Single Nucleotide Polymorphism in Mismatch Repair Pathway Genes, and its Computational Analysis in Lung Cancer Patients

Abstract

Background: Lung cancer is the leading, cause of cancer mortality globally, and the critical risk factors are smoking and occupational exposure. Molecular alterations in the repair pathway genes may lead to improper repair and, ultimately, carcinogenesis. Objective: To evaluate the role of single nucleotide polymorphic variants of DNA mismatch repair (MMR) genes i.e. MLH1 (rs1800734), MSH3 (rs26279), MSH6 (rs3136228, rs1800932, rs1042821) and MSH2 (rs63749993, rs2303425, rs2303426, rs4987188, rs2303428, and rs17217772) towards lung cancer susceptibility. Methodology: The study was designed to find out any association between genetic polymorphism of mismatch repair pathway genes and risk of developing lung cancer. This study recruited 500 lung cancer patients from the Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research (PGIMER), and 500 healthy controls. These cancer patients were further subdivided in to smaller subgroups based on histology of lung cancer, gender, smoking status (Yes/No and Heavy smokers/ Light smokers). Genomic DNA from lung cancer subjects was genotyped using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) for each polymorphic site under study. Logistic regression was used to analyze subjects to determine any association towards development of lung cancer. Classification and Regression tree (CART) analysis was carried to understand SNP-SNP (single nucleotide polymorphism) interaction. Overall survival analysis was carried out using Kaplan-Meier survival analysis and Cox-regression analysis. Toxicity associated with different chemotherapeutic regimen was also evaluated. Further different computational tools were used to analyze the nsSNPs to evaluate their effect on structure and stability of the protein. Results: For MLH1 polymorphism, reduced risk of developing Adenocarcinoma (ADCC) in patients harboring variant (p= 0.0007) and combined type genotype (p=0.008) was reported. Further, the heterozygous type and combined type genotype of heavy smokers reported a 2-fold (p=0.001) and 1.8-fold increased risk of lung cancer development (p=0.007). For MSH3 polymorphism, females, patients harboring heterozygous type (GA) genotype have a 2-fold increased risk (p=0.04) for the development of lung cancer. For MSH6 rs3136228 polymorphism, decreased risk of developing lung cancer in the combined genotype (p= 0.03) and 1.4-fold increased risk of developing lung cancer was reported in individuals carrying mutant type genotype (p=0.03). Segregation based on histological subtypes, subjects carrying the heterozygous genotype (GT) and combined (GT+GG) genotype reported a decreased risk of developing adenocarcinoma (p=0.03). For MSH6 (rs1800932) polymorphism, our study reported that patients suffering from Small cell lung cancer (SCLC) and harboring heterozygous genotype (AG) have a significant increase in median survival time (MST) (p=0.03). Furthermore, for MSH6 rs1042821 polymorphism, patients undergoing docetaxel chemotherapeutic regimen with CT genotype reported a significant increase in MST (p= 0.03). Four MSH2 (118T>C (rs2303425), 1032G>A (rs4987188), T>C/-6 (rs2303428), and Asn127Ser (rs17217772)) polymorphisms reported a strong propensity towards the risk of developing lung cancer in subjects having heterozygous and mutant genotype. T>C/-6 polymorphism showed a 13-fold (Pcorr=0.0006) 10 increased risk of developing lung cancer in patients carrying the variant allele. Our study also found that in 118T>C polymorphism, minor allele C was significantly associated with poor survival (MST=3.13, p=0.03) in lung cancer patients. In lung cancer patients undergoing paclitaxel therapy, heterozygous carriers for the Asn127Ser polymorphism were shown to significantly reduce survival (p=0.02). For 2063T>G polymorphism undergoing irinotecan therapy, subjects with a mutant genotype showed poor survival (p=0.0004). Six out of eleven SNPs of four genes (MLH1, MSH2, MSH3, and MSH6) located in the coding region were considered for computational analysis. For MSH2 protein, rs63749993 and rs17217772 polymorphism was shown to be deleterious by SIFT, PROVEAN, PANTHER, Polyphen-2, PhDSNP and SNP&GO whereas I-mutant and MuPRO predicted it to have decreased stability. rs4987188 polymorphism was predicted to have damaging effect by PANTHER, Polyphen-2 only and I-mutant and MuPro predicted it to have decreased stability. rs1800932 (MLH1), rs1042821 (MSH6) and rs26279 (MSH3) was shown to have damaging effect by PANTHER, Polyphen-2 whereas only rs1800932, rs1042821 were predicted to have increased stability by I-mutant and MuPRO. Conclusion: MLH1 (rs1800734), MSH6 (rs3136228), MSH2 (rs2303425, rs4987188, rs2303428 and rs17217772) and MSH3 (rs26279) polymorphism were associated with modulating the risk of lung cancer in North Indian population. Computational analysis of nsSNPs selected for this study showed that they play a key role in altering the structure of protein