Identifying disease-related functional regulatory variants in open chromatin

描述（由申请人提供）：在过去的两年中，疾病相关代谢和心血管性状的全基因组关联（GWA）研究已经成功鉴定了70多个位点，其中包含数十个DNA变异，这些变异被强烈证实与一个性状相关，尽管可能的功能变异涉及极少数位点。成功识别这些潜在的变异将有助于理解基因调控的生物学途径和疾病相关机制。了解增加2型糖尿病、肥胖、不健康胆固醇水平和相关特征易感性的变异，将提供有关心血管疾病发展和治疗的生物学和临床数据，并为高危人群提供具体的生活方式改变建议，从而对公众健康产生重大影响。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中，如启动子、增强子、抑制子和绝缘子。大量证据表明，许多调控元件位于开放染色质区域，在活性状态下不与核小体结合。成功的全基因组鉴定开放染色质的实验方法包括dna酶超敏（DHS），甲醛辅助鉴定调节元件（FAIRE），组蛋白修饰标记和染色质免疫沉淀（ChIP）与调节蛋白。重要的是，这些数据现在可用于几种医学上相关的细胞类型。在本应用中，我们首先关注胰岛和肝细胞最有可能涉及的基因座，对于这些基因座，我们建议分析现有的重测序数据，以开发潜在功能变异的综合列表，根据序列注释和相关组织开放染色质的全基因组图谱对变异进行优先排序，并测试高优先级的预测调节变异对启动子、增强子、抑制子、或者绝缘体活度。等位基因特异性结合转录因子和其他调节蛋白将使用电泳迁移位移（EMSA）和超位移测定和等位基因特异性ChIP进行验证。在转录上表现出等位基因特异性作用的变体将在开放染色质区域中进行差异表征测试。通过这项工作，我们希望确定与代谢和心血管特征相关的可能的功能调节变异，并为评估代谢和心血管特征的未来动物模型和人类生理学研究中的生物学影响提出可测试的假设。

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.