A genetic approach to defining the Ttc21b interactome in mammalian ciliopathies

定义哺乳动物纤毛病中 Ttc21b 相互作用组的遗传学方法

基本信息

批准号：
9415039
负责人：
Rolf W Stottmann
金额：
$ 30.03万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2020-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9415039
关键词：
Address Adult Affect Alleles Area Bioinformatics Biology Candidate Disease Gene Cells Cellular Membrane Cilia Cilium Microtubule Congenic Strain Congenital Abnormality Craniofacial Abnormalities Data Defect Developmental Biology Disease Embryo Ethylnitrosourea Face Felis catus Functional disorder Gene Proteins Gene-Modified Genes Genetic Genetic Techniques Genetic study Goals Human Human Genetics In Vitro Inbred Strains Mice Incidence Interphase Cell Intervention Kidney Knockout Mice Knowledge Lead Lung Meiotic Recombination Microcephaly Microtubules Mission Modeling Molecular Mouse Strains Mus Mutagenesis Mutate Mutation Nervous system structure Organ Organelles Organogenesis Outcome Pathology Pathway interactions Patients Perinatal mortality demographics Phenotype Population Prosencephalon Protein Binding Domain Proteins Public Health Publishing Quantitative Trait Loci Research Scaffolding Protein Severities Signal Transduction Skeleton Solid Testing Therapeutic Intervention Tissues United States National Institutes of Health Work base burden of illness ciliopathy cohort craniofacial craniofacial tissue gene product genetic approach in vivo innovation insight interest mouse model mutant next generation sequencing novel patient population positional cloning public health relevance scaffold trafficking

项目摘要

DESCRIPTION (provided by applicant): Ciliopathies are a spectrum of diseases resulting from defects in primary cilia function affecting 1:800 people. Primary cilia are microtubule based organelles found on almost all cells and crucial for proper signal transduction of a number of molecular pathways. Ciliopathies affect a wide range of tissues including the nervous system, craniofacial tissues, skeleton, kidneys, lungs and digestive organs. These manifest as both congenital and adult-onset defects. Genetic studies of ciliopathy patients show TTC21B (tetratricopeptide repeat domain-containing protein1B) is the most commonly mutated cilia gene identified to date. In addition to defects associated with loss of just TTC21B, mutations in trans with a number of other ciliary genes lead to ciliopathies. We have recently shown loss of Ttc21b in the mouse leads to perinatal lethality and organogenesis defects. We also note some of these phenotypes are dependent on the specific inbred mouse strain background. The TTC21B protein is large with many protein-protein interaction domains and important for intraflagellar transport and regulating signal transduction in the cilium. All of these data together lead us to the central hypothesis that TTC21B serves as a network hub for scaffolding and trafficking activities essential for proper cilia form and function. The goal of this application is identify genes and proteins interacting with Ttc21b: the Ttc21b "interactome," and begin to understand how these interact in the cell. The rationale for the project is that a more complete understanding of how TTC21B acts is likely to give insight to a range of ciliopathies. We will address this hypothesis and achieve these goals with the following three specific aims: 1) identify chromosomal regions containing genes modifying the Ttc21bnull/null phenotype in the B6 and FVB mouse strains, 2) identify novel genetic interactions with Ttc21b using a forward genetic approach, and 3) study functional mechanisms of genes interacting with Ttc21b. The first aim will utilize a QTL analysis to identify loci regulating the strain specific phenotypes we see in Ttc21bnull/null embryos. The second aim will take a forward genetic, ENU mutagenesis approach to identify novel interactions with TTC21B in an unbiased manner. The third aim will recapitulate interactions identified in humans or previously identified in mouse. After verifying these interactions yield ciliopathy phenotype(s), we will perform further analyses in vitro and in vivo to study the molecular mechanisms of ciliary dysfunction. These studies will focus on ciliary trafficking and Shh signal transduction. The significance of this project is that these studies wil together dramatically increase our understanding of how TTC21B acts within the primary cilium and why perturbation of function leads to ciliopathic disease. These studies will fill an important gap in our knowledge and identify possible areas for therapeutic intervention. These advances are not specific to TTC21B but are likely going to be largely applicable to multiple areas of primary cilia biology inter- est. The innovation of this project lies in the application of unbiase genetic techniques to identify the Ttc21b interactome in close concert with solid molecular studies to determine the underlying mechanism(s).

描述（由申请人提供）：纤毛病是由于影响1：800人的主要纤毛功能的缺陷而导致的一系列疾病。原发性纤毛是几乎所有细胞上发现的基于微管的细胞器，对于正确信号转导了许多分子途径，至关重要。纤毛病影响包括神经系统，颅面组织，骨骼，肾脏，肺部和消化器官在内的各种组织。这些表现为先天性和成人发作的缺陷。纤毛病患者的遗传研究表明TTC21B（四肽重复结构域的蛋白1b）是迄今为止最常见的纤毛基因。除了与仅损失TTC21b相关的缺陷外，具有许多其他纤毛基因的反式突变导致纤毛病。我们最近显示了小鼠中TTC21b的丧失导致围产期致死性和器官发生缺陷。我们还注意到，其中一些表型取决于特定的近交小鼠应变背景。 TTC21b蛋白具有许多蛋白质 - 蛋白质相互作用结构域的大型，对于flagellar的转运和调节纤毛中信号转导至关重要。所有这些数据共同提出了一个核心假设，即TTC21B是脚手架和运输活动的网络中心，这对于适当的纤毛形式和功能至关重要。该应用的目的是识别与TTC21B相互作用的基因和蛋白质：TTC21B“ Interactome”，并开始理解这些相互作用在细胞中的相互作用。该项目的理由是，对TTC21B行为的方式有了更全面的了解，可能会深入了解一系列纤毛病。我们将通过以下三个特定目的来解决这一假设并实现这些目标：1）确定包含基因的染色体区域，这些基因在B6和FVB小鼠菌株中修改了TTC21BNULL/NULL表型，2）使用远期遗传学方法与TTC21B相互作用，并与TTC21B相互作用，与TTC21B相互作用，与TTC21B相互作用。第一个目标将利用QTL分析来识别调节特定菌株表型的基因座我们请参阅TTC21BNULL/NAULL胚胎中的内容。第二个目的将采用一种正向的遗传，ENU诱变方法，以公正的方式鉴定与TTC21B的新型相互作用。第三个目标将概括在人类或以前在小鼠中鉴定出的相互作用。在验证这些相互作用产生纤毛病表型后，我们将在体外和体内进行进一步的分析，以研究纤毛功能障碍的分子机制。这些研究将集中于睫状运输和SHH信号转导。该项目的意义在于，这些研究将大大增加我们对TTC21B在原发性纤毛内的作用以及功能扰动导致纤毛性疾病的理解。这些研究将填补重要的在我们的知识上差距，并确定治疗干预的可能领域。这些进步不是特定于TTC21B的特定，但很可能主要适用于原发性纤毛生物学的多个领域。该项目的创新在于应用无轴基遗传技术来识别TTC21B相互作用与固体分子研究中的近距离相互作用，以确定基础机制（S）。