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，从细胞表面伸出，执行不同的生物学功能，包括全细胞运动，流体运动，化学，机械和光敏，以及有性生殖。与它们严格的进化保守性一致，纤毛或鞭毛的缺陷与广泛的人类疾病有关。淋巴结纤毛的缺失或功能障碍会扰乱左右轴的决定，而感觉纤毛的缺陷会导致多囊肝和肾病。同样地，室管膜纤毛的功能障碍引起脑积水，并且蛋白质沿着连接纤毛的光感受器的缺陷运输沿着导致视网膜变性。尽管它们具有深远的重要性，纤毛及其在人类生理学中的作用只是最近才获得了更广泛的关注，大多数哺乳动物细胞具有纤毛能力的事实一直未得到充分的重视。在这里，我们建议对哺乳动物基因组和蛋白质组进行全面分析，以确定和验证参与纤毛结构和功能的人类蛋白质的部分，并确定它们对人类遗传疾病的贡献。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纤毛，运动纤毛或两者的功能所需的蛋白质。第二，集中在感觉纤毛表达的蛋白质，我们将询问他们的直接参与纤毛功能，通过确定其在纤毛细胞的亚细胞定位。第三，我们将进行高密度定位的近亲家庭与建立纤毛障碍，如Bardet-Biedl综合征（BBS）和肾单位结核（NPH），并确定每个家系的基因组中的所有区域，显示纯合性的下降。然后，我们将交叉我们的计算和实验睫状蛋白质组数据与我们的遗传图谱信息，并确定新的BBS和NPH基因。最后，为了研究功能障碍的机制，我们将抑制哺乳动物纤毛细胞中新疾病基因的mRNA信息，并模拟纤毛形态，鞭毛内运输和纤毛生命周期中转录调控功能丧失突变的后果。这些研究将进一步显着我们的纤毛，研究最少的细胞器之一，但主要的生理重要性的功能的理解，并提供新的工具来解剖人类遗传疾病的分子基础。

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.