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

原发性纤毛运动障碍(PCD)是一种遗传异质性疾病，其特征是由于粘液纤毛清除减少而导致的慢性破坏性呼吸道疾病。根据PCD变异的不同，这种疾病可能与偏侧缺陷和男性不育有关。已有38个基因参与了PCD的发病过程。这些基因中的大多数已经由我们的实验室识别和表征。PCD的诊断很困难，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的异常，如外动力蛋白臂或肾小管组织的缺陷。然而，通过透射电子显微镜观察发现，导致PCD伴径向辐条(RS)缺陷的基因缺陷缺乏偏侧性缺陷和标志性缺陷。因此，诊断主要依赖于少数已知的导致PCD伴RS缺陷的基因缺陷的遗传学发现和/或仅在专门中心才有的异常纤毛搏动模式的记录。使用高速视频显微镜(HSVM)，我们已经证明RS的缺陷导致非常微小的呼吸纤毛搏动异常，幅度降低，产生僵硬的纤毛搏动模式。此外，我们还证明了高分辨率免疫荧光显微镜分析(IF)的使用可以通过显示隐性错义PCD患者的睫状轴丝中缺少RS头部成分以及RSPH4A、RSPH1和RSPH9功能缺失突变来辅助PCD的诊断。通过突变分析检测到的不确定意义的DNA变异(如错义等位基因)的致病性的验证尤为重要。在这里，我们想要详细地分析对照和突变的睫状轴丝的人类RS结构的组成，以了解正常的组成和提高PCD患者的诊断水平。除了已知的遗传缺陷的特征外，我们还将识别导致RS组成异常的新的遗传缺陷。在我们未发表的初步工作中，我们已经在编码RS成分的两个新基因中发现了突变。我们的工作将有助于PCD的诊断(HSVM、IF、基因测试)，我们将建立用于新治疗策略的细胞模型。因此，这一建议的目标如下：1.对已知和新的RS缺陷进行基因表征，以改进基因检测。RS功能和成分的遗传缺陷(已知的和新的)的分子表征3.对照和突变诱导的多能干细胞(IPSCs)的产生和这些IPSCs向呼吸道上皮细胞的分化以验证已识别的遗传变异4。与其他PCD变异相比，PCD变异与RS复合体缺陷蛋白的基因型/表型相关性5。RS与相关组分之间特定蛋白质相互作用的表征

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