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

先天性肾脏和尿路异常（CAKUT）是儿童慢性肾脏疾病的最常见原因。关于CAKUT的遗传基础和分子发病机制，还有很多有待研究。在本项目中，我们将在细胞和动物模型以及患者中详细研究三个CAKUT（候选）基因在CAKUT发病机制中的作用。该研究是在与汉诺威医学院（MHH）儿科肾脏、肝脏和代谢疾病部门密切合作的背景下进行的，该部门为德国儿童和青少年提供最大的肾脏移植项目，并与MHH的分子生物学研究所密切合作，该研究所在小鼠泌尿生殖道发育研究方面具有长期的专业知识，这允许(i)对大量严重影响的CAKUT患者及其家属进行遗传分析；（ii）在小鼠模型中对已鉴定基因进行深入表征，（iii）根据遗传发现对患者进行反向表型分析。在这次合作中，申请人及其同事最近确定并详细描述了新的CAKUT相关基因TBC1D1和LIFR。在该研究的项目1中，将使用RNA原位杂交、免疫荧光和表达研究，重点分析一种新的Lifr依赖表型，即Lifr-/-胚胎输尿管尿路上皮细胞脱皮增加，以进一步表征Lifr在CAKUT发病机制中的作用。随后对携带LIFR变异的CAKUT患者进行反向表型分析，将探索这种表型是否也存在于这些患者中。由于所有的Lifr-/-小鼠和一名具有致病性Lifr变异的CAKUT患者也表现出隐睾症，因此在项目2中，申请人将研究与隐睾症相关的基因，以及另一种编码在相同途径中起作用的蛋白质的基因。我们在一小群CAKUT患者中发现了这些基因中罕见的可能致病的变异。在这里，(i)将通过这两个基因的靶向测序来分析至少300名CAKUT患者和家庭成员，（ii）将通过测定突变型与野生型蛋白的蛋白质半衰期和转录因子活性来分析检测到的变体的致病性，（iii）将研究发育中的小鼠泌尿生殖系统和人类胎儿，新生儿和成人组织中的表达。（iv）基因敲除小鼠将使用杂合基因敲除小鼠和具有特征的泌尿生殖系统来繁殖，(v)携带变异的患者的反向表型将阐明CAKUT与隐睾症之间的关系。在本项目中，我们将通过对三种CAKUT候选基因的体外和体内详细表征，旨在阐明新的CAKUT致病机制，更好地了解CAKUT患者的表型复杂性，从而最终改善患者的护理。

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.