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

慢性肾脏疾病是全球疾病负担的主要贡献者(约10%)，产生高额的医疗费用，并与心血管死亡率增加有关。尽管激素抵抗型肾病综合征(SRNS)是一种罕见的疾病，但它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，还没有治愈的方法。SRNS最常见的组织学表现是局灶节段性肾小球硬化(FSGS)，在肾移植中复发的风险为33%，从而再次导致ESKD。SRNS的病因和发病机制几十年来一直扑朔迷离。然而，对肾病综合征单基因病因的识别从根本上改变了SRNS的概念，到目前为止已经发现了55个致病基因。肾病综合征表现为肾足细胞功能障碍，并导致大量蛋白尿，导致低蛋白血症和水肿。到目前为止，已鉴定的SRNS基因编码的蛋白质参与了相应的蛋白质簇，这些蛋白质簇相应地聚集在确定的足细胞功能的结构成分或信号通路上，从而阐明了SRNS发病的足细胞概念。最近的一项研究还发现，一类意想不到的蛋白质--核孔素--是引起SRNS的蛋白质。核孔蛋白是一种高度保守的真核蛋白质，参与核孔复合体的形成以及蛋白质和其他分子的核运输，但几乎不参与任何其他发病机制。这一发现为进一步解开SRN的谜团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体的问题。因此，为了识别导致新的SRN的基因，拟议的研究遵循了一种公正和有偏见的方法。新的基因将使用最先进的遗传技术，如纯合性作图和全外显子测序(WES)，包括在一个无与伦比的约5000名SRNS患者(约1000个家庭)的队列中进行三重测序。此外，现有的WES数据将被专门过滤，以识别参与核孔复合体的其他SRNS基因。一旦识别出新的SRNS基因，将通过在已建立的足细胞检测中测试特定突变的危害性，应用功能研究来表征其基因产物的亚细胞蛋白质定位、蛋白质-蛋白质相互作用和基因-表型相关性。此外，还将应用基于细胞的斑马鱼和小鼠疾病模型，使用RNA转基因、CRISPR/Cas9技术和蛋白质表达。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病因和发病机制，并可能为(个体化)治疗的发展开辟新的道路。

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.