Discovery and functional characterization of full-penetrance single-gene causes of steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis

类固醇抵抗性肾病综合征和局灶节段性肾小球硬化症的全外显率单基因病因的发现和功能表征

• 批准号: 404527522
• 负责人: Dr. Florian Buerger
• 金额: --
• 依托单位: Klinik für Kinder- und Jugendmedizin
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/404527522?language=en
• 关键词: Discovery; functional; characterization; full; penetrance

Chronic kidney disease is a major contributor to the global burden of disease (approx. 10%), generates high amounts of healthcare costs and is associated with increased cardiovascular mortality. Although a rare disease, steroid-resistant nephrotic syndrome (SRNS) constitutes the second most frequent cause of end-stage kidney disease (ESKD) in children and young adults. To date, no curative treatment is available. The most frequent histologic representation of SRNS is focal segmental glomerulosclerosis (FSGS), which carries a risk of 33% for reoccurrence in renal transplants, thereby leading again to ESKD. The etiology and pathomechanism of SRNS have been elusive for decades. However, the identification of single-gene causes of nephrotic syndrome has fundamentally changed the concept of SRNS and has so far revealed 55 disease-causing genes. Nephrotic syndrome represents a dysfunction of renal podocyte cells and causes significant proteinuria resulting in hypoalbuminemia and edema. To date, the identified SRNS genes encode proteins that participate in protein clusters that correspondingly converge on defined structural components or signaling pathways of podocyte function, thus elucidating the podocytic concept of SRNS pathogenesis. A very recent study has also implicated an unexpected class of proteins - nucleoporins - as SRNS causing proteins. Nucleoporins are highly conserved eukaryotic proteins involved in formation of the nuclear pore complex and nuclear transport of proteins and other molecules, but scarcely implicated in any other pathogenesis. This finding offers a novel and promising approach to further unravel the conundrum of SRNS and raises questions on why podocytes appear to be especially dependent on a proper functioning nuclear pore complex.To identify new SRNS causing genes the proposed study therefore follows an unbiased as well as a biased approach. Novel genes will be identified using state of the art genetic techniques, such as homozygosity mapping and whole exome sequencing (WES) including trio sequencing in an unparalleled cohort of ~5000 SRNS patients (~1000 families). Also, existing WES data will be specifically filtered to identify other SRNS genes that participate in the nuclear pore complex.Upon identification of new SRNS genes functional studies will be applied to characterize their gene products for subcellular protein localization, protein-protein interaction and genotype-phenotype correlations by testing for deleteriousness of the specific mutations in an established podocyte assay. Also, cell-based as well as zebrafish and mouse disease models using RNA transfection, CRISPR/Cas9 technology and protein expression will therefore be applied.The expected results of the proposed project will give rise to a better understanding of causes and pathogenesis of steroid-resistant nephrotic syndrome and may open new inroads into development of (individual) treatment.

慢性肾脏疾病是全球疾病负担的主要贡献者（约100%）。10%），产生大量的医疗保健费用，并与心血管死亡率增加有关。虽然是一种罕见的疾病，但类固醇耐药肾病综合征（SRNS）是儿童和年轻人终末期肾病（ESKD）的第二大常见原因。到目前为止，还没有治愈性的治疗方法。SRNS最常见的组织学表现是局灶节段性肾小球硬化（FSGS），其在肾移植中复发的风险为33%，从而再次导致ESKD。SRNS的病因和病理机制几十年来一直难以捉摸。然而，对肾病综合征单基因病因的鉴定从根本上改变了SRNS的概念，迄今已揭示了55个致病基因。肾病综合征代表肾足细胞功能障碍，并引起显著的蛋白尿，导致低白蛋白血症和水肿。到目前为止，所确定的SRNS基因编码的蛋白质参与蛋白质簇，相应地收敛于定义的结构组件或信号通路的足细胞功能，从而阐明了足细胞的概念SRNS发病机制。最近的一项研究也暗示了一类意想不到的蛋白质-核孔蛋白-作为SRNS引起的蛋白质。核孔蛋白是一种高度保守的真核生物蛋白质，参与核孔复合物的形成和蛋白质及其他分子的核转运，但几乎不参与任何其他发病机制。这一发现提供了一种新的和有前途的方法，以进一步解开SRNS的难题，并提出了问题，为什么足细胞似乎是特别依赖于一个正常运作的核孔complex.To确定新的SRNS引起的基因，因此，拟议的研究遵循一个公正的，以及有偏见的方法。将使用最先进的遗传技术来识别新基因，例如纯合性图谱和全外显子组测序（WES），包括在约5000名SRNS患者（约1000个家庭）的无与伦比的队列中进行三重测序。此外，现有的WES数据将被专门筛选，以确定其他SRNS基因参与核孔complex.Upon鉴定新的SRNS基因的功能研究将被应用到表征其基因产物的亚细胞蛋白定位，蛋白质-蛋白质相互作用和基因型-表型相关性，通过测试在一个既定的足细胞测定特定突变的危险性。此外，基于RNA转染、CRISPR/Cas9技术和蛋白表达的细胞模型、斑马鱼和小鼠疾病模型也将因此得到应用。拟议项目的预期结果将有助于更好地了解类固醇耐药肾病综合征的原因和发病机制，并可能为（个体）治疗的发展开辟新的进展。