Search for new MODY genes and molecular studies of their function in beta cells

描述（由申请人提供）：尽管在治疗方面取得了许多进展，但糖尿病仍然是慢性肾衰竭、成人失明和截肢的主要原因，也是心脏病、中风和出生缺陷的主要危险因素。为了减少这种疾病的负担，迫切需要更好地了解调节b细胞胰岛素分泌和外周组织胰岛素作用的分子机制，以便开发新的干预措施。我们的策略是通过对年轻人成熟型糖尿病（MODY）的遗传学研究来获得这方面的知识，MODY是早发性糖尿病的一种常染色体显性形式。这种方法导致在之前的资助期间将酪氨酸激酶BLK鉴定为以前未被识别的b细胞功能调节剂，其作为响应于葡萄糖的胰岛素合成和分泌的刺激剂。在这项资助更新申请中，我们建议继续这项研究，寻求进一步了解BLK调节b细胞功能的分子机制，并通过定位克隆位于染色体4q32上的新MODY基因，我们在MODY家族的Joslin集合中观察到显着的连锁。我们的具体目标是：1.研究MODY基因BLK影响b细胞功能的分子机制。我们将进行体外和体内研究，系统地研究BLK基因对b细胞功能，增殖和存活的影响。我们将首先研究MIN6 b细胞中BLK功能获得或丧失的影响。然后，我们将研究现有的全球BLK KO小鼠以及我们将在此期间创建的诱导性b细胞特异性BLK KO小鼠。将通过详细的生理学研究对小鼠进行表型分析，以表征其葡萄糖稳态和b细胞功能。2.鉴定和功能性表征染色体4q32上新MODY基因座的序列变异。我们将使用下一代测序技术来编制一个由糖尿病连锁单倍型在4q32携带的罕见变异的综合目录。从这些，我们将选择一组候选的变异，有一个在硅片上的可能性很高，参与这些家庭的MODY的病因。然后，我们将通过功能研究确定这些变异中的哪些与糖尿病有因果关系。我们的建议有几个重要特点。首先，它是基于广泛的初步数据有关的作用，BLK在b细胞生理学和展示的可行性，使用下一代测序定位克隆。其次，它利用了已知MODY基因未解释的MODY家族的最大可用集合之一。第三，它建立在遗传流行病学家和b细胞分子生理学家之间正在进行的合作基础上，具有互补的专业知识。最后，也是最重要的是，我们的建议具有特别高的转化价值，可能导致新的药物靶点的鉴定，对开发新的干预措施以遏制正在进行的糖尿病流行具有重要意义。 公共卫生相关性：该项目的目标是确定参与MODY发展的基因，并了解这些基因影响葡萄糖稳态的机制。MODY的新遗传标记的可用性将有助于这种形式的糖尿病的诊断，并对治疗优化产生影响（例如，用于将先前未诊断的MODY从胰岛素治疗转移到口服药物）。也许更重要的是，了解基因的功能可能指向尚未发现的调节胰岛素分泌和作用的分子途径，这反过来可能为开发新药以预防或治疗常见的多因素形式的2型糖尿病提供新的靶点。

DESCRIPTION (provided by applicant): Despite many advances in treatment, diabetes continues to be the leading cause of chronic renal failure, adult blindness and limb amputation, and a major risk factor for heart disease, stroke and birth defects. To decrease the burden of this disease, a better understanding of the molecular mechanisms regulating insulin secretion by b-cell and insulin action in peripheral tissues is urgently needed, so that new interventions can be developed. Our strategy is to gain this knowledge through genetic studies of maturity-onset diabetes of the young (MODY) - an autosomal dominant form of early-onset diabetes. This approach has led during the previous funding period to the identification of the tyrosine kinase BLK as a previously unrecognized modulator of b-cell function that acts as a stimulator of insulin synthesis and secretion in response to glucose. In this grant renewal application, we propose to continue this research by seeking further insights onto the molecular mechanisms through which BLK modulates b-cell function and by pursuing the positional cloning of a new MODY gene placed on chromosome 4q32, where we have observed significant linkage in the Joslin collection of MODY families. Our specific aims are: 1. To investigate the molecular mechanisms by which the MODY gene BLK impacts b-cell function. We will perform in vitro and in vivo studies to systematically examine the effects of the BLK gene on b-cell function, proliferation, and survival. We will first investigate the effects of gain or loss of function of BLK in MIN6 b-cells. We will then study an existing global BLK KO mouse as well as an inducible-b-cell-specific BLK KO mouse that we will create during this grant. Mice will be phenotyped through detailed physiological studies to characterize their glucose homeostasis and b-cell function. 2. To identify and functionally characterize the sequence variants underlying the new MODY locus on chromosome 4q32. We will use next-gen sequencing technologies to compile a comprehensive catalog of the rare variants carried by the diabetes-linked haplotypes at 4q32. From these, we will select a set of candidate variants that have a high in silico likelihood of being involved in the etiology of MODY in these families. We will then determine which of these variants are causally linked to diabetes by means of functional studies. Several important features distinguish our proposal. First, it is based on extensive preliminary data concerning the role of BLK in b-cell physiology and demonstrating the feasibility of using next-gen sequencing for positional cloning. Second, it takes advantage of one of the largest available collections of MODY families unaccounted for by known MODY genes. Third, it builds on the ongoing collaboration between a genetic epidemiologist and a b-cell molecular physiologist with complementary expertise. Finally, and most importantly, our proposal has an especially high translational value, potentially leading to the identification of novel drug targets, with critical implications for the development of new interventions to curb the on-going diabetes epidemic. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify genes involved in the development of MODY and understand the mechanisms through which these affect glucose homeostasis. Availability of new genetic markers of MODY would facilitate the diagnosis of this form of diabetes, with implications for treatment optimization (e.g., for transferring previously undiagnosed MODY from insulin therapy to oral medications). Perhaps more importantly, understanding the function of the genes may point to as yet undiscovered molecular pathways regulating insulin secretion and action, which may in turn suggest novel targets for the development of new drugs to prevent or treat common, multifactorial forms of type 2 diabetes.