Genetically unsolved families with congenital anomalies of the kidney and urinary tract: reanalysis of whole-exome data, genome-wide high-resolution analysis using genome imaging and functional characterization of identified candidate genes

• Applicant: Dr. Helge Martens
• Subject Area: Human Genetics
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 459305231

The term congenital anomalies of the kidney and urinary tract (CAKUT) is used to subsume malformations caused by defects in the morphogenesis of the kidney and the urinary tract. CAKUT are a common cause of end-stage kidney disease in children and adolescents. To date, about 60 genes are known to cause CAKUT in an isolated form or combined with mild extrarenal phenotypes, if mutated. Despite exome-wide sequencing, causative variants in these genes have been reported in less than 15% of CAKUT patients. This low detection rate illustrates the need to identify new CAKUT-associated genes. Furthermore, there is evidence that re-analysis of data from whole-exome sequencing increases the detection rate e.g. because of new clinical, genetic or functional evidence on the pathogenicity of variants, newly identified candidate genes and improved bioinformatic algorithms and tools. Therefore, existing whole-exome data of 100 genetically unsolved CAKUT families will be subjected to profound reanalysis in the proposed project (work package 1). Thereby, we aim to identify new CAKUT-relevant genes and to determine the detection rate after re-analysis of whole-exome data of CAKUT patients (aim 1). Using current methods, 10-15% of CAKUT patients were found to harbor structural variants such as microdeletions or microduplications, which often cannot be reliably detected by whole-exome sequencing. Genome imaging is a new technology that enables optical mapping of DNA and genome-wide detection of structural variants with a resolution of up to 500 base pairs by analyzing extremely long linear DNA molecules. In 35 CAKUT families that could not be genetically solved by reanalysis of whole-exome data, genome imaging will be performed (work package 2). This technique will be used for the first time in a cohort of CAKUT patients to identify structural variants that could not or only insufficiently be detected with NGS methods (aim 2). Finally, CAKUT candidate genes identified in work packages 1 and 2 will be functionally characterized. To this end, the effect of their loss of function on tubulomorphogenesis and cyst formation in a cell model after their knockout using CRISPR-Cas9 genomic engineering and rescue experiments with wild-type or variant expression constructs will be investigated (work package 3). These data will provide evidence on whether these candidate genes play a role in kidney development and whether selected identified variants may be pathogenic (aim 3). Altogether, the proposed project should expand our knowledge about the genetic basis of CAKUT using state-of-the-art methods.

• DFG Programme: Research Grants