Functional Studies of Novel Genes Mutated in Primary Ciliary Dyskinesia

描述（由申请方提供）：原发性纤毛运动障碍（PCD）是一种常染色体隐性遗传疾病，由破坏纤毛功能并导致粘膜纤毛清除缺陷（MCC）的突变引起。粘膜纤毛清除是一种重要的先天防御机制，受损的MCC有助于几种气道疾病的发病机制，包括PCD、哮喘、囊性纤维化（CF）和慢性阻塞性肺病（COPD）。PCD在遗传上是异质的，候选基因的测序、纯合性作图以及最近的全外显子组测序现在已经在十几个引起PCD的基因中鉴定出突变。这些遗传学研究不仅迅速推进了PCD的诊断，而且扩展了PCD的定义，以前未诊断的呼吸道疾病患者实际上可能具有PCD的变体形式。我们已经确定了7个导致PCD的新基因突变。虽然这些基因中的一些已经在具有“典型”PCD表型的患者中被鉴定，并且编码作为纤毛轴丝的已知组分的蛋白质，但是其他基因已经在具有“非典型”PCD表型的患者中被鉴定，并且突变的蛋白质是完全未表征的。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，精子相关抗原1（SPAG 1）、放射状辐条头同源物1（RSPH 1）和生长停滞特异性蛋白2样2（GAS 2L 2）的功能，它们中的每一种都与不同的临床表型相关。为了更全面地了解遗传变异对粘膜纤毛清除的作用及其在疾病中的作用，必须了解这些基因的功能。 基因.我们将使用不同的模型系统和各种技术来研究这些基因的功能。首先，我们将使用定量RT-PCR和免疫染色来研究正常蛋白在人气道上皮（HAE）细胞的分化培养物中的表达和定位。然后，我们将使用shRNA技术敲低新基因的表达，并使用一种新方法直接培养PCD患者的鼻上皮细胞样本。将通过电子显微镜检查纤毛的结构缺陷，并测量纤毛搏动频率、波形、粘液纤毛转运和一氧化氮产生，以确定缺失蛋白的作用。与SPAG 1，RSPH 1和GAS 2L 2相互作用的蛋白质将使用生化交联技术，免疫沉淀和质谱法进行鉴定。最后，将研究在RIM 1基因中具有缺失的小鼠，以确定该基因的缺失对体内粘膜纤毛清除和疾病发病机制的影响。这些研究将导致对这些蛋白质在纤毛结构和功能，粘膜纤毛清除， 和呼吸系统健康，并可能导致开发用于各种呼吸系统疾病的新治疗方法，包括PCD、哮喘、CF和COPD。

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.