Abstract
Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function variation in ZFHX3 as 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 of ZFHX3. We used data mining, RNA and protein analysis to identify the subcellular localization and spatiotemporal expression of ZFHX3 in multiple in vitro models. 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 with ZFHX3 haploinsufficiency using DNA methylation analysis on whole blood extracted DNA of six individuals with ZFHX3 PTVs and four with a (partial) deletion of ZFHX3. A reversed genetic approach characterized the ZFHX3 orthologue in Drosophila melanogaster.
Loss-of-function variation of ZFHX3 consistently 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, ZFHX3 haploinsufficiency associates with a specific DNA methylation profile in leukocyte-derived DNA. The target genes of ZFHX3 are implicated in neuron and axon development.
In Drosophila melanogaster, zfh2, considered to be the ZFHX3 orthologue, 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 in ZFHX3 are 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.
Competing Interest Statement
Lindsay Rhodes is an employee of GeneDx, LLC. Xia Wang is a co-founder and employee of AiLife Diagnostics.
Funding Statement
Financial support has been provided by grants 1520518N, G044615N and G055422N from the Research Foundation Flanders (FWO) and BOF STA 201909009 from the Special Research Fund (BOF) from Ghent University. B.S. received funding from the government of Canada through Genome Canada and the Ontario Genomics Institute (OGI 188). M.R.P.B is supported by a doctoral grant of the Marguerite Marie Delacroix Foundation. E.Z.J. was supported by a doctoral grant of the Research Foundation Flanders. B.C. is a senior clinical investigator of the Research Foundation Flanders. MHW is supported by NIH NICHD K23 HD102589, NIH NHGRI R21 HG012397 and an Early Career Award from the Thrasher Research Fund. Sequencing and analysis for patient 15 were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics and was funded by the National Human Genome Research Institute, the National Eye Institute, and the National Heart, Lung and Blood Institute grant UM1 HG008900, and in part by National Human Genome Research Institute grants U01 HG0011755 and R01 HG009141.
Author Declarations
I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.
Yes
The details of the IRB/oversight body that provided approval or exemption for the research described are given below:
Legal guardians of affected probands gave informed consent for genomic investigations and publication of pseudonymized data following the rules of the Helsinki declaration and according to EC2019/1430 of Ghent University Hospital and/or local ethical committees of the referring centers and the Western University Research Ethics Board (REB 106302).
I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.
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Data Availability
Public data was used from: https://apps.kaessmannlab.org/evodevoapp/ https://www.gtexportal.org/home/ http://www.brainspan.org https://bioinf.eva.mpg.de/shiny/sample-apps/scApeX/ https://shcheglovitov.shinyapps.io/u_brain_browser/ https://gnomad.broadinstitute.org/about https://doi.org/doi:10.17989/ENCSR259WOE The analyzed IP-MS data and the overlapping peaks between endogenous and ectopic ZFHX3 ChIP-seq is available in the Supplementary Material. Some of the DNA methylation datasets used in this study are publicly available and may be obtained from gene expression omnibus (GEO) using the following accession numbers. GEO: GSE116992, GSE66552, GSE74432, GSE97362, GSE116300, GSE95040, GSE 104451, GSE125367, GSE55491, GSE108423, GSE116300, GSE 89353, GSE52588, GSE42861, GSE85210, GSE87571, GSE87648, GSE99863, and GSE35069. These include DNA methylation data from patients with Kabuki syndrome, Sotos syndrome, CHARGE syndrome, immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, Williams-Beuren syndrome, Chr7q11.23 duplication syndrome, BAFopathies, Down syndrome, a large cohort of unresolved subjects with developmental delays and congenital abnormalities, and also several large cohorts of DNA methylation data from the general population. Remaining methylation data is not publicly available due to institutional and ethics restrictions.
https://apps.kaessmannlab.org/evodevoapp/
https://www.gtexportal.org/home/
https://bioinf.eva.mpg.de/shiny/sample-apps/scApeX/
https://shcheglovitov.shinyapps.io/u_brain_browser/
https://gnomad.broadinstitute.org/about
https://doi.org/doi:10.17989/ENCSR259WOE