Molecular characterization of defects of the central pair complex of cilia causing Primary Ciliary Dyskinesia (PCD)

导致原发性纤毛运动障碍 (PCD) 的纤毛中央对复合体缺陷的分子特征

• 批准号: 269498644
• 负责人: Professor Dr. Heymut Omran
• 金额: --
• 依托单位: Klinik für Kinder- und Jugendmedizin - Allgemeine Pädiatrie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/269498644?language=en
• 关键词: Molecular; characterization; defects; central; pair

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic airway disease, randomization of left-right (LR) body asymmetry and male infertility due to defects of motile cilia/flagella function. Currently PCD diagnosis mainly depends on demonstration of ultrastructural defects of respiratory motile cilia. In contrast to most forms of PCD, defects of the central pair (CP) complex cannot be identified by conventional TEM and neither is HSVM suitable for confirming a diagnosis. Therefore PCD types with defects of the CP are difficult to diagnose.Recently we demonstrated that several forms of PCD can be diagnosed by immunofluorescence microscopy (IF). The analysis of proteins associated with the CP revealed that SPEF2 is not detectable by IF in the cilia of HYDIN mutant respiratory cells. Genetic analysis of individuals in which SPEF2 could not be detected in respiratory cilia revealed the identification of SPEF2 as PCD-causing gene as well as HYDIN mutations in additional 15 individuals.Due to the fact that the newly identified mutations in HYDIN, SPEF2 and STK36 as well as most likely in all genes encoding parts of the CP complex, do not lead to a situs inversus or situs ambiguous and that structural defects of the CP apparatus are not regularly detectable with routine TEM, additional knowledge about the composition of the CP complex is of great importance to improve PCD diagnostic procedures and patient care. The objectives of this current proposal are therefore as follows: 1. Characterization of the human CP composition including novel components of the CP complex2. Translation of the results into the clinics by development of novel diagnostic tools such as immunofluorescence (IF) microscopy to identify specific CP complex defects3. Identification and characterization of novel mutations in genes encoding elements of the CP complexes in human PCD individuals4. Functional characterization of novel CP defects using Air-liquid interface (ALI) cell culture of primary respiratory epithelial cells to study cilia morphology, ciliary beat pattern, and mucociliary transport by particle tracking analysis 5. Genotype/phenotype correlation of PCD patients with defective proteins of the CP complexFollowing this strategy we expect, that we will 1) succeed to be able to detect several CP components and also novel CP complex defects in PCD patients and 2) succeed to characterize the composition of the CP complex in human cilia and cell type specific differences in more detail. These data will improve our understanding of normal cilia biology and which will give additional insights into the pathogenesis of PCD, thus improving diagnosis and genetic counseling of affected individuals. In addition we hope that – based upon this knowledge – we will be able to develop novel therapeutic strategies for PCD.

原发性纤毛运动障碍（PCD）是一种遗传异质性疾病，其特征是慢性气道疾病、随机左右（LR）身体不对称以及由于纤毛/鞭毛运动功能缺陷导致的男性不育。目前PCD的诊断主要依靠呼吸运动纤毛超微结构缺陷的显示。与大多数形式的PCD相反，中心对（CP）复合物的缺陷不能通过传统的TEM识别，HSVM也不适合确诊。因此，有CP缺陷的PCD类型很难诊断。最近，我们证明了几种形式的PCD可以通过免疫荧光显微镜（IF）诊断。与CP相关的蛋白分析显示，在HYDIN突变体呼吸细胞的纤毛中，IF检测不到SPEF2。对呼吸纤毛中未检测到SPEF2的个体进行遗传分析，发现SPEF2是导致pcd的基因，另外15个个体存在HYDIN突变。由于新发现的HYDIN、SPEF2和STK36突变，以及很可能编码CP复合物部分的所有基因的突变，不会导致位置反转或位置模糊，并且常规TEM无法定期检测到CP装置的结构缺陷，因此关于CP复合物组成的额外知识对于改善PCD诊断程序和患者护理非常重要。因此，本提案的目标如下：人类CP组成的表征，包括CP复合物的新组分2。通过开发新的诊断工具（如免疫荧光（IF）显微镜）将结果转化为临床，以识别特定的CP复合物缺陷3。人类PCD个体CP复合物元件编码基因新突变的鉴定和表征利用气液界面（ALI）细胞培养原代呼吸道上皮细胞，研究纤毛形态、纤毛跳动模式和纤毛黏液运输的功能表征根据这一策略，我们预计，我们将1)成功地能够检测PCD患者的几种CP成分和新的CP复合物缺陷；2)成功地表征人类纤毛中CP复合物的组成和细胞类型特异性差异。这些数据将提高我们对正常纤毛生物学的理解，并将为PCD的发病机制提供更多的见解，从而改善受影响个体的诊断和遗传咨询。此外，我们希望-基于这些知识-我们将能够开发新的治疗策略PCD。