A novel neurodevelopmental syndrome caused by loss-of-function of the Zinc Finger Homeobox 3 (ZFHX3) gene

Abstract

AbstractNeurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function variation inZFHX3as a novel cause for syndromic intellectual disability (ID). ZFHX3, previously known as ATBF1, is a zinc-finger homeodomain transcription factor involved in multiple biological processes including cell differentiation and tumorigenesis.Through international collaboration, we collected clinical and morphometric data (Face2Gene) of 41 individuals with protein truncating variants (PTVs) or (partial) deletions ofZFHX3. We used data mining, RNA and protein analysis to identify the subcellular localization and spatiotemporal expression of ZFHX3 in multiplein vitromodels. We identified the DNA targets of ZFHX3 using ChIP seq. Immunoprecipitation followed by mass spectrometry indicated potential binding partners of endogenous ZFHX3 in neural stem cells that were subsequently confirmed by reversed co-immunoprecipitation and western blot. We evaluated a DNA methylation profile associated withZFHX3haploinsufficiency using DNA methylation analysis on whole blood extracted DNA of six individuals withZFHX3PTVs and four with a (partial) deletion ofZFHX3. A reversed genetic approach characterized theZFHX3orthologue inDrosophila melanogaster.Loss-of-function variation ofZFHX3consistently associates with (mild) ID and/or behavioural problems, postnatal growth retardation, feeding difficulties, and recognizable facial characteristics, including the rare occurrence of cleft palate.Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation in neural stem cells and SH-SY5Y cells, ZFHX3 interacts with the chromatin remodelling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex. In line with a role for chromatin remodelling,ZFHX3haploinsufficiency associates with a specific DNA methylation profile in leukocyte-derived DNA. The target genes of ZFHX3 are implicated in neuron and axon development.InDrosophila melanogaster, zfh2,considered to be theZFHX3orthologue, is expressed in the third instar larval brain. Ubiquitous and neuron-specific knockdown of zfh2 results in adult lethality underscoring a key role for zfh2 in development and neurodevelopment.Interestingly, ectopic expression of zfh2 as well as ZFHX3 in the developing wing disc results in a thoracic cleft phenotype.Collectively, our data shows that loss-of-function variants inZFHX3are a cause of syndromic ID, that associates with a specific DNA methylation profile. Furthermore, we show that ZFHX3 participates in chromatin remodelling and mRNA processing.