The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes
Amjad Horani, Deepesh Kumar Gupta, Jian Xu, Huihui Xu, Lis del Carmen Puga-Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody
Amjad Horani, Deepesh Kumar Gupta, Jian Xu, Huihui Xu, Lis del Carmen Puga-Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody
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Resource and Technical Advance Genetics Pulmonology

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.

Authors

Amjad Horani, Deepesh Kumar Gupta, Jian Xu, Huihui Xu, Lis del Carmen Puga-Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody

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Figure 2

The effect of NULL and MIS allele dose on airway cilia function and ultrastructure.

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The effect of NULL and MIS allele dose on airway cilia function and ultr...
(A) Schematic of primary culture airway cells and ex vivo isolated trachea mounts used for analysis of cilia function by cilia beat frequency (CBF), transport, and ultrastructure by transmission electron microscopy (TEM). (B) CBF of cultured cells from WT/WT and WT/NULL littermates, with mean ± SEM shown as 17.47 ± 0.80 Hz and 16.0 ± 0.47 Hz, respectively. (C) CBF of cultured cells from WT/WT, WT/MIS, MIS/MIS, and MIS/NULL littermates, with mean ± SEM frequency of 16.22 ± 0.29 Hz, 16.39 ± 0.33 Hz, 7.25 ± 0.53 Hz, and 0.06 ± 0.06 Hz, respectively. CBF was assessed in 5 random areas per Transwell; 5 Transwell cultures were performed for each genotype per experiment. n = 2–4 independent experiments comprising unique mice. (D and E) Mucociliary transport speed and bead displacement on the surface of isolated trachea from WT/WT, WT/MIS, and MIS/MIS mice, with mean ± SEM speeds shown as 39.04 ± 5.02 Hz, 39.29 ± 2.86 Hz, and 7.16 ± 0.45 m/sec, respectively. Mean bead displacement over 5 seconds is shown as 89.13 ± 4.42 μm, 86.82 ± 4.80 μm, 26.00 ± 1.36 μm, respectively (n = 8–10 animals per genotype). (F) Representative TEM images of airway cilia from WT/WT, MIS/MIS, and MIS/NULL mice. Arrows indicate presence (magenta) or absence (green) of outer and inner dynein arms (ODA, IDA). Scale bar: 100 nm. (G) Quantitation of outer dynein motor protein complexes detected by TEM of WT/WT, MIS/MIS, and MIS/NULL cilia cross section, with mean ± SEM shown number of ODA 6.0 ± 0.4, 3.8 ± 0.2, and 0.8 ± 0.4, respectively. (H) Quantification of inner dynein arms in ciliary axonemes from cultured tracheal epithelial cells from WT/WT, MIS/MIS, and MIS/NULL, with mean ± SEM shown number of IDA 3.8 ± 04, 0.3 ± 0.0, and 0.3 ± 0.2, respectively. n = 4 animals per genotype, n = 5–10 cross sections per genotype. *P < 0.05, **P < 0.01, ***P < 0.001 determined using Kruskal-Wallis test with Dunn’s multiple-comparison test.