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

DiGeorge 或 22q11.2 综合征发育性肾脏和尿路异常的遗传和分子病因学。

• 批准号: 9906751
• 负责人: Jeremiah Martino
• 金额: $ 7.3万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906751
• 关键词: 22q11.2; Ablation; Accounting; Adaptor Signaling Protein; Address; Affect; Alternative Splicing; Animal Model; Animals; Binding; Biochemical; Biological Assay; CRKL gene; Cells; Childhood; Chromosomes; Complex; Coupled; DNA Sequence Alteration; Data; Defect; Development; DiGeorge Syndrome; Diagnostic; Disease; Dysplasia; End stage renal failure; Etiology; Event; Exons; Family; Genes; Genetic; Genetic Models; Goals; Human; Human Genetics; Hydronephrosis; Impairment; Kidney; Kidney Failure; Lead; Life; Light; Metanephric Diverticulum; Methodology; Modeling; Molecular; Morphogenesis; Mus; Mutant Strains Mice; Mutate; Mutation; Neurologic; Organogenesis; Outcome; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Play; Point Mutation; Pregnancy; Prenatal Diagnosis; Protein Family; Protein Isoforms; Proteins; RNA Splicing; Regulation; Role; SH3 Domains; Signal Transduction; Structure; Syndrome; System; Terminator Codon; Testing; Therapeutic; Tissues; Transcript; Ultrasonography; Ureter; Urinary tract; Variant; Vertebrates; Work; base; case control; cell type; congenital anomaly; design; developmental genetics; differential expression; experimental study; falls; genomic data; human disease; imaging study; in vivo; innovation; insight; interdisciplinary approach; kidney cell; kidney malformation; malformation; member; microdeletion; mouse model; nephrogenesis; novel; prenatal; response; spatiotemporal; src Homology Region 2 Domain; transcriptome sequencing; transcriptomics; urinary tract obstruction

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.

项目摘要 全世界高达50%的儿童终末期肾衰竭病例属于以下范围： 先天性肾脏和泌尿道异常（CAKUT）。虽然CAKUT的遗传基础仍然存在， 最近的人类研究开始揭示疾病的发病机制。我们的研究 使用基于家族的分析以及病例对照分析与功能模型相结合， 脊椎动物已经确定了多个基因，当在人类中突变时，导致CAKUT。有趣的是， 在约30%的DiGeorge综合征患者中描述了CAKUT表型， 染色体22q11.2是DiGeorge综合征最常见的原因，构成了最常见的 人类的微缺失综合征到目前为止，我们的工作已经表明，单倍不足和点突变， CRKL是在22q11.2位点发现的基因之一，在DiGeorge中驱动肾脏和尿路畸形 综合征和散发性CAKUT;然而，人类疾病发生的因果机制仍然未知。 在小鼠和人类中，CRKL以至少两种主要转录物同种型存在，提高了CRKL的表达。 CRKL及其结合伴侣在调节肾脏和泌尿道中的复杂调节的可能性 发展尽管一些动物研究已经表明，在小鼠中操纵Crkl可以导致 尽管在肾表型中，没有一个解决了不同Crkl同种型的作用。这就留下了未解之谜 问题是每个变异是如何参与的，以及它们在调节细胞增殖中发挥什么样的组织和细胞特异性作用。 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 一种利用几种小鼠模型的方法，其中一种或两种转录本将在小鼠中被遗传消除。 组织特异性的方式。因此，本文提出的实验测试了中心假设，即 Crk 1的两种同种型不同地调节肾和尿道发育的特定事件， 或者彼此独立地或者通过调节彼此的活性。我的主要目标是 发现同一基因的不同亚型如何对发育产生多方面的影响。 肾脏和泌尿道。在目标1和目标2中，我将解决有关精确时空的关键问题， 和潜在的差异，每个剪接变体的表达模式，每个转录本的发育作用， 在肾脏（目的1）和泌尿道（目的2），和一个亚型的发展需求超过 另在目标3中，将使用传统和转录组学/RNAseq方法来确定关键的 发育信号级联受体内每种Crkl同种型丢失的影响， 通过生物化学和组织化学分析验证。最终，通过使用发育遗传学 和计算方法来设计一个新的分析框架，整合表型，遗传和 单细胞转录组学数据，我打算，a）改进新CAKUT基因的搜索，和B）应用发现 致力于解决更广泛的，未解决的细胞自主性和对遗传损伤的二次反应问题。