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

描述(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功能衰竭、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风险因素。为了减轻这种疾病的负担，迫切需要更好地了解调控b细胞胰岛素分泌的分子机制和胰岛素在外周组织中的作用，以便开发新的干预措施。我们的策略是通过对青年成熟型糖尿病(MODY)的遗传学研究来获得这一知识--一种常染色体显性形式的早发性糖尿病。在之前的资金支持期间，这种方法导致酪氨酸激酶BLK被确认为一种以前未被认识到的b细胞功能调节器，它作为胰岛素合成和分泌的刺激器对葡萄糖做出反应。在这次拨款续期申请中，我们建议通过寻求对BLK调节b细胞功能的分子机制的进一步深入了解来继续这项研究，并通过定位克隆位于染色体4q32上的新MODY基因，我们在该染色体上观察到MODY家族的Joslin集合中存在显著的连锁。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，系统地检测BLK基因对b细胞功能、增殖和存活的影响。我们将首先研究在MIN6b细胞中BLK功能的获得或丧失的影响。然后，我们将研究现有的全球BLK KO小鼠以及我们将在此次赠款期间创建的可诱导的b细胞特异性BLK KO小鼠。将通过详细的生理研究对小鼠进行表型鉴定，以确定它们的葡萄糖稳态和b细胞功能。2.鉴定染色体4q32上新的MODY基因座的序列变异并对其进行功能鉴定。我们将使用下一代测序技术，在4q32汇编糖尿病相关单倍型携带的罕见变异的全面目录。从中，我们将选择一组候选变异，这些变异在这些家庭中参与MODY的病因学的可能性很高。然后，我们将通过功能研究来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的特点。首先，它基于大量关于BLK在b细胞生理学中的作用的初步数据，并证明了使用下一基因测序进行位置克隆的可行性。其次，它利用了已知MODY基因无法解释的最大可用MODY家族集合之一。第三，它建立在遗传流行病学家和具有互补专业知识的b细胞分子生理学家之间持续合作的基础上。最后，也是最重要的是，我们的建议具有特别高的翻译价值，可能导致确定新的药物靶点，对开发新的干预措施以遏制正在进行的糖尿病流行具有关键影响。 与公众健康相关：该项目的目标是确定参与MODY发展的基因，并了解这些基因通过哪些机制影响血糖稳态。新的MODY遗传标记的出现将促进这种形式的糖尿病的诊断，并对治疗优化产生影响(例如，将以前未确诊的MODY从胰岛素治疗转移到口服药物治疗)。也许更重要的是，了解这些基因的功能可能会指向迄今尚未发现的调节胰岛素分泌和作用的分子途径，这反过来可能会为开发预防或治疗常见的多因素2型糖尿病的新药提供新的靶点。

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.