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NIH Public Access Author Manuscript Nat Genet. Author manuscript; available in PMC 2011 July 8. NIH-PA Author Manuscript Published in final edited form as: Nat Genet. 2011 January ; 43(1): 79–84. doi:10.1038/ng.727. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation Anita Becker-Heck1,2,3,#, Irene Zohn4,5,#, Noriko Okabe6,#, Andrew Pollock4,#, Kari Baker Lenhart6, Jessica Sullivan-Brown6, Jason McSheene6, Niki T. Loges1,2, Heike Olbrich2, Karsten Haeffner1, Manfred Fliegauf1, Judith Horvath1,7, Richard Reinhardt8, Kim G. Nielsen9, June K Marthin9, Gyorgy Baktai10, Kathryn V. Anderson11, Robert Geisler12,%, Lee Niswander4,*, Heymut Omran1,2,*, and Rebecca D. Burdine6,* 1Department of Pediatrics, University Hospital Freiburg, Freiburg, Germany 2Klinik und Poliklinik für Kinder- und Jugendmedizin -Allgemeine Pädiatrie -Universitätsklinikum Münster, Germany 3 NIH-PA Author Manuscript Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany 4Howard Hughes Medical Institute, Department of Pediatrics, University of Colorado Denver USA 5Center for Neuroscience Research, Children's Research Institute, Children’s National Medical Center, USA 6Department of Molecular Biology, Princeton University, USA 7National Medical Center, Budapest, Hungary 8Genome Centre Cologne at MPI for Plant Breeding Research, Köln, Germany 9Pediatric Pulmonary Service and Cystic Fibrosis Centre Copenhagen University Hospital, Denmark 10Pediatric Institute Svabhegy, Budapest, Hungary 11Developmental Biology Program, Sloan-Kettering Institute, New York, USA 12Max Planck Institute for Developmental Biology, Department of Genetics, Tübingen, Germany NIH-PA Author Manuscript Abstract Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with abnormal function of motile cilia. Approximately half of PCD patients also have alterations in the left-right organization of internal organ positioning including situs inversus and situs ambiguous (Kartagener’s Syndrome, KS). Here we identify an uncharacterized coiled-coil domain containing protein (CCDC40) essential for correct left-right patterning in mouse, zebrafish and humans. Ccdc40 is expressed in tissues that contain motile cilia and mutation of Ccdc40 results in cilia with reduced ranges of motility. Importantly, we demonstrate that CCDC40 deficiency causes a novel PCD variant characterized by misplacement of central pair microtubules and defective axonemal *Corresponding authors how jointly supervised this work. #These authors contributed equally %Current address: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany Author contributions. Studies in mice were conducted by I.Z., A.P., A.B-H., H.O., K.V.A. and L.N. Studies in zebrafish were conducted by N.O., K.B.L., J.S-B., J.M., R.G. and R.D.B. Studies with patient samples were conducted by A.B-H., N.T.L., H.O., K.H., M.F., J.H., R.R., K.G.N., J.K.M. G.B. and H.O. The manuscript was prepared by A.B-H, I.E.Z., L.N., H.O. and R.D.B. The authors have no competing financial interests. Becker-Heck et al. Page 2 assembly of inner dynein arms (IDAs) and dynein regulator complexes (DRCs). CCDC40 localizes to motile cilia and the apical cytoplasm and is responsible for axonemal recruitment of NIH-PA Author Manuscript CCDC39, which is also mutated in a similar PCD variant. Underlying defects in cilia ultrastructure are responsible for altered ciliary beat in PCD patients. The core structure of the cilium is the axoneme: nine peripheral microtubule doublets with or without a central pair of microtubles (9+2 or 9+0), interconnected by outer and inner dynein arms (ODAs and IDAs), radial spokes, nexin links and a central sheath 1. Coordinated activation of the ODAs and IDAs generates the ciliary beat. Most of the characterized PCD variants exhibit mutations in genes that encode dynein arm components such as DNAI1, DNAI2, DNAH5, DNAH11, and TXNDC3 2. Mutations in genes encoding cytoplasmic proteins such as C14orf104 (KTU) and LRRC50 also affect assembly of dynein arm complexes in the cytoplasm in a poorly understood process 3–6. In our forward genetic screens to identify genes required for normal development of the mouse embryo 7–8, we isolated a mutant which exhibits left-right patterning defects. Greater than one-third of homozygous links (lnks) mutant embryos (39%, n=172) display laterality defects at E11.5–15.5 (Fig. 1a–d) including situs inversus (8%) or left isomerism (19%) based on lung lobation patterns. The majority of homozygous lnks mutant pups die before weaning due to unknown causes. In two homozygous lnks mutant pups that were examined, NIH-PA Author Manuscript no kidney cysts were detected but hydrocephalus was noted (Supplementary Fig. 1). These observations resemble findings obtained in Mdnah5 deficient mice, a mouse model for PCD where ependymal cilia motility is important to prevent hydrocephalus 9. The lnks mutation was mapped to a 0.3 MB region of mouse chromosome 11 (Fig. 1e) that included the uncharacterized Coiled-coil domain containing 40 (Ccdc40) gene. Coiled-coil domains typically function in homodimerization and are present in a number of proteins involved in intracellular transport 10. Ccdc40 is specifically expressed in the embryonic node and midline tissues (Fig. 1f–i), key tissues that control left-right patterning. Upon sequencing the 3378 base pair Ccdc40 transcript from lnks mutant mice, a C to A transversion was identified (Fig. 1j). This nonsense mutation converts Valine792 to a stop codon in the middle of the coiled-coil domain, truncating the predicted 1125 amino acid protein (Fig.1j,k). In zebrafish embryos, ccdc40 is expressed in tissues that contain motile cilia including Kupffer’s vesicle, floorplate, pronephric tubules and otic vesicle (Fig. 2; and data not shown). To explore the evolutionary conserved role of ccdc40 in left-right patterning, we designed two different antisense morpholino oligonucleotides (MOs) against zebrafish ccdc40. Both MOs disrupt splicing of the ccdc40 transcript (Supplementary Fig. 2) and produce similar phenotypes upon injection (Fig. 2e,g,i). Injection of MO resulted in a curly- NIH-PA Author Manuscript tail down phenotype characteristic of other zebrafish mutants with laterality defects. Uninjected control embryos exhibited predominantly situs solitus (SS) at the 48 hpf stage with normal rightward looping of the heart, liver on the left and pancreas on the right side of the midline. By contrast, injection of either MO resulted in laterality defects: either reversed organ patterning, situs inversus (SI; 15–24%), or randomized organ patterning, heterotaxia (HTX; 13–19%). Both laterality and curly-tail down phenotypes could be rescued by co- injection of ccdc40 mRNA (Fig. 2h,j). ccdc40 maps to zebrafish chromosome 6 in a region associated with the zebrafish mutant locke (lok), previously described as having a strong curly-tail down phenotype, laterality defects and pronephric cysts, without defects in sensory cilia or presence of hydrocephalus 11–12 (and J. S-B and R.D.B. unpublished). The locke phenotype is indistinguishable from knockdown of Ccdc40 in zebrafish (Fig. 2f,g,i). We sequenced genomic DNA from lokto237b mutants and found a C to T transition within the 3370 base pair transcript that changes Glutamine778 to a stop codon (Fig. 2d). Nat Genet. Author manuscript; available in PMC 2011 July 8. Becker-Heck et al. Page 3 The laterality defects observed in mouse zebrafish mutants combined with expression of the transcript in the node/Kupffer’s vesicle suggest that Ccdc40 may act to regulate cilia NIH-PA Author Manuscript function (Fig. 3). Indeed, scanning electron microscopy (SEM) revealed that the length of the cilia projecting from the nodal pit cells in lnks mutants is drastically reduced (Fig. 3a,b,e,f). Similarly, cilia were shorter in Kupffer’s vesicle and the pronephric tubules of ccdc40 morphants compared to uninjected controls (Fig. 3c,d,g,h). These results indicate that Ccdc40 is required for proper formation or maintenance of cilia. Based on the cilia and laterality phenotypes in mouse and zebrafish ccdc40 mutants, we considered CCDC40, a strong candidate gene for human PCD. All coding CCDC40 exons and the adjacent intron-exon boundaries were amplified by PCR in a cohort of 26 PCD patients displaying a similar axonemal defect (see below). Sequence analyses revealed CCDC40 loss-of-function mutations in 17 PCD patients (Supplementary Fig. 3 and Table 1). Segregation analyses in all PCD families with CCDC40 mutations were consistent with autosomal recessive inheritance (Supplementary Fig. 4). Furthermore, in 15 affected individuals originating from 13 families, sequence analyses identified mutations on both CCDC40 alleles. However, in two families a mutation on the second allele was not identified by this approach. We addressed whether large deletions involving CCDC40 might be present on the other allele in these patients. Indeed segregation analysis of single nucleotide polymorphisms (SNPs) identified by sequence analysis of PCR products NIH-PA Author Manuscript provided evidence for parental non-contribution suggestive of heterozygous CCDC40 deletion in family OP-43. SNP segregation was consistent with the interpretation that the three affected individuals inherited a large genomic deletion involving at least exon 1 from the mother and the point mutation