Molecular characterization of defects of the nexin-dynein regulatory complex causing Primary Ciliary Dyskinesia (PCD)

• Applicants: Dr. Heike Olbrich; Professor Dr. Heymut Omran
• Subject Area: Pediatric and Adolescent Medicine
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433807262

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder with an autosomal-recessive inheritance pattern. Only rarely other modes of inheritance such as X-linked transmission are observed. PCD is characterized by recurrent airway infections causing progressive lung damage. These chronic infections are triggered by dysfunction of multiple motile cilia lining the respiratory epithelium and as consequence decreased mucociliary clearance. We previously demonstrated that mutations in N-DRC associated genes encoding CCDC39 and CCDC40 result in distinct ciliary beating abnormalities and characteristic ultrastructural defects detected by electron microscopy (TEM) and immunofluorescence analyses (IF). In contrast, mutations in genes encoding for N-DRC components such as GAS8, CCDC164 or CCDC65 frequently result in only very subtle abnormalities of ciliary beating and ultrastructure. Consistently, those PCD variants are not readily identified by standard diagnostic tools. Patients might be “overlooked” in the diagnostic setup because the subtle ciliary defects in affected individuals do not meet current diagnostic criteria for PCD. Therefore, there is urgent need to expand our knowledge about the composition of the human N-DRC and N-DRC related complexes and to improve genetic analyses for the benefit of so far underdiagnosed patients. In the previous project, we already performed an IF screen focusing on N-DRC related ciliary defects. We identified 149 patients with isolated N-DRC or N-DRC associated defects based on the staining pattern. We were able to identify the underlying genetic defect in 52 individuals using a step wise approach including targeted panel sequencing and whole exome sequencing (WES), leaving 97 individuals undiagnosed on genetic level. Here, we aim to identify the underlying genetic defect in those genetically undiagnosed 97 individuals. We plan to perform WES trio analyses on samples, which have already been analyzed by the diagnostic targeted panel sequencing approach. Whole genome analyses (WGS) will be applied as trio analysis to 40 families which are genetically unsolved despite previous WES analyses. Transcript analyses using RNAseq is planned for 40 samples derived from PCD patients and controls in triplicates. Results will be correlated to findings obtained by WES and especially WGS for functional evaluation of identified genes and genetic variants. Candidate genes will be evaluated for ciliary localization and interaction with axonemal components by IF and protein interaction studies in control and mutant cilia. Since we expect only subtle beating defects, mucociliary clearance capacity of respiratory epithelial grown at air-liquid interface (ALI) will be assessed. Careful genotype/phenotype correlation will be performed to enable the improvement of diagnostic procedures and to facilitate identification of individuals with N-DRC and N-DRC associated defects for the benefit of patients.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Peter Nürnberg