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1.
Genet Med ; 25(2): 100333, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36480001

RESUMEN

PURPOSE: Sub-Saharan Africa bears the highest burden of epilepsy worldwide. A presumed proportion is genetic, but this etiology is buried under the burden of infections and perinatal insults in a setting of limited awareness and few options for testing. Children with developmental and epileptic encephalopathies (DEEs) are most severely affected by this diagnostic gap in Africa, because the rate of actionable findings is highest in DEE-associated genes. METHODS: We tested 234 genetically naive South African children diagnosed with/possible DEE using gene panels, exome sequencing, and chromosomal microarray. Statistical comparison of electroclinical features in children with and children without candidate variants was performed to identify characteristics most likely predictive of a positive genetic finding. RESULTS: Of the 41 (of 234) children with likely/pathogenic variants, 26 had variants supporting precision therapy. Multivariate regression modeling highlighted neonatal or infantile-onset seizures and movement abnormalities as predictive of a positive genetic finding. We used this, coupled with an emphasis on precision medicine outcomes, to propose the pragmatic "Think-Genetics" strategy for early recognition of a possible genetic etiology. CONCLUSION: Our findings emphasize the importance of an early genetic diagnosis in DEE. We designed the Think-Genetics strategy for early recognition, appropriate interim management, and genetic testing for DEE in resource-constrained settings.


Asunto(s)
Epilepsia , Medicina de Precisión , Niño , Recién Nacido , Humanos , Configuración de Recursos Limitados , Epilepsia/diagnóstico , Epilepsia/epidemiología , Epilepsia/genética , Pruebas Genéticas , África
2.
Brain ; 145(6): 1939-1948, 2022 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-35773235

RESUMEN

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.


Asunto(s)
Epilepsias Parciales , Epilepsia , Megalencefalia , Epilepsia/genética , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Megalencefalia/genética , Proteínas del Tejido Nervioso/genética , Proteínas Supresoras de Tumor/genética
3.
J Biol Chem ; 295(30): 10380-10393, 2020 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-32503841

RESUMEN

Voltage-gated sodium channel (VGSC) ß1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell-cell and cell-matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC ß1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that ß1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that ß1 subunits undergo regulated intramembrane proteolysis via the activity of ß-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, ß1-ICD, which modulates transcription. Here, we report that ß1 subunits are phosphorylated by FYN kinase. Moreover, we show that ß1 subunits are S-palmitoylated. Substitution of a single residue in ß1, Cys-162, to alanine prevented palmitoylation, reduced the level of ß1 polypeptides at the plasma membrane, and reduced the extent of ß1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of ß1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of ß1-p.C162A to WT levels. Despite these observations, palmitoylation-null ß1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC ß subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.


Asunto(s)
Membrana Celular/metabolismo , Lipoilación , Procesamiento Proteico-Postraduccional , Proteolisis , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/metabolismo , Sustitución de Aminoácidos , Animales , Membrana Celular/genética , Células HEK293 , Humanos , Hidrazonas/farmacología , Ratones , Mutación Missense , Naftoles/farmacología , Fosforilación , Proto-Oncogenes Mas , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genética
4.
Hum Mutat ; 41(6): 1138-1144, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32227660

RESUMEN

CACNA1H genetic variants were originally reported in a childhood absence epilepsy cohort. Subsequently, genetic testing for CACNA1H became available and is currently offered by commercial laboratories. However, the current status of CACNA1H as a monogenic cause of epilepsy is controversial, highlighted by ClinGen's recent reclassification of CACNA1H as disputed. We analyzed published CACNA1H variants and those reported in ClinVar and found none would be classified as pathogenic or likely pathogenic per the American College of Medical Genetics classification criteria. Moreover, Cacna1h did not modify survival in a Dravet Syndrome mouse model. We observed a mild increase in susceptibility to hyperthermia-induced seizures in mice with reduced Cacna1h expression. Overall, we conclude that there is limited evidence that CACNA1H is a monogenic cause of epilepsy in humans and that this gene should be removed from commercial genetic testing panels to reduce the burden of variants of uncertain significance for healthcare providers, families and patients with epilepsy.


Asunto(s)
Canales de Calcio Tipo T/genética , Epilepsia Tipo Ausencia/genética , Animales , Modelos Animales de Enfermedad , Pruebas Genéticas , Humanos , Ratones , Convulsiones/genética
5.
Ann Neurol ; 86(6): 899-912, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31600826

RESUMEN

OBJECTIVE: Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. METHODS: We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. RESULTS: Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. INTERPRETATION: Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.


Asunto(s)
Variación Genética/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Trastornos del Neurodesarrollo/genética , Canales de Potasio Shab/genética , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Trastornos del Neurodesarrollo/diagnóstico , Estructura Secundaria de Proteína , Canales de Potasio Shab/química
6.
Am J Med Genet A ; 182(6): 1460-1465, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32267060

RESUMEN

Congenital disorders of glycosylation (CDG) are metabolic disorders that affect the glycosylation of proteins and lipids. Since glycosylation affects all organs, CDG show a wide spectrum of phenotypes. We present a patient with microcephaly, dysmorphic facies, congenital heart defect, focal epilepsy, infantile spasms, skeletal dysplasia, and a type 1 serum transferrin isoelectrofocusing due to a novel CDG caused by a homozygous variant in the oligosaccharyltransferase complex noncatalytic subunit (OSTC) gene involved in glycosylation and confirmed by serum transferrin electrophoresis.


Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Epilepsia/genética , Hexosiltransferasas/genética , Proteínas de la Membrana/genética , Trastornos Congénitos de Glicosilación/complicaciones , Trastornos Congénitos de Glicosilación/patología , Epilepsia/complicaciones , Epilepsia/patología , Femenino , Humanos , Lactante , Masculino , Mutación/genética , Fenotipo , Transferrina/genética , Secuenciación del Exoma
7.
J Neurogenet ; 32(4): 295-312, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30247086

RESUMEN

The technological advancement of next-generation sequencing has greatly accelerated the pace of variant discovery in epilepsy. Despite an initial focus on autosomal dominant epilepsy due to the tractable nature of variant discovery with trios under a de novo model, more and more variants are being reported in families with epilepsies consistent with autosomal recessive (AR) inheritance. In this review, we touch on the classical AR epilepsy variants such as the inborn errors of metabolism and malformations of cortical development. However, we also highlight recently reported genes that are being identified by next-generation sequencing approaches and online 'matchmaking' platforms. Syndromes mainly characterized by seizures and complex neurodevelopmental disorders comorbid with epilepsy are discussed as an example of the wide phenotypic spectrum associated with the AR epilepsies. We conclude with a foray into the future, from the application of whole-genome sequencing to identify elusive epilepsy variants, to the promise of precision medicine initiatives to provide novel targeted therapeutics specific to the individual based on their clinical genetic testing.


Asunto(s)
Epilepsia/genética , Predisposición Genética a la Enfermedad/genética , Genes Recesivos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos
8.
Epilepsia ; 58(8): e111-e115, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28556246

RESUMEN

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage-gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic-clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain-dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T-type calcium channel family, as an epilepsy modifier in the Scn2aQ54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a+/- Dravet phenotype. Scn1a+/- mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia-induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a+/- epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.


Asunto(s)
Canales de Calcio Tipo T/genética , Epilepsias Mioclónicas/genética , Mutación/genética , Animales , Animales Recién Nacidos , Canales de Calcio Tipo T/metabolismo , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsias Mioclónicas/complicaciones , Epilepsias Mioclónicas/etiología , Fiebre/complicaciones , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Grabación en Video
9.
Epilepsia ; 57(6): e103-7, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27112236

RESUMEN

More than 1,200 mutations in neuronal voltage-gated sodium channel (VGSC) genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2a(Q54) transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2a(Q54) phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2a(Q54) mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2a(Q54) mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2a(Q54) phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2a(Q54) seizure phenotype. Scn2a(Q54) mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.


Asunto(s)
Canales de Calcio Tipo T/metabolismo , Epilepsia/genética , Epilepsia/fisiopatología , Regulación de la Expresión Génica/genética , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Animales , Canales de Calcio Tipo T/genética , Modelos Animales de Enfermedad , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo
10.
Handb Exp Pharmacol ; 221: 51-89, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24737232

RESUMEN

Voltage-gated sodium channel ß1 and ß2 subunits were discovered as auxiliary proteins that co-purify with pore-forming α subunits in brain. The other family members, ß1B, ß3, and ß4, were identified by homology and shown to modulate sodium current in heterologous systems. Work over the past 2 decades, however, has provided strong evidence that these proteins are not simply ancillary ion channel subunits, but are multifunctional signaling proteins in their own right, playing both conducting (channel modulatory) and nonconducting roles in cell signaling. Here, we discuss evidence that sodium channel ß subunits not only regulate sodium channel function and localization but also modulate voltage-gated potassium channels. In their nonconducting roles, VGSC ß subunits function as immunoglobulin superfamily cell adhesion molecules that modulate brain development by influencing cell proliferation and migration, axon outgrowth, axonal fasciculation, and neuronal pathfinding. Mutations in genes encoding ß subunits are linked to paroxysmal diseases including epilepsy, cardiac arrhythmia, and sudden infant death syndrome. Finally, ß subunits may be targets for the future development of novel therapeutics.


Asunto(s)
Activación del Canal Iónico , Sodio/metabolismo , Subunidades beta de Canales de Sodio Activados por Voltaje/metabolismo , Animales , Humanos , Potenciales de la Membrana , Canales de Potasio con Entrada de Voltaje/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Subunidades beta de Canales de Sodio Activados por Voltaje/química
11.
bioRxiv ; 2024 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-38895336

RESUMEN

Efforts to resolve the functional impact of variants of uncertain significance (VUS) have lagged behind the identification of new VUS; as such, there is a critical need for scalable VUS resolution technologies. Computational variant effect predictors (VEPs), once trained, can predict pathogenicity for all missense variants in a gene, set of genes, or the exome. Existing tools have employed information on known pathogenic and benign variants throughout the genome to predict pathogenicity of VUS. We hypothesize that taking a gene-specific approach will improve pathogenicity prediction over globally-trained VEPs. We tested this hypothesis using the gene TSC2, whose loss of function results in tuberous sclerosis, a multisystem mTORopathy affecting about 1 in 6,000 individuals born in the United States. TSC2 has been identified as a high-priority target for VUS resolution, with (1) well-characterized molecular and patient phenotypes associated with loss-of-function variants, and (2) more than 2,700 VUS already documented in ClinVar. We developed Tuberous sclerosis classifier to Resolve variants of Uncertain Significance in T SC2 (TRUST), a machine learning model to predict pathogenicity of TSC2 missense VUS. To test whether these predictions are accurate, we further introduce curated loci prime editing (cliPE) as an accessible strategy for performing scalable multiplexed assays of variant effect (MAVEs). Using cliPE, we tested the effects of more than 200 TSC2 variants, including 106 VUS. It is highly likely this functional data alone would be sufficient to reclassify 92 VUS with most being reclassified as likely benign. We found that TRUST's classifications were correlated with the functional data, providing additional validation for the in silico predictions. We provide our pathogenicity predictions and MAVE data to aid with VUS resolution. In the near future, we plan to host these data on a public website and deposit into relevant databases such as MAVEdb as a community resource. Ultimately, this study provides a framework to complete variant effect maps of TSC1 and TSC2 and adapt this approach to other mTORopathy genes.

12.
J Biol Chem ; 287(46): 39061-9, 2012 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-22992729

RESUMEN

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked ß subunit (ß1 or ß3), and one disulfide-linked ß subunit (ß2 or ß4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-ß2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding ß2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant ß2 cDNA constructs to investigate the cysteine residue(s) responsible for α-ß2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and ß2 subunits. Loss of α-ß2 covalent complex formation disrupts the targeting of ß2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal ß2 subcellular localization in vivo. WT ß2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant ß2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-ß2 covalent association via a single, extracellular disulfide bond is required for ß2 targeting to specialized neuronal subcellular domains and for ß2 association with the neuronal cytoskeleton within those domains.


Asunto(s)
Cisteína/química , Canal de Sodio Activado por Voltaje NAV1.1/química , Animales , Encéfalo/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Técnicas de Cocultivo , Citoesqueleto/metabolismo , Disulfuros/química , Epítopos/química , Células HEK293 , Hipocampo/metabolismo , Humanos , Inmunohistoquímica/métodos , Mutación , Canal de Sodio Activado por Voltaje NAV1.1/genética , Neuronas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Ratas , Células de Schwann , Canales de Sodio/química
13.
Proc Natl Acad Sci U S A ; 107(5): 2283-8, 2010 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-20133873

RESUMEN

Voltage-gated Na(+) channel (VGSC) beta1 subunits regulate cell-cell adhesion and channel activity in vitro. We previously showed that beta1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that beta1-mediated neurite outgrowth requires Na(+) current (I(Na)) mediated by Na(v)1.6. In addition, beta1 is required for high-frequency action potential firing. Transient I(Na) is unchanged in Scn1b (beta1) null CGNs; however, the resurgent I(Na), thought to underlie high-frequency firing in Na(v)1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Na(v)1.6 is reduced in Scn1b null cerebellar neurons. In place of Na(v)1.6 at the AIS, we observed an increase in Na(v)1.1, whereas Na(v)1.2 was unchanged. This indicates that beta1 is required for normal localization of Na(v)1.6 at the AIS during the postnatal developmental switch to Na(v)1.6-mediated high-frequency firing. In agreement with this, beta1 is normally expressed with alpha subunits at the AIS of P14 CGNs. We propose reciprocity of function between beta1 and Na(v)1.6 such that beta1-mediated neurite outgrowth requires Na(v)1.6-mediated I(Na), and Na(v)1.6 localization and consequent high-frequency firing require beta1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.


Asunto(s)
Cerebelo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Canales de Sodio/metabolismo , Potenciales de Acción/fisiología , Animales , Axones/metabolismo , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Neurológicos , Complejos Multiproteicos , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/química , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Transducción de Señal , Canales de Sodio/química , Canales de Sodio/deficiencia , Canales de Sodio/genética , Tetrodotoxina/toxicidad , Subunidad beta-1 de Canal de Sodio Activado por Voltaje
14.
bioRxiv ; 2023 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-37205386

RESUMEN

Pathogenic loss-of-function SCN1A variants cause a spectrum of seizure disorders. We previously identified variants in individuals with SCN1A -related epilepsy that fall in or near a poison exon (PE) in SCN1A intron 20 (20N). We hypothesized these variants lead to increased PE inclusion, which introduces a premature stop codon, and, therefore, reduced abundance of the full-length SCN1A transcript and Na v 1.1 protein. We used a splicing reporter assay to interrogate PE inclusion in HEK293T cells. In addition, we used patient-specific induced pluripotent stem cells (iPSCs) differentiated into neurons to quantify 20N inclusion by long and short-read sequencing and Na v 1.1 abundance by western blot. We performed RNA-antisense purification with mass spectrometry to identify RNA-binding proteins (RBPs) that could account for the aberrant PE splicing. We demonstrate that variants in/near 20N lead to increased 20N inclusion by long-read sequencing or splicing reporter assay and decreased Na v 1.1 abundance. We also identified 28 RBPs that differentially interact with variant constructs compared to wild-type, including SRSF1 and HNRNPL. We propose a model whereby 20N variants disrupt RBP binding to splicing enhancers (SRSF1) and suppressors (HNRNPL), to favor PE inclusion. Overall, we demonstrate that SCN1A 20N variants cause haploinsufficiency and SCN1A -related epilepsies. This work provides insights into the complex control of RBP-mediated PE alternative splicing, with broader implications for PE discovery and identification of pathogenic PE variants in other genetic conditions.

15.
J Neurosci ; 31(41): 14577-91, 2011 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-21994374

RESUMEN

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, ß1 and ß1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of ß1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to ß1B. We show that wild-type ß1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of ß1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and ß1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, ß1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that ß1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of ß1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that ß1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.


Asunto(s)
Epilepsia/genética , Mutación/genética , Canales de Sodio/genética , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Arginina/genética , Biotinilación/métodos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Cerebelo , Cricetinae , Cricetulus , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Glicina/genética , Humanos , Inmunoprecipitación/métodos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.3 , Neuritas/metabolismo , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Saxitoxina/farmacocinética , Canales de Sodio/deficiencia , Estadísticas no Paramétricas , Transfección/métodos , Tritio/farmacocinética , Subunidad beta-1 de Canal de Sodio Activado por Voltaje
16.
Breast Cancer Res Treat ; 134(2): 603-15, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22678159

RESUMEN

Voltage-gated Na(+) channels (VGSCs) are heteromeric membrane protein complexes containing pore-forming α subunits and smaller, non-pore-forming ß subunits. VGSCs are classically expressed in excitable cells, including neurons and muscle cells, where they mediate action potential firing, neurite outgrowth, pathfinding, and migration. VGSCs are also expressed in metastatic cells from a number of cancers. The Na(v)1.5 α subunit (encoded by SCN5A) is expressed in breast cancer (BCa) cell lines, where it enhances migration and invasion. We studied the expression of SCN5A in BCa array data, and tested the effect of the VGSC-blocking anticonvulsant phenytoin (5,5-diphenylhydantoin) on Na(+) current, migration, and invasion in BCa cells. SCN5A was up-regulated in BCa samples in several datasets, and was more highly expressed in samples from patients who had a recurrence, metastasis, or died within 5 years. SCN5A was also overexpressed as an outlier in a subset of samples, and associated with increased odds of developing metastasis. Phenytoin inhibited transient and persistent Na(+) current recorded from strongly metastatic MDA-MB-231 cells, and this effect was more potent at depolarized holding voltages. It may thus be an effective VGSC-blocking drug in cancer cells, which typically have depolarized membrane potentials. At a concentration within the therapeutic range used to treat epilepsy, phenytoin significantly inhibited the migration and invasion of MDA-MB-231 cells, but had no effect on weakly metastatic MCF-7 cells, which do not express Na(+) currents. We conclude that phenytoin suppresses Na(+) current in VGSC-expressing metastatic BCa cells, thus inhibiting VGSC-dependent migration and invasion. Together, our data support the hypothesis that SCN5A is up-regulated in BCa, favoring an invasive/metastatic phenotype. We therefore propose that repurposing existing VGSC-blocking therapeutic drugs should be further investigated as a potential new strategy to improve patient outcomes in metastatic BCa.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Fenitoína/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Área Bajo la Curva , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/secundario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Potenciales de la Membrana/efectos de los fármacos , Terapia Molecular Dirigida , Canal de Sodio Activado por Voltaje NAV1.5/genética , Invasividad Neoplásica , Curva ROC
17.
Exp Neurol ; 311: 247-256, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30347190

RESUMEN

Dravet syndrome is a severe, early-onset epileptic encephalopathy frequently resulting from de novo mutations of SCN1A. Mice with heterozygous deletion of Scn1a (Scn1a+/-) model many features of Dravet syndrome, including spontaneous seizures and premature lethality. Scn1a+/- mice exhibit variable phenotype penetrance and expressivity dependent upon the strain background. On the 129S6/SvEvTac (129) strain, Scn1a+/- mice do not display an overt phenotype. However Scn1a+/- mice on the [129S6xB6]F1 strain (F1.Scn1a+/-) exhibit juvenile-onset spontaneous seizures and premature lethality. QTL mapping identified several modifier loci responsible for strain-dependent differences in survival of Scn1a+/- mice, but these loci do not account for all the observed phenotypic variance. Global RNA-seq analysis was performed to identify additional genes and pathways that may contribute to variable phenotypes. Hippocampal gene expression was analyzed in wild-type (WT) and Scn1a+/- mice on both F1 and 129 strains, at two time points during disease development. There were few gene expression differences between 129.WT and 129.Scn1a+/- mice and approximately 100 genes with small expression differences (6-36%) between F1.WT and F1.Scn1a+/- mice. Strain-specific gene expression differences were more pronounced, with dozens of genes with >1.5-fold expression differences between 129 and F1 strains. Age-specific and seizure-related gene expression differences were most prominent, with hundreds of genes with >2-fold differences in expression identified between groups with and without seizures, suggesting potential differences in developmental trajectory and/or homeostatic plasticity during disease onset. Global expression differences in the context of Scn1a deletion may account for strain-dependent variation in seizure susceptibility and survival observed in Scn1a+/- mice.


Asunto(s)
Modelos Animales de Enfermedad , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Perfilación de la Expresión Génica/métodos , Canal de Sodio Activado por Voltaje NAV1.1/biosíntesis , Canal de Sodio Activado por Voltaje NAV1.1/genética , Animales , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Especificidad de la Especie
18.
Neurol Genet ; 3(6): e198, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29264390

RESUMEN

OBJECTIVE: To perform functional characterization of a potentially pathogenic KCNB1 variant identified by clinical exome sequencing of a proband with a neurodevelopmental disorder that included epilepsy and centrotemporal spikes on EEG. METHODS: Whole-exome sequencing identified the KCNB1 variant c.595A>T (p.Ile199Phe). Biochemical and electrophysiologic experiments were performed to determine whether this variant affected protein expression, trafficking, and channel functional properties. RESULTS: Biochemical characterization of the variant suggested normal protein expression and trafficking. Functional characterization revealed biophysical channel defects in assembled homotetrameric and heterotetrameric channels. CONCLUSIONS: The identification of the KCNB1 variant c.595A>T (p.Ile199Phe) in a neurodevelopmental disorder that included epilepsy with centrotemporal spikes expands the phenotypic spectrum of epilepsies associated with KCNB1 variants. The KCNB1-I199F variant exhibited partial loss of function relative to the wild-type channel. This defect is arguably less severe than previously reported KCNB1 variants, suggesting the possibility that the degree of KCNB1 protein dysfunction may influence disease severity.

19.
Int J Biochem Cell Biol ; 41(5): 1216-27, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19041953

RESUMEN

Voltage-gated Na(+) channels (VGSCs), predominantly the 'neonatal' splice form of Na(v)1.5 (nNa(v)1.5), are upregulated in metastatic breast cancer (BCa) and potentiate metastatic cell behaviours. VGSCs comprise one pore-forming alpha subunit and one or more beta subunits. The latter modulate VGSC expression and gating, and can function as cell adhesion molecules of the immunoglobulin superfamily. The aims of this study were (1) to determine which beta subunits were expressed in weakly metastatic MCF-7 and strongly metastatic MDA-MB-231 human BCa cells, and (2) to investigate the possible role of beta subunits in adhesion and migration. In both cell lines, the beta subunit mRNA expression profile was SCN1B (encoding beta1)>>SCN4B (encoding beta4)>SCN2B (encoding beta2); SCN3B (encoding beta3) was not detected. MCF-7 cells had much higher levels of all beta subunit mRNAs than MDA-MB-231 cells, and beta1 mRNA was the most abundant. Similarly, beta1 protein was strongly expressed in MCF-7 and barely detectable in MDA-MB-231 cells. In MCF-7 cells transfected with siRNA targeting beta1, adhesion was reduced by 35%, while migration was increased by 121%. The increase in migration was reversed by tetrodotoxin (TTX). In addition, levels of nNa(v)1.5 mRNA and protein were increased following beta1 down-regulation. Stable expression of beta1 in MDA-MB-231 cells increased functional VGSC activity, process length and adhesion, and reduced lateral motility and proliferation. We conclude that beta1 is a novel cell adhesion molecule in BCa cells and can control VGSC (nNa(v)1.5) expression and, concomitantly, cellular migration.


Asunto(s)
Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/metabolismo , Activación del Canal Iónico/fisiología , Canales de Sodio/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Humanos , Activación del Canal Iónico/genética , Canales de Sodio/genética , Células Tumorales Cultivadas
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