ZMIZ1 Variants Cause a Syndromic Neurodevelopmental Disorder

Abstract

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.

Keywords: ZMIZ1; intellectual disability; neurodevelopmental disorder; neuronal positioning; transcriptional coactivation.

Figure 1

Topographical Images of Subjects with ZMIZ1 Variants

Figure 2

Domain Organization of ZMIZ1 and Its Variants from Affected Subjects (A) Schematic representation of ZMIZ1 and 10 variants identified in 12 families. See Table 2 for DNA sequence changes in the subjects. The splice variant c.3097−2A>G and the t(10;19) (q22.3;q13.33) translocation are not represented here. Abbreviations: TPR, tetratricopeptide repeat; SUMO, SUMO acceptor site; NLS, nuclear localization signal; MIZ, SP-RING/MIZ domain; TAD, transactivation domain. (B) Hydrophobic cluster analysis (HCA) of the alanine-rich region with predictions of intrinsically disordered regions from IUPRED performed with the Medor Metaserver. Symbols are used to represent amino acids with peculiar structural properties (star for proline, black diamond for glycine, square for threonine, and dotted square for serine, which may be either exposed or buried). Phase transition or phase separation has been put forward as a general mechanism for the transient formation of cellular bodies that behaves as liquid droplet. The occurrence of stochastic liquid-liquid phase transitions within a cell nucleus provides an interesting framework to explain recent experimental observations on the transcriptional regulation in higher eukaryotes.

Figure 3

In Vitro and In Vivo Effects of ZMIZ1 Variants (A) Effect of ZMIZ1 variants on DHT-induced AR activity, as determined by the ARE-luciferase assay. HEK293T cells were cultivated with 1 nM DHT or without DHT (ethanol as vehicle) and transiently transfected with 30 ng of ZMIZ1 expression constructs in combination with 5 ng of pEGFP-ARwt (wild-type AR expression plasmid), 150 ng of pARE-luc (androgen response element (ARE)-driven luciferase reporter plasmid), and 20 ng of pRenilla-luc (renilla luciferase expression vector for normalization). The total amount of plasmids per well was kept constant by adding empty vectors as needed. Control transfections without the pEGFP-ARwt or the ZMIZ1 vectors were also performed. Transfection experiments were repeated three times in quadruplicate. Relative luciferase units (RLUs) are presented as the mean ± SEM. Two-tailed t test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (B) Effect of ZMIZ1 variants on neuronal migration and positioning. cDNA constructions were coelectroporated with an RFP-encoding reporter construction (dt-Tomato) in progenitor cells adjacent to the ventricle of E14.5 embryonic mouse cortices. Analyses were performed at E18.5. Images show coronal sections of E18.5 brains electroporated at E14.5 with either a control vector, WT ZMIZ1 or one of the three mutated forms of ZMIZ1. Sections were stained with 4′,6-diamidino-2-phenylindole (DAPI). Abbreviations: CP, cortical plate; IZ, intermediate zone; and VZ/SVZ, ventricular zone/subventricular zone. Scale bar, 50 μm. Histograms present the quantification of the percentage of fluorescent neurons in four different regions: upper and lower CP, IZ, and VZ/SVZ. Data are presented as the mean ± SEM, two-way ANOVA with Tukey’s multiple comparisons test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 compared to an empty vector control. (C) Morphological features of cells accumulated in the IZ with the three ZMIZ1 variants. Arrow shows cells with long and abnormally oriented processes. Double arrow shows a bipolar cell with a leading process oriented toward the VZ. Scale bar, 50 μm.