Study of the Genetic polymorphism and Methylation profile in the Wnt signaling pathway with risk for occurrence of Lung cancer in North Indian population

Abstract

Background: Wnt pathway has been implicated in the process of lung carcinogenesis. The genetic differences and methylation profile in the Wnt antagonist genes (DKK, Axin2 and sFRP) may lead to chronic activation of this cascade. Objectives: To evaluate the role of Single nucleotide polymorphic variants of DKK3 (rs2291599, rs3206824 and rs7391689), DKK2 (rs447372, rs419558 and rs17037102) and DKK4 (rs2073664) genes, Axin2 (148 C/T, 1365 G/A, 432 T/C, 956+16 A/G, 1712+19 G/T, 1386 C/T and 2062 C/T), sFRP3 ( rs7775 and rs228326) and sFRP4 ( rs1802073 and rs1802074). Also, to detect the methylation status of sFRP1, DKK2, DKK3 and Axin2 gene. Methodology: Genotyping of genomic DNA was carried out using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) for each polymorphic site under study. Total number of subjects genotyped in case of DKK, Axin2 and sFRP genetic variants were 600 (300 cases and 300 controls), 608 (303 cases and 305 controls) and 610 (300 cases and 310 controls) respectively. Further association analysis was carried out using logistic regression approach to obtain adjusted odds ratio and statistical significance. MDR and CART analysis were applied to evaluate high order interactions between the SNP’s of same gene. Also, MDR and CART was also used to analyze the high order interaction between the genetic variants of different genes. Overall Survival was estimated using Kaplan meier survival analysis and adjusted hazards ratio was obtained using cox regression method. DNA isolated from plasma samples of 100 lung cancer patients were screened for the methylation status of promoter region of sFRP1, DKK2, DKK3 and Axin2 gene using methylation specific PCR. Further, the methylation status was correlated with the clincopathological parameters such as stage, primary tumor extension, lymph node involvement and distant metastasis using logistic regression approach. Results: DKK4 (rs2073664) did not confer any effect on lung cancer susceptibility. Also, the two variants of DKK3 namely rs3206824 and rs2291599 didn’t show any association except rs7396187 which showed a protective effect (OR=0.63, p=0.01). However, the subjects with heterozygous genotype of DKK2 rs17037102 and rs419558 were found to have a higher risk of developing lung cancer. DKK3 rs3206824 and DKK2 rs419558 showed a two-fold increased risk of developing lung cancer (p=0.008). None of the polymorphic site was found to be associated with the clinic-pathological features. Three out of seven studied polymorphic sites studied for Axin2 gene showed a strong protective effect in subjects having mutant genotype for Axin2 148 C/T and heterozygous genotype for 1365 G/A towards lung cancer risk. The other important finding was the significant association of Axin2 148 C/T in SQCC patients having variant (TT) genotype. Unfavorable genotypes of the three DKK2 variants collectively (rs447372, rs419558 and rs17037102) exhibited a highly decreased rate of death (HR’=0.37, p=0.03). ADCC patients carrying the heterozygous (CT) genotype for DKK4 rs2073664 showed a better OS (log rank p=0.01). ADCC subjects having TT genotype for Axin2 148 showed a better prognosis (HR’=0.48, p=0.003) and SCLC patients with TT genotype for Axin2 1386 showed a poor prognosis (HR’=2.33, p=0.02). Survival tree analysis depicted DKK4 rs2073664 as the major contributor. Findings from the present study revealed that the rate of methylation of promoters in plasma of lung cancer patients for sFRP1, DKK3, Axin2 and DKK2 was found to be 24%, 45%, 25% and 10% respectively. Conclusion: This preliminary study gave an insight that about the potential of genetic differences in Wnt antagonist genes to modulate lung cancer susceptibility. Genetic variations in Wnt antagonist, DKK2 (rs419558 and rs17037102) were certainly playing a crucial role in modulating susceptibility of North Indians as compared to DKK3 and DKK4. Axin2 148 was also found to play a major. Survival analysis revealed that a cumulative effect of three variants of DKK2 gene along with DKK4 rs2073664 can jointly predict the survival of lung cancer patients.