CFAP300 Loss-of-Function Mutations with Primary Ciliary Dyskinesia: Evidence from Ex Vivo and ALI Cultures

Abstract

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach, we integrated molecular genetic testing, transmission electron microscopy, the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein, the complete loss of outer and inner dynein arms and immotile cilia. Air-liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating, contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia, underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD, offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows.

Keywords: CFAP300; air–liquid interface culture; immunofluorescence; primary ciliary dyskinesia; transmission electron microscopy.

Figure 1

Molecular and ultrastructural analysis in PCD patients (PCD1, PCD2, PCD3) and a healthy donor (HC1). (A) Sanger sequencing results for the CFAP3000 gene mutation region. (B) Transmission electron microscopy of a cilia. Arrows and circles indicate the presence of ODA and IDA in a healthy donor and their absence in three PCD patients. Scale bar 50 nm. (C) Transmission electron microscopy of a spermatozoon from a PCD2 patient. Arrows and circles indicate the absence of ODA and IDA. Scale bar 50 nm.

Figure 2

Characterization of nasal epithelial cells and ALI cultures. (A) A phase-contrast image of the cells isolated during brush biopsy at the zero passage. Scale bar 200 μm. (B) Representative fluorescence images of nasal epithelial cells stained for epithelial cell markers (cytokeratin 5 and TP63). Scale bar 100 μm. (C) Representative fluorescence images of cilia (β-tubulin) in ALI culture at 24 d of ciliogenesis in vitro. Nuclei were stained with DAPI (blue). Scale bar 10 μm.

Figure 3

Fluorescence images of cilia in ALI cultures of patients with loss-of-function mutations in the CFAP300 gene and a healthy donor (HC1) stained for CFAP300 and β-tubulin. Nuclei were stained with DAPI (blue). Scale bar 25 μm.