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

原发性睫状体运动障碍(PCD)是一种遗传性异质性疾病，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。目前PCD的诊断主要依靠显示呼吸运动纤毛的超微结构缺陷。与大多数形式的PCD不同，中心对(CP)复合体的缺陷不能用常规的透射电子显微镜识别，HSVM也不适合确认诊断。因此，伴有CP缺陷的PCD类型很难诊断。最近，我们证明了免疫荧光显微镜(IF)可以诊断几种形式的PCD。对CP相关蛋白质的分析表明，在HYDIN突变的呼吸道细胞的纤毛中，IF不能检测到SPEF2。对呼吸道纤毛中未检测到SPEF2的个体的遗传分析显示，SPEF2是PCD的致病基因，另外还有15名个体存在HYDIN突变。由于新发现的HYDIN、SPEF2和STK36以及最有可能在CP复合体的所有编码基因中的突变不会导致位置反转或位置模糊，而且CP装置的结构缺陷不能用常规的透射电子显微镜定期检测到，因此关于CP复合体的组成的额外知识对于改进PCD的诊断程序和患者护理非常重要。因此，本提案的目的如下：1.鉴定包括CP络合物的新组分的人CP组合物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复合体缺陷3。人类PCD个体CP复合体元件编码基因新突变的鉴定和特征4。利用原代呼吸道上皮细胞的气液界面(ALI)细胞培养来研究纤毛形态、纤毛节拍模式和粘液纤毛运输的功能鉴定5.具有CP复合体缺陷蛋白的PCD患者的基因型/表型相关性遵循这一策略，我们将1)成功地检测到PCD患者中的几个CP组分和新的CP复合体缺陷，2)成功地更详细地表征人类纤毛的CP复合体的组成和细胞类型的特异性差异。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，从而改善对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--我们将能够为PCD开发新的治疗策略。

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.