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

项目摘要：肥胖和2型糖尿病（T2 D）流行病交织在一起， 注意脂肪组织的病理变化。然而，肥胖只是几种肥胖中的一种- 与T2 D相关的表型。与总脂肪量无关的内脏脂肪储存是T2 D的预测因子。 此外，T2 D和肥胖表型的潜在遗传结构存在重叠， 表明有共同的发育途径在最近的GWAS荟萃分析中，我们发现了几个新的 与有害的异位脂肪沉积相关的候选基因，包括E2泛素连接酶UBE 2 E2， 通过其与内脏脂肪相关的非编码SNP的基因组接近性来确定候选者。UBE 2 E2具有 也被T2 D的GWAS识别。这与代谢有害的身体脂肪相结合 分布和T2 D提供了优先考虑UBE 2 E2进行其他功能研究的理由。我们现在已经 证明切除非编码DNA的100 bp区域，包括前导UBE 2 E2相关SNP， 减弱UBE 2 E2的表达，在离体脂肪形成测定中UBE 2 E2功能丧失显著 抑制脂肪细胞分化，并且在UBE 2 E2小鼠模型中出现葡萄糖稳态受损 功能丧失这些初步数据告诉我们的中心假设：非编码基因的遗传变异 UBE 2 E2基因座中的区域导致部分UBE 2 E2功能丧失， 表现为有利于异位脂肪沉积的专性转移和T2 DM易感性。我们建议 用两个相互关联的特异性信号分析了GWAS信号的分子和生化机制， 目标。在目标1中，我们将用规则间隔的短回文序列询问UBE 2 E2基因座， 重复序列（Crispr）基因组编辑。我们将编辑前导SNP进入人类脂肪来源的干细胞， UBE 2 E2表达和脂肪细胞分化。通过补充方法，我们将扫描UBE 2 E2 基因座与Crispr筛选，以确定推定的调控区域及其接近疾病相关的SNP。 我们还将进行结构-功能研究，突变关键的UBE 2 E2功能结构域，以表征 UBE 2 E2调节脂肪细胞发育的生化机制。在目标2中，我们将利用 UBE 2 E2功能丧失的鼠模型，以测试人类特征-内脏肥胖和T2 D-是否 此外，这些表型是否可归因于脂肪细胞发育的缺陷。 我们的实验室已经开发出利用稳定同位素在体内精确定量脂肪形成的方法 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。我们将 将我们对脂肪生成的定量与对贮库特异性肥胖的严格表征结合起来， 全身代谢分析。既然GWAS已经确定了大量的候选基因， 功能表征是一个主要瓶颈。该项目将建立一个管道， 应用于与脂肪表型和心脏代谢疾病易感性相关的变体的功能验证。

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.