Genetic and Functional Studies of Human Ciliary Syndromes

描述(由申请人提供)： 纤毛和鞭毛是古老的、进化上保守的细胞器，它们从细胞表面突出，发挥不同的生物学作用，包括全细胞运动、液体运动、化学、机械和光感觉以及有性繁殖。纤毛或鞭毛的缺陷与它们严格的进化保守一致，与广泛的人类疾病有关。结节纤毛丢失或功能障碍扰乱左右轴的确定，而感觉性纤毛缺陷会导致多囊肝肾疾病。同样，室管膜纤毛功能障碍会导致脑积水，而沿着连接纤毛的光感受器的蛋白质运输缺陷会导致视网膜退化。尽管纤毛具有深远的重要性，但纤毛及其在人类生理学中的作用直到最近才得到更广泛的关注，而大多数哺乳动物细胞具有纤毛能力的事实被低估了。在这里，我们建议对哺乳动物基因组和蛋白质组进行全面的分析，以识别和验证与纤毛结构和功能有关的人类蛋白质组分，并确定它们对人类遗传病的贡献。首先，我们将使用选择性进化保守结合微阵列分析来区分感觉性纤毛和/或运动性纤毛功能所需的蛋白质。其次，聚焦于感觉纤毛表达的蛋白质，我们将通过确定它们在纤毛细胞中的亚细胞定位来询问它们与纤毛功能的直接关系。第三，我们将对Bardet-Biedl综合征(BBS)和肾病综合征(NPH)等已确定的中介疾病的近亲家庭进行高密度图谱绘制，并为每个家系确定基因组中按血统显示纯合的所有区域。然后，我们将把我们的计算和实验睫毛蛋白质组数据与我们的遗传图谱信息相交，并识别新的BBS和NPH基因。最后，为了研究纤毛功能障碍的机制，我们将抑制哺乳动物纤毛细胞中新疾病基因的mRNA信息，并模拟纤毛生活周期中纤毛形态、鞭毛内运输和转录调控功能突变丢失的后果。这些研究将大大加深我们对纤毛的功能的了解，纤毛是目前研究最少的细胞器之一，但具有重要的生理意义，并为剖析人类遗传病的分子基础提供了新的工具。

DESCRIPTION (provided by applicant): Cilia and flagella are ancient, evolutionarily conserved organelles that project from cell surfaces to perform diverse biological roles, including whole-cell locomotion, movement of fluid, chemo-, mechano- and photosensation, and sexual reproduction. Consistent with their stringent evolutionary conservation defects in cilia or flagella are associated with a wide range of human diseases. Loss or dysfunction of nodal cilia perturbs left-right axis determination, whereas sensory cilia defects lead to polycystic liver and kidney disease. Likewise, dysfunction of ependymal cilia cause hydrocephalus, and defective transportation of proteins along the photoreceptor connecting cilium leads to retinal degeneration. Despite their profound importance, cilia and their roles in human physiology have only recently gained broader attention and the fact that most mammalian cells have the capacity to ciliate has been under-appreciated. Here we propose to perform a comprehensive analysis of the mammalian genome and proteome to identify and validate the fraction of human proteins involved in ciliary structure and function, and to determine their contribution to human genetic disease. First, we will use selective evolutionary conservation coupled with microarray analyses to differentiate between proteins necessary for the function of sensory cilia, motile cilia, or both. Second, focusing on proteins expressed in sensory cilia, we will interrogate their direct involvement in ciliary function by determining their subcellular localization in ciliated cells. Third, we will perform high-density mapping of consanguineous families with established ciliation disorders such as Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) and determine for each pedigree all regions in the genome that display homozygosity by descent. We will then intersect our computational and experimental ciliary proteome data with our genetic mapping information and identify novel BBS and NPH genes. Finally, to investigate the mechanism of dysfunction, we will suppress the mRNA message of novel disease genes in mammalian ciliated cells and model the consequences of loss of function mutations in ciliary morphology, intraflagellar transport and transcriptional regulation during the ciliary life cycle. These studies will further significantly our understanding of the function of the cilium, one of the least-studied cellular organelles, yet of major physiological importance, and provide novel tools to dissect the molecular basis of human genetic disease.