Molecular characterization of defects of the nexin-dynein regulatory complex causing Primary Ciliary Dyskinesia (PCD)

原发性纤毛运动障碍（PCD）是一种遗传异质性疾病，具有常染色体隐性遗传模式。很少观察到其他遗传模式，如x连锁遗传。PCD的特点是反复气道感染，引起进行性肺损伤。这些慢性感染是由呼吸上皮内的多个活动纤毛功能障碍引起的，结果是纤毛黏液清除减少。我们之前已经证明，编码CCDC39和CCDC40的N-DRC相关基因突变导致明显的纤毛跳动异常和特征性的超微结构缺陷，这些缺陷是通过电子显微镜（TEM）和免疫荧光分析（IF）检测到的。相比之下，编码N-DRC成分的基因如GAS8、CCDC164或CCDC65的突变通常只会导致纤毛跳动和超微结构的非常细微的异常。一贯地，这些PCD变体不容易被标准诊断工具识别。患者可能在诊断设置中被“忽视”，因为受影响个体的细微纤毛缺陷不符合当前PCD的诊断标准。因此，迫切需要扩大我们对人类N-DRC和N-DRC相关复合物组成的了解，并改进遗传分析，以造福迄今未被诊断的患者。在之前的项目中，我们已经对N-DRC相关的纤毛缺陷进行了中频筛查。我们根据染色模式确定了149例孤立的N-DRC或N-DRC相关缺陷患者。我们能够使用包括靶向小组测序和全外显子组测序（WES）在内的分步方法识别52个人的潜在遗传缺陷，留下97个人在遗传水平上未被诊断。在这里，我们的目标是在那些基因未诊断的97个人中确定潜在的遗传缺陷。我们计划对样本进行WES三重奏分析，这些样本已经通过诊断靶向小组测序方法进行了分析。全基因组分析（WGS）将作为三重奏分析应用于40个家庭，这些家庭在遗传上没有得到解决。计划使用RNAseq对来自PCD患者和对照者的40份样本进行转录分析。结果将与WES，特别是WGS的结果相关联，用于鉴定基因和遗传变异的功能评估。候选基因将通过控制和突变纤毛的IF和蛋白质相互作用研究来评估纤毛定位和与轴突成分的相互作用。由于我们预计只有细微的跳动缺陷，我们将评估生长在气液界面（ALI）的呼吸上皮细胞的纤毛粘液清除能力。将进行仔细的基因型/表型相关性，以改进诊断程序，并促进N-DRC和N-DRC相关缺陷个体的识别，从而使患者受益。

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder with an autosomal-recessive inheritance pattern. Only rarely other modes of inheritance such as X-linked transmission are observed. PCD is characterized by recurrent airway infections causing progressive lung damage. These chronic infections are triggered by dysfunction of multiple motile cilia lining the respiratory epithelium and as consequence decreased mucociliary clearance. We previously demonstrated that mutations in N-DRC associated genes encoding CCDC39 and CCDC40 result in distinct ciliary beating abnormalities and characteristic ultrastructural defects detected by electron microscopy (TEM) and immunofluorescence analyses (IF). In contrast, mutations in genes encoding for N-DRC components such as GAS8, CCDC164 or CCDC65 frequently result in only very subtle abnormalities of ciliary beating and ultrastructure. Consistently, those PCD variants are not readily identified by standard diagnostic tools. Patients might be “overlooked” in the diagnostic setup because the subtle ciliary defects in affected individuals do not meet current diagnostic criteria for PCD. Therefore, there is urgent need to expand our knowledge about the composition of the human N-DRC and N-DRC related complexes and to improve genetic analyses for the benefit of so far underdiagnosed patients. In the previous project, we already performed an IF screen focusing on N-DRC related ciliary defects. We identified 149 patients with isolated N-DRC or N-DRC associated defects based on the staining pattern. We were able to identify the underlying genetic defect in 52 individuals using a step wise approach including targeted panel sequencing and whole exome sequencing (WES), leaving 97 individuals undiagnosed on genetic level. Here, we aim to identify the underlying genetic defect in those genetically undiagnosed 97 individuals. We plan to perform WES trio analyses on samples, which have already been analyzed by the diagnostic targeted panel sequencing approach. Whole genome analyses (WGS) will be applied as trio analysis to 40 families which are genetically unsolved despite previous WES analyses. Transcript analyses using RNAseq is planned for 40 samples derived from PCD patients and controls in triplicates. Results will be correlated to findings obtained by WES and especially WGS for functional evaluation of identified genes and genetic variants. Candidate genes will be evaluated for ciliary localization and interaction with axonemal components by IF and protein interaction studies in control and mutant cilia. Since we expect only subtle beating defects, mucociliary clearance capacity of respiratory epithelial grown at air-liquid interface (ALI) will be assessed. Careful genotype/phenotype correlation will be performed to enable the improvement of diagnostic procedures and to facilitate identification of individuals with N-DRC and N-DRC associated defects for the benefit of patients.