The Association Between DNA Double-strand Break Gene XRCC2 And XRCC5 SNPs And Lung Cancer

Objective Lung cancer is one of the most common malignant tumors in the world. Smoking, along with occupational exposure, is the major cause of lung cancer. The relative risk for lung cancer in current smokers is 10 to 15 times higher than non-smokers. However, only a small number of cigarette smokers develop lung cancer, suggesting inter-individual differences in susceptibility. It has been hypothesized that these differences may be due to genetic variations in DNA repair. Many of carcinogens may result in DNA damage. However, genomic integrity can still be restored through DNA repair mechanisms. Repair of DNA double-strand breaks (DSB) involves homologous recombination (HR) and non-homology end joining (NHEJ) pathways. These pathways include several proteins such as RAD51, some of X-ray repair cross complementing (XRCC), which are important for maintenance of genomic stability. Lung cancer patients have been found to have lower DNA repair capacity compared with healthy individuals. Molecular epidemiology studies have demonstrated that the single nucleotide polymorphism(SNP)of DNA repair gene may alter DNA repair capacity which is an important foctor influencing on individual′s susceptibility to lung cancer,staging and lymph nodes metastasis of cancer, and sensitivity of cancer cell towards chemotherapy. For the DSB-R pathway, it has been reported the variant allele of the DSB gene XRCC2 (Arg188His) is associated with significantly increased risk of NSCLC, the variant allele of the XRCC9 (Thr297Ile) and ATR (Thr211Met) genes may play a protective role and are associated with a significantly decreased risk of developing NSCLC. The correct functioning of XRCC genes involved in both HR and NHEJ is important for genetic stability. Many studies of humans and mice with XRCC gene disruptions have confirmed that these genes are likely to contribute to cancer induction and/or progression, those affecting NHEJ show increased lymphoid tumours, while those affecting HR lead to breast cancer and perhaps to gynaecological tumours. DSB repair genes XRCC2 and XRCC5 play an important role in the early stage of HR and NHEJ. To study the effects of the XRCC2 and XRCC5 SNPs on the development of lung cancer, we investigated the distribution of the XRCC2 C41657T, G4234C SNP and XRCC5 G74582A, C74468A SNPs among the population and lung cancer patients from the North Chineses, with the aim of understanding the association between the SNPs and susceptibility of lung cancer and its influence on the staging and lymph nodes metastasis. The chemosensitivity of lung cancer cell was detected by MTT method analysing the role of SNPs on the drugs.Methods1. Three hundred and fifty-two patients with lung cancer and 404 healthy controls were recruited in the hospital case-control study. Five ml of venous blood from each subject was drawn into the Vacutainer tube containing EDTA. Genomic DNA was extracted using proteinase K digestion followed by a salting out procedure. Information on gender, age, smoking habit and family history of cancer was obtained from patients and healthy controls by interview following sampling. The XRCC2 C41657T, G4234C and XRCC5 C74468A, G74582A genotypes were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and primer-introduced restriction analysis- polymerase chain reaction(PIRA-PCR), respectively. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated using an unconditional logistic regression model and adjusted by age, genger and smoking status accordingly. The relationship between the SNPs and the staging and lymph node metastasis was analysed.2. One hundred and fifty lung cancer patients treated with operation. The sensitivity of lung cancer cell to cisplatin (DDP) and carboplatin (CBP) were detected by MTT and the patients were genotyped for polymorphisms in XRCC2 and XRCC5 genes. Polymorphisms were detected by PCR-RFLP or PIRA-PCR method. The effecs of these polymorphisms on the response of lung cancer to the drugs was investigated3. Statistical analysis was performed using SPSS11.5 software package. P<0.05 was considered significant for all statistical analyses. The haplotype frequencies were estimated by using EH linkage software.Results1. The frequency of smokers in lung cancer patients (66.8%) was significantly higher than that in healthy controls (44.6%) (χ2=37.46,P<0.001). Smoking may increase the risk of developing lung cancer (age and gender adjusted OR=3.44,95% CI =2.37～4.98).2. Frequency of the T allele in lung cancer patients was 20.6%, which was significantly higher than that in healthy controls (14.5%) (χ2=9.83,P=0.002). The distribution of genotypes (C/C,C/T,T/T) in the overall lung cancer patients was also significantly different from that in healthy controls(χ2=9.45,P=0.009). Compared with C/C genotype, the C/T genotype and combination of C/T and T/T genotype both increased the risk of lung cancer (age, gender and smoking status adjusted OR=1.48 and 1.53, 95%CI=1.06~2.06 and 1.11~2.11, respectively). Stratification analysis according to pathological type showed that compared with C/C genotype, the C/T genotype and combination of C/T and T/T both increased the risk of adenocarcinoma (age, gender and smoking status adjusted OR=1.85 and 1.89, 95%CI=1.18～2.90 and 1.22～2.92, respec-tively). When stratified by smoking status, compared with the C/C genotype, the C/T genotype and combination of C/T and T/T genotype both increased the risk of lung cancer in non-smokers (age and gender adjusted OR= 1.84 and 1.71, 95%CI= 1.12～3.04 and 1.05～2.78, respectively). However, when stratified by age, lymphatic metastasis and clinical staging, XRCC2 C41657T SNP was not associated with lymphatic metastasis and clinical staging.3. The genotype and allele distribution of the XRCC2 G4234C SNP in the patients with lung cancer was not significantly different from that in healthy controls (all P>0.05). Stratification analysis according to pathological type showed that compared with G/G genotype, the G/C genotype and combination of G/C and C/C increased the risk of small cell lung cancer (age, gender and smoking status adjusted OR= 1.83 and 1.99; 95%CI=1.01~3.30 and 1.13～3.52, respectively). Stratification analysis according to age showed that compared with G/G genotype, the G/C genotype and combination of G/C and C/C increased the lung cancer risk of the old (age≥60) ( gender and smoking status adjusted OR=1.84 and 1.90, 95%CI=1.07～3.15 and 1.17～3.22). However, when stratified by smoking status, lymphatic metastasis and clinical staging, XRCC2 G4234C SNP was not associated with lymphatic metastasis and clinical staging.4. The combined effect of XRCC2 C41657T and G4234C SNPs on lung cancer was analyzed by EH software. The four haplotypes distribution of the XRCC2 SNP in the patients with lung cancer was significantly different from that in healthy controls (χ2=10.98, P=0.012). Frequency of the 41657T/4234G haplotype in lung cancer patients was 17.6%, which was significantly higher than that in healthy controls (12.6%) (χ2=8.63,P=0.003). Compared with the 41657C/4234G haplotype, the 41657T/4234G haplotype had higher risk in developing lung cancer (OR=1.54,95%CI=1.15～2.05).5. The allelotype and genotype distributions of the XRCC5 C74468A SNP in the lung cancer patients were not significantly different from that in healthy controls (P>0.05). Stratification analysis according to histological type,smoking status,age and lymphatic metastasis showed that XRCC5 C74468A SNP had no significant influence on the risk of lung cancer. However, when stratified by clinical staging, frequency of the XRCC5 C74468A C/C genotype and individuals with A allelotyoe (A/C+A/A) were significantly different between patients with stageⅠandⅡstage disease. Compared with the C/C genotype, lung cancer patients with A allele (A/C+A/A genotype) were not prone to devleop fromⅠstage toⅡstage disease (age, gender, smoking status and histological type adjusted OR=0.45,95%CI=0.25～0.82).6. The allelotype and genotype distributions of the XRCC5 G74582A SNP in the lung cancer patients were not significantly different from that in healthy controls (P>0.05). When stratified by histological type, smoking status, age, lymphatic metastasis and clinical staging, XRCC5 G74582A SNP had no significant effect on the risk of lung cancer.7. The combined effect of XRCC5 C74468A and G74582A SNPs on lung cancer was analyzed by EH software. It was indicated that the 74468C/74582A haplotype was the most frequent haplotype in the population, with a frequency of 53.0%. The distribution of the four haplotypes in lung cancer patients was not clearly different from that in healthy controls (P>0.05).8. Among 150 lung cancer cell from operative patients, there were 60 (40%)resistant samples , 78(52%)sensitive and 12(8%)high sensitive to CBP, respectively. The sensitive rate of CBP was 60%. There are 66 samples resistance , 76 senstivity and 8 high senstivity to DDP, respectively. The sensitive rate of CBP was 56%. The sensitive rate for CBP and DDP was similar (χ2=1.11,P=0.574).9. The sensitive rate of lung cancer cell to CBP and DDP in patients with XRCC2 41657T allele (C/T+T/T genotype) was 70.2% and 66.7%, compared to 53.7% for CBP (χ2=3.97,P=0.046) and 49.5% for DDP (χ2=4.25,P=0.039 ) in patients with C/C genotype. The sensitivity to CBP and DDP in the patients with at least one T allele was 2.06 times (pathological type adjusted OR=2.06,95%CI=1.02～4.18 ) and 2.07 times (pathological type adjusted OR=2.07,95%CI=1.04～4.14 ) higher than that in the patients with C/C genotype.10. The sensitive rate of lung cancer cell to CBP and DDP in patients with XRCC2 4234C allele (G/C+C/C genotype) was 61.9% and 64.3%%. The sensitive rate to CBP and DDP in patients with G/G genotype was 59.3% and 52.8%. There was no significantly difference between sensitive group and resistant group (CBPχ2=0.09,P=0.766 and DDPχ2=1.63,P=0.202 ).11. According to haplotype of XRCC2, the sensitive rate of lung cancer cell to CBP was 70.4% with 41657T/4234G , 70.0% with 41657T/4234C, 58.3% with 41657C/4234C and 57.0% with 41657C/4234G, respectively. The distributions of four haplotype between sensitive group and resistant group were not different (P>0.05). The sensitive rate to DDP was 68.5% with 41657T/4234G , 80.0% with 41657T/4234C, 63.8% with 41657C/4234C and 50.0% with 41657C/4234G, respectively. The distributions of four haplotype between sensitive group and resistant group were significantly difference (χ2=9.60, P=0.022). The sensitivity to DDP in patients with 41657T/4234G was 2.18 times higher than that in patients with 41657C/4234G(OR=2.18,95%CI=1.15～4.12).12. The sensitive rate of lung cancer cell to CBP and DDP in patients with XRCC5 G74582A G allele (A/G+G/G genotype) was 57.9% and 61.8%. The sensitive rate to CBP and DDP in patients with A/A genotype was 62.2% and 50.0%. There was no significantly difference between sensitive group and resistant group(CBPχ2=0.28,P=0.594 and DDPχ2=2.13,P=0.144 ).13. The sensitive rate of lung cancer cell to CBP and DDP in patients with XRCC5 C74468A A allele (A/C+A/A genotype) was 52.9% and 62.7%. The sensitive rate to CBP and DDP in patients with C/C genotype was 63.6% and 52.5%. There was no significantly difference between sensitive group and resistant group (CBPχ2=1.60,P=0.205 and DDPχ2=1.43,P=0.231 ).Conclusions1. Smoking greatly increases the risk of developing lung cancer.2. The polymorphism of XRCC2 C41657T is associated with the increased risk of lung cancer especially to the adenocarcinoma. Compared with C/C genotype, People with the combination of C/T and T/T of XRCC2 C41657T and C/T genotype have significantly increased risk for lung cancer, especially for adenocarcinoma.3. The combination of XRCC2 C41657T C/T and T/T genotype and C/T genotype both increase the risk of lung cancer in non-smokers .4. The polymorphism of XRCC2 G4234C is associated with the increased risk of small cell lung cancer. Compared with G/G genotype, the combination of G/C and C/C of XRCC2 G4234C and G/C genotype increase the risk of small cell lung cancer. 5. Compared with G/G genotype, the combination of G/C and C/C of XRCC2 G4234C and G/C genotype increase the lung cancer risk of the old (age≥60).6. Compared with 41657C/4234G haplotype the 41657T/4234G haplotype significantly increase the risk of developing lung cancer.7. XRCC2 C41657T and G4234C SNP are not associated with lymphatic metastasis and clinical stage of lung cancer.8. Lung cancer patients with XRCC5 C74468A A allele (A/C+A/A genotype) are not prone to devleop from clinical stageⅠto stageⅡ.9. The XRCC5 G74582A polymorphism is not associated with susceptibility in lung cancer.10. All the haplotypes of XRCC5 C74468A and G74582A are not associated with risk of lung cancer.11. The XRCC2 C41657T SNP was associated with the sensitivity to CBP and DDP. The sensitive rate to the drugs of the cancer cell with the combination of C/T+T/T is higher than that with C/C genotype,12. In the subject with 41657T/4234G the sensitivity to the drugs of the cancer cell is higher than that with 41657C/4234G haplotypes,13. The XRCC5 G74582A, C74468A SNP are not associated with the sensitivity to CBP and DDP.