Abstract
Aim: The present study aimed to evaluate the contribution of X-ray repair cross-complementing group 7 (XRCC7) G6721T (rs7003908) genetic polymorphism and smoking habit on the risk of lung cancer in Taiwanese. Materials and Methods: In this hospital-based case–control study, association of single nucleotide polymorphism XRCC7 G6721T with lung cancer risk were examined among 358 patients with lung cancer and 716 age- and gender-matched healthy controls. The genetic–lifestyle interaction was also investigated. Results: The results showed that the percentages of TT, GT and GG genotypes for XRCC7 G6721T were differentially distributed as 60.9%, 34.9% and 4.2% in the group of patients with lung cancer and 48.7%, 43.3% and 8.0% in the non-cancer control group, respectively (p=3.6*10−7). We further stratified the populations by gender and smoking behavior to investigate their combinatorial effects with XRCC7 G6721T genotype on lung cancer risk. The results showed that the GG genotype of XRCC7 G6721T had a protective effect on lung cancer susceptibility which was obvious among males and smokers (p=2.2×10−4 and 3.1×10−4, respectively). Conclusion: The GG and GT genotypes of XRCC7 rs7003908 compared to the TT genotype had a protective effect on lung cancer risk in Taiwan, particularly among males and smokers.
Lung cancer is keeping its throne as the leading cause of cancer death both worldwide and in Taiwan (1). In literature, it is reported that various carcinogens contained in cigarette smoke may produce reactive oxygen species that can induce DNA adducts and strand breaks (2). However, there were also some studies that showed that only 10-15% of all smokers actually develop lung cancer during their lifetime, suggesting that individual susceptibility to carcinogens in cigarette smoke can vary among different populations (3, 4). From the twentieth century, mounting evidence has shown that individual differences in susceptibility may be inherited in genes encoding DNA repair proteins, which may be closely associated with personal cancer risk (5-10).
Homologous recombination (HR) and non-homologous end-joining (NHEJ) are the two important pathways for removing such double-strand breaks (DSBs) induced by endogenous and exogenous carcinogens. HR, which acts during the transition of S to G2 phases of the cell cycle, entails copying the missing information from an undamaged homologous chromosome. In NHEJ, which acts during all phases of the cell cycle, the broken DNA termini are first processed to make them compatible and then sealed by a ligation step. Among the NHEJ DNA repair proteins, X-ray repair cross-complementing group 7 (XRCC7) (MIM: 600899; Genbank accession no: NM_001469) plays a central role, encoding the catalytic subunit of DNA-activated protein kinase (DNA-PKcs) of NHEJ pathway (11). HR and NHEJ are error-free and error-prone pathways, respectively. It should be noted that NHEJ is the dominant subpathway for DSB repair in human cells (11). DNA-PKcs is recruited to the site of DSBs by the KU70/KU80 heterodimer to form an active DNA–PK complex that is essential for the progression of the NHEJ pathway (12). Deficiencies in DNA-PK activity are clinically significant and mice with inactivated components of DNA-PK show severe combined immunodeficiency, as well as hypersensitivity to ionizing radiation (13, 14). Genetic variation G6721T of XRCC7 (rs7003908) is located in the intron 8 of the gene. It is speculated that this polymorphism may regulate splicing and cause mRNA instability (12). Recently, an increasing number of studies investigating the G6721T polymorphism of XRCC7 in association with several types of cancer have been published (15-21), however, few have been focused on lung cancer.
As for the overall DSB repair system, some previous reports showed that the genotypes of DSB repair genes, especially genes involved in NHEJ, may interact with personal lifestyle, such as smoking, in determining the relative risk of lung cancer for individual Taiwanese (22-24). Therefore, in this study, we aimed to reveal the genotypic frequencies of genotypes of G6721T polymorphism of XRCC7, focusing on the association of XRCC7 genotypes with lung cancer susceptibility among Taiwanese never and ever smokers.
Materials and Methods
Investigated population and sample collection. Three hundred and fifty-eight patients diagnosed with lung cancer were recruited at the Outpatient Clinics of General Surgery between 2005-2008 at the China Medical University Hospital, Taichung, Taiwan. The clinical characteristics of patients, including histological details, were all graded and defined by expert surgeons. All participants voluntarily completed a self-administered questionnaire and provided their peripheral blood samples. Twice as many healthy volunteers without lung cancer as controls were selected by matching for age, gender and personal habits after initial random sampling from the Health Examination Cohort of our hospital. The exclusion criteria of the controls included previous malignancy, metastasized cancer from other or unknown origin, and any genetic or familial diseases. Our study was approved by the Institutional Review Board of the China Medical University Hospital (DMR100-IRB-284) and written-informed consent was obtained from all participants.
Genotyping conditions. Genomic DNA was extracted from peripheral blood leucocytes using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) and stored as previously published (25-27). The paired primers used for G6721T polymorphism of XRCC7 were: forward 5’-TGGTGCTCAGCTTCTGGCTT-3’, and reverse 5’-CATCCCTGCCAGCTCTTCTG-3’. The XRCC7 G6721T polymerase chain reaction (PCR) conditions were one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min.
Restriction fragment length polymorphism (RFLP) conditions. After the PCR process of XRCC7 G6721T, the resultant 301 bp PCR product was mixed with 2 U TaqI and incubated for 3 h at 65°C in CutSmart™ Buffer (New England BioLabs, Taipei, Taiwan, ROC). The G form PCR products could be further digested, while the T form could not. Two fragments 235 bp and 66 bp were present if the product was the digestible G form. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The genotype analysis was performed by two researchers independently and blindly. Five percent of the samples were randomly selected for direct sequencing and the results were 100% concordant.
Statistical analyses. All 716 of the controls and 358 cases with genotypic and clinical data were analyzed. Pearson's Chi-square test was used to compare the distribution of the genotypes between cases and controls. Data were recognized as significant when the statistical p-value was less than 0.05. The lung cancer risk associated with the genotypes was estimated as an odds ratio (ORs) and 95% confidence intervals (CIs) by unconditional logistic regression with adjustment for the effect of possible confounders such as age and gender.
Results
The frequency distributions of selected characteristics for the 358 lung cancer patients and 716 non-cancer controls are summarized in Table I. Since we applied frequency matching to select the healthy controls, distributions of age and gender were comparable between the cases and the controls (Table I). The cases had a slightly higher percentage of smokers (81.8%) than don the controls (78.6%) (p>0.05) (Table I).
The distributions of the XRCC7 G6721T genotypes among the controls and the lung cancer patients are provided and analyzed in Table II. The genotypes of XRCC7 G6721T were differently distributed between lung cancer and healthy control groups (p=3.6×10−7) (Table II). Statistically speaking, the XRCC7 G6721T heterozygous GT and homozygous GG genotypes were significantly associated with decreased lung cancer risk (OR=0.65, 95% CI=0.49-0.84, p=0.0016; OR=0.42, 95% CI=0.23-0.76, p=0.0040, respectively).
The gender ratio of patients with lung cancer in Taiwan is about two males to one female. We are interested in the genotypic contribution of XRCC7 G6721T to gender difference in lung cancer susceptibility. After stratification by the gender, it was found that the genotypes of XRCC7 G6721T were differently distributed among males (p=2.2×10−4) but not females (p=0.6134) (Table III).
The interaction of the genotype of XRCC7 G6721T and the smoking behavior of the participants was of great interest since lung cancer is a smoking-related cancer. The results in Table IV show that the genotypic distribution of the variant genotypes of XRCC7 G6721T was significantly different between lung cancer and control groups who were ever smokers (p=3.1×10−4), but not different in the case among the non-smokers (p=0.6885) (Table IV). Overall, it seemed that there was an interaction between XRCC7 G6721T genotype and smoking lifestyle in regard to lung cancer susceptibility.
Discussion
As the catalytic subunit of the DNA-PK complex, XRCC7 play a role in NHEJ via recognition and repair of DNA DSBs (28). It was reported that mice with inactivated components of DNA-PK had severe combined immunodeficiency and hypersensitivity to ionizing radiation (13, 14). In addition, cells defective in DNA-PK components are hypersensitive to ionizing radiation killing due to an inability to repair DSBs effectively (29). The polymorphic site XRCC7 G6721T (rs.7003908), located in intron 8, may regulate alternative splicing and cause mRNA instability (12), associated with altered cancer risk (15-18, 20, 21).
In the present study, we report that the GT and GG genotypes were associated with reduced lung cancer risk in a moderate (controls:cases=716:358) Taiwanese population (Table II). The prevalence of the major T allele of the XRCC7 G6721T polymorphism in our controls (70.4%) was similar to those reported from other Han populations (15-18, 20, 21). There is no report regarding the role of XRCC7 genotype as a genetic marker for lung cancer. In Taiwan, lung cancer is the most prevalent type of cancer and of highest mortality.
There is a gender difference and there were about twice as many males as females suffering from lung cancer. Smoking is also a risk factor for lung cancer worldwide. Therefore, the present study was also aimed to investigate the interaction of XRCC7 genotypes with gender and smoking lifestyle on lung cancer risk in Taiwan. We found that the association between XRCC7 genotype and lung cancer risk was obvious among the males (Table III). As for the females, the frequency of wild-type TT genotype was also higher in the case group (55.8%) than the control group (50.4%), but the difference did not reach the statistically significant level (Table III). As for the smoking lifestyle, the association between XRCC7 genotypes with lung cancer risk was obvious, especially among ever smokers (Table IV). However, there was no such differential genotypic distribution for the non-smokers. Although this XRCC7 genetic variation was not found to directly result in an amino acid coding change, it might influence the expression level or stability of XRCC7 protein, as well as its function in NHEJ and genome-protecting capacity. Overall, males carrying a T allele for XRCC7 G6721T may have lower capacity than those carrying G allele in DSB-removal capacity, leading to higher susceptibility to smoking-induced lung cancer development.
The current study has several limitations. Firstly, the power of the hospital-based case–control study could be enhanced by enlarging the sample size. Secondly, other confounding factors, such as obesity, alcohol drinking, lifestyle and pollutant exposure status, were not taken into consideration and adjusted for. Further studies could be performed to reveal the contribution of XRCC7 to lung carcinogenesis such as measuring the expression alterations at mRNA and protein levels for XRCC7 in tumor sites from patients with lung cancer, comparing with the non-tumor sites.
In conclusion, our findings suggested that the G allele of XRCC7 G6721T was associated with lower lung cancer risk, especially among males and smokers.
Acknowledgements
The Authors declare no interest conflict with any person or company. We appreciate Hong-Xue Ji, Liang-Yi Lin, Chieh-Lun Hsiao, Chia-En Miao and Tissue-bank of China Medical University Hospital for their excellent technical assistance. This study was supported by research grants from Terry Fox Cancer Research Foundation and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW103-TDU-B-212-113002).
Footnotes
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↵* These Authors contributed equally to this study.
- Received August 21, 2014.
- Revision received September 23, 2014.
- Accepted September 26, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved