CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

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 asymmetry. 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.

Figure 1

Positional identification of CCDC39 as the gene that underlies PCD in Bobtails. (a) Old English Sheepdog (Bobtail). Representative TEM images of disorganized cilia identified in nasal mucosal biopsies of cases (PCD) and normal cilia from a healthy dog (CTR). (b) Positional identification of the p.Arg96X alteration in CCDC39. Homozygosity mapping identified a genome-wide significant signal on chromosome CFA34, corresponding to a 15-Mb segment shared homozygous- by-descent by 5 affected animals and encompassing 151 annotated protein-coding genes, of which 10 were included in the ciliome or cilia proteome database (or both). Sequencing CCDC39 in affected individuals revealed a C>T transition in the third exon of the main isoform, creating a stop codon that causes nonsense-mediated RNA decay. d, disease.

Figure 2

Expression and functional studies in mouse and zebrafish. (a) Whole-mount in situ hybridization analysis of mouse Ccdc39 in mouse embryos. Ccdc39 expression is restricted to the node in embryos at embryonic day (E) 7.75–8.0 (arrowheads). In E16.5 mouse embryonic sections, Ccdc39 (arrowheads) is expressed in ciliated cells of the upper and lower airways. (b) Dose-response curve of ccdc39 translation-blocking morpholino (tb-MO). Wildtype zebrafish embryos were injected with increasing concentrations of tb-MO and were scored live at 36 h post fertilization for heart looping (left, right, no loop). (c) Dose-response curve of ccdc39 splice-blocking MO. Scoring was conducted as in b. (d) Quantification of spaw staining in embryo batches injected with 4 ng ccdc39 morpholino or 4 ng ccdc39 morpholino plus 25 pg wildtype (WT) human CCDC39 mRNA (n = 24–30 embryos per injection). (e) Representative spaw RNA in situ staining in 14 somite-stage embryos. In wildtype embryos, spaw is expressed in the left lateral plate mesoderm (lpm; left); however, ccdc39 morphant embryos showed bilateral (center) or, in most cases, undetectable spaw expression (right).

Figure 3

Ultrastructural and mutational analysis of human PCD cases with axonemal disorganization. (a) Electron microscopy of respiratory cilia from an individual (DCP85) who is homozygous for the CCDC39 mutation resulting in the p.Glu731AsnfsX31 alteration. It shows the absence of inner dynein arms in all ciliary sections, associated with a range of other, heterogeneous defects: isolated absence of the nine inner dynein arms (1), axonemal disorganization with mislocalized peripheral doublet associated with either a displacement of the central pair (2), an absence of the central pair (3), or supernumerary central pairs (4). Magnification of the axoneme from a normal cilium is shown in the upper right panel with presence of inner dynein arms (black arrow), nexin links (white arrow) and radial spokes (short arrow). The axonemal disorganization found in cases is associated with defects of inner dynein arms (black flash), nexin links (white flash) and radial spokes (star) that are better seen after magnification (lower right panel). Scale bar, 0.2 μm. (b) Unambiguous disease-causing CCDC39 mutations detected in PCD cases with axonemal disorganization. Exonic organization of the human CCDC39 cDNA (top) and domain organization model of the corresponding protein (bottom). The 20 coding exons are indicated by empty or hashed boxes, depicting translated or untranslated sequences, respectively. ‘SMC_N’ and ‘SMC_Prok_B’ refer to domains homologous to the N terminus of SMC (structural maintenance of chromosomes) proteins and to the common bacterial type SMC protein, respectively. The predicted coiled-coil domains of the protein are indicated by green rectangles. The canine p.Arg96X alteration is shown in green. The splice mutation leading to the inclusion of pseudo-exon 9 is underlined.

Figure 4

Subcellular localization of CCDC39 in respiratory epithelial cells from individuals with PCD carrying CCDC39 mutations. Axoneme-specific antibodies against acetylated α-tubulin (green) were used as the control. Nuclei were stained with Hoechst 33342 (blue). (a) In respiratory epithelial cells from healthy probands, CCDC39 (red) localized predominantly along the entire length of the axonemes, as well as to the apical cytoplasm. (b–e) In respiratory epithelial cells from individuals OP-736 II2 (b), OP-736 II1 (c), OP-122 (d) and OP-18 II1 (e) carrying CCDC39 loss-of function mutations, CCDC39 was absent from the axoneme and markedly reduced in the apical cytoplasm. White scale bars, 5 μm.

Figure 5

Subcellular localization of DNAH5, DNALI1 and GAS11 in respiratory epithelial cells from individuals with PCD carrying CCDC39 mutations. Immunofluorescence analyses of human respiratory epithelial cells using antibodies to the outer dynein arm heavy chain DNAH5 (a), the inner dynein arm component DNALI1 (b) and the DRC component GAS11 (c). Axoneme-specific antibodies to acetylated α-tubulin (a) or α/β-tubulin (b, c) were used as controls. Nuclei were stained with Hoechst 33342 (blue). The localization of DNAH5 (red) in respiratory epithelial cells from case OP-736 II1 was unchanged (a). DNALI1 (green) localized along the entire length of the axonemes of respiratory epithelial cells from healthy probands (b). In epithelial cells from case OP-736 II1, DNALI1 (green) was absent from the ciliary axonemes (b). In respiratory epithelial cells from healthy probands, GAS11 (green) localizes along the entire length of the axonemes (c). In respiratory epithelial cells from case OP-736 II1, GAS11 (green) was targeted to the ciliary base, where it accumulated (c). White scale bars, 5 μm.