Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the “g” inner dyneins (DHC7 and DHC3) and the “d” inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.

Each fraction was resuspended in 50 μL 1% acetonitrile/1% formic acid. 5 μL was analyzed by LC-MS (HCD for MS/MS) with a Dionex RSLCnano HPLC coupled to a Velos Pro OrbiTrap mass spectrometer (Thermo Scientific) using a 2h gradient. Peptides were resolved using 75 μm x 25 cm PepMap C18 column (Thermo Scientific).

Data Analysis

All MS/MS samples were analyzed using Proteome Discoverer 2.1 (Thermo Scientific). The Sequest HT search engine in the Proteome Discover was set to search Chlamydomonas database (Uniprot.org). The digestion enzyme was set as trypsin. The HCD MS/MS spectra were searched with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 20 ppm. Oxidation of methionine was specified as a variable modification, while carbamidomethyl of cysteine and TMT labeling was designated at lysine residues or peptide N-termini were specified in Proteome Discoverer as static modifications.

MS/MS based peptide and protein identifications and quantification results was initially generated in Proteome Discover 2.1 and later uploaded to Scaffold (version Scaffold_4.8.2 Proteome Software Inc., Portland, OR) for final TMT quantification and data visualization. Normalized and scaled protein/peptide abundance ratios were calculated against the abundance value of the four wild-type controls. Peptide identifications were accepted if they could be established at greater than 80.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.