CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

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Abstract

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality1. Cilia motility defects cause primary ciliary dyskinesia (PCD, MIM244400), a disorder affecting 1:15,000–30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive ciliary bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhans (smhtn222) encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a-regulated gene product. Screening 146 unrelated PCD families identified individuals in six families with reduced outer dynein arms who carried mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103/Pr46b functions as a tightly bound, axoneme-associated protein. These results identify Ccdc103 as a dynein arm attachment factor that causes primary ciliary dyskinesia when mutated.

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Figure 1: The smh mutation causes cilia paralysis and absent dynein arms.
Figure 2: Pedigrees of families carrying CCDC103 loss-of-function mutations.
Figure 3: Functional assay of human CCDC103 mutant alleles in smh embryos.
Figure 4: Localization of DNAH5, DNAI2 and DNAH9 in respiratory epithelial cells from a patient with PCD carrying the CCDC103 loss-of-function mutation.
Figure 5: Ccdc103 homodimers assemble with dynein light chain 2 in the cytoplasm and bind tightly to cilia axonemes.

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Acknowledgements

We thank the patients and their families for their participation and acknowledge support from the PCD Family Support Group (UK) and the German patient support group 'Kartagener Syndrom und Primaere Ciliaere Dyskinesie e.V.' . We thank R. Mark Gardiner and E. Moya for their help and involvement in the study. We thank R. Hirst and A. Rutman for electron microscopy. We thank A. Heer, C. Warmt, D. Nergenau, R.S. Patel-King and L. Luo for excellent technical assistance. This work was funded by US National Institutes of Health grants DK053093 (I.A.D.) and GM051293 (S.M.K.), National Research Service Award fellowship grant F32DK083868 (J.R.P.), an American Heart Association Established Investigator Award (A.F.S.), Deutsche Forschungsgemeinschaft (DFG) grants (DFG Om 6/4, GRK1109 and SFB592), EU grant SYSCILIA, IZKF-project (Om2/009/12), and Kindness for Kids grant (H. Omran), the Medical Research Council UK, the Wellcome Trust and the Fondation Milena Carvajal–ProKartagener.

Author information

J.R.P., Y.L., C.A.-T., A.G., N.H., A.K.-Z., R.D.B., F.O., A.F.S., N.P. and I.A.D. performed genetic mapping and Ccdc103 characterization in zebrafish. A.B.-H., V.H.C., D.A.A.-M., N.T.L., E.S., M.S., H. Olbrich, C.W., K.H., R.C., C.O., E.M.K.C., R.R., H.M.M. and H. Omran conducted studies with human patient samples. S.M.K. performed Chlamydomonas studies. J.R.P., A.B.-H., S.M.K., H. Omran and I.A.D. prepared the manuscript.

Correspondence to Stephen M King or Heymut Omran or Iain A Drummond.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–7 and Supplementary Figures 1–16. (PDF 1406 kb)

Supplementary Video 1

Pronephric cilia motility in wildtype 2.5 dpf proximal tubules. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. Anterior is to the left. (MOV 3690 kb)

Supplementary Video 2

Pronephric cilia paralysis in schmalhans 2.5 dpf mutant proximal tubules. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. Anterior is to the left. (MOV 1799 kb)

Supplementary Video 3

Olfactory placode cilia motility in wildtype 3 dpf embryos. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. (MOV 786 kb)

Supplementary Video 4

Olfactory placode cilia paralysis in schmalhans mutant 3 dpf embryos. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. (MOV 1461 kb)

Supplementary Movie 5

Spinal canal cilia motility in wildtype 3 dpf embryos. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. Anterior is to the left. (MOV 3088 kb)

Supplementary Video 6

Spinal canal cilia paralysis in schmalhans mutant 3 dpf embryos. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. Anterior is to the left. (MOV 2619 kb)

Supplementary Video 7

Restoration of olfactory placode cilia motility in genotypically mutant schmalhans embryo by ccdc103 mRNA injection. Movies were acquired at 240 frames per second (fps) and slowed to 15 fps for viewing. (MOV 1971 kb)

Supplementary Video 8

Pronephric cilia paralysis in ccdc103 morpholino knockdown 2.5 dpf proximal tubules. Movies were acquired at 250 frames per second (fps) and slowed to 15 fps for viewing. Anterior is to the upper right corner. (MOV 2272 kb)

Supplementary Video 9

Normal human ciliated nasal epithelium show robust cilia motility and wave form. Video was acquired at 256 frames per second and the ciliary beat pattern was evaluated by slow motion playback at 30 fps. (MOV 532 kb)

Supplementary Video 10

Human ciliated nasal epithelium cells from patient OP-1193 II1 carrying the homozygous p.Gly128fs25* mutation. Cilia are completely paralysed. Video was acquired at 125 frames per second and the ciliary beat pattern was evaluated by slow motion playback at 30 fps. (MOV 760 kb)

Supplementary Video 11

Human ciliated nasal epithelium cells from patient OP-32II1 carrying the p.His154Pro CCDC103 missense mutation. Cilia show reduced beat amplitude. Video was acquired at 125 frames per second and the ciliary beat pattern was evaluated by slow motion playback at 30 fps. (MOV 290 kb)

Supplementary Video 12

Human ciliated nasal epithelium cells from patient OP-32II2 carrying the p.His154Pro CCDC103 missense mutation. Cilia are paralysed or show reduced beat frequency, reduced beat amplitude and lack of beat coordination. Video was acquired at 125 frames per second and the ciliary beat pattern was evaluated by slow motion playback at 30 fps. (MOV 653 kb)

Supplementary Video 13

Normal human ciliated nasal epithelium cells show robust cilia motility and wave form. Video was acquired at 125 frames per second and the ciliary beat pattern was evaluated by slow motion playback at 30 fps. (MOV 183 kb)

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Panizzi, J., Becker-Heck, A., Castleman, V. et al. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms. Nat Genet 44, 714–719 (2012) doi:10.1038/ng.2277

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