【作者】 梁戈玉；

【导师】 浦跃朴； 尹立红；

【作者基本信息】 东南大学 ， 劳动卫生与环境卫生学， 2005， 博士

【摘要】 肺癌严重威胁人类健康,是我国发病率和死亡率上升最快的恶性肿瘤之一。本研究采用配对病例-对照的流行病学研究方法,检测比较了汉族人群CYP1A1、CYP1B1、CYP2C19、CYP2D6、CYP2E1、GSTM1、GSTT1、GSTP1、mEH、NAT2、NQO1、XPA、XPD、XRCC1、XRCC3、hOGG1等代谢酶和修复酶基因的遗传多态性分布及其与该人群肺癌易感性之间的关系,并调查分析了汉族人群肺癌发生的环境危险因素,通过多因素的统计学分析与单纯病例研究方法,初步确定了基因-基因,基因-环境在汉族人群肺癌发生中存在的交互作用。同时还探讨了两个新发展的SNP快速检测技术——diASA-AMP技术和双色荧光杂交芯片技术在人群中进行肺癌相关基因筛检的可行性,以期为肺癌病因学研究,高危人群筛检,以及肺癌的一级预防提供理论依据和技术平台。本论文具体内容如下:1.代谢酶基因多态性和肺癌易感性关系的研究按配对病例-对照研究原则选择原发性肺癌患者及对照227对,每例收集血液3ml,提取基因组DNA。运用PCR-RFLP、diASA-AMP及双色荧光杂交芯片技术对CYP1A1、CYP1B1、CYP2C19、CYP2D6、CYP2E1、GSTM1、GSTT1、GSTP1、mEH、NAT2、NQO1等代谢酶基因进行多态性分析。研究结果表明,携带CYP1A1突变基因型,GSTT1缺失基因型, NAT2 M3等位基因可增加患肺鳞癌的危险性,OR值分别为2.07(95%CI=1.10-3.92),1.90(95%CI=1.09- 3.30),1.63(95%CI=1.03-2.59)。携带CYP2C19突变基因型者患肺癌的危险性增加2.07倍(95%CI =1.34-3.17)。经组织病理学分类后分析发现,CYP2C19突变基因型可同时增加患肺鳞癌和腺癌的危险性,但以患肺鳞癌的危险性增加较多(OR=2.25,95%CI=1.14-4.44)。CYP1B1、CYP2D6、CYP2E1、GSTM1、GSTP1、mEH、NQO1基因在肺癌组和与对照组之间的基因型分布无显著性差异。CYP1B1基因多态性与汉族人群肺癌易感性的关系为首次报道。2.修复酶基因多态性和肺癌易感性关系的研究运用PCR-RFLP、diASA-AMP及双色荧光杂交芯片技术对XPA、XPD、XRCC1、XRCC3、hOGG1修复酶基因进行多态性分析。研究结果表明,携带XRCC1突变基因型可增加患肺腺癌的危险性(OR=1.91, 95%CI=1.12-3.23)。携带XPD突变基因型者患肺癌的危险性增加3.13倍(95%CI=1.78-5.49)。经组织病理学分类后分析发现,XPD突变基因型可同时增加患肺鳞癌和腺癌的危险性, OR值分别为3.20(95%CI=1.57-6.51)和3.00(95%CI=1.19-7.56)。XPA、XRCC3、hOGG1基因在肺癌组和与对照组之间的基因型分布无显著性差异。XPA与XRCC3基因多态性与汉族人群肺癌易感性的关系为首次研究,其中XPA A23G位点突变频率在汉族人群的分布为首次报道。3.代谢酶基因与修复酶基因多态性在肺癌发生中的交互作用研究应用Logistic多元回归模型分析基因-基因交互作用,在α=0.05水平上发现,CYP1A1突变基因型与GSTM1缺失基因型(OR=2.23),CYP2C19突变基因型与NQO1突变基因型(OR=2.61),mEH-exon3突变基因型与NQO1突变基因型(OR=2.47),CYP2C19突变基因型与XPA突变基因型之间对肺癌的发生存在协同作用(OR=4.55),同时携带两种突变基因型者患肺癌的危险性比单独携带其中任何一个突变基因型明显增加。4.肺癌环境危险因素的病例对照研究对所有病例-对照以回顾性问卷询问的方式进行肺癌环境危险因素的流行病学调查,主要内容包括:一般社会学特征、肺部疾病史、吸烟状况、烹调油烟接触状况、职业接触史、煤烟接触状况、家族肿瘤史等19项环境危险因素。结合组织病理学的诊断结果,通过单因素及多因素Logistic回归统计分析,对江苏汉族人群肺癌发生的危险因素进行筛检和危险度评估。研究结果表明,结果表明肺部疾病史、吸烟指数、职业有害物质接触史是肺鳞癌发生的主要环境危险因素,同时消除这些因素其发病可减少67.24%;肺部疾病史、吸烟指数、烹饪时厨房充满油烟味、煤炉使用年限、肿瘤家族史是肺腺癌发生的主要环境危险因素,同时消除这些因素其发病可减少58.92%。5.基因-环境在肺癌发生中交互作用的单纯病例研究应用单纯病例研究分析了基因-环境交互作用,结果表明,CYP1A1基因多态性、hOGG1基因多态性分别与吸烟对肺癌的发生存在有协同作用。携带CYP1A1突变基因型或hOGG1突变基因型而又同时吸烟者发生肺癌的危险性明显增加,OR值分别为2.29(95%CI=1.31-3.98)和2.30(95%CI=1.00-5.27)。GSTP1基因多态性、XPD基因多态性分别与煤炉使用年限对肺癌的发生存在有协同作用。携带GSTP1突变基因型或XPD突变基因型可增加接触煤烟不超过20年者肺癌发生的危险性,OR值分别为2.23(95%CI=1.09-4.54)和3.12(95%CI=1.42-6.85)。6.人工修饰双等位基因特异性引物扩增法(diASA- AMP)在肺癌易感基因筛选中的应用本研究首次应用课题协作组成员华东医学生物技术研究所周国华教授等发明的一种新的快速基因多态性检测方法-diASA-AMP技术(发明专利公开号:1446928)对汉族人群肺癌相关易感基因CYP1B1、GSTP1、hOGG1和XPD进行检测和筛选,并对该技术应用于人群SNP筛检的可行性进行了论证。结果表明对照组各等位基因频率(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%),与现有的中国人资料结果相近(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%)。CYP1B1、GSTP1、hOGG1基因各随机选取15例样本进行测序,结果与diASA-AMP法结果完全相符。XPD基因随机抽取115例样本进行PCR-RFLP分型,结果两种检测方法有4例不相符,二者检测相同结果的吻合率为96.5%,经χ2检验两种方法的基因型分型结果具有很好的关联性和一致性(Kappa=0.90,95%CI=0.81-1.00)。7.双色荧光杂交芯片在肺癌易感基因筛选中的应用本研究首次应用课题协作组成员东南大学吴健雄实验室陆祖宏教授等创建的一种新的高通量基因多态性检测方法-双色荧光杂交芯片技术对汉族人群肺癌相关易感基因CYP1A1、XRCC3进行了检测和筛选,并对该技术应用于人群SNP筛检的可行性进行了论证。结果表明152例样本的CYP1A1基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符。XRCC3基因在对照组的等位基因频率(C 94.1%, T 5.9%)与现有的中国人资料结果一致(C 94.1%-95.2%,T 4.8%-5.9%)。XRCC3基因随机抽取125例样本进行PCR-RFLP分型,结果两种检测方法有2例不相符,二者检测相同结果的吻合率为98.4%,经χ2检验两种方法的基因型分型结果具有很好的关联性和一致性(Kappa=0.97,95%CI=0.92-1.01)。综合分析上述研究结果,可获得以下初步结论:1. CYP1A1、CYP2C19、GSTT1、NAT2、XPD、XRCC1基因与汉族人群肺癌易感性相关。其中CYP1A1突变基因型,GSTT1缺失基因型,NAT2 M3等位基因增加了患肺鳞癌的危险性,XRCC1突变基因型增加了患肺腺癌的危险性,CYP2C19突变基因型、XPD突变基因型同时增加患肺鳞癌和肺腺癌的危险性。CYP1A1与GSTM1,CYP2C19与NQO1,mEH-exon3与NQO1,CYP2C19与XPA基因对肺癌的发生存在交互作用,同时携带两种突变基因型个体更容易发生肺癌。2.肺部疾病史、吸烟指数、职业有害物质接触史是汉族人群肺鳞癌发生的主要环境危险因素,肺部疾病史、吸烟指数、烹饪时厨房充满油烟味、煤炉使用年限、肿瘤家族史是肺腺癌发生的主要环境危险因素。3. CYP1A1、hOGG1突变基因型与吸烟对肺癌的发生存在协同作用,GSTP1、XPD突变基因型与不超过20年煤炉使用年限对肺癌的发生存在有协同作用,两种因素同时存在时均使肺癌患病危险性升高。基因-基因,基因-环境之间的联合检测有助于筛选肺癌发生的高危人群。4. DiASA-AMP技术与双色荧光杂交芯片技术具有较高的特异性和准确度。与传统PCR-RFLP方法相比,diASA-AMP技术简便、快速、经济,适用于较大规模人群SNP位点的快速检测;双色荧光杂交芯片技术具有高通量和自动化的特点,在大规模人群SNP筛检中具有良好的发展前景。更多还原

【Abstract】 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.更多还原