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Functional Studies of Novel Genes Mutated in Primary Ciliary Dyskinesia

原发性纤毛运动障碍新突变基因的功能研究

基本信息

批准号：
8480072
负责人：
LAWRENCE E OSTROWSKI
金额：
$ 36.18万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-15 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8480072
关键词：
Affect Air Antigens Asthma Bacteria Biochemical Biological Models Biopsy Breathing Bronchiectasis Candidate Disease Gene Cell Fractionation Cells Chronic Chronic Obstructive Airway Disease Chronic Sinusitis Cilia Coughing Cystic Fibrosis Defect Defense Mechanisms Development Diagnosis Disease Docking Dynein ATPase Electron Microscopy Epithelial Cells Evaluation Exhibits Frequencies Gases Genes Genetic Genetic Transcription Genetic Variation Goals Growth-Arrest-Specific Protein 2 Head Health Homologous Gene Human Immunoprecipitation Incidence Lead Lentivirus Vector Liquid substance Lung diseases Maps Mass Spectrum Analysis Measurement Measures Methods Molecular Molecular Mechanisms of Action Mucociliary Clearance Mucous body substance Mus Mutate Mutation Neonatal Nitric Oxide Nose Otitis Media Pathogenesis Patients Phase Phenotype Primary Ciliary Dyskinesias Production Proteins Proteomics Radial Rare Diseases Recombinant Proteins Regulation Reporting Research Respiratory distress Reverse Transcriptase Polymerase Chain Reaction Role Sampling Situs Inversus Structure Symptoms Techniques Technology Time Transmission Electron Microscopy Variant Video Microscopy Virus Western Blotting arm cilium biogenesis clinical phenotype crosslink disease-causing mutation early onset exome sequencing gene function human tissue immunocytochemistry improved in vivo knock-down mutant novel novel therapeutics protein complex protein expression public health relevance respiratory respiratory examination small hairpin RNA sperm cell

项目摘要

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.

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疾病，由破坏纤毛功能并导致粘液纤毛清除（MCC）缺陷的突变引起。粘膜纤毛清除是一种重要的先天防御机制，受损的 MCC 会导致多种气道疾病的发病机制，包括 PCD、哮喘、囊性纤维化 (CF) 和慢性阻塞性肺病 (COPD)。 PCD 具有遗传异质性，候选基因测序、纯合性图谱以及最近的全外显子组测序现已鉴定出十多个导致 PCD 的基因的突变。这些遗传学研究不仅迅速推进了 PCD 的诊断，而且正在扩大 PCD 的定义，以前未诊断出呼吸系统疾病的患者实际上可能患有 PCD 的变异形式。我们已经鉴定出 7 个导致 PCD 的新基因突变。虽然其中一些基因已在具有“典型”PCD表型的患者中被鉴定出并编码已知的睫状轴丝成分的蛋白质，但其他基因已在具有“非典型”PCD表型的患者中被鉴定出，并且突变的蛋白质完全未被表征。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正确组装、活动或调节中发挥不同作用的基因突变所致。因此，本提案的目标是研究三种新的 PCD 致病基因：精子相关抗原 1 (SPAG1)、径向辐条头同源物 1 (RSPH1) 和生长停滞特异性蛋白 2 样 2 (GAS2L2) 的功能，每个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粘膜纤毛清除的作用及其在疾病中的作用，有必要了解这些功能基因。我们将使用不同的模型系统和各种技术研究这些基因的功能。首先，我们将使用定量 RT-PCR 和免疫染色研究人气道上皮 (HAE) 细胞分化良好的培养物中正常蛋白的表达和定位。然后，我们将使用 shRNA 技术来抑制新基因的表达，并使用一种新方法直接培养来自 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，并测量纤毛跳动频率、波形、粘液纤毛运输和一氧化氮的产生，以确定缺失蛋白质的作用。将使用生化交联技术、免疫沉淀和质谱法来鉴定与 SPAG1、RSPH1 和 GAS2L2 相互作用的蛋白质。最后，我们将对 Rsph1 基因缺失的小鼠进行研究，以确定该基因的缺失对粘膜纤毛清除和体内疾病发病机制有何影响。这些研究将加深人们对这些蛋白质在纤毛结构和功能、粘液纤毛清除、和呼吸系统健康，并可能导致针对多种呼吸系统疾病（包括 PCD、哮喘、CF 和 COPD）的新治疗方法的开发。