CFAP300 mutation causing primary ciliary dyskinesia in Finland

Abstract

Primary ciliary dyskinesia (PCD) is a rare genetic condition characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair-like organelles protruding out of the cell that are responsible for removal of mucus from the airways and organizing internal organ positioning during embryonic development. PCD is caused by mutations in genes coding for structural or assembly proteins in motile cilia. Thus far mutations in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported, thus highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics. In this study, we identified a recurrent loss-of-function mutation c.198_200delinsCC in CFAP300 causing lack of the protein product. PCD patients homozygous for the identified CFAP300 mutation have immotile airway epithelial cilia associated with missing dynein arms in their ciliary axonemes. Furthermore, using super resolution microscopy we demonstrate that CFAP300 is transported along cilia in normal human airway epithelial cells suggesting a role for CFAP300 in dynein complex transport in addition to preassembly in the cytoplasm. Our results highlight the importance of CFAP300 in dynein arm assembly and improve diagnostics of PCD in Finland.

Keywords: CFAP300; diagnostics; dynein arm preassembly; motile cilia; primary ciliary dyskinesia.

FIGURE 1

Segregation of CFAP300 mutations. Patients I-1 and II-1 were homozygous for the mutation while parents were heterozygous. (A). Patient I-1 family with carrier parents and one carrier brother and two unaffected siblings. The mutation site is indicated in carrier and unaffected siblings and in patient I-1. (B). Patient II-1 family with carrier parents and brother. (C). Sanger sequencing result for patient III-1.

FIGURE 2

The effect of the CFAP300 variant on protein function. (A). The CFAP300 variant (C).198_200delinsCC results in premature stop codon at 76 aa and protein product. (B). Clustal Omega alignment of the control and PCD patient CFAP300 protein sequence. (C). Structural changes in the CFAP300 protein folding based on Phyre2. (D). Cytoplasmic localization of the CFAP300 protein product appears missing in the PCD patient (patient II-1). αTubulin was used as a marker for cilia and Dapi as a nuclear stain. Scale bar is 10 µm.

FIGURE 3

Localization of axonemal proteins in airway cilia of a control and PCD patient (patient II-1) with CFAP300 mutations. (A). High number of cilia cross sections were present in the nasal brushing sample of a PCD patient with CFAP300 c.198_200delinsCC mutations, but the orientation of the basal foot (red arrows) was disorganized compared to control. (B). The main finding in TEM of cilia cross sections in a patient with the CFAP300 variant was lack of dynein arms (black arrows). (C). ODA (DNAH5 and DNAI1) and IDA (DNAH7) proteins were missing from cilia in the patient airway epithelial cilia, while strong staining is present in the control sample. Radial spoke protein RSPH4A is present in the patient cilia. αTubulin was used as a marker for cilia and Dapi as a nuclear stain. Scale bar is 10 µm.

FIGURE 4

CFAP300 is transported along the motile airway epithelial cilium. (A). In normal airway epithelial cells most of the CFAP300 protein is localized in the cytoplasm, but some of the protein appears to be transported into the cilium. (B). No CFAP300 staining was detected in the patient airway epithelial cells.