Sequencing-based innovative approaches to study FSGS

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Steroid resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS) are a leading cause of end-stage renal failure in children, adolescents and adults. Sequencing technologies have allowed for the identification of several SRNS genes in children but the genetic basis of SRNS/FSGS in adolescents and adults is less clear. Up to now, the predominant application of short-read based sequencing techniques results in preferential detection of point mutations and small sized deletions or insertions while larger structural aberrations, gene rearrangements, and mutations in genomic repeats frequently escape detection. To overcome these limitations in genetic diagnostics, we will combine short-read based high-capacity next generation sequencing (WES/WGS) with new computational and advanced technologies for structural variation analyses. Modern sequencing technologies can impact the management of kidney disease far beyond the level of genomic variant detection and genetic counseling. Cell free DNA (cfDNA), released by injured tissues, can be detected and sequenced with high sensitivity, and, more importantly, the epigenetic signature of cfDNA can be used to identify the tissue or cell type of origin in a noninvasive way. In glomerular cells, single-cell RNAseq uncovered significant heterogeneity within the podocytes as well as the endothelial cells. Recently, spatial reconstruction of tissue architecture and cellular neighborhood was made possible by computational technologies. The overarching aim of this proposal is to use innovative approaches based on modern genomic and transcriptomic technologies to gain deeper insights into the pathomechanisms underlying FSGS and recurrent disease (FSGS-R). Specifically, (1) we will perform comprehensive genomic analyses using biosamples from the FOrMe registry as well as from a cohort of pediatric and adult FSGS patients, that we continuously collect from our joint diagnostic and research activities, and integrate this with already existing and (deep) clinical and generated experimental data, (2) we aim to establish cfDNA from blood as a biomarker to monitor podocyte injury in MCD/FSGS/FSGS-R patients in real time using tissue- and cell-specific methylation marks, and (3) we will develop innovative spatial transcriptomic approaches based on available single-cell RNA datasets and integrate those with RNAscope data of cell marker transcripts. In summary, we aim to significantly improve the detection of genetic causes or predispositions for FSGS on multiple levels and to enhance our understanding of the pathomechanisms leading to disease development and progression up to the level of single cells. This will ultimately contribute to discoveries able to drive relevant changes in clinical practice by precision diagnostics and open the way to establishment of individualized therapies.