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。只有很少的其他模式的遗传，如X连锁传播观察。PCD的特征在于引起进行性肺损伤的反复气道感染。这些慢性感染是由内衬呼吸道上皮的多个运动纤毛功能障碍引起的，结果是粘膜纤毛清除率降低。我们以前证明，N-DRC相关基因编码CCDC 39和CCDC 40的突变导致不同的纤毛跳动异常和特征性的超微结构缺陷检测电子显微镜（TEM）和免疫荧光分析（IF）。相比之下，编码N-DRC组分如GAS 8、CCDC 164或CCDC 65的基因突变通常仅导致纤毛跳动和超微结构的非常细微的异常。因此，这些PCD变体不容易通过标准诊断工具识别。患者可能会被“忽视”的诊断设置，因为在受影响的个人微妙的睫状体缺陷不符合目前的诊断标准的PCD。因此，迫切需要扩大我们对人类N-DRC和N-DRC相关复合物的组成的了解，并改善遗传分析，以使迄今诊断不足的患者受益。在之前的项目中，我们已经进行了IF筛选，重点关注N-DRC相关的睫状体缺陷。我们根据染色模式确定了149例孤立性N-DRC或N-DRC相关缺陷患者。我们能够使用逐步方法（包括靶向面板测序和全外显子组测序（WES））在52个个体中鉴定潜在的遗传缺陷，留下97个个体在遗传水平上未诊断。在这里，我们的目标是确定潜在的遗传缺陷，在这些基因未确诊的97个人。我们计划对已经通过诊断靶向组测序方法分析的样本进行WES三重分析。全基因组分析（WGS）将作为三重分析应用于40个家族，这些家族尽管之前进行了WES分析，但遗传学上仍未解决。计划使用RNAseq对来自PCD患者和对照的40个样品进行转录物分析，一式三份。结果将与WES，特别是WGS获得的结果相关，用于鉴定基因和遗传变异的功能评价。候选基因将通过IF和蛋白质相互作用研究在对照和突变纤毛中评价纤毛定位和与轴丝组分的相互作用。由于我们预期只有轻微的搏动缺陷，因此将评估在气液界面（ALI）生长的呼吸道上皮的粘膜纤毛清除能力。将进行仔细的基因型/表型相关性分析，以改善诊断程序，并促进识别N-DRC和N-DRC相关缺陷的个体，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.