Loss-of-function of the Zinc Finger Homeobox 4 (ZFHX4) gene underlies a neurodevelopmental disorder

Abstract

Abstract8q21.11 microdeletions encompassing the gene encoding transcription factor ZFHX4, have previously been associated by us with a syndromic form of intellectual disability, hypotonia, decreased balance and hearing loss. Here, we report on 57 individuals, 52 probands and 5 affected family members, with protein truncating variants (n=36), (micro)deletions (n=20) or an inversion (n=1) affectingZFHX4with variable developmental delay and intellectual disability, distinctive facial characteristics, morphological abnormalities of the central nervous system, behavioral alterations, short stature, hypotonia, and occasionally cleft palate and anterior segment dysgenesis. The phenotypes associated with 8q21.11 microdeletions andZFHX4intragenic loss-of-function variants largely overlap, identifying ZFHX4 as the main driver for the microdeletion syndrome, although leukocyte-derived DNA shows a mild common methylation profile for (micro)deletions only. We identify ZFHX4 as a transcription factor that is increasingly expressed during human brain development and neuronal differentiation. Furthermore, ZFHX4 interacting factors identified via IP-MS in neural progenitor cells, suggest an important role for ZFHX4 in cellular and developmental pathways, especially during histone modifications, cytosolic transport and development. Additionally, using CUT&RUN, we observed that ZFHX4 binds with the promoter regions of genes with crucial roles in embryonic, neuron and axon development. Since loss-of-function variants inZFHX4are found with consistent dysmorphic facial features, we investigated whether the disruption ofzfhx4causes craniofacial abnormalities in zebrafish. First-generation (F0)zfhx4crispant zebrafish, (mosaic) mutant forzfhx4loss-of-function variants, have significantly shorter Meckel’s cartilages and smaller ethmoid plates compared to control zebrafish. Furthermore, behavioral assays show a decreased movement frequency in thezfhx4crispant zebrafish in comparison with control zebrafish larvae. Although further research is needed, ourin vivowork suggests a role for zfhx4 in facial skeleton patterning, palatal development and behavior.