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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 KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

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. METHODS: Whole cell patch clamp and dynamic action potential clamp were used to study T226M Nav 1.1 channels expressed in mammalian cells. Computational modeling was used to explore the neuronal scale mechanisms that account for altered action potential firing. RESULTS: T226M channels exhibited hyperpolarizing shifts of the activation and inactivation curves and enhanced fast inactivation. Dynamic action potential clamp hybrid simulation showed that model neurons containing T226M conductance displayed a left shift in rheobase relative to control. At current stimulation levels that produced repetitive action potential firing in control model neurons, depolarization block and cessation of action potential firing occurred in T226M model neurons. Fully computationally simulated neuron models recapitulated the findings from dynamic action potential clamp and showed that heterozygous T226M models were also more susceptible to depolarization block. INTERPRETATION: From a biophysical perspective, the T226M mutation produces gain of function. Somewhat paradoxically, our data suggest that this gain of function would cause interneurons to more readily develop depolarization block. This "functional dominant negative" interaction would produce a more profound disinhibition than seen with haploinsufficiency that is typical of Dravet syndrome and could readily explain the more severe phenotype of patients with T226M mutation. Ann Neurol 2019;85:514-525.

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 SLC2A1, causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function. To test this, we measured glucose transport by GLUT1 variants identified in population controls and patients with mild to severe epilepsies. Controls were reference sequence from the NCBI and 4 population missense variants chosen from public reference control databases. Nine variants associated with epilepsies or movement disorders, with normal intellect in all individuals, formed the mild group. The severe group included 5 missense variants associated with classical GLUT1 encephalopathy. GLUT1 variants were expressed in Xenopus laevis oocytes, and glucose uptake was measured to determine kinetics (Vmax) and affinity (Km). RESULTS: Disease severity inversely correlated with rate of glucose transport between control (Vmax = 28 ± 5), mild (Vmax = 16 ± 3), and severe (Vmax = 3 ± 1) groups, respectively. Affinities of glucose binding in control (Km = 55 ± 18) and mild (Km = 43 ± 10) groups were not significantly different, whereas affinity was indeterminate in the severe group because of low transport rates. Simplified analysis of glucose transport at high concentration (100 mM) was equally effective at separating the groups. CONCLUSIONS: Disease severity can be partly explained by the extent of GLUT1 dysfunction. This simple Xenopus oocyte assay complements genetic and clinical assessments. In prenatal diagnosis, this simple oocyte glucose uptake assay could be useful because standard clinical assessments are not available.

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. Electroclinical, neuroimaging, and genetic data were reviewed. Two patients had novel variants in the KCNT1 gene that were modeled in Xenopus oocytes with channel properties characterized using electrophysiology recordings. RESULTS: Three of four patients were treated with quinidine early in their disease course, prior to 6 months of age. No significant side effects were noted with quinidine therapy. In addition, there were no significant changes in electroencephalography (EEG)-confirmed seizure burden during therapy, and patients had near hourly seizures before, during, and after treatment. Two patients had previously reported gain-of-function mutations, which demonstrated sensitivity to high levels of quinidine in the oocyte assay. Two patients with novel variants, showed characteristic gain-of-function and were thus predicted to be pathogenic. Of interest, these variants were essentially insensitive to high levels of quinidine. SIGNIFICANCE: Patients had no reported benefit to quinidine therapy despite age at treatment initiation. Pharmacogenetic results in oocytes were consistent with clinical treatment failure in 2 patients, suggesting that single-dose pharmacologic assessment may be helpful in predicting which patients are exceedingly unlikely to achieve benefit with quinidine. In the 2 patients who had a lack of therapeutic benefit despite sensitivity to high concentrations of quinidine with in vitro oocyte assay, it is likely that the achievable exposu

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8a(V929F)). Here, we confirmed the prior suggestion that Scn8a(V929F) exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8a(V929F) is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2(V752F) as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2(V752F) functional analysis revealed an overall gain-of-function and for NaV1.6(V929F) revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2(V752F) due to "functional silencing" of NaV1.6(V929F) suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6(V929F) loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as a