Antisense oligonucleotide therapy for KCNT1 encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.

SCN1A gain of function in early infantile encephalopathy.

Objective: To elucidate the biophysical basis underlying the distinct and severe clinical presentation in patients with the recurrent missense SCN1A variant, p.Thr226Met. Patients with this variant show a well-defined genotype-phenotype correlation and present with developmental and early infantile epileptic encephalopathy that is far more severe than typical SCN1A Dravet syndrome.

Development of a rapid functional assay that predicts GLUT1 disease severity.

Objective: To examine the genotype to phenotype connection in glucose transporter type 1 (GLUT1) deficiency and whether a simple functional assay can predict disease outcome from genetic sequence alone.

Methods: GLUT1 deficiency, due to mutations in , causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function.

Lack of response to quinidine in KCNT1-related neonatal epilepsy.

Objective: To evaluate the clinical efficacy and safety of quinidine in patients with KCNT1-related epilepsy of infancy with migrating focal seizures (EIMFS) in the infantile period and to compare with the effect of quinidine on mutant channels in vitro.

Methods: We identified 4 patients with EIMFS with onset in the neonatal period, pathogenic variants in the KCNT1 gene, and lack of response to AEDs. Patients were prospectively enrolled, treated with quinidine, and monitored according to a predefined protocol.

Clinical and molecular characterization of -related severe early-onset epilepsy.

Objective: To characterize the phenotypic spectrum, molecular genetic findings, and functional consequences of pathogenic variants in early-onset epilepsy.

Methods: We identified a cohort of 31 patients with epilepsy of infancy with migrating focal seizures (EIMFS) and screened for variants in using direct Sanger sequencing, a multiple-gene next-generation sequencing panel, and whole-exome sequencing. Additional patients with non-EIMFS early-onset epilepsy in whom we identified variants on local diagnostic multiple gene panel testing were also included.

Method of derivation and differentiation of mouse embryonic stem cells generating synchronous neuronal networks.

Background: Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties.

Electrophysiological and gene expression characterization of the ontogeny of nestin-expressing cells in the adult mouse midbrain.

The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin+neural precursor cells (NPCs) have a propensity to generate new neurons here.

Rare and common epilepsies converge on a shared gene regulatory network providing opportunities for novel antiepileptic drug discovery.

Background: The relationship between monogenic and polygenic forms of epilepsy is poorly understood and the extent to which the genetic and acquired epilepsies share common pathways is unclear. Here, we use an integrated systems-level analysis of brain gene expression data to identify molecular networks disrupted in epilepsy.

Results: We identified a co-expression network of 320 genes (M30), which is significantly enriched for non-synonymous de novo mutations ascertained from patients with monogenic epilepsy and for common variants associated with polygenic epilepsy.

Dominant KCNA2 mutation causes episodic ataxia and pharmacoresponsive epilepsy.

Objective: To identify the genetic basis of a family segregating episodic ataxia, infantile seizures, and heterogeneous epilepsies and to study the phenotypic spectrum of KCNA2 mutations.

Methods: A family with 7 affected individuals over 3 generations underwent detailed phenotyping. Whole genome sequencing was performed on a mildly affected grandmother and her grandson with epileptic encephalopathy (EE).

A targeted resequencing gene panel for focal epilepsy.

Objectives: We report development of a targeted resequencing gene panel for focal epilepsy, the most prevalent phenotypic group of the epilepsies.

Methods: The targeted resequencing gene panel was designed using molecular inversion probe (MIP) capture technology and sequenced using massively parallel Illumina sequencing.

Results: We demonstrated proof of principle that mutations can be detected in 4 previously genotyped focal epilepsy cases.

PRIMA1 mutation: a new cause of nocturnal frontal lobe epilepsy.

Objective: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene.

Methods: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation.

'Neonatal' Nav1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour.

Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.

GABRA1 and STXBP1: novel genetic causes of Dravet syndrome.

Objective: To determine the genes underlying Dravet syndrome in patients who do not have an SCN1A mutation on routine testing.

Methods: We performed whole-exome sequencing in 13 SCN1A-negative patients with Dravet syndrome and targeted resequencing in 67 additional patients to identify new genes for this disorder.

Results: We detected disease-causing mutations in 2 novel genes for Dravet syndrome, with mutations in GABRA1 in 4 cases and STXBP1 in 3.

KCNT1 gain of function in 2 epilepsy phenotypes is reversed by quinidine.

Objective: Mutations in KCNT1 have been implicated in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and epilepsy of infancy with migrating focal seizures (EIMFS). More recently, a whole exome sequencing study of epileptic encephalopathies identified an additional de novo mutation in 1 proband with EIMFS. We aim to investigate the electrophysiological and pharmacological characteristics of hKCNT1 mutations and examine developmental expression levels.

Computational Analysis of Amiloride Analogue Inhibitors of B3 RNA Polymerase.

(CVB3) is a picornavirus that is responsible for a significant proportion of human myocarditis. However, no antiviral treatment is currently available to treat this disease or indeed any picornaviral infections. Previously it was shown that amiloride and its derivative 5-(-ethyl--isopropyl)amiloride inhibit the enzymatic activity of CVB3 RNA polymerase (3D).

Viral targets of acylguanidines.

Acylguanidines are a new class of antiviral compounds with the unique ability to target both RNA polymerase and transmembrane proteins of viruses from different families. Importantly, they inhibit proteins which are not targeted by existing antiviral therapies, for example, Vpu of HIV type 1, p7 of hepatitis C virus, E of severe acute respiratory syndrome coronavirus and RNA-dependent RNA polymerase of coxsackievirus B3. BIT225, developed by Biotron Limited, is the first acylguanidine in clinical trials against HIV type 1 and hepatitis C virus.

Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase.

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E.

Augmented currents of an HCN2 variant in patients with febrile seizure syndromes.

The genetic architecture of common epilepsies is largely unknown. HCNs are excellent epilepsy candidate genes because of their fundamental neurophysiological roles. Screening in subjects with febrile seizures and genetic epilepsy with febrile seizures plus revealed that 2.

Heat opens axon initial segment sodium channels: a febrile seizure mechanism?

Objective: A number of hypotheses have been put forward as to why humans respond to fever by seizing. The current leading hypotheses are that respiratory alkalosis produces an as yet unidentified change in neural excitability or that inflammatory mediators potentiate excitatory synaptic transmission. However, it is well known that ion channel gating rates increase with increased temperature.

Amiloride derivatives inhibit coxsackievirus B3 RNA replication.

Amiloride derivatives are known blockers of the cellular Na(+)/H(+) exchanger and the epithelial Na(+) channel. More recent studies demonstrate that they also inhibit ion channels formed by a number of viral proteins. We previously reported that 5-(N-ethyl-N-isopropyl)amiloride (EIPA) modestly inhibits intracellular replication and, to a larger extent, release of human rhinovirus 2 (HRV2) (E.

A childhood epilepsy mutation reveals a role for developmentally regulated splicing of a sodium channel.

Seizure susceptibility is high in human infants compared to adults, presumably because of developmentally regulated changes in neural excitability. Benign familial neonatal-infantile seizures (BFNIS), characterized by both early onset and remission, are caused by mutations in the gene encoding a human sodium channel (NaV1.2).

Ion transport blockers inhibit human rhinovirus 2 release.

Picornavirus replication causes leakage of cytoplasmic K+ and an influx of Na+ and Ca2+. In this study, we have explored the possibility that a blockade of Ca2+ and Na+ influx would reduce rhinovirus production and/or release. The Ca2+-channel blockers, verapamil and diltiazem, as well as the blocker of Na+/H+ exchange and the epithelial Na+ channel, EIPA, inhibited both virus production and release.

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae.

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited.