Exome sequencing identifies a recurrent de novo ZSWIM6 mutation associated with acromelic frontonasal dysostosis

Abstract

Acromelic frontonasal dysostosis (AFND) is a rare disorder characterized by distinct craniofacial, brain, and limb malformations, including frontonasal dysplasia, interhemispheric lipoma, agenesis of the corpus callosum, tibial hemimelia, preaxial polydactyly of the feet, and intellectual disability. Exome sequencing of one trio and two unrelated probands revealed the same heterozygous variant (c.3487C>T [p. Arg1163Trp]) in a highly conserved protein domain of ZSWIM6; this variant has not been seen in the 1000 Genomes data, dbSNP, or the Exome Sequencing Project. Sanger validation of the three trios confirmed that the variant was de novo and was also present in a fourth isolated proband. In situ hybridization of early zebrafish embryos at 24 hr postfertilization (hpf) demonstrated telencephalic expression of zswim6 and onset of midbrain, hindbrain, and retinal expression at 48 hpf. Immunohistochemistry of later-stage mouse embryos demonstrated tissue-specific expression in the derivatives of all three germ layers. qRT-PCR expression analysis of osteoblast and fibroblast cell lines available from two probands was suggestive of Hedgehog pathway activation, indicating that the ZSWIM6 mutation associated with AFND may lead to the craniofacial, brain and limb malformations through the disruption of Hedgehog signaling.

Figure 1

Classic Craniofacial Phenotype Represented by Individual 1 (A) Anteroposterior view of the facial features of individual 1. Note the severe frontonasal dysplasia with ocular hypertelorism, widely spaced nasal alae remnants, and carp-shaped mouth. (B) Coronal MRI demonstrates a large interhemispheric lipoma. (C and D) Craniofacial CT demonstrates (C) anterior cranium bifidum, an interfrontal bone, a true median cleft of the maxilla, complete disruption of nasal structures, and (D) symmetric parietal foramina.

Figure 2

Sanger Validation, Evolutionary Comparative Analysis, Genomic Structure, and Predicted Protein Regions (A) Sanger sequencing of ZSWIM6 confirms the exon 14 c.3487C>T mutation that leads to the amino acid substitution p.Arg1163Trp in all four cases. The CADD score for this substitution is 22.9, suggesting that it has moderate impact on protein function. (B) Alignments of amino acids spanning 25 residues on either side of p.Arg1163Trp (yellow) in diverse species, from sea squirts (XP_002126311) to Homo sapiens (NP_065979.1), show that this region is highly conserved. Divergent residues are marked in red, and conservative substitutions are marked in green. (C) ZSWIM6 is composed of 14 exons that encode the protein, and only one transcript has been detected in vivo. The de novo substitution p.Arg1163Trp is indicated by the black arrow.

Figure 3

In Silico Protein Modeling (A) One-dimensional analyses of the predicted functional relevance of protein domains in ZSWIM6. Arginine 1163 occurs in a highly conserved domain from 1148–1215 with similarity to Sin3. Substitution to tryptophan is predicted to have severe effects on protein function. Regions with detectable similarity to non-ZSWIM proteins are denoted at the top. One-dimensional analyses are plotted from the ZSWIM6 N terminus (residue 1, left) to C terminus (1215, right). Plots are shown for percent identity among 97 orthologous proteins (red), predicted functional importance (blue, measured by MFS), relative sequence entropy (orange, measured by HMMRE) and predicted impact of substitution (measured by HUSCY; the mean and standard deviation for all possible single-nucleotide variations are shown in black and gray). At bottom, predicted disordered regions (as measured by DISpro) are shown in purple, domain boundaries (determined by DOMpro) in black, and exon borders as pink lines. Scores for p.Arg1163 and p.Arg1163Trp are highlighted in red and black circles. (B) Protein modeling of amino acid residues 1148–1215 of the wild-type (left) and mutant (right) shows that the p.Arg1163Trp substitution (arrow) dramatically alters the structure and hydrophobicity of the protein.

Figure 4

Zswim6 RNA In Situ Staining in Zebrafish Zebrafish zswim6 expression in the embryonic brain. The NCBI zebrafish zswim6 (RefSeq accession number NM_001136487.1) was used for creation of a zswim6 cDNA clone from 24 hr wild-type cDNA; PCR primers zswim6 F 5′-GCTTTTCCTCTCCGTATCCCG-3′ and zswim6 R 5′-GTATCCGCCCTGAGACAGGA-3′ were used. Zebrafish RNA in situ hybridizations were performed as previously described. (A–D) Fluorescent RNA in situ hybridization patterns for zswim6 (green) and dlx2a (red) are shown along with their overlap (merge) at 24 hpf (A and B) and 48 hpf (C and D). Images are shown in lateral view (A and C) and dorsal view (B and D); anterior is to the left. At 24 hpf, zswim6 shows enhanced expression in the dorsal telencephalon (A, arrow). At 48 hpf, zswim6 shows enhanced expression in a telencephalic domain that borders dlx2a expression in the subpallium (C, arrow), in the midbrain and hindbrain (C, arrowheads), and in the retina (D, arrow). An asterisk (^∗) indicates yolk background stain.

Figure 5

ZSWIM6 Localization during Mouse Development (A) Immunohistochemistry demonstrating discrete localization of ZSWIM6 in fiber cells of the developing lens of an E13.5 mouse embryo (arrow); polyclonal rabbit anti-human ZSWIM6 antibody (ab122301, Abcam) was used at a dilution of 1:100 and incubated on the sections overnight at 4°C. Immunoreactivity was detected with ImmPACT DAB (SK-4105, Vector Labs), followed by counterstaining with Hematoxylin QS (H-3404, Vector Labs). Slides were dehydrated, mounted in Permount media, and photographed with a Leica DM 4000B digital microscope with Leica Application Suite v 4.3.0 software. (B) Neighboring-section control of (A) without primary antibody. Postnatal day 0 mouse expression of ZSWIM6 in stereocilia of the outer hair cells of the cochlea (C, arrow), subpopulation of cells in the outer root sheath of the hair follicle (D, arrow), odontoblasts and ameloblasts of the tooth (E, arrows), and variable expression in skeletal muscle (F, arrows) are shown.