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OBJECTIVE: Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. METHODS: We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents. RESULTS: Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20. SIGNIFICANCE: Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.
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Neurônios , Receptores de N-Metil-D-Aspartato , Serina-Treonina Quinases TOR , Animais , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Ratos , Neurônios/metabolismo , Feminino , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/fisiopatologia , Modelos Animais de Doenças , Ratos Sprague-Dawley , Técnicas de Patch-Clamp , Malformações do Desenvolvimento Cortical do Grupo I/genética , Displasia Cortical Focal , EpilepsiaAssuntos
Gagueira , Humanos , Gagueira/genética , Peptidil-Prolil Isomerase F , Chaperonas MolecularesRESUMO
The epilepsy of infancy with migrating focal seizures (EIMFS; previously called Malignant migrating partial seizures of infancy) are early-onset epileptic encephalopathies (EOEE) that associate multifocal ictal discharges and profound psychomotor retardation. EIMFS have a genetic origin and are mostly caused by de novo mutations in the KCNT1 gene, and much more rarely in the KCNT2 gene. KCNT1 and KCNT2 respectively encode the KNa1.1 (Slack) and KNa1.2 (Slick) subunits of the sodium-dependent voltage-gated potassium channel KNa. Functional analyses of the corresponding mutant homomeric channels in vitro suggested gain-of-function effects. Here, we report two novel, de novo truncating mutations of KCNT2: one mutation is frameshift (p.L48Qfs43), is situated in the N-terminal domain, and was found in a patient with EOEE (possibly EIMFS); the other mutation is nonsense (p.K564*), is located in the C-terminal region, and was found in a typical EIMFS patient. Using whole-cell patch-clamp recordings, we have analyzed the functional consequences of those two novel KCNT2 mutations on reconstituted KNa1.2 homomeric and KNa1.1/KNa1.2 heteromeric channels in transfected chinese hamster ovary (CHO) cells. We report that both mutations significantly impacted on KNa function; notably, they decreased the global current density of heteromeric channels by ~25% (p.K564*) and ~55% (p.L48Qfs43). Overall our data emphasize the involvement of KCNT2 in EOEE and provide novel insights into the role of heteromeric KNa channel in the severe KCNT2-related epileptic phenotypes. This may have important implications regarding the elaboration of future treatment.
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Formerly idiopathic, focal epilepsies (IFE) are self-limiting, "age-related" diseases that mainly occur during critical developmental periods. Childhood epilepsy with centrotemporal spikes, or Rolandic epilepsy (RE), is the most frequent form of IFE. Together with the Landau-Kleffner syndrome and the epileptic Encephalopathy related to Status Epilepticus during slow Sleep syndrome (ESES), RE is part of a single and continuous spectrum of childhood epilepsies and epileptic encephalopathies with acquired cognitive, behavioral and speech and/or language impairment, known as the epilepsy-aphasia spectrum (EAS). The pathophysiology has long been attributed to an elusive and complex interplay between brain development and maturation processes on the one hand, and susceptibility genes on the other hand. Studies based on the variable combination of molecular cytogenetics, Sanger and next-generation sequencing tools, and functional assays have led to the identification and validation of genetic mutations in the GRIN2A gene that can directly cause various types of EAS disorders. The recent identification of GRIN2A defects in EAS represents a first and major break-through in our understanding of the underlying pathophysiological mechanisms. In this review, we describe the current knowledge on the genetic architecture of IFE.
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Afasia/genética , Epilepsia Rolândica/genética , Síndrome de Landau-Kleffner/genética , Mutação/genética , Receptores de N-Metil-D-Aspartato/genética , Criança , Eletroencefalografia/métodos , Humanos , Síndrome de Landau-Kleffner/diagnóstico , LinhagemRESUMO
OBJECTIVE: Glutamate-gated N-methyl-d-aspartate receptors (NMDARs) are instrumental to brain development and functioning. Defects in the GRIN2A gene, encoding the GluN2A subunit of NMDARs, cause slow-wave sleep (SWS)-related disorders of the epilepsy-aphasia spectrum (EAS). The as-yet poorly understood developmental sequence of early EAS-related phenotypes, and the role of GluN2A-containing NMDARs in the development of SWS and associated electroencephalographic (EEG) activity patterns, were investigated in Grin2a knockout (KO) mice. METHODS: Early social communication was investigated by ultrasonic vocalization (USV) recordings; the relationship of electrical activity of the cerebral cortex with SWS was studied using deep local field potential or chronic EEG recordings at various postnatal stages. RESULTS: Grin2a KO pups displayed altered USV and increased occurrence of high-voltage spindles. The pattern of slow-wave activity induced by low-dose isoflurane was altered in Grin2a KO mice in the 3rd postnatal week and at 1 month of age. These alterations included strong suppression of the delta oscillation power and an increase in the occurrence of the spike-wave bursts. The proportion of SWS and the sleep quality were transiently reduced in Grin2a KO mice aged 1 month but recovered by the age of 2 months. Grin2a KO mice also displayed spontaneous spike-wave discharges, which occurred nearly exclusively during SWS, at 1 and 2 months of age. SIGNIFICANCE: The impaired vocal communication, the spike-wave discharges occurring almost exclusively in SWS, and the age-dependent alteration of SWS that were all seen in Grin2a KO mice matched the sleep-related and age-dependent manifestations seen in children with EAS, hence validating the Grin2a KO as a reliable model of EAS disorders. Our data also show that GluN2A-containing NMDARs are involved in slow-wave activity, and that the period of postnatal brain development (postnatal day 30) when several anomalies peaked might be critical for GluN2A-dependent, sleep-related physiological and pathological processes.
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Receptores de N-Metil-D-Aspartato/fisiologia , Sono de Ondas Lentas/fisiologia , Sono/fisiologia , Vocalização Animal , Animais , Animais Recém-Nascidos/fisiologia , Eletroencefalografia , Feminino , Masculino , Camundongos/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/metabolismo , Vocalização Animal/fisiologiaRESUMO
OBJECTIVE: The epilepsy-aphasia spectrum (EAS) is a heterogeneous group of age-dependent childhood disorders characterized by sleep-activated discharges associated with infrequent seizures and language, cognitive, and behavioral deficits. Defects in the GRIN2A gene, encoding a subunit of glutamate-gated N-methyl-d-aspartate (NMDA) receptors, represent the most important cause of EAS identified so far. Neocortical or thalamic lesions were detected in a subset of severe EAS disorders, and more subtle anomalies were reported in patients with so-called "benign" phenotypes. However, whether brain structural alterations exist in the context of GRIN2A defects is unknown. METHODS: Magnetic resonance diffusion tensor imaging (MR-DTI) was used to perform longitudinal analysis of the brain at 3 developmental timepoints in living mice genetically knocked out (KO) for Grin2a. In addition, electroencephalography (EEG) was recorded using multisite extracellular electrodes to characterize the neocortical activity in vivo. RESULTS: Microstructural alterations were detected in the neocortex, the corpus callosum, the hippocampus, and the thalamus of Grin2a KO mice. Most MR-DTI alterations were detected at a specific developmental stage when mice were aged 30 days, but not at earlier (15 days) or later (2 months) ages. EEG analysis detected epileptiform discharges in Grin2a KO mice in the third postnatal week. SIGNIFICANCE: Grin2a KO mice replicated several anomalies found in patients with EAS disorders. Transient structural alterations detected by MR-DTI recalled the age-dependent course of EAS disorders, which in humans start during childhood and show variable outcome at the onset of adolescence. Together with the epileptiform discharges detected in young Grin2a KO mice, our data suggested the existence of early anomalies in the maturation of the neocortical and thalamocortical systems. Whereas the possible relationship of those anomalies with sleep warrants further investigations, our data suggest that Grin2a KO mice may serve as an animal model to study the neuronal mechanisms of EAS disorders and to design new therapeutic strategies.
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Encéfalo/patologia , Síndrome de Landau-Kleffner/genética , Síndrome de Landau-Kleffner/patologia , Mutação/genética , Receptores de N-Metil-D-Aspartato/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/diagnóstico por imagem , Encéfalo/crescimento & desenvolvimento , Ondas Encefálicas/genética , Eletroencefalografia , Genótipo , Processamento de Imagem Assistida por Computador , Síndrome de Landau-Kleffner/diagnóstico por imagem , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/diagnóstico por imagem , Transtornos do Neurodesenvolvimento , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Congenital cytomegalovirus (CMV) infections represent one leading cause of neurodevelopmental disorders. Recently, we reported on a rat model of CMV infection of the developing brain in utero, characterized by early and prominent infection and alteration of microglia-the brain-resident mononuclear phagocytes. Besides their canonical function against pathogens, microglia are also pivotal to brain development. Here we show that CMV infection of the rat fetal brain recapitulated key postnatal phenotypes of human congenital CMV including increased mortality, sensorimotor impairment reminiscent of cerebral palsy, hearing defects, and epileptic seizures. The possible influence of early microglia alteration on those phenotypes was then questioned by pharmacological targeting of microglia during pregnancy. One single administration of clodronate liposomes in the embryonic brains at the time of CMV injection to deplete microglia, and maternal feeding with doxycyxline throughout pregnancy to modify microglia in the litters' brains, were both associated with dramatic improvements of survival, body weight gain, sensorimotor development and with decreased risk of epileptic seizures. Improvement of microglia activation status did not persist postnatally after doxycycline discontinuation; also, active brain infection remained unchanged by doxycycline. Altogether our data indicate that early microglia alteration, rather than brain CMV load per se, is instrumental in influencing survival and the neurological outcomes of CMV-infected rats, and suggest that microglia might participate in the neurological outcome of congenital CMV in humans. Furthermore this study represents a first proof-of-principle for the design of microglia-targeted preventive strategies in the context of congenital CMV infection of the brain.
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Numerous epilepsy genes have been identified in the last years, mostly in the (rare) monogenic forms and thanks to the increased availability and the decreased cost of next-generation sequencing approaches. Besides the somehow expected group of epilepsy genes encoding various ion channel subunits (e.g. sodium or potassium channel subunits, or GABA receptors, or glutamate-gated NMDA receptors), more diversity has emerged recently, with novel epilepsy genes encoding proteins playing a wide range of physiological roles at the cellular and molecular levels, such as synaptic proteins, members of the mTOR pathway, or proteins involved in chromatin remodeling. The overall picture is somehow complicated: one given epilepsy gene can be associated with more than one epileptic phenotype, and with variable degrees of severity, from the benign to the severe forms (e.g. epileptic encephalopathies), and with various comorbid conditions such as migraine or autism spectrum of disorders. Conversely, one given epileptic syndrome may be associated with different genes, some of which have obvious links with each other (e.g. encoding different subunits of the same receptor) while other ones have no clear relationships. Also genomic copy number variations have been detected, some of which, albeit rare, may confer high risk to epilepsy. Whereas translation from gene identification to targeted medicine still remains challenging, progress in epilepsy genetics is currently revolutionizing genetic-based diagnosis and genetic counseling. Epilepsy gene identification also represents a key entry point to start in deciphering the underlying pathophysiological mechanisms via the design and the study of the most pertinent cellular and animal models - which may in turn provide proofs-of-principle for future applications in human epilepsies.
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Epilepsia/genética , Predisposição Genética para Doença , Feminino , Humanos , Masculino , Mutação , FenótipoRESUMO
The functional study of reconstituted NMDA receptors (NMDARs) in host cells requires that the corresponding vectors for the expression of the NMDAR subunits are co-transfected with high efficiency. Magnetofection™ is a technology used to deliver nucleic acids to cells. It is driven and site-specifically guided by the attractive forces of magnetic fields acting on magnetic nanoparticles that are associated with nucleic acid vectors. In magnetofection™, cationic lipids form self-assembled complexes with the nucleic acid vectors of interest. Those complexes are then associated with magnetic nanoparticles that are concentrated at the surface of cultured cells by applying a permanent magnetic field. Magnetofection™ is a simple method to transfect cultured cells with high transfection rates. Satisfactory expression levels are obtained with very low amounts of nucleic acid vector. Moreover, incubation time with host cells is less than 1 h, as compared with the several hours needed with standard transfection assays.
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Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Vetores Genéticos/genética , Células HEK293 , Humanos , Nanopartículas de Magnetita , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , TransfecçãoRESUMO
Genetic variants of the glutamate activated N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunit GluN2A are associated with the hyperexcitable states manifested by epileptic seizures and interictal discharges in patients with disorders of the epilepsy-aphasia spectrum (EAS). The variants found in sporadic cases and families are of different types and include microdeletions encompassing the corresponding GRIN2A gene as well as nonsense, splice-site and missense GRIN2A defects. They are located at different functional domains of GluN2A and no clear genotype-phenotype correlation has emerged yet. Moreover, GluN2A variants may be associated with phenotypic pleiotropy. Deciphering the consequences of pathogenic GRIN2A variants would surely help in better understanding of the underlying mechanisms. This emphasizes the need for functional studies to unravel the basic functional properties of each specific NMDAR variant. In the present study, we have used patch-clamp recordings to evaluate kinetic changes of mutant NMDARs reconstituted after co-transfection of cultured cells with the appropriate expression vectors. Three previously identified missense variants found in patients or families with disorders of the EAS and situated in the N-terminal domain (p.Ile184Ser) or in the ligand-binding domain (p.Arg518His and p.Ala716Thr) of GluN2A were studied in both the homozygous and heterozygous conditions. Relative surface expression and current amplitude were significantly reduced for NMDARs composed of mutant p.Ile184Ser and p.Arg518His, but not p.Ala716His, as compared with wild-type (WT) NMDARs. Amplitude of whole-cell currents was still drastically decreased when WT and mutant p.Arg518His-GluN2A subunits were co-expressed, suggesting a dominant-negative mechanism. Activation times were significantly decreased in both homozygous and heterozygous conditions for the two p.Ile184Ser and p.Arg518His variants, but not for p.Ala716His. Deactivation also significantly increased for p.Ile184Ser variant in the homozygous but not the heterozygous state while it was increased for p.Arg518His in both states. Our data indicate that p.Ile184Ser and p.Arg518His GluN2A variants both impacted on NMDAR function, albeit differently, whereas p.Ala716His did not significantly influence NMDAR kinetics, hence partly questioning its direct and strong pathogenic role. This study brings new insights into the functional impact that GRIN2A variants might have on NMDAR kinetics, and provides a mechanistic explanation for the neurological manifestations seen in the corresponding human spectrum of disorders.
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The term idiopathic focal epilepsies of childhood (IFE) is not formally recognised by the ILAE in its 2010 revision (Berg et al., 2010), nor are its members and boundaries precisely delineated. The IFEs are amongst the most commonly encountered epilepsy syndromes affecting children. They are fascinating disorders that hold many "treats" for both clinicians and researchers. For example, the IFEs pose many of the most interesting questions central to epileptology: how are functional brain networks involved in the manifestation of epilepsy? What are the shared mechanisms of comorbidity between epilepsy and neurodevelopmental disorders? How do focal EEG discharges impact cognitive functioning? What explains the age-related expression of these syndromes? Why are EEG discharges and seizures so tightly locked to slow-wave sleep? In the last few decades, the clinical symptomatology and the respective courses of many IFEs have been described, although they are still not widely appreciated beyond the specialist community. Most neurologists would recognise the core syndromes of IFE to comprise: benign epilepsy of childhood with centro-temporal spikes or Rolandic epilepsy (BECTS/RE); Panayiotopoulos syndrome; and the idiopathic occipital epilepsies (Gastaut and photosensitive types). The Landau-Kleffner syndrome and the related (idiopathic) epilepsy with continuous spikes and waves in sleep (CSWS or ESES) are also often included, both as a consequence of the shared morphology of the interictal discharges and their potential evolution from core syndromes, for example, CSWS from BECTS. Atypical benign focal epilepsy of childhood also has shared electro-clinical features warranting inclusion. In addition, a number of less well-defined syndromes of IFE have been proposed, including benign childhood seizures with affective symptoms, benign childhood epilepsy with parietal spikes, benign childhood seizures with frontal or midline spikes, and benign focal seizures of adolescence. The term "benign" is often used in connection with the IFEs and is increasingly being challenged. Certainly most of these disorders are not associated with the devastating cognitive and behavioural problems seen with early childhood epileptic encephalopathies, such as West or Dravet syndromes. However, it is clear that specific, and sometimes persistent, neuropsychological deficits in attention, language and literacy accompany many of the IFEs that, when multiplied by the large numbers affected, make up a significant public health problem. Understanding the nature, distribution, evolution, risk and management of these is an important area of current research. A corollary to such questions regarding comorbidities is the role of focal interictal spikes and their enduring impact on cognitive functioning. What explains the paradox that epilepsies characterised by abundant interictal epileptiform abnormalities are often associated with very few clinical seizures? This is an exciting area in both clinical and experimental arenas and will eventually have important implications for clinical management of the whole child, taking into account not just seizures, but also adaptive functioning and quality of life. For several decades, we have accepted an evidence-free approach to using or not using antiepileptic drugs in IFEs. There is huge international variation and only a handful of studies examining neurocognitive outcomes. Clearly, this is a situation ready for an overhaul in practice. Fundamental to understanding treatment is knowledge of aetiology. In recent years, there have been several significant discoveries in IFEs from studies of copy number variation, exome sequencing, and linkage that prompt reconsideration of the "unknown cause" classification and strongly suggest a genetic aetiology. The IFE are strongly age-related, both with regards to age of seizure onset and remission. Does this time window solely relate to a similar age-related gene expression, or are there epigenetic factors involved that might also explain low observed twin concordance? The genetic (and epigenetic) models for different IFEs, their comorbidities, and their similarities to other neurodevelopmental disorders deserve investigation in the coming years. In so doing, we will probably learn much about normal brain functioning. This is because these disorders, perhaps more than any other human brain disease, are disorders of functional brain systems (even though these functional networks may not yet be fully defined). In June 2012, an international group of clinical and basic science researchers met in London under the auspices of the Waterloo Foundation to discuss and debate these issues in relation to IFEs. This Waterloo Foundation Symposium on the Idiopathic Focal Epilepsies: Phenotype to Genotype witnessed presentations that explored the clinical phenomenology, phenotypes and endophenotypes, and genetic approaches to investigation of these disorders. In parallel, the impact of these epilepsies on children and their families was reviewed. The papers in this supplement are based upon these presentations. They represent an updated state-of-the-art thinking on the topics explored. The symposium led to the formation of international working groups under the umbrella of "Luke's Idiopathic Focal Epilepsy Project" to investigate various aspects of the idiopathic focal epilepsies including: semiology and classification, genetics, cognition, sleep, high-frequency oscillations, and parental resources (see www.childhood-epilepsy.org). The next sponsored international workshop, in June 2014, was on randomised controlled trials in IFEs and overnight learning outcome measures.
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Epilepsias Parciais/fisiopatologia , Criança , HumanosRESUMO
BACKGROUND: Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. OBJECTIVES AND METHODS: In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. RESULTS: Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. CONCLUSION: In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.
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Encéfalo/embriologia , Infecções por Citomegalovirus/patologia , Microglia/patologia , Muromegalovirus/patogenicidade , Animais , Linhagem da Célula , Infecções por Citomegalovirus/imunologia , Citometria de Fluxo , Ativação de Macrófagos , Microglia/imunologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Genetic generalized epilepsy (GGE), formerly known as idiopathic generalized epilepsy, is the most common form of epilepsy and is thought to have predominant genetic etiology. GGE are clinically characterized by absence, myoclonic, or generalized tonic-clonic seizures with electroencephalographic pattern of bilateral, synchronous, and symmetrical spike-and-wave discharges. Despite their strong heritability, the genetic basis of generalized epilepsies remains largely elusive. Nevertheless, recent advances in genetic technology have led to the identification of numerous genes and genomic defects in various types of epilepsies in the past few years. In the present study, we performed whole-exome sequencing in a family with GGE consistent with the diagnosis of eyelid myoclonia with absences. We found a nonsense variant (c.196C>T/p.(Arg66*)) in RORB, which encodes the beta retinoid-related orphan nuclear receptor (RORß), in four affected family members. In addition, two de novo variants (c.218T>C/p.(Leu73Pro); c.1249_1251delACG/p.(Thr417del)) were identified in sporadic patients by trio-based exome sequencing. We also found two de novo deletions in patients with behavioral and cognitive impairment and epilepsy: a 52-kb microdeletion involving exons 5-10 of RORB and a larger 9q21-microdeletion. Furthermore, we identified a patient with intellectual disability and a balanced translocation where one breakpoint truncates RORB and refined the phenotype of a recently reported patient with RORB deletion. Our data support the role of RORB gene variants/CNVs in neurodevelopmental disorders including epilepsy, and especially in generalized epilepsies with predominant absence seizures.
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Deficiências do Desenvolvimento/genética , Epilepsia Generalizada/genética , Membro 2 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Adulto , Criança , Pontos de Quebra do Cromossomo , Deleção Cromossômica , Códon sem Sentido , Deficiências do Desenvolvimento/diagnóstico , Epilepsia Generalizada/diagnóstico , Exoma , Éxons , Feminino , Humanos , Masculino , Linhagem , Síndrome , Translocação GenéticaRESUMO
BACKGROUND: Heterozygous dominant mutations of PRRT2 have been associated with various types of paroxysmal neurological manifestations, including benign familial infantile convulsions and paroxysmal kinesigenic dyskinesia. The phenotype associated with biallelic mutations is not well understood as few cases have been reported. METHODS: PRRT2 screening was performed by Sanger sequencing and quantitative multiplex PCR of short fluorescent fragments. A CGH array was used to characterise the size of the deletion at the 16p11.2 locus. RESULTS: Five patients with homozygous or compound heterozygous deleterious PRRT2 gene mutations are described. These patients differ from those with a single mutation by their overall increased severity: (1) the combination of at least three different forms of paroxysmal neurological disorders within the same patient and persistence of paroxysmal attacks; (2) the occurrence of uncommon prolonged episodes of ataxia; and (3) the association of permanent neurological disorders including learning difficulties in four patients and cerebellar atrophy in 2. CONCLUSIONS: Our observations expand the phenotype related to PRRT2 insufficiency, and highlight the complexity of the phenotype associated with biallelic mutations, which represents a severe neurological disease with various paroxysmal disorders and frequent developmental disabilities.
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Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Adolescente , Adulto , Fatores Etários , Alelos , Ataxia/genética , Atrofia/genética , Encefalopatias/genética , Criança , Pré-Escolar , Coreia/genética , Cromossomos Humanos Par 16/genética , Feminino , Deleção de Genes , Genes/genética , Humanos , Lactente , Deficiências da Aprendizagem/genética , Masculino , Reação em Cadeia da Polimerase Multiplex , Fenótipo , Adulto JovemRESUMO
N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated cation channels that are expressed throughout the brain and play essential role in brain functioning. Diversity of the subunits and of their spatio-temporal expression imparts distinct functional properties for the particular NMDAR in a particular brain region and developmental stage. Mutations in NMDARs may have pathological consequences and actually lead to various neurological disorders. Recent human genetic studies as highlighted here show the existence of multiple alterations in NMDARs subunits genes in several usual and common brain diseases, such as intellectual disability, autism spectrum disorders (ASD), or epilepsy. Relation of a particular mutation to the corresponding alteration of NMDARs function may provide an avenue to the targeted therapy for the pharmacological treatment of the disorders.
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Transtornos Mentais/genética , Doenças do Sistema Nervoso/genética , Receptores de N-Metil-D-Aspartato/genética , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Encefalopatias/tratamento farmacológico , Encefalopatias/genética , Encefalopatias/fisiopatologia , Humanos , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/fisiopatologia , Terapia de Alvo Molecular , Mutação , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/fisiopatologiaRESUMO
Inherited and de novo genomic imbalances at chromosome 16p11.2 are associated with autism spectrum disorders (ASD), but the causative genes remain unknown. Among the genes located in this region, PRRT2 codes for a member of the synaptic SNARE complex that allows the release of synaptic vesicles. PRRT2 is a candidate gene for ASD since homozygote mutations are associated with intellectual disability and heterozygote mutations cause benign infantile seizures, paroxysmal dyskinesia, or hemiplegic migraine. Here, we explored the contribution of PRRT2 mutations in ASD by screening its coding part in a large sample of 1578 individuals including 431 individuals with ASD, 186 controls and 961 individuals from the human genome Diversity Panel. We detected 24 nonsynonymous variants, 1 frameshift (A217PfsX8) and 1 in-frame deletion of 6 bp (p.A361_P362del). The frameshift mutation was observed in a control with no history of neurological or psychiatric disorders. The p.A361_P362del was observed in two individuals with autism from sub-Saharan African origin. Overall, the frequency of PRRT2 deleterious variants was not different between individuals with ASD and controls. Remarkably, PRRT2 displays a highly significant excess of nonsynonymous (pN) vs synonymous (pS) mutations in Asia (pN/pSâ=â4.85) and Europe (pN/pSâ=â1.62) compared with Africa (pN/pSâ=â0.26; Asia vs Africa: Pâ=â0.000087; Europe vs Africa Pâ=â0.00035; Europe vs Asia Pâ=âPâ=â0.084). We also showed that whole genome amplification performed through rolling cycle amplification could artificially introduce the A217PfsX8 mutation indicating that this technology should not be performed prior to PRRT2 mutation screening. In summary, our results do not support a role for PRRT2 coding sequence variants in ASD, but provide an ascertainment of its genetic variability in worldwide populations that should help researchers and clinicians to better investigate the role of PRRT2 in human diseases.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Estudos de Casos e Controles , Cromossomos Humanos Par 16 , Variação Genética , Heterozigoto , Humanos , MutaçãoRESUMO
OBJECTIVES: Rolandic epilepsies (REs) represent the most frequent epilepsy in childhood. Patients may experience cognitive, speech, language, reading, and behavioral issues. The genetic origin of REs has long been debated. The participation of rare copy number variations (CNVs) in the pathophysiology of various human epilepsies has been increasingly recognized. However, no systematic search for microdeletions or microduplications has been reported in RE so far. METHODS: Array comparative genomic hybridization (aCGH) and quantitative polymerase chain reaction (qPCR) were used to analyze the genomic status of a series of 47 unrelated RE patients who displayed various types of electroclinical manifestations. RESULTS: Thirty rare CNVs were detected in 21 RE patients. Two CNVs were de novo, 12 were inherited, and 16 were of unknown inheritance. Each CNV was unique to one given patient, except for a 16p11.2 duplication found in two patients. The CNVs of highest interest comprised or disrupted strong candidate or confirmed genes for epileptic and other neurodevelopmental disorders, including BRWD3, GRIN2A, KCNC3, PRKCE, PRRT2, SHANK1, and TSPAN7. SIGNIFICANCE: Patients with REs showed rare microdeletions and microduplications with high frequency and heterogeneity. Whereas only a subset of all genomic alterations found here may actually participate in the phenotype, the novel de novo events as well as several inherited CNVs contain or disrupt genes, some of which are likely to influence the emergence, the presentation, or the comorbidity of RE. The future screening of cohorts of larger size will help in detecting more de novo or recurrent events and in appreciating the possible enrichment of specific CNVs in patients with RE.
Assuntos
Epilepsia Rolândica/diagnóstico , Epilepsia Rolândica/genética , Estudos de Associação Genética/métodos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Feminino , Heterogeneidade Genética , Humanos , MasculinoRESUMO
Epilepsy-aphasia syndromes (EAS) are a group of rare, severe epileptic encephalopathies of unknown etiology with a characteristic electroencephalogram (EEG) pattern and developmental regression particularly affecting language. Rare pathogenic deletions that include GRIN2A have been implicated in neurodevelopmental disorders. We sought to delineate the pathogenic role of GRIN2A in 519 probands with epileptic encephalopathies with diverse epilepsy syndromes. We identified four probands with GRIN2A variants that segregated with the disorder in their families. Notably, all four families presented with EAS, accounting for 9% of epilepsy-aphasia cases. We did not detect pathogenic variants in GRIN2A in other epileptic encephalopathies (n = 475) nor in probands with benign childhood epilepsy with centrotemporal spikes (n = 81). We report the first monogenic cause, to our knowledge, for EAS. GRIN2A mutations are restricted to this group of cases, which has important ramifications for diagnostic testing and treatment and provides new insights into the pathogenesis of this debilitating group of conditions.
Assuntos
Síndrome de Landau-Kleffner/genética , Mutação , Receptores de N-Metil-D-Aspartato/genética , Eletroencefalografia , Feminino , Humanos , Síndrome de Landau-Kleffner/diagnóstico , Masculino , Linhagem , FenótipoRESUMO
Epileptic encephalopathies are severe brain disorders with the epileptic component contributing to the worsening of cognitive and behavioral manifestations. Acquired epileptic aphasia (Landau-Kleffner syndrome, LKS) and continuous spike and waves during slow-wave sleep syndrome (CSWSS) represent rare and closely related childhood focal epileptic encephalopathies of unknown etiology. They show electroclinical overlap with rolandic epilepsy (the most frequent childhood focal epilepsy) and can be viewed as different clinical expressions of a single pathological entity situated at the crossroads of epileptic, speech, language, cognitive and behavioral disorders. Here we demonstrate that about 20% of cases of LKS, CSWSS and electroclinically atypical rolandic epilepsy often associated with speech impairment can have a genetic origin sustained by de novo or inherited mutations in the GRIN2A gene (encoding the N-methyl-D-aspartate (NMDA) glutamate receptor α2 subunit, GluN2A). The identification of GRIN2A as a major gene for these epileptic encephalopathies provides crucial insights into the underlying pathophysiology.
Assuntos
Epilepsias Parciais/genética , Síndrome de Landau-Kleffner/genética , Mutação , Receptores de N-Metil-D-Aspartato/genética , Substituição de Aminoácidos , Linhagem Celular , Eletroencefalografia , Feminino , Expressão Gênica , Genótipo , Humanos , Masculino , Linhagem , FenótipoRESUMO
Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.