【作者】 滑峰；

【导师】 周清华； 陈军；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 博士

【摘要】 肺癌是世界范围内的常见恶性肿瘤。随着工业化和城市化的进程,我国肺癌的防控形势也越趋严峻。积极开展肺癌预防在21世纪的肿瘤防控战略中具有重要意义。对肺癌的大量研究已经证实肺癌是一种典型的环境相关疾病,吸烟和大气污染等是肺癌的主要危险因素。但是肺癌的群体流行病学研究也表明了不同个体间对共同的环境致癌因素具有不同的易感性。随着人类基因组计划的完成,能够反映个体间基因差异的单核苷酸多态性(single neucleotide polymorphism, SNP)受到广泛重视并成为重要的功能基因组学研究内容。充分利用SNP公共数据库的信息,研究肺癌相关候选基因,寻找与肺癌发生具有相关性的SNP位点是研究肺癌易感性分子标志物的有效策略。肺癌的发生与发展是一个多因素介入的多步骤过程。烟草等各种环境致癌物在体内要经过一系列代谢酶的复杂作用,体外实验发现谷胱甘肽-S-转移酶P1(glutathione-S-transferase P1, GSTP1)、髓过氧化物酶(Myeloperoxidase, MPO)等代谢酶基因的多态性影响了烟草致癌物的代谢,由此推论个体对肺癌的易感性在一定程度上可能取决于外来化学致癌物在不同个体内的代谢差异,特别是在癌变的始动阶段,这种差异可能对肺癌的发生起着关键作用。关于慢性病发病机制的一个学说认为常见基因变异引起常见疾病。我们假设：参与烟草等环境致癌物代谢的基因常见多态性与肺癌的发生具有一定相关性,而具有相关性的这种常见多态性位点在理论上有望成为筛选肺癌易感人群的分子标志物。为了验证上述假设,我们在肺癌的病因、表现、发病机制的认识基础上选择了在人体中具有代谢烟草前致癌物作用并在肺组织中充分表达的代谢酶GSTP1、细胞色素P450酶2A13(cytochrome P450 2A13, CYP2A13)、MPO作为候选基因,采用分子流行病学病例-对照研究方法研究其常见多态性与肺癌易感性的关系。国际人类基因组单体型图计划(International HapMap project)的顺利进行为开展中国人群基因多态性的研究提供了大量有用信息。我们在充分利用SNP公共数据库信息的基础上,对上述基因进行了整体分析,综合运用“以序列为基础”和“以图谱为基础”的选点策略,选择了基本可以覆盖上述基因常见多态性信息的SNP位点(GSTP1的单体型标签SNPs rs762803、rs749174、rs1695、rs4891,CYP2A13的标签SNPs rs8192789和rs1645690,MPO的标签SNPsrs7208693),采用了Taqman探针基因分型技术对比分析了266例中国汉族肺癌人群及307例年龄、性别相匹配的健康对照者的基因型分布,并应用TA克隆测序技术验证了Taqman探针基因分型技术的准确性,对基因型分型结果应用单元多因素logistic回归模型统计分析其与肺癌易感性的关系。对得到广泛研究的GSTP1 rs1695位点及MPO G-463A位点应用Meta分析的方法进行了系统评价。在对CYP2A13的基因rs8192789位点分析中我们发现了同源序列干扰的现象,进一步应用TA克隆测序技术研究了同源序列对CYP2A13基因SNP研究的影响。结果显示我们得到的基因型数据与HapMap数据库中公布的北京汉族人群基因型数据是一致的。MPO rs7208693与肺癌不具有相关性；CYP2A13的标签SNPs rs1645690与非吸烟人群的肺癌发生具有明显相关性(adjust OR=0.448,95%CI0.208～0.967,p=0.041)；GSTP1的单体型标签SNPs rs762803、rs749174、rs1695、rs4891在两组人群的分布均具有差异性,利用Phase 2软件构建了其单体型,分析发现一个常见单体型CACA与吸烟人群的肺癌易感性具有关联(adjustOR=1.53,95%CI 1.04～2.25,p=0.033),由其构成的双倍型与吸烟人群的相关性更趋明显。对GSTP1进行的以单体型为基础的分析表明其符合常染色体隐性遗传模式。对MPO G-463A的Meta分析表明这两个位点与肺癌易感性不具有明显相关性。对CYP2A13的基因部分序列克隆测序的结果表明,CYP2A13的同源序列可能干扰了关于SNP的研究,dbSNP数据库中一些上传的位点可能是不存在的。综上所述,我们充分利用了SNP公共数据库的信息,首次研究了上述三个基因常见多态性与肺癌易感性的关系,发现了一个与肺癌具有关联的位于CYP2A13基因的危险多态性位点以及位于GSTP1基因的一个危险单体型,其有望成为筛查肺癌易感人群的分子标记物,即肺癌易感性SNPs。更多还原

【Abstract】 Lung cancer is the most common malignant tumor all over the world. With the development of the industry and urbanization process in Chian, we also face the more and more serious state to control and prevent the tumor. It is very intersting for the control and prevention of cancer to deal with lung cancer in the 21 century. It has been well established that lung caner is an enviroment-associated disease caused by mainly tabacco smoking and air pollution. Otherwise, it also indicate that variant susceptibility exist between individuals in the common carcinogen-enviroment exposure by the epidemiologic study. With the complement of human genomic project, the study on the single neucleotide polymorphism (SNP), which can reflect the variance between individuals, was think highly of one most important post-genomic project. To research the candidated gene and look for the SNPs associated with lung caner susecptibility on the basis of information of SNP database is a valid method.The carcinogenesis and process of lung cancer is an multi-step caused by multitude factors. The carcinogens such as tobacco smoking,will go through complex process by a series of carcinogen-metabolizing enzymes. It has been found that the polymorphisms of some metabolizing enzymes, such as glutathione-S-transferase PI (GSTP1), Myeloperoxidase(MPO) can effect the metabolazing for the tobacco carcinogens. So, it can be infered that The genetic variations at carcinogen-metabolizing enzymes may lead to interindividual differences at the level of internal carcinogenic dose and lead to differential risk in the individuals with similar exposures, and especially in the initial step, the variations may play an important role. In the light of common disease-common variant hypothesis, we hypothesis also that, there is association between the common polymorphisms of some metabolazing enzymes gene and lung cancer susceptibility, and the common gene polymorphisms may be the candidated biomarkers for the scan of the risk individuals.To validat the hypothesis, we select the metabolizing enzyme gene GSTP1, MPO and cytochrome P450 2A13(CYP2A13) as the candidated gene which play an important role in tobacco carcinoges metabolization and expressed in human lung tissue, to investigate the association between common polymorphisms and lung cancer risk in Chinese population, using a case-control analysis of 266 subjects with lung cancer and 307 controls with no personal history of the disease. With the process of International HapMap project, we can retrieve a lot of information for the research on Chinese gene polymorphisms. We analysed the 3 gene thoroughly according to the SNP data retrieved from the database and select the SNPs to study using the sequence-based and HapMap-based method syntheticly. The tagSNPs rs7208693 for MPO, tagSNPs rs1645690 and rs8192789 for CYP2A13, and the haplotype-tagging SNPs rs1695, rs4891, rs762803, rs749174 for GSTP1 were selected, these SNPs can reflect the common polymorphisms of the 3 genes. Taqman assays was used to genotype the the genetic polymorphisms for the 266 patients and 307 controls, and the genotyping method was validated by TA cloning and sequencing. Unconditional logistic regression method was used to evaluate the distribution of the genetic polymorphisms between the 2 groups and the association with lung cancer after adjusting for confounders. We evaluated the relationship between MPO G-463A, GSTP1 rs1695 and lung cancer risk by Meta analysis.We found the interference caused by homologous sequences during the study for the rs8192789 of CYP2A13, so we furtherly researched the interfering effect to CYP2A13 SNP study caused by homologous sequences using TA cloning and sequencing method.The frequency of the 6 SNPs we tested was consistent with the data of Han-Chinese of Beijing released by HapMap database. The tagSNPs rs1645690 of CYP2A13 showed association between lung caner risk and non-smokers(adjust OR =0.448,95%CI 0.208～0.967,p=0.041), not the tagSNPs rs7208693 of MPO. The all 4 Haplotype-tagging SNPs, rs1695, rs4891, rs762803 and rs749174, showed association with lung cancer susceptibility in the smokers. Phase 2 sofrware was used to reconsrtuct the haplotype for GSTP1, and the haplotype(CACA) showed an increased risk of lung cancer among the smokers(adjust OR=1.53,95% CI 1.04～2.25,p=0.033). Furthermore, the diplotype analyses also demonstrated the significant association beween the risk haplotype and lung cancer. The risk haplotypes cosegregate with one or more biologically functional polymorphisms and it corresponds to a recessive inheritance mode. The results of Meta analysis indicate that the polymorphisms of MPO G-463A and GSTP1 rs1695 were not significantly associated with lung cancer risk. We cloned and sequenced the sequence of CYP2A13 gene, and the outcome indicate that the homologous sequences may interfere with the SNP study and some published SNPs in dbSNPs may not exist.In conclusion, our study firstly investigated the association between the common polymorhpisms of the 3 candidated genes and lung cancer risk according to the SNPs information published. We found a risk SNPs of CYP2A13 and a risk haplotype of GSTP1 ssociated with lung caner, and these polymorphisms may be candidated SNP markers for lung cancer susceptibility in Chinese, which can be defined lung cancer risk SNPs更多还原