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

• Applicant: Professor Dr. Heymut Omran
• Subject Area: Pediatric and Adolescent Medicine
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 425347732

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 In our unpublished preliminary work we already found mutations in two new genes encoding RS components.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

• DFG Programme: Research Grants