GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 19, Number 7, 2015 ª Mary Ann Liebert, Inc. Pp. 387–393 DOI: 10.1089/gtmb.2015.0001
DNA Repair Genes XRCC1 and ERCC1 Polymorphisms and the Risk of Sporadic Breast Cancer in Han Women in the Gansu Province of China Gongjian Zhu,1,2 Lan Wang,1 Hongyun Guo,1 Lingeng Lu,3 Suisheng Yang,1 Tao Wang,1 Huan Guo,1 Haitao Wang,1 Jianping Min,1 Kai Yang,1 Xuezhong Chen,1 Yuanqiang Liu,1 Zhiping Wang,4 and Haixiang Su1
Aims: Polymorphisms in DNA damage repair genes may affect DNA repair capacity and modulate breast cancer susceptibility. In this study, we aimed to analyze two polymorphisms for each of the DNA repair genes X-ray repair cross-complementing group 1 (XRCC1) rs25487 and rs1799782 and excision repair cross-complementing group 1 (ERCC1) rs3212964 and rs11615, to evaluate their associations with the risk of sporadic breast cancer in Han women in the Gansu Province of China. Methods: Genotypes were determined by a polymerase chain reaction-based approach for 101 patients with breast cancer and in 101 disease-free controls. Results: We found that individuals with the AA genotype at XRCC1 rs25487 had a significantly increased risk of breast cancer compared with GG genotype ( p < 0.001, odds ratio [OR] = 6.39, 95% confidence interval [CI]: 2.18–18.65). The dominant model showed that the combined rs25487 genotypes (AA + AG) increased the disease risk ( p < 0.001, OR = 3.17, 95% CI: 1.76–5.72). However, no statistical associations were found between rs1799782 in XRCC1, or rs3212964 and rs11615 in ERCC1 and the risk of disease. In haplotype analysis, the GC haplotype in XRCC1 conferred an increased risk ( p < 0.001) with a 4.78-fold increase for each copy (95% CI: 2.52–8.72). Significant associations were also shown between the single nucleotide polymorphisms (SNPs) and the status of estrogen receptor (ER), progesterone receptor (PR), and HER-2. Conclusions: The results suggest that the XRCC1 rs25487 polymorphism may increase the risk of breast cancer.
Introduction
B
reast cancer is the most common malignancy affecting women, and is the leading cause of cancer-related death in females worldwide (Shu et al., 2003). However, its etiology is extremely complex, and genetic factors are indicated to be associated with the risk (Xu et al., 2012). Recently, genome-wide association studies have suggested that genetic variations may lead to the susceptibility to breast cancer (Easton et al., 2007). DNA damage repair systems are essential to human health in response to endogenous and exogenous carcinogens and mutagens (Berwick and Vineis, 2000). The genes involved in DNA repair and the maintenance of genome integrity play a crucial role in providing protection against mutations that lead to cancer ( Jiricny and Nystrom-Lahti, 2000; Dufloth et al., 2005). Epidemiological studies have shown that the inheritance of genetic variants at
one or more loci results in reduced DNA repair capacity and an increase in the risk of cancer (Terry et al., 2004; Kostrzewska-Poczekaj et al., 2013). There are two major types of DNA repair pathways. For single-strand DNA breaks, these include the base excision repair (BER) and nucleotide excision repair (NER) systems; for double-strand DNA breaks, there are two principle mechanisms: homologous recombination and nonhomologous end joining (Hegde et al., 2008; Jackson and Bartek, 2009; Friedman and Stivers, 2010). It is known that X-ray repair cross-complementing group 1 (XRCC1) is crucial to the BER system, which repairs DNA damage due to ionizing radiation (Schreiber et al., 2006; Smith et al., 2008). Deletion of XRCC1 gene in mice results in an embryonic lethal phenotype (Tebbs et al., 1999). Chinese hamster ovary cell lines with mutations in XRCC1 have shown a reduced ability to repair single-strand DNA breaks and concomitant cellular
1
Gansu Provincial Academy of Medical Sciences, Gansu Provincial Cancer Hospital, Lanzhou, People’s Republic of China. School of Life Sciences, Lanzhou University, Lanzhou, China. 3 Department of Chronic Disease Epidemiology, School of Public Health, School of Medicine, Yale Cancer Center, Yale University, New Haven, Connecticut. 4 Institute of Urology, The Second Hospital of Lanzhou University, Lanzhou, China. 2
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hypersensitivity to ionizing radiation and alkylating agents (Shen et al., 1998). Another important component, excision repair cross complementation 1 (ERCC1), encodes ERCC1 protein involved in the NER system, removing bulky lesions from DNA caused by chemical or physical factors such as toxic chemicals or ultraviolet light (Smith et al., 2008). Single nucleotide polymorphism (SNP) is a single nucleotide change in a DNA sequence, which may change DNA or RNA conformations (Lu et al., 2012, 2013) or lead to amino acid substitution (Roberts et al., 2011), thereby resulting in corresponding phenotypes. SNPs may cause human diseases and/or contribute to the risk of diseases, and their identification may help us discover new ways to diagnose, treat, and prevent human diseases (Ulrich et al., 2003). Given that the importance of DNA repair machinery in human health, understanding the associations between SNPs in the DNA repair-associated genes and the likelihood of developing cancer may have implications in the prevention and diagnosis of cancer (Xing et al., 2002). There are many identified SNPs in the XRCC1 and ERCC1 genes, some of which are reported to be implicated in carcinogenesis (Zheng et al., 2012; Alsbeih et al., 2013; Wang et al., 2013). However, the results are inconsistent in different organs and in different ethnic groups. In this case–control study, the two tag SNPs in each of XRCC1 (rs25487, rs1799782) and ERCC1 (rs3212964 and rs11615) were analyzed, respectively. To the best of our knowledge, until now there have been no reports investigating the association between these SNPs and breast cancer in Han women in the Gansu Province of China. We also evaluated the relationships between the polymorphisms of XRCC1 and ERCC1 and tumor clinicopathological features, including tumor grade, estrogen receptor (ER) and progesterone receptor (PR) status, tumor size, and nodal status in a breast cancer case–control study. Materials and Methods Subjects
This study included 101 breast cancer cases and 101 disease-free controls. All histologically confirmed incident cases (mean age 45.7 – 7.4 years) were recruited from the Department of Breast Surgery of the Gansu Provincial Cancer Hospital. Pathology slides (or tissue blocks) from all patients were obtained from the original pathology departments and reviewed by two independent pathologists. All cases were classified according to the 2001 WHO classification (Alsheikh et al., 2001). Detailed clinical and pathological information on these patients is shown in Table 1. Exclusion criteria included metastasized cancer from other organs and previous radiotherapy or chemotherapy. The controls were frequency matched to cases by age (mean age 45.5 – 7.6 years) and region, and were randomly selected from the volunteers without prior cancer and other major medical conditions during routine medical fitness examination. Both cases and controls were from the Gansu province in northwest China between 2010 and 2012. At recruitment, each subject signed a written informed consent and provided 5 mL of venous blood, which was stored at - 80C. This study was approved by the Gansu Academy of Medical Science and the Cancer Hospital Ethical Review Board.
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Table 1. Clinicopathological Features of Participants Feature Age (years) £ 40 41–50 51–60 > 60 Histological type IDC Intraductal carcinoma Mucinous adenocarcinoma Others Tumor size (cm) TZ £ 2 2 < TZ £ 5 TZ > 5 Unknown LN involvement Positive Negative Unknown ER Positive Negative Unknown PR Positive Negative Unknown P53 Positive Negative Unknown HER-2 Positive Negative Unknown Ki-67 Positive Negative Unknown
Case, n (%) 28 50 19 4
(27.72) (49.50) (18.81) (3.96)
86 1 3 11
(85.15) (0.99) (2.97) (10.89)
7 61 21 12
(6.93) (60.40) (20.79) (11.88)
Control, n (%) 22 58 18 3
(21.78) (57.43) (17.82) (2.97)
22 (21.78) 47 (46.53) 32 (31.69) 56 (55.45) 41 (40.59) 4 (3.96) 51 (50.50) 47 (46.53) 3 (2.97) 49 (48.52) 44 (43.56) 8 (7.92) 23 (22.77) 74 (73.27) 4 (3.96) 91 (90.10) 5 (4.95) 5 (4.95)
ER, estrogen receptor; HER-2, human epidermal growth factor receptor 2; IDC, infiltrating duct carcinoma; LN, lymph node; PR, progesterone receptor; TZ, tumor size.
SNP selection and genotyping
All SNPs in the candidate genes were selected from HapMap CHB database (phase 2, build 35) (www.hapmap.org) using the Tagger program implemented in HaploView version 4.1. The tag SNPs were chosen based on the following criteria: (1) minor allele frequency ‡ 5%; (2) pair-wise c2 ‡ 0.8; (3) spanning 2 kb upstream of the 5¢ end and 1 kb downstream of the 3¢ end of each gene. The XRCC1 rs25487 and rs1799782, ERCC1 rs3212964 and rs11615 were finally selected. Genomic DNA was extracted from frozen whole blood using the universal genomic DNA Extraction Kit VER.3.0 (Biotech). Genotyping was performed using the polymerase chain reaction (PCR)–restriction fragment length polymorphism method. The primer sequences of the four SNPs were
XRCC1 AND ERCC1 POLYMORPHISMS AND THE RISK OF BREAST CANCER
rs25487 (F: 5¢-TTGTGCTTTCTCTGTGTCCA-3¢, R: 5¢TCCTCCAGCCTTTTCTGATA-3¢), rs1799782 (F: 5¢-ATG AGAGCGCCAACTCTCTGAGGTC-3¢, R: 5¢-GCTGAG CCCCAAGACCCTTTCACTC-3¢), rs3212964 (F: 5¢-TCAC ATCCTCTCTCCCGTAGGGATC-3¢, R: 5¢-AGAGGAGGG ACTGAGCCT-3¢), rs11615 (F: 5¢-TGTGGGAAGAGGT GCGA-3¢, R: 5¢-CCAGCACATAGTCGGGAATTGC-3¢). The polymorphic region was amplified by PCR using a S1000 Thermal Cycler (BIO-RAD) in a 25 mL reaction solution containing 0.2 mg of genomic DNA, 12.5 mL of 2 · GoTaq Green Master Mix (Promega Corporation), and a pair of primers at a final concentration of 100 nM. Annealing temperatures were as follows: rs25487 at 58C, rs1799782 at 58.3C, rs3212964 at 58.3C, and rs11615 at 54.9C. The lengths of the PCR products were 615bp (rs25487), 341bp (rs1799782), and 214bp (rs3212964 and rs11615). The PCR products were digested with restriction enzymes (Promega) according to the manufacturer’s instructions and analyzed using 3% agarose gel electrophoresis. Restriction enzymes of each SNP were MspI (rs25487 and rs1799782), TaqI (rs3212964), and TaiI (rs11615). The lengths of the digested fragments of each SNP were rs25487 (G: 375 + 240 bp, A: 615 bp); rs1799782 (C: 297 + 44 bp, T: 341 bp); rs3212964 (G: 190 + 24 bp, A: 214 bp); rs11615 (C: 173 + 41 bp, T: 214 bp). To validate the accuracy of the genotyping results, 10% samples of each SNP were randomly selected to be tested twice by different persons. Additionally, 5% random samples of each SNP were confirmed by direct sequencing, and the reproducibility of both was 99%. Determination of clinicopathological features
Histology, grade, and hormone receptor status of cases were assessed using immunohistochemistry. Tumors with an Allred score ‡ 3 and nuclear staining of more than 10% of the tumor cells were classified as ER-positive or PR-positive (Ishikawa et al., 2011). P53 was defined as positive when more than 25% (median value) of the tumor cells were positive for nuclear staining. For HER-2 expression, four grades were evaluated based on the percentage of positive cells and color intensity: 0, colorless or < 10% cells with membrane staining; 1 + , > 10% cells with discontinuous membrane staining; 2 + , 10–30% cells with intact membrane staining; 3 + , > 30% cells with strong membrane staining. Samples with scores of 0 and 1 + were considered as negative and 3 + samples were considered as positive. Samples with scores of 2 + were subjected to further analysis by fluorescence in situ hybridization assay to confirm HER-2 expression. Ki67 status was expressed in terms of the percentage of positive cells with a threshold of 14% (Cheang et al., 2009). Statistical analysis
Genotype frequencies of SNPs were tested for Hardy– Weinberg equilibrium (HWE). We used HaploView 4.1 and Phase 2.1 to tag all common haplotypes and their frequencies in cases and controls. Associations between four SNPs and breast cancer risk were estimated by odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression with adjustment for age. The associations were also analyzed by different genetic models (codominant, dominant, and recessive models). In the codominant model, homozygotes for the major allele were the reference group,
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and heterozygotes and minor allele homozygotes were compared to the reference group. In the dominant model, the combination of heterozygotes and minor allele homozygotes were also compared to the reference group. The recessive model was run with minor allele homozygotes versus heterozygotes and major allele homozygotes. A Bayesian approach was applied to reconstruct haplotypes and estimate their frequencies. The associations between clinical features and the haplotypes were analyzed by using the Chi-square test. Unconditional logistical regression model was used to estimate ORs and their 95% CIs by treating each haplotype as a continuous variable. Bonferroni correction was used to adjust for the multiple comparisons. A cutoff of 0.0125 (0.05/4 = 0.0125) was used for the significance of a p-value in the multiple comparison. Statistical analyses were performed using SPSS 16.0 software. Results
The genetic models of four SNPs are shown in Table 2. No deviation from HWE was found in the genotype distributions of the four SNPs in controls ( p > 0.05). XRCC1 and ERCC1 gene polymorphisms and the risk of breast cancer
In XRCC1 rs25487, compared with GG genotype, AA genotype showed an association with a significantly increased risk of breast cancer ( p < 0.001, OR = 6.39, 95% CI: 2.18–18.65). Moreover, in the dominant model, combined rs25487 genotypes (AA + AG) conferred an increased risk of breast cancer ( p < 0.001, OR = 3.17, 95% CI: 1.76–5.72). This association still remained statistically significant after the correction using the Bonferroni test (corrected p < 0.01 and p < 0.01, respectively). However, no association with the risk of breast cancer was observed for rs1799782 in XRCC1 and other two SNPs (rs3212964 and rs11615) in ERCC1. XRCC1 and ERCC1 gene polymorphisms and clinicopathological features
In the cases, we also analyzed the associations of XRCC1 and ERCC1 polymorphisms with clinicopathological features, including lymph node metastasis, tumor size, and the statuses of ER, PR, HER-2, and P53. The results are shown in Table 3. We found in rs3212964, women carrying AG genotype were less likely to have ER-positive and P53 status tumors compared with women with GG genotype ( p = 0.02, p = 0.04, respectively), and the rs3212964 was associated with P53 status in the recessive model ( p = 0.04). However, these associations were not statistically significant after the Bonferroni correction. In rs11615, compared with the CC genotype, CT genotype had a lower frequency in both ER- and PR-positive cases ( p = 0.003; p = 0.001, respectively), and significant association was also found in the dominant model of both ER- and PRpositive cases ( p = 0.002; p < 0.001, respectively). Furthermore, the rs25487 was associated with HER-2-positive in the recessive model ( p = 0.009). After the Bonferroni correction, these associations were still significant. XRCC1 and ERCC1 haplotypes
We further analyzed the distribution of haplotypes in cases and controls. All the frequencies of haplotypes are greater
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Table 2. Genotype Frequencies of XRCC1 and ERCC1 Gene Polymorphisms in Controls and Cases and Their Associations with Breast Cancer Genotype
Case, n (%)
Control, n (%)
OR (95% CI)a
GG AG AA Dominantb Recessivec CC CT TT Dominant Recessive
13 (12.87) 74 (73.27) 14 (13.86)
46 (45.55) 47 (46.53) 8 (7.92)
53 (52.48) 42 (41.58) 6 (5.94)
51 (50.50) 45 (44.55) 5 (4.95)
Reference 1.05 (0.40–2.71) 6.39 (2.18–18.65) 3.17 (1.76–5.72) 2.02 (0.82–5.01) Reference 0.89 (0.50–1.58) 1.14 (0.33–3.98) 0.91 (0.53–1.59) 1.20 (0.35–4.09)
GG AG AA Dominant Recessive CC CT TT Dominant Recessive
35 (34.65) 45 (44.55) 21 (20.79)
33 (32.67) 46 (45.55) 22 (21.78)
56 (55.44) 41 (43.56) 4 (5.00)
63 (62.38) 33 (32.67) 5 (4.95)
SNP XRCC1 rs25487 (missense)
rs1799782 (missense)
ERCC1 rs3212964 (Intron)
rs11615 (synon)
Reference 1.07 (0.51–2.22) 0.91 (0.44–1.90) 0.89 (0.45–1.76) 0.99 (0.57–1.74) Reference 1.43 (0.79–2.56) 0.87 (0.22–3.42) 1.35 (0.77–2.38) 0.76 (0.20–2.93)
p-Value
pc
0.93 < 0.001 < 0.001 0.13
NS < 0.01 < 0.01 NS
0.69 0.84 0.75 0.77
NS NS NS NS
0.86 0.81 0.74 0.99
NS NS NS NS
0.24 0.84 0.30 0.69
NS NS NS NS
a
ORs were adjusted for age. The dominant model: comparing the combination of heterozygotes and minor allele homozygotes with the major allele homozygotes. The recessive model: comparing minor allele homozygotes with the combination of heterozygotes and major allele homozygotes. CI, confidence interval; ERCC1, excision repair cross complementation 1; NS, not significant; OR, odds ratio; pc, corrected p-value (after Bonferroni multiple adjustment); SNP, single nucleotide polymorphism; XRCC1, X-ray repair cross-complementing group 1. b c
Table 3. Associations of Clinical Features and Polymorphisms in XRCC1 and ERCC1 Genes No. (%) Clinical features ER
SNP
Genotype
Positive
Negative
OR (95% CI)a
rs11615
CC CT TT Dominantb Recessivec AA AG GG Dominant Recessive CC CT TT Dominant Recessive AA AG GG Dominant Recessive AA AG GG Dominant Recessive
36 (64.29) 19 (33.93) 1 (1.78)
18 (45.00) 19 (47.50) 3 (7.50)
12 (21.43) 20 (35.71) 24 (42.86)
7 (16.67) 25 (59.52) 10 (23.81)
34 (66.67) 16 (31.37) 1 (0.20)
21 (43.75) 25 (52.08) 2 (4.17)
6 (10.00) 29 (48.33) 25 (41.67)
2(4.88) 17 (41.46) 22 (53.66)
13 (0.26) 22 (0.44) 15 (0.30)
7 (14.89) 23 (48.94) 17 (36.17)
Reference 0.40 (0.22–0.73) 0.26 (0.04–1.51) 0.39 (0.21–0.71) 0.41 (0.07–2.34) Reference 0.32 (0.14–0.72) 0.85 (0.36–2.00) 0.48 (0.23–1.03) 1.88 (1.00–3.53) Reference 0.36 (0.20–0.65) 0.16 (0.02–1.51) 0.34 (0.19–0.63) 0.26 (0.03–2.38) Reference 0.74 (0.21–2.60) 0.35 (0.10–1.19) 0.48 (0.14–1.60) 0.45 (0.25–0.81) Reference 0.45 (0.21–0.95) 0.54 (0.24–1.21) 0.48 (0.24–0.98) 0.48 (0.24–0.98)
rs3212964
PR
HER-2
P53
a
rs11615
rs25487
rs3212964
p-Value
pc
0.003 0.13 0.002 0.32
0.01 NS 0.008 NS
0.006 0.71 0.06 0.05
0.02 NS NS NS
0.001 0.11 0.001 0.23
0.004 NS 0.004 NS
0.64 0.09 0.23 0.009
NS NS NS 0.04
0.04 0.13 0.77 0.04
NS NS NS NS
ORs were adjusted for age. The dominant model: comparing the combination of heterozygotes and minor allele homozygotes with the major allele homozygotes. The recessive model: comparing minor allele homozygotes with the combination of heterozygotes and major allele homozygotes. ER, estrogen receptor; PR, progesterone receptor. b c
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Table 4. Frequency Distributions of Haplotypes of XRCC1 and ERCC1 in Cases and Controls Gene XRCC1
Haplotype
Frequency
Cases
Controls
p-Valuea
GC AC GT AT AC GC GT AT
0.37 0.36 0.23 0.04 0.41 0.36 0.21 0.02
0.46 0.27 0.24 0.03 0.37 0.37 0.24 0.03
0.29 0.44 0.21 0.06 0.45 0.34 0.19 0.02
< 0.001 0.27 0.47 0.15 0.11 0.53 0.22 0.52
b
ERCC1c
OR (95% CI) 4.78 1.68 0.75 0.86 1.33 1.19 0.87 0.81
(2.52–8.72) (0.82–3.47) (0.46–1.23) (0.53–1.40) (0.86–2.07) (0.65–2.20) (0.56–1.35) (0.54–1.23)
Pc < 0.01 NS NS NS NS NS NS NS
a
p-Value calculated by Fisher’s test. The order of SNPs in XRCC1 is rs25487, rs1799782. c The order of SNPs in ERCC1 is rs3212964, rs11615. b
than 1% (Table 4). Four haplotypes were constructed in XRCC1 based on the two tag SNPs (rs25487 and rs1799782), as well as other four haplotypes were constructed in ERCC1 based on the tag SNPs (rs3212964, rs11615). Haplotype GC in XRCC1 had a higher frequency in cases than in controls ( p < 0.001). The individuals carrying haplotype GC had an elevated risk of breast cancer than those carrying other haplotypes; the adjusted OR was 4.78 (95% CI: 2.52–8.72). After the Bonferroni correction, the haplotype GC was still significantly associated with breast cancer (corrected p < 0.01). However, no significant differences were found for other haplotypes in XRCC1 and all haplotypes in ERCC1. We also analyzed the association between haplotypes and clinical features in the subgroup of the cases. The results are shown in Table 5. In the ERCC1 gene, the frequency of GC haplotypes were higher in ER- and PR-positive cases ( p < 0.001; p = 0.003, respectively), and GT haplotype had a lower frequency in ER-positive cases ( p < 0.001). Moreover, AT haplotype had a lower frequency in PR-positive cases ( p < 0.001). However, no significant association was observed between haplotypes in XRCC1 gene and clinical features of the cases (data not shown). Discussion
In our study, we investigated the association of genetic polymorphisms in both XRCC1 and ERCC1 with the risk of breast cancer. We found that the XRCC1 rs25487 (Arg399Gln) allele was associated with an increased risk for sporadic breast cancer among the Chinese woman of the Gansu province. In contrast, neither rs3212964 nor rs11615 polymorphisms of the ERCC1 gene was associated with risk of breast cancer in this study population. XRCC1 is involved in the repair of DNA base damage and single-strand DNA breaks by binding DNA ligase III at its
carboxyl and DNA polymerase b and poly (ADP-ribose) polymerase at the site of the damaged DNA (Caldecott et al., 1996). Furthermore, XRCC1 is exclusively required for DNA BER, strand-break repair, and maintenance of genetic stability (Whitehouse et al., 2001). A reduced DNA repair capacity could lead to a predisposition to accumulated DNA damage, mutations, and subsequently developing diseases such as cancer (Rowe and Glazer, 2010). Polymorphisms in XRCC1 have been indicated to have a contributive role in DNA adduct formation and an increased risk of cancer development (Mahjabeen et al., 2013). The Arg399Gln (rs25487) polymorphism has been studied in several epidemiological studies in relation to various cancers. It has been recently reported that the variant allele of XRCC1 rs25487 was significantly associated with an increased risk of glioma (Zhou et al., 2011), head, neck (Azad et al., 2012; Mahjabeen et al., 2013), pancreatic (Nakao et al., 2012), and cervical carcinoma (Roszak et al., 2011), and breast cancer (Mitra et al., 2008; Roberts et al., 2011). These findings are consistent with our study among Chinese women in the Gansu province. In addition, we found that cases had a higher frequency of haplotype GC in XRCC1 than controls. To our knowledge, this phenomenon has not been reported previously in breast cancer, but in pancreatic cancer among Japanese population (Nakao et al., 2012). It is suggested that XRCC1 mediates the repair of aflatoxin-induced DNA damage, thereby associating with cancer risk (Hruban et al., 2010). Higher levels of aflatoxin B1-DNA adducts were observed in the individuals with the Arg399Gln allele, which are consistent with the increased levels of DNA damage due to a reduced BER function (Bachmann et al., 2009). Taken together, the findings suggest that DNA repair-related XRCC1 gene may play important roles in protection against gene mutation and cancer initiation.
Table 5. Clinical Features and Haplotypes of ERCC1 Clinical features ER PR P53 a
Haplotypea
Frequency
Negative
Positive
p-Value
pc
GC GT GC GT AT AT
0.36 0.22 0.36 0.21 0.03 0.21
0.27 0.27 0.29 0.26 0.04 0.26
0.43 0.17 0.43 0.16 0.02 0.17
< 0.001 < 0.001 0.003 0.02 < 0.001 0.02
< 0.01 < 0.01 0.01 NS < 0.01 NS
The order of SNPs in ERCC1 is rs3212964, rs11615.
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ASE-1 is located in an anti-sense orientation and overlaps with the gene for ERCC1 and is possibly involved with the RNA polymerase I transcription complex. Recent studies show that BER mechanism of NER system is involved in repairing DNA lesions induced by the low concentrations of N-nitrosodiethylamine (Aiub et al., 2004). However, we failed to observe any association between the genetic polymorphisms in the ERCC1 gene and breast cancer risk in our study. One possible explanation is that ERCC1 is one component of the NER complex network involved in the repair of radiation-induced and platinum-induced inter- and intrastrand crosslinks (Gillet and Scharer, 2006; Markowitz and Bertagnolli, 2009). Other components in the NER system may compensate the ERCC1 deficiency to repair bulky DNA damage. Another possibility is due to the low statistical power due to the relatively small sample size in this study. Thus, caution should be made in interpreting these findings since it is well known that more than 20 genes are involved in the NER pathway, which cooperates with each other. In analyzing the associations of clinical features and the SNPs, we found that XRCC1 and ERCC1 gene polymorphisms were associated with the clinicopathological features of the patients. For rs25487, rs11615, and rs3212964, statistically significant associations were found with the status of ER, PR, HER-2, and P53. It has been shown that steroid hormone receptors influence the disease-free and overall survival of breast cancer patients, and are considered to be predictive markers of endocrine therapy (Bauer et al., 2007; Lee et al., 2007). The expression of HER-2 and P53 were associated with the tumor metastasis and poor outcomes in breast cancer (Beenken et al., 2001; Yamashita et al., 2006). Taken together, the findings in our study suggest that these three SNPs may be potential markers to predict the prognosis of breast cancer and the effectiveness of pharmaceutical treatment. Conclusion
In conclusion, the variants of rs25487 in XRCC1 as well as a haplotype are likely involved in the risk of breast cancer. Some SNPs in XRCC1 and ERCC1 as well as some haplotypes were also associated with clinical characteristics of breast cancer. However, because this study has a relatively small sample size, the statistical power is limited. It would be necessary to validate these findings in a population-based prospective study. Acknowledgment
The authors are grateful for the financial support from the Technology Research and Development Program of the Gansu Provincial Science and Technology Department of China (No. 1011FKCA089). Author Disclosure Statement
No competing financial interests exist. References
Aiub CA, Mazzei JL, Pinto LF, Felzenszwalb I (2004) Participation of BER and NER pathways in the repair of DNA lesions induced at low N-nitrosodiethylamine concentrations. Toxicol Lett 154:133–142.
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Address correspondence to: Haixiang Su, MD, PhD Gansu Provincial Academy of Medical Sciences Gansu Provincial Cancer Hospital 2 Xiaoxihu East Street Lanzhou 730050 People’s Republic of China E-mail: [email protected]
DNA Repair Genes XRCC1 and ERCC1 Polymorphisms and the Risk of Sporadic Breast Cancer in Han Women in the Gansu Province of China Gongjian Zhu,1,2 Lan Wang,1 Hongyun Guo,1 Lingeng Lu,3 Suisheng Yang,1 Tao Wang,1 Huan Guo,1 Haitao Wang,1 Jianping Min,1 Kai Yang,1 Xuezhong Chen,1 Yuanqiang Liu,1 Zhiping Wang,4 and Haixiang Su1
Aims: Polymorphisms in DNA damage repair genes may affect DNA repair capacity and modulate breast cancer susceptibility. In this study, we aimed to analyze two polymorphisms for each of the DNA repair genes X-ray repair cross-complementing group 1 (XRCC1) rs25487 and rs1799782 and excision repair cross-complementing group 1 (ERCC1) rs3212964 and rs11615, to evaluate their associations with the risk of sporadic breast cancer in Han women in the Gansu Province of China. Methods: Genotypes were determined by a polymerase chain reaction-based approach for 101 patients with breast cancer and in 101 disease-free controls. Results: We found that individuals with the AA genotype at XRCC1 rs25487 had a significantly increased risk of breast cancer compared with GG genotype ( p < 0.001, odds ratio [OR] = 6.39, 95% confidence interval [CI]: 2.18–18.65). The dominant model showed that the combined rs25487 genotypes (AA + AG) increased the disease risk ( p < 0.001, OR = 3.17, 95% CI: 1.76–5.72). However, no statistical associations were found between rs1799782 in XRCC1, or rs3212964 and rs11615 in ERCC1 and the risk of disease. In haplotype analysis, the GC haplotype in XRCC1 conferred an increased risk ( p < 0.001) with a 4.78-fold increase for each copy (95% CI: 2.52–8.72). Significant associations were also shown between the single nucleotide polymorphisms (SNPs) and the status of estrogen receptor (ER), progesterone receptor (PR), and HER-2. Conclusions: The results suggest that the XRCC1 rs25487 polymorphism may increase the risk of breast cancer.
Introduction
B
reast cancer is the most common malignancy affecting women, and is the leading cause of cancer-related death in females worldwide (Shu et al., 2003). However, its etiology is extremely complex, and genetic factors are indicated to be associated with the risk (Xu et al., 2012). Recently, genome-wide association studies have suggested that genetic variations may lead to the susceptibility to breast cancer (Easton et al., 2007). DNA damage repair systems are essential to human health in response to endogenous and exogenous carcinogens and mutagens (Berwick and Vineis, 2000). The genes involved in DNA repair and the maintenance of genome integrity play a crucial role in providing protection against mutations that lead to cancer ( Jiricny and Nystrom-Lahti, 2000; Dufloth et al., 2005). Epidemiological studies have shown that the inheritance of genetic variants at
one or more loci results in reduced DNA repair capacity and an increase in the risk of cancer (Terry et al., 2004; Kostrzewska-Poczekaj et al., 2013). There are two major types of DNA repair pathways. For single-strand DNA breaks, these include the base excision repair (BER) and nucleotide excision repair (NER) systems; for double-strand DNA breaks, there are two principle mechanisms: homologous recombination and nonhomologous end joining (Hegde et al., 2008; Jackson and Bartek, 2009; Friedman and Stivers, 2010). It is known that X-ray repair cross-complementing group 1 (XRCC1) is crucial to the BER system, which repairs DNA damage due to ionizing radiation (Schreiber et al., 2006; Smith et al., 2008). Deletion of XRCC1 gene in mice results in an embryonic lethal phenotype (Tebbs et al., 1999). Chinese hamster ovary cell lines with mutations in XRCC1 have shown a reduced ability to repair single-strand DNA breaks and concomitant cellular
1
Gansu Provincial Academy of Medical Sciences, Gansu Provincial Cancer Hospital, Lanzhou, People’s Republic of China. School of Life Sciences, Lanzhou University, Lanzhou, China. 3 Department of Chronic Disease Epidemiology, School of Public Health, School of Medicine, Yale Cancer Center, Yale University, New Haven, Connecticut. 4 Institute of Urology, The Second Hospital of Lanzhou University, Lanzhou, China. 2
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hypersensitivity to ionizing radiation and alkylating agents (Shen et al., 1998). Another important component, excision repair cross complementation 1 (ERCC1), encodes ERCC1 protein involved in the NER system, removing bulky lesions from DNA caused by chemical or physical factors such as toxic chemicals or ultraviolet light (Smith et al., 2008). Single nucleotide polymorphism (SNP) is a single nucleotide change in a DNA sequence, which may change DNA or RNA conformations (Lu et al., 2012, 2013) or lead to amino acid substitution (Roberts et al., 2011), thereby resulting in corresponding phenotypes. SNPs may cause human diseases and/or contribute to the risk of diseases, and their identification may help us discover new ways to diagnose, treat, and prevent human diseases (Ulrich et al., 2003). Given that the importance of DNA repair machinery in human health, understanding the associations between SNPs in the DNA repair-associated genes and the likelihood of developing cancer may have implications in the prevention and diagnosis of cancer (Xing et al., 2002). There are many identified SNPs in the XRCC1 and ERCC1 genes, some of which are reported to be implicated in carcinogenesis (Zheng et al., 2012; Alsbeih et al., 2013; Wang et al., 2013). However, the results are inconsistent in different organs and in different ethnic groups. In this case–control study, the two tag SNPs in each of XRCC1 (rs25487, rs1799782) and ERCC1 (rs3212964 and rs11615) were analyzed, respectively. To the best of our knowledge, until now there have been no reports investigating the association between these SNPs and breast cancer in Han women in the Gansu Province of China. We also evaluated the relationships between the polymorphisms of XRCC1 and ERCC1 and tumor clinicopathological features, including tumor grade, estrogen receptor (ER) and progesterone receptor (PR) status, tumor size, and nodal status in a breast cancer case–control study. Materials and Methods Subjects
This study included 101 breast cancer cases and 101 disease-free controls. All histologically confirmed incident cases (mean age 45.7 – 7.4 years) were recruited from the Department of Breast Surgery of the Gansu Provincial Cancer Hospital. Pathology slides (or tissue blocks) from all patients were obtained from the original pathology departments and reviewed by two independent pathologists. All cases were classified according to the 2001 WHO classification (Alsheikh et al., 2001). Detailed clinical and pathological information on these patients is shown in Table 1. Exclusion criteria included metastasized cancer from other organs and previous radiotherapy or chemotherapy. The controls were frequency matched to cases by age (mean age 45.5 – 7.6 years) and region, and were randomly selected from the volunteers without prior cancer and other major medical conditions during routine medical fitness examination. Both cases and controls were from the Gansu province in northwest China between 2010 and 2012. At recruitment, each subject signed a written informed consent and provided 5 mL of venous blood, which was stored at - 80C. This study was approved by the Gansu Academy of Medical Science and the Cancer Hospital Ethical Review Board.
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Table 1. Clinicopathological Features of Participants Feature Age (years) £ 40 41–50 51–60 > 60 Histological type IDC Intraductal carcinoma Mucinous adenocarcinoma Others Tumor size (cm) TZ £ 2 2 < TZ £ 5 TZ > 5 Unknown LN involvement Positive Negative Unknown ER Positive Negative Unknown PR Positive Negative Unknown P53 Positive Negative Unknown HER-2 Positive Negative Unknown Ki-67 Positive Negative Unknown
Case, n (%) 28 50 19 4
(27.72) (49.50) (18.81) (3.96)
86 1 3 11
(85.15) (0.99) (2.97) (10.89)
7 61 21 12
(6.93) (60.40) (20.79) (11.88)
Control, n (%) 22 58 18 3
(21.78) (57.43) (17.82) (2.97)
22 (21.78) 47 (46.53) 32 (31.69) 56 (55.45) 41 (40.59) 4 (3.96) 51 (50.50) 47 (46.53) 3 (2.97) 49 (48.52) 44 (43.56) 8 (7.92) 23 (22.77) 74 (73.27) 4 (3.96) 91 (90.10) 5 (4.95) 5 (4.95)
ER, estrogen receptor; HER-2, human epidermal growth factor receptor 2; IDC, infiltrating duct carcinoma; LN, lymph node; PR, progesterone receptor; TZ, tumor size.
SNP selection and genotyping
All SNPs in the candidate genes were selected from HapMap CHB database (phase 2, build 35) (www.hapmap.org) using the Tagger program implemented in HaploView version 4.1. The tag SNPs were chosen based on the following criteria: (1) minor allele frequency ‡ 5%; (2) pair-wise c2 ‡ 0.8; (3) spanning 2 kb upstream of the 5¢ end and 1 kb downstream of the 3¢ end of each gene. The XRCC1 rs25487 and rs1799782, ERCC1 rs3212964 and rs11615 were finally selected. Genomic DNA was extracted from frozen whole blood using the universal genomic DNA Extraction Kit VER.3.0 (Biotech). Genotyping was performed using the polymerase chain reaction (PCR)–restriction fragment length polymorphism method. The primer sequences of the four SNPs were
XRCC1 AND ERCC1 POLYMORPHISMS AND THE RISK OF BREAST CANCER
rs25487 (F: 5¢-TTGTGCTTTCTCTGTGTCCA-3¢, R: 5¢TCCTCCAGCCTTTTCTGATA-3¢), rs1799782 (F: 5¢-ATG AGAGCGCCAACTCTCTGAGGTC-3¢, R: 5¢-GCTGAG CCCCAAGACCCTTTCACTC-3¢), rs3212964 (F: 5¢-TCAC ATCCTCTCTCCCGTAGGGATC-3¢, R: 5¢-AGAGGAGGG ACTGAGCCT-3¢), rs11615 (F: 5¢-TGTGGGAAGAGGT GCGA-3¢, R: 5¢-CCAGCACATAGTCGGGAATTGC-3¢). The polymorphic region was amplified by PCR using a S1000 Thermal Cycler (BIO-RAD) in a 25 mL reaction solution containing 0.2 mg of genomic DNA, 12.5 mL of 2 · GoTaq Green Master Mix (Promega Corporation), and a pair of primers at a final concentration of 100 nM. Annealing temperatures were as follows: rs25487 at 58C, rs1799782 at 58.3C, rs3212964 at 58.3C, and rs11615 at 54.9C. The lengths of the PCR products were 615bp (rs25487), 341bp (rs1799782), and 214bp (rs3212964 and rs11615). The PCR products were digested with restriction enzymes (Promega) according to the manufacturer’s instructions and analyzed using 3% agarose gel electrophoresis. Restriction enzymes of each SNP were MspI (rs25487 and rs1799782), TaqI (rs3212964), and TaiI (rs11615). The lengths of the digested fragments of each SNP were rs25487 (G: 375 + 240 bp, A: 615 bp); rs1799782 (C: 297 + 44 bp, T: 341 bp); rs3212964 (G: 190 + 24 bp, A: 214 bp); rs11615 (C: 173 + 41 bp, T: 214 bp). To validate the accuracy of the genotyping results, 10% samples of each SNP were randomly selected to be tested twice by different persons. Additionally, 5% random samples of each SNP were confirmed by direct sequencing, and the reproducibility of both was 99%. Determination of clinicopathological features
Histology, grade, and hormone receptor status of cases were assessed using immunohistochemistry. Tumors with an Allred score ‡ 3 and nuclear staining of more than 10% of the tumor cells were classified as ER-positive or PR-positive (Ishikawa et al., 2011). P53 was defined as positive when more than 25% (median value) of the tumor cells were positive for nuclear staining. For HER-2 expression, four grades were evaluated based on the percentage of positive cells and color intensity: 0, colorless or < 10% cells with membrane staining; 1 + , > 10% cells with discontinuous membrane staining; 2 + , 10–30% cells with intact membrane staining; 3 + , > 30% cells with strong membrane staining. Samples with scores of 0 and 1 + were considered as negative and 3 + samples were considered as positive. Samples with scores of 2 + were subjected to further analysis by fluorescence in situ hybridization assay to confirm HER-2 expression. Ki67 status was expressed in terms of the percentage of positive cells with a threshold of 14% (Cheang et al., 2009). Statistical analysis
Genotype frequencies of SNPs were tested for Hardy– Weinberg equilibrium (HWE). We used HaploView 4.1 and Phase 2.1 to tag all common haplotypes and their frequencies in cases and controls. Associations between four SNPs and breast cancer risk were estimated by odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression with adjustment for age. The associations were also analyzed by different genetic models (codominant, dominant, and recessive models). In the codominant model, homozygotes for the major allele were the reference group,
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and heterozygotes and minor allele homozygotes were compared to the reference group. In the dominant model, the combination of heterozygotes and minor allele homozygotes were also compared to the reference group. The recessive model was run with minor allele homozygotes versus heterozygotes and major allele homozygotes. A Bayesian approach was applied to reconstruct haplotypes and estimate their frequencies. The associations between clinical features and the haplotypes were analyzed by using the Chi-square test. Unconditional logistical regression model was used to estimate ORs and their 95% CIs by treating each haplotype as a continuous variable. Bonferroni correction was used to adjust for the multiple comparisons. A cutoff of 0.0125 (0.05/4 = 0.0125) was used for the significance of a p-value in the multiple comparison. Statistical analyses were performed using SPSS 16.0 software. Results
The genetic models of four SNPs are shown in Table 2. No deviation from HWE was found in the genotype distributions of the four SNPs in controls ( p > 0.05). XRCC1 and ERCC1 gene polymorphisms and the risk of breast cancer
In XRCC1 rs25487, compared with GG genotype, AA genotype showed an association with a significantly increased risk of breast cancer ( p < 0.001, OR = 6.39, 95% CI: 2.18–18.65). Moreover, in the dominant model, combined rs25487 genotypes (AA + AG) conferred an increased risk of breast cancer ( p < 0.001, OR = 3.17, 95% CI: 1.76–5.72). This association still remained statistically significant after the correction using the Bonferroni test (corrected p < 0.01 and p < 0.01, respectively). However, no association with the risk of breast cancer was observed for rs1799782 in XRCC1 and other two SNPs (rs3212964 and rs11615) in ERCC1. XRCC1 and ERCC1 gene polymorphisms and clinicopathological features
In the cases, we also analyzed the associations of XRCC1 and ERCC1 polymorphisms with clinicopathological features, including lymph node metastasis, tumor size, and the statuses of ER, PR, HER-2, and P53. The results are shown in Table 3. We found in rs3212964, women carrying AG genotype were less likely to have ER-positive and P53 status tumors compared with women with GG genotype ( p = 0.02, p = 0.04, respectively), and the rs3212964 was associated with P53 status in the recessive model ( p = 0.04). However, these associations were not statistically significant after the Bonferroni correction. In rs11615, compared with the CC genotype, CT genotype had a lower frequency in both ER- and PR-positive cases ( p = 0.003; p = 0.001, respectively), and significant association was also found in the dominant model of both ER- and PRpositive cases ( p = 0.002; p < 0.001, respectively). Furthermore, the rs25487 was associated with HER-2-positive in the recessive model ( p = 0.009). After the Bonferroni correction, these associations were still significant. XRCC1 and ERCC1 haplotypes
We further analyzed the distribution of haplotypes in cases and controls. All the frequencies of haplotypes are greater
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Table 2. Genotype Frequencies of XRCC1 and ERCC1 Gene Polymorphisms in Controls and Cases and Their Associations with Breast Cancer Genotype
Case, n (%)
Control, n (%)
OR (95% CI)a
GG AG AA Dominantb Recessivec CC CT TT Dominant Recessive
13 (12.87) 74 (73.27) 14 (13.86)
46 (45.55) 47 (46.53) 8 (7.92)
53 (52.48) 42 (41.58) 6 (5.94)
51 (50.50) 45 (44.55) 5 (4.95)
Reference 1.05 (0.40–2.71) 6.39 (2.18–18.65) 3.17 (1.76–5.72) 2.02 (0.82–5.01) Reference 0.89 (0.50–1.58) 1.14 (0.33–3.98) 0.91 (0.53–1.59) 1.20 (0.35–4.09)
GG AG AA Dominant Recessive CC CT TT Dominant Recessive
35 (34.65) 45 (44.55) 21 (20.79)
33 (32.67) 46 (45.55) 22 (21.78)
56 (55.44) 41 (43.56) 4 (5.00)
63 (62.38) 33 (32.67) 5 (4.95)
SNP XRCC1 rs25487 (missense)
rs1799782 (missense)
ERCC1 rs3212964 (Intron)
rs11615 (synon)
Reference 1.07 (0.51–2.22) 0.91 (0.44–1.90) 0.89 (0.45–1.76) 0.99 (0.57–1.74) Reference 1.43 (0.79–2.56) 0.87 (0.22–3.42) 1.35 (0.77–2.38) 0.76 (0.20–2.93)
p-Value
pc
0.93 < 0.001 < 0.001 0.13
NS < 0.01 < 0.01 NS
0.69 0.84 0.75 0.77
NS NS NS NS
0.86 0.81 0.74 0.99
NS NS NS NS
0.24 0.84 0.30 0.69
NS NS NS NS
a
ORs were adjusted for age. The dominant model: comparing the combination of heterozygotes and minor allele homozygotes with the major allele homozygotes. The recessive model: comparing minor allele homozygotes with the combination of heterozygotes and major allele homozygotes. CI, confidence interval; ERCC1, excision repair cross complementation 1; NS, not significant; OR, odds ratio; pc, corrected p-value (after Bonferroni multiple adjustment); SNP, single nucleotide polymorphism; XRCC1, X-ray repair cross-complementing group 1. b c
Table 3. Associations of Clinical Features and Polymorphisms in XRCC1 and ERCC1 Genes No. (%) Clinical features ER
SNP
Genotype
Positive
Negative
OR (95% CI)a
rs11615
CC CT TT Dominantb Recessivec AA AG GG Dominant Recessive CC CT TT Dominant Recessive AA AG GG Dominant Recessive AA AG GG Dominant Recessive
36 (64.29) 19 (33.93) 1 (1.78)
18 (45.00) 19 (47.50) 3 (7.50)
12 (21.43) 20 (35.71) 24 (42.86)
7 (16.67) 25 (59.52) 10 (23.81)
34 (66.67) 16 (31.37) 1 (0.20)
21 (43.75) 25 (52.08) 2 (4.17)
6 (10.00) 29 (48.33) 25 (41.67)
2(4.88) 17 (41.46) 22 (53.66)
13 (0.26) 22 (0.44) 15 (0.30)
7 (14.89) 23 (48.94) 17 (36.17)
Reference 0.40 (0.22–0.73) 0.26 (0.04–1.51) 0.39 (0.21–0.71) 0.41 (0.07–2.34) Reference 0.32 (0.14–0.72) 0.85 (0.36–2.00) 0.48 (0.23–1.03) 1.88 (1.00–3.53) Reference 0.36 (0.20–0.65) 0.16 (0.02–1.51) 0.34 (0.19–0.63) 0.26 (0.03–2.38) Reference 0.74 (0.21–2.60) 0.35 (0.10–1.19) 0.48 (0.14–1.60) 0.45 (0.25–0.81) Reference 0.45 (0.21–0.95) 0.54 (0.24–1.21) 0.48 (0.24–0.98) 0.48 (0.24–0.98)
rs3212964
PR
HER-2
P53
a
rs11615
rs25487
rs3212964
p-Value
pc
0.003 0.13 0.002 0.32
0.01 NS 0.008 NS
0.006 0.71 0.06 0.05
0.02 NS NS NS
0.001 0.11 0.001 0.23
0.004 NS 0.004 NS
0.64 0.09 0.23 0.009
NS NS NS 0.04
0.04 0.13 0.77 0.04
NS NS NS NS
ORs were adjusted for age. The dominant model: comparing the combination of heterozygotes and minor allele homozygotes with the major allele homozygotes. The recessive model: comparing minor allele homozygotes with the combination of heterozygotes and major allele homozygotes. ER, estrogen receptor; PR, progesterone receptor. b c
XRCC1 AND ERCC1 POLYMORPHISMS AND THE RISK OF BREAST CANCER
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Table 4. Frequency Distributions of Haplotypes of XRCC1 and ERCC1 in Cases and Controls Gene XRCC1
Haplotype
Frequency
Cases
Controls
p-Valuea
GC AC GT AT AC GC GT AT
0.37 0.36 0.23 0.04 0.41 0.36 0.21 0.02
0.46 0.27 0.24 0.03 0.37 0.37 0.24 0.03
0.29 0.44 0.21 0.06 0.45 0.34 0.19 0.02
< 0.001 0.27 0.47 0.15 0.11 0.53 0.22 0.52
b
ERCC1c
OR (95% CI) 4.78 1.68 0.75 0.86 1.33 1.19 0.87 0.81
(2.52–8.72) (0.82–3.47) (0.46–1.23) (0.53–1.40) (0.86–2.07) (0.65–2.20) (0.56–1.35) (0.54–1.23)
Pc < 0.01 NS NS NS NS NS NS NS
a
p-Value calculated by Fisher’s test. The order of SNPs in XRCC1 is rs25487, rs1799782. c The order of SNPs in ERCC1 is rs3212964, rs11615. b
than 1% (Table 4). Four haplotypes were constructed in XRCC1 based on the two tag SNPs (rs25487 and rs1799782), as well as other four haplotypes were constructed in ERCC1 based on the tag SNPs (rs3212964, rs11615). Haplotype GC in XRCC1 had a higher frequency in cases than in controls ( p < 0.001). The individuals carrying haplotype GC had an elevated risk of breast cancer than those carrying other haplotypes; the adjusted OR was 4.78 (95% CI: 2.52–8.72). After the Bonferroni correction, the haplotype GC was still significantly associated with breast cancer (corrected p < 0.01). However, no significant differences were found for other haplotypes in XRCC1 and all haplotypes in ERCC1. We also analyzed the association between haplotypes and clinical features in the subgroup of the cases. The results are shown in Table 5. In the ERCC1 gene, the frequency of GC haplotypes were higher in ER- and PR-positive cases ( p < 0.001; p = 0.003, respectively), and GT haplotype had a lower frequency in ER-positive cases ( p < 0.001). Moreover, AT haplotype had a lower frequency in PR-positive cases ( p < 0.001). However, no significant association was observed between haplotypes in XRCC1 gene and clinical features of the cases (data not shown). Discussion
In our study, we investigated the association of genetic polymorphisms in both XRCC1 and ERCC1 with the risk of breast cancer. We found that the XRCC1 rs25487 (Arg399Gln) allele was associated with an increased risk for sporadic breast cancer among the Chinese woman of the Gansu province. In contrast, neither rs3212964 nor rs11615 polymorphisms of the ERCC1 gene was associated with risk of breast cancer in this study population. XRCC1 is involved in the repair of DNA base damage and single-strand DNA breaks by binding DNA ligase III at its
carboxyl and DNA polymerase b and poly (ADP-ribose) polymerase at the site of the damaged DNA (Caldecott et al., 1996). Furthermore, XRCC1 is exclusively required for DNA BER, strand-break repair, and maintenance of genetic stability (Whitehouse et al., 2001). A reduced DNA repair capacity could lead to a predisposition to accumulated DNA damage, mutations, and subsequently developing diseases such as cancer (Rowe and Glazer, 2010). Polymorphisms in XRCC1 have been indicated to have a contributive role in DNA adduct formation and an increased risk of cancer development (Mahjabeen et al., 2013). The Arg399Gln (rs25487) polymorphism has been studied in several epidemiological studies in relation to various cancers. It has been recently reported that the variant allele of XRCC1 rs25487 was significantly associated with an increased risk of glioma (Zhou et al., 2011), head, neck (Azad et al., 2012; Mahjabeen et al., 2013), pancreatic (Nakao et al., 2012), and cervical carcinoma (Roszak et al., 2011), and breast cancer (Mitra et al., 2008; Roberts et al., 2011). These findings are consistent with our study among Chinese women in the Gansu province. In addition, we found that cases had a higher frequency of haplotype GC in XRCC1 than controls. To our knowledge, this phenomenon has not been reported previously in breast cancer, but in pancreatic cancer among Japanese population (Nakao et al., 2012). It is suggested that XRCC1 mediates the repair of aflatoxin-induced DNA damage, thereby associating with cancer risk (Hruban et al., 2010). Higher levels of aflatoxin B1-DNA adducts were observed in the individuals with the Arg399Gln allele, which are consistent with the increased levels of DNA damage due to a reduced BER function (Bachmann et al., 2009). Taken together, the findings suggest that DNA repair-related XRCC1 gene may play important roles in protection against gene mutation and cancer initiation.
Table 5. Clinical Features and Haplotypes of ERCC1 Clinical features ER PR P53 a
Haplotypea
Frequency
Negative
Positive
p-Value
pc
GC GT GC GT AT AT
0.36 0.22 0.36 0.21 0.03 0.21
0.27 0.27 0.29 0.26 0.04 0.26
0.43 0.17 0.43 0.16 0.02 0.17
< 0.001 < 0.001 0.003 0.02 < 0.001 0.02
< 0.01 < 0.01 0.01 NS < 0.01 NS
The order of SNPs in ERCC1 is rs3212964, rs11615.
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ASE-1 is located in an anti-sense orientation and overlaps with the gene for ERCC1 and is possibly involved with the RNA polymerase I transcription complex. Recent studies show that BER mechanism of NER system is involved in repairing DNA lesions induced by the low concentrations of N-nitrosodiethylamine (Aiub et al., 2004). However, we failed to observe any association between the genetic polymorphisms in the ERCC1 gene and breast cancer risk in our study. One possible explanation is that ERCC1 is one component of the NER complex network involved in the repair of radiation-induced and platinum-induced inter- and intrastrand crosslinks (Gillet and Scharer, 2006; Markowitz and Bertagnolli, 2009). Other components in the NER system may compensate the ERCC1 deficiency to repair bulky DNA damage. Another possibility is due to the low statistical power due to the relatively small sample size in this study. Thus, caution should be made in interpreting these findings since it is well known that more than 20 genes are involved in the NER pathway, which cooperates with each other. In analyzing the associations of clinical features and the SNPs, we found that XRCC1 and ERCC1 gene polymorphisms were associated with the clinicopathological features of the patients. For rs25487, rs11615, and rs3212964, statistically significant associations were found with the status of ER, PR, HER-2, and P53. It has been shown that steroid hormone receptors influence the disease-free and overall survival of breast cancer patients, and are considered to be predictive markers of endocrine therapy (Bauer et al., 2007; Lee et al., 2007). The expression of HER-2 and P53 were associated with the tumor metastasis and poor outcomes in breast cancer (Beenken et al., 2001; Yamashita et al., 2006). Taken together, the findings in our study suggest that these three SNPs may be potential markers to predict the prognosis of breast cancer and the effectiveness of pharmaceutical treatment. Conclusion
In conclusion, the variants of rs25487 in XRCC1 as well as a haplotype are likely involved in the risk of breast cancer. Some SNPs in XRCC1 and ERCC1 as well as some haplotypes were also associated with clinical characteristics of breast cancer. However, because this study has a relatively small sample size, the statistical power is limited. It would be necessary to validate these findings in a population-based prospective study. Acknowledgment
The authors are grateful for the financial support from the Technology Research and Development Program of the Gansu Provincial Science and Technology Department of China (No. 1011FKCA089). Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Haixiang Su, MD, PhD Gansu Provincial Academy of Medical Sciences Gansu Provincial Cancer Hospital 2 Xiaoxihu East Street Lanzhou 730050 People’s Republic of China E-mail: [email protected]
