Mutations in ZMYND10, a gene essential for proper axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.

Figure 1

Family Segregation Analysis, Cilia Ultrastructure, and Localization of Variants within the Predicted ZMYND10 (A) Pedigree structure and segregation analysis of the six PCD-affected families harboring ZMYND10 mutations. Filled symbols indicate affected individuals, an asterisk indicates the laterality defect, and a slash indicates that an individual is deceased. Plus signs indicate a normal allele, and “NA” indicates that the result is not available. (B) TEM shows that the nasal respiratory epithelial cell cilia of ZMYND10-mutant cases have either fewer (red arrows indicate remnant arms) or no (black arrowheads) IDAs and ODAs in comparison to those of the control. Scale bar represents 100 nm. (C) Location of ZMYND10 variants. Green boxes indicate LxxLL motifs, the blue box represents the ZF-MYND domain, and the amino acid positions are shown. Conservation across species of residues affected by the three missense variants is shown with proteins (corresponding accession numbers are shown in parentheses) from the following species: H. sapiens (RefSeq NP_056980.2), P. troglodytes (RefSeq XP_516479.2), M. musculus (RefSeq NP_444483.2), B. taurus (RefSeq NP_001035638.1), C. lupus (RefSeq XP_533818.1), D. rerio (RefSeq NP_956691.1), X. leavis (RefSeq NP_001090272.2), D. melanogaster (RefSeq NP_648625.1), T. brucei (RefSeq XP_828897.1), T. thermophila (RefSeq XP_001026696.1), and C. reinhardtii (Phytozome accession Cre08.g358750.t1.3). The transcript annotations are based on the Augustus update u11.6 annotation of Joint Genome Institute assembly v.5.

Figure 2

Restricted Expression of ZMYND10 within Motile Ciliated Tissues (A and B) Expression of Zmynd10 mRNA in embryonic day 18.5 mouse embryos by whole-mount in situ hybridization reveals specific expression in the nasal epithelium (A, arrow) and the bronchi (B, arrows) of the lung (indicated by an asterisk). Scale bars represent 0.5 mm. (C) Expression of Zmynd10 (CG11253) in Drosophila embryos by whole-mount in situ hybridization reveals expression restricted to differentiating Ch neurons. (D) The Drosophila Zmynd10-mVenus fusion gene (green) is expressed in Ch neurons (lch5, v’ch5, and vchAB), but not other ciliated sensory neurons (marked by FUTSCH, magenta) in the embryo. (E) In the pupal antenna, the Zmynd10-mVenus fusion gene is strongly expressed in the Ch neurons of Johnston’s Organ (“JO”), but not the olfactory receptor neurons (“ORNs”).

Figure 3

Zmynd10-Mutant Flies Have Sensory Defects and Loss of the Axonemal Dynein Arms (A) A box and whisker plot of climbing assay for proprioceptive defects. Twenty 2- to 7-day-old flies were placed in a measuring cylinder, and then after a 1 min recovery period, they were banged to the bottom of the cylinder, and the height climbed in 10 s was then recorded. Homozygous mutant Zmynd10^EY10886 flies performed significantly less well in this assay than did the “wild-type” revertants (in which the P element had been lost by excision) (p < 0.0001). The Zmynd10-mVenus fusion transgene completely restored the climbing behavior of homozygous mutant flies (“rescue”). Significance was determined by a Kruskal-Wallis test (H = 35.48; n = 44, 42, and 28; p < 0.0001) followed by a Dunn’s test. (B and C) The lch5 neuron cluster from an embryonic abdominal segment stained with FUTSCH (magenta) and anti-HRP, which detects the cilium at this stage (green). In mutant embryos, the cilia are present and appear grossly normal in length. (D) Schematic of embryonic Ch neuron. (E and F) In Ch neurons in pupal antennae, Zmynd10-mVenus expression (green) is largely cytoplasmic relative to nompC and GT335 (distal and proximal ciliary markers, magenta). (G) Schematic of pupal Ch neurons. (H and I) TEM of adult antennal Ch neuron cilia. A control with ODA and IDA complexes (white and black arrows, respectively) is indicated in (H). A Zmynd10 mutant, showing loss of some ODA and IDA complexes (white and black arrowheads, respectively) is shown in (I). Primary antibodies used were mAb-22C10 (1:200), RbAb-HRP (1:500), RbAb-GFP (1:500) (Molecular Probes), mAb-NompC (1:100), and GT335 (Sigma, 1:200). Secondary antibodies were from Molecular Probes. Scale bars represent 100 nm.

Figure 4

Zmynd10-Mutant Male Flies Lack Motile Sperm (A) In a dissected wild-type adult (5-day-old) testis, sperm bundles (“SBs”) can be seen, the mature sperm are transferred to the seminal vesicle (“SV”), and motile sperm (“MS”) can be seen emerging from this structure. (B) In a Zmynd10-mutant fly, sperm bundles are still observed in the testis, but the seminal vesicle is empty and no motile sperm are seen emerging. There is also some disruption of sperm-bundle coiling at the proximal end of the testis, which might be a secondary effect of sperm immotility. (C–E) TEM transverse sections of sperm bundles in a testis. (C) In a control fly, yellow arrows point to dynein arms. Also visible (red arrows) are the inner pair and outer accessory microtubules, which have an electron-dense core (the luminal filament). Scale bar represents 20 nm. (D) In the mutant, there is loss of dynein arms (cyan arrows) and ectopic luminal filaments in the A microtubule of the doublets (red arrows). Scale bar represents 20 nm. (E) In the mutant, some flagella have fragmented axonemes (arrows). Scale bar represents 100 nm. (F) Yeast two-hybrid assay of human ZMYND10 (“Z”) and LRRC6 (“L”). In each sector, the Bait:Prey combinations are indicated. Also present are positive (p53) and negative (Lamin) controls. (G) A summary of yeast two-hybrid results of the interaction between LRRC6 and mutated or truncated forms of ZMYND10 shows that interaction (+) is not affected by the PCD-linked protein variants but relies on the presence of the MYND domain. Variants were introduced by site-directed mutagenesis with the use of the Strataclone Quickchange 2 kit (Stratagene). (H) A box and whisker blot shows the progeny produced by single male flies. Zmynd10-mutant males (“mutant”) were infertile, but fertility was restored with the rescue transgene (“rescue”). A two-tailed Mann-Whitney test shows that restoration of fertility was significantly less with a rescue transgene containing the p.Val14Gly missense variant (“rescue V14G”) (U = 586; n = 40, 40; p = 0.0385). For fertility analysis, 40 2- to 5-day-old males were crossed individually to wild-type females. After 2 days of prelaying, flies were transferred to new vials and were allowed to lay eggs for 3 days. Progeny from the latter were counted.