| Objective:Breast cancer is the most common invasive cancer in women. At present, breast cancer has become a public health epidemic with an obviously increasing incidence, and it is estimated that mortality rate and 5-year prevalence of breast cancer reach 13.7% and 33.9% worldwide, respectively. In China, breast cancer has ranked the first killer for women with its incidence increased by an increment of 3% per year, and it often happens in younger age. It has been well established that breast cancer is characterized by family accumulation and genetic predisposition, and family studies found that the risk for those with first-degree relatives of affected individuals was more than two times higher than the risk of general population. The exact cause of breast cancer so far remains unclear, and it is regarded as a multifactorial disease to which genetic and environmental factors contribute interactively. Evidence is growing indicating that DNA damage repair system plays a crucially important role in the initiation and progression of breast cancer. To generate more information, we adopted a pathway-based case-control method to examine the association of seven single nucleotide polymorphisms within five genes of DNA damage repair pathway, both individually and interactively, with the risk of having breast cancer in a large Northern Han Chinese female population. By doing so, this study is expected to establish the contributory role of DNA damage repair system in the pathogenesis and prediction of breast cancer and the underlying molecular regulatory mechanisms.Methods: This was a hospital-based case-control genetic association study. All study subjects were consecutively recruited from Chengde Medical College Affiliated Hospital between September 2009 and May 2013, and they are unrelated Han Chinese female. This study involved 606 patients with sporadic breast cancer who had no prior history of any cancers(except for skin cancer), reported no family history of breast cancer, and were for their first time diagnosed as invasive ductal carcinoma based on pathological confirmation. Meanwhile, a total of 633 unrelated heathy subjects without clinical evidence of breast cancer and without family history of any cancer expect for skin cancer formed the control group. At the rime of enrollment, both breast cancer patients and healthy controls underwent clinical examinations and baseline characteristics such as age, family history of cancer, age at menarche, menopausal status, tumor size(T1 to T4), tumor grade(G1 to G3) and lymph node(positive or negative) were collected before enrollment. Approval of this study was obtained from the Ethics Committee of Chengde Medical College, and each study subject read and signed the informed consent before entering this study. On the basis of DNA damage repair system, we selected five relevant genes, including X-ray repair complementing defective repair in Chinese hamster cells 1(XRCC1) gene(rs1799782 and rs25487), X-ray repair complementing defective repair in Chinese hamster cells 2(XRCC2) gene(rs3218536), X-ray repair complementing defective repair in Chinese hamster cells 3(XRCC3) gene(rs861539), xeroderma pigmentosum, complementation group A(XPA) gene(rs1800975) and PEX nuclease multifunctional DNA repair enzyme 1(APEX1) gene(rs1760944 and rs1130409). EDTA blood samples were obtained from all study subjects at the time of enrollment. Genomic DNA was isolated from peripheral blood leukocytes by using TIANamp Blood DNA Kit(Tiangen Biotect Co., Beijing, China). The polymerase chain reaction- ligase detection reaction(PCR-LDR) method was adopted to determine the genotypes of seven examined polymorphisms in this study. Statistical analyses were completed with SAS version 8.1 and STATA version 12.0, as well some professional softwares including Haplo.Stata version 1.6.11, Haploview version 4.2, expression quantitative trait loci(e QTL) Genevar version 3.3.0 and multifactor dimensionality reduction(MDR) version 2.0.Results: This study involved a total of 1,239 study subjects, including 606 breast cancer patients and 633 healthy controls. All study subjects were Han Chinese females. There was no difference in age distributions between breast cancer patients(mean ± standard deviation: 54.36 ± 12.33 years) and controls(55.15 ± 9.38 years)(P < 0.05). The percentage of breast cancer patients who had family history of cancer was 10.56% and there was no reported family history of cancer in all healthy controls. The average age at menarche was 14.60(standard deviation: 1.63) years. No deviations from Hardy-Weinberg equilibrium were noted in healthy controls for all seven polymorphisms examined(all P > 0.05). The genotypes and alleles of XRCC1 gene rs25487 polymorphism(GG, GA and AA: 52.48%, 34.49% and 13.04% in breast cancer patients and 54.82%, 40.13% and 5.06% in healthy controls, P < 0.0005; A allele: 30.28% in patients and 25.12% in controls, P = 0.004) and XPA gene rs1800975 polymorphism(AA, AG and GG: 33.17%, 44.22% and 22.61% in breast cancer patients and 24.80%, 47.24% and 27.96% in healthy controls, P = 0.003; A allele: 44.72% in patients and 51.58% in controls, P = 0.001) differed significantly between the two groups, even after the false discovery rate(FDR) correction and the Bonferroni correction(Bonferroni significance threshold P = 0.05 divided by the total number of examined polymorphisms(n = 7): P = 0.007). The power to reject the null hypothesis of no differences in mutant allele frequencies of XRCC1 gene rs25487 polymorphism and XPA gene rs1800975 polymorphism between the two groups was 81.9% and 92.8%, respectively. The odds of having breast cancer for XRCC1 gene rs25487 polymorphism was significant under both additive(before adjustment: OR = 1.29; 95% CI: 1.08- 1.53; P = 0.005; after adjustment: OR = 1.28; 95% CI: 1.07- 1.51; P = 0.006) and recessive(before adjustment: OR = 2.86; 95% CI: 1.81- 4.39; P < 0.001; after adjustment: OR = 2.82; 95% CI: 1.84- 4.32; P < 0.001) models, and remained significant after the FDR and Bonferroni correction. Moreover, the risk of XPA gene rs1800975 polymorphism for breast cancer was significantly reduced by 23%(before adjustment: OR = 0.78; 95% CI: 0.68- 0.92; P = 0.001; after adjustment: OR = 0.77; 95% CI: 0.67- 0.90; P = 0.001) and 34%(before adjustment: OR = 0.65; 95% CI: 0.51- 0.83; P = 0.001; after adjustment: OR= 0.66; 95% CI: 0.52- 0.85; P = 0.001) under both additive and dominant models, respectively, even with a Bonferroni corrected alpha of 0.05 / 7. After prediction from expression quantitative trait loci(e QTL) analysis, expression level of XRCC1 gene was slightly higher for rs25487 polymorphism CC genotype than the TT genotype(P = 0.0713), and that of APEX1 gene was higher for rs1760944 polymorphism TT genotype than the GG genotype(P = 0.079). The most common allele combination was C – G – G – C – G – G- G(alleles in order of rs1799782, rs25487, rs3218536, rs861539, rs1800975, rs1760944 and rs1130409 polymorphisms), which was overrepresented in controls(6.66% versus 2.96% in breast cancer patients, P = 0.002, significant at a Bonferroni corrected alpha of 0.05 / 11). Further, a data-mining analytical approach MDR was adopted to explore the potential interactions of multiple polymorphisms of five DNA repair genes, by showing that the best single-locus model incorporated XPA gene rs1800975 polymorphism with cross-validation consistency of 7, and out of seven MDR models, the two-locus model including rs1800975 and rs25487 polymorphisms was the best, with the maximal testing accuracy of 0.654 and the maximal cross-validation consistency of 10 out of 10, which was significant at 0.001, indicating that a model this good or better was observed one out of 1000 permutations and thus unlikely hinged on the null hypothesis of null association.Conclusions: Via a panel of single-locus analysis, expression quantitative trait loci analysis, linkage disequilibrium analysis, allele combination analysis and interaction analysis, our findings provide clear evidence that XRCC1 gene rs25487 polymorphism and XPA gene rs1800975 polymorphism might exert both independent and interactive effects on the development of breast cancer. As breast cancer is a multifactorial complex disorder, large well-designed longitudinal studies attempting to account for high-order gene-gene and gene-environment interactions, as well as in-vitro and in-vivo studies seeking to provide biological or clinical implications of DNA damage repair genes in susceptibility to breast cancer, are required in future investigation. |
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