Molecular characterization of radial spoke composition and defects in Primary Ciliary Dyskinesia

原发性纤毛运动障碍的径向辐条成分和缺陷的分子特征

• 批准号: 425347732
• 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
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/425347732?language=en
• 关键词: Molecular; characterization; radial; spoke; composition

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic destructive airway disease due to reduced muco-ciliary clearance. Depending on the PCD variant the disease can be associated with laterality defects and male infertility. 38 genes have been shown to be involved in the pathogenesis of PCD. Most of those genes have been identified and characterized by our laboratory. PCD diagnosis is difficult because ultrastructural defects documented by transmission electron microscopy (TEM) can only detect major abnormalities such as defects of the outer dynein arms or tubular organization. However, gene defects responsible for PCD with radial spoke (RS) defects lack laterality defects and hallmark defects by TEM. Therefore, diagnosis mainly relies on genetic findings of the few known gene defects responsible for PCD with RS defects and/or documentation of abnormal ciliary beat patterns only available in specialized centers.Using high-speed videomicroscopy (HSVM) we have shown that defects of the RS cause very subtle respiratory cilia beating abnormalities with reduced amplitude creating a stiff beat pattern. In addition we demonstrated that the use of high-resolution immunofluorescence microscopy analysis (IF) can aid PCD diagnosis by demonstrating the absence of RS head components from the ciliary axonemes of PCD individuals with recessive missense as well as loss-of function mutations in RSPH4A, RSPH1 and RSPH9. The validation of the pathogenicity of DNA variants of uncertain significance (e.g. missense alleles) detected by mutational analysis is especially important.Here, we want to analyze the composition of human RS structures in detail in control and mutant ciliary axonemes to understand normal composition and improve diagnostics in PCD individuals. Besides characterization of known genetic defects we will identify novel genetic defects responsible for abnormal RS composure. In our unpublished preliminary work we already found mutations in two new genes encoding RS components. Our work will aid PCD diagnostics (HSVM, IF, genetic testing) and we will establish cellular models used for new therapeutic strategies. The objectives of this current proposal are therefore as follows: 1. Genetic characterization of known and novel RS defects to improve genetic testing2. Molecular characterization of genetic defects (known and novel) for RS function and composition 3. Generation of control and mutant induced pluripotent stem cells (iPSCs) and differentiation of those iPSCs to respiratory epithelial cells to validate identified genetic variants4. Genotype/phenotype correlation of PCD variants with defective proteins of the RS complex in comparison to other PCD variants5. Characterization of selected protein interactions between RS and related components

原发性纤毛运动障碍（PCD）是一种遗传异质性疾病，其特征是由于粘膜纤毛清除率降低而导致的慢性破坏性气道疾病。根据PCD变异，该疾病可能与偏侧缺陷和男性不育有关。有38个基因参与了PCD的发病机制。这些基因中的大多数已经被我们的实验室鉴定和表征。PCD诊断是困难的，因为透射电子显微镜（TEM）记录的超微结构缺陷只能检测到主要异常，如外部动力蛋白臂或管状组织的缺陷。然而，负责PCD与径向辐条（RS）缺陷的基因缺陷缺乏偏侧性缺陷和通过TEM的标志缺陷。因此，诊断主要依赖于少数已知的基因缺陷的遗传结果，负责PCD与RS缺陷和/或文件异常纤毛跳动模式，只有在专门centers.Using高速视频显微镜（HSVM），我们已经表明，RS的缺陷导致非常微妙的呼吸纤毛跳动异常，幅度降低，创造一个僵硬的跳动模式。此外，我们证明了使用高分辨率免疫荧光显微镜分析（IF）可以帮助PCD诊断，通过证明从PCD个体的纤毛轴丝与隐性错义以及RSPH 4A，RSPH 1和RSPH 9的功能缺失突变的RS头组件的情况下。通过突变分析检测到的不确定意义的DNA变体（例如错义等位基因）的致病性的验证是特别重要的。在这里，我们要详细分析控制和突变纤毛轴丝中的人类RS结构的组成，以了解正常组成和改善PCD个体的诊断。除了表征已知的遗传缺陷，我们将确定新的遗传缺陷异常RS镇静负责。在我们未发表的初步工作中，我们已经在两个编码RS组分的新基因中发现了突变。我们的工作将有助于PCD诊断（HSVM，IF，基因检测），我们将建立用于新治疗策略的细胞模型。因此，本提案的目标如下：1. 已知和新的RS缺陷的遗传表征，以改善遗传检测2。 RS功能和组成的遗传缺陷（已知和新）的分子表征3. 产生对照和突变体诱导多能干细胞（iPSC）并将这些iPSC分化为呼吸道上皮细胞以验证鉴定的遗传变体4。 与其他PCD变体相比，具有RS复合物缺陷蛋白的PCD变体的基因型/表型相关性5。 RS和相关组分之间的选定蛋白质相互作用的表征