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最常见的原因。关于CAKUT的遗传学基础和分子发病机制仍有许多需要了解。在拟议的项目中，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。这项研究是在与汉诺威医学院(MHH)提供德国最大的儿童和青少年肾脏移植计划的儿科肾脏、肝脏和代谢病学系以及MHH在小鼠泌尿生殖道发育研究方面拥有长期专业知识的分子生物学研究所密切合作的背景下进行的，该研究所可以(I)对大量严重受影响的CAKUT患者及其家属进行基因分析，(Ii)在小鼠模型中深入表征已确定的基因，(Iii)根据遗传学结果对患者进行反向表型分析。在这项合作中，申请人和同事最近已经详细确定并表征了新的CAKUT相关基因TBC1D1和LIFR。在拟议研究的项目1中，将利用RNA原位杂交、免疫荧光和表达研究对一种新的依赖LIFR的表型进行重点分析，即LIFR-/-胚胎输尿管上皮细胞脱屑增加，以进一步表征LIFR在CAKUT发病中的作用。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LIFR/-小鼠和一名携带致病LIFR变异的CAKUT患者也表现出隐睾症，在项目2中，申请者将研究与隐睾症相关的基因，以及另一种编码作用于同一途径的蛋白质的基因。我们在一小部分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)将通过对这两个基因的定向测序来分析至少300名CAKUT患者及其家属，(Ii)将通过测定突变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异的致病性，(Iii)将研究CAKUT在发育中的小鼠泌尿生殖系统以及人类胎儿、新生儿和成人组织中的表达，(Iv)将利用杂合基因敲除小鼠和具有特定特征的泌尿生殖系统培育出基因敲除小鼠，(V)携带变异基因的患者的反向表型鉴定将阐明CAKUT与隐睾症的关系。在这个项目中，通过对三个CAKUT候选基因的详细的体外和体内表征，我们旨在阐明导致CAKUT的新的致病机制，并更好地了解CAKUT患者的表型复杂性，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.