Abstract
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
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Acknowledgements
This work was supported by grants from the European Union (LUPA IP) and from the Police Scientifique Fédérale de Belgique (GENFUNC PAI) (to M.G.), from the Legs Poix from the Chancellerie des Universités, the Assistance Publique-Hôpitaux de Paris (PHRC AOM06053, P060245) and the Agence Nationale pour la Recherche (ANR-05-MRAR-022-01) (to S.A.), the US National Institutes of Health (HD04260, DK072301 and DK075972 (to N.K.) and DK079541 (to E.E.D.)) and by grants from the “Deutsche Forschungsgemeinschaft” DFG Om 6/4, GRK1104, SFB592, and the European Community (EU-CILIA; SYS-CILIA) (to H.O.). A.C.M. is a fellow from the FRIA. N.K. is the Jean and George W. Brumley Professor. Y.M. benefits from a postdoctoral fellowship to study abroad from the Japanese Society for the Promotion of Science (JSPS). We thank the Bobtail breeders for assistance; patients and their family members whose cooperation made this study possible; all referring physicians; the German patient support group “Kartagener Syndrom und Primaere Ciliaere Dyskinesie e.V.”; K. Nakamura and the GIGA-R genomics platform for their contribution to sequencing; E. Ostrander for samples from healthy Old English Sheepdogs; the Unité de Recherche Clinique (URC) Est (AP-HP, Hôpital Saint-Antoine, Paris, France) for support; and A. Heer, C. Reinhard, C. Kopp, K. Sutter, M. Petry, C. Tessmer, A.-M. Vojtek and S. Franz for technical assistance.
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The positional cloning of CCDC39 in the dog was performed by A.-C.M. and G.B. Genome-wide SNP genotyping was conducted at CNG under supervision of M. Lathrop and D.Z. Mining the ciliome databases was conducted by E.E.D. Experiments in the zebrafish were conducted by E.E.D. In situ hybridization in the mouse was conducted by A.K. and H.O. qRT-PCR on human samples was conducted by M. Legendre, P.D., G.M. and H.T. Sequencing of CCDC39 in human subjects, including high-throughput sequencing, was conducted by A.-C.M., G.B., Y.M., A.B.-H., M. Legendre, E.E., P.D., G.M. and H.T. Identifying the p.Glu390SerfsX6 mutation was realized by M. Legendre, P.D., G.M. and H.T. Haplotype analysis to determine founder status of CCDC39 mutations was conducted by M. Legendre and P.D. TEM analysis was conducted by M.J. (dog), E.E. and D.E. (French cohort), and by K.G.N., J.K.M., H.O. and routine laboratories (German cohort). High-resolution immunofluorescence analyses were done by A.B.-H., M.F., J.H. and N.T.L. Immunoblotting analyses were done by A.B.-H. High-speed video analyses were conducted by H.O., N.T.L. and A.B.-H. Monoclonal anti-GAS11 antibody was produced by A.B.-H. and H.Z. Polyclonal anti-GAS11 antibodies were provided by K.H. and R.C. Clinical examination and collection of the canine PCD cases was conducted by F.B., C.C. and S.D. M.G., A.-S.L., N.K., H.O. and S.A. designed experiments, analyzed data and wrote the manuscript. All remaining authors as well as H.O., K.G.N. and J.K.M. examined and contributed samples from individuals with PCD or heterotaxia.
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Supplementary Text and Figures
Supplementary Note, Supplementary Figures 1–9 and Supplementary Table 1 (PDF 4139 kb)
Supplemental Video 1
Ciliary beating pattern of PCD patient OP122 with CCDC39 mutations assessed by high-speed videomicroscopy analyses of respiratory cells obtained by nasal brushing biopsy (AVI 1118 kb)
Supplemental Video 2
Ciliary beating pattern of a healthy control individual assessed by high-speed videomicroscopy analyses of respiratory cells obtained by nasal brushing biopsy (AVI 4582 kb)
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Merveille, AC., Davis, E., Becker-Heck, A. et al. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nat Genet 43, 72–78 (2011). https://doi.org/10.1038/ng.726
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DOI: https://doi.org/10.1038/ng.726
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