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

慢性肾脏疾病是造成全球疾病负担的一个主要因素（约占全球疾病负担的一半）。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，但类固醇抵抗性肾病综合征（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，还没有有效的治疗方法。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发现从根本上改变了SRNS的概念，迄今已发现55个致病基因。肾病综合征表现为肾足细胞功能障碍，引起大量蛋白尿，导致低白蛋白血症和水肿。迄今为止，已鉴定的SRNS基因编码的蛋白质参与相应的聚集于足细胞功能的定义结构成分或信号通路的蛋白质簇，从而阐明了SRNS发病机制的足细胞概念。最近的一项研究也暗示了一种意想不到的蛋白质——核孔蛋白——是引起SRNS的蛋白质。核孔蛋白是一种高度保守的真核蛋白，参与核孔复合物的形成和蛋白质和其他分子的核运输，但几乎不涉及任何其他发病机制。这一发现为进一步解开SRNS之谜提供了一种新颖而有希望的方法，并提出了为什么足细胞似乎特别依赖于正常功能的核孔复合物的问题。因此，为了确定新的SRNS引起基因，拟议的研究遵循无偏见和有偏见的方法。新基因的鉴定将使用最先进的遗传技术，如纯合子作图和全外显子组测序（WES），包括对5000名SRNS患者（1000个家庭）进行三重奏测序。此外，现有的WES数据将被专门筛选，以确定参与核孔复合物的其他SRNS基因。在鉴定新的SRNS基因后，功能研究将应用于表征其基因产物的亚细胞蛋白定位，蛋白-蛋白相互作用和基因型-表型相关性，通过在既定的足细胞试验中检测特定突变的有害性。此外，基于细胞的以及使用RNA转染、CRISPR/Cas9技术和蛋白质表达的斑马鱼和小鼠疾病模型也将被应用。拟议项目的预期结果将有助于更好地了解类固醇抵抗性肾病综合征的病因和发病机制，并可能为（个体）治疗的发展开辟新的途径。

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.