Genetic and Molecular Etiology of Developmental Kidney and Urinary Tract Abnormalities in the DiGeorge, or 22q11.2, Syndrome.

项目总结 全世界高达50%的儿童终末期肾功能衰竭病例属于 先天性肾和尿路畸形(CAKUT)。尽管CAKUT的遗传基础仍然存在 令人难以捉摸的是，最近的人类研究开始揭示疾病的发病机制。来自我们的研究 小组使用基于家庭的分析和病例对照分析相结合的功能建模 脊椎动物已经发现了多种基因，当这些基因在人类身上发生突变时，就会导致CAKUT。有趣的是， 约30%的DiGeorge综合征患者描述了CAKUT表型，并且缺失了 染色体22q11.2是DiGeorge综合征最常见的原因，构成了最常见的 人类的微缺失综合症。到目前为止，我们的工作已经表明，单倍体不足和点突变 位于22q11.2基因座的CRKL基因是导致DiGeorge患者肾脏和尿路畸形的基因之一 综合征和散发性CAKUT；然而，人类疾病发生的原因机制仍不清楚。 在小鼠和人类中，CRKL至少以两种主要转录异构体的形式存在，提高了 CRKL及其结合伴侣(S)在调节肾脏和尿路中复杂调控的可能性 发展。尽管一些动物研究表明，在小鼠体内操纵Crk1可以导致 肾脏的表型，没有一个涉及不同的Crk1亚型的作用。这个问题没有得到回答 关于每个变体是如何参与的，以及它们在组织和细胞特异性调控中扮演什么角色的问题 发育信号级联。为了回答这些问题，我们设计了一个多学科的 一种利用几个小鼠模型的方法，其中一个或两个转录本将在 一种组织特有的方式。因此，这里提出的实验验证了中心假设，即 CRK1的两种亚型对肾脏和尿路发育的特定事件有不同的调节作用， 或者彼此独立，或者通过调节彼此的活动。我的主要目标是 探索同一基因的不同亚型如何在发育过程中产生多方面的影响 肾脏和尿路。在目标1和2中，我将解决有关精确时空的关键问题， 以及潜在的差异，每个剪接变体的表达模式，每个转录本的发育作用 在肾脏(目标1)和尿路(目标2)，以及一个亚型以上的发育要求 又一个。在目标3中，将使用传统和转录/RNAseq方法来确定关键字 体内每个Crk1亚型缺失对发育信号级联的影响 通过生化和组织化学检测得到证实。最终，通过使用发育遗传学 和计算方法来设计一种新的分析框架，该框架集成了表型、遗传和 单细胞转录数据，我打算，a)改进对新CAKUT基因的搜索，b)应用发现 旨在解决更广泛的、悬而未决的问题，如细胞自主性和对遗传侮辱的二次反应。

PROJECT SUMMARY Up to 50% of worldwide cases of pediatric end-stage kidney failure fall within the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). Although the genetic bases of CAKUT remain elusive, recent human studies are starting to shed light into the pathogenesis of disease. Studies from our group using a combination of family-based as well as case-control analyses coupled to functional modeling in vertebrates have identified multiple genes that, when mutated in humans, lead to CAKUT. Interestingly, CAKUT phenotypes are described in ~30% of patients with DiGeorge Syndrome, and deletions on chromosome 22q11.2 are the most common cause of DiGeorge syndrome, constituting the most common microdeletion syndrome in humans. To date, our work has shown that haploinsufficiency and point mutations in CRKL, one of the genes found at the 22q11.2 locus, drive kidney and urinary tract malformations in DiGeorge syndrome and sporadic CAKUT; however, the causal mechanisms of human disease occur are still unknown. In both in mice and humans, CRKL exists as at least two main transcripts isoforms, raising the possibility of a complex regulation of CRKL and its binding partner(s) in regulating kidney and urinary tract development. Although some animal studies have shown that manipulation of Crkl in the mouse can lead to kidney phenotypes, none have addressed the role of different Crkl isoforms. This leaves unanswered questions of how each variant is involved and what tissue- and cell-specific roles they play in the modulation of developmental signaling cascades. In an attempt to answer these questions, we devised a multidisciplinary approach that makes use of several mouse models, where one or both transcripts will be genetically ablated in a tissue-specific manner. The experiments proposed herein therefore test the central hypothesis that the two isoforms of Crkl differentially regulate specific events of kidney and urinary tract development, either independently of one another or by modulating the activity of each other. My main goal is to discover how different isoforms of the same gene can have multifaceted effects on the development of the kidney and urinary tract. In Aims 1 and 2, I will address key questions concerning the precise spatiotemporal, and potentially differential, expression pattern of each splice variant, the developmental role of each transcript in the kidney (Aim 1) and urinary tract (Aim 2), and the developmental requirements of one isoform over another. In Aim 3, traditional and transcriptomic/RNAseq approaches will be used to identify key developmental signaling cascades that are affected by the loss of each Crkl isoform in vivo, with findings verified through biochemical and histochemical assays. Ultimately, through the use of developmental genetics and computational approaches to design a novel analytical framework that integrates phenotypic, genetic and single-cell transcriptomic data, I intend to, a) refine searches for novel CAKUT genes, and b) apply findings toward addressing broader, unsolved questions of cell autonomy and secondary responses to genetic insults.