Loss of function of the zinc finger homeobox 4 gene, ZFHX4, underlies a neurodevelopmental disorder

Abstract

8q21.11 microdeletions involving ZFHX4 have previously been associated with a syndromic form of intellectual disability, hypotonia, unstable gait, and hearing loss. We report on 63 individuals-57 probands and 6 affected family members-with protein-truncating variants (n = 41), (micro)deletions (n = 21), or an inversion (n = 1) affecting ZFHX4. Probands display 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 and ZFHX4 intragenic loss-of-function (LoF) variants largely overlap, although leukocyte-derived DNA shows a mild common methylation profile for (micro)deletions. ZFHX4 shows increased expression during human brain development and neuronal differentiation. Furthermore, ZFHX4-interacting factors identified via immunoprecipitation followed by mass spectrometry (IP-MS) suggest an important role for ZFHX4 in cellular pathways, especially during histone modifications, protein trafficking, signal transduction, cytosolic transport, and development. Additionally, using CUT&RUN, we observed that ZFHX4 binds the promoter of genes with crucial roles in embryonic, neuronal, and axonal development. Moreover, we investigated whether the disruption of zfhx4 causes craniofacial abnormalities in zebrafish. First-generation (F0) zfhx4 crispant zebrafish, a (mosaic) mutant for zfhx4 LoF variants, have significantly shorter Meckel's cartilage and smaller ethmoid plates compared to control zebrafish. Behavioral assays showed a decreased movement frequency in the zfhx4 crispant zebrafish in comparison with controls. Furthermore, structural abnormalities were found in the zebrafish hindbrain. In conclusion, our findings delineate a ZFHX4-associated neurodevelopmental disorder and suggest a role for zfhx4 in facial skeleton patterning, palatal development, and behavior.

Keywords: 8q21.11 microdeletion; ZFHX4; craniofacial development; methylation profile; neurodevelopmental disorder; neurogenesis; ocular anomalies; orofacial cleft; transcription factor; transcription regulation.

Graphical abstract

Figure 1

Front profile pictures and clinical representation of individuals with a deletion or PTV affecting ZFHX4 (A) Rows 1 and 2: frontal photographs of probands with a deletion containing ZFHX4. ^∗Proband 1 has an insertion breakpoint within ZFHX4. Rows 3 and 4: frontal photographs of probands with a ZFHX4 protein-truncating variant (PTV). °Proband 57 has compound heterozygous PTVs affecting ZFHX4. (B) Clinical presentation of individuals with ZFHX4 aberration. De-identified face mask of images. Top left: a total of 31 frontal photographs of 24 individuals with a deletion affecting ZFHX4. Bottom left: a total of 26 facial photographs of 18 individuals with predicted LoF variants in ZFHX4. Top right: matched healthy individuals. Note that no significant differences are shown in the facial features of both affected groups (i.e., ZFHX4 deletion vs. ZFHX4 PTV). The consent forms of the patients for the publication of the clinical photographs are available for this study.

Figure 2

Overview of ZFHX4 aberrations in 57 probands and 6 affected family members (A) (Micro)deletions identified in probands 1–21 (#) are represented by the red bars and the duplication in proband 15 by the yellow bar, all affecting ZFHX4 (light blue box). RefSeq coding genes are indicated in blue. Genomic positions are according to hg38; ZFHX4 is located on the reverse strand. The lightning bolts indicate the breakpoint positions of the inversion. (B) Nonsense, frameshift, and splice variants in ZFHX4 are identified in probands 22–57 and visualized with IBS 2.0. Protein and gene structure of the canonical transcript (GenBank: NM_024721.5) are shown on the top and bottom, respectively. The majority of PTVs are located in exons 2 and 10. The variants in probands 22–34, 36, 37, and 39–57 (Table S2) are shown from the N to the C terminus of the protein: p.Glu84^∗, p.Ser319Profs^∗3, p.Pro331Leufs^∗36, p.Ser341Ilefs^∗30, p.Ser341Ilefs^∗30, p.Leu408Argfs^∗7, p.Leu411Alafs^∗3, p.Leu411Alafs^∗3, p.Gly630Aspfs^∗16, p.Tyr655^∗, p.Gln681^∗, p.Gln681^∗, p.Trp767^∗, p.Leu903Tyrfs^∗18, p.Cys962Alafs^∗27, p.Arg1078Profs^∗19, p.Gly1114Glufs^∗14, p.?, p.Arg1384Glyfs^∗57, p.His1443Thrfs^∗8, p.Gln1712Valfs^∗64, p.Phe1739Serfs^∗37, p.Gln1797^∗, p.Gln1836Argfs^∗4, p.Lys1886^∗, p.Lys1886Glufs^∗33, p.Arg1899^∗, p.Cys1951Trpfs^∗2, p.Cys1951Trpfs^∗2, p.Gln2258Serfs^∗69, p.Thr2349Glnfs^∗43, p.Thr2349Glnfs^∗43, p.Gly2549Thrfs^∗35, p.Gly2549Thrfs^∗35, p.(Gly2549Thrfs^∗35, p.Gln2664^∗, p.His2874Serfs^∗30, p.His2874Serfs^∗30, p.His2874Serfs^∗30, and p.Trp2176^∗/p.Asn2188Lysfs^∗12. As the effect on the protein is not known for the splice variants in probands 35 and 38, only the c. notation is mentioned. Top: LoF variants are present in the ZFHX4 cohort and located in different domains of ZFHX4. Blue dots represent the frameshift variants, the yellow dots represent the nonsense variant, and the brown-gray dot represents the variants of proband 57, which contains a nonsense p.Trp2176^∗ and a frameshift p.Asn2188^∗fs mutation. The dot has been placed on the position of the nonsense variant p.Trp2176^∗. Bottom: coding exons are indicated as light blue boxes, and the UTRs are represented with white boxes. All c. notations can be found in Table S2.

Figure 3

Hi-C on proband 1 fibroblasts reveals disrupted chromatin structure at inversion breakpoints Hi-C contact frequency matrix for control and proband 1 fibroblasts (inversion indicated by red arrows). Close-up of left and right breakpoint regions, including observed/expected (O/E) contact frequencies for control and proband 1.

Figure 4

Deletion of a ZFHX4 region is associated with a mild methylation profile (A) The Euclidean clustering plot depicts ZFHX4 deletion samples in red, and the matched controls are shown in blue. The rows represent selected probes for the identified methylation pattern, while the columns represent training ZFHX4 deletion samples and controls. A clear separation between ZFHX4 deletion samples and controls was observed. Methylation levels are color coded: blue for 0 beta values and red for 1 beta values, with intensity indicating methylation level intensity. (B) The MDS plot demonstrates the differentiation between ZFHX4 deletion and control samples based on selected probes. The graph shows two dimensions of multidimensional scaling plots (x axis = coordinate 1, y axis = coordinate 2) representing the pairwise distance across the ZFHX4 deletion samples with controls. ZFHX4 deletion samples are represented by red circles and controls by blue circles. The plot shows individuals with ZFHX4 deletion cluster away from the controls. (C) Utilizing SVM classification trained with ZFHX4 deletion samples against matched controls, as well as 75% of other control samples and samples from other disorders (blue), in addition to the remaining 25% of the database samples used for testing (gray), produced significant findings. Except for the ZFHX4 deletion samples (training), all other conditions and ZFHX4 testing samples (ZFHX4_truncating) exhibited significantly low MVP scores, indicating an enhancement in the specificity of the model. The ZFHX4 testing samples, which encompass truncating variants (ZFHX4_truncating), a large duplication variant (ZFHX4_duplication), an inversion variant (ZFHX4 _inversion), and an intragenic deletion (ZFHX4_in_frame_del), were included, enabling the model to predict the class probability for ZFHX4 samples. ADCADN, cerebellar ataxia, deafness, and narcolepsy, autosomal dominant; ARTHS, Arboleda-Tham syndrome; ATRX, alpha-thalassemia/intellectual development syndrome, X-linked; AUTS18, autism, susceptibility to 18; BEFAHRS, Beck-Fahrner syndrome; BFLS, Börjeson-Forssman-Lehmann syndrome; BIS, blepharophimosis intellectual disability SMARCA2 syndrome; CdLS, Cornelia de Lange syndrome1–4; CHARGE, CHARGE syndrome; 16p11.2del, 16p11.2 deletion syndrome; CSS_c.6200, Coffin-Siris syndrome (c.6232G>A [GenBank: NM_006015.4 (ARID1A)] [p.Glu2078Lys]) (c.6254T>G [GenBank: NM_006015.4 (ARID1A)] p.Leu2085Arg) (c.6133T>C [GenBank: NM_017519.2 (ARID1B)] [p.Cys2045Arg]); CSS4_c.2650, Coffin-Siris syndrome (c.2656A>G [GenBank: NM_001128849.1 (SMARCA4)] [p.Met886Val]); CSS9, Coffin-Siris syndrome-9; Down, Down syndrome; Dup7, Williams-Beuren duplication syndrome (7q11.23 duplication syndrome); DYT28, Dystonia-28, childhood onset; EEOC, epileptic encephalopathy, childhood onset; FLHS, Floating-Harbour syndrome; GADEVS, Gabriele de Vries syndrome; GTPTS, Genitopatellar syndrome; HMA, HVDAS_C, Helsmoortel-Van der Aa syndrome (ADNP syndrome [central]); HVDAS_T, Helsmoortel-Van der Aa syndrome (ADNP syndrome [terminal]); ICF1 immunodeficiency, centromeric instability, facial anomalies syndrome 1; ICF2_3_4, immunodeficiency, centromeric instability, facial anomalies syndrome 2, 3, and 4; IDDSELD, intellectual developmental disorder with seizures and language delay; Kabuki, Kabuki syndromes 1 and 2; KDM2B, KDM2B-related syndrome; KDM4B, KDM4B-related syndrome; KDVS, Koolen de Vries syndrome; Kleefstra, Kleefstra syndrome 1; LLS, Luscan-Lumish syndrome; MKHK_ID4, Menke-Hennekam syndrome-1, 2; MLASA2, myopathy, lactic acidosis, and sideroblastic anemia-2; MRD23, intellectual developmental disorder, autosomal dominant 23; MRD51, intellectual developmental disorder, autosomal dominant 51; MRX93, intellectual developmental disorder, X-linked, XLID93; MRX97, intellectual developmental disorder, X-linked 97, XLID97; MRXSA, Armfield syndrome; MRXSCJ, syndromic X-linked intellectual disability, Claes-Jensen type; MRXSN, syndromic X-linked intellectual disability, Nascimento type; MRXSSR, syndromic X-linked intellectual disability, Snyder-Robinson type; PHMDS, Phelan-McDermid syndrome; PRC2, RENS1, Renpenning syndrome; RMNS, Rahman syndrome; RSTS, Rubinstein-Taybi syndrome-1, 2; RSTS1, Rubinstein-Taybi syndrome-1; RSTS2, Rubinstein-Taybi syndrome-2; SBBYSS, Say-Barber-Biesecker-Young-Simpson syndrome; Sotos, Sotos syndrome; TBRS, Tatton-Brown-Rahman syndrome; VCFS_comp, velocardiofacial syndrome; VCFS_core, velocardiofacial syndrome; WDSTS, Wiedemann-Steiner syndrome; WHS, Wolf-Hirschhorn syndrome; Williams, Williams-Beuren deletion syndrome (7q11.23 deletion syndrome).

Figure 5

ZFHX4 expression increases during human neuronal differentiation and brain and craniofacial development (A) ZFHX4 expression levels during in vitro neural differentiation. RNA was sampled at the following stages: iPSCs, NSC (neural stem cells), neural progenitor cells (NPCs), and direct differentiated neurons (Ns). An increasing expression is observed upon differentiation. The median expression value is indicated by the middle horizontal line. The whiskers indicate the minimum and maximum values. CNRQ, calibrated normalized relative quantity. (B) Endogenous ZFHX4 (399 kDa) levels in HEK293T, iPSCs, and NPCs as detected with western blot using the ZFHX4 antibody (top) and a vinculin antibody as a loading control (bottom). (C) ZFHX4 levels in developing human brain. ZFHX4 expression in the developing human brain (normalized RPKM data) shows higher expression during early prenatal development followed by decreased expression upon brain maturation. Data were obtained from BrainSpan: http://www.brainspan.org. (D) Human embryonic craniofacial tissue bulk RNA-seq is visualized in a plot, showing an increase in ZFHX4 in craniofacial samples from five Carnegie stages (CS13, CS14, CS15, CS17, and CS22).

Figure 6

ZFHX4 interacts with 46 enriched proteins involved in axonogenesis, cell differentiation, and neuron differentiation (A) The enriched ZFHX4-interacting proteins in NPCs shown in a volcano plot. For each detected protein, the log2 fold change (FC) between the ZFHX4 IP-enriched samples and the IgG controls is shown on the x axis, while the −log10 of the p value is shown on the y axis. The horizontal dashed line indicates the −log10 value of a p value equal to 0.05. Proteins with an FDR < 0.05, a log2 FC > 2, and a log2 FC < −2 are, respectively, highlighted in blue and red. (B) Hierarchical clustering of potentially interesting proteins. The proteins shown to be differentially abundant between groups are visualized in a heatmap after non-supervised hierarchical clustering of Z scored log2 PG.MaxLFQ intensities. The heatmap color scale from blue to red represents the level of abundance. The interacting factors that are absent in the IgG control, significant but not absent in the IgG control, and significantly enriched in the protein and absent in the IgG control are listed in Tables S8 and S9. (C) ZFHX4-interacting factors associated to their biological processes with EnrichR.

Figure 7

CUT&RUN sequencing data show ZFHX4 binding to genes involved in axonogenesis, the regulation of the nervous system development, neurogenesis, and neural migration (A) Annotation of CUT&RUN consensus peaks reveals ZFHX4 binding to promoter regions. Left: distribution of the ZFHX4 consensus peaks relative to transcription start sites (TSSs). Half of the peaks are found between 0 and 1 kb of the TSS. Right: pie chart visualizing the genomic annotation and the percentage of the peaks that overlap a TSS, 5′ UTR, 3′ UTR, or exonic, intronic, downstream, or distal intergenic regions. This shows that 55.62% of ZFHX4 consensus peaks are found near gene promoters. (B) ZFHX4 binds to genes involved in axonogenesis, forebrain, and nervous system development, including the regulation of the neurogenesis and synapse organization and cell and neuron differentiation. The Gene Ontology (GO) dot plot displays the top 20 enriched biological processes (BPs) ranked by gene ratio (the number of genes related to GO term/total number of significant genes) and the p-adjusted values for these terms (color, p = 1 × 10⁻⁷–1 × 10⁻⁵). The size of the dot represents the gene counts per BP. (C) ZFHX4 plays a key role in embryonic, neuron, and axon developmental pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) dot plot provides all enriched biological pathways ranked by gene ratio (# of genes related to GO term/total number of significant genes) and the p-adjusted values for these terms (color). The size of the dot represents the gene counts per pathway.

Figure 8

Zebrafish zfhx4 crispants showed both craniofacial and behavioral changes (A) Gene structure of the zebrafish zfhx4 and ZFHX4. Top: the coding exons of ZFHX4 are shown as light gray boxes and the CDS (coding sequence) as light blue boxes, and untranslated regions are in red (5′ UTR) and green (3′ UTR). Bottom: the coding exons of zfhx4 are shown as light gray boxes and the CDS as light blue boxes, and untranslated regions are in red (5′ UTR) and green (3′ UTR). (B) Representative craniofacial images for zfhx4 and scrambled zebrafish crispants are shown. Left: microscopic ventral view scrambled (top) and zfhx4 crispants (bottom) of Alcian blue-stained craniofacial structures at 5 dpf reveal smaller ethmoid plates and shorter Meckel’s cartilage in the F0 zfhx4 crispant larvae. Right: adapted image from the representation of the cartilage structures of the zebrafish neurocranium (dark blue) at 5 dpf by lines. The red lines and circle show the measurements taken and normalized with the length of the head (purple line). (C) Left: the surface area of the ethmoid plate was measured and normalized to the head length of the larvae. Right: the length of the individual Meckel’s cartilage structures was measured and normalized to the head length of the larvae. The ethmoid plate and the Meckel’s cartilage showed significant differences between crispant larvae compared to scrambled larvae (^∗∗∗p ≤ 0.001 and ^∗∗∗∗p ≤ 0.0001). (D) zfhx4 crispants show a consistently lower movement frequency compared to scrambled control larvae during the complete behavior experiment, but the trends during light and dark cycles remain similar. Dark periods are indicated with gray boxes. Data are represented as mean ± SEM (n = 60/genotype).