RESUMEN
Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
Asunto(s)
Empalme Alternativo , Canal de Sodio Activado por Voltaje NAV1.6 , Isoformas de Proteínas , Canal de Sodio Activado por Voltaje NAV1.6/genética , Humanos , Isoformas de Proteínas/genética , Empalme Alternativo/genética , Tetrodotoxina/farmacología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Mutación , Línea Celular , AnimalesRESUMEN
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.
Asunto(s)
Epilepsia , Canal de Sodio Activado por Voltaje NAV1.2 , Fenotipo , Humanos , Canal de Sodio Activado por Voltaje NAV1.2/genética , Femenino , Masculino , Preescolar , Niño , Lactante , Adolescente , Epilepsia/genética , Adulto , Adulto Joven , Mutación , Trastorno Autístico/genética , Índice de Severidad de la EnfermedadRESUMEN
Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described altered sensitivity and cooperativity of the voltage sensor and impaired capacity for repetitive firing of neurons. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1R306C mouse model and characterized the molecular and phenotypic effects. Consistent with the in vitro studies, neurons from Kcnb1R306C mice showed altered excitability. Heterozygous and homozygous R306C mice exhibited hyperactivity, altered susceptibility to chemoconvulsant-induced seizures, and frequent, long runs of slow spike wave discharges on EEG, reminiscent of the slow spike and wave activity characteristic of Lennox Gastaut syndrome. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies.
Asunto(s)
Trastorno del Espectro Autista , Encefalopatías , Epilepsia , Animales , Ratones , Trastorno del Espectro Autista/patología , Encefalopatías/patología , Epilepsia/patología , Mutación , Fenotipo , ConvulsionesRESUMEN
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.
Asunto(s)
Hemiplejía , Mutación Missense , Humanos , Hemiplejía/diagnóstico , Hemiplejía/genética , Secuenciación del Exoma , Mutación , ATPasa Intercambiadora de Sodio-Potasio/genética , Proteínas de Unión al GTP/genética , Proteínas Supresoras de Tumor/genética , Canal de Sodio Activado por Voltaje NAV1.2/genéticaRESUMEN
Pathogenic variants in SCN8A , which encodes the voltage-gated sodium (Na V ) channel Na V 1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of Na V 1.6 (Na V 1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (Na V 1.6N, Na V 1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous Na V current to obviate the need for TTX-resistance mutations. Expression of Na V 1.6N or Na V 1.6A in ND7/LoNav cells generated Na V currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
RESUMEN
Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.
Asunto(s)
Trastorno del Espectro Autista , Epilepsia , Trastornos del Neurodesarrollo , Humanos , Trastorno del Espectro Autista/genética , Epilepsia/genética , Células HEK293 , Canal de Sodio Activado por Voltaje NAV1.2/genética , Trastornos del Neurodesarrollo/genética , FenotipoRESUMEN
It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.
Asunto(s)
Edición Génica , Canal de Sodio Activado por Voltaje NAV1.2 , Canales de Sodio Activados por Voltaje , Edición Génica/métodos , Mutagénesis/genética , Mutación , Mutación Missense/genéticaRESUMEN
Pathogenic variants in neuronal voltage-gated sodium (Na V ) channel genes including SCN2A , which encodes Na V 1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of Na V 1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of Na V 1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of Na V channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.
RESUMEN
Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function.
Asunto(s)
Trastorno del Espectro Autista , Animales , Trastorno del Espectro Autista/genética , Calcio/metabolismo , Humanos , Ratones , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Permeabilidad , Convulsiones/genética , Sodio/metabolismo , Canales de Sodio/genéticaRESUMEN
KEY POINTS: The recurrent SCN2A mutation R853Q is associated with developmental and epileptic encephalopathy with typical onset after the first months of life. Heterologously expressed R853Q channels exhibit an overall loss-of-function as a result of multiple defects in time- and voltage-dependent channel properties. A previously unrecognized enhancement of slow inactivation is conferred by the R853Q mutation and is a major driver of loss-of-function. Enhanced slow inactivation is potentiated in the canonical splice isoform of the channel and this may explain the later onset of symptoms associated with R853Q. ABSTRACT: Mutations in voltage gated sodium (NaV ) channel genes, including SCN2A (encoding NaV 1.2), are associated with diverse neurodevelopmental disorders with or without epilepsy that present clinically with varying severity, age-of-onset and pharmacoresponsiveness. We examined the functional properties of the most recurrent SCN2A mutation (R853Q) to determine whether developmentally-regulated alternative splicing impacts dysfunction severity and to investigate effects of the mutation on slow inactivation. We engineered the R853Q mutation into neonatal and adult NaV 1.2 splice isoforms. Channel constructs were heterologously co-expressed in HEK293T cells with human ß1 and ß2 subunits. Whole-cell patch clamp recording was used to compare time- and voltage-dependent properties of mutant and wild-type channels. The R853Q mutation exhibits an overall loss-of-function attributed to multiple functional defects including a previously undiscovered enhancement of slow inactivation. The mutation exhibited altered voltage dependence of activation and inactivation, slower recovery from inactivation and decreased channel availability during high-frequency depolarizations. More notable were effects on slow inactivation, including a 10-fold slower rate of recovery from slow inactivation exhibited by mutant channels. The impairments in fast inactivation properties were more severe in the neonatal splice isoform, whereas slow inactivation was more pronounced in the splice isoform of the channel expressed predominantly in later childhood. Enhanced later-onset slow inactivation may be a primary driver of the later onset of neurological features associated with this mutation.
Asunto(s)
Epilepsia , Canal de Sodio Activado por Voltaje NAV1.2 , Niño , Epilepsia/genética , Células HEK293 , Humanos , Mutación , Canal de Sodio Activado por Voltaje NAV1.2/genética , Técnicas de Placa-ClampRESUMEN
Patch clamp recording enabled a revolution in cellular electrophysiology, and is useful for evaluating the functional consequences of ion channel gene mutations or variants associated with human disorders called channelopathies. However, due to massive growth of genetic testing in medical practice and research, the number of known ion channel variants has exploded into the thousands. Fortunately, automated methods for performing patch clamp recording have emerged as important tools to address the explosion in ion channel variants. In this chapter, we present our approach to harnessing automated electrophysiology to study a human voltage-gated potassium channel gene (KCNQ1), which harbors hundreds of mutations associated with genetic disorders of heart rhythm including the congenital long-QT syndrome. We include protocols for performing high efficiency electroporation of heterologous cells with recombinant KCNQ1 plasmid DNA and for automated planar patch recording including data analysis. These methods can be adapted for studying other voltage-gated ion channels.
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Canal de Potasio KCNQ1 , Canales de Potasio con Entrada de Voltaje , Fenómenos Electrofisiológicos , Electrofisiología , Humanos , Canales IónicosRESUMEN
Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here, using a combination of in silico analysis, random and site-directed mutagenesis, we investigated the cause for instability of human NaV1.1 cDNA. We identified nucleotide sequences within the NaV1.1 coding region that resemble prokaryotic promoter-like elements, which are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability. We further demonstrated that mutations disrupting these elements mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the latent reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.
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Escherichia coli/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.6/genética , Regiones Promotoras Genéticas , Ingeniería de Proteínas/métodos , Secuencia de Bases , Clonación Molecular/métodos , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Células HEK293 , Humanos , Potenciales de la Membrana/fisiología , Mutagénesis Sitio-Dirigida/métodos , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Técnicas de Placa-Clamp , Plásmidos/química , Plásmidos/metabolismo , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: We identified a novel de novo SCN2A variant (M1879T) associated with infantile-onset epilepsy that responded dramatically to sodium channel blocker antiepileptic drugs. We analyzed the functional and pharmacological consequences of this variant to establish pathogenicity, and to correlate genotype with phenotype and clinical drug response. METHODS: The clinical and genetic features of an infant boy with epilepsy are presented. We investigated the effect of the variant using heterologously expressed recombinant human NaV 1.2 channels. We performed whole-cell patch clamp recording to determine the functional consequences and response to carbamazepine. RESULTS: The M1879T variant caused disturbances in channel inactivation including substantially depolarized voltage dependence of inactivation, slower time course of inactivation, and enhanced resurgent current that collectively represent a gain-of-function. Carbamazepine partially normalized the voltage dependence of inactivation and produced use-dependent block of the variant channel at high pulsing frequencies. Carbamazepine also suppresses resurgent current conducted by M1879T channels, but this effect was explained primarily by reducing the peak transient current. Molecular modeling suggests that the M1879T variant disrupts contacts with nearby residues in the C-terminal domain of the channel. INTERPRETATION: Our study demonstrates the value of conducting functional analyses of SCN2A variants of unknown significance to establish pathogenicity and genotype-phenotype correlations. We also show concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically in a case of SCN2A-associated epilepsy.
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Anticonvulsivantes/farmacología , Carbamazepina/farmacología , Epilepsia , Canal de Sodio Activado por Voltaje NAV1.2/genética , Edad de Inicio , Fenómenos Electrofisiológicos/fisiología , Epilepsia/diagnóstico , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/fisiopatología , Estudios de Asociación Genética , Humanos , Lactante , MasculinoRESUMEN
OBJECTIVE: Pathogenic variants in SCN3A, encoding the voltage-gated sodium channel subunit Nav1.3, cause severe childhood onset epilepsy and malformation of cortical development. Here, we define the spectrum of clinical, genetic, and neuroimaging features of SCN3A-related neurodevelopmental disorder. METHODS: Patients were ascertained via an international collaborative network. We compared sodium channels containing wild-type versus variant Nav1.3 subunits coexpressed with ß1 and ß2 subunits using whole-cell voltage clamp electrophysiological recordings in a heterologous mammalian system (HEK-293T cells). RESULTS: Of 22 patients with pathogenic SCN3A variants, most had treatment-resistant epilepsy beginning in the first year of life (16/21, 76%; median onset, 2 weeks), with severe or profound developmental delay (15/20, 75%). Many, but not all (15/19, 79%), exhibited malformations of cortical development. Pathogenic variants clustered in transmembrane segments 4 to 6 of domains II to IV. Most pathogenic missense variants tested (10/11, 91%) displayed gain of channel function, with increased persistent current and/or a leftward shift in the voltage dependence of activation, and all variants associated with malformation of cortical development exhibited gain of channel function. One variant (p.Ile1468Arg) exhibited mixed effects, with gain and partial loss of function. Two variants demonstrated loss of channel function. INTERPRETATION: Our study defines SCN3A-related neurodevelopmental disorder along a spectrum of severity, but typically including epilepsy and severe or profound developmental delay/intellectual disability. Malformations of cortical development are a characteristic feature of this unusual channelopathy syndrome, present in >75% of affected individuals. Gain of function at the channel level in developing neurons is likely an important mechanism of disease pathogenesis. ANN NEUROL 2020;88:348-362.
Asunto(s)
Encéfalo/diagnóstico por imagen , Epilepsia/diagnóstico por imagen , Epilepsia/genética , Canal de Sodio Activado por Voltaje NAV1.3/genética , Trastornos del Neurodesarrollo/diagnóstico por imagen , Trastornos del Neurodesarrollo/genética , Canales de Sodio/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Feto/diagnóstico por imagen , Variación Genética/genética , Células HEK293 , Humanos , Lactante , MasculinoRESUMEN
Epileptic encephalopathies are severe forms of infantile-onset epilepsy often complicated by severe neurodevelopmental impairments. Some forms of early-onset epileptic encephalopathy (EOEE) have been associated with variants in SCN2A, which encodes the brain voltage-gated sodium channel NaV1.2. Many voltage-gated sodium channel genes, including SCN2A, undergo developmentally regulated mRNA splicing. The early onset of these disorders suggests that developmentally regulated alternative splicing of NaV1.2 may be an important consideration when elucidating the pathophysiological consequences of epilepsy-associated variants. We hypothesized that EOEE-associated NaV1.2 variants would exhibit greater dysfunction in a splice isoform that is prominently expressed during early development. We engineered five EOEE-associated NaV1.2 variants (T236S, E999K, S1336Y, T1623N, and R1882Q) into the adult and neonatal splice isoforms of NaV1.2 and performed whole-cell voltage clamp to elucidate their functional properties. All variants exhibited functional defects that could enhance neuronal excitability. Three of the five variants (T236S, E999K, and S1336Y) exhibited greater dysfunction in the neonatal isoform compared with those observed in the adult isoform. Computational modeling of a developing cortical pyramidal neuron indicated that T236S, E999K, S1336Y, and R1882Q showed hyperexcitability preferentially in immature neurons. These results suggest that both splice isoform and neuronal developmental stage influence how EOEE-associated NaV1.2 variants affect neuronal excitability.
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Empalme Alternativo/genética , Encefalopatías/genética , Epilepsia/genética , Variación Genética/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Encéfalo/metabolismo , Humanos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: We identified a novel de novo KCNT1 variant in a patient with early-infantile epileptic encephalopathy (EIEE) and status dystonicus, a life-threatening movement disorder. We determined the functional consequences of this variant on the encoded KNa 1.1 channel to investigate the molecular mechanisms responsible for this disorder. METHODS: A retrospective case review of the proband is presented. We performed manual and automated electrophysiologic analyses of the KCNT1-L437F variant expressed heterologously in Chinese hamster ovary (CHO) cells in the presence of channel activators/blockers. RESULTS: The KCNT1-L437F variant, identified in a patient with refractory EIEE and status dystonicus, confers a gain-of-function channel phenotype characterized by instantaneous, voltage-dependent activation. Channel openers do not further increase L437F channel function, suggesting maximal activation, whereas channel blockers similarly block wild-type and variant channels. We further demonstrated that KCNT1 current can be measured on a high-throughput automated electrophysiology platform with potential value for future screening of novel and repurposed pharmacotherapies. INTERPRETATION: A novel pathogenic variant in KCNT1 associated with early-onset, medication-refractory epilepsy and dystonia causes gain-of-function with rapid activation kinetics. Our findings extend the genotype-phenotype relationships of KCNT1 variants to include severe dystonia.
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Distonía/genética , Proteínas del Tejido Nervioso/genética , Canales de potasio activados por Sodio/genética , Espasmos Infantiles/genética , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Distonía/diagnóstico por imagen , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Mutación , Fenotipo , Espasmos Infantiles/diagnóstico por imagenRESUMEN
Full Document: Wall BW, Ash P, Keram E, et al: AAPL Practice Resource for the Forensic Psychiatric Evaluation of Competence to Stand Trial Update 2018. Journal of the American Academy of Psychiatry and the Law Online Supplement 2018, 46 (3). Available at: http://www.jaapl.org/content/46/3_Supplement.
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Psiquiatría Forense/legislación & jurisprudencia , Competencia Mental/legislación & jurisprudencia , Competencia Mental/psicología , Competencia Cultural , Ética Médica , Humanos , Entrevista Psicológica , Registros MédicosRESUMEN
OBJECTIVE: De novo mutations of SCN8A, encoding the voltage-gated sodium channel NaV 1.6, have been associated with a severe infant onset epileptic encephalopathy. Individuals with SCN8A encephalopathy have a mean age of seizure onset of 4-5 months, with multiple seizure types that are often refractory to treatment with available drugs. Anecdotal reports suggest that high-dose phenytoin is effective for some patients, but there are associated adverse effects and potential for toxicity. Functional characterization of several SCN8A encephalopathy variants has shown that elevated persistent sodium current is one of several common biophysical defects. Therefore, specifically targeting elevated persistent current may be a useful therapeutic strategy in some cases. METHODS: The novel sodium channel modulator GS967 has greater preference for persistent as opposed to peak current and nearly 10-fold greater potency than phenytoin. We evaluated the therapeutic effect of GS967 in the Scn8aN1768D/+ mouse model carrying an SCN8A patient mutation that results in elevated persistent sodium current. We also performed patch clamp recordings to assess the effect of GS967 on peak and persistent sodium current and excitability in hippocampal neurons from Scn8aN1768D/+ mice. RESULTS: GS967 potently blocked persistent sodium current without affecting peak current, normalized action potential morphology, and attenuated excitability in neurons from heterozygous Scn8aN1768D/+ mice. Acute treatment with GS967 provided dose-dependent protection against maximal electroshock-induced seizures in Scn8aN1768D/+ and wild-type mice. Chronic treatment of Scn8aN1768D/+ mice with GS967 resulted in lower seizure burden and complete protection from seizure-associated lethality observed in untreated Scn8aN1768D/+ mice. Protection was achieved at a chronic dose that did not cause overt behavioral toxicity or sedation. SIGNIFICANCE: Persistent sodium current modulators like GS967 may be an effective precision targeting strategy for SCN8A encephalopathy and other functionally similar channelopathies when elevated persistent sodium current is the primary dysfunction.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.6/genética , Piridinas/uso terapéutico , Triazoles/uso terapéutico , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Anticonvulsivantes/farmacología , Encefalopatías/complicaciones , Encefalopatías/genética , Modelos Animales de Enfermedad , Esquema de Medicación , Electrochoque/efectos adversos , Epilepsia/etiología , Epilepsia/genética , Epilepsia/patología , Femenino , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Uso Fuera de lo Indicado , Fenitoína/farmacología , Fenitoína/uso terapéutico , Piridinas/farmacología , Triazoles/farmacologíaRESUMEN
We identified SCN1A variants in 2 infants who died of sudden infant death syndrome (SIDS) with hippocampal abnormalities from an exome sequencing study of 10 cases of SIDS but no history of seizures. One harbored SCN1A G682V, and the other had 2 SCN1A variants in cis: L1296M and E1308D, a variant previously associated with epilepsy. Functional evaluation in a heterologous expression system demonstrated partial loss of function for both G682V and the compound variant L1296M/E1308D. Our cases represent a novel association between SCN1A and SIDS, extending the SCN1A spectrum from epilepsy to SIDS. Our findings provide insights into SIDS and support genetic evaluation focused on epilepsy genes in SIDS.