Genetic Analysis of Organ Patterning Defects in Ciliopathies

摘要： 纤毛疾病是一组不断扩大的人类疾病，其细胞学基础可以追溯到 纤毛一种叫做纤毛的小细胞器的形成或功能纤毛几乎存在于每一种细胞类型中 脊椎动物的身体和纤毛疾病可以包括多个器官和组织的缺陷。令人惊讶的是， 纤毛病的范围从不孕不育到新生儿死亡，但这些疾病背后的遗传学 人们对不同的表型知之甚少。确定调节纤毛的基因和遗传交互作用 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。 我们已经将携带Ift56基因自发突变(HOP)的小鼠发展成一种新的 明确睫毛病变严重程度的遗传控制。Ift56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需的。我们最近发现，虽然Ift56hop 在Balb鼠的背景下，表型是活的和温和的，在B6背景下是致命的。表型 HOP-B6突变模型在人类中的Vacterl关联，并与Ift27突变的纤毛病变重叠 表型。我们的目标是1)利用HOP-B6突变体作为了解Vacterl疾病的一种手段， 2)确定Ift56在哺乳动物纤毛中的功能；3)分离与Ift56突变相互作用的遗传位点 以阐明纤毛疾病背后的遗传图景。 我们的第一个目标是分析Ift56-B6突变体的组织缺陷。组织学方法结合起来 通过基因表达分析和MicroCT成像将提供对信号和发育的关键洞察 HOP-B6突变体中由Ift56控制的图案化事件和Vacterl相关缺陷 AIM2探索了Ift56的纤毛作用，以及这些作用在B6和Balb背景下是如何改变的。vbl.使用 候选和蛋白质组学方法，我们将确定一组需要Ift56功能的蛋白质 纤毛内的本地化和贩运。最后，测试了Ift56对IFTB复数完整性的要求。 在我们的第三个目标中，我们定位、分离和验证B6和Balb中的HOP表型修饰物 背景。我们还将研究Ift27和HOP突变体之间的遗传相互作用以及它们的影响 Ift27表型的Balb修饰符背景。这些实验将确定关键的遗传修饰因素 并为纤毛疾病患者的重新测序提供新的靶点。 总之，拟议的研究将揭示Ift56的保守功能，Ift56是一种关键的纤毛蛋白，通过定义 Ift56如何调控纤毛和器官发生。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疾病严重程度的独特见解。这项研究的结果 将对纤毛生物学和纤毛疾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.