Functional evaluation of a new GWAS locus that links visceral adiposity and type 2 diabetes

关联内脏肥胖和 2 型糖尿病的新 GWAS 位点的功能评估

• 批准号: 10338084
• 负责人: Matthew Steinhauser
• 金额: $ 39.13万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-26 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10338084
• 关键词: Adipose tissue; Affect; Agonist; Attention; Attenuated; Autopsy; Biochemical; Biological Assay; Body fat; CRISPR screen; Candidate Disease Gene; Cardiometabolic Disease; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data; Defect; Deposition; Development; Diabetes Mellitus; Disease; Disease susceptibility; Dissection; Epidemic; Etiology; Evaluation; Excision; Fatty acid glycerol esters; Future; Genes; Genetic; Genetic Predisposition to Disease; Genetic Transcription; Genetic Variation; Genomics; Glucose; Goals; High Fat Diet; Histologic; Human; Human Genetics; Impairment; Insulin; Insulin Resistance; Laboratories; Lead; Ligase; Link; Liver; Measurement; Measures; Meta-Analysis; Metabolic; Metabolic Diseases; Methods; Modeling; Molecular; Monoubiquitination; Mus; Mutate; Nature; Non-Insulin-Dependent Diabetes Mellitus; Nucleic Acid Regulatory Sequences; Obesity; PPARG gene; Pathologic; Pathway interactions; Pattern; Pharmaceutical Preparations; Phenotype; Polyubiquitination; Population; Predisposition; Publishing; Regulation; Reporter; Risk; Role; Scanning; Signal Transduction; Structure; System; Testing; Thiazolidinediones; Tracer; Untranslated RNA; Validation; Variant; Visceral; Visceral fat; Wild Type Mouse; Work; adipocyte differentiation; base; blood glucose regulation; causal variant; developmental disease; diabetes risk; epigenomics; experience; genetic architecture; genome editing; genome wide association study; genome-wide; improved; in vivo; in vivo imaging; insulin sensitivity; lead candidate; lipid biosynthesis; loss of function; mass spectrometric imaging; mouse model; novel; novel therapeutics; programs; protein function; stable isotope; stem cells; subcutaneous; therapeutic target; trait; ubiquitin ligase

Project Summary: The intertwined obesity and type 2 diabetes mellitus (T2D) epidemics have focused attention on pathological changes in adipose tissue. However, obesity represents just one of several adiposity- related phenotypes linked to T2D. Visceral fat storage independent of total fat mass is a predictor of T2D. Moreover, there is an overlap in the underlying genetic architecture of T2D and adiposity phenotypes, suggesting shared developmental pathways. In a recent GWAS meta-analysis, we discovered several novel candidate genes linked to deleterious ectopic fat deposition, including the E2 ubiquitin ligase UBE2E2, a lead candidate identified by its genomic proximity to non-coding SNPs associated with visceral fat. UBE2E2 has also been identified by GWAS of T2D. This combined association with a metabolically deleterious body fat distribution and T2D provided rationale to prioritize UBE2E2 for additional functional studies. We have now demonstrated that excision of 100bp regions of non-coding DNA, inclusive of lead UBE2E2-associated SNPs, attenuates expression of UBE2E2, that UBE2E2 loss-of-function in ex vivo adipogenesis assays dramatically inhibits adipocyte differentiation, and that impaired glucose homeostasis arises in a mouse model of UBE2E2 loss-of-function. These preliminary data inform our central hypothesis: that genetic variation in non-coding regions in the UBE2E2 locus results in partial UBE2E2 loss-of-function, impaired adipocyte development as manifested by an obligate shift in favor of ectopic fat deposition, and predisposition to T2DM. We propose to dissect the molecular and biochemical mechanisms underlying the GWAS signals with two interrelated specific aims. In Aim 1, we will interrogate the UBE2E2 locus with Clustered regularly interspaced short palindromic repeats (Crispr) genome editing. We will edit lead SNPs into human adipose derived stem cells and quantify UBE2E2 expression and adipocyte differentiation. With a complementary approach, we will scan the UBE2E2 locus with a Crispr screen to identify putative regulatory regions and their proximity to disease-related SNPs. We will also perform structure-function studies, mutating critical UBE2E2 functional domains, to characterize the biochemical mechanism by which UBE2E2 regulates adipocyte development. Then in Aim 2, we will utilize a murine model of UBE2E2 loss of function to test whether the human traits – visceral adiposity and T2D – are recapitulated and moreover whether these phenotypes are attributable to a defect in adipocyte development. Our laboratory has developed methods of precisely quantifying adipogenesis, in vivo, utilizing stable isotope tracers and state-of-the-art mass spectrometric imaging, which we will utilize to quantify adipogenesis. We will couple our quantification of adipogenesis with rigorous characterization of depot specific adiposity and systemic metabolic profiling. Now that GWAS have identified large numbers of candidate genes, their functional characterization is a major bottleneck. This project will establish a pipeline that can be broadly applied to functional validation of variants related to fat phenotypes and cardiometabolic disease susceptibility.

项目概述：肥胖症和2型糖尿病(T2D)疫情相互交织 关注脂肪组织的病理变化。然而，肥胖只是几种肥胖中的一种-- 与T2D连锁的相关表型。内脏脂肪储存独立于总脂肪量是T2D的预测因子。 此外，T2D和肥胖症表型的潜在遗传结构存在重叠， 暗示着共同的发展道路。在最近的一次GWAS荟萃分析中，我们发现了几种新的 与有害的异位脂肪沉积有关的候选基因，包括E2泛素连接酶UBE2E2，一种铅 通过其基因组与与内脏脂肪相关的非编码SNPs的相似性来确定候选基因。UBE2E2具有 也被T2D的GWAS鉴定。这与新陈代谢有害的身体脂肪相结合 分布和T2D为UBE2E2优先进行额外的功能研究提供了理论基础。我们现在有了 证实了100bp的非编码DNA区域的切除，包括与UBE2E2相关的先导SNPs， 抑制UBE2E2的表达，UBE2E2在体外成脂试验中功能丧失显著 抑制脂肪细胞分化，在UBE2E2小鼠模型中出现葡萄糖稳态受损 功能丧失。这些初步数据提供了我们的中心假设：非编码的遗传变异 UBE2E2基因座上的区域导致部分UBE2E2功能丧失，脂肪细胞发育受损，如 表现为倾向于异位脂肪沉积，并易患T2 DM。我们建议 用两个相互关联的特异体剖析了Gwas信号背后的分子和生化机制 目标。在目标1中，我们将用成簇的规则间隔短回文来询问UBE2E2基因座 重复(Crispr)基因组编辑。我们将把铅SNPs编辑成人类脂肪来源的干细胞，并量化 UBE2E2表达与脂肪细胞分化通过一种补充方法，我们将扫描UBE2E2 带有Crispr屏幕的基因座，用于识别可能的调控区域及其与疾病相关的SNPs的接近程度。 我们还将进行结构-功能研究，突变关键的UBE2E2功能结构域，以表征 UBE2E2调控脂肪细胞发育的生化机制。然后在目标2中，我们将利用 一种UBE2E2功能丧失的小鼠模型，以测试人类特征-内脏肥胖和T2D-是否 简而言之，这些表型是否可归因于脂肪细胞发育缺陷。 我们的实验室已经开发出利用稳定的同位素在体内精确定量脂肪生成的方法。 示踪剂和最先进的质谱学成像，我们将利用它们来量化脂肪生成。我们会 将我们对脂肪生成的量化与仓库特异性肥胖症和 全身性代谢特征。现在Gwas已经确定了大量的候选基因，他们的 功能表征是一个主要的瓶颈。该项目将建立一条可以广泛应用的管道 应用于与脂肪表型和心脏代谢性疾病易感性相关的变异的功能验证。