De Novo Loss-of-Function Mutations in CHD2 Cause a Fever-Sensitive Myoclonic Epileptic Encephalopathy Sharing Features with Dravet Syndrome

Variants of the SCN1A gene encoding the neuronal voltage-gated sodium channel NaV1.1 cause over 85% of all cases of Dravet syndrome, a severe and often pharmacoresistent epileptic encephalopathy with mostly infantile onset. But with the increased availability of genetic testing for patients with epilepsy, variants in SCN1A have now also been described in a range of other epilepsy phenotypes. The vast majority of these epilepsy-associated variants are de novo, and most are either nonsense variants that truncate the channel or missense variants that are presumed to cause loss of channel function. However, biophysical analysis has revealed a significant subset of missense mutations that result in increased excitability, further complicating approaches to precision pharmacotherapy for patients with SCN1A variants and epilepsy. We describe clinical and biophysical data of a familial SCN1A variant encoding the NaV1.1 L1624Q mutant. This substitution is located on the extracellular linker between S3 and S4 of Domain IV of NaV1.1 and is a rare case of a familial SCN1A variant causing an autosomal dominant frontal lobe epilepsy. We expressed wild-type (WT) and L1642Q channels in CHO cells. Using patch-clamp to characterize channel properties at several temperatures, we show that the L1624Q variant increases persistent current, accelerates fast inactivation onset and decreases current density. While SCN1A-associated epilepsy is typically considered a loss-of-function disease, our results put L1624Q into a growing set of mixed gain and loss-of-function variants in SCN1A responsible for epilepsy.

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Dravet syndrome associated mutations in GABRA1, GABRB2 and GABRG2 define the genetic landscape of defects of GABAA receptors

Brain Communications ◽

10.1093/braincomms/fcab033 ◽

2021 ◽

Author(s):

Ciria C Hernandez ◽

XiaoJuan Tian ◽

Ningning Hu ◽

Wangzhen Shen ◽

Mackenzie A Catron ◽

...

Keyword(s):

Gabaa Receptor ◽

De Novo ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

First Year ◽

Genes Encoding ◽

Genetic Landscape ◽

Clonic Seizures ◽

Trafficking Defects ◽

First Year Of Life

Abstract Dravet syndrome is a rare, catastrophic epileptic encephalopathy that begins in the first year of life, usually with febrile or afebrile hemiclonic or generalized tonic-clonic seizures followed by status epilepticus. De novo variants in genes that mediate synaptic transmission such as SCN1A and PCDH19 are often associated with Dravet syndrome. Recently, GABAA receptor subunit genes (GABRs) encoding α1 (GABRA1), β3 (GABRB3) and γ2 (GABRG2), but not β2 (GABRB2) or β1 (GABRB1), subunits are frequently associated with Dravet syndrome or Dravet syndrome-like phenotype. We performed next generation sequencing on 870 patients with Dravet syndrome and identified nine variants in three different GABRs. Interestingly, the variants were all in genes encoding the most common GABAA receptor, the α1β2γ2 receptor. Mutations in GABRA1 (c.644T>C, p.L215P; c.640C>T, p.R214C; c.859G>A; V287I; c.641G>A, p.R214H) and GABRG2 (c.269C>G, p.T90R; c.1025C>T, p.P342L) presented as de novo cases, while in GABRB2 two variants were de novo (c.992T>C, p.F331S; c.542A>T, p.Y181F) and one was autosomal dominant and inherited from the maternal side (c.990_992del, p.330_331del). We characterized the effects of these GABR variants on GABAA receptor biogenesis and channel function. We found that defects in receptor gating were the common deficiency of GABRA1 and GABRB2 Dravet syndrome variants, while mainly trafficking defects were found with the GABRG2 (c.269C>G, p.T90R) variant. It seems that variants in α1 and β2 subunits are less tolerated than in γ2 subunits, since variant α1 and β2 subunits express well but were functionally deficient. This suggests that all of these GABR variants are all targeting GABR genes that encode the assembled α1β2γ2 receptor, and regardless of which of the three subunits are mutated, variants in genes coding for α1, β2 and γ2 receptor subunits make them candidate causative genes in the pathogenesis of Dravet syndrome.

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Dravet Variant SCN1AA1783V Impairs Interneuron Firing Predominantly by Altered Channel Activation

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.754530 ◽

2021 ◽

Vol 15 ◽

Author(s):

Nikolas Layer ◽

Lukas Sonnenberg ◽

Emilio Pardo González ◽

Jan Benda ◽

Ulrike B. S. Hedrich ◽

...

Keyword(s):

Current Density ◽

Input Resistance ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

Slow Inactivation ◽

Loss Of Function ◽

Peak Current Density ◽

Channel Activation ◽

Peak Current

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in inhibitory interneurons. Recently, a new conditional mouse model expressing the recurrent human p.(Ala1783Val) missense variant has become available. In this study, we provided an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Based on these data, simulated interneuron (IN) firing properties in a conductance-based single-compartment model suggested surprisingly similar firing deficits for NaV1.1A1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaV1.1A1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.Ala1783V in vitro confirmed a predicted IN firing deficit and revealed an accompanying reduction of interneuronal input resistance while demonstrating normal excitability of pyramidal neurons. Altered input resistance was fed back into the model for further refinement. Taken together these data demonstrate that primary loss of function (LOF) gating properties accompanied by altered membrane characteristics may match effects of full haploinsufficiency on the neuronal level despite maintaining physiological peak current density, thereby causing DS.

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Channelopathy of Dravet Syndrome and Potential Neuroprotective Effects of Cannabidiol

Journal of Central Nervous System Disease ◽

10.1177/11795735211048045 ◽

2021 ◽

Vol 13 ◽

pp. 117957352110480

Author(s):

Changqing Xu ◽

Yumin Zhang ◽

David Gozal ◽

Paul Carney

Keyword(s):

Gene Mutations ◽

Premature Death ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

Hcn Channels ◽

Loss Of Function ◽

Action Mechanism ◽

Neuroprotective Effects ◽

Thermally Induced ◽

Spontaneous Seizures

Dravet syndrome (DS) is a channelopathy, neurodevelopmental, epileptic encephalopathy characterized by seizures, developmental delay, and cognitive impairment that includes susceptibility to thermally induced seizures, spontaneous seizures, ataxia, circadian rhythm and sleep disorders, autistic-like behaviors, and premature death. More than 80% of DS cases are linked to mutations in genes which encode voltage-gated sodium channel subunits, SCN1A and SCN1B, which encode the Nav1.1α subunit and Nav1.1β1 subunit, respectively. There are other gene mutations encoding potassium, calcium, and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels related to DS. One-third of patients have pharmacoresistance epilepsy. DS is unresponsive to standard therapy. Cannabidiol (CBD), a non-psychoactive phytocannabinoid present in Cannabis, has been introduced for treating DS because of its anticonvulsant properties in animal models and humans, especially in pharmacoresistant patients. However, the etiological channelopathiological mechanism of DS and action mechanism of CBD on the channels are unclear. In this review, we summarize evidence of the direct and indirect action mechanism of sodium, potassium, calcium, and HCN channels in DS, especially sodium subunits. Some channels’ loss-of-function or gain-of-function in inhibitory or excitatory neurons determine the balance of excitatory and inhibitory are associated with DS. A great variety of mechanisms of CBD anticonvulsant effects are focused on modulating these channels, especially sodium, calcium, and potassium channels, which will shed light on ionic channelopathy of DS and the precise molecular treatment of DS in the future.

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Loss–of–function variants in the KCNQ5 gene are associated with genetic generalized epilepsies

10.1101/2021.04.20.21255696 ◽

2021 ◽

Author(s):

Johanna Krueger ◽

Julian Schubert ◽

Josua Kegele ◽

Audrey Labalme ◽

Miaomiao Mao ◽

...

Keyword(s):

Mammalian Cells ◽

De Novo ◽

Surface Expression ◽

Epileptic Encephalopathy ◽

Dominant Negative ◽

Dominant Negative Effect ◽

K Channel ◽

Loss Of Function ◽

Normal Surface ◽

Missense Variants

Objective: De novo missense variants in KCNQ5, encoding the voltage–gated K+ channel KV7.5, have been described as a cause of developmental and epileptic encephalopathy (DEE) or intellectual disability (ID). We set out to identify disease–related KCNQ5 variants in genetic generalized epilepsy (GGE) and their underlying mechanisms. Methods: 1292 families with GGE were studied by next-generation sequencing. Whole–cell patch–clamp recordings, biotinylation and phospholipid overlay assays were performed in mammalian cells combined with docking and homology modeling. Results: We identified three deleterious heterozygous missense variants, one truncation and one splice site alteration in five independent families with GGE with predominant absence seizures, two variants were also associated with mild to moderate ID. All three missense variants displayed a strongly decreased current density indicating a loss–of–function (LOF). When mutant channels were co–expressed with wild–type (WT) KV7.5 or KV7.5 and KV7.3 channels, three variants also revealed a significant dominant–negative effect on WT channels. Other gating parameters were unchanged. Biotinylation assays indicated a normal surface expression of the variants. The p.Arg359Cys variant altered PI(4,5)P2–interaction, presumably in the non–conducting preopen–closed state. Interpretation: Our study indicates that specific deleterious KCNQ5 variants are associated with GGE, partially combined with mild to moderate ID. The disease mechanism is a LOF partially with dominant–negative effects through functional, rather than trafficking deficits. LOF of KV7.5 channels will reduce the M–current, likely resulting in increased excitability of KV7.5–expressing neurons. Further studies on a network level are necessary to understand which circuits are affected and how the variants induce generalized seizures.

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Astrocyte Ca2+ Signaling is Facilitated in an Scn1a+/− Mouse Model of Dravet Syndrome

10.1101/2021.05.18.444602 ◽

2021 ◽

Author(s):

Kouya Uchino ◽

Wakana Ikezawa ◽

Yasuyoshi Tanaka ◽

Masanobu Deshimaru ◽

Kaori Kubota ◽

...

Keyword(s):

Mouse Model ◽

Sodium Channel ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

Heterozygous Mutation ◽

Loss Of Function ◽

Cell Type ◽

Voltage Gated ◽

A Subunit ◽

The Brain

Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A, which encodes a subunit of the voltage-gated sodium channel, Nav1.1, in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a. We found that the slope of spontaneous Ca2+ spiking was increased without a change in amplitude in Scn1a+/− astrocytes. In addition, ATP-induced transient Ca2+ influx and the slope of Ca2+ spiking were also increased in Scn1a+/− astrocytes. These data indicate that perturbed Ca2+ dynamics in astrocytes may be involved in the pathogenesis of DS.

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NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype in a novel zebrafish model for Dravet Syndrome

10.1101/675082 ◽

2019 ◽

Author(s):

Wout J. Weuring ◽

Sakshi Singh ◽

Linda Volkers ◽

Martin Rook ◽

Ruben H. van ‘t Slot ◽

...

Keyword(s):

Treatment Strategies ◽

Epileptic Seizures ◽

Selective Inhibition ◽

Epileptic Encephalopathy ◽

Model Systems ◽

Dravet Syndrome ◽

Loss Of Function ◽

Zebrafish Model ◽

Neuronal Inhibition ◽

Increased Sensitivity

AbstractDravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous knock-down models. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the convulsant pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and our newly synthesized NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of burst movements. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems, preferably human derived.

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Antisense oligonucleotides increase Scn1a expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome

Science Translational Medicine ◽

10.1126/scitranslmed.aaz6100 ◽

2020 ◽

Vol 12 (558) ◽

pp. eaaz6100 ◽

Cited By ~ 6

Author(s):

Zhou Han ◽

Chunling Chen ◽

Anne Christiansen ◽

Sophina Ji ◽

Qian Lin ◽

...

Keyword(s):

Mouse Model ◽

Antisense Oligonucleotides ◽

De Novo ◽

Nuclear Gene ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

Unexpected Death ◽

Α Subunit ◽

Gene And Protein Expression ◽

Electrographic Seizures

Dravet syndrome (DS) is an intractable developmental and epileptic encephalopathy caused largely by de novo variants in the SCN1A gene, resulting in haploinsufficiency of the voltage-gated sodium channel α subunit NaV1.1. Here, we used Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, nonproductive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human cell lines, as well as in mouse brain. We show that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy (SUDEP) in the F1:129S-Scn1a+/− × C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and NaV1.1 protein was confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS.

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Changing Landscape of Dravet Syndrome Management: An Overview

Neuropediatrics ◽

10.1055/s-0040-1701694 ◽

2020 ◽

Vol 51 (02) ◽

pp. 135-145 ◽

Cited By ~ 1

Author(s):

Debopam Samanta

Keyword(s):

Motor Impairment ◽

Current Treatment ◽

Epileptic Encephalopathy ◽

Dravet Syndrome ◽

Loss Of Function ◽

Unexpected Death ◽

Autistic Behavior ◽

Behavioral Abnormalities ◽

Treatment Paradigm ◽

Antisense Oligonucleotide Therapy

AbstractDravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is a severe developmental and epileptic encephalopathy caused by loss-of-function mutations in one copy of SCN1A (haploinsufficiency), located on chromosome 2q24, with decreased function of Nav1.1 sodium channels in GABAergic inhibitory interneurons. Pharmacoresistant seizures in DS start in the infancy in the form of hemiclonic febrile status epilepticus. Later, other intractable seizure types develop including myoclonic seizures. Early normal development in infancy evolves into moderate to severe intellectual impairment, motor impairment, behavioral abnormalities, and later a characteristic crouching gait. Clobazam, valproate, levetiracetam, topiramate, zonisamide, ketogenic diet, and vagus nerve stimulation had been shown to be effective, but even with polytherapy, only 10% of patients get adequate seizure control. The author provides a narrative review of the current treatment paradigm as well as recent advances in the management of DS based on a comprehensive literature review (MEDLINE using PubMed and OvidSP vendors with appropriate keywords to incorporate recent evidence), personal practice, and experience. In recent years, the treatment paradigm of DS is changing with the approval of pharmaceutical-grade cannabidiol oil and stiripentol. Another novel antiepileptic drug (AED), fenfluramine, had also shown excellent efficacy in phase 3 studies of DS. However, these AEDs primarily control seizures without addressing the underlying pathogenesis and other important common comorbidities such as cognitive impairment, autistic behavior, neuropsychiatric abnormalities, and motor impairment including crouching gait. Several agents targeted for DS are in the developmental stage: TAK935, lorcaserin, clemizole, huperzine analog, ataluren, selective sodium channel modulators and activators, antisense oligonucleotide therapy, and adenoviral vector therapy. As DS is associated with a high risk of sudden unexpected death in epilepsy, seizure detection devices can be used in this population for testing and clinical validation of these devices.

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Biallelic Mutations in SLC1A2; an Additional Mode of Inheritance for SLC1A2-Related Epilepsy

Neuropediatrics ◽

10.1055/s-0037-1606370 ◽

2017 ◽

Vol 49 (01) ◽

pp. 059-062 ◽

Cited By ~ 6

Author(s):

Mirjana Gusic ◽

Roman Günthner ◽

Bader Alhaddad ◽

Reka Kovacs-Nagy ◽

Christine Makowski ◽

...

Keyword(s):

De Novo ◽

Epileptic Seizures ◽

Epileptic Encephalopathy ◽

Genetic Mechanism ◽

Loss Of Function ◽

De Novo Mutations ◽

Functional Studies ◽

Additional Mode ◽

Biallelic Mutations ◽

Mode Of Inheritance

AbstractRecently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2-related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism.

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