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

导致原发性纤毛运动障碍 (PCD) 的连接蛋白-动力蛋白调节复合物缺陷的分子特征

• 批准号: 433807262
• 负责人: Dr. Heike Olbrich
• 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/433807262?language=en
• 关键词: Molecular; characterization; defects; nexin; dynein

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.

原发性纤毛运动障碍（PCD）是一种具有常染色体隐性遗传模式的遗传异质性疾病。很少观察到其他遗传模式，例如 X 连锁遗传。 PCD 的特点是反复发生气道感染，导致进行性肺损伤。这些慢性感染是由呼吸道上皮内衬的多个运动纤毛功能障碍引起的，从而导致粘液纤毛清除能力降低。我们之前证明，编码 CCDC39 和 CCDC40 的 N-DRC 相关基因的突变会导致明显的纤毛跳动异常以及通过电子显微镜 (TEM) 和免疫荧光分析 (IF) 检测到的特征性超微结构缺陷。相比之下，编码 N-DRC 成分（例如 GAS8、CCDC164 或 CCDC65）的基因突变通常只会导致纤毛跳动和超微结构的非常微妙的异常。始终如一的是，标准诊断工具不容易识别这些 PCD 变体。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的 PCD 诊断标准。因此，迫切需要扩大我们对人类 N-DRC 和 N-DRC 相关复合物的组成的了解，并改进遗传分析，以造福迄今为止诊断不足的患者。在之前的项目中，我们已经针对 N-DRC 相关睫状体缺陷进行了 IF 筛查。我们根据染色模式识别出 149 名患有孤立性 N-DRC 或 N-DRC 相关缺陷的患者。我们采用包括靶向组测序和全外显子组测序 (WES) 在内的逐步方法，能够识别 52 名个体的潜在遗传缺陷，从而使 97 名个体在基因水平上未得到诊断。在这里，我们的目标是确定 97 名未经基因诊断的个体的潜在遗传缺陷。我们计划对样本进行 WES 三重分析，这些样本已经通过诊断靶向面板测序方法进行了分析。全基因组分析 (WGS) 将作为三重分析应用于 40 个家族，尽管之前进行了 WES 分析，但这些家族在遗传上仍未得到解决。计划使用 RNAseq 对来自 PCD 患者和对照的 40 个样本进行三次转录分析。结果将与 WES 尤其是 WGS 获得的结果相关联，以对已识别的基因和遗传变异进行功能评估。将通过对照和突变纤毛中的 IF 和蛋白质相互作用研究来评估候选基因的纤毛定位和与轴丝成分的相互作用。由于我们预计只有细微的跳动缺陷，因此将评估气液界面（ALI）上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/表型关联，以改进诊断程序并促进识别具有 N-DRC 和 N-DRC 相关缺陷的个体，从而造福于患者。