Dynamic BTB-domain filaments promote clustering of ZBTB proteins.

The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.

A novel heterozygous ZBTB18 missense mutation in a family with non-syndromic intellectual disability.

Intellectual disability (ID) is a common neurodevelopmental disorder characterized by significantly impaired adaptive behavior and cognitive capacity. High throughput sequencing approaches have revealed the genetic etiologies for 25-50% of ID patients, while inherited genetic mutations were detected in <5% cases. Here, we investigated the genetic cause for non-syndromic ID in a Han Chinese family. Whole genome sequencing was performed on identical twin sisters diagnosed with ID, their respective children, and their asymptomatic parents. Data was filtered for rare variants, and in silico prediction tools were used to establish pathogenic alleles. Candidate mutations were validated by Sanger sequencing. In silico modeling was used to evaluate the mutation's effects on the protein encoded by a candidate coding gene. A novel heterozygous variant in the ZBTB18 gene c.1323C>G (p.His441Gln) was identified. This variant co-segregated with affected individuals in an autosomal dominant pattern and was not detected in asymptomatic family members. Molecular studies reveal that a p.His441Gln substitution disrupts zinc binding within the second zinc finger and disrupts the capacity for ZBTB18 to bind DNA. This is the first report of an inherited ZBTB18 mutation for ID. This study further validates WGS for the accurate molecular diagnosis of ID.

The genetic basis of neurocranial size and shape across varied lab mouse populations.

Brain and skull tissues interact through molecular signalling and mechanical forces during head development, leading to a strong correlation between the neurocranium and the external brain surface. Therefore, when brain tissue is unavailable, neurocranial endocasts are often used to approximate brain size and shape. Evolutionary changes in brain morphology may have resulted in secondary changes to neurocranial morphology, but the developmental and genetic processes underlying this relationship are not well understood. Using automated phenotyping methods, we quantified the genetic basis of endocast variation across large genetically varied populations of laboratory mice in two ways: (1) to determine the contributions of various genetic factors to neurocranial form and (2) to help clarify whether a neurocranial variation is based on genetic variation that primarily impacts bone development or on genetic variation that primarily impacts brain development, leading to secondary changes in bone morphology. Our results indicate that endocast size is highly heritable and is primarily determined by additive genetic factors. In addition, a non-additive inbreeding effect led to founder strains with lower neurocranial size, but relatively large brains compared to skull size; suggesting stronger canalization of brain size and/or a general allometric effect. Within an outbred sample of mice, we identified a locus on mouse chromosome 1 that is significantly associated with variation in several positively correlated endocast size measures. Because the protein-coding genes at this locus have been previously associated with brain development and not with bone development, we propose that genetic variation at this locus leads primarily to variation in brain volume that secondarily leads to changes in neurocranial globularity. We identify a strain-specific missense mutation within Akt3 that is a strong causal candidate for this genetic effect. Whilst it is not appropriate to generalize our hypothesis for this single locus to all other loci that also contribute to the complex trait of neurocranial skull morphology, our results further reveal the genetic basis of neurocranial variation and highlight the importance of the mechanical influence of brain growth in determining skull morphology.

Prenatal diagnosis of a likely pathogenic variant in ZBTB18: Natural evolution of fetal phenotype including the long bones and corpus callosum.

Pathogenic variants in ZBTB18 gene have been described only postnatally with a variable phenotypic spectrum that includes intellectual disability, microcephaly, hypotonia, poor growth, corpus callosum abnormalities, seizures, and dysmorphic facial features. These features overlap with the phenotype of 1q43-q44 deletion syndrome (OMIM #612337). There are several genes within the 1q43-q44 deletion region, and ZBTB18 is of particular interest due to its known involvement in neuronal differentiation and migration. We describe here a fetus presenting with an intrauterine growth restriction, diminished long bones growth, single umbilical artery, and a short corpus callosum. On mid pregnancy ultrasound, all biometric parameters including the corpus callosum were relatively small but still within the normal range. Only a targeted follow-up during the third trimester, including neurosonographic and MRI exams, revealed the full extent of the malformation, leading to amniocentesis and a genetic workup that led to the identification of a de novo likely pathogenic variant in ZBTB18 gene. This is the first description of the evolving phenotype of a ZBTB18-related disorder in a fetus, which emphasizes the challenging diagnosis of subtle findings, that mandates a high level of clinical suspicion and a targeted follow-up throughout pregnancy.

CircTP63 promotes hepatocellular carcinoma progression by sponging miR-155-5p and upregulating ZBTB18.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear. METHODS: qRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18. RESULTS: circTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression. CONCLUSION: Our data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

General population ZBTB18 missense variants influence DNA binding and transcriptional regulation.

Genetic variation of the multi-zinc finger BTB domain transcription factor ZBTB18 can cause a spectrum of human neurodevelopmental disorders, but the underlying mechanisms are not well understood. Recently, we reported that pathogenic, de novo ZBTB18 missense mutations alter its DNA-binding specificity and gene regulatory functions, leading to human neurodevelopmental disease. However, the functional impact of the general population ZBTB18 missense variants is unknown. Here, we investigated such variants documented in the Genome Aggregation Database (gnomAD) to discover that ZBTB gene family members are intolerant to loss-of-function and missense mutations, but not synonymous mutations. We studied ZBTB18 as a protein-DNA complex to find that general population missense variants are rare, and disproportionately map to non-DNA-contact residues, in contrast to the majority of disease-associated variants that map to DNA-contact residues, essential to motif binding. We studied a selection of variants (n = 12), which spans the multi-zinc finger region to find 58.3% (7/12) of variants displayed altered DNA binding, 41.6% (5/12) exhibited altered transcriptional activity in a luciferase reporter assay, 33.3% (4/12) exhibited altered DNA binding and transcriptional activity, whereas 33.3% (4/12) displayed a negligible functional impact. Our results demonstrate that general population variants, while rare, can influence ZBTB18 function, with potential consequences for neurodevelopment, homeostasis, and disease.

Disease-associated missense variants in ZBTB18 disrupt DNA binding and impair the development of neurons within the embryonic cerebral cortex.

The activities of DNA-binding transcription factors, such as the multi-zinc-finger protein ZBTB18 (also known as RP58, or ZNF238), are essential to coordinate mammalian neurodevelopment, including the birth and radial migration of newborn neurons within the fetal brain. In humans, the majority of disease-associated missense mutations in ZBTB18 lie within the DNA-binding zinc-finger domain and are associated with brain developmental disorder, yet the molecular mechanisms explaining their role in disease remain unclear. To address this, we developed in silico models of ZBTB18, bound to DNA, and discovered that half of the missense variants map to residues (Asn461, Arg464, Glu486) predicted to be essential to sequence-specific DNA contact, whereas others map to residues (Leu434, Tyr447, Arg495) with limited contributions to DNA binding. We studied pathogenic variants to residues with close (N461S) and limited (R495G) DNA contact and found that each bound DNA promiscuously, displayed altered transcriptional regulatory activity in vitro, and influenced the radial migration of newborn neurons in vivo in different ways. Taken together, our results suggest that altered transcriptional regulation could represent an important pathological mechanism for ZBTB18 missense variants in brain developmental disease.

Differential Expression of Genes for Ubiquitin Ligases in Medulloblastoma Subtypes.

Using publically available datasets on gene expression in medulloblastoma (MB) subtypes, we selected genes for ubiquitin ligases and identified statistically those that best predicted each of the four major MB subgroups as separate disease entities. We identify a gene coding for an ubiquitin ligase, ZNRF3, whose overexpression alone can predict the WNT subgroup for 100% in the Pfister dataset. For the SHH subgroup, we identify a gene for a regulatory subunit of the protein phosphatase 2A (PP2A), PPP2R2C, as the major predictor among the E3 ligases genes. The ubiquitin and ubiquitin-like conjugation database (UUCD) lists PPP2R2C as coding for a Cullin Ring ubiquitin ligase adaptor. For group 3 MBs, the best ubiquitin ligase predictor was PPP2R2B, a gene which codes for another regulatory subunit of the PP2A holoenzyme. For group 4, the best E3 gene predictors were MID2, ZBTB18, and PPP2R2A, which codes for a third PP2A regulatory subunit. Heatmap analysis of the E3 gene data shows that expression of ten genes for ubiquitin ligases can be used to classify MBs into the four major consensus subgroups. This was illustrated by analysis of gene expression of ubiquitin ligases of the Pfister dataset and confirmed in the dataset of Cavalli. We conclude that genes for ubiquitin ligases can be used as genetic markers for MB subtypes and that the proteins coded for by these genes should be investigated as subtype specific therapeutic targets for MB.

Further evidence that de novo missense and truncating variants in ZBTB18 cause intellectual disability with variable features.

Identification of rare genetic variants in patients with intellectual disability (ID) has been greatly accelerated by advances in next generation sequencing technologies. However, due to small numbers of patients, the complete phenotypic spectrum associated with pathogenic variants in single genes is still emerging. Among these genes is ZBTB18 (ZNF238), which is deleted in patients with 1q43q44 microdeletions who typically present with ID, microcephaly, corpus callosum (CC) abnormalities, and seizures. Here we provide additional evidence for haploinsufficiency or dysfunction of the ZBTB18 gene as the cause of ID in five unrelated patients with variable syndromic features who underwent whole exome sequencing revealing separate de novo pathogenic or likely pathogenic variants in ZBTB18 (two missense alterations and three truncating alterations). The neuroimaging findings in our cohort (CC hypoplasia seen in 4/4 of our patients who underwent MRI) lend further support for ZBTB18 as a critical gene for CC abnormalities. A similar phenotype of microcephaly, CC agenesis, and cerebellar vermis hypoplasia has been reported in mice with central nervous system-specific knockout of Zbtb18. Our five patients, in addition to the previously described cases of de novo ZBTB18 variants, add to knowledge about the phenotypic spectrum associated with ZBTB18 haploinsufficiency/dysfunction.

A de novo nonsense mutation in ZBTB18 plus a de novo 15q13.3 microdeletion in a 6-year-old female.

ZBTB18 has been proposed as candidate gene for microcephaly and abnormalities of the corpus callosum based on overlapping microdeletions of 1q43q44. More recently, de novo mutations of ZBTB18 have been identified in patients with syndromic and non-syndromic intellectual disability. Heterozygous microdeletions of 15q13.3 encompassing the candidate gene CHRNA7 are associated with developmental delay or intellectual disability with speech problems, hypotonia, and seizures. They are characterized by significant variability and reduced penetrance. We report on a patient with a de novo ZBTB18 nonsense mutation and a de novo 15q13.3 microdeletion, both in a heterozygous state, identified by next generation sequencing and array-CGH. The 6-year-old girl showed global developmental delay, absent speech, therapy-refractory seizures, ataxia, muscular hypotonia, and discrete facial dysmorphisms. Almost all of these features have been reported for both genetic aberrations, but the severity could hardly been explained by the microdeletion 15q13.3 alone. We assume an additive effect of haploinsufficiency of ZBTB18 and CHRNA7 in our patient. Assembling the features of our patient and the published patients, we noted that only one of them showed mild anomalies of the corpus callosum. Moreover, we hypothesize that nonsense mutations of ZBTB18 are associated with a more severe phenotype than missense mutations. This report indicates that haploinsufficiency of additional genes beside ZBTB18 causes the high frequency of corpus callosum anomalies in patients with microdeletions of 1q43q44 and underlines the importance of an NGS-based molecular diagnostic in complex phenotypes.

p21/Zbtb18 repress the expression of cKit to regulate the self-renewal of hematopoietic stem cells.

The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21-tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT+ HSCs exhibited increased self-renewal capacity compared to p21-tdT- HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT+ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.

Analyses of Genes Critical to Tumor Survival Reveal Potential 'Supertargets': Focus on Transcription.

The identification of mechanisms that underlie the biology of individual tumors is aimed at the development of personalized treatment strategies. Herein, we performed a comprehensive search of genes (termed Supertargets) vital for tumors of particular tissue origin. In so doing, we used the DepMap database portal that encompasses a broad panel of cell lines with individual genes knocked out by CRISPR/Cas9 technology. For each of the 27 tumor types, we revealed the top five genes whose deletion was lethal in the particular case, indicating both known and unknown Supertargets. Most importantly, the majority of Supertargets (41%) were represented by DNA-binding transcription factors. RNAseq data analysis demonstrated that a subset of Supertargets was deregulated in clinical tumor samples but not in the respective non-malignant tissues. These results point to transcriptional mechanisms as key regulators of cell survival in specific tumors. Targeted inactivation of these factors emerges as a straightforward approach to optimize therapeutic regimens.

Identification of ZBTB18 as a novel colorectal tumor suppressor gene through genome-wide promoter hypermethylation analysis.

BACKGROUND: Cancer initiation and progression are driven by genetic and epigenetic changes. Although genome/exome sequencing has significantly contributed to the characterization of the genetic driver alterations, further investigation is required to systematically identify cancer driver genes regulated by promoter hypermethylation. RESULTS: Using genome-wide analysis of promoter methylation in 45 colorectal cancer cell lines, we found that higher overall methylation levels were associated with microsatellite instability (MSI), faster proliferation and absence of APC mutations. Because epigenetically silenced genes could represent important oncogenic drivers, we used mRNA expression profiling of colorectal cancer cell lines and primary tumors to identify a subset of 382 (3.9%) genes for which promoter methylation was negatively associated with gene expression. Remarkably, a significant enrichment in zinc finger proteins was observed, including the transcriptional repressor ZBTB18. Re-introduction of ZBTB18 in colon cancer cells significantly reduced proliferation in vitro and in a subcutaneous xenograft mouse model. Moreover, immunohistochemical analysis revealed that ZBTB18 is frequently lost or reduced in colorectal tumors, and reduced ZBTB18 expression was found to be associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. CONCLUSIONS: We identified a set of 382 genes putatively silenced by promoter methylation in colorectal cancer that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZBTB18 has tumor suppressor activity and is a novel prognostic marker for patients with locally advanced colorectal cancer.

Nervous system regulated by POZ domain Krüppel-like zinc finger (POK) family transcription repressor RP58.

The POZ domain Krüppel-like zinc finger transcription repressor (POK family) contains many important molecules, including RP58, Bcl6 and PLZF. They function as transcription repressors via chromatin remodelling and histone deacetylation and are known to be involved in the development and tumourigenesis of various organs. Furthermore, they are important in the formation and function of the nervous system. This review summarizes the role of the POK family transcription repressors in the nervous system. We particularly targeted Rp58 (also known as Znf238, Znp238 and Zbtb18), a sequence-specific transcriptional repressor that is strongly expressed in developing glutamatergic projection neurons in the cerebral cortex. It regulates various physiological processes, including neuronal production, neuronal migration and neuronal maturation. Human studies suggest that reduced RP58 levels are involved in cognitive function impairment and brain tumour formation. This review particularly focuses on the mechanisms underlying RP58-mediated neuronal development and function. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc.

Structure-Based Approaches to Classify the Functional Impact of ZBTB18 Missense Variants in Health and Disease.

The Cys2His2 type zinc finger is a motif found in many eukaryotic transcription factor proteins that facilitates binding to genomic DNA so as to influence cellular gene expression. One such transcription factor is ZBTB18, characterized as a repressor that orchestrates the development of mammalian tissues including skeletal muscle and brain during embryogenesis. In humans, it has been recognized that disease-associated ZBTB18 missense variants mapping to the coding sequence of the zinc finger domain influence sequence-specific DNA binding, disrupt transcriptional regulation, and impair neural circuit formation in the brain. Furthermore, general population ZBTB18 missense variants that influence DNA binding and transcriptional regulation have also been documented within this domain; however, the molecular traits that explain why some variants cause disease while others do not are poorly understood. Here, we have applied five structure-based approaches to evaluate their ability to discriminate between disease-associated and general population ZBTB18 missense variants. We found that thermodynamic integration and Residue Scanning in the Schrodinger Biologics Suite were the best approaches for distinguishing disease-associated variants from general population variants. Our results demonstrate the effectiveness of structure-based approaches for the functional characterization of missense alleles to DNA binding, zinc finger transcription factor protein-coding genes that underlie human health and disease.

RP58 Represses Transcriptional Programs Linked to Nonneuronal Cell Identity and Glioblastoma Subtypes in Developing Neurons.

How mammalian neuronal identity is progressively acquired and reinforced during development is not understood. We have previously shown that loss of RP58 (ZNF238 or ZBTB18), a BTB/POZ-zinc finger-containing transcription factor, in the mouse brain leads to microcephaly, corpus callosum agenesis, and cerebellum hypoplasia and that it is required for normal neuronal differentiation. The transcriptional programs regulated by RP58 during this process are not known. Here, we report for the first time that in embryonic mouse neocortical neurons a complex set of genes normally expressed in other cell types, such as those from mesoderm derivatives, must be actively repressed in vivo and that RP58 is a critical regulator of these repressed transcriptional programs. Importantly, gene set enrichment analysis (GSEA) analyses of these transcriptional programs indicate that repressed genes include distinct sets of genes significantly associated with glioma progression and/or pluripotency. We also demonstrate that reintroducing RP58 in glioma stem cells leads not only to aspects of neuronal differentiation but also to loss of stem cell characteristics, including loss of stem cell markers and decrease in stem cell self-renewal capacities. Thus, RP58 acts as an in vivo master guardian of the neuronal identity transcriptome, and its function may be required to prevent brain disease development, including glioma progression.

CtBP2 interacts with ZBTB18 to promote malignancy of glioblastoma.

OBJECTIVE: To investigate how the interaction of CtBP2 with ZBTB18 affect glioblastoma (GBM). METHODS: Western blotting was performed to detect CtBP2 and ZBTB18 expression in GBM and normal brain tissues (NBT). U-87 MG cells were transfected with ZBTB18 CRISPR activation plasmid, CtBP2 shRNA with/without ZBTB18 shRNA. The biological characteristics were detected by EdU assay, MTT, Wound-healing, Transwell, TUNEL staining, and Flow cytometry. Furthermore, U-87 MG cells transfected with CtBP2 shRNA and/or ZBTB18 shRNA were injected into the flank region of mice and the tumor volume was measured. The mRNA and protein expression was quantified by qRT-PCR or Western blotting. RESULTS: GBM tissues exhibited increased CtBP2 expression and decreased ZBTB18 expression, which demonstrated a negative correlation in GBM tissues and showed the combined effect on prognosis. Based on immunoprecipitation and immunofluorescence, there was an interaction between CtBP2 and ZBTB18 in U-87 MG cells. CtBP2 shRNA counteracted the effect of ZBTB18 shRNA on inhibiting U-87 MG cell apoptosis, as well as promoting cell proliferation and viability with increased EMT, invasion and migration. Meanwhile, CtBP2 shRNA interact with ZBTB18 to block cells at phase G0/G1 and suppress SHH-GLI1 pathway. CtBP2 shRNA decreased tumor volume, increase ZBTB18 expression in tumor tissues, and inhibit SHH-GLI1 pathway in mice, which could be reversed by ZBTB18 shRNA. CONCLUSION: CtBP2 elevation and ZBTB18 down-regulation were found in GBM, both of which were associated with prognosis of GBM patients. CtBP2 interacted with ZBTB18 to affect biological characteristics of GBM cells, and the tumor growth, which may be related to the SHH-GLI1 pathway.

The Role of AKT3 Copy Number Changes in Brain Abnormalities and Neurodevelopmental Disorders: Four New Cases and Literature Review.

Microdeletions at 1q43-q44 have been described as resulting in a clinically recognizable phenotype of intellectual disability (ID), facial dysmorphisms and microcephaly (MIC). In contrast, the reciprocal microduplications of 1q43-q44 region have been less frequently reported and patients showed a variable phenotype, including macrocephaly. Reports of a large number of patients with copy number variations involving this region highlighted the AKT3 gene as a likely key player in head size anomalies. We report four novel patients with copy number variations in the 1q43-q44 region: one with a larger deletion (3.7Mb), two with smaller deletions affecting AKT3 and SDCCAG8 genes (0.16 and 0.18Mb) and one with a quadruplication (1Mb) that affects the entire AKT3 gene. All patients with deletions presented MIC without structural brain abnormalities, whereas the patient with quadruplication had macrocephaly, but his carrier father had normal head circumference. Our report also includes a comparison of phenotypes in cases with 1q43-q44 duplications to assist future genotype-phenotype correlations. Our observations implicate AKT3 as a contributor to ID/development delay (DD) and head size but raise doubts about its straightforward impact on the latter aspect of the phenotype in patients with 1q43-q44 deletion/duplication syndrome.

Toward clinical and molecular understanding of pathogenic variants in the ZBTB18 gene.

BACKGROUND: Patients with pathogenic variants in ZBTB18 present with Intellectual Disability (ID) with frequent co-occurrence of corpus callosum (CC) anomalies, hypotonia, microcephaly, growth problems and variable facial dysmorphologies. These features illustrate a key role for ZBTB18 in brain development. METHODS: Patients with a pathogenic variant in ZBTB18 were detected by diagnostic whole exome sequencing (WES) performed in our center. We reviewed the literature and used GeneMatcher to include other cases. YASARA and WHAT IF were used to provide insight into the structural effect of missense variants located in the C2H2 zinc finger domains of the ZBTB18 protein. RESULTS: We give a complete overview of pathogenic variants in ZBTB18 detected to date, showing inconsistent presence of clinical features, including CC anomalies. We present four new cases with a de novo pathogenic variant in the ZBTB18 gene, including the fourth case in which a de novo p.Arg464His variant was found. CONCLUSION: Homology modeling of protein structure points to a variable degree of impaired DNA binding caused by missense variants in these domains probably leading to Loss of Function (LoF). Putative partial LoF may present with a less distinctive phenotype than complete LoF, as seen in truncating variants, which presents with an extensive variability in the phenotypic spectrum. Our data do not support a clear genotype to phenotype correlation.

FOXG1 Orchestrates Neocortical Organization and Cortico-Cortical Connections.

The hallmarks of FOXG1 syndrome, which results from mutations in a single FOXG1 allele, include cortical atrophy and corpus callosum agenesis. However, the etiology for these structural deficits and the role of FOXG1 in cortical projection neurons remain unclear. Here we demonstrate that Foxg1 in pyramidal neurons plays essential roles in establishing cortical layers and the identity and axon trajectory of callosal projection neurons. The neuron-specific actions of Foxg1 are achieved by forming a transcription complex with Rp58. The Foxg1-Rp58 complex directly binds and represses Robo1, Slit3, and Reelin genes, the key regulators of callosal axon guidance and neuronal migration. We also found that inactivation of one Foxg1 allele specifically in cortical neurons was sufficient to cause cerebral cortical hypoplasia and corpus callosum agenesis. Together, this study reveals a novel gene regulatory pathway that specifies neuronal characteristics during cerebral cortex development and sheds light on the etiology of FOXG1 syndrome. VIDEO ABSTRACT.