RESUMO
OBJECTIVE: Variants in several potassium channel genes, including KCNA1 and KCNA2, cause Developmental and Epileptic Encephalopathies (DEEs). We investigated whether variants in KCNA3, another mammalian homologue of the Drosophila shaker family and encoding for Kv1.3 subunits, can cause DEE. METHODS: Genetic analysis of study individuals was performed by routine exome or genome sequencing, usually of parent-offspring trios. Phenotyping was performed via a standard clinical questionnaire. Currents from wild-type and/or mutant Kv1.3 subunits were investigated by whole-cell patch-clamp upon their heterologous expression. RESULTS: Fourteen individuals, each carrying a de novo heterozygous missense variant in KCNA3, were identified. Most (12/14; 86%) had DEE with marked speech delay with or without motor delay, intellectual disability, epilepsy, and autism spectrum disorder. Functional analysis of Kv1.3 channels carrying each variant revealed heterogeneous functional changes, ranging from "pure" loss-of-function (LoF) effects due to faster inactivation kinetics, depolarized voltage-dependence of activation, slower activation kinetics, increased current inactivation, reduced or absent currents with or without dominant-negative effects, to "mixed" loss- and gain-of-function (GoF) effects. Compared to controls, Kv1.3 currents in lymphoblasts from 1 of the proband displayed functional changes similar to those observed upon heterologous expression of channels carrying the same variant. The antidepressant drug fluoxetine inhibited with similar potency the currents from wild-type and 1 of the Kv1.3 GoF variant. INTERPRETATION: We describe a novel association of de novo missense variants in KCNA3 with a human DEE, and provide evidence that fluoxetine might represent a potential targeted treatment for individuals carrying variants with significant GoF effects. ANN NEUROL 2024;95:365-376.
Assuntos
Transtorno do Espectro Autista , Epilepsia Generalizada , Epilepsia , Animais , Humanos , Fluoxetina , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/complicações , Mutação de Sentido Incorreto/genética , Mamíferos , Canal de Potássio Kv1.3/genéticaRESUMO
Developmental and epileptic encephalopathies (DEEs) are neurodevelopmental diseases characterized by refractory epilepsy, distinct electroencephalographic and neuroradiological features, and various degrees of developmental delay. Mutations in KCNQ2, KCNQ3, and, more rarely, KCNQ5 genes encoding voltage-gated potassium channel subunits variably contributing to excitability control of specific neuronal populations at distinct developmental stages have been associated to DEEs. In the present work, the clinical features of two DEE patients carrying de novo KCNQ5 variants affecting the same residue in the pore region of the Kv7.5 subunit (G347S/A) are described. The in vitro functional properties of channels incorporating these variants were investigated with electrophysiological and biochemical techniques to highlight pathophysiological disease mechanisms. Currents carried by Kv7.5 G347 S/A channels displayed: 1) large (>10 times) increases in maximal current density, 2) the occurrence of a voltage-independent component, 3) slower deactivation kinetics, and 4) hyperpolarization shift in activation. All these functional features are consistent with a gain-of-function (GoF) pathogenetic mechanism. Similar functional changes were also observed when the same variants were introduced at the corresponding position in Kv7.2 subunits. Nonstationary noise analysis revealed that GoF effects observed for both Kv7.2 and Kv7.5 variants were mainly attributable to an increase in single-channel open probability, without changes in membrane abundance or single-channel conductance. The mutation-induced increase in channel opening probability was insensitive to manipulation of membrane levels of the critical Kv7 channel regulator PIP2. These results reveal a pathophysiological mechanism for KCNQ5-related DEEs, which might be exploited to implement personalized treatments.
Assuntos
Epilepsia Resistente a Medicamentos , Mutação com Ganho de Função , Canais de Potássio KCNQ , Adolescente , Criança , Epilepsia Resistente a Medicamentos/genética , Feminino , Humanos , Canais de Potássio KCNQ/genética , Masculino , Mutação , Fenótipo , ProbabilidadeRESUMO
Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.
Assuntos
Barreira Hematoencefálica , Carbamatos , Epilepsia , Fenilenodiaminas , Animais , Ratos , Células Endoteliais , Ácido Caínico/toxicidade , EncéfaloRESUMO
OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.
Assuntos
Deficiência Intelectual , Neuroblastoma , Humanos , Células HEK293 , Fenótipo , Genótipo , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/genética , Canais de Potássio Ativados por Sódio/genéticaRESUMO
Variable phenotypes, including developmental encephalopathy with (DEE) or without seizures and myoclonic epilepsy and ataxia due to potassium channel mutation, are caused by pathogenetic variants in KCNC1, encoding for Kv3.1 channel subunits. In vitro, channels carrying most KCNC1 pathogenic variants display loss-of-function features. Here, we describe a child affected by DEE with fever-triggered seizures, caused by a novel de novo heterozygous missense KCNC1 variant (c.1273G>A; V425M). Patch-clamp recordings in transiently transfected CHO cells revealed that, compared to wild-type, Kv3.1 V425M currents (1) were larger, with membrane potentials between -40 and +40 mV; (2) displayed a hyperpolarizing shift in activation gating; (3) failed to inactivate; and (4) had slower activation and deactivation kinetics, consistent with a mixed functional pattern with prevalent gain-of-function effects. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild-type and mutant Kv3.1 channels. Treatment of the proband with fluoxetine led to a rapid and prolonged clinical amelioration, with the disappearance of seizures and an improvement in balance, gross motor skills, and oculomotor coordination. These results suggest that drug repurposing based on the specific genetic defect may provide an effective personalized treatment for KCNC1-related DEEs.
Assuntos
Epilepsias Mioclônicas , Convulsões Febris , Cricetinae , Animais , Fluoxetina/uso terapêutico , Cricetulus , Medicina de Precisão , Mutação com Ganho de Função , Convulsões/genética , Epilepsias Mioclônicas/tratamento farmacológico , Epilepsias Mioclônicas/genéticaRESUMO
KCNQ2-encoded Kv7.2 subunits play a critical role in balancing neuronal excitability. Mutations in KCNQ2 are responsible for highly-heterogenous epileptic and neurodevelopmental phenotypes ranging from self-limited familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). Pathogenic KCNQ2 variants cluster at the voltage sensor domain (VSD), the pore domain, and the C-terminal tail. Although several knock-in mice harboring Kcnq2 pore variants have been developed, no mouse line carrying Kcnq2 voltage-sensor mutations has been described. KCNQ2-R207W is an epilepsy-causing mutation located in the VSD, mainly affecting voltage-dependent channel gating. To study the physiological consequence of Kcnq2 VSD dysfunction, we generated a Kcnq2-R207W mouse line and analyzed the pathological and pharmacological phenotypes of mutant mice. As a result, both homozygous (Kcnq2RW/RW) and heterozygous (Kcnq2RW/+) mice were viable. While Kcnq2RW/RW mice displayed a short lifespan, growth retardation, and spontaneous seizures, Kcnq2RW/+ mice survived and developed normally, although only a fraction (9/64; 14%) of them showed behavioral- and ECoG-confirmed spontaneous seizures. Kcnq2RW/+ mice displayed increased susceptibility to evoked seizures, which was dramatically ameliorated by treatment with the novel KCNQ opener pynegabine (HN37). Our results show that the Kcnq2-R207W mouse line, the first harboring a Kcnq2 voltage-sensor mutation, exhibits a unique epileptic phenotype with both spontaneous seizures and increased susceptibility to evoked seizures. In Kcnq2-R207W mice, the potent KCNQ opener HN37, currently in clinical phase I, shows strong anticonvulsant activity, suggesting it may represent a valuable option for the severe phenotypes of KCNQ2-related epilepsy.
Assuntos
Epilepsia , Canal de Potássio KCNQ2 , Animais , Camundongos , Canal de Potássio KCNQ2/genética , Epilepsia/genética , Fenótipo , Mutação/genética , Convulsões/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
A wide phenotypic spectrum of neurological diseases is associated with KCNA1 (Kv1.1) variants. To investigate the molecular basis of such a heterogeneous clinical presentation and identify the possible correlation with in vitro phenotypes, we compared the functional consequences of three heterozygous de novo variants (p.P403S, p.P405L, and p.P405S) in Kv1.1 pore region found in four patients with severe developmental and epileptic encephalopathy (DEE), with those of a de novo variant in the voltage sensor (p.A261T) identified in two patients with mild, carbamazepine-responsive, focal epilepsy. Patch-clamp electrophysiology was used to investigate the functional properties of mutant Kv1.1 subunits, both expressed as homomers and heteromers with wild-type Kv1.1 subunits. KCNA1 pore mutations markedly decreased (p. P405S) or fully suppressed (p. P403S, p. P405L) Kv1.1-mediated currents, exerting loss-of-function (LoF) effects. By contrast, channels carrying the p.A261T variant exhibited a hyperpolarizing shift of the activation process, consistent with a gain-of-function (GoF) effect. The present results unveil a novel correlation between in vitro phenotype (GoF vs LoF) and clinical course (mild vs severe) in KCNA1-related phenotypes. The excellent clinical response to carbamazepine observed in the patients carrying the A261T variant suggests an exquisite sensitivity of KCNA1 GoF to sodium channel inhibition that should be further explored.
Assuntos
Epilepsia , Carbamazepina/uso terapêutico , Epilepsia/tratamento farmacológico , Epilepsia/genética , Humanos , Canal de Potássio Kv1.1/genética , Mutação/genética , FenótipoRESUMO
Retigabine is a first-in-class potassium channel opener approved for patients with epilepsy. Unfortunately, several side effects have limited its use in clinical practice, overshadowing its beneficial effects. Multiple studies have shown that retigabine acts by enhancing the activity of members of the voltage-gated KCNQ (Kv7) potassium channel family, particularly the neuronal KCNQ channels KCNQ2-KCNQ5. However, it is currently unknown whether retigabine's action in neurons is mediated by all KCNQ neuronal channels or by only a subset. This knowledge is necessary to elucidate retigabine's mechanism of action in the central nervous system and its adverse effects and to design more effective and selective retigabine analogs. In this study, we show that the action of retigabine in excitatory neurons strongly depends on the presence of KCNQ3 channels. Deletion of Kcnq3 severely limited the ability of retigabine to reduce neuronal excitability in mouse CA1 and subiculum excitatory neurons. In addition, we report that in the absence of KCNQ3 channels, retigabine can enhance CA1 pyramidal neuron activity, leading to a greater number of action potentials and reduced spike frequency adaptation; this finding further supports a key role of KCNQ3 channels in mediating the action of retigabine. Our work provides new insight into the action of retigabine in forebrain neurons, clarifying retigabine's action in the nervous system.NEW & NOTEWORTHY Retigabine has risen to prominence as a first-in-class potassium channel opener approved by the Food and Drug Administration, with potential for treating multiple neurological disorders. Here, we demonstrate that KCNQ3 channels are the primary target of retigabine in excitatory neurons, as deleting these channels greatly diminishes the effect of retigabine in pyramidal neurons. Our data provide the first indication that retigabine controls neuronal firing properties primarily through KCNQ3 channels.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Anticonvulsivantes/farmacologia , Região CA1 Hipocampal/efeitos dos fármacos , Carbamatos/farmacologia , Canal de Potássio KCNQ3/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Células Piramidais/efeitos dos fármacos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-ClampRESUMO
Seizures are the most common neurological manifestation in the newborn period, with an estimated incidence of 1.8-3.5 per 1000 live births. Prolonged or intractable seizures have a detrimental effect on cognition and brain function in experimental animals and are associated with adverse long-term neurodevelopmental sequelae and an increased risk of post-neonatal epilepsy in humans. The developing brain is particularly susceptible to the potentially severe effects of epilepsy, and epilepsy, especially when refractory to medications, often results in a developmental and epileptic encephalopathy (DEE) with developmental arrest or regression. DEEs can be primarily attributed to genetic causes. Given the critical role of potassium (K+) currents with distinct subcellular localization, biophysical properties, modulation, and pharmacological profile in regulating intrinsic electrical properties of neurons and their responsiveness to synaptic inputs, it is not too surprising that genetic research in the past two decades has identified several K+ channel genes as responsible for a large fraction of DEE. In the present article, we review the genetically determined epileptic channelopathies affecting three members of the Kv7 family, namely Kv7.2 (KCNQ2), Kv7.3 (KCNQ3), and Kv7.5 (KCNQ5); we review the phenotypic spectrum of Kv7-related epileptic channelopathies, the different genetic and pathogenetic mechanisms, and the emerging genotype-phenotype correlations which may prove crucial for prognostic predictions, disease management, parental counseling, and individually tailored therapeutic attempts.
Assuntos
Canalopatias/genética , Canalopatias/patologia , Canal de Potássio KCNQ1/genética , Neurônios/patologia , Convulsões/genética , Convulsões/patologia , Animais , HumanosRESUMO
In the first two decades of the 21st century, there have been three outbreaks of severe respiratory infections caused by highly pathogenic coronaviruses (CoVs) around the world: the severe acute respiratory syndrome (SARS) by the SARS-CoV in 2002-2003, the Middle East respiratory syndrome (MERS) by the MERS-CoV in June 2012, and Coronavirus Disease 2019 (COVID-19) by the SARS-CoV-2 presently affecting most countries In all of these, fatalities are a consequence of a multiorgan dysregulation caused by pulmonary, renal, cardiac, and circulatory damage; however, COVID patients may show significant neurological signs and symptoms such as headache, nausea, vomiting, and sensory disturbances, the most prominent being anosmia and ageusia. The neuroinvasive potential of CoVs might be responsible for at least part of these symptoms and may contribute to the respiratory failure observed in affected patients. Therefore, in the present manuscript, we have reviewed the available preclinical evidence on the mechanisms and consequences of CoVs-induced CNS damage, and highlighted the potential role of CoVs in determining or aggravating acute and long-term neurological diseases in infected individuals. We consider that a widespread awareness of the significant neurotropism of CoVs might contribute to an earlier recognition of the signs and symptoms of viral-induced CNS damage. Moreover, a better understanding of the cellular and molecular mechanisms by which CoVs affect CNS function and cause CNS damage could help in planning new strategies for prognostic evaluation and targeted therapeutic intervention.
Assuntos
Betacoronavirus , Encéfalo/virologia , Infecções por Coronavirus/epidemiologia , Doenças do Sistema Nervoso/epidemiologia , Pneumonia Viral/epidemiologia , Animais , Encéfalo/fisiopatologia , COVID-19 , Infecções por Coronavirus/fisiopatologia , Humanos , Doenças do Sistema Nervoso/fisiopatologia , Pandemias , Pneumonia Viral/fisiopatologia , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/fisiopatologiaRESUMO
OBJECTIVE: Recent reports have described single individuals with neurodevelopmental disability (NDD) harboring heterozygous KCNQ3 de novo variants (DNVs). We sought to assess whether pathogenic variants in KCNQ3 cause NDD and to elucidate the associated phenotype and molecular mechanisms. METHODS: Patients with NDD and KCNQ3 DNVs were identified through an international collaboration. Phenotypes were characterized by clinical assessment, review of charts, electroencephalographic (EEG) recordings, and parental interview. Functional consequences of variants were analyzed in vitro by patch-clamp recording. RESULTS: Eleven patients were assessed. They had recurrent heterozygous DNVs in KCNQ3 affecting residues R230 (R230C, R230H, R230S) and R227 (R227Q). All patients exhibited global developmental delay within the first 2 years of life. Most (8/11, 73%) were nonverbal or had a few words only. All patients had autistic features, and autism spectrum disorder (ASD) was diagnosed in 5 of 11 (45%). EEGs performed before 10 years of age revealed frequent sleep-activated multifocal epileptiform discharges in 8 of 11 (73%). For 6 of 9 (67%) recorded between 1.5 and 6 years of age, spikes became near-continuous during sleep. Interestingly, most patients (9/11, 82%) did not have seizures, and no patient had seizures in the neonatal period. Voltage-clamp recordings of the mutant KCNQ3 channels revealed gain-of-function (GoF) effects. INTERPRETATION: Specific GoF variants in KCNQ3 cause NDD, ASD, and abundant sleep-activated spikes. This new phenotype contrasts both with self-limited neonatal epilepsy due to KCNQ3 partial loss of function, and with the neonatal or infantile onset epileptic encephalopathies due to KCNQ2 GoF. ANN NEUROL 2019;86:181-192.
Assuntos
Transtorno Autístico/diagnóstico , Transtorno Autístico/genética , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Mutação com Ganho de Função/genética , Canal de Potássio KCNQ3/genética , Sequência de Aminoácidos , Criança , Pré-Escolar , Variação Genética/genética , Humanos , Canal de Potássio KCNQ3/química , Masculino , Estrutura Secundária de Proteína , Adulto JovemRESUMO
De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations.
Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Epilepsia/genética , Gabapentina/uso terapêutico , Canal de Potássio KCNQ2/genética , Idade de Início , Animais , Células CHO , Carbamatos/uso terapêutico , Células Cultivadas , Criança , Cricetinae , Cricetulus , Eletroencefalografia , Feminino , Humanos , Mutação , Fenilenodiaminas/uso terapêutico , Medicina de Precisão , Ratos , Resultado do TratamentoRESUMO
The symptoms of chronic urticaria, be it chronic spontaneous urticaria (CSU) or chronic inducible urticaria (CindU), are mediated primarily by the actions of histamine on H1 receptors located on endothelial cells (the weal) and on sensory nerves (neurogenic flare and pruritus). Thus, second-generation H1 antihistamines (sgAHs) are the primary treatment of these conditions. However, many patients are poorly responsive to licensed doses of antihistamines. In these patients, the current EAACI/GA2 LEN/EDF/WAO guideline for urticaria suggests updosing of sgAHs up to fourfold. However, such updosing is off-label and the responsibility resides with the prescribing physician. Therefore, the safety of the drug when used above its licensed dose is of paramount importance. An important aspect of safety is potential cardiotoxicity. This problem was initially identified some 20 years ago with cardiotoxic deaths occurring with astemizole and terfenadine, two early sgAHs. In this review, we discuss the mechanisms and assessments of potential cardiotoxicity of H1 antihistamines when updosed to four times their licensed dose. In particular, we have focused on the potential of H1 antihistamines to block hERG (human Ether-a-go-go-Related Gene) voltage-gated K+ channels, also known as Kv11.1 channels according to the IUPHAR classification. Blockade of these channels causes QT prolongation leading to torsade de pointes that may possibly degenerate into ventricular fibrillation and sudden death. We considered in detail bilastine, cetirizine, levocetirizine, ebastine, fexofenadine, loratadine, desloratadine, mizolastine and rupatadine and concluded that all these drugs have an excellent safety profile with no evidence of cardiotoxicity even when updosed up to four times their standard licensed dose, provided that the prescribers carefully consider and rule out potential risk factors for cardiotoxicity, such as the presence of inherited long QT syndrome, older age, cardiovascular disorders, hypokalemia and hypomagnesemia, or the use of drugs that either have direct QT prolonging effects or inhibit sgAH metabolism.
Assuntos
Cardiotoxicidade , Urticária Crônica , Canal de Potássio ERG1 , Antagonistas dos Receptores Histamínicos H1 , Torsades de Pointes , Fatores Etários , Cardiotoxicidade/imunologia , Cardiotoxicidade/patologia , Cardiotoxicidade/prevenção & controle , Urticária Crônica/tratamento farmacológico , Urticária Crônica/imunologia , Urticária Crônica/patologia , Canal de Potássio ERG1/imunologia , Feminino , Antagonistas dos Receptores Histamínicos H1/efeitos adversos , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Humanos , Síndrome do QT Longo/imunologia , Síndrome do QT Longo/patologia , Masculino , Fatores de Risco , Torsades de Pointes/induzido quimicamente , Torsades de Pointes/imunologia , Torsades de Pointes/patologiaRESUMO
Variants in several potassium channel genes have been found in developmental and epileptic encephalopathies (DEE). We report on 2 females with de novo variants in KCNT2 with West syndrome followed by Lennox-Gastaut syndrome or with DEE with migrating focal seizures. After in vitro analysis suggested quinidine-responsive gain-of-function effects, we treated 1 of the girls with quinidine add-on therapy and achieved marked clinical improvements. This suggests that the new spectrum of KCNT2-related disorders do not only share similar phenotypic and in vitro functional and pharmacological features with previously known KCNT1-related disorders, but also represents a further example for possible precision medicine approaches. Ann Neurol 2018;83:1198-1204.
Assuntos
Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Canais de Potássio/genética , Espasmos Infantis/genética , Adolescente , Criança , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Recém-Nascido , Potenciais da Membrana/genética , Modelos Moleculares , Transtornos do Neurodesenvolvimento/complicações , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Espasmos Infantis/complicações , TransfecçãoRESUMO
The development of patient-specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot-Marie-Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post-mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post-mitotic neurons within 6-8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post-mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient-derived iPSCs into post-mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1-CMT disease.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , ATPase Trocadora de Sódio-Potássio/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , LinhagemRESUMO
Kv7.2 subunits encoded by the KCNQ2 gene provide a major contribution to the M-current (IKM), a voltage-gated K+ current crucially involved in the regulation of neuronal excitability. Heterozygous missense variants in Kv7.2 are responsible for epileptic diseases characterized by highly heterogeneous genetic transmission and clinical severity, ranging from autosomal-dominant Benign Familial Neonatal Seizures (BFNS) to sporadic cases of severe epileptic and developmental encephalopathy (DEE). Here, we describe a patient with neonatal onset DEE, carrying a previously undescribed heterozygous KCNQ2 c.418G > C, p.Glu140Gln (E140Q) variant. Patch-clamp recordings in CHO cells expressing the E140Q mutation reveal dramatic loss of function (LoF) effects. Multistate structural modelling suggested that the E140Q substitution impeded an intrasubunit electrostatic interaction occurring between the E140 side chain in S2 and the arginine at position 210 in S4 (R210); this interaction is critically involved in stabilizing the activated configuration of the voltage-sensing domain (VSD) of Kv7.2. Functional results from coupled charge reversal or disulfide trapping experiments supported such a hypothesis. Finally, retigabine restored mutation-induced functional changes, reinforcing the rationale for the clinical use of Kv7 activators as personalized therapy for DEE-affected patients carrying Kv7.2 LoF mutations.
Assuntos
Encefalopatias/genética , Deficiências do Desenvolvimento/genética , Predisposição Genética para Doença , Variação Genética , Canal de Potássio KCNQ2/genética , Domínios e Motivos de Interação entre Proteínas/genética , Espasmos Infantis/genética , Substituição de Aminoácidos , Biomarcadores , Encefalopatias/diagnóstico , Encefalopatias/terapia , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/terapia , Eletroencefalografia , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Canal de Potássio KCNQ2/química , Mutação com Perda de Função , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Neuroimagem , Conformação Proteica , Espasmos Infantis/diagnóstico , Espasmos Infantis/terapia , Relação Estrutura-Atividade , Avaliação de SintomasRESUMO
Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 µM) was higher than that of ICA-27243 (IC50~5 µM) and (S)-1 (IC50~7 µM). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.
Assuntos
Sinalização do Cálcio , Capsaicina/farmacologia , Canais de Potássio KCNQ/metabolismo , Células Receptoras Sensoriais/metabolismo , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/metabolismo , Animais , Bradicinina/farmacologia , Cálcio/metabolismo , Carbamatos/farmacologia , Linhagem Celular , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Fenilenodiaminas/farmacologia , Ratos , Células Receptoras Sensoriais/efeitos dos fármacosRESUMO
Variants in KCNQ2 encoding for Kv 7.2 neuronal K+ channel subunits lead to a spectrum of neonatal-onset epilepsies, ranging from self-limiting forms to severe epileptic encephalopathy. Most KCNQ2 pathogenic variants cause loss-of-function, whereas few increase channel activity (gain-of-function). We herein provide evidence for a new phenotypic and functional profile in KCNQ2-related epilepsy: infantile spasms without prior neonatal seizures associated with a gain-of-function gene variant. With use of an international registry, we identified four unrelated patients with the same de novo heterozygous KCNQ2 c.593G>A, p.Arg198Gln (R198Q) variant. All were born at term and discharged home without seizures or concern of encephalopathy, but developed infantile spasms with hypsarrhythmia (or modified hypsarrhythmia) between the ages of 4 and 6 months. At last follow-up (ages 3-11 years), all patients were seizure-free and had severe developmental delay. In vitro experiments showed that Kv7.2 R198Q subunits shifted current activation gating to hyperpolarized potentials, indicative of gain-of-function; in neurons, Kv 7.2 and Kv 7.2 R198Q subunits similarly populated the axon initial segment, suggesting that gating changes rather than altered subcellular distribution contribute to disease molecular pathogenesis. We conclude that KCNQ2 R198Q is a model for a new subclass of KCNQ2 variants causing infantile spasms and encephalopathy, without preceding neonatal seizures. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.
Assuntos
Encefalopatias/genética , Canal de Potássio KCNQ2/genética , Mutação/genética , Espasmos Infantis/genética , Animais , Arginina/genética , Células CHO , Células Cultivadas , Criança , Pré-Escolar , Cricetulus , Glutamina/genética , Hipocampo/citologia , Humanos , Lactente , Estudos Longitudinais , Potenciais da Membrana/genética , Modelos Moleculares , Neurônios/fisiologia , Ratos , TransfecçãoRESUMO
OBJECTIVE: To analyze whether KCNQ2 R201C and R201H variants, which show atypical gain-of-function electrophysiologic properties in vitro, have a distinct clinical presentation and outcome. METHODS: Ten children with heterozygous, de novo KCNQ2 R201C or R201H variants were identified worldwide, using an institutional review board (IRB)-approved KCNQ2 patient registry and database. We reviewed medical records and, where possible, interviewed parents and treating physicians using a structured, detailed phenotype inventory focusing on the neonatal presentation and subsequent course. RESULTS: Nine patients had encephalopathy from birth and presented with prominent startle-like myoclonus, which could be triggered by sound or touch. In seven patients, electroencephalography (EEG) was performed in the neonatal period and showed a burst-suppression pattern. However, myoclonus did not have an EEG correlate. In many patients the paroxysmal movements were misdiagnosed as seizures. Seven patients developed epileptic spasms in infancy. In all patients, EEG showed a slow background and multifocal epileptiform discharges later in life. Other prominent features included respiratory dysfunction (perinatal respiratory failure and/or chronic hypoventilation), hypomyelination, reduced brain volume, and profound developmental delay. One patient had a later onset, and sequencing indicated that a low abundance (~20%) R201C variant had arisen by postzygotic mosaicism. SIGNIFICANCE: Heterozygous KCNQ2 R201C and R201H gain-of-function variants present with profound neonatal encephalopathy in the absence of neonatal seizures. Neonates present with nonepileptic myoclonus that is often misdiagnosed and treated as seizures. Prognosis is poor. This clinical presentation is distinct from the phenotype associated with loss-of-function variants, supporting the value of in vitro functional screening. These findings suggest that gain-of-function and loss-of-function variants need different targeted therapeutic approaches.
Assuntos
Canal de Potássio KCNQ2/genética , Mioclonia/genética , Polimorfismo de Nucleotídeo Único/genética , Espasmos Infantis/genética , Anticonvulsivantes/uso terapêutico , Arginina/genética , Pré-Escolar , Cisteína/genética , Eletroencefalografia , Feminino , Histidina/genética , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Mioclonia/diagnóstico por imagem , Mioclonia/tratamento farmacológico , Mioclonia/fisiopatologia , Fenótipo , Sistema de Registros , Transtornos Respiratórios/etiologia , Transtornos Respiratórios/genéticaRESUMO
The KCNT1 gene encodes for subunits contributing to the Na(+)-activated K(+) current (KNa), expressed in many cell types. Mutations in KCNT1 have been found in patients affected with a wide spectrum of early-onset epilepsies, including Malignant Migrating Partial Seizures in Infancy (MMPSI), a severe early-onset epileptic encephalopathy characterized by pharmacoresistant focal seizures migrating from one brain region or hemisphere to another and neurodevelopment arrest or regression, resulting in profound disability. In the present study we report identification by whole exome sequencing (WES) of two de novo, heterozygous KCNT1 mutations (G288S and, not previously reported, M516V) in two unrelated MMPSI probands. Functional studies in a heterologous expression system revealed that channels formed by mutant KCNT1 subunits carried larger currents when compared to wild-type KCNT1 channels, both as homo- and heteromers with these last. Both mutations induced a marked leftward shift in homomeric channel activation gating. Interestingly, the KCNT1 blockers quinidine (3-1000µM) and bepridil (0.03-10µM) inhibited both wild-type and mutant KCNT1 currents in a concentration-dependent manner, with mutant channels showing higher sensitivity to blockade. This latter result suggests two genotype-tailored pharmacological strategies to specifically counteract the dysfunction of KCNT1 activating mutations in MMPSI patients.