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

原发性纤毛运动障碍（PCD）是一种遗传性异质性疾病，其特征是由于纤毛粘膜清除减少而导致的慢性破坏性气道疾病。根据PCD变异，该疾病可能与侧性缺陷和男性不育有关。38个基因已被证实与PCD的发病机制有关。这些基因中的大多数已经被我们的实验室识别和表征。PCD的诊断是困难的，因为通过透射电子显微镜（TEM）记录的超微结构缺陷只能检测到主要异常，如外动力臂或管状组织的缺陷。然而，基因缺陷导致的PCD与径向辐条（RS）缺陷缺乏横向缺陷和标志缺陷。因此，诊断主要依赖于少数已知的导致PCD合并RS缺陷的基因缺陷的遗传发现和/或仅在专业中心提供的异常纤毛搏动模式的文件。使用高速视频显微镜（HSVM），我们发现RS缺陷导致非常细微的呼吸纤毛跳动异常，其振幅降低，形成僵硬的跳动模式。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PCD个体的纤毛轴突中缺乏RS头部成分，这些PCD个体具有隐性错意以及RSPH4A， RSPH1和RSPH9的功能缺失突变。通过突变分析检测不确定意义的DNA变异（如错义等位基因）的致病性验证尤为重要。在这里，我们希望详细分析控制和突变纤毛轴突中人类RS结构的组成，以了解PCD个体的正常组成和提高诊断水平。除了表征已知的遗传缺陷，我们将确定新的遗传缺陷负责异常RS组成。在我们未发表的初步工作中，我们已经发现了两个编码RS成分的新基因的突变。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：已知和新的RS缺陷的遗传特征以改进基因检测。RS功能和组成的遗传缺陷（已知的和新的）的分子特征生成对照和突变诱导多能干细胞（iPSCs）并将这些iPSCs分化为呼吸道上皮细胞以验证鉴定的遗传变异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与相关组分之间选定蛋白相互作用的表征

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