Functional Studies of Novel Genes Mutated in Primary Ciliary Dyskinesia

描述（由申请人提供）：原发性纤毛运动障碍（PCD）是一种常染色体隐性遗传病，由破坏纤毛功能的突变引起，导致纤毛粘膜清除（MCC）缺陷。粘膜纤毛清除是一种重要的先天防御机制，MCC受损有助于多种气道疾病的发病机制，包括PCD、哮喘、囊性纤维化（CF）和慢性阻塞性肺疾病（COPD）。PCD是遗传异质性的，候选基因测序、纯合子作图以及最近的全外显子组测序现在已经确定了十几个导致PCD的基因突变。这些基因研究不仅迅速推进了PCD的诊断，而且扩大了PCD的定义，以前未确诊的呼吸系统疾病患者实际上可能患有不同形式的PCD。我们已经确定了导致PCD的7个新基因的突变。虽然其中一些基因已在“典型”PCD表型患者中被鉴定出来，并编码已知纤毛轴突成分的蛋白质，但其他基因已在“非典型”PCD表型患者中被鉴定出来，并且突变的蛋白质完全不具有特征。我们的假设是，在PCD中观察到的不同临床表型是由于在纤毛的适当组装、活动或调节中发挥不同作用的基因突变。因此，本研究的目的是研究三种新的PCD致病基因的功能，即精子相关抗原1 （SPAG1）、径向辐头同源基因1 （RSPH1）和生长停滞特异性蛋白2-样2 (GAS2L2)，每一种基因都与不同的临床表型相关。为了更全面地了解遗传变异对黏毛清除的作用及其在疾病中的作用，有必要了解这些功能

DESCRIPTION (provided by applicant): Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by mutations that disrupt ciliary function and result in defective mucociliary clearance (MCC). Mucociliary clearance is a critical innate defense mechanism, and impaired MCC contributes to the pathogenesis of several airway diseases, including PCD, asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). PCD is genetically heterogeneous, and sequencing of candidate genes, homozygosity mapping, and more recently, whole-exome sequencing, have now identified mutations in over a dozen genes that cause PCD. These genetic studies are not only rapidly advancing the diagnosis of PCD, but are expanding the definition of PCD, and patients with previously undiagnosed respiratory disease may actually have variant forms of PCD. We have identified mutations in 7 new genes that cause PCD. While some of these genes have been identified in patients with a "typical" PCD phenotype and encode proteins that are known components of the ciliary axoneme, others have been identified in patients with an "atypical" PCD phenotype, and the mutated proteins are completely uncharacterized. Our hypothesis is that the different clinical phenotypes observed in PCD are due to mutations in genes that perform different roles in the proper assembly, activity, or regulation of cilia. Therefore, the goal of this proposal is to investigate the function of thre novel PCD causing genes, sperm associated antigen 1 (SPAG1), radial spoke head homolog 1 (RSPH1), and growth arrest-specific protein 2-like 2 (GAS2L2), each of which is associated with a different clinical phenotype. To more completely understand the role of genetic variation on mucociliary clearance and its role in disease, it is essential to understand the functions of these genes. We will investigate the function of these genes using different model systems and a variety of techniques. First, we will study the expression and localization of the normal proteins in well- differentiated cultures of human airway epithelial (HAE) cells, using quantitative RT-PCR and immunostaining. We will then use shRNA technology to knock down expression of the novel genes and a new method to culture samples of nasal epithelial cells directly from PCD patients. Cilia will be examined by electron microscopy for structural defects, and measurements of ciliary beat frequency, waveform, mucociliary transport, and nitric oxide production will be performed to determine the role of the missing protein. Proteins that interact with SPAG1, RSPH1, and GAS2L2 will be identified using biochemical crosslinking techniques, immunoprecipitation, and mass spectrometry. Finally, mice that have a deletion in the Rsph1 gene will be studied to determine what effects the absence of this gene has on mucociliary clearance and disease pathogenesis in vivo. These studies will lead to an increased understanding of the role of these proteins in cilia structure and function, mucociliary clearance, and respiratory health, and may lead to the developments of new therapeutic treatments for a variety of respiratory diseases, including PCD, asthma, CF, and COPD.