A Recurrent De Novo Nonsense Variant in ZSWIM6 Results in Severe Intellectual Disability without Frontonasal or Limb Malformations

Abstract

A recurrent de novo missense variant within the C-terminal Sin3-like domain of ZSWIM6 was previously reported to cause acromelic frontonasal dysostosis (AFND), an autosomal-dominant severe frontonasal and limb malformation syndrome, associated with neurocognitive and motor delay, via a proposed gain-of-function effect. We present detailed phenotypic information on seven unrelated individuals with a recurrent de novo nonsense variant (c.2737C>T [p.Arg913Ter]) in the penultimate exon of ZSWIM6 who have severe-profound intellectual disability and additional central and peripheral nervous system symptoms but an absence of frontonasal or limb malformations. We show that the c.2737C>T variant does not trigger nonsense-mediated decay of the ZSWIM6 mRNA in affected individual-derived cells. This finding supports the existence of a truncated ZSWIM6 protein lacking the Sin3-like domain, which could have a dominant-negative effect. This study builds support for a key role for ZSWIM6 in neuronal development and function, in addition to its putative roles in limb and craniofacial development, and provides a striking example of different variants in the same gene leading to distinct phenotypes.

Keywords: ZSWIM6; autism; de novo; epilepsy; exome sequencing; genomics; intellectual disability; nonsense-mediated decay; recurrent; ubiquitination.

Figure 1

ZSWIM6 Domain Structure and Variants and Molecular Data on mRNA Encoding the p.Arg913Ter Variant (A) Diagram showing predicted ZSWIM6 protein domains (lower) relative to exonic structure (upper) and positions of the p.Arg913Ter and p.Gln874Ter variants reported here (in red and black, respectively) and the previously reported p.Arg1163Trp recurrent missense variant located within the C-terminal Sin3-like domain (information adapted from Tischfield et al.⁷). For clarity, the correspondence between the exon boundaries and the protein sequence is depicted only for the final three exons. Introns not drawn to scale. (B) The mRNA encoding p.Arg913Ter is not degraded by nonsense-mediated decay (NMD) in LCLs. Sequence chromatograms showing normal and p.Arg913Ter-encoding reverse-transcribed mRNAs (cDNA) from LCLs (3 × 10⁶) cultured in the presence or absence of 100 μg/mL NMD inhibitor cycloheximide (CHX) for 6 hr and genomic DNA (gDNA) of the affected individual and his unaffected brother. The cDNA of affected individual 1 and genomic DNA of affected individual 1 and his unaffected brother were PCR amplified using hZSWIM6_2633F and hZSWIM6_2871R (cDNA); and hZSWIM6_gDNA_F and hZSWIM6_gDNA_R (gDNA: Table S2) and subjected to Sanger sequencing. Presence of the variant mRNA in the affected individual’s LCLs cultured without CHX indicates absence of NMD. Normal/variant nucleotides are boxed. (C) ZSWIM6 expression levels are not reduced in affected individual 1 relative to his unaffected brother and is consistent with four unrelated male control subjects. Total RNA from affected individual 1, his unaffected brother, and four unrelated normal male LCL pellets was extracted using a RNeasy Mini Kit (QIAGEN) and RNase-Free DNase set (QIAGEN) according to the manufacturers’ protocols. The cDNA was synthesized from total RNA using Super Script RT III Reverse Transcription kit (Life Technologies) following the manufacturer’s protocol. ZSWIM6 expression was determined by real-time quantitative PCR from cDNA using SYBR Green reagent (Bio-Rad Laboratories) and hZSWIM6_2633F and hZSWIM6_2871R primers (Table S2) and normalized to HPRT1 expression assayed alongside using HPRT1-F and HPRT1-R primers (Table S2). Each sample was assayed twice independently in triplicate. Error bars indicate standard deviation. The significance was calculated by Student’s t test.

Figure 2

Facial and Neurological Features of Individuals with the p.Arg913Ter ZSWIM6 Variant (A) Facial features of individuals 1 to 7. In early childhood frequent features include fine, arched eyebrows, short nose with depressed bridge and blunt tip, broad columella, thick everted lower lip vermillion, widely spaced teeth, downturned corners to mouth, mouth often held open, and esotropia. Individuals 1 and 6 were noted with age to develop prominent forehead and supraorbital ridges, thick eyebrows, and thicker everted lower lip vermillion, reminiscent of the facial appearance of older individuals with Coffin-Lowry syndrome. This was not the case for individual 7. (B) Sural nerve biopsy from individual 1 demonstrating demyelination and abnormal accumulation of neurofilaments within numerous myelinated axons. (a) Low-power electron micrograph showing three fibers containing proliferated neurofilaments (arrows) among thinly myelinated fibers which demonstrate the normal density of axonal filaments. (b) Accumulated neurofilaments [n] in an axon with thick and folded myelin. (c) Demyelinated axon [d] packed with neurofilaments that displace other axonal organelles. (d) High magnification of (c) showing mitochondria and other organelles in septa between neurofilaments. (e) Demyelinated axon with proliferated neurofilaments in distinct whorled bundles. (f) High magnification of proliferated neurofilament bundle. The individual filaments measure 9–15 nm in diameter.