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

Abstract

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality. 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 bending. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci. Here we show that the zebrafish cilia paralysis mutant schmalhans (smh(tn222)) 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.

Figure 1. The schmalhans mutation causes cilia paralysis and absent dynein arms

(A) Wildtype sibling and schmalhans mutant embryos at 52 hpf with inset magnified view of smh pronephric cyst. (B) Quantification of in situ hybridization results showing left-right asymmetry defects in 48 hpf (myl7(cmlc2), ins, and foxa3) and 18 hpf (pitx2) smh embryos and siblings (sib). SI, Situs Inversus. SS, Situs Solitus. (C–D) Electron micrographs of cilia axonemes in a wild-type (C) and smh (D) pronephric duct. Outer dynein arms are highlighted by black arrowheads, inner dynein arms are indicated by white arrowheads. Scale bars in C and D = 100 nm. (E–F) Still images from wildtype (E; Supplementary Movie 1) and schmalhans mutant (F; Supplementary Movie 2) pronephric cilia showing position of line scan used to generate kymographs (insets in (E) and (F)). Scale bars in E and F = 5 μm. (G) Chromatogram showing the C to T change at base 79 in ccdc103 resulting in Q to Stop at amino acid 27. (H) Schematic representation of the Ccdc103 proteins resulting from zebrafish (Dr) wild-type (WT) and smh (Q27Stop) coding sequence. (I,J) Rescue of axis curvature in a mutant clutch (I; white asterisks) by injection of 20 pg ccdc103 synthetic mRNA (J). (K) Frequency of axis curvature defects in smh −/−, smh −/− + ccdc103 mRNA, and smh −/− + Q27Stop ccdc103 mutant mRNA demonstrates mutant rescue and partial dominant-negative effect of Q27Stop smh ccdc103 mRNA. Red bars indicate the percent of embryos from an incross of smh heterozygotes that show the smh phenotype (Mendelian ~25% in uninjected embryos); blue bars indicate percent wild-type embryos; number of embryos in each class is indicated in the bars.

Figure 2. Pedigrees of families carrying CCDC103 loss of function mutations

Pedigrees of the consanguineous families UCL-120, OP-1192 and OP-1193. Affected children are represented by black symbols; those with situs inversus are denoted with an asterisk (*). The affected individuals from all three families carry a homozygous loss-of-function mutation (c.383_384insG) predicting a premature stop of translation (p.Gly128fs25*). The parents (UCL-120I1 and I2, as well as OP-1193I1 and I2) are heterozygous carriers for the mutation. Segregation of the mutant allele is consistent with autosomal recessive inheritance.

Figure 3. Functional assay of human CCDC103 mutant alleles in smh embryos

(A) Graphical representation of zebrafish (green) and human (yellow) ccdc103 mutant alleles. Mutation position is noted in red. (B) Frequency of axis curvature observed in smh clutches alone, or injected with 35 pg synthetic RNA encoding the indicated form of human CCDC103 at 36 hpf. smh clutches alone exhibited the expected 25% Mendelian ratio of affected homozygous mutants (yellow bar). (C) Frequency of cilia paralysis observed in smh clutches alone, or injected with 35 pg synthetic RNA encoding the indicated form of human CCDC103 at 52 hpf. (D) Western blot detection of myc epitope-tagged Ccdc103 protein expression in 24 hpf whole-embryo extracts from CCDC103 mRNA injected embryos (in B and C). CCDC103 monomers (m) and dimers (d) are noted with arrowheads. Anti-alpha tubulin was used as a loading control. (E) Electron micrograph pronephric duct cilia from a 52 hpf smh −/− embryo that received injection of synthetic RNA encoding wildtype human Ccdc103. Outer dynein arms are marked with black arrowheads; white arrows mark inner dynein arms. Scale bar = 100 nm.

Figure 4. Localization of DNAH5, DNAI2 and DNAH9 in respiratory epithelial cells from a PCD patient carrying the CCDC103 loss-of-function mutation

(A–C) Immunofluorescence analyses of human respiratory epithelial cells using specific antibodies directed against the outer dynein arm heavy chains DNAH5 (A) and DNAH9 (C) as well as the outer dynein arm intermediate chain DNAI2 (B). As control, axoneme specific antibodies against acetylated α-tubulin (A, C) or α/β-tubulin (B) were used. Nuclei were stained with Hoechst 33342 (blue). (A) In respiratory epithelial cells from healthy probands, DNAH5 (red) localizes along the entire length of the axonemes. In respiratory epithelial cells from patient OP-1192 carrying the CCDC103 loss-of-function mutation DNAH5 (red) localization is restricted to the proximal part of the axoneme and shows mis-localization to subapical cytoplasm and the perinuclear region (white arrowheads). (B) DNAI2 (green) is localized along the entire length of the axonemes of healthy probands. In contrast in respiratory cells of patient OP-1192 DNAI2 (green) localizes solely to the proximal ciliary axonemes (white arrowhead denotes cilia tips devoid of DNAI2 green fluorescence). (C) DNAH9 (red) localization is restricted to distal ciliary axonemes of respiratory epithelial cells from healthy probands, because it is only present in type2 ODA complexes. In the patient OP-1192 DNAH9 is completely missing (white arrowhead) , consistent with altered assembly of type-2 ODA complexes. (D) Transmission electron microscopy of respiratory cilia showing normal outer- and inner-dynein arms in epithelial cells from a healthy proband. Respiratory cilia of two affected siblings carrying both identical homozygous CCDC103 loss-of-function mutations display variable defects of outer dynein arms. Cilia from patient UCL-120II2 display severe defects of the outer- and inner-dynein arms, whereas in cilia from the other affected sibling (UCL-120II3) seems to have remnant outer dynein arms. White scale bars (A–C) are 5μm. Black scale bars (D) are 0.1μm.

Figure 5. Ccdc103 homodimers assemble with dynein light chain 2 in the cytoplasm and bind tightly to cilia axonemes

(A) Ccdc103 (red) is expressed both in the cytoplasm (red arrowhead) and in anti-acetylated tubulin-positive (green) zebrafish olfactory placode cilia (yellow arrowhead) in 52 hpf zebrafish embryos. Dashed white line indicates the dimensions of a single olfactory placode multiciliated cell. Scale bar = 10 μm. See Supplementary Movie 7 for comparison live image of olfactory placode cilia. Ccdc103 staining was not observed in smh morpholino knockdown embryos (Supplementary figure 11). (B) Fractionation of Chlamydomonas flagella demonstrates Ccdc103/Pr46b is present in flagella and remains tightly associated with 0.6 M NaCl extracted axonemes (asterisk). Ccdc103/Pr46b migrates both as a monomer (m) and a dimer (d). (C) Ccdc103/Pr46b monomers (m) and dimers (d) co-purify with dynein light chain 2 (LC2) in a high molecular weight fraction (440,000 - 2,000,000 D, right panel) of Chlamydomonas cytoplasm and are also present in a lower molecular weight cytoplasmic fraction (<440,000 D, left panel). (D) Circular dichroism spectroscopic analysis of recombinant Ccdc103/Pr46b reveals strong alpha helical content and robust resistance to heat denaturation. (E) Gel filtration of recombinant Ccdc103/Pr46b demonstrates a mixture of dimer and monomer peaks. Single Gel lane (left) shows Ni²⁺ column eluate containing recombinant His10-Ccdc103/Pr46b in total eluate protein. Pooled fractions from a superose 6 column (left chromatogram; black bar) fractionated on a Superdex 200 column (main chromatogram) revealed a mixture of monomer and dimer sized protein peaks. Western blotting for Ccdc103 confirmed Ccdc103 monomers and dimers in Superdex 200 fractions (See Supplementary figure 16). (F) Mutant smh/Ccdc103 protein disrupts Ccdc103 dimer formation in vivo. Western blotting using anti-c-myc antisera to detect expression of myc-tagged full length zebrafish Ccdc103 mRNA (18 pg) when co-injected into wildtype embryos with increasing amounts (gradient indicated) of truncated smh Q27Stop protein. This revealed Ccdc103 dimers when co-expressed with low amounts of smh/Ccdc103 truncated protein (18 pg mRNA) but primarily monomers when co-expressed with high amounts of smh/Ccdc103 mRNA (74 pg). Anti-tubulin was used as a loading control.