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.

Understanding the impact of ZBTB18 missense variation on transcription factor function in neurodevelopment and disease.

Mutations to genes that encode DNA-binding transcription factors (TFs) underlie a broad spectrum of human neurodevelopmental disorders. Here, we highlight the pathological mechanisms arising from mutations to TF genes that influence the development of mammalian cerebral cortex neurons. Drawing on recent findings for TF genes including ZBTB18, we discuss how functional missense mutations to such genes confer non-native gene regulatory actions in developing neurons, leading to cell-morphological defects, neuroanatomical abnormalities during foetal brain development and functional impairment. Further, we discuss how missense variation to human TF genes documented in the general population endow quantifiable changes to transcriptional regulation, with potential cell biological effects on the temporal progression of cerebral cortex neuron development and homeostasis. We offer a systematic approach to investigate the functional impact of missense variation in brain TFs and define their direct molecular and cellular actions in foetal neurodevelopment, tissue homeostasis and disease states.

Further evidence for distinct traits associated with RBM10 missense variants.

A case of a missense RBM10 variant in an adult with mild to moderate intellectual disability.

The atypical Rho GTPase Rnd2 is critical for dentate granule neuron development and anxiety-like behavior during adult but not neonatal neurogenesis.

Despite the central role of Rho GTPases in neuronal development, their functions in adult hippocampal neurogenesis remain poorly explored. Here, by using a retrovirus-based loss-of-function approach in vivo, we show that the atypical Rho GTPase Rnd2 is crucial for survival, positioning, somatodendritic morphogenesis, and functional maturation of adult-born dentate granule neurons. Interestingly, most of these functions are specific to granule neurons generated during adulthood since the deletion of Rnd2 in neonatally-born granule neurons only affects dendritogenesis. In addition, suppression of Rnd2 in adult-born dentate granule neurons increases anxiety-like behavior whereas its deletion in pups has no such effect, a finding supporting the adult neurogenesis hypothesis of anxiety disorders. Thus, our results are in line with the view that adult neurogenesis is not a simple continuation of earlier processes from development, and establish a causal relationship between Rnd2 expression and anxiety.

Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression.

Our understanding of the transcriptional programme underpinning adult hippocampal neurogenesis is incomplete. In mice, under basal conditions, adult hippocampal neural stem cells (AH-NSCs) generate neurons and astrocytes, but not oligodendrocytes. The factors limiting oligodendrocyte production, however, remain unclear. Here, we reveal that the transcription factor NFIX plays a key role in this process. NFIX is expressed by AH-NSCs, and its expression is sharply upregulated in adult hippocampal neuroblasts. Conditional ablation of Nfix from AH-NSCs, coupled with lineage tracing, transcriptomic sequencing and behavioural studies collectively reveal that NFIX is cell-autonomously required for neuroblast maturation and survival. Moreover, a small number of AH-NSCs also develop into oligodendrocytes following Nfix deletion. Remarkably, when Nfix is deleted specifically from intermediate progenitor cells and neuroblasts using a Dcx-creERT2 driver, these cells also display elevated signatures of oligodendrocyte gene expression. Together, these results demonstrate the central role played by NFIX in neuroblasts within the adult hippocampal stem cell neurogenic niche in promoting the maturation and survival of these cells, while concomitantly repressing oligodendrocyte gene expression signatures.

Quantitative neurogenetics: applications in understanding disease.

Neurodevelopmental and neurodegenerative disorders (NNDs) are a group of conditions with a broad range of core and co-morbidities, associated with dysfunction of the central nervous system. Improvements in high throughput sequencing have led to the detection of putative risk genetic loci for NNDs, however, quantitative neurogenetic approaches need to be further developed in order to establish causality and underlying molecular genetic mechanisms of pathogenesis. Here, we discuss an approach for prioritizing the contribution of genetic risk loci to complex-NND pathogenesis by estimating the possible impacts of these loci on gene regulation. Furthermore, we highlight the use of a tissue-specificity gene expression index and the application of artificial intelligence (AI) to improve the interpretation of the role of genetic risk elements in NND pathogenesis. Given that NND symptoms are associated with brain dysfunction, risk loci with direct, causative actions would comprise genes with essential functions in neural cells that are highly expressed in the brain. Indeed, NND risk genes implicated in brain dysfunction are disproportionately enriched in the brain compared with other tissues, which we refer to as brain-specific expressed genes. In addition, the tissue-specificity gene expression index can be used as a handle to identify non-brain contexts that are involved in NND pathogenesis. Lastly, we discuss how using an AI approach provides the opportunity to integrate the biological impacts of risk loci to identify those putative combinations of causative relationships through which genetic factors contribute to NND pathogenesis.

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.

Transcriptional regulation of intermediate progenitor cell generation during hippocampal development.

During forebrain development, radial glia generate neurons through the production of intermediate progenitor cells (IPCs). The production of IPCs is a central tenet underlying the generation of the appropriate number of cortical neurons, but the transcriptional logic underpinning this process remains poorly defined. Here, we examined IPC production using mice lacking the transcription factor nuclear factor I/X (Nfix). We show that Nfix deficiency delays IPC production and prolongs the neurogenic window, resulting in an increased number of neurons in the postnatal forebrain. Loss of additional Nfi alleles (Nfib) resulted in a severe delay in IPC generation while, conversely, overexpression of NFIX led to precocious IPC generation. Mechanistically, analyses of microarray and ChIP-seq datasets, coupled with the investigation of spindle orientation during radial glial cell division, revealed that NFIX promotes the generation of IPCs via the transcriptional upregulation of inscuteable (Insc). These data thereby provide novel insights into the mechanisms controlling the timely transition of radial glia into IPCs during forebrain development.

Infantile neurodegenerative disorder associated with mutations in TBCD, an essential gene in the tubulin heterodimer assembly pathway.

Mutation in a growing spectrum of genes is known to either cause or contribute to primary or secondary microcephaly. In primary microcephaly the genetic determinants frequently involve mutations that contribute to or modulate the microtubule cytoskeleton by causing perturbations of neuronal proliferation and migration. Here we describe four patients from two unrelated families each with an infantile neurodegenerative disorder characterized by loss of developmental milestones at 9­24 months of age followed by seizures, dystonia and acquired microcephaly. The patients harboured homozygous missense mutations (A475T and A586V) in TBCD, a gene encoding one of five tubulin-specific chaperones (termed TBCA-E) that function in concert as a nanomachine required for the de novo assembly of the α/ß tubulin heterodimer. The latter is the subunit from which microtubule polymers are assembled. We found a reduced intracellular abundance of TBCD in patient fibroblasts to about 10% (in the case of A475T) or 40% (in the case of A586V) compared to age-matched wild type controls. Functional analyses of the mutant proteins revealed a partially compromised ability to participate in the heterodimer assembly pathway. We show via in utero shRNA-mediated suppression that a balanced supply of tbcd is critical for cortical cell proliferation and radial migration in the developing mouse brain. We conclude that TBCD is a novel functional contributor to the mammalian cerebral cortex development, and that the pathological mechanism resulting from the mutations we describe is likely to involve compromised interactions with one or more TBCD-interacting effectors that influence the dynamics and behaviour of the neuronal cytoskeleton.

Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3.

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole-exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator-like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging-negative focal epilepsy.

TUBB5 and its disease-associated mutations influence the terminal differentiation and dendritic spine densities of cerebral cortical neurons.

The microtubule cytoskeleton is critical for the generation and maturation of neurons in the developing mammalian nervous system. We have previously shown that mutations in the ß-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities in humans. While it is known that TUBB5 is necessary for the proper generation and migration of neurons, little is understood of the role it plays in neuronal differentiation and connectivity. Here, we report that perturbations to TUBB5 disrupt the morphology of cortical neurons, their neuronal complexity, axonal outgrowth, as well as the density and shape of dendritic spines in the postnatal murine cortex. The features we describe are consistent with defects in synaptic signaling. Cellular-based assays have revealed that TUBB5 substitutions have the capacity to alter the dynamic properties and polymerization rates of the microtubule cytoskeleton. Together, our studies show that TUBB5 is essential for neuronal differentiation and dendritic spine formation in vivo, providing insight into the underlying cellular pathology associated with TUBB5 disease states.

The zinc finger transcription factor RP58 negatively regulates Rnd2 for the control of neuronal migration during cerebral cortical development.

The zinc finger transcription factor RP58 (also known as ZNF238) regulates neurogenesis of the mouse neocortex and cerebellum (Okado et al. 2009; Xiang et al. 2011; Baubet et al. 2012; Ohtaka-Maruyama et al. 2013), but its mechanism of action remains unclear. In this study, we report a cell-autonomous function for RP58 during the differentiation of embryonic cortical projection neurons via its activities as a transcriptional repressor. Disruption of RP58 expression alters the differentiation of immature neurons and impairs their migration and positioning within the mouse cerebral cortex. Loss of RP58 within the embryonic cortex also leads to elevated mRNA for Rnd2, a member of the Rnd family of atypical RhoA-like GTPase proteins important for cortical neuron migration (Heng et al. 2008). Mechanistically, RP58 represses transcription of Rnd2 via binding to a 3'-regulatory enhancer in a sequence-specific fashion. Using reporter assays, we found that RP58 repression of Rnd2 is competed by proneural basic helix-loop-helix transcriptional activators. Finally, our rescue experiments revealed that negative regulation of Rnd2 by RP58 was important for cortical cell migration in vivo. Taken together, these studies demonstrate that RP58 is a key player in the transcriptional control of cell migration in the developing cerebral cortex.

Reinforcing the association between distal 1q CNVs and structural brain disorder: A case of a complex 1q43-q44 CNV and a review of the literature.

Copy Number Variations (CNVs) comprising the distal 1q region 1q43-q44 are associated with neurological impairments, structural brain disorder, and intellectual disability. Here, we report an extremely rare, de novo case of a 1q43-q44 deletion with an adjacent duplication, associated with severe seizures, microcephaly, agenesis of the corpus callosum, and pachygyria, a consequence of defective neuronal migration disorder. We conducted a literature survey to find that our patient is only the second case of such a 1q43-q44 CNV ever to be described. Our data support an association between 1q43-q44 deletions and microcephaly, as well as an association between 1q43-q44 duplications and macrocephaly. We compare and contrast our findings with previous studies reporting on critical 1q43-q44 regions and their constituent genes associated with seizures, microcephaly, and corpus callosum abnormalities [Ballif et al., 2012; Hum Genet 131:145-156; Nagamani et al., 2012; Eur J Hum Genet 20:176-179]. Taken together, our study reinforces the association between 1q43-q44 CNVs and brain disorder.

WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression.

The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression.

During corticogenesis, late-born callosal projection neurons (CPNs) acquire their laminar position through glia-guided radial migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of CPNs are poorly defined. Here, we show that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching the cortical plate and have morphological defects during migration. Interestingly, we observed that the rate of neuronal migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This gradient is strongly impaired in COUP-TFI(-/-) brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. We show that COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring correct Rnd2 levels in COUP-TFI(-/-) brains cell-autonomously rescues neuron radial migration and morphological transitions. We also observed impairments in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by lowering Rnd2 expression levels. Thus, our data demonstrate that COUP-TFI modulates late-born neuron migration and favours proper differentiation of CPNs by finely regulating Rnd2 expression levels.

Neurogenin 2 controls cortical neuron migration through regulation of Rnd2.

Motility is a universal property of newly generated neurons. How cell migration is coordinately regulated with other aspects of neuron production is not well understood. Here we show that the proneural protein neurogenin 2 (Neurog2), which controls neurogenesis in the embryonic cerebral cortex, directly induces the expression of the small GTP-binding protein Rnd2 (ref. 3) in newly generated mouse cortical neurons before they initiate migration. Rnd2 silencing leads to a defect in radial migration of cortical neurons similar to that observed when the Neurog2 gene is deleted. Remarkably, restoring Rnd2 expression in Neurog2-mutant neurons is sufficient to rescue their ability to migrate. Our results identify Rnd2 as a novel essential regulator of neuronal migration in the cerebral cortex and demonstrate that Rnd2 is a major effector of Neurog2 function in the promotion of migration. Thus, a proneural protein controls the complex cellular behaviour of cell migration through a remarkably direct pathway involving the transcriptional activation of a small GTP-binding protein.

A Critical Review of the Impact of Candidate Copy Number Variants on Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder (NDD) influenced by genetic, epigenetic, and environmental factors. Recent advancements in genomic analysis have shed light on numerous genes associated with ASD, highlighting the significant role of both common and rare genetic mutations, as well as copy number variations (CNVs), single nucleotide polymorphisms (SNPs) and unique de novo variants. These genetic variations disrupt neurodevelopmental pathways, contributing to the disorder's complexity. Notably, CNVs are present in 10%-20% of individuals with autism, with 3%-7% detectable through cytogenetic methods. While the role of submicroscopic CNVs in ASD has been recently studied, their association with genomic loci and genes has not been thoroughly explored. In this review, we focus on 47 CNV regions linked to ASD, encompassing 1,632 genes, including protein-coding genes and long non-coding RNAs (lncRNAs), of which 659 show significant brain expression. Using a list of ASD-associated genes from SFARI, we detect 17 regions harboring at least one known ASD-related protein-coding gene. Of the remaining 30 regions, we identify 24 regions containing at least one protein-coding gene with brain-enriched expression and a nervous system phenotype in mouse mutants, and one lncRNA with both brain-enriched expression and upregulation in iPSC to neuron differentiation. This review not only expands our understanding of the genetic diversity associated with ASD but also underscores the potential of lncRNAs in contributing to its etiology. Additionally, the discovered CNVs will be a valuable resource for future diagnostic, therapeutic, and research endeavors aimed at prioritizing genetic variations in ASD.

Perturbations in cortical development and neuronal network excitability arising from prenatal exposure to benzodiazepines in mice.

During brain development, many factors influence the assembly and final positioning of cortical neurons, and this process is essential for proper circuit formation and normal brain function. Among many important extrinsic factors that guide the maturation of embryonic cortical neurons, the secreted neurotransmitter GABA has been proposed to influence both their migratory behaviour and their terminal differentiation. The full extent of the short-term and long-term changes in brain patterning and function caused by modulators of the GABA system is not known. In this study, we specifically investigated whether diazepam, a commonly used benzodiazepine that modulates the GABAA receptor, alters neuronal positioning in vivo, and whether this can lead to lasting effects on brain function. We found that fetal exposure to diazepam did not change cell positioning within the embryonic day (E)14.5 mouse cerebral cortex, but significantly altered neuron positioning within the E18.5 cortex. In adult mice, diazepam treatment affected the distribution of cortical interneurons that express parvalbumin or calretinin, and also led to a decrease in the numbers of calretinin-expressing interneurons. In addition, we observed that neonatal exposure to diazepam altered the sensitivity of mice to a proconvulsant challenge. Therefore, exposure of the fetal brain to benzodiazepines has consequences for the positioning of neurons and cortical network excitability.