RESUMEN
SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture. At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro. Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE. In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.
Asunto(s)
Células Madre Pluripotentes Inducidas , Espasmos Infantiles , Humanos , Masculino , Células Madre Pluripotentes Inducidas/metabolismo , Convulsiones/genética , Espasmos Infantiles/genética , Espasmos Infantiles/metabolismo , Fenotipo , Neuronas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genéticaRESUMEN
De novo variants in the NaV1.2 voltage-gated sodium channel gene SCN2A are among the major causes of developmental and epileptic encephalopathies (DEE). Based on their biophysical impact on channel conductance and gating, SCN2A DEE variants can be classified into gain-of-function (GoF) or loss-of-function (LoF). Clinical and functional data have linked early seizure onset DEE to the GoF SCN2A variants, whereas late seizure onset DEE is associated with the loss of SCN2A function. This study aims to assess the impact of GoF and LoF SCN2A variants on cultured neuronal network activity and explore their modulation by selected antiseizure medications (ASM). To this end, primary cortical cultures were generated from two knock-in mouse lines carrying variants corresponding to human GoF SCN2A p.R1882Q and LoF p.R853Q DEE variant. In vitro neuronal network activity and responses to ASM were analyzed using multielectrode array (MEA) between 2 and 4 weeks in culture. The SCN2A p.R1882Q neuronal cultures showed significantly greater mean firing and burst firing. Their network synchronicity was also higher. In contrast, the SCN2A p.R853Q cultures showed lower mean firing rate, and burst firing events were less frequent. The network synchronicity was also lower. Phenytoin and levetiracetam reduced the excitability of GoF cultures, while retigabine showed differential and potentially beneficial effects on cultures with both GoF and LoF variants. We conclude that in vitro neuronal networks harboring SCN2A GoF or LoF DEE variants present with distinctive phenotypes and responses to ASM.
RESUMEN
Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is an important cause of drug-resistant epilepsy. A significant subset of individuals diagnosed with MOGHE display somatic mosaicism for loss-of-function variants in SLC35A2, which encodes the UDP-galactose transporter. We developed a mouse model to investigate how disruption of this transporter leads to a malformation of cortical development. We used in utero electroporation and CRISPR/Cas9 to knockout Slc35a2 in a subset of layer 2/3 cortical neuronal progenitors in the developing brains of male and female fetal mice to model mosaic expression. Mosaic Slc35a2 knockout was verified through next-generation sequencing and immunohistochemistry of GFP-labelled transfected cells. Histology of brain tissue in mosaic Slc35a2 knockout mice revealed the presence of upper layer-derived cortical neurons in the white matter. Reconstruction of single filled neurons identified altered dendritic arborisation with Slc35a2 knockout neurons having increased complexity. Whole-cell electrophysiological recordings revealed that Slc35a2 knockout neurons display reduced action potential firing, increased afterhyperpolarisation duration and reduced burst-firing when compared with control neurons. Mosaic Slc35a2 knockout mice also exhibited significantly increased epileptiform spiking and increased locomotor activity. We successfully generated a mouse model of mosaic Slc35a2 deficiency, which recapitulates features of the human phenotype, including impaired neuronal migration. We show that knockout in layer 2/3 cortical neuron progenitors is sufficient to disrupt neuronal excitability, increase epileptiform activity and cause hyperactivity in mosaic mice. Our mouse model provides an opportunity to further investigate the disease mechanisms that contribute to MOGHE and facilitate the development of precision therapies.
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Neuronas , Animales , Femenino , Masculino , Ratones , Potenciales de Acción/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Dendritas/metabolismo , Dendritas/patología , Modelos Animales de Enfermedad , Epilepsia/genética , Epilepsia/patología , Epilepsia/metabolismo , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/patología , Ratones Noqueados , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/deficiencia , Proteínas de Transporte de Monosacáridos/metabolismo , Mosaicismo , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.
Asunto(s)
Canales Iónicos Sensibles al Ácido/biosíntesis , Canales Iónicos Sensibles al Ácido/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Ratones Noqueados , Isquemia Miocárdica/terapia , Daño por Reperfusión Miocárdica/terapia , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Polimorfismo de Nucleótido Simple/fisiología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Venenos de Araña/farmacologíaRESUMEN
Genetic variation in voltage-gated sodium (NaV) channels is a significant contributor to neurodevelopmental disorders. NaV channel alpha subunits are encoded by the SCNxA family and four are predominately expressed in the brain: SCN1A, SCN2A, SCN3A, and SCN8A. Gene expression is developmentally regulated, and they are known to express functionally distinct transcript variants. Precision therapies targeting these genes and their transcript variants are currently in preclinical development, yet the developmental expression of these transcripts in the human brain is yet to be fully understood. Additionally, the functional consequences of some mutations differ depending on the studied channel isoform, suggesting differential transcript variant expression can affect disease prognoses. We characterise the expression of the four SCNxAs and their transcript variants in human, Rhesus monkey and mouse brain using publicly available RNA-sequencing data and analysis tools, demonstrating that this approach can be used to answer important biological questions of gene and transcript developmental regulation. We find that gene expression and transcript variant regulation are conserved across species at similar developmental stages and determine the developmental milestones for transcript variant expression. Our study provides a guide to researchers testing therapies and clinicians advising prognoses based on the expression of channel isoforms.
Asunto(s)
Encéfalo/embriología , Mutación , Canales de Sodio/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Macaca mulatta , Ratones , Canales de Sodio/genéticaRESUMEN
OBJECTIVE: We aimed to characterize the phenotypic spectrum and functional consequences associated with variants in the gene GABRB2, coding for the γ-aminobutyric acid type A (GABAA ) receptor subunit ß2. METHODS: We recruited and systematically evaluated 25 individuals with variants in GABRB2, 17 of whom are newly described and 8 previously reported with additional clinical data. Functional analysis was performed using a Xenopus laevis oocyte model system. RESULTS: Our cohort of 25 individuals from 22 families with variants in GABRB2 demonstrated a range of epilepsy phenotypes from genetic generalized epilepsy to developmental and epileptic encephalopathy. Fifty-eight percent of individuals had pharmacoresistant epilepsy; response to medications targeting the GABAergic pathway was inconsistent. Developmental disability (present in 84%) ranged from mild intellectual disability to severe global disability; movement disorders (present in 44%) included choreoathetosis, dystonia, and ataxia. Disease-associated variants cluster in the extracellular N-terminus and transmembrane domains 1-3, with more severe phenotypes seen in association with variants in transmembrane domains 1 and 2 and the allosteric binding site between transmembrane domains 2 and 3. Functional analysis of 4 variants in transmembrane domains 1 or 2 (p.Ile246Thr, p.Pro252Leu, p.Ile288Ser, p.Val282Ala) revealed strongly reduced amplitudes of GABA-evoked anionic currents. INTERPRETATION: GABRB2-related epilepsy ranges broadly in severity from genetic generalized epilepsy to developmental and epileptic encephalopathies. Developmental disability and movement disorder are key features. The phenotypic spectrum is comparable to other GABAA receptor-encoding genes. Phenotypic severity varies by protein domain. Experimental evidence supports loss of GABAergic inhibition as the mechanism underlying GABRB2-associated neurodevelopmental disorders. ANN NEUROL 2021;89:573-586.
Asunto(s)
Epilepsia/fisiopatología , Trastornos del Movimiento/fisiopatología , Trastornos del Neurodesarrollo/fisiopatología , Receptores de GABA-A/genética , Adolescente , Adulto , Animales , Ataxia/genética , Ataxia/fisiopatología , Atetosis/genética , Atetosis/fisiopatología , Niño , Preescolar , Corea/genética , Corea/fisiopatología , Estudios de Cohortes , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/fisiopatología , Epilepsia Refractaria/genética , Epilepsia Refractaria/fisiopatología , Distonía/genética , Distonía/fisiopatología , Epilepsia/genética , Femenino , Genotipo , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/fisiopatología , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , Oocitos , Técnicas de Placa-Clamp , Fenotipo , Dominios Proteicos/genética , Xenopus laevis , Adulto JovenRESUMEN
Pathogenic variants in HCN1 are associated with developmental and epileptic encephalopathies. The recurrent de novo HCN1 M305L pathogenic variant is associated with severe developmental impairment and drug-resistant epilepsy. We engineered the homologue Hcn1 M294L heterozygous knock-in (Hcn1M294L) mouse to explore the disease mechanism underlying an HCN1 developmental and epileptic encephalopathy. The Hcn1M294L mouse recapitulated the phenotypic features of patients with the HCN1 M305L variant, including spontaneous seizures and a learning deficit. Active epileptiform spiking on the electrocorticogram and morphological markers typical of rodent seizure models were observed in the Hcn1M294L mouse. Lamotrigine exacerbated seizures and increased spiking, whereas sodium valproate reduced spiking, mirroring drug responses reported in a patient with this variant. Functional analysis in Xenopus laevis oocytes and layer V somatosensory cortical pyramidal neurons in ex vivo tissue revealed a loss of voltage dependence for the disease variant resulting in a constitutively open channel that allowed for cation 'leak' at depolarized membrane potentials. Consequently, Hcn1M294L layer V somatosensory cortical pyramidal neurons were significantly depolarized at rest. These neurons adapted through a depolarizing shift in action potential threshold. Despite this compensation, layer V somatosensory cortical pyramidal neurons fired action potentials more readily from rest. A similar depolarized resting potential and left-shift in rheobase was observed for CA1 hippocampal pyramidal neurons. The Hcn1M294L mouse provides insight into the pathological mechanisms underlying hyperexcitability in HCN1 developmental and epileptic encephalopathy, as well as being a preclinical model with strong construct and face validity, on which potential treatments can be tested.
Asunto(s)
Encefalopatías/metabolismo , Modelos Animales de Enfermedad , Epilepsia/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , Animales , Encefalopatías/genética , Epilepsia/genética , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Masculino , Ratones , Ratones Mutantes , Mutación , Neuronas/patología , Canales de Potasio/genética , Células Piramidales/metabolismo , Xenopus laevisRESUMEN
Epilepsy is a common and serious neurologic disease with a strong genetic component. Genetic studies have identified an increasing collection of disease-causing genes. The impact of these genetic discoveries is wide reaching-from precise diagnosis and classification of syndromes to the discovery and validation of new drug targets and the development of disease-targeted therapeutic strategies. About 25% of genes identified in epilepsy encode ion channels. Much of our understanding of disease mechanisms comes from work focused on this class of protein. In this study, we review the genetic, molecular, and physiologic evidence supporting the pathogenic role of a number of different voltage- and ligand-activated ion channels in genetic epilepsy. We also review proposed disease mechanisms for each ion channel and highlight targeted therapeutic strategies.
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Epilepsia/genética , Canales Iónicos/genética , Animales , Anticonvulsivantes/farmacología , Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Epilepsia/metabolismo , Humanos , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Terapia Molecular DirigidaRESUMEN
OBJECTIVE: To elucidate the biophysical basis underlying the distinct and severe clinical presentation in patients with the recurrent missense SCN1A variant, p.Thr226Met. Patients with this variant show a well-defined genotype-phenotype correlation and present with developmental and early infantile epileptic encephalopathy that is far more severe than typical SCN1A Dravet syndrome. METHODS: Whole cell patch clamp and dynamic action potential clamp were used to study T226M Nav 1.1 channels expressed in mammalian cells. Computational modeling was used to explore the neuronal scale mechanisms that account for altered action potential firing. RESULTS: T226M channels exhibited hyperpolarizing shifts of the activation and inactivation curves and enhanced fast inactivation. Dynamic action potential clamp hybrid simulation showed that model neurons containing T226M conductance displayed a left shift in rheobase relative to control. At current stimulation levels that produced repetitive action potential firing in control model neurons, depolarization block and cessation of action potential firing occurred in T226M model neurons. Fully computationally simulated neuron models recapitulated the findings from dynamic action potential clamp and showed that heterozygous T226M models were also more susceptible to depolarization block. INTERPRETATION: From a biophysical perspective, the T226M mutation produces gain of function. Somewhat paradoxically, our data suggest that this gain of function would cause interneurons to more readily develop depolarization block. This "functional dominant negative" interaction would produce a more profound disinhibition than seen with haploinsufficiency that is typical of Dravet syndrome and could readily explain the more severe phenotype of patients with T226M mutation. Ann Neurol 2019;85:514-525.
Asunto(s)
Epilepsias Mioclónicas/genética , Mutación con Ganancia de Función/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Espasmos Infantiles/genética , Animales , Células CHO , Cricetulus , Bases de Datos Genéticas , Epilepsias Mioclónicas/diagnóstico , Humanos , Espasmos Infantiles/diagnósticoRESUMEN
PURPOSE OF REVIEW: Recent publications point to an increasingly important role of variants in genes encoding GABAA receptor subunits associated with both common and rare forms of epilepsies. The aim of this review is to give an overview of the current clinical phenotypes, genetic findings and pathophysiological mechanisms related to GABAA receptor variants. RECENT FINDINGS: Early work showed that inherited variants in GABRG2 and GABRA1 cause relatively mild forms of monogenic epilepsies in large families. More recent studies have revealed that de novo variants in several GABAA receptor genes cause severe developmental and epileptic encephalopathies, inherited variants cause remarkably variable phenotypes within the same pedigrees ranging from asymptomatic carriers to developmental and epileptic encephalopathies, and variants in all GABAA receptor genes are enriched in common forms of epilepsy, namely rolandic epilepsy and genetic generalized epilepsy. Analyses from cellular expression systems and mouse models suggest that all variants cause a loss of GABAA receptor function resulting in GABAergic disinhibition. SUMMARY: Genetic studies have revealed a crucial role of the GABAergic system in the underlying pathogenesis of various forms of common and rare epilepsies. Our understanding of functional consequences of GABAA receptor variants provide an opportunity to develop precision-based therapeutic strategies that are hopefully free from the side-effect burden seen with currently available GABAergic drugs.
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Epilepsia/genética , Receptores de GABA-A/genética , Animales , Epilepsia/fisiopatología , Humanos , RatonesRESUMEN
Genetic generalized epilepsy (GGE) is a common epilepsy syndrome that encompasses seizure disorders characterized by spike-and-wave discharges (SWDs). Pacemaker hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are considered integral to SWD genesis, making them an ideal gene candidate for GGE. We identified HCN2 missense variants from a large cohort of 585 GGE patients, recruited by the Epilepsy Phenome-Genome Project (EPGP), and performed functional analysis using two-electrode voltage clamp recordings from Xenopus oocytes. The p.S632W variant was identified in a patient with idiopathic photosensitive occipital epilepsy and segregated in the family. This variant was also independently identified in an unrelated patient with childhood absence seizures from a European cohort of 238 familial GGE cases. The p.V246M variant was identified in a patient with photo-sensitive GGE and his father diagnosed with juvenile myoclonic epilepsy. Functional studies revealed that both p.S632W and p.V246M had an identical functional impact including a depolarizing shift in the voltage dependence of activation that is consistent with a gain-of-function. In contrast, no biophysical changes resulted from the introduction of common population variants, p.E280K and p.A705T, and the p.R756C variant from EPGP that did not segregate with disease. Our data suggest that HCN2 variants can confer susceptibility to GGE via a gain-of-function mechanism.
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ADN Complementario/genética , Epilepsia Generalizada/genética , Epilepsia/genética , Mutación con Ganancia de Función/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Electrofisiología , Femenino , Humanos , Masculino , Modelos Biológicos , LinajeRESUMEN
OBJECTIVE: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. METHODS: We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-clamp to explore biophysical properties of wild-type and mutant KV 3.1 channels. RESULTS: Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus; 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type KV 3.1, increasing channel availability. INTERPRETATION: MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type KV 3.1 subunit-containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics. Ann Neurol 2017;81:677-689.
Asunto(s)
Ataxia , Disfunción Cognitiva/etiología , Epilepsias Mioclónicas , Calor , Canales de Potasio Shaw/metabolismo , Adolescente , Adulto , Edad de Inicio , Ataxia/complicaciones , Ataxia/diagnóstico por imagen , Ataxia/genética , Ataxia/fisiopatología , Electroencefalografía , Epilepsias Mioclónicas/complicaciones , Epilepsias Mioclónicas/diagnóstico por imagen , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Femenino , Células HEK293 , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mutación , Linaje , Canales de Potasio Shaw/genética , Síndrome , Adulto JovenRESUMEN
Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.
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Encefalopatías/genética , Fisura del Paladar/genética , Discapacidades del Desarrollo/genética , Epilepsia/genética , Facies , Discapacidad Intelectual/genética , Nistagmo Patológico/genética , Receptores de GABA-A/genética , Adolescente , Adulto , Animales , Niño , Preescolar , Femenino , Variación Genética , Humanos , Masculino , Microcefalia/genética , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Técnicas de Placa-Clamp , Linaje , Receptores de GABA-A/metabolismo , Síndrome , Xenopus laevis , Adulto Joven , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Epileptic encephalopathies are severe disorders emerging in the first days to years of life that commonly include refractory seizures, various types of movement disorders, and different levels of developmental delay. In recent years, many de novo occurring variants have been identified in individuals with these devastating disorders. To unravel disease mechanisms, the functional impact of detected variants associated with epileptic encephalopathies is investigated in a range of cellular and animal models. This review addresses efforts to advance and use such models to identify specific molecular and cellular targets for the development of novel therapies. We focus on ion channels as the best-studied group of epilepsy genes. Given the clinical and genetic heterogeneity of epileptic encephalopathy disorders, experimental models that can reflect this complexity are critical for the development of disease mechanisms-based targeted therapy. The convergence of technological advances in gene sequencing, stem cell biology, genome editing, and high throughput functional screening together with massive unmet clinical needs provides unprecedented opportunities and imperatives for precision medicine in epileptic encephalopathies.
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Encefalopatías/genética , Encefalopatías/terapia , Sistemas de Liberación de Medicamentos/tendencias , Epilepsia/genética , Epilepsia/terapia , Predisposición Genética a la Enfermedad/genética , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticonvulsivantes/administración & dosificación , Encefalopatías/epidemiología , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Modelos Animales de Enfermedad , Epilepsia/epidemiología , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/prevención & control , Terapia Genética/tendencias , HumanosRESUMEN
OBJECTIVE: Mutations in KCNQ2 and KCNQ3, encoding the voltage-gated potassium channels KV 7.2 and KV 7.3, are known to cause benign familial neonatal seizures mainly by haploinsufficiency. Here, we set out to determine the disease mechanism of 7 de novo missense KCNQ2 mutations that were recently described in patients with a severe epileptic encephalopathy including pharmacoresistant seizures and pronounced intellectual disability. METHODS: Mutations were inserted into the KCNQ2 cDNA. Potassium currents were recorded using 2-microelectrode voltage clamping, and surface expression was analyzed by a biotinylation assay in cRNA-injected Xenopus laevis oocytes. RESULTS: We observed a clear loss of function for all mutations. Strikingly, 5 of 7 mutations exhibited a drastic dominant-negative effect on wild-type KV 7.2 or KV 7.3 subunits, either by globally reducing current amplitudes (3 pore mutations) or by a depolarizing shift of the activation curve (2 voltage sensor mutations) decreasing potassium currents at the subthreshold level at which these channels are known to critically influence neuronal firing. One mutation significantly reduced surface expression. Application of retigabine, a recently marketed KV 7 channel opener, partially reversed these effects for the majority of analyzed mutations. INTERPRETATION: The development of severe epilepsy and cognitive decline in children carrying 5 of the 7 studied KCNQ2 mutations can be related to a dominant-negative reduction of the resulting potassium current at subthreshold membrane potentials. Other factors such as genetic modifiers have to be postulated for the remaining 2 mutations. Retigabine or similar drugs may be used as a personalized therapy for this severe disease.
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Epilepsia Benigna Neonatal/genética , Predisposición Genética a la Enfermedad/genética , Canal de Potasio KCNQ2/genética , Canales de Potasio con Entrada de Voltaje/genética , Animales , Carbamatos/farmacología , Epilepsia Benigna Neonatal/fisiopatología , Humanos , Canal de Potasio KCNQ2/efectos de los fármacos , Canal de Potasio KCNQ2/fisiología , Potenciales de la Membrana/genética , Mutación Missense , Oocitos , Fenilendiaminas/farmacología , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/fisiología , XenopusAsunto(s)
Epilepsia Generalizada , Epilepsia , Discapacidad Intelectual , Humanos , Receptores de GABA-A , ConvulsionesAsunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Epilepsia/genética , Epilepsia/fisiopatología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Reflejo Anormal/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Femenino , Humanos , Hiperekplexia/genética , Masculino , Mutación , Linaje , Fenotipo , Estado Epiléptico/genéticaRESUMEN
Many developmental and epileptic encephalopathies (DEEs) result from variants in cation channel genes. Using mRNA transfection, we generated and characterised an induced pluripotent stem cell (iPSC) line from the fibroblasts of a male late-onset DEE patient carrying a heterozygous missense variant (E1211K) in Nav1.2(SCN2A) protein. The iPSC line displays features characteristic of the human iPSCs, colony morphology and expression of pluripotency-associated marker genes, ability to produce derivatives of all three embryonic germ layers, and normal karyotype without SNP array-detectable abnormalities. We anticipate that this iPSC line will aid in the modelling and development of precision therapies for this debilitating condition.
Asunto(s)
Encefalopatías , Células Madre Pluripotentes Inducidas , Humanos , Masculino , Células Madre Pluripotentes Inducidas/metabolismo , Mutación Missense , Heterocigoto , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genéticaRESUMEN
The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.
Asunto(s)
Activación del Canal Iónico , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Treonina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Secuencia Conservada , Cricetinae , Cricetulus , Humanos , Canal de Potasio KCNQ2/química , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/química , Canal de Potasio KCNQ3/genética , Potenciales de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Treonina/química , Treonina/genéticaRESUMEN
Missense mutations in SCN2A, encoding the brain sodium channel NaV 1.2, have been described in benign familial neonatal-infantile seizures (BFNIS), a self-limiting disorder, whereas several SCN2A de novo nonsense mutations have been found in patients with more severe phenotypes including epileptic encephalopathy. We report a family with BFNIS originating from Madagascar. Onset extended from 3 to 9 months of age. Interictal EEGs were normal. In two patients, ictal electroencephalography (EEG) studies showed partial seizure patterns with secondary generalization in one. Seizures remitted before 18 months of age, with or without medication. Intellectual development was normal. A novel missense mutation of SCN2A, c.4766A>G/p.Tyr1589Cys, was found in a highly conserved region of NaV 1.2 (D4/S2-S3). Functional studies using heterologous expression in tsA201 cells and whole-cell patch clamping revealed a depolarizing shift of steady-state inactivation, increased persistent Na(+) current, a slowing of fast inactivation and an acceleration of its recovery, thus a gain-of-function. Using an action potential waveform in a voltage-clamp experiment we indicated an increased inward Na(+) current at subthreshold voltages, which can explain a neuronal hyperexcitability. Our results suggest that this mutation induces neuronal hyperexcitability, resulting in infantile epilepsy with favorable outcome.