Single Nucleotide Polymorphisms in the Base Excision Repair and Double Strand Break Repair pathway genes in Lung Cancer patients and their correlation with clinical outcomes

Abstract

DNA repair act as guards to the genomic integrity. Molecular alterations in the repair pathways genes may lead to improper repair and ultimately carcinogenesis. Objectives To evaluate the role of single nucleotide polymorphic variants of BER (Base Excision Repair) genes OGG1 (OGG1 Ser326Cys), MUTYH(MUTYH Gln324 His (G/C)), XRCC1 (XRCC1 Arg399Gln, XRCC1 Arg194Trp), XRCC1 Pro206Pro, XRCC1 Arg280His, XRCC1 Gln632Gln), and DSBR(Double Strand Break Repair) genes XRCC3 (XRCC3 Thr241 Met), XRCC4 (XRCC4 intron 3 I/D), XRCC6 (XRCC6 61G>C), XRCC7 (XRCC7 6721G>T). Methodology Genotyping of genomic DNA was carried out using PCR-RFLP (polymerase chain reactionrestriction fragment length polymorphism) for each polymorphic site under study. Total number of subjects genotyped in case of OGG1 and MUTYH were 656 (330 cases,326 controls); in XRCC1 were 655 (330 cases, 325 controls) and in case of XRCC6 and XRCC7 were 650 (330 cases, 320 controls) respectively. Following this association analysis was carried out using logistic regression to obtain adjusted odds ratio and significance. Data mining analysis was performed including both Multi-dimensionality reduction (MDR) and Classification and Regression tree (CART) analysis to find the possible interaction between interacting snp-snp and gene-gene. Overall survival analysis was performed using Kaplan meier survival analysis and Cox-regression analysis which gave adjusted hazards ratio. Results OGG1 presented a high risk towards lung cancer (CG: OR=2.44, p=0.0003; CG+GG: OR=1.88,p=0.0093). On the same lines adenocarcinoma patients in case of OGG1 were at high risk of acquiring lung cancer (CG: OR=4.72, p=0.0002; CG+GG: OR=3.63, p=0.0018). Single allelic carriers for MUTYH gene imposed a high risk towards overall lung cancer susceptibility and for all the three histology. Gene-environmental interaction revealed that non-smokers both in case of OGG1 and MUTYH acquired a high risk towards lung cancer. Evaluation of clinicopathological factors revealed that OGG1 showed a strong correlation with lymph node involved. For the five polymorphic variants of XRCC1 statistical analysis revealed XRCC1 Gln632Gln (OR=2.67, p=<0.001) depicted an overall high risk towards lung cancer. Histological subdivision revealed carriers of mutant genotype in case of XRCC1 Arg399Gln imposed a protective effect iii towards SQCC subtype. Likewise, mutant genotype in XRCC1 Pro206Pro implied a protective effect for SCLC subtype (OR=0.29, p=0.0017) on the contrary XRCC1 Gln632Gln showed a high risk in SQCC diseased group (OR=4.16, p=<0.0001). Combination of XRCC1 Gln632Gln with other SNPs revealed XRCC1 Gln632Gln with Arg194Trp (OR=2.10, p=0.03) and Pro206Pro (OR=5.6, p<0.0004) increased an overall risk towards lung cancer. Haplotype analysis illustrated haplotype block 11 (CGAGG) carrying minor allele for XRCC1 206 was associated with the highest risk towards lung cancer on the contrary block 4 (CAGAG) carrying mutant allele for XRCC1 399 significantly decreased the risk. Multi-dimensionality reduction (MDR) results showed the three factor model comprising XRCC1 206, 632, 280 as the best model (CVC=10, prediction error= 0.34). Further Classification and Regression tree (CART) analysis revealed terminal node 1 carrying mutant genotype of XRCC1 632 and wild type of XRCC1 280 represented the highest risk group. The DSBR gene XRCC4 intron3 did not show any significant role in modulating the susceptibility towards lung cancer. However, when association analysis was carried out for smokers and non-smokers, the non-smoker individuals with the combined variant genotype were found to be at two times higher risk of developing cancer (OR=1.9, p=0.04). The statistical evaluation of XRCC6 61C>G and XRCC7 6721G>T did not show any significant impact in modulating the susceptibility towards lung cancer. SCLC (Small cell lung cancer) subtype posed an amplified risk towards lung cancer in case of XRCC7 6721G>T (OR=4.11, p=0.0040). Gene-environment interaction analysis revealed that non-smokers with heterozygous genotype (CG) in case of XRCC6 61C¿G showed a strong protective effect (OR=0.38, p=0.01) towards lung cancer. Survival analysis for stratified chemotherapeutic drug regimens revealed, administration of cisplatin/carboplatin+ pemtrexed for OGG1Ser326Cys showed a better survival (MST CG vs. CC: 9.1 vs. 0.56, p=<0.0001; HR =0.051, p=0.0025). Whereas, MU- TYH Gln324His showed a smaller survival for mutant genotype (CC)(MST CC vs. GG: 4.0 vs. 9.4, p=0.05; HR= 1.75, p=0.26). The mutant genotype for XRCC1 399 is observed to have a better survival (MST=9.6). Histological stratification did not reveal any association for any SNP except for SCLC subtype in XRCC1 632 with an increased death rate (HR=3.08, p=0.02). On stratification according to chemotherapy regimen administered; cisplatin/carboplatin+docetaxel was observed to increase survival for XRCC1 399 mutant genotype (H.R=0.26, p=0.05). Cisplatin/carboplatin+irinotecan increased survival in both heterozygotes and combined variants (HR=0.22, p=0.014; H.R=0.23, p=0.012). Subjects with SCLC subtype for polymorphic variant XRCC6 61C>G revealed a poor prognosis as high death rate was obtained. Multiple gene interaction, showed that the three factor model with CVC 10/10 and prediction error =0.33 was the best interacting model determining the lung cancer risk. The second best model was the iv four factor model (CVC=9/10, Prediction error=0.29). In CART analysis XRCC1 632 (H+M) came out to be the significant factor, XRCC1 632(H+M)/MUTYH 324(H+M) XRCC1 206(W) showed a twenty fold risk of acquiring lung cancer. Survival tree analysis depicted OGG1Ser326Cys as the major contributor. Conclusion This preliminary study provides an insight into the potential of genetic differences in BER and DSBR genes to modulate lung cancer susceptibility. BER genes OGG1 and MUTYH were certainly modifying risk of lung cancer in North Indian population. XRCC1 632 acted as an important. risk modifier of lung cancer. Furthermore, polymorphic XRCC6 and XRCC7 were not modifying both overall lung cancer susceptibility and survival.