Genetic Analysis of Organ Patterning Defects in Ciliopathies

摘要： 纤毛病是一组不断扩大的人类疾病，其细胞基础可以追溯到细胞内的缺陷。 纤毛的形成或功能。纤毛几乎存在于每一种细胞类型中， 脊椎动物体，因此纤毛病可包括多个器官和组织中的缺陷。引人注目的是， 纤毛病变的范围很广，从不育到新生儿死亡，但这些疾病背后的遗传学 不同的表型知之甚少。定义调控纤毛的基因和遗传相互作用 功能对于了解这些疾病的病因以及开发未来的治疗方法至关重要。 我们已经将Ift 56基因中携带自发突变（hop）的小鼠培育成一种新的模型， 确定纤毛病变严重程度的遗传控制。ift 56是一种高度保守的纤毛定位蛋白， 在几种模式生物中纤毛功能所必需的。我们最近发现， 表型在Balb小鼠背景下是活的和温和的，在B6背景下是致死的。表型 hop-B6突变模型在人类中的VEGIL相关性，并与Ift 27突变型纤毛病变重叠 表型。我们的目标是：1）利用hop-B6突变体作为了解VEGF-L疾病的手段， 2)确定Ift 56在哺乳动物纤毛中的功能，以及3）分离与Ift 56突变相互作用的遗传基因座 以阐明纤毛病的遗传背景。 我们的第一个目标是分析Ift 56-B6突变体的组织缺陷。组织学方法结合 基因表达分析和microCT成像将提供关键的洞察信号和发育 hop-B6突变体中由Ift 56和VEGIL相关缺陷控制的模式化事件 Aim 2探讨了Ift 56的纤毛作用，以及这些在B6和Balb背景中是如何改变的。使用 候选人和蛋白质组学方法，我们将确定一组蛋白质，需要Ift 56的功能，其 纤毛内的定位和运输。最后，我们测试了Ift 56对IFTB复合体完整性的要求。 在我们的第三个目标中，我们在B6和Balb中映射、分离和验证了啤酒花表型的修饰符。 背景我们还将研究Ift 27和啤酒花突变体之间的遗传相互作用以及 Balb修饰子背景对Ift 27表型的影响。这些实验将确定关键的遗传修饰剂 的纤毛病变的严重程度，并提供了新的目标，在纤毛病变患者的重新测序。 总之，拟议的研究将揭示Ift 56的保守功能，一个关键的纤毛蛋白，通过定义， Ift 56如何调节纤毛和器官发生。更广泛地说，这些研究有机会揭示 对基因组中的遗传变异如何改变纤毛病变严重程度的独特见解。本研究结果 将为纤毛生物学和纤毛病提供关键和新颖的见解，并为未来的诊断奠定基础。 和治疗策略。

Abstract: Ciliopathies are an expanding group of human disorders whose cellular basis can be traced to defects in the formation or function of small, cellular organelles called cilia. Cilia are found on almost every cell type in the vertebrate body and thus ciliopathies can encompass defects in multiple organs and tissues. Strikingly, ciliopathies span a spectrum, ranging from infertility to neonatal lethality but the genetics underlying these divergent phenotypes are poorly understood. Defining the genes and genetic interactions that regulate cilia function is essential to understanding the etiology of these diseases as well as for developing future therapies. We have developed mice carrying a spontaneous mutation (hop) in the Ift56 gene into a new model for defining the genetic control of ciliopathy severity. Ift56 is a highly conserved cilia-localized protein that is required for cilia function in several model organisms. We recently discovered that while the Ift56hop phenotype is viable and mild on the Balb mouse background, it is lethal on the B6 background. The phenotype of the hop-B6 mutant models VACTERL Association in humans and overlaps with the Ift27 mutant ciliopathy phenotype. Our objectives are to 1) utilize the hop-B6 mutant as a means to understand VACTERL disorders, 2) define the function of Ift56 in mammalian cilia, and 3) isolate genetic loci that interact with the Ift56 mutation in order to elucidate the genetic landscape underlying ciliopathies. Our First Aim focuses on analyzing the tissue defects in Ift56-B6 mutants. Histological approaches coupled with gene expression analysis and microCT imaging will provide key insight into signaling and developmental patterning events controlled by Ift56 and the VACTERL-associated defects in hop-B6 mutants Aim2 explores the ciliary roles of Ift56, and how these are altered in the B6 and Balb backgrounds. Using candidate and proteomic approaches, we will determine the set of proteins that require Ift56 function for their localization and trafficking within cilia. Finally, we test the requirement of Ift56 for IFTB complex integrity. In our Third Aim we map, isolate and validate modifiers of the hop phenotype in the B6 and Balb backgrounds. We will also examine genetic interactions between Ift27 and hop mutants as well as the effects of the Balb modifier background on the Ift27 phenotype. These experiments will identify key genetic modifiers of ciliopathy severity and provide new targets for resequencing in ciliopathy patients. Together, the proposed studies will uncover the conserved functions of Ift56, a key cilia protein, by defining how Ift56 regulates cilia and organogenesis. More broadly, these studies have the opportunity to uncover unique insights into how genetic variants across the genome modify ciliopathy severity. Results from this study will shed key and novel insights into cilia biology and ciliopathies, and lay the groundwork for future diagnostic and therapeutic strategies.