Characterization of three candidate genes and selected variants thereof associated with congenital anomalies of the kidneys and urinary tract (CAKUT) using in vitro and in vivo models

• Applicant: Professorin Dr. Ruthild Weber, since 11/2022
• Subject Area: Human Genetics
• Term: from 2018 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 393047016

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. Much is still to be learned regarding the genetic basis and the molecular pathogenesis of CAKUT. In the proposed project, the role of three CAKUT (candidate) genes in CAKUT pathogenesis will be studied in detail in cellular and animal models as well as in patients. The study is set in the context of close collaborations with the Department of Pediatric Kidney, Liver and Metabolic Diseases at Hannover Medical School (MHH) offering the largest kidney transplantation program for children and adolescents in Germany, and with the Institute of Molecular Biology at MHH with a longstanding expertise in the study of murine urogenital tract development, which allows (i) the genetic analysis of a high number of severely affected CAKUT patients and their families, (ii) the in-depth characterization of identified genes in mouse models, (iii) the reverse phenotyping of patients based on the genetic findings. In this collaboration, the applicant and colleagues have recently identified and characterized in detail the new CAKUT associated genes TBC1D1 and LIFR. In project 1 of the proposed study, a focused analysis of a novel LIFR-dependent phenotype, that is increased desquamation of cells from the urothelium in the ureter of Lifr-/- embryos, will be performed using RNA in situ hybridization, immunofluorescence, and expression studies to further characterize the role of LIFR in CAKUT pathogenesis. Subsequent reverse phenotyping of CAKUT patients carrying LIFR variants will explore whether this phenotype is also present in these patients. Since all Lifr-/- mice and a CAKUT patient with a pathogenic LIFR variant also displayed cryptorchidism, in project 2, the applicant will study a gene associated with cryptorchidism, and another gene encoding a protein acting in the same pathway. We have found rare probably pathogenic variants in these genes in a small cohort of CAKUT patients. Here, (i) at least 300 CAKUT patients and family members will be analyzed by targeted sequencing of these two genes, (ii) pathogenicity of detected variants will be analyzed by determining protein half-life and transcription factor activity of mutant versus wildtype proteins, (iii) the expression in the developing murine urogenital system and human fetal, neonatal and adult tissues will be studied, (iv) knockout mice will be bred using heterozygous knockout mice and urogenital systems characterized, and (v) reverse phenotyping of patients carrying variants will elucidate the relationship between CAKUT and cryptorchidism. In this project, by detailed in vitro and in vivo characterization of three CAKUT candidate genes, we aim to elucidate new CAKUT causing pathomechanisms and to better understand phenotype complexity in CAKUT patients to ultimately improve patient care.

• DFG Programme: Research Grants
• Ehemalige Antragstellerin: Dr. Anne Christians, until 10/2022