Identifizierung und funktionelle Charakterisierung monogenetischer Ursachen des steroidresistenten nephrotischen Syndroms und der fokal-segmentalen Glomerulosklerose.
Allgemeine Genetik und funktionelle Genomforschung
Nephrologie
Zusammenfassung der Projektergebnisse
Nephrotic syndrome (NS) is the second leading cause of chronic kidney disease in the first three decades of life. NS manifests with edema, proteinuria, and hypoalbuminemia, and is caused by a disruption of the glomerular filtration barrier, primarily affecting the podocyte foot processes. The most commonly observed histologic manifestation in monogenic NS is focal segmental glomerulosclerosis (FSGS). The majority of monogenic NS cases is found to be steroid resistant (SRNS). Genes, that if mutated can cause NS, are predominantly expressed in the glomerular podocyte, and variants in their encoded proteins impair podocyte structure and function. Using gene panel or exome sequencing (ES), mutations in one of the 59 known NS genes can be identified in up to 30% of patients, leaving up to 70% genetically unsolved. Thus, the primary aim of this study was to identify novel SRNS disease genes using ES and validate their candidate status with subsequent in vitro and in vivo studies. In a second step, this study aimed to specifically identify candidate genes encoding proteins of the nuclear pore complex (NPC), for which recently first genes had been implicated in monogenic forms of NS. As no NPC genes reaching all criteria of confidence to pursue functional studies could be identified, the studied disease population was extended to congenital anomalies of the kidneys and urinary tract (CAKUT) and two candidate genes for NS and one candidate gene for CAKUT were functionally studied. During the funding period ES data of about 100 families from different renal diseases with childhood onset, mainly NS and CAKUT, were analyzed. Using this approach multiple candidate genes were identified of which three were functionally studied. (1) Recessive NOS1AP variants in two families with early-onset NS were discovered by ES. In human podocytes overexpression of wildtype (WT) NOS1AP, but not patient-derived variants, increased active CDC42 levels and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, the formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), an established intermediate phenotype of NS, which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes could also be rescued by constitutively active CDC42Q61L or the formin DIAPH3. In a next step, the NOS1AP patient missense variant was modelled in knock-in human kidney organoids which revealed malformed glomeruli with increased apoptosis. Finally, a mouse model with an in-frame deletion of exon 3 was studied. Nos1apEx3-/Ex3- mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement and glomerulosclerosis. (2) Hemizygous variants in ARHGEF6 in 9 male individuals from 6 families with CAKUT were identified using ES. The discovered 6 genetic variants comprised of nonsense, missense and splice site variants as well as small deletions. Wild type ARHGEF6, but not truncating patient variants, increased active levels of RAC1 and CDC42 in HEK293T cells. In IMCD3 cells wild type ARHGEF6, but not patient variants, increased fibronectin-induced cell spreading in co-overexpression with PARVA. Finally, modelling ARHGEF6 deficiency in a mouse model revealed increased frequencies of CAKUT (duplex kidneys and hypodysplasia) in both hemizygous and homozygous allele carriers. (3) By ES in 1,382 individuals with NS or FSGS, rare homozygous mutations in the gene SEMA3G in three individuals from three unrelated families with nephrotic syndrome or FSGS were identified. Single-cell RNA sequencing from murine and human kidneys showed high predominant expression in glomerular podocytes. Immunofluorescence staining in rat glomeruli demonstrated strong linear anti-Sema3g signals adjacent to podocyte marker synaptopodin. In summary, exome sequencing in a defined disease cohort of renal diseases is viable approach to identifying novel disease-causing genes. Studying these candidate genes and the patient-derived variants in in vitro and in vivo greatly furthers the understanding of the underlying pathomechanisms and might eventually lead to specific therapeutic approaches.
Projektbezogene Publikationen (Auswahl)
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ADCK4 Deficiency Destabilizes the Coenzyme Q Complex, Which Is Rescued by 2,4-Dihydroxybenzoic Acid Treatment. J Am Soc Nephrol 31, 1191-1211 (2020)
E. Widmeier, S. Yu, A. Nag, Y. W. Chung, M. Nakayama, L. Fernandez-Del-Rio, H. Hugo, D. Schapiro, F. Buerger, W. I. Choi, M. Helmstadter, J. W. Kim, J. H. Ryu, M. G. Lee, C. F. Clarke, F. Hildebrandt and H. Y. Gee
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Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations. Am J Hum Genet 107, 727-742 (2020)
D. M. Connaughton, ..., F. Hildebrandt
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Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice. Sci. Adv. 2021; 7 : eabe1386
A. J. Majmundar, F. Buerger, T. A. Forbes, V. Klämbt, R. Schneider, K. Deutsch, T. M. Kitzler,, S. E. Howden,, M. Scurr, K. Sin Tan, M. Krzeminski, E. Widmeier, D. A. Braun, E. Lai, I. Ullah, A. Amar, A. Kolb, K. Eddy, C. H. Chen, D. Salmanullah, R. Dai, M. Nakayama, I. Ottlewski, C. M. Kolvenbach, A. C. Onuchic-Whitford,, Y. Mao, N. Mann, M. M. Nabhan, S. Rosen, J. D. Forman-Kay,, N. A. Soliman, A. Heilos, R. Kain, C. Aufricht, S. Mane, R. P. Lifton, S., M. H. Little,, and F. Hildebrandt
