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.

Studying Disease-Associated UBE3A Missense Variants Using Enhanced Sampling Molecular Simulations.

Missense variants in UBE3A underlie neurodevelopmental conditions such as Angelman Syndrome and Autism Spectrum Disorder, but the underlying molecular pathological consequences on protein folding and function are poorly understood. Here, we report a novel, maternally inherited, likely pathogenic missense variant in UBE3A (NM_000462.4(UBE3A_v001):(c.1841T>C) (p.(Leu614Pro))) in a child that presented with myoclonic epilepsy from 14 months, subsequent developmental regression from 16 months, and additional features consistent with Angelman Syndrome. To understand the impact of p.(Leu614Pro) on UBE3A, we used adiabatic biased molecular dynamics and metadynamics simulations to investigate conformational differences from wildtype proteins. Our results suggest that Leu614Pro substitution leads to less efficient binding and substrate processing compared to wildtype. Our results support the use of enhanced sampling molecular simulations to investigate the impact of missense UBE3A variants on protein function that underlies neurodevelopment and human disorders.

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.

Understanding and responding to remote mental health help-seeking by gay, bisexual and other men who have sex with men (GBMSM) in the U.K. and Republic of Ireland: a mixed-method study conducted in the context of COVID-19.

Background: Gay, bisexual and other men who have sex with men (GBMSM) are at far greater risk of experiencing poor mental health (MH) than wider society. This disparity was exacerbated by additional 'unique to sexual minority status' COVID-19 stressors. Objective: This sequential, mixed-methods study examined remote MH help-seeking among GBMSM in the U.K. and Ireland during the first COVID-19 lockdown. Methods and Results: Quantitative survey data (n = 1368), analysed with logistic regression, suggested GBMSM experiencing moderate-to-severe anxiety and those with a past MH diagnosis were most likely to seek MH support. Thematic analysis of qualitative interview (n = 18) data identified multiple barriers and enablers to GBMSM seeking remote MH help, with the help primarily sought from GBMSM-facing organisations and generic online resources. Finally, the behaviour change wheel was used to generate theoretically informed recommendations to promote MH help-seeking among GBMSM in Scotland. Implications: We discuss how applying these recommendations in the short, medium and long term will begin to address GBMSM's MH needs, post COVID-19.

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.

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.

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.

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.

Brain-Enriched Coding and Long Non-coding RNA Genes Are Overrepresented in Recurrent Neurodevelopmental Disorder CNVs.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with substantial phenotypic and etiological heterogeneity. Although 10%-20% of ASD cases are attributable to copy number variation (CNV), causative genomic loci and constituent genes remain unclarified. We have developed SNATCNV, a tool that outperforms existing tools, to identify 47 recurrent ASD CNV regions from 19,663 cases and 6,479 controls documented in the AutDB database. Analysis of ASD CNV gene content using FANTOM5 shows that constituent coding genes and long non-coding RNAs have brain-enriched patterns of expression. Notably, such enrichment is not observed for regions identified by using other tools. We also find evidence of sexual dimorphism, one locus uniquely comprising a single lncRNA gene, and correlation of CNVs to distinct clinical and behavioral traits. Finally, we analyze a large dataset for schizophrenia to further demonstrate that SNATCNV is an effective, publicly available tool to define genomic loci and causative genes for multiple CNV-associated conditions.

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.

Atypical nested 22q11.2 duplications between LCR22B and LCR22D are associated with neurodevelopmental phenotypes including autism spectrum disorder with incomplete penetrance.

BACKGROUND: Chromosome 22q11.2 is susceptible to genomic rearrangements and the most frequently reported involve deletions and duplications between low copy repeats LCR22A to LCR22D. Atypical nested deletions and duplications are rarer and can provide a valuable opportunity to investigate the dosage effects of a smaller subset of genes within the 22q11.2 genomic disorder region. METHODS: We describe thirteen individuals from six families, each with atypical nested duplications within the central 22q11.2 region between LCR22B and LCR22D. We then compared the molecular and clinical data for patients from this study and the few reported atypical duplication cases, to the cases with larger typical duplications between LCR22A and LCR22D. Further, we analyzed genes with the nested region to identify candidates highly enriched in human brain tissues. RESULTS: We observed that atypical nested duplications are heterogeneous in size, often familial, and associated with incomplete penetrance and highly variable clinical expressivity. We found that the nested atypical duplications are a possible risk factor for neurodevelopmental phenotypes, particularly for autism spectrum disorder (ASD), speech and language delay, and behavioral abnormalities. In addition, we analyzed genes within the nested region between LCR22B and LCR22D to identify nine genes (ZNF74, KLHL22, MED15, PI4KA, SERPIND1, CRKL, AIFM3, SLC7A4, and BCRP2) with enriched expression in the nervous system, each with unique spatiotemporal patterns in fetal and adult brain tissues. Interestingly, PI4KA is prominently expressed in the brain, and this gene is included either partially or completely in all of our subjects. CONCLUSION: Our findings confirm variable expressivity and incomplete penetrance for atypical nested 22q11.2 duplications and identify genes such as PI4KA to be directly relevant to brain development and disorder. We conclude that further work is needed to elucidate the basis of variable neurodevelopmental phenotypes and to exclude the presence of a second disorder. Our findings contribute to the genotype-phenotype data for atypical nested 22q11.2 duplications, with implications for genetic counseling.

Pleiotropic Effects of GLP-1 and Analogs on Cell Signaling, Metabolism, and Function.

The incretin hormone Glucagon-Like Peptide-1 (GLP-1) is best known for its "incretin effect" in restoring glucose homeostasis in diabetics, however, it is now apparent that it has a broader range of physiological effects in the body. Both in vitro and in vivo studies have demonstrated that GLP-1 mimetics alleviate endoplasmic reticulum stress, regulate autophagy, promote metabolic reprogramming, stimulate anti-inflammatory signaling, alter gene expression, and influence neuroprotective pathways. A substantial body of evidence has accumulated with respect to how GLP-1 and its analogs act to restore and maintain normal cellular functions. These findings have prompted several clinical trials which have reported GLP-1 analogs improve cardiac function, restore lung function and reduce mortality in patients with obstructive lung disease, influence blood pressure and lipid storage, and even prevent synaptic loss and neurodegeneration. Mechanistically, GLP-1 elicits its effects via acute elevation in cAMP levels, and subsequent protein kinase(s) activation, pathways well-defined in pancreatic ß-cells which stimulate insulin secretion in conjunction with elevated Ca2+ and ATP. More recently, new studies have shed light on additional downstream pathways stimulated by chronic GLP-1 exposure, findings which have direct relevance to our understanding of the potential therapeutic effects of longer lasting analogs recently developed for clinical use. In this review, we provide a comprehensive description of the diverse roles for GLP-1 across multiple tissues, describe downstream pathways stimulated by acute and chronic exposure, and discuss novel pleiotropic applications of GLP-1 mimetics in the treatment of human disease.

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.

Correction to: Rp58 and p27kip1 coordinate cell cycle exit and neuronal migration within the embryonic mouse cerebral cortex.

CORRECTION: After publication of the original article [1] it was realised that there were errors in figures 2a,b,f,g, which arose as a result of preparing figures from data collected and analysed at the same time as the work reported in [2] (Supplementary Figure 1 of [2]). An updated Fig. 2 is included with this Correction.

A review of structural brain abnormalities in Pallister-Killian syndrome.

BACKGROUND: Pallister-Killian syndrome (PKS) is a rare multisystem developmental syndrome usually caused by mosaic tetrasomy of chromosome 12p that is known to be associated with neurological defects. METHODS: We describe two patients with PKS, one of whom has bilateral perisylvian polymicrogyria (PMG), the other with macrocephaly, enlarged lateral ventricles and hypogenesis of the corpus callosum. We have also summarized the current literature describing brain abnormalities in PKS. RESULTS: We reviewed available cases with intracranial scans (n = 93) and found a strong association between PKS and structural brain abnormalities (77.41%; 72/93). Notably, ventricular abnormalities (45.83%; 33/72), abnormalities of the corpus callosum (25.00%; 18/72) and cerebral atrophy (29.17%; 21/72) were the most frequently reported, while macrocephaly (12.5%; 9/72) and PMG (4.17%; 3/72) were less frequent. To further understand how 12p genes might be relevant to brain development, we identified 63 genes which are enriched in the nervous system. These genes display distinct temporal as well as region-specific expression in the brain, suggesting specific roles in neurodevelopment and disease. Finally, we utilized these data to define minimal critical regions on 12p and their constituent genes associated with atrophy, abnormalities of the corpus callosum, and macrocephaly in PKS. CONCLUSION: Our study reinforces the association between brain abnormalities and PKS, and documents a diverse neurogenetic basis for structural brain abnormalities and impaired function in children diagnosed with this rare disorder.

Rp58 and p27kip1 coordinate cell cycle exit and neuronal migration within the embryonic mouse cerebral cortex.

BACKGROUND: During the development of the mammalian cerebral cortex, newborn postmitotic projection neurons are born from local neural stem cells and must undergo radial migration so as to position themselves appropriately to form functional neural circuits. The zinc finger transcriptional repressor Rp58 (also known as Znf238 or Zbtb18) is critical for coordinating corticogenesis, but its underlying molecular mechanism remains to be better characterised. FINDINGS: Here, we demonstrate that the co-expression of Rp58 and the cyclin dependent kinase inhibitor (CDKI) p27kip1 is important for E14.5-born cortical neurons to coordinate cell cycle exit and initiate their radial migration. Notably, we find that the impaired radial positioning of Rp58-deficient cortical neurons within the embryonic (E17.5) mouse cortex, as well as their multipolar to bipolar transition from the intermediate zone to the cortical plate can be restored by forced expression of p27kip1 in concert with suppression of Rnd2, a downstream target gene of Rp58. Furthermore, the restorative effects of p27kip1 and Rnd2 abrogation are reminiscent of suppressing RhoA signalling in Rp58-deficient cells. CONCLUSIONS: Our findings demonstrate functional interplay between a transcriptional regulator and a CDKI to mediate neuroprogenitor cell cycle exit, as well as to promote radial migration through a molecular mechanism consistent with suppression of RhoA signalling.

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.