Individual Promoter SNP and CpG Methylation Signatures

描述（由申请人提供）：肺癌发生的关键早期代谢步骤包括致癌物原生物活化和失活。因此，可以设想个体对致癌的易感性部分是由于个体的前致癌物代谢的特定模式。我们的一般假设是，致癌代谢酶水平的高I期和低II期表达不平衡，确定了肺癌风险特别高的个体吸烟者。在这一提议中，我们假设启动子区域SNP单倍型和表观遗传CpG甲基化特征的遗传个体间差异赋予了基因表达表型个体功能上的重要差异。本实验室和其他实验室先前的研究发现，人类肺部I期和II期致癌物代谢基因表达水平存在显著的个体间差异。我们最近获得的初步证据表明，在个体人类受试者中，观察到的人类肺部致癌物代谢酶表达表型的变化可能是由于：1}共同启动子区域单核苷酸多态性（SNP）单倍型；和/或2)启动子CpG甲基化的变化。在本次R21提交中，我们拟探讨这两种类型的调节因子在mRNA水平上如何影响致癌物质代谢途径中所选择的指标基因CYP1B1和GSTP1的定量基因表达。这些调节区特征的功能后果将在体外和原位来源的人肺上皮中得到验证。采用的新工具将包括实验室的rna特异性适应实时定量RT-PCR，以及最近开发的亚硫酸盐基因组测序标签适应，生成详细的甲基化光谱而无需克隆，每种工具都适用于人类气道生物标本。将与烟草暴露、激素和其他临床变量进行比较。因此，本研究旨在验证显著的启动子区域特征，这些特征既对致癌物代谢表型有机制影响，又适用于基于基因组DNA的肺癌易感性高通量筛查策略。

DESCRIPTION (provided by applicant): Key early metabolic steps in lung carcinogenesis include procarcinogen bioactivation and inactivation. Individual susceptibility to carcinogenesis can therefore be envisioned to be due, in part, to an individual's specific pattern of procarcinogen metabolism. Our general hypothesis is that high phase I and low phase II expression imbalance in carcinogen-metabolizing enzyme levels, identifies individual smokers at particularly high risk for lung cancer. In this proposal, we hypothesize that heritable inter-individual differences in promoter region SNP haplotype and epigenetic CpG methylation features confer upon the individual functionally important differences in gene expression phenotype. Previous studies from this and other laboratories have identified substantial inter-individual variation in phase I and phase II carcinogen metabolism gene expression levels in human lung. We have recently generated preliminary evidence that across individual human subjects, the observed variation of expression phenotype for carcinogen metabolism enzyme expression in human lung may plausibly result from: 1} common promoter region single nucleotide polymorphism (SNP) haplotypes; and/or 2) variation in promoter CpG methylation. In this R21 submission, we propose to explore how each of these two types of regulatory factors impacts on quantitative gene expression at the mRNA level, for the selected index genes CYP1B1 and GSTP1 in the carcinogen metabolism pathway. Functional consequences of these regulatory region features will be verified both in vitro, and from in-situ - derived human lung epithelium. Novel tools employed will include the laboratory's RNA-specific adaptation of real-time quantitative RT-PCR, and the recently developed tag-adaptation of bisulfite genomic sequencing, generating detailed methylation spectra without the need for cloning, each applied to human airway biospecimens. Comparison with tobacco exposure, hormonal and other clinical variables will be made. This proposal is therefore aimed at verifying salient promoter region characteristics that both mechanistically impact on carcinogen metabolism phenotype, and are amenable to genomic DNA based high throughput screening strategies for lung cancer susceptibility.