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

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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.