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Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.
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Encefalopatias , Camundongos , Animais , Convulsões/genética , Convulsões/terapiaRESUMO
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.
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Epilepsias Mioclônicas/genética , Mutação com Ganho de Função/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Espasmos Infantis/genética , Animais , Células CHO , Cricetulus , Bases de Dados Genéticas , Epilepsias Mioclônicas/diagnóstico , Humanos , Espasmos Infantis/diagnósticoRESUMO
OBJECTIVE: To examine the genotype to phenotype connection in glucose transporter type 1 (GLUT1) deficiency and whether a simple functional assay can predict disease outcome from genetic sequence alone. METHODS: GLUT1 deficiency, due to mutations in SLC2A1, causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function. To test this, we measured glucose transport by GLUT1 variants identified in population controls and patients with mild to severe epilepsies. Controls were reference sequence from the NCBI and 4 population missense variants chosen from public reference control databases. Nine variants associated with epilepsies or movement disorders, with normal intellect in all individuals, formed the mild group. The severe group included 5 missense variants associated with classical GLUT1 encephalopathy. GLUT1 variants were expressed in Xenopus laevis oocytes, and glucose uptake was measured to determine kinetics (Vmax) and affinity (Km). RESULTS: Disease severity inversely correlated with rate of glucose transport between control (Vmax = 28 ± 5), mild (Vmax = 16 ± 3), and severe (Vmax = 3 ± 1) groups, respectively. Affinities of glucose binding in control (Km = 55 ± 18) and mild (Km = 43 ± 10) groups were not significantly different, whereas affinity was indeterminate in the severe group because of low transport rates. Simplified analysis of glucose transport at high concentration (100 mM) was equally effective at separating the groups. CONCLUSIONS: Disease severity can be partly explained by the extent of GLUT1 dysfunction. This simple Xenopus oocyte assay complements genetic and clinical assessments. In prenatal diagnosis, this simple oocyte glucose uptake assay could be useful because standard clinical assessments are not available.
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BACKGROUND: Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties. Therefore, strategies to improve correspondence of derived neuronal cultures with primary neurons need to be developed to validate the use of stem cell-derived neuronal cultures as in vitro models. NEW METHOD: By combining serum-free derivation of ESCs from mouse blastocysts with neuronal differentiation of ESCs in morphogen-free adherent culture we generated neuronal networks with properties recapitulating those of mature primary cortical cultures. RESULTS: After 35days of differentiation ESC-derived neurons developed network activity very similar to that of mature primary cortical neurons. Importantly, ESC plating density was critical for network development. COMPARISON WITH EXISTING METHOD(S): Compared to the previously published methods this protocol generated more synchronous neuronal networks, with high similarity to the networks formed in mature primary cortical culture. CONCLUSION: We have demonstrated that ESC-derived neuronal networks recapitulating key properties of mature primary cortical networks can be generated by optimising both stem cell derivation and differentiation. This validates the approach of using ESC-derived neuronal cultures for disease modelling and in vitro drug screening.
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Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Embrionárias Murinas/fisiologia , Neurogênese , Neurônios/fisiologia , Potenciais de Ação , Animais , Animais Recém-Nascidos , Blastômeros/citologia , Blastômeros/fisiologia , Contagem de Células , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Sincronização Cortical/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microeletrodos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurônios/citologiaRESUMO
OBJECTIVE: To characterize the phenotypic spectrum, molecular genetic findings, and functional consequences of pathogenic variants in early-onset KCNT1 epilepsy. METHODS: We identified a cohort of 31 patients with epilepsy of infancy with migrating focal seizures (EIMFS) and screened for variants in KCNT1 using direct Sanger sequencing, a multiple-gene next-generation sequencing panel, and whole-exome sequencing. Additional patients with non-EIMFS early-onset epilepsy in whom we identified KCNT1 variants on local diagnostic multiple gene panel testing were also included. When possible, we performed homology modeling to predict the putative effects of variants on protein structure and function. We undertook electrophysiologic assessment of mutant KCNT1 channels in a xenopus oocyte model system. RESULTS: We identified pathogenic variants in KCNT1 in 12 patients, 4 of which are novel. Most variants occurred de novo. Ten patients had a clinical diagnosis of EIMFS, and the other 2 presented with early-onset severe nocturnal frontal lobe seizures. Three patients had a trial of quinidine with good clinical response in 1 patient. Computational modeling analysis implicates abnormal pore function (F346L) and impaired tetramer formation (F502V) as putative disease mechanisms. All evaluated KCNT1 variants resulted in marked gain of function with significantly increased channel amplitude and variable blockade by quinidine. CONCLUSIONS: Gain-of-function KCNT1 pathogenic variants cause a spectrum of severe focal epilepsies with onset in early infancy. Currently, genotype-phenotype correlations are unclear, although clinical outcome is poor for the majority of cases. Further elucidation of disease mechanisms may facilitate the development of targeted treatments, much needed for this pharmacoresistant genetic epilepsy.
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Epilepsias Parciais/genética , Epilepsias Parciais/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Idade de Início , Animais , Anticonvulsivantes , Pré-Escolar , Simulação por Computador , Epilepsias Parciais/epidemiologia , Epilepsias Parciais/terapia , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Genéticos , Modelos Moleculares , Proteínas do Tecido Nervoso/antagonistas & inibidores , Oócitos , Fenótipo , Bloqueadores dos Canais de Potássio/uso terapêutico , Canais de Potássio Ativados por Sódio , Quinidina/uso terapêutico , Relação Estrutura-Atividade , XenopusRESUMO
The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin+neural precursor cells (NPCs) have a propensity to generate new neurons here. We sought to confirm this by administering tamoxifen to adult NesCreERT2/R26eYFP transgenic mice, which permanently labelled adult nestin-expressing cells and their progeny with enhanced yellow fluorescent protein (eYFP). eYFP+ midbrain cells were then characterized 1-32weeks later in acutely prepared brain slices using whole-cell patch clamp electrophysiology combined with single-cell RT-qPCR. Most eYFP+ cells exhibited a mature neuronal phenotype with large amplitude fast action potentials (APs), spontaneous post-synaptic currents (sPSCs), and expression of 'mature' neuronal genes (NeuN, Gad1, Gad2 and/or VGLUT2). This was the case even at the earliest time-point following tamoxifen (i.e. 1week). In comparison to neighboring eYFP- (control) cells, eYFP+ cells discharged more APs per unit current injection, and had faster AP time-to-peak, hyperpolarized resting membrane potential, smaller membrane capacitance and shorter duration sPSCs. eYFP+ cells were also differentiated from eYFP- cells by increased expression of 'immature' pro-neuronal genes (Pax6, Ngn2 and/or Msx1). However, further analyses failed to reveal evidence of a place of birth, neuronal differentiation, maturation and integration indicative of classical neurogenesis. Thus our findings do not support the notion that nestin+NPCs in the adult SNc and midbrain generate new neurons via classical neurogenesis. Rather, they raise the possibility that mature neurons express nestin under unknown circumstances, and that this is associated with altered physiology and gene expression.
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Envelhecimento/fisiologia , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Mesencéfalo/citologia , Nestina/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Forma Celular , Proteínas Luminescentes/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Análise de Componente Principal , Reação em Cadeia da Polimerase em Tempo Real , Análise de Célula ÚnicaRESUMO
BACKGROUND: The relationship between monogenic and polygenic forms of epilepsy is poorly understood and the extent to which the genetic and acquired epilepsies share common pathways is unclear. Here, we use an integrated systems-level analysis of brain gene expression data to identify molecular networks disrupted in epilepsy. RESULTS: We identified a co-expression network of 320 genes (M30), which is significantly enriched for non-synonymous de novo mutations ascertained from patients with monogenic epilepsy and for common variants associated with polygenic epilepsy. The genes in the M30 network are expressed widely in the human brain under tight developmental control and encode physically interacting proteins involved in synaptic processes. The most highly connected proteins within the M30 network were preferentially disrupted by deleterious de novo mutations for monogenic epilepsy, in line with the centrality-lethality hypothesis. Analysis of M30 expression revealed consistent downregulation in the epileptic brain in heterogeneous forms of epilepsy including human temporal lobe epilepsy, a mouse model of acquired temporal lobe epilepsy, and a mouse model of monogenic Dravet (SCN1A) disease. These results suggest functional disruption of M30 via gene mutation or altered expression as a convergent mechanism regulating susceptibility to epilepsy broadly. Using the large collection of drug-induced gene expression data from Connectivity Map, several drugs were predicted to preferentially restore the downregulation of M30 in epilepsy toward health, most notably valproic acid, whose effect on M30 expression was replicated in neurons. CONCLUSIONS: Taken together, our results suggest targeting the expression of M30 as a potential new therapeutic strategy in epilepsy.
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Descoberta de Drogas , Epilepsia do Lobo Temporal/genética , Epilepsia/genética , Redes Reguladoras de Genes/genética , Animais , Anticonvulsivantes/uso terapêutico , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Epilepsia/patologia , Epilepsia do Lobo Temporal/patologia , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Herança Multifatorial/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genéticaRESUMO
OBJECTIVE: To identify the genetic basis of a family segregating episodic ataxia, infantile seizures, and heterogeneous epilepsies and to study the phenotypic spectrum of KCNA2 mutations. METHODS: A family with 7 affected individuals over 3 generations underwent detailed phenotyping. Whole genome sequencing was performed on a mildly affected grandmother and her grandson with epileptic encephalopathy (EE). Segregating variants were filtered and prioritized based on functional annotations. The effects of the mutation on channel function were analyzed in vitro by voltage clamp assay and in silico by molecular modeling. KCNA2 was sequenced in 35 probands with heterogeneous phenotypes. RESULTS: The 7 family members had episodic ataxia (5), self-limited infantile seizures (5), evolving to genetic generalized epilepsy (4), focal seizures (2), and EE (1). They had a segregating novel mutation in the shaker type voltage-gated potassium channel KCNA2 (CCDS_827.1: c.765_773del; p.255_257del). A rare missense SCN2A (rs200884216) variant was also found in 2 affected siblings and their unaffected mother. The p.255_257del mutation caused dominant negative loss of channel function. Molecular modeling predicted repositioning of critical arginine residues in the voltage-sensing domain. KCNA2 sequencing revealed 1 de novo mutation (CCDS_827.1: c.890G>A; p.Arg297Gln) in a girl with EE, ataxia, and tremor. CONCLUSIONS: A KCNA2 mutation caused dominantly inherited episodic ataxia, mild infantile-onset seizures, and later generalized and focal epilepsies in the setting of normal intellect. This observation expands the KCNA2 phenotypic spectrum from EE often associated with chronic ataxia, reflecting the marked variation in severity observed in many ion channel disorders.
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Anticonvulsivantes/uso terapêutico , Ataxia/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Canal de Potássio Kv1.2/genética , Mutação/genética , Farmacogenética , Idoso , Animais , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Lactente , Masculino , Potenciais da Membrana/genética , Pessoa de Meia-Idade , Modelos Químicos , Oócitos , Xenopus laevis , Adulto JovemRESUMO
OBJECTIVES: We report development of a targeted resequencing gene panel for focal epilepsy, the most prevalent phenotypic group of the epilepsies. METHODS: The targeted resequencing gene panel was designed using molecular inversion probe (MIP) capture technology and sequenced using massively parallel Illumina sequencing. RESULTS: We demonstrated proof of principle that mutations can be detected in 4 previously genotyped focal epilepsy cases. We searched for both germline and somatic mutations in 251 patients with unsolved sporadic or familial focal epilepsy and identified 11 novel or very rare missense variants in 5 different genes: CHRNA4, GRIN2B, KCNT1, PCDH19, and SCN1A. Of these, 2 were predicted to be pathogenic or likely pathogenic, explaining â¼0.8% of the cohort, and 8 were of uncertain significance based on available data. CONCLUSIONS: We have developed and validated a targeted resequencing panel for focal epilepsies, the most important clinical class of epilepsies, accounting for about 60% of all cases. Our application of MIP technology is an innovative approach that will be advantageous in the clinical setting because it is highly sensitive, efficient, and cost-effective for screening large patient cohorts. Our findings indicate that mutations in known genes likely explain only a small proportion of focal epilepsy cases. This is not surprising given the established clinical and genetic heterogeneity of these disorders and underscores the importance of further gene discovery studies in this complex syndrome.
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Epilepsias Parciais/genética , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Análise de Sequência de DNA/métodos , Caderinas/genética , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , Protocaderinas , Receptores de N-Metil-D-Aspartato/genética , Receptores Nicotínicos/genéticaRESUMO
OBJECTIVE: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene. METHODS: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation. RESULTS: Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system. INTERPRETATION: PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease.
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Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.
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Processamento Alternativo , Comportamento Animal , Predisposição Genética para Doença/genética , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Convulsões/genética , Potenciais de Ação , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Modelos Animais de Doenças , Éxons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Neurônios/citologia , Neurônios/metabolismo , Pentilenotetrazol/efeitos adversos , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: Mutations in KCNT1 have been implicated in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and epilepsy of infancy with migrating focal seizures (EIMFS). More recently, a whole exome sequencing study of epileptic encephalopathies identified an additional de novo mutation in 1 proband with EIMFS. We aim to investigate the electrophysiological and pharmacological characteristics of hKCNT1 mutations and examine developmental expression levels. METHODS: Here we use a Xenopus laevis oocyte-based automated 2-electrode voltage clamp assay. The effects of quinidine (100 and 300 µM) are also tested. Using quantitative reverse transcriptase polymerase chain reaction, the relative levels of mouse brain mKcnt1 mRNA expression are determined. RESULTS: We demonstrate that KCNT1 mutations implicated in epilepsy cause a marked increase in function. Importantly, there is a significant group difference in gain of function between mutations associated with ADNFLE and EIMFS. Finally, exposure to quinidine significantly reduces this gain of function for all mutations studied. INTERPRETATION: These results establish direction for a targeted therapy and potentially exemplify a translational paradigm for in vitro studies informing novel therapies in a neuropsychiatric disease.
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Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Quinidina/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Oócitos , Técnicas de Patch-Clamp , Canais de Potássio Ativados por Sódio , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Xenopus laevisRESUMO
OBJECTIVE: To determine the genes underlying Dravet syndrome in patients who do not have an SCN1A mutation on routine testing. METHODS: We performed whole-exome sequencing in 13 SCN1A-negative patients with Dravet syndrome and targeted resequencing in 67 additional patients to identify new genes for this disorder. RESULTS: We detected disease-causing mutations in 2 novel genes for Dravet syndrome, with mutations in GABRA1 in 4 cases and STXBP1 in 3. Furthermore, we identified 3 patients with previously undetected SCN1A mutations, suggesting that SCN1A mutations occur in even more than the currently accepted â¼ 75% of cases. CONCLUSIONS: We show that GABRA1 and STXBP1 make a significant contribution to Dravet syndrome after SCN1A abnormalities have been excluded. Our results have important implications for diagnostic testing, clinical management, and genetic counseling of patients with this devastating disorder and their families.
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Epilepsias Mioclônicas/genética , Predisposição Genética para Doença/genética , Proteínas Munc18/genética , Mutação/genética , Receptores de GABA-A/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Adulto JovemRESUMO
Coxsackievirus B3 (CVB3) is a picornavirus that is responsible for a significant proportion of human myocarditis. However, no antiviral treatment is currently available to treat this disease or indeed any picornaviral infections. Previously it was shown that amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride inhibit the in vitro enzymatic activity of CVB3 RNA polymerase (3Dpol). Here we measure and compare the inhibitory activity of ten amiloride analogues against CVB3 3Dpol. We show that replacement of the 3,5-diaminopyrazinyl moiety of amiloride causes loss of the inhibitory activity, whereas modifications at the 5-amino and guanidino groups increase or decrease potency. Importantly, a combination of substitutions at both the 5-amino and guanidino groups produced a compound that was more potent than its singly modified precursors. The compounds were computationally-docked into available crystal structures of CVB3 3Dpol in order to obtain a structural explanation for the activities of the analogues. To create a robust model which explained the biological activity, optimization of one of the CVB3 3Dpol crystal structures to take into account active site flexibility was necessary, together with the use of consensus docking from two different docking algorithms. This robust predictive 3D atomic model provides insights into the interactions required for inhibitor binding and provides a promising basis for the development of more potent inhibitors against this important therapeutic target.
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Acylguanidines are a new class of antiviral compounds with the unique ability to target both RNA polymerase and transmembrane proteins of viruses from different families. Importantly, they inhibit proteins which are not targeted by existing antiviral therapies, for example, Vpu of HIV type 1, p7 of hepatitis C virus, E of severe acute respiratory syndrome coronavirus and RNA-dependent RNA polymerase of coxsackievirus B3. BIT225, developed by Biotron Limited, is the first acylguanidine in clinical trials against HIV type 1 and hepatitis C virus. In this article we focus on the mechanisms of inhibition of viral proteins by acylguanidines.
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Antivirais/farmacologia , Guanidinas/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , HumanosRESUMO
Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.
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Amilorida/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/enzimologia , Inibidores Enzimáticos/farmacologia , Amilorida/metabolismo , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação ProteicaRESUMO
OBJECTIVE: A number of hypotheses have been put forward as to why humans respond to fever by seizing. The current leading hypotheses are that respiratory alkalosis produces an as yet unidentified change in neural excitability or that inflammatory mediators potentiate excitatory synaptic transmission. However, it is well known that ion channel gating rates increase with increased temperature. Furthermore, skeletal and cardiac sodium channel activation can be temperature sensitive in some situations. We measured the temperature sensitivity of the brain sodium channel, Na(V)1.2, to determine whether febrile temperatures might produce a direct increase in neuronal excitability. METHODS: The effect of temperature on Na(V)1.2 electrophysiological properties was measured in a transfected mammalian cell line. The subcellular location of Na(V)1.2 in the mouse brain was ascertained using antibodies against Na(V)1.2 and ankyrin-G. Computer simulation of a hippocampal granule cell model was used to predict the effect of temperature on action potential firing. RESULTS: As well as the expected increase in gating rates, the voltage dependence of activation became 7.6 mV more negative when the temperature was increased from 37 degrees C to 41 degrees C. Na(V)1.2 was localized to the axon initial segment in hippocampal and cortical neurons. Computer simulation showed that increased gating rates and the more negative activation dramatically increase neuronal excitability. INTERPRETATION: The direct effect of heat on ion channels localized to the site of action potential initiation potentially causes a profound increase in neuronal excitability. This is likely to contribute to febrile seizure genesis.
Assuntos
Axônios/fisiologia , Temperatura Alta , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Convulsões Febris/fisiopatologia , Canais de Sódio/metabolismo , Potenciais de Ação/fisiologia , Envelhecimento , Animais , Animais Recém-Nascidos , Anquirinas/metabolismo , Linhagem Celular , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiopatologia , Simulação por Computador , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiopatologia , Humanos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2 , TransfecçãoRESUMO
Amiloride derivatives are known blockers of the cellular Na(+)/H(+) exchanger and the epithelial Na(+) channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E. V. Gazina, D. N. Harrison, M. Jefferies, H. Tan, D. Williams, D. A. Anderson and S. Petrou, Antiviral Res. 67:98-106, 2005). Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.
Assuntos
Amilorida/farmacologia , Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Amilorida/análogos & derivados , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Humanos , Proteínas Virais/antagonistas & inibidoresRESUMO
Seizure susceptibility is high in human infants compared to adults, presumably because of developmentally regulated changes in neural excitability. Benign familial neonatal-infantile seizures (BFNIS), characterized by both early onset and remission, are caused by mutations in the gene encoding a human sodium channel (NaV1.2). We analyzed neonatal and adult splice forms of NaV1.2 with a BFNIS mutation (L1563V) in human embryonic kidney cells. Computer modeling revealed that neonatal channels are less excitable than adult channels. Introduction of the mutation increased excitability in the neonatal channels to a level similar to adult channels. By contrast, the mutation did not affect the adult channel variant. This "adult-like" increased excitability is likely to be the mechanism underlying BFNIS in infants with this mutation. More generally, developmentally regulated NaV1.2 splicing may be one mechanism that counters the normally high excitability of neonatal neurons and helps to reduce seizure susceptibility in normal human infants.
Assuntos
Epilepsia/genética , Potenciais da Membrana/genética , Proteínas do Tecido Nervoso/genética , Splicing de RNA/fisiologia , Canais de Sódio/genética , Adulto , Linhagem Celular Transformada , Simulação por Computador , Análise Mutacional de DNA , Relação Dose-Resposta à Radiação , Estimulação Elétrica , Humanos , Lactente , Modelos Biológicos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2 , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/metabolismoRESUMO
Picornavirus replication causes leakage of cytoplasmic K+ and an influx of Na+ and Ca2+. In this study, we have explored the possibility that a blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release. The Ca2+-channel blockers, verapamil and diltiazem, as well as the blocker of Na+/H+ exchange and the epithelial Na+ channel, EIPA, inhibited both virus production and release. The effect on virus release was more pronounced than the effect on production, thus raising the possibility that rhinovirus release may serve as a target for antiviral agents. Unexpectedly, our results also showed that the antiviral activity of the Ca2+-channel blockers was not due to the block of Ca2+ influx. Similarly, the antiviral activity of EIPA appeared to be unrelated to the blockade of cellular Na+/H+ exchanger or the epithelial Na+ channel. Potential alternative mechanisms of the antiviral activity of these compounds are discussed.