RESUMO
Neonatal intensive care unit (NICU), particularly in treating developmental and epileptic encephalopathy (DEE) and metabolic epilepsy (ME), requires a deep understanding of their complex etiologies and treatment responses. After excluding treatable cases such as infectious or autoimmune encephalitis, our focus shifted to a more challenging subgroup of 59 patients for in-depth genetic analysis using exome sequencing (ES). The ES analysis identified 40 genetic abnormalities, significantly including de novo variants. Notably, we found structural variation as duplications in regions 2q24.3, including SCN1A and SCN2A were observed in 7 cases. These genetic variants, impacting ion channels, glucose transport, transcription regulation, and kinases, play a crucial role in determining medication efficacy. More than one-third (34.2%) of patients with DEE had an unfavorable response to anti-seizure medications (ASMs) in the chronic phase. However, since the ketogenic supplementary diet showed a positive effect, more than three-quarters (80%) of these drug-resistant patients improved during a 3-month follow-up. In contrast, the ME had a lower adverse reaction rate of 9.1% (2/22) to specialized medications, yet there were 5 fatalities and 10 cases with unidentified genetic etiologies. This study suggests the potential of categorizing drug-resistant variants and that a ketogenic diet could be beneficial in managing DEE and ME. It also opens new perspectives on the mechanisms of the ketogenic diet on the discovered genetic variants.
Assuntos
Genótipo , Humanos , Feminino , Masculino , Epilepsia/tratamento farmacológico , Epilepsia/genética , Sequenciamento do Exoma , Recém-Nascido , Dieta Cetogênica , Resultado do Tratamento , Lactente , Anticonvulsivantes/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Resistência a Medicamentos/genética , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Epilepsia Resistente a Medicamentos/genéticaRESUMO
The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.
Assuntos
Proteínas de Ciclo Celular , Quinase 3 da Glicogênio Sintase , Canal de Sódio Disparado por Voltagem NAV1.2 , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-akt , Humanos , Células HEK293 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ciclo Celular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Proteínas Tirosina Quinases/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Sodium channel blockers (SCBs) have traditionally been utilized as anti-seizure medications by primarily targeting the inactivation process. In a drug discovery project aiming at finding potential anticonvulsants, we have identified arbidol, originally an antiviral drug, as a potent SCB. In order to evaluate its anticonvulsant potential, we have thoroughly examined its biophysical properties as well as its effects on animal seizure models. EXPERIMENTAL APPROACH: Patch clamp recording was used to investigate the electrophysiological properties of arbidol, as well as the binding and unbinding kinetics of arbidol, carbamazepine and lacosamide. Furthermore, we evaluated the anticonvulsant effects of arbidol using three different seizure models in male mice. KEY RESULTS: Arbidol effectively suppressed neuronal epileptiform activity by blocking sodium channels. Arbidol demonstrated a distinct mode of action by interacting with both the fast and slow inactivation of Nav1.2 channels compared with carbamazepine and lacosamide. A kinetic study suggested that the binding and unbinding rates might be associated with the specific characteristics of these three drugs. Arbidol targeted the classical binding site of local anaesthetics, effectively inhibited the gain-of-function effects of Nav1.2 epileptic mutations and exhibited varying degrees of anticonvulsant effects in the maximal electroshock model and subcutaneous pentylenetetrazol model but had no effect in the pilocarpine-induced status epilepticus model. CONCLUSIONS AND IMPLICATIONS: Arbidol shows promising potential as an anticonvulsant agent, providing a unique mode of action that sets it apart from existing SCBs.
Assuntos
Anticonvulsivantes , Antivirais , Indóis , Convulsões , Bloqueadores dos Canais de Sódio , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Masculino , Antivirais/farmacologia , Convulsões/tratamento farmacológico , Camundongos , Indóis/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Lacosamida/farmacologia , Humanos , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Células HEK293 , Carbamazepina/farmacologia , SulfetosRESUMO
Dysfunction of voltage-gated sodium channel Nav1.2 causes various epileptic disorders, and inhibition of the channel has emerged as an attractive therapeutic strategy. However, currently available Nav1.2 inhibitors exhibit low potency and limited structural diversity. In this study, a novel series of pyrimidine-based derivatives with Nav1.2 inhibitory activity were designed, synthesized, and evaluated. Compounds 14 and 35 exhibited potent activity against Nav1.2, boasting IC50 values of 120 and 65 nM, respectively. Compound 14 displayed favorable pharmacokinetics (F = 43%) following intraperitoneal injection and excellent brain penetration potency (B/P = 3.6). Compounds 14 and 35 exhibited robust antiepileptic activities in the maximal electroshock test, with ED50 values of 3.2 and 11.1 mg/kg, respectively. Compound 35 also demonstrated potent antiepileptic activity in a 6 Hz (32 mA) model, with an ED50 value of 18.5 mg/kg. Overall, compounds 14 and 35 are promising leads for the development of new small-molecule therapeutics for epilepsy.
Assuntos
Anticonvulsivantes , Epilepsia , Canal de Sódio Disparado por Voltagem NAV1.2 , Pirimidinas , Animais , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Anticonvulsivantes/farmacologia , Anticonvulsivantes/química , Anticonvulsivantes/síntese química , Anticonvulsivantes/uso terapêutico , Anticonvulsivantes/farmacocinética , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Relação Estrutura-Atividade , Humanos , Modelos Animais de Doenças , Masculino , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Descoberta de Drogas , Eletrochoque , Simulação de Acoplamento MolecularRESUMO
OBJECTIVE: Epilepsy patients exhibit considerable differences in their response to sodium valproate (VPA) therapy, a phenomenon that might be attributed to individual genetic variances. The role of genetic variations, specifically in sodium channels encoded by SCN1A and SCN2A genes, in influencing the effectiveness of VPA in treating epilepsy is still debated. This research focuses on examining the impact of these genetic polymorphisms on the efficacy of VPA therapy among pediatric epilepsy patients in China. METHODS: Five single nucleotide polymorphisms (SNPs), including SCN1A (rs10188577, rs2298771, rs3812718) and SCN2A (rs2304016, rs17183814), were genotyped in 233 epilepsy patients undergoing VPA therapy. The associations between genotypes and the antiepileptic effects of VPA were assessed, with 128 patients categorized as VPA responders and 105 as VPA non-responders. RESULTS: In the context of VPA monotherapy, SCN1A rs2298771 and SCN2A rs17183814 were found to be significantly associated with VPA response (P< 0.05). CONCLUSION: Our study suggests the findings of this investigation indicate that the polymorphisms SCN1A rs2298771 and SCN2A rs17183814 could potentially act as predictive biomarkers for the responsiveness to VPA among Chinese epilepsy patients.
Assuntos
Anticonvulsivantes , Epilepsia , Canal de Sódio Disparado por Voltagem NAV1.1 , Canal de Sódio Disparado por Voltagem NAV1.2 , Polimorfismo de Nucleotídeo Único , Ácido Valproico , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Ácido Valproico/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Criança , Masculino , Feminino , Epilepsia/tratamento farmacológico , Epilepsia/genética , Anticonvulsivantes/uso terapêutico , Pré-Escolar , China , Povo Asiático/genética , Adolescente , Resultado do Tratamento , Genótipo , Lactente , População do Leste AsiáticoRESUMO
Variants in voltage-gated sodium channel (VGSC) genes are implicated in seizures, epilepsy, and neurodevelopmental disorders, constituting a significant aspect of hereditary epilepsy in the Chinese population. Through retrospective analysis utilizing next-generation sequencing (NGS), we examined the genotypes and phenotypes of VGSC-related epilepsy cases from a cohort of 691 epilepsy subjects. Our findings revealed that 5.1% of subjects harbored VGSC variants, specifically 22 with SCN1A, 9 with SCN2A, 1 with SCN8A, and 3 with SCN1B variants; no SCN3A variants were detected. Among these, 14 variants were previously reported, while 21 were newly identified. SCN1A variant carriers predominantly presented with Dravet Syndrome (DS) and Genetic Epilepsy with Febrile Seizures Plus (GEFS + ), featuring a heightened sensitivity to fever-induced seizures. Statistically significant disparities emerged between the SCN1A-DS and SCN1A-GEFS+ groups concerning seizure onset and genetic diagnosis age, incidence of status epilepticus, mental retardation, anti-seizure medication (ASM) responsiveness, and familial history. Notably, subjects with SCN1A variants affecting the protein's pore region experienced more frequent cluster seizures. All SCN2A variants were of de novo origin, and 88.9% of individuals with SCN2A variations exhibited cluster seizures. This research reveals a significant association between variations in VGSC-related genes and the clinical phenotype diversity of epilepsy subjects in China, emphasizing the pivotal role of NGS screening in establishing accurate disease diagnoses and guiding the selection of ASM.
Assuntos
Epilepsia , Genótipo , Canal de Sódio Disparado por Voltagem NAV1.1 , Canal de Sódio Disparado por Voltagem NAV1.2 , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Adulto Jovem , China/epidemiologia , População do Leste Asiático/genética , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/epidemiologia , Epilepsia/genética , Epilepsia/epidemiologia , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Fenótipo , Estudos Retrospectivos , Convulsões Febris/genética , Convulsões Febris/epidemiologiaRESUMO
ABSTRACT: The SCN2A gene encodes a subunit that forms part of voltage-gated sodium channels in the brain. Gain-of-function mutations are associated with epilepsy as well as numerous movement/motor abnormalities. Loss-of-function mutations may also cause epilepsy in addition to a variety of neurodevelopmental anomalies, including autism and intellectual disability. The occurrence of catatonia has also been described in 1 previous report that involved a 4-year-old boy. We describe a 20-year-old intellectually disabled female patient who developed recurrent catatonic symptoms in her teenage years that remitted with electroconvulsive therapy. This is only the second report of catatonia occurring in relation to an SCN2A mutation and the first involving a female. Moreover, this case is unique given our patient's later age of symptom onset and given that her symptoms responded well to electroconvulsive therapy.
Assuntos
Catatonia , Eletroconvulsoterapia , Canal de Sódio Disparado por Voltagem NAV1.2 , Humanos , Catatonia/terapia , Catatonia/genética , Feminino , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Adulto Jovem , Deficiência Intelectual/genética , Deficiência Intelectual/terapia , MutaçãoRESUMO
SCN2A loss-of-function variants cause a range of neurodevelopmental disorders. Here, we present a protocol to induce severe Scn2a insufficiency in mice. We describe steps for intracerebroventricular (ICV) antisense oligonucleotide (ASO) injection that causes a selective downregulation of Scn2a and ASO-mediated mRNA degradation. We then detail procedures for qPCR and western blot protocol to measure Scn2a mRNA and protein. This protocol can be used as a mouse model for behavioral and in vivo two-photon Ca2+ imaging.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.2 , Oligonucleotídeos Antissenso , Animais , Camundongos , Modelos Animais de Doenças , Injeções Intraventriculares , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel Nav1.2 are rare, with clinically heterogeneous expressions that include epilepsy, autism and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relationship to channel function, 81 patients (36 female, 44%, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their Nav1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal onset, n = 27; infant onset, n = 18; and later onset n = 24; and autism without seizures, n = 12) was strongly correlated with a non-seizure severity index (P = 0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (P = 0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland Adaptive Behavior composite score of 49.5 (>3 standard deviations below the norm-referenced mean of the test). Epileptic spasms were significantly more common in infant-onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (P = 0.007). Primary phenotype was also strongly correlated with variant function (P < 0.0001); gain-of-function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy and to severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups, representing: (i) primarily later-onset epilepsy with moderate loss-of-function variants and low severity indices; (ii) mostly infant-onset epilepsy with moderate loss-of-function variants but higher severity indices; and (iii) late-onset and autism only, with the lowest severity indices (mostly zero) and severe/complete loss-of-function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relationship between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on Nav1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance towards clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes.
Assuntos
Epilepsia , Canal de Sódio Disparado por Voltagem NAV1.2 , Fenótipo , Humanos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Feminino , Masculino , Pré-Escolar , Criança , Lactente , Adolescente , Epilepsia/genética , Adulto , Adulto Jovem , Mutação , Transtorno Autístico/genética , Índice de Gravidade de DoençaRESUMO
Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.
Assuntos
Transtorno do Espectro Autista , Modelos Animais de Doenças , Microglia , Canal de Sódio Disparado por Voltagem NAV1.2 , Organoides , Sinapses , Animais , Microglia/metabolismo , Humanos , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Sinapses/metabolismo , Organoides/metabolismo , Neurônios/metabolismo , Encéfalo/metabolismo , Masculino , Camundongos Knockout , Hipocampo/metabolismo , Transmissão Sináptica , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Encefalopatias , Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação de Sentido Incorreto , Heterozigoto , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/genéticaRESUMO
Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or abnormally strong reactions to sensory stimuli. Such hypersensitivity can be overwhelming, causing high levels of distress that contribute markedly to the negative aspects of the disorder. Here, we identify a mechanism that underlies hypersensitivity in a sensorimotor reflex found to be altered in humans and in mice with loss of function in the ASD risk-factor gene SCN2A. The cerebellum-dependent vestibulo-ocular reflex (VOR), which helps maintain one's gaze during movement, was hypersensitized due to deficits in cerebellar synaptic plasticity. Heterozygous loss of SCN2A-encoded NaV1.2 sodium channels in granule cells impaired high-frequency transmission to Purkinje cells and long-term potentiation, a form of synaptic plasticity important for modulating VOR gain. VOR plasticity could be rescued in mice via a CRISPR-activator approach that increases Scn2a expression, demonstrating that evaluation of a simple reflex can be used to assess and quantify successful therapeutic intervention.
Assuntos
Transtorno do Espectro Autista , Cerebelo , Canal de Sódio Disparado por Voltagem NAV1.2 , Plasticidade Neuronal , Animais , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Camundongos , Plasticidade Neuronal/fisiologia , Cerebelo/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Humanos , Reflexo Vestíbulo-Ocular/fisiologia , Masculino , Células de Purkinje/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Neocórtex , Animais , Camundongos , Anquirinas/genética , Anquirinas/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/metabolismo , Dendritos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Neocórtex/metabolismo , Células Piramidais/fisiologiaRESUMO
PURPOSE: There are limited psychometric data on outcome measures for children with Developmental Epileptic Encephalopathies (DEEs), beyond measuring seizures, and no data to describe meaningful change. This study aimed to explore parent perceptions of important differences in functional abilities that would guide their participation in clinical trials. METHODS: This was a descriptive qualitative study. Semi-structured one-on-one interviews were conducted with 10 families (15 parent participants) with a child with a SCN2A-DEE [8 male, median (range) age 7.5 (4.5-21)] years. Questions and probes sought to understand the child's functioning across four domains: gross motor, fine motor, communication, and activities of daily living. Additional probing questions sought to identify the smallest differences in the child's functioning for each domain that would be important to achieve, if enrolling in a traditional therapy clinical trial or in a gene therapy trial. Data were analyzed with directed content analysis. RESULTS: Expressed meaningful differences appeared to describe smaller developmental steps for children with more limited developmental skills and more complex developmental steps for children with less limited skills and were different for different clinical trial scenarios. Individual meaningful changes were described as important for the child's quality of life and to facilitate day-to-day caring. CONCLUSION: Meaningful change thresholds have not been evaluated in the DEE literature. This study was a preliminary qualitative approach to inform future studies that will aim to determine quantitative values of change, applicable to groups and within-person, to inform interpretation of specific clinical outcome assessments in individuals with a DEE.
Assuntos
Atividades Cotidianas , Epilepsia , Criança , Humanos , Masculino , Qualidade de Vida/psicologia , Pais , Pesquisa Qualitativa , Canal de Sódio Disparado por Voltagem NAV1.2RESUMO
Alternating hemiplegia of childhood (AHC) is a rare neurodevelopment disorder that is typically characterized by debilitating episodic attacks of hemiplegia, seizures, and intellectual disability. Over 85% of individuals with AHC have a de novo missense variant in ATP1A3 encoding the catalytic α3 subunit of neuronal Na+/K+ ATPases. The remainder of the patients are genetically unexplained. Here, we used next-generation sequencing to search for the genetic cause of 26 ATP1A3-negative index patients with a clinical presentation of AHC or an AHC-like phenotype. Three patients had affected siblings. Using targeted sequencing of exonic, intronic, and flanking regions of ATP1A3 in 22 of the 26 index patients, we found no ultra-rare variants. Using exome sequencing, we identified the likely genetic diagnosis in 9 probands (35%) in five genes, including RHOBTB2 (n = 3), ATP1A2 (n = 3), ANK3 (n = 1), SCN2A (n = 1), and CHD2 (n = 1). In follow-up investigations, two additional ATP1A3-negative individuals were found to have rare missense SCN2A variants, including one de novo likely pathogenic variant and one likely pathogenic variant for which inheritance could not be determined. Functional evaluation of the variants identified in SCN2A and ATP1A2 supports the pathogenicity of the identified variants. Our data show that genetic variants in various neurodevelopmental genes, including SCN2A, lead to AHC or AHC-like presentation. Still, the majority of ATP1A3-negative AHC or AHC-like patients remain unexplained, suggesting that other mutational mechanisms may account for the phenotype or that cases may be explained by oligo- or polygenic risk factors.
Assuntos
Hemiplegia , Mutação de Sentido Incorreto , Humanos , Hemiplegia/diagnóstico , Hemiplegia/genética , Sequenciamento do Exoma , Mutação , ATPase Trocadora de Sódio-Potássio/genética , Proteínas de Ligação ao GTP/genética , Proteínas Supressoras de Tumor/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genéticaRESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Espasmos Infantis , Humanos , Masculino , Células-Tronco Pluripotentes Induzidas/metabolismo , Convulsões/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Fenótipo , Neurônios/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genéticaRESUMO
Dysfunction in the gene SCN2A, which encodes the voltage-gated sodium channel Nav1.2, is strongly associated with neurodevelopmental disorders including autism spectrum disorder and intellectual disability (ASD/ID). This dysfunction typically manifests in these disorders as a haploinsufficiency, where loss of one copy of a gene cannot be compensated for by the other allele. Scn2a haploinsufficiency affects a range of cells and circuits across the brain, including associative neocortical circuits that are important for cognitive flexibility and decision-making behaviors. Here, we tested whether Scn2a haploinsufficiency has any effect on a dynamic foraging task that engages such circuits. Scn2a +/- mice and wild-type (WT) littermates were trained on a choice behavior where the probability of reward between two options varied dynamically across trials and where the location of the high reward underwent uncued reversals. Despite impairments in Scn2a-related neuronal excitability, we found that both male and female Scn2a +/- mice performed these tasks as well as wild-type littermates, with no behavioral difference across genotypes in learning or performance parameters. Varying the number of trials between reversals or probabilities of receiving reward did not result in an observable behavioral difference, either. These data suggest that, despite heterozygous loss of Scn2a, mice can perform relatively complex foraging tasks that make use of higher-order neuronal circuits.
Assuntos
Haploinsuficiência , Canal de Sódio Disparado por Voltagem NAV1.2 , Animais , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Masculino , Feminino , Comportamento Animal , Aprendizagem , Recompensa , Tomada de Decisões , Humanos , Modelos AnimaisRESUMO
OBJECTIVE: SCN2A gene pathogenic variants are associated with a wide phenotypic spectrum, encompassing epilepsy, developmental delay, and autism spectrum disorder. Researches conducted in Denmark have revealed a disease frequency of approximately 1/78,608 (0.0012%) live births in this population. We estimated the frequency of SCN2A-related disorder in the birth cohort of Brescia and its province between 2002 and 2021. METHODS: Frequency was calculated by ratio between patients with SCN2A pathogenic variant and the total number of live births at the Regional Epilepsy Center of Brescia, between 2002 and 2021. The number of births in Brescia and province was obtained from the Italian National Institute of Statistics (ISTAT). RESULTS: A frequency of 11/23,2678 births (0.0047%) was found. In comparison with Danish data, we noticed a higher frequency of the pathogenic variant in our population, even considering the same time frame (0.0035% of subjects born between 2006 and 2014). CONCLUSION: The frequency of SCN2A pathogenic variant among live births in Brescia and its Province between 2006 and 2014 was about three times that of Danish population; this difference was about four times if we consider the period from 2002 to 2021. More studies are needed to further delineate the frequency of SCN2A pathogenic variant in Italian population.
Assuntos
Transtorno do Espectro Autista , Epilepsia , Humanos , Transtorno do Espectro Autista/genética , Fenótipo , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Epilepsia/epidemiologia , Epilepsia/genéticaRESUMO
A range of epilepsies, including the most severe group of developmental and epileptic encephalopathies (DEEs), are caused by gain-of-function variants in voltage-gated channels. Here we report the generation and characterisation of an iPSC cell line from the fibroblasts of a girl with early infantile DEE carrying heterozygous missense gain-of-function mutation (R1882Q) in Nav1.2(SCN2A) protein, using transient transfection with a single mRNA molecule. The established iPSC line displays typical human primed pluripotent stem cell characteristics: typical colony morphology and robust expression of pluripotency-associated marker genes, ability to give rise to derivatives of all three embryonic germ layers, and normal karyotype without any SNP array-detectable copy number variations. We anticipate that this iPSC line will be useful for the development of neuronal hyperactivity-caused human stem cell-based DEE models, advancing both understanding and potential therapy development for this debilitating condition.