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Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain significance in approximately 30% of tested individuals. We aimed to assess genotype-phenotype correlations in adults with bronchiectasis, clinical suspicion of PCD, and inconclusive whole-exome sequencing results using transmission electron microscopy (TEM) and ciliary image averaging by the PCD Detect software. We recruited 16 patients with VUS in CCDC39 , CCDC40 , CCDC103 , DNAH5 , DNAH5 / CCDC40 , DNAH8 / HYDIN , DNAH11 , and DNAI1 as well as variants in the PCD candidate genes DNAH1 , DNAH7 , NEK10 , and NME5 . We found normal ciliary ultrastructure in eight patients with VUS in CCDC39 , DNAH1 , DNAH7 , DNAH8 / HYDIN , DNAH11 , and DNAI1 . In six patients with VUS in CCDC40 , CCDC103 , DNAH5 , and DNAI1 , we identified a corresponding ultrastructural hallmark defect. In one patient with homozygous variant in NME5 , we detected a central complex defect supporting clinical relevance. Using TEM as a targeted approach, we established important genotype-phenotype correlations and definite PCD in a considerable proportion of patients. Overall, the PCD Detect software proved feasible in support of TEM.

Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in DNAH5 and NME5 , respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.