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

基本信息

批准号：
10044898
负责人：
Matthew Steinhauser
金额：
$ 29.56万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/10044898
关键词：
Adipose tissue Affect Agonist Attention Attenuated Autopsy Biochemical Biological Assay Body fat CRISPR screen Candidate Disease Gene 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 Image 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 cardiometabolism 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 metabolic profile mouse model novel novel therapeutics programs protein function stable isotope stem cells subcutaneous therapeutic target trait ubiquitin ligase

项目摘要

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.

项目摘要：肥胖和 2 型糖尿病 (T2D) 流行病相互交织，引起了人们的关注关注脂肪组织的病理变化然而，肥胖只是多种肥胖症之一。与 T2D 相关的相关表型独立于总脂肪量的内脏脂肪储存是 T2D 的预测因子。此外，T2D 和肥胖表型的潜在遗传结构存在重叠，在最近的 GWAS 荟萃分析中，我们发现了几部小说。与有害异位脂肪沉积相关的候选基因，包括 E2 泛素连接酶 UBE2E2，这是一种先导基因通过其与内脏脂肪相关的非编码 SNP 的基因组邻近性鉴定出候选者。 T2D 的 GWAS 也发现了这种与代谢有害的身体脂肪的关联。分布和 T2D 为优先考虑 UBE2E2 进行其他功能研究提供了理由。切除非编码 DNA 的 100bp 区域，包括与 UBE2E2 相关的先导 SNP，减弱 UBE2E2 的表达，在离体脂肪生成测定中 UBE2E2 功能丧失显着抑制脂肪细胞分化，UBE2E2 小鼠模型中葡萄糖稳态受损这些初步数据告诉我们的中心假设：非编码的遗传变异。 UBE2E2 基因座中的区域导致部分 UBE2E2 功能丧失，脂肪细胞发育受损表现为有利于异位脂肪沉积的必然转变以及 T2DM 的易感性。通过两个相互关联的特定信号剖析 GWAS 信号背后的分子和生化机制在目标 1 中，我们将询问具有规则间隔的短回文簇的 UBE2E2 基因座。我们将通过重复 (Crispr) 基因组编辑将先导 SNP 编辑到人类脂肪干细胞中并进行定量。通过补充方法，我们将扫描 UBE2E2 表达和脂肪细胞分化。基因座与 Crispr 屏幕识别假定的监管区域及其与疾病相关的 SNP 的接近程度。我们还将进行结构功能研究，突变关键的 UBE2E2 功能域，以表征然后在目标 2 中，我们将利用 UBE2E2 调节脂肪细胞发育的生化机制。 UBE2E2 功能丧失的小鼠模型，用于测试人类特征（内脏肥胖和 T2D）是否与概括一下，此外，这些表型是否可归因于脂肪细胞发育的缺陷。我们的实验室开发了利用稳定同位素精确量化体内脂肪生成的方法示踪剂和最先进的质谱成像，我们将利用它们来量化脂肪生成。将我们对脂肪生成的量化与对仓库特异性肥胖的严格表征结合起来现在 GWAS 已经鉴定出系统性的大量候选基因，以及它们的代谢谱。功能表征是一个主要瓶颈，该项目将建立一个可以广泛应用的管道。应用于与脂肪表型和心脏代谢疾病易感性相关的变异的功能验证。