A genetic approach to defining the Ttc21b interactome in mammalian ciliopathies

描述（由申请人提供）：纤毛病是由原发性纤毛功能缺陷引起的一系列疾病，影响1:80人。初级纤毛是基于微管的细胞器，几乎存在于所有细胞中，对许多分子途径的正确信号转导至关重要。纤毛病影响广泛的组织，包括神经系统、颅面组织、骨骼、肾脏、肺和消化器官。这些表现为先天性和成人性缺陷。对纤毛病患者的遗传学研究表明TTC21B（四肽重复结构域蛋白1b）是迄今为止发现的最常见的突变纤毛基因。除了与TTC21B缺失相关的缺陷外，许多其他纤毛基因的反式突变也会导致纤毛病。我们最近发现Ttc21b在小鼠体内的缺失会导致围产期死亡和器官发生缺陷。我们还注意到，其中一些表型依赖于特定的近交系小鼠株背景。TTC21B蛋白很大，具有许多蛋白-蛋白相互作用结构域，在鞭毛内运输和调节纤毛信号转导中起重要作用。所有这些数据结合在一起，我们得出了一个中心假设，即TTC21B是支撑和贩运活动的网络枢纽，对正确的纤毛形态和功能至关重要。该应用程序的目标是识别与Ttc21b相互作用的基因和蛋白质：Ttc21b“相互作用组”，并开始了解这些基因和蛋白质如何在细胞中相互作用。该项目的基本原理是，更全面地了解TTC21B如何起作用，可能会对一系列纤毛病有更深入的了解。我们将通过以下三个具体目标来解决这一假设并实现这些目标：1)在B6和FVB小鼠品系中鉴定含有修饰Ttc21bnull/null表型的基因的染色体区域，2)使用正向遗传方法鉴定与Ttc21b的新的遗传相互作用，以及3)研究与Ttc21b相互作用的基因的功能机制。第一个目标是利用QTL分析来确定调节菌株特异性表型的位点

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).