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Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia

Abstract

Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.

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Figure 1: CCNO mutations cause a chronic airway disease with progressive loss of respiratory function.
Figure 2: TEM photographs of respiratory epithelial cells of individuals with CCNO mutations show severe reduction in the numbers of motile cilia and basal bodies at the apical cell region and mislocalization of basal bodies and rootlets within the cytoplasm.
Figure 3: MMC generation is markedly reduced in CCNO-mutant respiratory cells.
Figure 4: Ccno acts downstream of mcidas.
Figure 5: Cnno morphants show a reduced number of extending cilia and the presence of undocked centrioles.

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Acknowledgements

We thank the affected individuals and their families for participating in this study and the German patient support group Kartagener Syndrom und Primaere Ciliaere Dyskinesie. We thank C. Westermann from the Pathology Department of the University of Münster and K. Sutter and C. Kopp from the Myological Laboratory of the University of Freiburg for electron microscopy assistance. We thank L. Overkamp, M. Herting, S. Helms, C. Vorspohl, B. Fukas and F.-J. Seesing for their technical assistance. We thank M. Joens and S. Dunn in the Waitt Advanced Biophotonics Center Core at the Salk Institute for assistance with electron microscopy. We thank the Faculty of Medicine, Health Sciences Center, Kuwait University and Ministry of Health in Kuwait for their assistance. This work was funded by Dubai Harvard Foundation for Medical Research Collaborative Research Grant (F.S.A.), General Facility Grant SRUL02/13 from the Health Science Center, Kuwait University, the Deutsche Forschungsgemeinschaft (DFG Om 6/4), the Interdisziplinaeres Zentrum für Klinische Forschung (IZKF Om2/009/12) Muenster (H. Omran), the European Community's Seventh Framework Programme FP7/2009 under grant agreement 241955, SYSCILIA (H. Omran) and BESTCILIA under grant agreement 305404 (H. Omran), the Schroeder Stiftung (H. Omran) and Kindness for Kids (H. Omran). Work reported here was supported by a grant, GM096021, to C.K.

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H. Omran designed the study. H. Omran, C.K. and J.W. wrote the paper. J.W. and H. Olbrich performed mutation analysis and prepared the figures. D.A.A.-M., H.E.S. and F.S.A. performed autozygosity mapping and mutational analysis in the Kuwaiti family and identified the CCNO mutation. D.A.A.-M. provided clinical data, the lung function analysis and nasal brushings of the Kuwaiti patients. N.T.L. and G.W.D. performed human protein chemistry experiments. P.P. and T.M. performed experiments with antibodies to Ccno in mouse tissue. C.K., C.-T.C. and L.M. performed Xenopus experiments. G.K. provided the electron microscopy pictures. C.W., B.H.A., M.J., M.B., M.G., S.S.-G., T.Z., C.K.-R., E.H. and H. Omran provided clinical data.

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Correspondence to Heymut Omran.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Pedigree and sequence files of the Kuwaiti family OP-1777.

Supplementary Figure 2 Homozygosity by descent mapping on chromosome 5.

The four affected, shown in the left four columns share a homozygous region (black) from 50,317,612 -65,419,300 bp. CCNO lies within that interval (Arrow) (yellow: heterozygous stage).

Supplementary Figure 3 ClustalW alignment of CCNO protein sequences of Homo sapiens, Mus musculus, Xenopus laevis, Oryzias latipes.

The position of the missense mutation resulting in exchange of an evolutionary conserved aminoacid residue is marked in blue c.638T>C; p.Leu213Pro.

Supplementary Figure 4 Pedigree and sequence files of OP-92.

The affected person show two mutations (a and b) in compound heterozygous status.

Supplementary Figure 5 Pedigree and sequence files of OP-151.

Supplementary Figure 6 Pedigree and sequence files of OP-642.

Supplementary Figure 7 Pedigree and sequence files of OP-857.

Supplementary Figure 8 Pedigree and sequence files of OP-971.

Supplementary Figure 9 Pedigree and sequence files of OP-1246.

Supplementary Figure 10 Pedigree and sequence files of OP-1367.

Supplementary Figure 11 Pedigree and sequence files of OP-1784.

Supplementary Figure 12 Pedigree and sequence file of BEL-1213.

Supplementary Figure 13 Xenopus data.

We studied the effects of morpholino-based knock-down of ccno on MMC generation in frog skin. The skin of ccno-morphants contained a normal number of multiciliated, mucus-secreting and proton secreting cells, indicating that ccno does not affect cell fate. Multiciliated cells (CCs) outer cells (OCs) and proton secreting cells (PSCs).

Supplementary Figure 14 Average number of centrioles per multiciliated cell in the skin of controls and ccno morphants.

Flag-ccno and MT-ccno RNAs lead to increased number of centrioles (basal bodies) per multiciliated cell.

Supplementary Figure 15 Centriole assembly.

(a-g) Xenopus embryos were injected at the two-four cells stage with RNAs encoding sass6-RFP, mRFP and cep152-GFP alone (control, a-b) or with mcidas-hgr RNA (c-d) or with both mcidas-hgr and the ccno-MO (e-f). Embryos were treated with Dexamethasone at stage 11 and then fixed 9 hrs later. Shown are confocal images of the skin (a-f). The average number of sass6-RFP and cep152-GFP foci per cell based on at least 240 cells taken from at least 8 embryos is presented (h) (h-n) Same experimental design as above, except that the tracer RNAs used were sass6-RFP/mRFP and deup1-GFP. The average number of sass6-RFP and deup1-GFP foci per cell based on at least 290 cells taken from 10 embryos is presented (n). Scale bar (10 microns) in panel a applies to all images. Error bars, standard deviation. Values obtained with mcidas-hgr plus ccno-morpholino differ in all cases significantly based on a two-tailed t-test from those obtained with mcidas-hgr alone (p<0.001).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1–4 (PDF 2384 kb)

Supplementary Video 1

Supplementary Video 1. High speed video microscopy (HVMA) of respiratory epithelial cells of affected individual OP-971 after in vitro ciliogenesis. (AVI 15106 kb)

Supplementary Video 2

Supplementary Video 2. High speed video microscopy (HVMA) of respiratory epithelial cells of affected individual OP-1246II3 after in vitro ciliogenesis (spheroid 1). (AVI 30148 kb)

Supplementary Video 3

Supplementary Video 3. High speed video microscopy (HVMA) of respiratory epithelial cells of affected individual OP-1246II3 after in vitro ciliogenesis (spheroid 2). (AVI 30148 kb)

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Wallmeier, J., Al-Mutairi, D., Chen, CT. et al. Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia. Nat Genet 46, 646–651 (2014). https://doi.org/10.1038/ng.2961

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