Sequencing-based innovative approaches to study FSGS

激素抵抗型肾病综合征(SRNS)和局灶节段性肾小球硬化(FSGS)是儿童、青少年和成人终末期肾功能衰竭的主要原因。测序技术已经能够识别儿童中的几个SRNS基因，但青少年和成年人中SRNS/FSGS的遗传基础不太清楚。到目前为止，短读测序技术的主要应用是优先检测点突变和小尺寸的缺失或插入，而较大的结构异常、基因重排和基因组重复序列的突变往往无法检测到。为了克服基因诊断中的这些限制，我们将把基于短读的大容量下一代测序(WES/WGS)与用于结构变异分析的新的计算和先进技术相结合。现代测序技术可以影响肾脏疾病的管理，远远超出基因组变异检测和遗传咨询的水平。损伤组织释放的细胞游离DNA(CfDNA)可以高灵敏度地进行检测和测序，更重要的是，cfDNA的表观遗传学特征可以用来非侵入性地识别起源的组织或细胞类型。在肾小球细胞中，单细胞RNAseq发现足细胞和内皮细胞内存在显著的异质性。最近，计算技术使组织结构和细胞邻域的空间重建成为可能。这项建议的总体目标是使用基于现代基因组和转录组技术的创新方法，以更深入地了解FSGS和复发性疾病(FSGS-R)的发病机制。具体地说，(1)我们将利用Forme登记以及一组儿科和成人FSGS患者的生物样本进行全面的基因组分析，我们将不断从我们的联合诊断和研究活动中收集这些样本，并将其与现有的和(深度)临床和生成的实验数据相结合；(2)我们的目标是从血液中建立cfDNA作为生物标记物，使用组织和细胞特有的甲基化标记实时监测MCD/FSGS/FSGS-R患者的足细胞损伤；(3)我们将基于现有的单细胞RNA数据集开发创新的空间转录分割方法，并将那些方法与细胞标记物的RNAScope数据整合。总之，我们的目标是在多个水平上显著提高对FSGS遗传原因或易感性的检测，并加强我们对导致疾病发展和进展到单细胞水平的病理机制的理解。这最终将有助于发现能够通过精确诊断推动临床实践中的相关变化，并为建立个性化治疗开辟道路。

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.