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 最多50％的全球小儿终末期肾脏衰竭病例属于 肾脏和尿路的先天异常（Cakut）。尽管卡库特的遗传基础仍然存在 难以捉摸的，最近的人类研究开始阐明疾病的发病机理。我们的研究 组结合了基于家庭的组合以及案例对照分析与功能建模相结合的组 脊椎动物已经确定了多个基因，这些基因在人类中突变后会导致cakut。有趣的是， 在约有30％的Digeorge综合征患者中描述了Cakut表型，并删除了 染色体22q11.2是迪吉尔戈综合症的最常见原因，构成了最常见的 人类中的微缺失综合征。迄今 CRKL是在Digeorge的22Q11.2基因座，驱动肾脏和尿路畸形的基因之一 综合征和零星cakut；但是，人类疾病的因果机制仍然未知。 在小鼠和人类中，CRKL至少是两个主要的成绩单同工型，从而提高了 在调节肾脏和尿路时，对CRKL及其结合伴侣进行复杂调节的可能性 发展。尽管一些动物研究表明，小鼠中CRKL的操纵可能导致 肾脏表型，没有人能解决不同CRKL同工型的作用。这留下了未解决的问题 关于每个变体的涉及方式以及它们在调制中扮演的组织和细胞特异性角色的问题 发育信号级联。为了回答这些问题，我们设计了一个多学科 使用几种鼠标模型的方法，其中一个或两个成绩单将在遗传上消失 组织特异性的方式。因此，本文提出的实验检验了中心假设 CRKL的两个同工型差异调节肾脏和尿路发展的特定事件， 要么是彼此独立的，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同工型如何对 肾脏和尿路。在AIMS 1和2中，我将解决有关精确时空的关键问题， 每个剪接变体的潜在差异，表达模式，每个转录本的发展作用 在肾脏（AIM 1）和尿路（AIM 2）中，以及一个同工型的发展要求 其他。在AIM 3中，传统和转录组/RNASEQ方法将用于识别密钥 发育信号传导级联反应受每个CRKL同工型在体内的影响，并带有发现 通过生化和组织化学测定验证。最终，通过使用发展遗传学 以及设计一个新颖的分析框架的计算方法，该框架整合了表型，遗传和 我打算，单细胞转录组数据，a）精炼搜索新型cakut基因，b）应用调查结果 要解决细胞自主权和对遗传侮辱的次要反应的更广泛的，未解决的问题。