De Novo Variants Found in Three Distinct Schizophrenia Populations Hit a Common Core Gene Network Related to Microtubule and Actin Cytoskeleton Gene Ontology Classes.

Schizophrenia (SZ) is a heterogeneous and debilitating psychiatric disorder with a strong genetic component. To elucidate functional networks perturbed in schizophrenia, we analysed a large dataset of whole-genome studies that identified SNVs, CNVs, and a multi-stage schizophrenia genome-wide association study. Our analysis identified three subclusters that are interrelated and with small overlaps: GO:0007017~Microtubule-Based Process, GO:00015629~Actin Cytoskeleton, and GO:0007268~SynapticTransmission. We next analysed three distinct trio cohorts of 75 SZ Algerian, 45 SZ French, and 61 SZ Japanese patients. We performed Illumina HiSeq whole-exome sequencing and identified de novo mutations using a Bayesian approach. We validated 88 de novo mutations by Sanger sequencing: 35 in French, 21 in Algerian, and 32 in Japanese SZ patients. These 88 de novo mutations exhibited an enrichment in genes encoding proteins related to GO:0051015~actin filament binding (p = 0.0011) using David, and enrichments in GO: 0003774~transport (p = 0.019) and GO:0003729~mRNA binding (p = 0.010) using Amigo. One of these de novo variant was found in CORO1C coding sequence. We studied Coro1c haploinsufficiency in a Coro1c+/- mouse and found defects in the corpus callosum. These results could motivate future studies of the mechanisms surrounding genes encoding proteins involved in transport and the cytoskeleton, with the goal of developing therapeutic intervention strategies for a subset of SZ cases.

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations.

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1,500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations, however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESC), including a knock-out and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and Western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR), and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-Seq analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry, and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative (DN) mode-of-action, wherein an increased level of AFF3 resulted in pathological effects. Methods: Evolutionary constraints suggest that other mode-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be deleterious variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous LoF or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the DN/DN or the LoF/LoF lines. While the same pathways are affected, only about one-third of the differentially expressed genes are common to these homozygote datasets, indicating that AFF3 LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

Dissecting the autism-associated 16p11.2 locus identifies multiple drivers in neuroanatomical phenotypes and unveils a male-specific role for the major vault protein.

BACKGROUND: Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus. RESULTS: We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp+/-;Mapk3+/- mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms. CONCLUSIONS: Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice.

Pathogenic variants in SMARCA1 cause an X-linked neurodevelopmental disorder modulated by NURF complex composition.

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SNF2H (SMARCA5) or SNF2L (SMARCA1) ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 were previously implicated in NDDs. Here, we describe 40 individuals from 30 families with de novo or maternally inherited pathogenic variants in SMARCA1. This novel NDD was associated with mild to severe ID/DD, delayed or regressive speech development, and some recurrent facial dysmorphisms. Individuals carrying SMARCA1 loss-of-function variants exhibited a mild genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptfsingle and double mouse knockouts revealed the importance of NURF composition and dosage for proper forebrain development. Finally, we propose that genetic alterations affecting different NURF components result in a NDD with a broad clinical spectrum.

Characterization of Vps13b-mutant mice reveals neuroanatomical and behavioral phenotypes with females less affected.

The vacuolar protein sorting-associated protein 13B (VPS13B) is a large and highly conserved protein. Disruption of VPS13B causes the autosomal recessive Cohen syndrome, a rare disorder characterized by microcephaly and intellectual disability among other features, including developmental delay, hypotonia, and friendly-personality. However, the underlying mechanisms by which VPS13B disruption leads to brain dysfunction still remain unexplained. To gain insights into the neuropathogenesis of Cohen syndrome, we systematically characterized brain changes in Vps13b-mutant mice and compared murine findings to 235 previously published and 17 new patients diagnosed with VPS13B-related Cohen syndrome. We showed that Vps13b is differentially expressed across brain regions with the highest expression in the cerebellum, the hippocampus and the cortex with postnatal peak. Half of the Vps13b-/- mice die during the first week of life. The remaining mice have a normal lifespan and display the core phenotypes of the human disease, including microcephaly, growth delay, hypotonia, altered memory, and enhanced sociability. Systematic 2D and 3D brain histo-morphological analyses reveal specific structural changes in the brain starting after birth. The dentate gyrus is the brain region with the most prominent reduction in size, while the motor cortex is specifically thinner in layer VI. The fornix, the fasciculus retroflexus, and the cingulate cortex remain unaffected. Interestingly, these neuroanatomical changes implicate an increase of neuronal death during infantile stages with no progression in adulthood suggesting that VPS13B promotes neuronal survival early in life. Importantly, whilst both sexes were affected, some neuroanatomical and behavioral phenotypes were less pronounced or even absent in females. We evaluate sex differences in Cohen patients and conclude that females are less affected both in mice and patients. Our findings provide new insights about the neurobiology of VPS13B and highlight previously unreported brain phenotypes while defining Cohen syndrome as a likely new entity of non-progressive infantile neurodegeneration.

Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.

YWHAE loss of function causes a rare neurodevelopmental disease with brain abnormalities in human and mouse.

PURPOSE: Miller-Dieker syndrome is caused by a multiple gene deletion, including PAFAH1B1 and YWHAE. Although deletion of PAFAH1B1 causes lissencephaly unambiguously, deletion of YWHAE alone has not clearly been linked to a human disorder. METHODS: Cases with YWHAE variants were collected through international data sharing networks. To address the specific impact of YWHAE loss of function, we phenotyped a mouse knockout of Ywhae. RESULTS: We report a series of 10 individuals with heterozygous loss-of-function YWHAE variants (3 single-nucleotide variants and 7 deletions <1 Mb encompassing YWHAE but not PAFAH1B1), including 8 new cases and 2 follow-ups, added with 5 cases (copy number variants) from literature review. Although, until now, only 1 intragenic deletion has been described in YWHAE, we report 4 new variants specifically in YWHAE (3 splice variants and 1 intragenic deletion). The most frequent manifestations are developmental delay, delayed speech, seizures, and brain malformations, including corpus callosum hypoplasia, delayed myelination, and ventricular dilatation. Individuals with variants affecting YWHAE alone have milder features than those with larger deletions. Neuroanatomical studies in Ywhae-/- mice revealed brain structural defects, including thin cerebral cortex, corpus callosum dysgenesis, and hydrocephalus paralleling those seen in humans. CONCLUSION: This study further demonstrates that YWHAE loss-of-function variants cause a neurodevelopmental disease with brain abnormalities.

Characterization of Two Mouse Chd7 Heterozygous Loss-of-Function Models Shows Dysgenesis of the Corpus Callosum and Previously Unreported Features of CHARGE Syndrome.

CHARGE syndrome is a rare congenital disorder frequently caused by mutations in the chromodomain helicase DNA-binding protein-7 CHD7. Here, we developed and systematically characterized two genetic mouse models with identical, heterozygous loss-of-function mutation of the Chd7 gene engineered on inbred and outbred genetic backgrounds. We found that both models showed consistent phenotypes with the core clinical manifestations seen in CHARGE syndrome, but the phenotypes in the inbred Chd7 model were more severe, sometimes having reduced penetrance and included dysgenesis of the corpus callosum, hypoplasia of the hippocampus, abnormal retrosplenial granular cortex, ventriculomegaly, hyperactivity, growth delays, impaired grip strength and repetitive behaviors. Interestingly, we also identified previously unreported features including reduced levels of basal insulin and reduced blood lipids. We suggest that the phenotypic variation reported in individuals diagnosed with CHARGE syndrome is likely due to the genetic background and modifiers. Finally, our study provides a valuable resource, making it possible for mouse biologists interested in Chd7 to make informed choices on which mouse model they should use to study phenotypes of interest and investigate in more depth the underlying cellular and molecular mechanisms.

Quantitative Neuroanatomical Phenotyping of the Embryonic Mouse Brain.

Congenital neurodevelopmental anomalies are present from birth and are characterized by an abnormal development of one or more structures of the brain. Brain structural anomalies are highly comorbid with neurodevelopmental and neuropsychiatric disorders such as intellectual disability, autism spectrum disorders, epilepsy, and schizophrenia, and 80% are of genetic origin. We aim to address an important neurobiological question: How many genes regulate the normal anatomy of the brain during development. To do so, we developed a quantitative approach for the assessment of a total of 106 neuroanatomical parameters in mouse mutant embryos at embryonic day 18.5 across two planes commonly used in brain anatomical studies, the coronal and sagittal planes. In this article we describe the techniques we developed and explain why ultrastandardized procedures involving embryonic mouse brains are even more of prime importance for morphological phenotyping than adult mouse brains. We focus our analysis on brain size anomalies and on the most frequently altered brain regions including the cortex, corpus callosum, hippocampus, ventricles, caudate putamen, and cerebellum. Our protocols allow a standardized histology pipeline from embryonic mouse brain preparation to sectioning, staining, and scanning and neuroanatomical analyses at well-defined positions on the coronal and sagittal planes. Together, our protocols will help scientists in deciphering congenital neurodevelopmental anomalies and anatomical changes between groups of mouse embryos in health and genetic diseases. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation and preparation of embryonic mouse brain samples Basic Protocol 2: Sectioning, staining, and scanning of embryonic mouse brain sections Basic Protocol 3: Coronal neuroanatomical measurements of embryonic mouse brain structures Basic Protocol 4: Sagittal neuroanatomical measurements of embryonic mouse brain structures.

Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development.

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.

Biallelic variants in TRAPPC10 cause a microcephalic TRAPPopathy disorder in humans and mice.

The highly evolutionarily conserved transport protein particle (TRAPP) complexes (TRAPP II and III) perform fundamental roles in subcellular trafficking pathways. Here we identified biallelic variants in TRAPPC10, a component of the TRAPP II complex, in individuals with a severe microcephalic neurodevelopmental disorder. Molecular studies revealed a weakened interaction between mutant TRAPPC10 and its putative adaptor protein TRAPPC2L. Studies of patient lymphoblastoid cells revealed an absence of TRAPPC10 alongside a concomitant absence of TRAPPC9, another key TRAPP II complex component associated with a clinically overlapping neurodevelopmental disorder. The TRAPPC9/10 reduction phenotype was recapitulated in TRAPPC10-/- knockout cells, which also displayed a membrane trafficking defect. Notably, both the reduction in TRAPPC9 levels and the trafficking defect in these cells could be rescued by wild type but not mutant TRAPPC10 gene constructs. Moreover, studies of Trappc10-/- knockout mice revealed neuroanatomical brain defects and microcephaly, paralleling findings seen in the human condition as well as in a Trappc9-/- mouse model. Together these studies confirm autosomal recessive TRAPPC10 variants as a cause of human disease and define TRAPP-mediated pathomolecular outcomes of importance to TRAPPC9 and TRAPPC10 mediated neurodevelopmental disorders in humans and mice.

A Positively Selected MAGEE2 LoF Allele Is Associated with Sexual Dimorphism in Human Brain Size and Shows Similar Phenotypes in Magee2 Null Mice.

A nonsense allele at rs1343879 in human MAGEE2 on chromosome X has previously been reported as a strong candidate for positive selection in East Asia. This premature stop codon causing ∼80% protein truncation is characterized by a striking geographical pattern of high population differentiation: common in Asia and the Americas (up to 84% in the 1000 Genomes Project East Asians) but rare elsewhere. Here, we generated a Magee2 mouse knockout mimicking the human loss-of-function mutation to study its functional consequences. The Magee2 null mice did not exhibit gross abnormalities apart from enlarged brain structures (13% increased total brain area, P = 0.0022) in hemizygous males. The area of the granular retrosplenial cortex responsible for memory, navigation, and spatial information processing was the most severely affected, exhibiting an enlargement of 34% (P = 3.4×10-6). The brain size in homozygous females showed the opposite trend of reduced brain size, although this did not reach statistical significance. With these insights, we performed human association analyses between brain size measurements and rs1343879 genotypes in 141 Chinese volunteers with brain MRI scans, replicating the sexual dimorphism seen in the knockout mouse model. The derived stop gain allele was significantly associated with a larger volume of gray matter in males (P = 0.00094), and smaller volumes of gray (P = 0.00021) and white (P = 0.0015) matter in females. It is unclear whether or not the observed neuroanatomical phenotypes affect behavior or cognition, but it might have been the driving force underlying the positive selection in humans.

Dissecting indirect genetic effects from peers in laboratory mice.

BACKGROUND: The phenotype of an individual can be affected not only by the individual's own genotypes, known as direct genetic effects (DGE), but also by genotypes of interacting partners, indirect genetic effects (IGE). IGE have been detected using polygenic models in multiple species, including laboratory mice and humans. However, the underlying mechanisms remain largely unknown. Genome-wide association studies of IGE (igeGWAS) can point to IGE genes, but have not yet been applied to non-familial IGE arising from "peers" and affecting biomedical phenotypes. In addition, the extent to which igeGWAS will identify loci not identified by dgeGWAS remains an open question. Finally, findings from igeGWAS have not been confirmed by experimental manipulation. RESULTS: We leverage a dataset of 170 behavioral, physiological, and morphological phenotypes measured in 1812 genetically heterogeneous laboratory mice to study IGE arising between same-sex, adult, unrelated mice housed in the same cage. We develop and apply methods for igeGWAS in this context and identify 24 significant IGE loci for 17 phenotypes (FDR < 10%). We observe no overlap between IGE loci and DGE loci for the same phenotype, which is consistent with the moderate genetic correlations between DGE and IGE for the same phenotype estimated using polygenic models. Finally, we fine-map seven significant IGE loci to individual genes and find supportive evidence in an experiment with a knockout model that Epha4 gives rise to IGE on stress-coping strategy and wound healing. CONCLUSIONS: Our results demonstrate the potential for igeGWAS to identify IGE genes and shed light into the mechanisms of peer influence.

SCA7 Mouse Cerebellar Pathology Reveals Preferential Downregulation of Key Purkinje Cell-Identity Genes and Shared Disease Signature with SCA1 and SCA2.

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease mainly characterized by motor incoordination because of progressive cerebellar degeneration. SCA7 is caused by polyglutamine expansion in ATXN7, a subunit of the transcriptional coactivator SAGA, which harbors histone modification activities. Polyglutamine expansions in specific proteins are also responsible for SCA1-SCA3, SCA6, and SCA17; however, the converging and diverging pathomechanisms remain poorly understood. Using a new SCA7 knock-in mouse, SCA7140Q/5Q, we analyzed gene expression in the cerebellum and assigned gene deregulation to specific cell types using published datasets. Gene deregulation affects all cerebellar cell types, although at variable degree, and correlates with alterations of SAGA-dependent epigenetic marks. Purkinje cells (PCs) are by far the most affected neurons and show reduced expression of 83 cell-type identity genes, including these critical for their spontaneous firing activity and synaptic functions. PC gene downregulation precedes morphologic alterations, pacemaker dysfunction, and motor incoordination. Strikingly, most PC genes downregulated in SCA7 have also decreased expression in SCA1 and SCA2 mice, revealing converging pathomechanisms and a common disease signature involving cGMP-PKG and phosphatidylinositol signaling pathways and LTD. Our study thus points out molecular targets for therapeutic development, which may prove beneficial for several SCAs. Furthermore, we show that SCA7140Q/5Q males and females exhibit the major disease features observed in patients, including cerebellar damage, cerebral atrophy, peripheral nerves pathology, and photoreceptor dystrophy, which account for progressive impairment of behavior, motor, and visual functions. SCA7140Q/5Q mice represent an accurate model for the investigation of different aspects of SCA7 pathogenesis.SIGNIFICANCE STATEMENT Spinocerebellar ataxia 7 (SCA7) is one of the several forms of inherited SCAs characterized by cerebellar degeneration because of polyglutamine expansion in specific proteins. The ATXN7 involved in SCA7 is a subunit of SAGA transcriptional coactivator complex. To understand the pathomechanisms of SCA7, we determined the cell type-specific gene deregulation in SCA7 mouse cerebellum. We found that the Purkinje cells are the most affected cerebellar cell type and show downregulation of a large subset of neuronal identity genes, critical for their spontaneous firing and synaptic functions. Strikingly, the same Purkinje cell genes are downregulated in mouse models of two other SCAs. Thus, our work reveals a disease signature shared among several SCAs and uncovers potential molecular targets for their treatment.

Haploinsufficiency of the HIRA gene located in the 22q11 deletion syndrome region is associated with abnormal neurodevelopment and impaired dendritic outgrowth.

The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira+/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira+/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.

Heterozygous Variants in KDM4B Lead to Global Developmental Delay and Neuroanatomical Defects.

KDM4B is a lysine-specific demethylase with a preferential activity on H3K9 tri/di-methylation (H3K9me3/2)-modified histones. H3K9 tri/di-demethylation is an important epigenetic mechanism responsible for silencing of gene expression in animal development and cancer. However, the role of KDM4B on human development is still poorly characterized. Through international data sharing, we gathered a cohort of nine individuals with mono-allelic de novo or inherited variants in KDM4B. All individuals presented with dysmorphic features and global developmental delay (GDD) with language and motor skills most affected. Three individuals had a history of seizures, and four had anomalies on brain imaging ranging from agenesis of the corpus callosum with hydrocephalus to cystic formations, abnormal hippocampi, and polymicrogyria. In mice, lysine demethylase 4B is expressed during brain development with high levels in the hippocampus, a region important for learning and memory. To understand how KDM4B variants can lead to GDD in humans, we assessed the effect of KDM4B disruption on brain anatomy and behavior through an in vivo heterozygous mouse model (Kdm4b+/-), focusing on neuroanatomical changes. In mutant mice, the total brain volume was significantly reduced with decreased size of the hippocampal dentate gyrus, partial agenesis of the corpus callosum, and ventriculomegaly. This report demonstrates that variants in KDM4B are associated with GDD/ intellectual disability and neuroanatomical defects. Our findings suggest that KDM4B variation leads to a chromatinopathy, broadening the spectrum of this group of Mendelian disorders caused by alterations in epigenetic machinery.

Trappc9 deficiency causes parent-of-origin dependent microcephaly and obesity.

Some imprinted genes exhibit parental origin specific expression bias rather than being transcribed exclusively from one copy. The physiological relevance of this remains poorly understood. In an analysis of brain-specific allele-biased expression, we identified that Trappc9, a cellular trafficking factor, was expressed predominantly (~70%) from the maternally inherited allele. Loss-of-function mutations in human TRAPPC9 cause a rare neurodevelopmental syndrome characterized by microcephaly and obesity. By studying Trappc9 null mice we discovered that homozygous mutant mice showed a reduction in brain size, exploratory activity and social memory, as well as a marked increase in body weight. A role for Trappc9 in energy balance was further supported by increased ad libitum food intake in a child with TRAPPC9 deficiency. Strikingly, heterozygous mice lacking the maternal allele (70% reduced expression) had pathology similar to homozygous mutants, whereas mice lacking the paternal allele (30% reduction) were phenotypically normal. Taken together, we conclude that Trappc9 deficient mice recapitulate key pathological features of TRAPPC9 mutations in humans and identify a role for Trappc9 and its imprinting in controlling brain development and metabolism.

A knock-in mouse model for KCNQ2-related epileptic encephalopathy displays spontaneous generalized seizures and cognitive impairment.

OBJECTIVE: Early onset epileptic encephalopathy with suppression-burst is one of the most severe epilepsy phenotypes in human patients. A significant proportion of cases have a genetic origin, and the most frequently mutated gene is KCNQ2, encoding Kv7.2, a voltage-dependent potassium channel subunit, leading to so-called KCNQ2-related epileptic encephalopathy (KCNQ2-REE). To study the pathophysiology of KCNQ2-REE in detail and to provide a relevant preclinical model, we generated and described a knock-in mouse model carrying the recurrent p.(Thr274Met) variant. METHODS: We introduced the p.(Thr274Met) variant by homologous recombination in embryonic stem cells, injected into C57Bl/6N blastocysts and implanted in pseudopregnant mice. Mice were then bred with 129Sv Cre-deleter to generate heterozygous mice carrying the p.(Thr274Met), and animals were maintained on the 129Sv genetic background. We studied the development of this new model and performed in vivo electroencephalographic (EEG) recordings, neuroanatomical studies at different time points, and multiple behavioral tests. RESULTS: The Kcnq2Thr274Met/+ mice are viable and display generalized spontaneous seizures first observed between postnatal day 20 (P20) and P30. In vivo EEG recordings show that the paroxysmal events observed macroscopically are epileptic seizures. The brain of the Kcnq2Thr274Met/+ animals does not display major structural defects, similar to humans, and their body weight is normal. Kcnq2Thr274Met/+ mice have a reduced life span, with a peak of unexpected death occurring for 25% of the animals by 3 months of age. Epileptic seizures were generally not observed when animals grew older. Behavioral characterization reveals important deficits in spatial learning and memory in adults but no gross abnormality during early neurosensory development. SIGNIFICANCE: Taken together, our results indicate that we have generated a relevant model to study the pathophysiology of KCNQ2-related epileptic encephalopathy and perform preclinical research for that devastating and currently intractable disease.

Conditional switching of KIF2A mutation provides new insights into cortical malformation pathogeny.

By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the flexibility of this model, we investigated RosaCre-, NestinCre- and NexCre-driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A.