Studies On Susceptiblity Genes Of Lung Cancer In Chinese Han Population And Rapid Detection Techniques Of Single Nucleotide Polymorphisms

The incidence and mortality of lung cancer have been increasing rapidly in recent years in China. In this study, a paired case-control epidemiological investigation was performed to determine the relationship between genetic polymorphisms of some metabolic and repair enzymes and susceptibility of lung cancer. CYP1A1, CYP1B1, CYP2C19, CYP2D6, CYP2E1, GSTM1, GSTT1, GSTP1, mEH, NAT2, NQO1, XPA, XPD, XRCC1, XRCC3 and hOGG1 genetic polymorphisms were detection by PCR- restriction fragment length polymorphism (PCR-RFLP) analysis and two new techniques for rapid genotyping of SNPs, that is di-allele-specific-amplification with artificially modified primer (diASA- AMP) and dual-color fluorescence hybridization chip. Logistic regression model was applied to screen 19 possible environmental risk factors and to analyze the gene-gene interaction to lung cancer. A case study was carried out to explore the possible gene-environment interaction in lung cancer development of Chinese Han population. Furthermore, detection efficiency and application foreground of two new methods, diASA-AMP and dual-color fluorescence hybridization chip, were evaluated in this study.The principal results were as follows:1. The relationship between the polymorphisms of metabolic enzyme genes and susceptibility to lung cancerA case-control epidemiological investigation was performed in which 227 hospital controls were matched to 227 original lung cancer cases by gender, ethnicity, and age(±5 years). Three milliliters of venous blood was collected from each patient and control. DNA was isolated from whole blood cells. The different gene alleles were determined by PCR-RFLP, diASA-AMP and dual-color fluorescence hybridization chips. The polymorphisms of CYP1A1, CYP1B1, CYP2C19, CYP2D6, CYP2E1, GSTM1, GSTT1, GSTP1, mEH, NAT2 and NQO1 genes were examined and analyzed statistically. The results showed that CYP1A1 mutation genotypes, GSTT1 null genotype and NAT2 M3 allele were related to the risk of lung squamous cell carcinoma and OR were 2.07(95%CI=1.10-3.92), 1.90(95%CI=1.09-3.30), 1.63(95%CI =1.03-2.59), respectively. The CYP2C19 mutation genotypes were associated with the risk of lung cancer (both squamous cell carcinoma and adenocarcinoma), especially in squamous cell carcinoma (OR=2.25,95%CI=1.14-4.44). It was shown that there was no difference in genotype distribution of CYP1B1, CYP2D6, CYP2E1, GSTM1, GSTP1, mEH and NQO1 between cases and controls. The relationship between genetic polymorphism of CYP1B1 andsusceptibility of lung cancer was first investigated in Chinese Han population.2. The relationship between the polymorphisms of DNA repair enzyme genes and susceptibility to lung cancerThe different gene alleles were determined by PCR-RFLP, diASA-AMP and dual-color fluorescence hybridization chips. The polymorphisms of XPA, XPD, XRCC1, XRCC3 and hOGG1 genes were examined and analyzed statistically. The results showed that XRCC1 mutation genotypes were related to the risk of lung adenocarcinoma (OR=1.91, 95%CI=1.12-3.23). The XPD mutation genotypes were associated with a 3.13-fold increase (95%CI=1.78-5.49) in risk of lung cancer. It was associated with risk both in lung squamous cell carcinoma (OR=3.20, 95%CI= 1.57-6.51) and in lung adenocarcinoma (OR=3.00,95%CI=1.19-7.56). It was shown that there was no difference in genotypes distribution of XPA, XRCC3 and hOGG1 between cases and controls. The relationship between genetic polymorphisms of XPA and XRCC3 and susceptibility of lung cancer was first investigated in Chinese Han population. The genotype distribution of XPA A23G mutation was first reported in Chinese Han population.3. Study on the gene-gene interaction between the polymorphisms of metabolic enzyme and DNA repair enzyme genes to lung cancerThe gene-gene interactions between the polymorphisms of metabolic enzyme and DNA repair enzyme genes were analyzed by multivariate logistic regression. The significant interactions of synergistic effect between CYP1A1 mutation genotype and GSTM1 null genotype (OR=2.23), CYP2C19 mutation genotype and NQO1 mutation genotype (OR=2.61), mEH-exon3 mutation genotype and NQO1 mutation genotype (OR=2.47), CYP2C19 mutation genotype and XPA mutation genotype (OR=4.55) were found respectively in the risk of lung cancer. Individuals with two mutation genotypes presented significantly higher risks to lung cancer than with only one of the two mutant genotypes.4. A case-control study on the environmental risk factors to lung cancerThe data of 19 environmental risk factors related to lung cancer were collected and analyzed statistically from each case and control, which included sociodemographic characteristics, lung disease history, smoking habit, cooking oil fume intake, occupational history, coal fume intake, and family history of cancer so on with a questionnaire. Single factor analysis and conditional multiple logistic regression were applied to screen environmental risk factors. The results showed that the environmental risk factors related with squamous cell lung carcinoma were lung disease history, smoking index and occupational exposure. Summary population attributable risk for the three factors above was 67.24%. The environmental risk factors related to lung adenocarcinoma were lung disease history, smoking index, heavy oil fume in cooking, using coal stove years, family history of cancer. Summary population attributable risk for the five factors above was 58.92%.5. A case-only study on the gene-environment interactions to lung cancerA case-only study was carried out to explore the possible gene-environment interaction in lung cancer development of Chinese Han population. Combination analysis of environmental risk factors and polymorphisms of metabolic enzyme and DNA repair genes showed that there was a synergistic effect on development of lung cancer between smoking and CYP1A1 mutation genotype(OR=2.29, 95%CI= 1.31-3.98) or hOGG1 mutation genotype(OR=2.30, 95%CI=1.00-5.27), respectively. A synergistic effect between GSTP1 mutation genotype or XPD mutation genotype and using coal stove no more than 20 years was also found in the risk of lung cancer, OR was 2.23(95%CI=1.09-4.54)and 3.12(95%CI= 1.42-6.85), respectively.6. Application of diASA-AMP method in the detection of genetic polymorphism related with lung caner susceptibilityDiASA-AMP was a new technique for rapid genotyping of SNPs, which was invention of our cooperation research group from professor Zhou 's laboratory in East China Research Institute for Medical and Biotechnics. DiASA-AMP method was evaluated for population screening of SNPs and applied to type CYP1B1, GSTP1, hOGG1 and XPD polymorphisms. The results showed that the allele frequencies of CYP1B1, GSTP1, hOGG1 and XPD in our control population (CYP1B1: C 84.1%, G 15.9%; GSTP1: A 77.1%, G 22.9%; hOGG1: C 39.4%, G 60.6%; XPD: A 93.3%, C 6.7%) were similar to other Chinese studies (CYP1B1: C 83.0%, G 17%; GSTP1: A 75.6%, G 24.4%; hOGG1: C 44.9%, G 55.1%; XPD: A 92.8%, C 7.2%). Furthermore, 15 samples of CYP1B1, GSTP1 and hOGG1 gene were randomly selected for DNA direct sequence. The sequencing results were consistent with diASA-AMP results. 115 samples of XPD gene were randomly selected for PCR-RFLP. The results of 4 samples were not match between diASA-AMP and PCR-RFLP. The accuracy frequency of diASA-AMP was 96.5%. The result ofχ2 test showed that diASA-AMP genotyping for SNP was a high consistent with PCR-RFLP (Kappa=0.90,95%CI=0.81-1.00).7. Application of dual-color fluorescence hybridization chip technique in the detection of genetic polymorphism related with lung caner susceptibilityDual-color fluorescence hybridization chip method was a new high-throughput approach for genotyping of SNPs for population study. It was invention of our cooperation group supervised by professor Lu of Laboratory of Molecular and Biomolecular Electronics (Southeast University). Dual-color fluorescence hybridization chip method was evaluated for population screening of SNPs and applied to type CYP1A1 and XRCC3 polymorphisms. The results showed that the results of CYP1A1 genotyping in 152 samples by dual-color fluorescence hybridization chip were consistent with PCR-RFLP results. The allele frequencies of XRCC3 gene in our control population (C 94.1%, T 5.9%) were similar to other Chinese studies (C 94.1%-95.2%, T 4.8%-5.9%). 125 samples of XPD gene were randomly selected for PCR-RFLP. The results of 2 samples were not match between dual-color fluorescence hybridization chip results and PCR-RFLP results. The accuracy frequency of dual-color fluorescence hybridization chip was 98.4%. The result ofχ2 test showed that dual-color fluorescence hybridization chip genotyping for SNP was a high consistent with PCR-RFLP (Kappa=0.97,95%CI=0.92-1.01).From all results described above, we can get preliminary conclusions as follows:1. CYP1A1, CYP2C19, GSTT1, NAT2, XPD and XRCC1 genetic polymorphisms were associated with the susceptibility to lung cancer in Chinese Han population. CYP1A1 mutation genotypes, GSTT1 null genotype and NAT2 M3 allele were related to the risk of lung squamous cell carcinoma. XRCC1 mutation genotypes were related to the risk of lung adenocarcinoma. The CYP2C19, XPD mutation genotypes were associated with risk of both lung squamous cell carcinoma and lung adenocarcinoma. The risk of lung cancer was increased significantly in individuals who carried high-risk genotypes in both CYP1A1 and GSTM1, both CYP2C19 and NQO1, both mEH-exon3 and NQO1, or both CYP2C19 and XPA.2. Lung disease history, smoking index and occupational exposure were environmental risk factors of squamous cell lung carcinoma. Lung disease history, smoking index, heavy oil fume in cooking, using coal stove years and family history of cancer were environmental risk factors of lung adenocarcinoma.3. There was a synergistic effect on development of lung cancer between CYP1A1 mutation genotype or hOGG1 mutation genotype and smoking, between GSTP1 mutation genotype or XPD mutation genotype and using coal stove no more than 20 years, respectively. The results showed that analysis of gene-gene and gene-environment interactions was helpful to identification of susceptible individuals and screening high-risk group.4. DiASA-AMP and dual-color fluorescence hybridization chip technique were specific and accurate on SNP genotyping. DiASA-AMP is a convenient, time-saving and cost-effective method compared with PCR-RFLP. It could be applied for a rapid detection of the gene polymorphisms related to tumor susceptibility in a middling size population. Dual-color fluorescence hybridization chip is an automatism and high-throughput tool, and is promising for SNP genotyping in a large population.