ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6

Abstract

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Figure 1

Homozygosity Mapping and WER in Family A4231 and Identification of 11 Different Homozygous or Compound-Heterozygous ZMYND10 Mutations in 13 Additional Unrelated PCD-Affected Families (A) For individual A4231, who has situs inversus, nonparametric LOD (NPL) scores from whole-genome mapping are plotted across the human genome. The x axis shows Affymetrix 250K StyI array SNP positions on human chromosomes concatenated from pter (left) to qter (right). Genetic distance is given in cM. Thirteen maximum NPL peaks (red circles) indicate candidate regions of homozygosity by descent. Note that the ZMYND10 locus (arrow head) is positioned within one of the maximum NPL peaks on chromosome 3p. (B) Homozygous ZMYND10 mutation detected in family A4231. Family number (underlined), mutation (arrowhead), and predicted translational changes are indicated (see also Table 1). Sequence trace is shown for the mutation above the normal control. (C) Exon structure of human ZMYND10 cDNA. The positions of the start codon (ATG) and the stop codon (TGA) are indicated. (D) Domain structure of ZMYND10. The C terminus contains a MYND (myeloid, Nervy and DEAF-1) domain. The blue bar delineates the region necessary for interaction with LRRC6 as identified in Figure 1F. (E) Ten homozygous or compound-heterozygous ZMYND10 mutations detected in 12 PCD-affected families. Family number (underlined), mutation, and predicted translational changes are indicated (see Table 1 and Figure S2). (F) Interaction between the wild-type (WT) and six ZMYND10 variants detected in human PCD (see Table 1) and LRRC6. FLAG-tagged ZMYND10 and Myc-tagged LRRC6 constructs were transfected into human embryonic kidney 293T (HEK293T) cells and coimmunoprecipitated with a FLAG antibody. Note that the three truncating protein alterations (p.Phe101Serfs^∗38, p.Gln323^∗, and p.Gln366^∗) abrogated interaction with LRRC6, whereas the three alterations resulting from missense mutations did not. (G) GST pull-down of purified ZMYND10 (MYND domain and full-length). Note that LRRC6 in rat lung lysates was pulled down by the full-length ZMYND10, but not by the MYND domain alone. (H) The purified CS domain of LRRC6 binds to the full-length ZMYND10, but not to the MYND domain of ZMYND10. Therefore, the MYND domain alone is not sufficient for the interaction with LRRC6. SM denotes size marker.

Figure 2

Loss of ZMYND10 Function Causes Structural and Functional Defects of Motile Cilia in Human Lung and in a Zebrafish Model of zmynd10 Knockdown (A–D) Compared to those of a normal control (A), ciliary axonemes from individuals A5014_188 (B), OI-143II2 (C), and OP-55II1 (D), who have PCD and ZYMYND10 mutations, lack IDAs and ODAs (arrows). (E–G) Images of respiratory epithelial cells from a healthy control and from PCD-affected individuals who carry ZMYND10 loss-of-function mutations. Cells were costained with antibodies against acetylated α-tubulin (green) and DNAH5 (red). Nuclei were stained with Hoechst 33342 (blue). In cells from a healthy control (E), DNAH5 localized to the axonemes of respiratory cilia. The yellow costaining within the ciliary axoneme indicates that both proteins colocalized within respiratory cilia. In respiratory cells of individuals OI-143II2 (F) and OP-55II1 (G), DNAH5 was not detectable in the ciliary axonemes, suggesting that ZMYND10 loss-of-function mutations led to defects in the ODA heavy-chain DNAH5. Rabbit polyclonal DNAH5 and DNALI1 antibodies were described previously. See also Figures S3 and S4. (H–M) zmynd10 knockdown in zebrafish replicated a ciliopathy phenotype. Compared to control-injected embryos (H), embryos injected with zmynd10-translation-blocking MOs (I) showed three otoliths (arrowheads), cystic pronephric glomeruli (arrows), and distended pronephric tubules (white bar). (J) A still image of a high-speed microvideo of pronephric cilia (J) shows the tubule outline (dashed line) and position of pixels (black line) sampled for the kymograph (J′; one second total duration). The double arrowhead denotes the approximate tubule lumen diameter. (K) A still image of a high-speed microvideo of zmynd10-morphant pronephric cilia shows a distended tubule outline (dashed line), pixels sampled for the kymograph (black line; K′, 1 s total duration), and a distended tubule lumen dimension (double-arrowhead line). (L) A still image of a high-speed microvideo of control olfactory placode cilia and position of pixels sampled for the kymograph (black line; L′, 1 s total duration). See also Movies S1, S2, S3, and S4. (M) A still image of high-speed microvideo of zmynd10-morphant olfactory placode cilia and position of pixels sampled for the kymograph (black line; M′, 1 s total duration). See also Movies S1, S2, S3, and S4. Scale bars represent 5 μm in (J)–(M).

Figure 3

Truncating Variants of LRRC6 Abrogate Interaction with ZMYND10 (A) For testing the effect of seven LRRC6 mutations detected in individuals with PCD, FLAG-tagged ZMYND10 and Myc-tagged LRRC6 constructs were transfected into HEK293T cells and coimmunoprecipitated with a FLAG antibody. Note that the five truncating protein alterations (p.Gln188^∗p.Lys200Glufs^∗3, p.Tro210Cysfs^∗12, p.Thr237Lysfs^∗7, and p.Ala298Profs^∗2) abrogated interaction with ZMYND10, whereas the two alterations resulting from missense mutations did not. (B) LRRC6 residues 1–204, which include the LRR, LRRCT, and CC domains, were not necessary for the interaction with ZMYND10. Coimmunoprecipitation was done as in Figure 3A. (C) GST pull-down of purified LRRC6 (CS domain and full-length). Note that the CS domain of LRRC6 was sufficient for pull-down of ZMYND10 in rat lung lysates. “SM” denotes size marker.

Figure 4

ZMYND10 and LRRC6 Colocalize with Centriolar Proteins in Rat Trachea (A) Coimmunofluorescence of ZMYND10 (Sigma) with acetylated tubulin (Sigma). (B–D) Coimmunofluorescence of ZMYND10 (Abnova) with SAS6 (spindle assembly abnormal protein 6) (Sigma) (B), PCM1 (pericentriolar material 1) (Cell Signaling Technology) (C), and LRRC6 (Novus) (D). (E–G) Coimmunofluorescence of LRRC6 (Sigma) with acetylated tubulin (E), SAS6 (F), and PCM1 (G). PCM1 is a component of centriolar satellites, which are electron-dense granules scattered around centrosomes. PCM1 also localizes to fibrous granules of ciliogenic cells, but not to deuterosomes. Scale bars represent 5 μm in (A)–(G).