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（T2 D）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参与罕见和常见形式的糖尿病， 噻唑烷二酮类药物（TZDs）是有效的，但会引起不希望的并发症。设计更好 靶向脂肪细胞分化/脂质储存的治疗受到以下不完全知识的阻碍： 这些通路与人类的T2 D相关，或者它们如何被调节以实现治疗 功效疾病相关的罕见编码变体直接识别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，这种关联可能在许多基因组中都可以观察到。 脂肪细胞分化和脂质储存途径中的基因。然而，罕见的变异关联 需要大数据集来检测，并且目前缺乏方法来识别哪些观察到的关联是 最有可能（a）代表与疾病的因果联系和（B）通过对感兴趣的途径的影响起作用。的 拟议的项目将解决这些差距的假设下，T2 D易感性罕见的编码变异 调节脂肪细胞分化/脂质储存途径的药物应该在体外损害这些过程， 在体内易发生“胰岛素抵抗特征”，即较高的T2 D风险、较高的BMI调整的空腹胰岛素 高甘油三酯水平、低臀围和低HDL水平。 具体目标1假设较大的外显子组数据集将识别新的基因水平的罕见编码变体 T2 D关联，并且基因功能的先验知识应该影响每个关联的可能性。 因果关系将测试150 K-600 K外显子组中的编码变体与T2 D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， 经验估计的关联的先验可能性。特异性目标2假设与以下相关的基因 体内胰岛素抵抗特征应该具有损害脂肪细胞分化/脂质的更高可能性 在体外消融时的储存，以及在这些基因内，只有未能补充效应的变体 在体外观察到的结果应该会增加体内T2 D风险。50个与基因水平T2 D相关的基因将被 通过人前脂肪细胞功能丧失实验和遗传互补实验筛选 将对5个基因中的每一个的50个变体进行研究，这些基因的消融会损害脂肪细胞分化/脂质 存储. 意义：T2 D相关编码变体对脂肪细胞分化或脂质的体外影响 储存将表明分子基因扰动以防止或治疗T2 D。这些和所有其他结果 该项目将通过NIDDK资助的AMP-T2 D知识门户向公众开放。的 所提出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.