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：交织在一起的肥胖和2型糖尿病（T2D）发作已集中注意脂肪组织的病理变化。但是，肥胖仅代表了几种肥胖之一 - 与T2D相关的相关表型。与总脂肪质量无关的内脏脂肪存储是T2D的预测因子。此外，T2D和肥胖表型的基本遗传结构存在重叠，暗示共同的发展途径。在最近的GWAS荟萃分析中，我们发现了几本小说与有害依托脂肪沉积相关的候选基因，包括E2泛素连接酶UBE2E2，铅候选者通过其与内脏脂肪相关的非编码SNP的基因组接近。 ube2e2具有也通过T2D的GWAS确定。这种与代谢删除的身体脂肪的结合结合分布和T2D为其他功能研究提供了优先级ube2e2的基本原理。我们现在有证明了非编码DNA的100bp区域的惊喜，包括铅UBE2E2相关的SNP， ube2e2的表达减弱，ube2e2在体内脂肪生成法中的功能丧失作用急剧抑制脂肪细胞分化，葡萄糖稳态受损，在Ube2e2的小鼠模型中产生功能丧失。这些初步数据为我们的中心假设提供了信息：非编码的遗传变异 UBE2E2基因座中的区域会导致部分UBE2E2功能丧失，脂肪细胞发育受损表现为强制转变，有利于生态脂肪沉积，而对T2DM的倾向也表现出来。我们建议剖析具有两个相互关联的特异性的GWAS信号的分子和生化机制目标。在AIM 1中，我们将使用群集的定期间隔短篇小学来询问UBE2E2基因座重复（CRISPR）基因组编辑。我们将将铅SNP编辑为人脂肪衍生的干细胞并进行量化 UBE2E2表达和脂肪细胞分化。通过完整的方法，我们将扫描UBE2E2 带有CRISPR屏幕的基因座，以识别推定的监管区域及其靠近与疾病相关的SNP。我们还将进行结构功能研究，突变关键的UBE2E2功能域，以表征 UBE2E2调节脂肪细胞发育的生化机制。然后在AIM 2中，我们将使用 UBE2E2失去功能的鼠模型，以测试人类特征（内脏肥胖和T2D）是否是此外，这些表型是否归因于脂肪细胞发育中的缺陷。我们的实验室已经开发了精确量化脂肪形成的方法，它利用稳定的同位素示踪剂和最先进的质谱成像，我们将用于量化脂肪形成。我们将将我们对成脂的定量进行逐渐定量，并严格表征沉积物特异性肥胖和系统性代谢分析。现在，GWAS已经确定了大量候选基因功能表征是主要的瓶颈。该项目将建立一个可以广泛的管道应用于与脂肪表型和心脏代谢疾病易感性有关的变体的功能验证。