Molecular Mechanisms of Insulin Resistance Associated Loci

 描述（由申请人提供） 2型糖尿病（T2 D）是由胰岛素敏感性受损（胰岛素抵抗，IR）和胰岛素分泌不足的不利交叉引起的。虽然在鉴定T2 D相关遗传变异方面取得了显著进展，但在定义特定的因果变异和破译因果变异与生物学之间的分子机制方面进展有限。大多数T2 D变体似乎通过对胰腺β细胞发育或胰岛素产生、加工或分泌的影响起作用。然而，越来越多的这些变体似乎与IR相关。这些IR相关变体特别重要，因为IR是T2 D和心血管疾病的强大风险因素，治疗选择非常有限。了解这些IR相关协会的机制现在是该领域取得进展的关键。我们组建了一个独特的研究小组，他们具有互补的专业知识，既可以加速发现影响IR风险的致病基因和变异，又可以进行功能研究，以获得对这些致病基因和变异导致疾病的机制的新见解。我们的初步研究基于正交数据优先考虑IR相关变体/基因座，包括来自我们独特的GWAS的结果，该GWAS具有基于“钳夹”的胰岛素敏感性测量以及IR变体的详细生理表征。在这个提议中，我们将采用方法学来鉴定因果变异，并全面研究这些变异调节疾病相关细胞类型（即脂肪和骨骼肌组织）中因果基因表达的机制。目标1中的研究将使用组蛋白修饰ChIP-Seq和ATAC-Seq方法确定胰岛素敏感组织中活性染色质的区域，研究致病变体和致病基因与等位基因特异性表达之间的局部相互作用，并在全基因组结构的背景下使用ChIA-PET鉴定致病的长程相互作用。在目标2中，体外报告基因研究将研究通过等位基因特异性转染报告基因研究和染色质免疫沉淀转录因子结合试验测定的致病变体和基因相互作用的具体机制。在目的3中，通过这些研究鉴定的转录元件的增强子活性，以及该增强子活性的因果变体修饰的相关性，将通过在相关细胞系中的基因组编辑，并在体内利用具有以下特征的转基因小鼠进行评估： 报告构建体特异性地对接到小鼠基因组中。总之，提出的研究将建立在GWAS发现的基础上，以鉴定与胰岛素抵抗基本相关的IR相关基因座中的因果变异和基因，这将促进开发用于治疗胰岛素抵抗的工具。 风险预测和疾病的早期治疗，并可能确定新的治疗靶点。

 DESCRIPTION (provided by applicant) Type 2 diabetes (T2D) results from the unfavorable intersection of impaired insulin sensitivity (insulin resistance, IR) and inadequate insulin secretion. While there has been phenomenal progress in identifying T2D associated genetic variants, there has been limited progress towards defining the specific causal variants and deciphering the molecular mechanisms between causal variation and biology. Most T2D variants appear to be acting through effects on pancreatic beta cell development or insulin production, processing or secretion. However, a growing number of these variants appear to relate to IR. These IR-associated variants are particularly important as IR is a powerful risk factor for T2D and cardiovascular disease with very limited therapeutic options. Understanding the mechanisms for these IR-related associations is now critical for progress in the field. We have assembled a unique group of investigators with complementary expertise both to accelerate the discovery of causal genes and variants that influence the risk for IR and to conduct functional studies to gain novel insight into the mechanisms by which these causal genes and variants contribute to disease. Our preliminary studies have prioritized IR related variants/loci based on orthogonal data including results from our unique GWAS of over 2,700 individuals with "clamp" based measures of insulin sensitivity as well as detailed physiologic characterization of IR variants. In this proposal, we wll employ methodology to identify causal variation and comprehensively investigate the mechanisms by which these variants modulate expression of causal genes in disease relevant cell types, i.e. adipose and skeletal muscle tissue. Studies in Aim 1 will determine regions of active chromatin in insulin sensitive tissues with histone modification ChIP-Seq and ATAC-Seq methods, investigate local interactions between the causal variant and causal gene with allele- specific expression, and identify causal long-range interactions in the context of whole genome architecture with ChIA-PET. In Aim 2, in vitro reporter gene studies will investigate the specific mechanisms for the causal variant and gene interactions as assayed by allele-specific transfection reporter gene studies and chromatin immunoprecipitation transcription factor binding assays. In Aim 3, the enhancer activity of transcriptional elements identified through these studies, and the relevance of causal variant modification of this enhancer activity, will be evaluated through genome editing in relevant cell lines, and in vivo utilizing transgenic mice with reporter constructs specifically docked into the mouse genome. Taken together, studies proposed will build on GWAS findings to identify causal variants and genes in IR associated loci that are fundamentally linked to insulin resistance, which will facilitate development of tools for risk prediction and early treatment of disease and potentially identify new therapeutic targets.