Monogenic forms of juvenile onset diabetes: towards novel insights in β-cell development, function and survival

与普通糖尿病的多因子遗传相比，一小部分青少年发病胰岛素治疗糖尿病（JOD）是由单基因突变引起的。到目前为止，已经确定了几个与单基因糖尿病有关的基因，这些基因影响β细胞的发育、功能和存活。该项目的总体目标是鉴定JOD的新单基因实体并对其进行深入的功能研究。这项联合提案的独特方法是由在人类遗传学和功能生物学方面具有互补记录的两个合作伙伴促成的。在第一阶段，我们将扩展正在进行的基因研究，对高度选定的单基因糖尿病患者和家庭进行研究，以确定致病基因。我们将对我们的外显子组测序数据中出现的选择基因进行扩展的大规模重测序，以遗传学上验证它们，以及已知的单基因糖尿病基因，在1000名JOD患者和对照组中进行大量收集。因此，我们将根据人群、家族结构和患者的临床特征确定这些基因的单基因致病突变。这将首次为各种环境下单基因对JOD的贡献提供可靠的估计。在初步研究中，我们已经确定了几个具有单基因贡献的基因，以及先前报道的基因中的新突变，验证了我们的遗传策略的力量。在第二阶段，同时开始，我们将对这些基因的选择组进行深入的功能研究，从两个基因开始：一个基因(X)，我们之前已经确定为一种新的单基因糖尿病（纯合突变状态：新生儿糖尿病，杂合状态：早发型2型糖尿病），以及一个基因(Y)，我们最近确认为一种单基因糖尿病基因，在8名不相关的JOD患者中发现了7个新的突变。首先，我们将使用人类胚胎干细胞衍生的be-cell平台扩展表征基因X的功能数据。其次，我们将使用最先进的表型模型，患者特异性诱导多能干细胞（hipsc）和基因组编辑的人类胚胎干细胞（hESCs）来研究基因Y的特定功能。后者将分化为胰腺β细胞进行详细的功能分析。第三，我们将利用iCRISPR平台在hESCs中进行基因组编辑，在这些基因中创建新发现的突变。因此，我们将开发一个完整的患者特异性细胞库，用于详细的细胞研究，旨在i)在培养皿中模拟糖尿病以确定疾病的分子基础，ii)寻找新的治疗可能性，以及iii)定义内分泌胰腺发育以及β细胞功能和维持所必需的完整分子网络。本研究将为糖尿病治疗中真正的“个体化医疗”奠定基础。

A small subset of juvenile onset insulin-treated diabetes (JOD) is caused by single gene mutations, contrasting with the general multifactorial inheritance of common diabetes. Several genes responsible for monogenic diabetes have been identified to date, and these affect β-cell development, function and survival. The overall aim of this project is to identify and genetically characterize new monogenic entities of JOD and perform in-depth functional studies of these genes. The unique approach of this joint proposal is fostered by two partners having complementary track records in human genetics and functional biology. In the first stage, we will extend ongoing genetic studies of highly selected patients and families enriched in monogenic forms of diabetes to identify causative genes. We will perform extended large-scale re-sequencing of selected genes emerging from our exome sequencing data to genetically validate them, as well as of already known monogenic diabetes genes, in a large collection of 1000 JOD patients and controls. Thereby, we will identify monogenic causative mutations in these genes depending on the population, familial structure and clinical characteristics of patients. This will provide for the first time a reliable estimation of monogenic contributions to JOD in various settings. In preliminary studies, we have already identified several genes with monogenic contributions, as well as new mutations in previously reported genes, validating the power of our genetic strategy. In the second stage, starting simultaneously, we will perform in-depth functional studies of selected groups of these genes, starting with two genes: one gene (X) that we have previously identified as mutated in a new form of monogenic diabetes (homozygous mutated status: neonatal diabetes, heterozygous status: early-onset type 2 diabetes), and one gene (Y) that we recently validated as a monogenic diabetes gene with the identification of 7 new mutations in 8 unrelated JOD patients. First, we will extend functional data that characterize gene X using a human embryonic stem cell-derived be-cell platform. Second, we will study the specific functions of gene Y using a state-of-the-art phenotyping model, patient-specific induced pluripotent stem cells (hiPSCs) and genome-edited human embryonic stem cells (hESCs). The latter will be differentiated into pancreatic β-cells for detailed functional analyses. Third, we will create the newly identified mutations in these genes by genomic editing in hESCs using an iCRISPR platform. Thus, we will develop a whole patient-specific cell-bank for detailed cellular investigations aiming to i) model diabetes in a dish to identify the molecular basis of the disease, ii) address novel therapeutic possibilities and iii) define a complete molecular network essential for endocrine pancreatic development as well as β-cell function and maintenance. This study will establish the basis for true “personalized medicine” in diabetes therapy.