The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

Abstract

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5 . Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

“…Imaging human cilia function. Cilia beat frequency was of cells on Transwell membranes was measured using a Nikon Ti inverted microscope enclosed in a temperature controlled environmental chamber held at 37 °C, using phase contrast and Hoffman modulation contrast lenses 20x (Plan Fluoro, Nikon) or 40x (NAMC3, Nikon) as described 92,104 . Images were recorded from at least 5 fields of each Transwell insert (24-well) using a high-speed video CMOS camera and processed using Sisson-Ammons Video Analysis software (Ammons Engineering).…”

“…Transport of beads on the surface of cultured airway cells was performed as previously described 104 . Fluorescent beads (Fluoresbrite, 2 mm diameter, Polysciences, #18338-5) were diluted 1:500 in PBS.…”

“…Images were capture at 10 frames per sec for 5 sec per image, for a total of 50 frames per image. Data were imported to ImageJ/FIJI software for analysis using Trackmate (v. 5.2.0) and the Linear Motion LAP Tracker and Track Statistics applications in ImageJ as described 104 .…”

“…For proteomic analysis, the cilia were collected in buffer (30 mM HEPES, 25 mM NaCl 2 , 5 mM MgSO 4 , 1 mM EGTA, 0.1 mM EDTA, 0.1 mM DTT, pH 7.3) and then the pellet was snap frozen on dry ice. Protein preparation and LC-MS analysis was performed as previously described 104 . MS/MS samples were normalized and analyzed using Proteome Discoverer 2.4 (Thermo Fisher Scientific).…”

“…Cilia isolation. For mass spectroscopy, ciliary axonemes were isolated from cultured airway cells by application of cilia buffer with minor modifications 104 . Cells cultured in 6-12 inserts of 12-well size Transwell, were washed with PBS then incubated for 1 min with 100 µL/well of deciliation buffer (20 mM Tris, 30 mM NaCl, 10 mM CaCl2, 1 mM EDTA, pH 7.5) with 0.1% Triton-X100 (Surfact-Amps X-100, Thermo Scientific #28314), and freshly made 1 mM dithiothreitol, 0.05 2mercaptoethanol, and proteinase inhibitor cocktail, Sigma #P2714) for 1 min on a vigorously moving rocker with orientation of the plate changed every 15 secs at RT.…”