Molecular characterization of outer dynein arm defects in Primary Ciliary Dyskinesia (PCD)

原发性睫状体运动障碍(PCD)是一种遗传异质性疾病，其特征是慢性呼吸道疾病、左右身体不对称的随机性以及由于运动纤毛/鞭毛功能缺陷而导致的男性不育。这种疾病通常会导致永久性的肺损伤，并可能进展为呼吸衰竭。目前的诊断主要依靠电子显微镜显示超微结构缺陷，这往往显示外动力蛋白臂(Oda)缺乏。我们证明，编码oda重链的DNAH5突变与一半的oda缺陷病例有关。通过使用高分辨率免疫荧光成像(IF)，我们在oda缺陷患者中发现了鼻气道细胞中两种不同类型的DNAH5错误定位，从而引入了一种新的PCD诊断工具。在这里，我们想要对一大批国际PCD患者进行遗传学特征分析，以确定是否存在已知的PCD基因DNAH5和DNAI1的突变。为了分析人类ODA的组成和生成，我们将产生针对其他尚未确定的oda重链的抗体，并应用蛋白质技术。为了提高对PCD致病机制和诊断的了解，我们将把遗传学结果与临床、超微结构和IF表现联系起来。为了评估新的治疗方案，我们将在体外进行纤毛发生，并在突变的呼吸道细胞中应用药物基因治疗。

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized bychronic airway disease, randomization of left-right body asymmetry and male infertility due todefects of motile cilia/flagella function. The disease often results in permanent lung damageand can progress to respiratory failure. Currently diagnosis mainly relies on demonstration ofultrastructural defects by electron microscopy, which often reveals outer dynein arm (ODA)deficiency. We demonstrated that mutations in DNAH5, encoding an ODA heavy chain, areresponsible for half of all ODA defect cases. With the use of high-resolution immunofluorescenceimaging (IF) we identified in patients with ODA defects two distinct types of DNAH5mislocalization in nasal airway cells, introducing a novel diagnostic tool in PCD. Here, wewant to genetically characterize a large international cohort of PCD patients for presence ofmutations in both of the known PCD genes, DNAH5 and DNAI1. To analyze composition andgeneration of human ODAs we will generate antibodies directed against other yet uncharacterizedODA heavy chains and apply protein techniques. To improve understanding ofthe disease causing mecanisms and diagnosis in PCD we will correlate genetic results withclinical, ultrastructural and IF findings. To evaluate novel therapeutic options we will performin vitro ciliogenesis and apply pharmaco-gene therapy in mutant respiratory cells.