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: 9760804
• 负责人: Jeremiah Martino
• 金额: $ 6.82万
• 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/9760804
• 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.

项目概要 全球高达 50% 的儿童终末期肾衰竭病例属于以下范围： 肾脏和泌尿道先天性异常（CAKUT）。尽管 CAKUT 的遗传基础仍然存在 最近难以捉摸的人类研究开始揭示疾病的发病机制。来自我们的研究 小组结合基于家庭的分析和病例对照分析以及功能建模 脊椎动物已经鉴定出多个基因，当这些基因在人类体内发生突变时，会导致 CAKUT。有趣的是， 约 30% 的 DiGeorge 综合征患者存在 CAKUT 表型，并且缺失 染色体 22q11.2 是 DiGeorge 综合征最常见的原因，也是最常见的 人类微缺失综合症。迄今为止，我们的工作已表明单倍体不足和点突变 CRKL 是在 22q11.2 位点发现的基因之一，可导致 DiGeorge 的肾脏和尿路畸形 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 都以至少两种主要转录本异构体的形式存在，从而提高了 CRKL 及其结合配偶体在肾脏和泌尿道调节中的复杂调节的可能性 发展。尽管一些动物研究表明，在小鼠体内操纵 Crkl 可以导致 肾脏表型方面，没有一个研究涉及不同 Crkl 亚型的作用。这没有答案 每个变体如何参与以及它们在调节中发挥什么组织和细胞特异性作用的问题 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 使用多种小鼠模型的方法，其中一个或两个转录本将在 组织特异性方式。因此，本文提出的实验检验了中心假设： Crkl 的两种亚型差异调节肾脏和泌尿道发育的特定事件， 要么彼此独立，要么通过调节彼此的活动。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 2 中，我将解决有关精确时空、 和潜在的差异，每个剪接变体的表达模式，每个转录物的发育作用 在肾脏（目标 1）和泌尿道（目标 2）中，以及一种异构体的发育需求超过 其他。在目标 3 中，将使用传统的转录组/RNAseq 方法来识别关键的 受体内每种 Crkl 同工型丢失影响的发育信号级联反应，研究结果 通过生化和组织化学测定进行验证。最终，通过发育遗传学的应用 和计算方法来设计一个新颖的分析框架，该框架整合了表型、遗传和 单细胞转录组数据，我打算 a) 完善对新 CAKUT 基因的搜索，b) 应用发现 致力于解决更广泛的、尚未解决的细胞自主性和对遗传损伤的二次反应的问题。