Effect of Rho GTPase-regulating protein DLC1 on podocytes
Human Genetics
Pediatric and Adolescent Medicine
Final Report Abstract
Steroid resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease (CKD) in the first three decades of life. To date, 63 genes have been identified to cause monogenic forms of SRNS if mutated. The characterization of the cellular function of these 63 genes has helped to delineate 12 distinct pathogenic pathways of this disease, revealing a central role for the podocyte in the pathogenesis of SRNS. Monogenic causation accounts for 11 - 29.5% of SRNS patients with onset before < 25 years, leaving up to 70% of cases genetically unresolved. Thus, one aim of this project was to identify novel SRNS candidate disease genes by whole exome sequencing (WES) and perform a functional in vitro and in vivo validation of a recently discovered SRNS gene, DLC1. As most of the envisioned work, proposed in aim 2/3 (functional characterization of DLC1) was just published when the project period started, an early adjustment of these scientific aims was necessary. During the funding period more than 150 WES datasets of families with SRNS, but also with other childhood onset renal diseases, such as congenital anomalies of the kidney and urinary tract (CAKUT), renal tubulopathies and ciliopathies were analyzed. Hereby, several potential candidate disease genes in multiple families were identified. These potential novel disease genes were functionally evaluated by performing in vitro and in vivo experiments. Immunofluorescence and immunohistochemistry was performed to analyze the endogenous expression of the protein in renal tissues, and also to compare subcellular localization of overexpressed wild type vs. mutant proteins within different renal cells. Cell migration, cell adhesion, small Rho-like GTPase activation assays or co-immunoprecipitation studies were employed in order to understand the impact of either loss-of-function or overexpression of the protein. Finally, a CRISPR/Cas9 knockout zebrafish line for a potential novel CAKUT gene was established, and knockout mice to elucidate the role of the gene for CAKUT were evaluated. In the following paragraph three of the projects are further described. By performing WES in a cohort of 17 patients with distal renal tubular acidosis, 3 potential candidate genes were discovered (ATP6V1C2, WDR72, SLC4A2) out of which 2 were functionally characterized in collaboration with Seth Alpers and Patricia Kane. Furthermore, we identified a homozygous mutation in the transcription factor gene TFCP2L1 in a patient with early onset CKD accompanied by a severe salt loosing distal tubulopathy. Tfcp2l1 encodes a transcription factor and has recently been shown to play a pivotal role in the maturation of the distal nephron and collecting duct in mice. We demonstrate, that Tfcp2l1 was expressed in the kidney throughout murine development and in the distal part of the mature nephron. Furthermore, in collaboration with John Barasch, we show that Tfcp2l1 alone was sufficient to induce tubulogenesis in rat metanephric explants. Additionally, we demonstrate, that the transcription factor TFCP2L1 only localizes to the nucleus when co-expressed with its binding partner and paralog UBP1. A mutant, representing the patient mutation, failed to form dimers with UBP1 and mislocalized in HEK293T cells compared to wild type, thus indicating loss-of-function for the identified variant. Finally, we propose a novel approach for identifying potential SRNS candidate genes and provide functional evidence for one identified variant. In this project we first validated two datasets by overlapping with known SRNS disease genes and then used these two datasets for overlap with a list of 120 WES-derived SRNS candidate genes that the Hildebrandt laboratory has discovered over the past 12 years. The two datasets were: 1) a list of 63 known murine NS/proteinuria genes, 2) 64 podocytic genes, downstream of the transcription factor WT1. Using this approach, we could identify 7 potential novel SRNS candidates and provided functional evidence for one of these genes.
Publications
- CAKUT and Autonomic Dysfunction Caused by Acetylcholine Receptor Mutations. Am J Hum Genet. 2019; Dec 5;105(6):1286-1293
Mann N, Kause F, Henze EK, Gharpure A, Shril S, Connaughton DM, Nakayama M, Klämbt V, Majmundar AJ, Wu CW, Kolvenbach CM, Dai R, Chen J, van der Ven AT, Ityel H, Tooley MJ, Kari JA, Bownass L, El Desoky S, De Franco E, Shalaby M, Tasic V, Bauer SB, Lee RS, Beckel JM, Yu W, Mane SM, Lifton RP, Reutter H, Ellard S, Hibbs RE, Kawate T, Hildebrandt F
(See online at https://doi.org/10.1016/j.ajhg.2019.10.004) - CRISPR-based diagnostics for transplantation medicine, Nature Biomedical Engineering, 2020 Apr 13
Kaminski MM, Alcantar M, Lape I, Greensmith R, Huske A, Valery J, Marty F, Klämbt V, Riella L, and Collins JJ
(See online at https://doi.org/10.1038/s41551-020-0546-5) - Phenotype expansion of heterozygous FOXC1 pathogenic variants towards involvement of CAKUT, Genetics in Medicine, 2020
Wu CHW, Mann N, Nakayama M, Connaughton DM, Dai R, Kolvenbach C, Kause F, Ottlewski I, Wang C, Klämbt V, Seltzsam S, Lai EW, Selvin A, Senguttu P, Bodamer O, Stein DR, ElDesoky S, Kari JA,Tasic V, Shril S, and Hildebrandt F
(See online at https://doi.org/10.1038/s41436-020-0844-z) - Whole exome sequencing identified ATP6V1C2 as a novel candidate gene for recessive distal renal tubular acidosis, Kidney International, 2020; Mar;97(3):567-579
Jobst-Schwan T, Klämbt V, Tarsio M, Heneghan JF, Majmundar AJ, Shril S, Buerger F, Ottlewski I, Shmukler B, Topaloglu R, Hashmi S, Emma F, Greco M, Laube G, Fathy HM, Pohl M, Gellermann J, Milosevic D, Baum M, Mane S, Lifton R, Kane P, Alper S, Hildebrandt F
(See online at https://doi.org/10.1016/j.kint.2019.09.026)
