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前致癌物生物活化和失活。 因此，可以设想，个体对致癌作用的易感性部分是由于个体的特定前致癌物代谢模式。我们的一般假设是，高阶段I和低阶段II的表达不平衡的致癌物质代谢酶水平，确定个别吸烟者特别高的肺癌风险。在这个建议中，我们假设，遗传个体间差异的启动子区域SNP单倍型和表观遗传CpG甲基化功能赋予个体功能上重要的差异，基因表达表型。本实验室和其他实验室的先前研究已经确定了人肺中I期和II期致癌物代谢基因表达水平的个体间差异。我们最近已经产生了初步证据，表明在个体人类受试者中，观察到的人肺中致癌物代谢酶表达的表达表型的变化可能是由以下原因引起的：1）共同的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。在此R21提交文件中，我们建议探索这两种类型的调节因子如何在mRNA水平上影响致癌物代谢途径中选定的指标基因CYP 1B 1和GSTP 1的定量基因表达。这些调控区功能的后果将在体外验证，并从原位衍生的人肺上皮细胞。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，以及最近开发的标签适应亚硫酸氢盐基因组测序，生成详细的甲基化谱，而无需克隆，每个应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，该提议旨在验证显著的启动子区域特征，这些特征既在机理上影响致癌物代谢表型，又适合于基于基因组DNA的肺癌易感性的高通量筛选策略。

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.