DOI: 10.5061/dryad.dv41ns1v6
title: Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia

description: 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&gt;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.
description: 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).
description: <i>Data Analysis
 </i> 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
 <i>Chlamydomonas </i>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.