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

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

Primary ciliary dyskinesia (PCD) is a chronic airway disease caused by impaired mucociliary clearance. It exhibits genetic heterogeneity and can manifest with laterality defects and male infertility. Diagnosing PCD is challenging and relies on high-speed video microscopy analysis (HVMA), transmission electron microscopy (TEM), high-resolution immunofluorescence (IF), and genetic analysis. PCD due to radial spoke (RS) dysfunction is particularly difficult to diagnose because 1) it does not result in situs anomalies, 2) RS defects are class II defects, meaning TEM findings alone cannot confirm PCD, and 3) subtle ciliary beating defects require HVMA expertise. This highlights the need for innovative diagnostic methods to detect RS-related PCD and improve treatment options. During the initial funding period, we analyzed genetic data from 1,236 individuals with PCD across 19 countries via the ERN-LUNG PCD Registry. We identified regional clusters of distinct DNA variants and significant genotype-phenotype correlations, particularly for RSPH genes. Our findings showed that RS gene defects (class II defects) do not exhibit hallmark ultrastructural abnormalities detectable by TEM, making HVMA and in vitro ciliogenesis essential for accurate diagnosis. A detailed analysis of our cohort led to the identification of several new pathogenic variants in previously reported RS complex genes. Specifically, we detected hotspot variants in RSPH9, RSPH1, and RSPH4A, as well as a common founder variant in RSPH9 in Arab-Bedouin PCD families. Additionally, pathogenic variants in RSPH3 and DNAJB13 were found, along with newly identified RS genes ROPN1L and RTDR1. For the first time, we demonstrated that RSPH1 and RSPH9 variants cause male infertility and showed that RSPH6A is a sperm-specific RS component, whose localization in sperm flagella depends on RSPH9. These research topics remain ongoing, and we aim to publish them soon. To avoid overlooking PCD-affected individuals, we must deepen our understanding of the RS complex's structure, composition, and function across various human ciliary tissues. Our preliminary data contribute to a better understanding of RS composition in human sperm, which is crucial for refining diagnostic methods and improving PCD management. Thus, the aims of this study are outlined as follows: 1. Identification of novel genetic defects in more than 15 RS-associated components linked to PCD 2. Molecular characterization of genetic defects (known and novel) for RS function and composition using our Biobank and antibody library 3. Genotype-phenotype correlation of PCD variants with defective RS complex proteins 4. Characterization of protein interactions between RS and related components 5. Comparison of the RS complex in human sperm vs. respiratory cilia 6. Validation of RS candidate proteins in motile cilia of Schmidtea via expression and/or knockdown analyses in control and mutant tissue.

• DFG Programme: Research Grants