RESUMO
KCNT1 (K+ channel subfamily T member 1) is a sodium-activated potassium channel highly expressed in the nervous system which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarisation following a train of action potentials. Gain of function mutations in the KCNT1 gene are the cause of neurological disorders associated with different forms of epilepsy. To gain insights into the underlying pathobiology we investigated the functional effects of 9 recently published KCNT1 mutations, 4 previously studied KCNT1 mutations, and one previously unpublished KCNT1 variant of unknown significance. We analysed the properties of KCNT1 potassium currents and attempted to find a correlation between the changes in KCNT1 characteristics due to the mutations and severity of the neurological disorder they cause. KCNT1 mutations identified in patients with epilepsy were introduced into the full length human KCNT1 cDNA using quick-change site-directed mutagenesis protocol. Electrophysiological properties of different KCNT1 constructs were investigated using a heterologous expression system (HEK293T cells) and patch clamping. All mutations studied, except T314A, increased the amplitude of KCNT1 currents, and some mutations shifted the voltage dependence of KCNT1 open probability, increasing the proportion of channels open at the resting membrane potential. The T314A mutation did not affect KCNT1 current amplitude but abolished its voltage dependence. We observed a positive correlation between the severity of the neurological disorder and the KCNT1 channel open probability at resting membrane potential. This suggests that gain of function KCNT1 mutations cause epilepsy by increasing resting potassium conductance and suppressing the activity of inhibitory neurons. A reduction in action potential firing in inhibitory neurons due to excessively high resting potassium conductance leads to disinhibition of neural circuits, hyperexcitability and seizures.
Assuntos
Epilepsia , Proteínas do Tecido Nervoso , Humanos , Canais de Potássio Ativados por Sódio/genética , Células HEK293 , Proteínas do Tecido Nervoso/metabolismo , Epilepsia/genética , Mutação , Potássio/metabolismoRESUMO
OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.
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PURPOSE: Our objective was to understand the range of parental views on whether and how to approach the issue of SUDEP with families, to clarify the optimal timing and formulation of the information, and to learn from parents the optimal counseling strategies in order to minimize the inherent emotional burden. METHODS: The principles of fundamental qualitative description were used to guide this descriptive and exploratory study of parental experiences and perceptions. Stratified purposeful sampling included parents (i) who lost children to SUDEP, (ii) of children with moderate to severe epilepsy, (iii) of children with mild epilepsy, and (iv) of children with new-onset epilepsy. Focus group and in-depth one-on-one interviews were conducted. The principles of directed content analysis were used to code and categorize the focus group and interview data. Key concepts from the interview guide were used as the first level of coding categories. Codes were subsequently collapsed into broader categories. RESULTS: There was full agreement, across both genders and regardless of seizure severity, that routine counseling about SUDEP should be provided by pediatric neurologists, during the appointment when the diagnosis of epilepsy is shared with parents, and with opportunities for short-term follow-up and discussions with clinical nurses or social workers. Parents described feeling overwhelmed, worried, and increasingly anxious when the risk of SUDEP was explained to them. Parents generally expressed a preference for receiving routine SUDEP counseling at the time of the diagnosis of epilepsy. Across all groups of parents, it was identified that SUDEP counseling should occur in a face-to-face interaction with the neurologist. In learning about SUDEP, parents expressed a need to be informed of the risk of SUDEP. There was group endorsement for receiving written information about SUDEP to reinforce the content shared by the neurologist. There was a consensus that it should be the parents' decision as to whether or not the child should be present at the meeting or when to inform the child about the risk of SUDEP. CONCLUSION: Participants in this study opined that all parents of children with epilepsy should receive routine SUDEP counseling and have access to ongoing professional support. Based on these findings, it is imperative for pediatric neurologists to refine their communication skills when counseling parents about SUDEP.
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Morte Súbita , Epilepsia/mortalidade , Pais/psicologia , Criança , Morte Súbita/epidemiologia , Morte Súbita/etiologia , Morte Súbita/prevenção & controle , Epilepsia/epidemiologia , Feminino , Pessoal de Saúde , Humanos , Masculino , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
PURPOSE: To further define the epilepsy phenotype in a cohort of children with 15q13.3 microdeletion syndrome. METHODS: We retrospectively reviewed the phenotypic spectrum of all children aged < 18 years with epilepsy and 15q13.3 microdeletion syndrome. RESULTS: Thirteen children were included, 69% were female. The median age of children in the cohort was 12 years (age range: 3 years-15 years). Median age at seizure onset was 4 years. Eleven children (85%) had intellectual disability. Nine of 13 children (69%) had a history of typical absence seizures with median age of onset at 5 years (2 had absence status epilepticus). Thirty-one percent (4/13) had focal with impaired awareness non-motor onset seizures. ILAE recognized absence epilepsy syndromes were diagnosed in 6/13 (46%). The remainder were classified as having genetic generalized epilepsies with overlap clinical features, combined or focal epilepsies. Electroencephalogram in the cohort showed generalized (85%) and focal epileptiform discharges (62%) and posterior dominant rhythm slowing (33%). One child had electrical status epilepticus of sleep. Neuroimaging was performed in 5 children (38%) and revealed abnormal findings in 3. Seizures were drug resistant in a third of the cohort. Valproate resulted in seizure freedom in 5 (42%). Oxcarbazepine caused clinical worsening in one child with combined seizure types. Two children tried cannabidiol and one tried the ketogenic diet; neither was effective. CONCLUSIONS: The epilepsy phenotype in children with 15q13.3 microdeletion syndrome is defined by childhood onset absence seizures, and may have atypical features such as, early onset absences, persistence into adolescence, status epilepticus, intellectual disability and treatment resistance. Focal seizures and focal EEG findings may be observed and should be treated cautiously, given the possibility of combined seizure types. Valproate appeared effective, although other treatments must be explored further.
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Epilepsia Tipo Ausência , Deficiência Intelectual , Criança , Pré-Escolar , Deleção Cromossômica , Transtornos Cromossômicos , Cromossomos Humanos Par 15 , Eletroencefalografia , Feminino , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Estudos Retrospectivos , ConvulsõesRESUMO
Standardization of the use of next-generation sequencing for the diagnosis of rare neurological disorders has made it possible to detect potential disease-causing genetic variations, including de novo variants. However, the lack of a clear pathogenic relevance of gene variants poses a critical limitation for translating this genetic information into clinical practice, increasing the necessity to perform functional assays. Genetic screening is currently recommended in the guidelines for diagnosis of hypomyelinating leukodystrophies (HLDs). HLDs represent a group of rare heterogeneous disorders that interfere with the myelination of the neurons in the central nervous system. One of the HLD-related genes is HSPD1, encoding the mitochondrial chaperone heat shock protein 60 (HSP60), which functions as folding machinery for the mitochondrial proteins imported into the mitochondrial matrix space. Disease-causing HSPD1 variants have been associated with an autosomal recessive form of fatal hypomyelinating leukodystrophy (HLD4, MitCHAP60 disease; MIM #612233) and an autosomal dominant form of spastic paraplegia, type 13 (SPG13; MIM #605280). In 2018, a de novo HSPD1 variant was reported in a patient with HLD. Here, we present another case carrying the same heterozygous de novo variation in the HSPD1 gene (c.139T > G, p.Leu47Val) associated with an HLD phenotype. Our molecular studies show that the variant HSP60 protein is stably present in the patient's fibroblasts, and functional assays demonstrate that the variant protein lacks in vivo function, thus confirming its disease association. We conclude that de novo variations of the HSPD1 gene should be considered as potentially disease-causing in the diagnosis and pathogenesis of the HLDs.
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Chaperonina 60/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Adulto , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Chaperonina 10/genética , Chaperonina 60/química , Criança , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Proteínas Mitocondriais/química , Modelos Moleculares , Mutação , Proteínas da Gravidez/genética , Conformação Proteica , Recidiva , Relação Estrutura-Atividade , Fatores Supressores Imunológicos/genéticaRESUMO
As the practice of pediatric neurology has continued to evolve and expand, concerns have emerged regarding a perceived trend toward increasing clinical demand and decreasing manpower. Quantitative data are lacking, however. To address this, a questionnaire was sent to hospital-based pediatric neurology units in Canada, with all 18 centers responding. A total of 63 full-time-equivalent pediatric neurologists were disproportionately spread across the country, giving an overall ratio of 2.1 per million population (or 1.1 per 100,000 children). Waiting times for nonurgent consultations showed a median of 12.5 weeks. The number of weekly clinics per population was not proportional to either the number of specialists per center or to the waiting times. The regional variations in the level of service do not correlate with the regional manpower figures, reflecting different individual profiles of clinical and academic activities.
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Serviços de Saúde da Criança/tendências , Neurologia , Pediatria , Adolescente , Canadá , Criança , Serviços de Saúde da Criança/organização & administração , Pré-Escolar , Humanos , Corpo Clínico Hospitalar/tendências , Neurologia/tendências , Pediatria/tendências , Encaminhamento e Consulta , Inquéritos e Questionários , Recursos HumanosRESUMO
BACKGROUND: Increasing evidence suggests that seizures and status epilepticus can be immune-mediated. We aimed to describe the clinical features of a new epileptic disorder, and to establish the target antigen and the effects of patients' antibodies on neuronal cultures. METHODS: In this observational study, we selected serum and CSF samples for antigen characterisation from 140 patients with encephalitis, seizures or status epilepticus, and antibodies to unknown neuropil antigens. The samples were obtained from worldwide referrals of patients with disorders suspected to be autoimmune between April 28, 2006, and April 25, 2013. We used samples from 75 healthy individuals and 416 patients with a range of neurological diseases as controls. We assessed the samples using immunoprecipitation, mass spectrometry, cell-based assay, and analysis of antibody effects in cultured rat hippocampal neurons with confocal microscopy. FINDINGS: Neuronal cell-membrane immunoprecipitation with serum of two index patients revealed GABAA receptor sequences. Cell-based assay with HEK293 expressing α1/ß3 subunits of the GABAA receptor showed high titre serum antibodies (>1:160) and CSF antibodies in six patients. All six patients (age 3-63 years, median 22 years; five male patients) developed refractory status epilepticus or epilepsia partialis continua along with extensive cortical-subcortical MRI abnormalities; four patients needed pharmacologically induced coma. 12 of 416 control patients with other diseases, but none of the healthy controls, had low-titre GABAA receptor antibodies detectable in only serum samples, five of them also had GAD-65 antibodies. These 12 patients (age 2-74 years, median 26.5 years; seven male patients) developed a broader spectrum of symptoms probably indicative of coexisting autoimmune disorders: six had encephalitis with seizures (one with status epilepticus needing pharmacologically induced coma; one with epilepsia partialis continua), four had stiff-person syndrome (one with seizures and limbic involvement), and two had opsoclonus-myoclonus. Overall, 12 of 15 patients for whom treatment and outcome were assessable had full (three patients) or partial (nine patients) response to immunotherapy or symptomatic treatment, and three died. Patients' antibodies caused a selective reduction of GABAA receptor clusters at synapses, but not along dendrites, without altering NMDA receptors and gephyrin (a protein that anchors the GABAA receptor). INTERPRETATION: High titres of serum and CSF GABAA receptor antibodies are associated with a severe form of encephalitis with seizures, refractory status epilepticus, or both. The antibodies cause a selective reduction of synaptic GABAA receptors. The disorder often occurs with GABAergic and other coexisting autoimmune disorders and is potentially treatable. FUNDING: The National Institutes of Health, the McKnight Neuroscience of Brain Disorders, the Fondo de Investigaciones Sanitarias, Fundació la Marató de TV3, the Netherlands Organisation for Scientific Research (Veni-incentive), the Dutch Epilepsy Foundation.
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Autoanticorpos/biossíntese , Autoantígenos/imunologia , Encefalite/imunologia , Receptores de GABA-A/metabolismo , Convulsões/imunologia , Estado Epiléptico/imunologia , Adolescente , Adulto , Idoso , Animais , Autoanticorpos/fisiologia , Autoantígenos/química , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Criança , Pré-Escolar , Encefalite/diagnóstico , Encefalite/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Neurópilo/química , Neurópilo/imunologia , Neurópilo/metabolismo , Ratos , Receptores de GABA-A/imunologia , Receptores de GABA-A/fisiologia , Convulsões/diagnóstico , Convulsões/metabolismo , Estado Epiléptico/diagnóstico , Estado Epiléptico/metabolismo , Adulto JovemRESUMO
Ovarian failure (OF) at age <40 years occurs in approximately 1% of all women. Other than karyotype abnormalities, very few genes are known to be associated with this ovarian dysfunction. We studied eight patients who presented with premature OF and white-matter abnormalities on magnetic resonance imaging. Neurological signs may be absent or present after OF. In seven patients, we report for the first time mutations in three of the five EIF2B genes (EIF2B2, -4, and -5) that were recently shown to cause childhood ataxia with central nervous system hypomyelination/vanishing white-matter disease leukodystrophy. The correlation we observed between the age at onset of the neurological deterioration and the severity of OF suggests a common pathophysiological pathway.