RESUMEN
OBJECTIVE: Mutations in KCNQ2 and KCNQ3, encoding the voltage-gated potassium channels KV 7.2 and KV 7.3, are known to cause benign familial neonatal seizures mainly by haploinsufficiency. Here, we set out to determine the disease mechanism of 7 de novo missense KCNQ2 mutations that were recently described in patients with a severe epileptic encephalopathy including pharmacoresistant seizures and pronounced intellectual disability. METHODS: Mutations were inserted into the KCNQ2 cDNA. Potassium currents were recorded using 2-microelectrode voltage clamping, and surface expression was analyzed by a biotinylation assay in cRNA-injected Xenopus laevis oocytes. RESULTS: We observed a clear loss of function for all mutations. Strikingly, 5 of 7 mutations exhibited a drastic dominant-negative effect on wild-type KV 7.2 or KV 7.3 subunits, either by globally reducing current amplitudes (3 pore mutations) or by a depolarizing shift of the activation curve (2 voltage sensor mutations) decreasing potassium currents at the subthreshold level at which these channels are known to critically influence neuronal firing. One mutation significantly reduced surface expression. Application of retigabine, a recently marketed KV 7 channel opener, partially reversed these effects for the majority of analyzed mutations. INTERPRETATION: The development of severe epilepsy and cognitive decline in children carrying 5 of the 7 studied KCNQ2 mutations can be related to a dominant-negative reduction of the resulting potassium current at subthreshold membrane potentials. Other factors such as genetic modifiers have to be postulated for the remaining 2 mutations. Retigabine or similar drugs may be used as a personalized therapy for this severe disease.
Asunto(s)
Epilepsia Benigna Neonatal/genética , Predisposición Genética a la Enfermedad/genética , Canal de Potasio KCNQ2/genética , Canales de Potasio con Entrada de Voltaje/genética , Animales , Carbamatos/farmacología , Epilepsia Benigna Neonatal/fisiopatología , Humanos , Canal de Potasio KCNQ2/efectos de los fármacos , Canal de Potasio KCNQ2/fisiología , Potenciales de la Membrana/genética , Mutación Missense , Oocitos , Fenilendiaminas/farmacología , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/fisiología , XenopusRESUMEN
The STX1B gene encodes the presynaptic protein syntaxin-1B, which plays a major role in regulating fusion of synaptic vesicles. Mutations in STX1B are known to cause epilepsy syndromes, such as genetic epilepsies with febrile seizures plus (GEFS+). Here, we reprogrammed skin fibroblasts from a female patient affected by GEFS+ to human induced pluripotent stem cells (iPSCs). The patient carries an InDel mutation (c.133_134insGGATGTGCATTG; p.Lys45delinsArgMetCysIleGlu and c.135_136AC > GA; p.Leu46Met), located in the regulatory Habc-domain of STX1B. Successful reprogramming of cells was confirmed by a normal karyotype, expression of several pluripotency markers and the potential to differentiate into all three germ layers.
Asunto(s)
Epilepsia , Células Madre Pluripotentes Inducidas , Humanos , Femenino , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Epilepsia/genética , Mutación INDEL , Fibroblastos , Diferenciación Celular , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
De novo mutations in the KCNA2 gene, encoding the voltage-gated potassium channel KV1.2, have been identified to cause early-onset developmental and epileptic encephalopathies (DEE). KV1.2 channels conduct delayed-rectifier type K+ currents and play a crucial role in action potential repolarization. In this study we reprogrammed fibroblasts from a 6-months-old male patient with DEE carrying a de novo point mutation (c.1120Aâ¯>â¯G, p.Thr374Ala) in KCNA2 to induced pluripotent stem cells. Their pluripotency was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.
Asunto(s)
Epilepsia/patología , Células Madre Pluripotentes Inducidas/citología , Canal de Potasio Kv.1.2/genética , Diferenciación Celular , Línea Celular , Reprogramación Celular , Epilepsia/genética , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
Developmental and epileptic encephalopathies (DEE) can be caused by mutations in the KCNA2 gene, coding for the voltage-gated K+ channel Kv1.2. This ion channel belongs to the delayed rectifier class of potassium channels and plays a role during the repolarization phase of an action potential. In this study we reprogrammed fibroblasts from a 30-year-old male patient with DDE carrying a point mutation (c.890Gâ¯>â¯A, p.Arg297Gln) in KCNA2 to induced pluripotent stem cells. Pluripotency state of the cells was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.
Asunto(s)
Diferenciación Celular , Epilepsia/genética , Fibroblastos/patología , Células Madre Pluripotentes Inducidas/patología , Canal de Potasio Kv.1.2/genética , Mutación , Trastornos del Neurodesarrollo/genética , Adulto , Células Cultivadas , Reprogramación Celular , Epilepsia/patología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Trastornos del Neurodesarrollo/patología , FenotipoRESUMEN
Most of our knowledge on human CNS circuitry and related disorders originates from model organisms. How well such data translate to the human CNS remains largely to be determined. Human brain slice cultures derived from neurosurgical resections may offer novel avenues to approach this translational gap. We now demonstrate robust preservation of the complex neuronal cytoarchitecture and electrophysiological properties of human pyramidal neurons in long-term brain slice cultures. Further experiments delineate the optimal conditions for efficient viral transduction of cultures, enabling 'high throughput' fluorescence-mediated 3D reconstruction of genetically targeted neurons at comparable quality to state-of-the-art biocytin fillings, and demonstrate feasibility of long term live cell imaging of human cells in vitro. This model system has implications toward a broad spectrum of translational studies, regarding the validation of data obtained in non-human model systems, for therapeutic screening and genetic dissection of human CNS circuitry.
Asunto(s)
Encéfalo/anatomía & histología , Encéfalo/fisiología , Técnicas de Cultivo de Órganos/métodos , Adulto , Encéfalo/patología , Encéfalo/fisiopatología , Humanos , Microscopía Intravital/métodos , Células Piramidales/patología , Células Piramidales/fisiologíaRESUMEN
Mutations in the KCNA2 gene, coding for the voltage-gated K+ channel Kv1.2, can cause developmental and epileptic encephalopathies. Kv1.2 channels play an important role in the repolarization phase of an action potential in nerve cells. Here, we reprogrammed human skin fibroblasts from a 13-year-old male patient with developmental and epileptic encephalopathy carrying a point mutation (c.982T>G, p.Leu328Val) in KCNA2 to human induced pluripotent stem cells (iPSCs) (HIHDNEi001-A). The cells maintained a normal karyotype and their pluripotency state was verified by the expression and staining of several pluripotency markers and capability to differentiate into all three germ layers.