Identifying disease-related functional regulatory variants in open chromatin

描述（由申请人提供）：在过去的两年中，针对疾病相关代谢和心血管性状的全基因组关联（GWA）研究已经成功地鉴定了70多个基因座，这些基因座含有数十种DNA变异，这些变异被强烈证实与性状相关，尽管极少数基因座涉及可能的功能变异。这些潜在变异的成功鉴定将有助于理解基因调控的生物学途径和疾病相关机制。了解变异增加对2型糖尿病、肥胖、不健康胆固醇水平和相关性状的易感性，将通过提供关于心血管疾病的发展和治疗的生物学和临床数据，并通过建议高危个体改变特定的生活方式，对公共卫生产生重大影响。我们假设，对于许多基因座，功能变异存在于非编码调控元件，如启动子，增强子，阻遏子和绝缘子。大量的证据表明，许多调控元件位于开放染色质区域，当处于活性状态时不被核小体结合。鉴定开放染色质的成功的全基因组实验方法包括DNA酶超敏反应（DHS）、甲醛辅助的调节元件鉴定（FAIRE）、组蛋白修饰的标记物和具有调节蛋白的染色质免疫沉淀（ChIP）。重要的是，这些数据现在可用于几种医学相关的细胞类型。在本申请中，我们最初关注胰岛和肝细胞最可能涉及的基因座，并且对于这些基因座，我们建议分析现有的重测序数据以开发潜在功能变体的全面列表，基于序列注释和来自相关组织的开放染色质的全基因组图谱对变体进行优先排序，并测试高优先级预测的调节变体对启动子、增强子、阻遏子或绝缘子活性的等位基因特异性作用。将使用电泳迁移率变动（EMSA）和超变动测定以及等位基因特异性ChIP验证等位基因与转录因子和其他调节蛋白的特异性结合。将测试对转录表现出等位基因特异性效应的变体在开放染色质区域中的差异表达。通过这项工作，我们希望确定可能与代谢和心血管性状相关的功能调节变体，并开发可验证的假设，用于评估未来动物模型和代谢和心血管性状的人体生理学研究的生物学影响。 公共卫生相关性：我们建议通过改变基因调控来确定影响心血管和代谢特征个体间变异的DNA变异。

DESCRIPTION (provided by applicant): In the past two years, genome-wide association (GWA) studies for disease-related metabolic and cardiovascular traits have successfully identified more than 70 loci containing dozens of DNA variants strongly confirmed to be associated with a trait, although a likely functional variant has been implicated for very few loci. Successful identification of these underlying variants will facilitate understanding of biological pathways and disease-related mechanisms of gene regulation. Knowledge of variants increasing susceptibility to type 2 diabetes, obesity, unhealthy cholesterol levels and related traits would substantially impact public health by providing biological and clinical data about development and treatment of cardiovascular disease and by advising specific lifestyle changes in at-risk individuals. We hypothesize that for numerous loci, functional variants are present in non-coding regulatory elements such as promoters, enhancers, repressors, and insulators. Substantial evidence indicates that many regulatory elements are located in regions of open chromatin, not bound by nucleosomes when in an active state. Successful genome-wide experimental approaches to identify open chromatin include DNase hypersensitivity (DHS), formaldehyde-assisted identification of regulatory elements (FAIRE), markers of histone modification, and chromatin immunoprecipitation (ChIP) with regulatory proteins. Importantly, these data are now available for several medically relevant cell types. In this application, we focus initially on loci for which pancreatic islets and hepatocytes are most likely to be implicated, and for these loci we propose to analyze existing resequencing data to develop comprehensive lists of potential functional variants, prioritize the variants based on sequence annotation and genome-wide maps of open chromatin from relevant tissues, and test high priority predicted regulatory variants for allele-specific effects on promoter, enhancer, repressor, or insulator activity. Allele- specific binding to transcription factors and other regulatory proteins will be validated using electrophoretic mobility shift (EMSA) and supershift assays and allele-specific ChIP. Variants exhibiting an allele-specific effect on transcription will be tested for differential representation in regions of open chromatin. Through this work we expect to identify likely functional regulatory variants associated with metabolic and cardiovascular traits and develop testable hypotheses for assessing the biological impact in future animal model and human physiology studies of metabolic and cardiovascular traits. PUBLIC HEALTH RELEVANCE: We propose to identify DNA variants that influence inter-individual variation in cardiovascular and metabolic traits by altering gene regulation.