In vivo and in vitro rare coding variant analyses to identify modulations of the adipocyte differentiation pathway that affect T2D risk

摘要 2型糖尿病(T2D)是一种高度流行的疾病，需要新的治疗方法。脂肪细胞 分化和脂质储存途径涉及罕见和常见形式的糖尿病，并被作为靶点 通过噻唑烷二酮(TZD)，这是有效的，但会造成不良的并发症。更好的设计 靶向脂肪细胞分化/脂肪储存的治疗因不完全了解哪些基因而受阻 在这些通路中与人类的T2D相关，或者它们可能被调节以实现治疗 功效。与疾病相关的罕见编码变体直接识别与人类疾病相关的基因调节， 我们最近对45,231个外显子的研究表明，这种关联很可能在许多 脂肪细胞分化和脂肪储存途径中的基因。然而，罕见的变异关联 需要大型数据集来检测，而目前缺乏方法来识别观察到的哪些关联 最有可能的是(A)表示与疾病的因果联系，以及(B)通过对感兴趣的途径的影响来发挥作用。这个 拟议的项目将在T2D易感性罕见编码变体的假设下解决这些差距 调节脂肪细胞分化/脂肪储存途径的物质应该会在体外和 在体内易患T2D风险较高、BMI调整后空腹胰岛素较高的“胰岛素抵抗信号” 更高的甘油三酯水平、更低的臀围和更低的高密度脂蛋白水平。 特定目标1假设更大的外显子组数据集将识别新的基因水平的稀有编码变体 T2D关联，且关于基因功能先验知识应该影响每个关联被 因果关系。150K-600K外显体的编码变异将被测试与T2D和胰岛素抵抗的关联， 每个基因的因果关联概率将通过一种新的方法来计算，以解释其 经验性地估计了先前关联的可能性。《特定目的2》假设与 体内的胰岛素抵抗信号应该有更高的可能性损害脂肪细胞的分化/脂质 在体外消融时的存储，以及这些基因中的存储，只有不能补充效应的变体 在体外观察会增加体内T2D的风险。具有基因水平T2D关联的50个基因将被 通过人前脂肪细胞功能丧失实验和遗传互补实验进行筛选 将对5个消融损伤脂肪细胞分化/脂肪的基因中的每一个进行50个变种 储藏室。 意义：T2D相关编码变体在体外对脂肪细胞分化或脂质的影响 储存将意味着分子基因的扰动，以保护或治疗T2D。这些结果和所有其他结果 该项目将通过NIDDK资助的AMP-T2D知识门户网站向公众开放。这个 拟议的方法也适用于其他生物过程和疾病。

Abstract Type 2 diabetes (T2D) is a highly prevalent disease for which new therapies are needed. The adipocyte differentiation and lipid storage pathways are involved in rare and common forms of diabetes and are targeted by thiazolidinediones (TZDs), which are efficacious but cause undesirable complications. Designing better therapies to target adipocyte differentiation/lipid storage is impeded by incomplete knowledge of which genes in these pathways are relevant to T2D in humans, or how they might be modulated to achieve therapeutic efficacy. Disease-associated rare coding variants directly identify human disease-relevant gene modulations, and our recent study of 45,231 exomes suggested that such associations are likely observable within many genes within the adipocyte differentiation and lipid storage pathways. However, rare variant associations require large datasets to detect, and methods are currently lacking to identify which observed associations are most likely to (a) represent causal links to disease and (b) act through effects on a pathway of interest. The proposed project will address these gaps under the hypothesis that T2D-susceptibility rare coding variants that modulate the adipocyte differentiation/lipid storage pathways should impair these processes in vitro and predispose in vivo to an “insulin resistance signature” of higher T2D risk, higher BMI-adjusted fasting insulin levels, higher triglyceride levels, lower hip circumference, and lower HDL levels. Specific aim 1 hypothesizes that larger exome datasets will identify new gene-level rare coding variant T2D associations, and that prior knowledge of gene function should affect the likelihood each association is causal. Coding variants in 150K-600K exomes will be tested for association with T2D and insulin resistance, and each gene's probability of causal association will be calculated by a new method to account for its empirically estimated prior likelihood of association. Specific aim 2 hypothesizes that genes associated with an insulin resistance signature in vivo should have a higher likelihood of impairing adipocyte differentiation/lipid storage when ablated in vitro, and that within these genes, only variants that fail to complement effects observed in vitro should increase T2D risk in vivo. Fifty genes with gene-level T2D associations will be screened via loss-of-function experiments in human pre-adipocytes, and genetic complementation experiments will be conducted for 50 variants in each of 5 genes whose ablation impairs adipocyte differentiation/lipid storage. Significance: T2D-associated coding variants with in vitro effects on adipocyte differentiation or lipid storage would suggest molecular gene perturbations to protect from or treat T2D. These and all other results of the project will be made publicly accessible through the NIDDK-funded AMP-T2D Knowledge Portal. The proposed approaches also apply to other biological processes and diseases.