis of polymorphisms in all known BER genes is needed to understand the various roles of the BER genes and their impact on breast cancer risk in more detail.
In conclusion, our study examined the possible correlation between breast cancer risk and polymorphisms of three DNA repair genes (OGG1, XRCC1, and APE1) involved in BER pathways. The results suggest that the XRCC1 399Gln allele is significantly associated with an increased risk for breast cancer in postmenopausal women and women with a high BMI (> 24 kg/m 2 ). Additionally, certain gene-gene interactions may also significantly increase the risk of breast cancer.