A genetic approach to defining the Ttc21b interactome in mammalian ciliopathies

描述（由申请人提供）：纤毛病是一系列由初级纤毛功能缺陷引起的疾病，影响1：800的人群。初级纤毛是基于微管的细胞器，存在于几乎所有细胞中，并且对于许多分子途径的适当信号转导至关重要。纤毛病影响广泛的组织，包括神经系统、颅面组织、骨骼、肾、肺和消化器官。这些表现为先天性和成人发病缺陷。对纤毛病患者的遗传学研究表明，TTC 21 B（含四肽重复结构域的蛋白1B）是迄今为止发现的最常见的纤毛突变基因。除了与TTC 21 B丢失相关的缺陷外，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，小鼠Ttc 21 b的缺失导致围产期死亡和器官发生缺陷。我们还注意到这些表型中的一些依赖于特定的近交系小鼠品系背景。TTC 21 B蛋白是一个大的蛋白质-蛋白质相互作用结构域和重要的鞭毛内运输和调节纤毛中的信号转导。所有这些数据共同导致我们的中心假设，TTC 21 B作为一个网络枢纽的脚手架和运输活动所必需的适当纤毛的形式和功能。该应用程序的目标是识别与Ttc 21 b相互作用的基因和蛋白质：Ttc 21 b“相互作用组”，并开始了解这些在细胞中如何相互作用。该项目的基本原理是，更全面地了解TTC 21 B的作用方式可能会深入了解一系列纤毛病变。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）鉴定B6和FVB小鼠品系中含有修饰Ttc 21 b无效/无效表型的基因的染色体区域，2）使用正向遗传学方法鉴定与Ttc 21 b的新型遗传相互作用，以及3）研究与Ttc 21 b相互作用的基因的功能机制。第一个目标是利用QTL分析来鉴定调节菌株特异性表型的基因座， 参见Ttc 21 bnull/null胚胎。第二个目标是采用正向遗传、ENU诱变方法，以无偏倚的方式鉴定与TTC 21 B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发现的相互作用。在证实这些相互作用产生纤毛病变表型后，我们将进行进一步的体外和体内分析，以研究纤毛功能障碍的分子机制。这些研究将集中在纤毛运输和Shh信号转导。该项目的意义在于，这些研究将极大地增加我们对TTC 21 B如何在初级纤毛内起作用以及为什么功能扰动导致纤毛疾病的理解。这些研究将填补重要的 我们的知识差距，并确定可能的治疗干预领域。这些进展并不是TTC 21 B特有的，但很可能在很大程度上适用于初级纤毛生物学兴趣的多个领域。该项目的创新之处在于应用无偏遗传技术来鉴定Ttc 21 b相互作用组，并与固体分子研究密切合作，以确定潜在的机制。

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).