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A recent study revealed that monoallelic missense or biallelic loss-of-function variants in the chloride voltage-gated channel 3 (CLCN3) cause neurodevelopmental disorders resulting in brain abnormalities. Functional studies suggested that some missense variants had varying gain-of-function effects on channel activity. Meanwhile, two patients with homozygous frameshift variants showed severe neuropsychiatric disorders and a range of brain structural abnormalities. Here we describe two patients with de novo CLCN3 variants affecting the same amino acid, Gly327 (p.(Gly327Ser) and p.(Gly327Asp)). They showed severe neurological phenotypes including global developmental delay, intellectual disability, hypotonia, failure to thrive, and various brain abnormalities. They also presented with characteristic brain and ophthalmological abnormalities, hippocampal and retinal degradation, which were observed in patients harboring homozygous loss-of-function variants. These findings were also observed in CLCN3-deficient mice, indicating that the monoallelic missense variant may also have a dominant negative effect. This study will expand the phenotypic spectrum of CLCN3-related disorders.

BackgroundO’Donnell-Luria-Rodan syndrome (ODLURO) is an autosomal-dominant neurodevelopmental disorder caused by pathogenic, mostly truncating variants in KMT2E. It was first described by O’Donnell-Luria et al in 2019 in a cohort of 38 patients. Clinical features encompass macrocephaly, mild intellectual disability (ID), autism spectrum disorder (ASD) susceptibility and seizure susceptibility.MethodsAffected individuals were ascertained at paediatric and genetic centres in various countries by diagnostic chromosome microarray or exome/genome sequencing. Patients were collected into a case cohort and were systematically phenotyped where possible.ResultsWe report 18 additional patients from 17 families with genetically confirmed ODLURO. We identified 15 different heterozygous likely pathogenic or pathogenic sequence variants (14 novel) and two partial microdeletions of KMT2E. We confirm and refine the phenotypic spectrum of the KMT2E-related neurodevelopmental disorder, especially concerning cognitive development, with rather mild ID and macrocephaly with subtle facial features in most patients. We observe a high prevalence of ASD in our cohort (41%), while seizures are present in only two patients. We extend the phenotypic spectrum by sleep disturbances.ConclusionOur study, bringing the total of known patients with ODLURO to more than 60 within 2 years of the first publication, suggests an unexpectedly high relative frequency of this syndrome worldwide. It seems likely that ODLURO, although just recently described, is among the more common single-gene aetiologies of neurodevelopmental delay and ASD. We present the second systematic case series of patients with ODLURO, further refining the mutational and phenotypic spectrum of this not-so-rare syndrome.

BACKGROUND: GLI3 encodes a zinc finger transcription factor that plays a role in the sonic hedgehog pathway. Germline pathogenic GLI3 variants are associated with Greig cephalopolysyndactyly and Pallister-Hall syndromes, two syndromes involving brain malformation and polydactyly. METHODS: We identified patients with pathogenic GLI3 variants and brain malformations in the absence of polydactyly or other skeletal malformation. RESULTS: Two patients were identified. Patient #1 is a 4-year-old boy with hypotonia and global developmental delay. Brain MRI showed a focal cortical dysplasia, but he had no history of seizures. Genetic testing identified a de novo likely pathogenic GLI3 variant: c.4453A>T, p.Asn1485Tyr. Patient #2 is a 4-year-old boy with hypotonia, macrocephaly, and global developmental delay. His brain MRI showed partial agenesis of the corpus callosum, dilatation of the right lateral ventricle, and absent hippocampal commissure. Genetic testing identified a de novo pathogenic GLI3 variant: c.4236_4237del, p.Gln1414AspfsTer21. Neither patient had polydactyly or any apparent skeletal abnormality. CONCLUSIONS: These patients widen the spectrum of clinical features that may be associated with GLI3 pathogenic variants to include hypotonia, focal cortical dysplasia, and other brain malformations, in the absence of apparent skeletal malformation. Further study is needed to determine if GLI3 pathogenic variants are a more common cause of focal cortical dysplasia or corpus callosum agenesis than presently recognized.

PurposeARF1was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum ofARF1-related neurodevelopmental disorder.MethodsWe collected detailed phenotypes of an international cohort of individuals (n=17) withARF1variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated.ResultsDe novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) inARF1were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder.ConclusionWe confirm the role ofARF1in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

OBJECTIVE: BCORL1, a transcriptional co-repressor, has a role in cortical migration, neuronal differentiation, maturation, and cerebellar development. We describe BCORL1 as a new genetic cause for major brain malformations. METHODS AND RESULTS: We report three patients from two unrelated families with neonatal onset intractable epilepsy and profound global developmental delay. Brain MRI of two siblings from the first family depicted hypoplastic corpus callosum and septal agenesis (ASP) in the older brother and unilateral perisylvian polymicrogyria (PMG) in the younger one. MRI of the patient from the second family demonstrated complete agenesis of corpus callosum (CC). Whole Exome Sequencing revealed a novel hemizygous variant in NM_021946.5 (BCORL1):c.796C>T (p.Pro266Ser) in the two siblings from the first family and the NM_021946.5 (BCORL1): c.3376G>A; p.Asp1126Asn variant in the patient from the second family, both variants inherited from healthy mothers. We reviewed the patients’ charts and MRIs and compared the phenotype to the other published BCORL1-related cases. Brain malformations have not been previously described in association with the BCORL1 phenotype. We discuss the potential influence of BCORL1 on brain development. CONCLUSIONS: We suggest that BCORL1 variants present with a spectrum of neurodevelopmental disorders and can lead to major brain malformations originating at different stages of fetal development. We suggest adding BCORL1 to the genetic causes of PMG, ASP, and CC dysgenesis.

PURPOSE Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy (DEE) with early-onset seizures and severe intellectual disability. METHODS By international collaboration we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders (NDDs). By western blotting we investigated the consequences of missense variants in vitro. RESULTS In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe DEE in 16 individuals. We now identified also de novo missense variants in the GTPase domain in six individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences.Furthermore, we observed bi-allelic splice-site and truncating variants in nine families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION By identifying phenotype-genotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying bi-allelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2 related disorders including both autosomal dominant and recessive NDDs.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using a neural network trained to distinguish disease-associated from dispensable genes, we classify 235 genes, including 121 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

Nonmuscle myosin II complexes are master regulators of actin dynamics that play essential roles during embryogenesis with vertebrates possessing 3 nonmuscle myosin II heavy chain genes, MYH9, MYH10, and MYH14. As opposed to MYH9 and MYH14, no recognizable disorder has been associated with MYH10. We sought to define the clinical characteristics and molecular mechanism of a novel autosomal dominant disorder related to MYH10.An international collaboration identified the patient cohort. CAS9-mediated knockout cell models were used to explore the mechanism of disease pathogenesis.We identified a cohort of 16 individuals with heterozygous MYH10 variants presenting with a broad spectrum of neurodevelopmental disorders and variable congenital anomalies that affect most organ systems and were recapitulated in animal models of altered MYH10 activity. Variants were typically de novo missense changes with clustering observed in the motor domain. MYH10 knockout cells showed defects in primary ciliogenesis and reduced ciliary length with impaired Hedgehog signaling. MYH10 variant overexpression produced a dominant-negative effect on ciliary length.These data presented a novel genetic cause of isolated and syndromic neurodevelopmental disorders related to heterozygous variants in the MYH10 gene with implications for disrupted primary cilia length control and altered Hedgehog signaling in disease pathogenesis.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl− channels and Cl−/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3−/− mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

Background Rhesus monkeys (Macaca mulatta) exhibit pronounced individual differences in social traits as measured by the macaque Social Responsiveness Scale-Revised. The macaque Social Responsiveness Scale was previously adapted from the Social Responsiveness Scale, an instrument designed to assess social and autistic trait variation in humans. To better understand potential biological underpinnings of this behavioral variation, we evaluated the trait-like consistency of several biological measures previously implicated in autism (e.g., arginine vasopressin, oxytocin, and their receptors, as well as ERK1/2, PTEN, and AKT(1–3) from the RAS-MAPK and PI3K-AKT pathways). We also tested which biological measures predicted macaque Social Responsiveness Scale-Revised scores. Methods Cerebrospinal fluid and blood samples were collected from N = 76 male monkeys, which, as a sample, showed a continuous distribution on the macaque Social Responsiveness Scale-Revised. In a subset of these subjects (n = 43), samples were collected thrice over a 10-month period. The following statistical tests were used: “Case 2A” intra-class correlation coefficients of consistency, principal component analysis, and general linear modeling. Results All biological measures (except AKT) showed significant test–retest reliability within individuals across time points. We next performed principal component analysis on data from monkeys with complete biological measurement sets at the first time point (n = 57), to explore potential correlations between the reliable biological measures and their relationship to macaque Social Responsiveness Scale-Revised score; a three-component solution was found. Follow-up analyses revealed that cerebrospinal fluid arginine vasopressin concentration, but no other biological measure, robustly predicted individual differences in macaque Social Responsiveness Scale-Revised scores, such that monkeys with the lowest cerebrospinal fluid arginine vasopressin concentration exhibited the greatest social impairment. Finally, we confirmed that this result held in the larger study sample (in which cerebrospinal fluid arginine vasopressin values were available from n = 75 of the subjects). Conclusions These findings indicate that cerebrospinal fluid arginine vasopressin concentration is a stable trait-like measure and that it is linked to quantitative social trait variation in male rhesus monkeys.

Summary BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands’ primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands’ fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.

International audience; The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and βIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and βIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.

International audience; Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

Stress plays a key role in modulating the development and expression of addictive behavior, and is a major cause of relapse following periods of abstinence. In this review we focus our attention on recent advances made in understanding how stress, aversive events, and drugs of abuse, cocaine in particular, interact directly with dopamine neurons in the ventral tegmental area, and how these interactions may be involved in stress-induced relapse. We start by outlining how dopamine neurons respond to aversive stimuli and stress, particularly in terms of firing activity and modulation of excitatory synaptic inputs. We then discuss some of the cellular mechanisms underlying the effects of cocaine on dopamine neurons, again with a selective focus on synaptic plasticity. Finally, we examine how the effects of stress and cocaine interact and how these cellular mechanisms in ventral tegmental area dopamine neurons may be engaged in stress-induced relapse.

SummaryA single exposure to drugs of abuse produces an NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA receptor (AMPAR) currents in DA neurons; however, the importance of LTP for various aspects of drug addiction is unclear. To test the role of NMDAR-dependent plasticity in addictive behavior, we genetically inactivated functional NMDAR signaling exclusively in DA neurons (KO mice). Inactivation of NMDARs results in increased AMPAR-mediated transmission that is indistinguishable from the increases associated with a single cocaine exposure, yet locomotor responses to multiple drugs of abuse were unaltered in the KO mice. The initial phase of locomotor sensitization to cocaine is intact; however, the delayed sensitization that occurs with prolonged cocaine withdrawal did not occur. Conditioned behavioral responses for cocaine-testing environment were also absent in the KO mice. These findings provide evidence for a role of NMDAR signaling in DA neurons for specific behavioral modifications associated with drug seeking behaviors.

Cocaine-induced plasticity of glutamatergic synaptic transmission in the ventral tegmental area (VTA) plays an important role in brain adaptations that promote addictive behaviors. However, the mechanisms responsible for triggering these synaptic changes are unknown. Here, we examined the effects of acute cocaine application on glutamatergic synaptic transmission in rat midbrain slices. Cocaine caused a delayed increase in NMDA receptor (NMDAR)-mediated synaptic currents in putative VTA dopamine (DA) cells. This effect was mimicked by a specific DA reuptake inhibitor and by a DA D1/D5receptor agonist. The effect of cocaine was blocked by a DA D1/D5receptor antagonist as well as by inhibitors of the cAMP/cAMP-dependent protein kinase A (PKA) pathway. Furthermore, biochemical analysis showed an increase in the immunoreactivity of the NMDAR subunits NR1 and NR2B and their redistribution to the synaptic membranes in VTA neurons. Accordingly, NMDAR-mediated EPSC decay time kinetics were significantly slower after cocaine, suggesting an increased number of NR2B-containing NMDARs. Finally, pharmacological analysis indicates that NR2B subunits might be incorporated in triheteromeric NR1/NR2A/NR2B complexes rather than in “pure” NR1/NR2B NMDA receptors. Together, our data suggest that acute cocaine increases NMDAR function in the VTA via activation of the cAMP/PKA pathway mediated by a DA D5-like receptor, leading to the insertion of NR2B-containing NMDARs in the membrane. These results provide a potential mechanism by which acute cocaine promotes synaptic plasticity of VTA neurons, which could ultimately lead to the development of addictive behaviors.

Potentiation of glutamate responses is a critical synaptic response to cocaine exposure in ventral tegmental area (VTA) neurons. However, the mechanism by which cocaine exposure promotes potentiation of NMDA receptors (NMDARs) and subsequently AMPA receptors (AMPARs) is not fully understood. In this study we demonstrate that repeated cocaine treatment causes loss of D2 dopamine receptor functional responses via interaction with lysosome-targeting G-protein-associated sorting protein1 (GASP1). We also show that the absence of D2 downregulation in GASP1-KO mice prevents cocaine-induced potentiation of NMDAR currents, elevation of the AMPA/NMDA ratio, and redistribution of NMDAR and AMPAR subunits to the membrane. As a pharmacological parallel, coadministration of the high-affinity D2 agonist, aripiprazole, reduces not only functional downregulation of D2s in response to cocaine but also potentiation of NMDAR and AMPAR responses in wild-type mice. Together these data suggest that functional loss of D2 receptors is a critical mechanism mediating cocaine-induced glutamate plasticity in VTA neurons.

Synaptic plasticity in the ventral tegmental area (VTA) has been implicated in the acquisition of a drug-dependent state. Even a single exposure to cocaine in naive animals is sufficient to trigger sustained changes on VTA glutamatergic synapses that resemble activity-dependent long-term potentiation (LTP) in other brain regions. However, an insight into its time course and mechanisms of action is limited. Here, we show that cocaine acts locally within the VTA to induce an LTP-like enhancement of AMPA receptor-mediated transmission that is not detectable minutes after drug exposure but is fully expressed within 3 h. This cocaine-induced LTP appears to be mediated via dopamine D5receptor activation of NMDA receptors and to require protein synthesis. Increased levels of high-conductance GluR1-containing AMPA receptors at synapses are evident at 3 h after cocaine exposure. Furthermore, our data suggest that cocaine-induced LTP might share the same molecular substrates for expression with activity-dependent LTP induced in the VTA by a spike-timing-dependent (STD) protocol, because we observed that STD LTP is significantly reduced or not inducible in VTA neurons previously exposed to cocainein vivoorin vitro.

Neurotrophins play an important role in modulating activity-dependent neuronal plasticity. In particular, threshold levels of brain-derived neurotrophic factor (BDNF) are required to induce long-term potentiation (LTP) in acute hippocampal slices. Conversely, the administration of exogenous BDNF prevents the induction of long-term depression (LTD) in the visual cortex. A long-standing missing link in the analysis of this modulatory role of BDNF was the determination of the time-course of endogenous BDNF secretion in the same organotypic preparation in which LTP and LTD are elicited. Here, we fulfilled this requirement in slices of perirhinal cortex. Classical theta-burst stimulation patterns evoking LTP lasting >180 min elicited a large increase in BDNF secretion that persisted 5-12 min beyond the stimulation period. Weaker theta-burst stimulation patterns leading only to the initial phase of LTP (≈35 min) were accompanied by a smaller increase in BDNF secretion lasting