Persistent sodium currents in SCN1A developmental and degenerative epileptic dyskinetic encephalopathy

Abstract

Pathogenic variants in the voltage-gated sodium channel gene (SCN1A) are amongst the most common genetic causes of childhood epilepsies. There is considerable heterogeneity in both the types of causative variants and associated phenotypes; a recent expansion of the phenotypic spectrum of SCN1A associated epilepsies now includes an early onset severe developmental and epileptic encephalopathy with regression and a hyperkinetic movement disorder. Herein, we report a female with a developmental and degenerative epileptic-dyskinetic encephalopathy, distinct and more severe than classic Dravet syndrome. Clinical diagnostics indicated a paternally inherited c.5053G>T; p. A1685S variant of uncertain significance in SCN1A. Whole-exome sequencing detected a second de novo mosaic (18%) c.2345G>A; p. T782I likely pathogenic variant in SCN1A (maternal allele). Biophysical characterization of both mutant channels in a heterologous expression system identified gain-of-function effects in both, with a milder shift in fast inactivation of the p. A1685S channels; and a more severe persistent sodium current in the p. T782I. Using computational models, we show that large persistent sodium currents induce hyper-excitability in individual cortical neurons, thus relating the severe phenotype to the empirically quantified sodium channel dysfunction. These findings further broaden the phenotypic spectrum of SCN1A associated epilepsies and highlight the importance of testing for mosaicism in epileptic encephalopathies. Detailed biophysical evaluation and computational modelling further highlight the role of gain-of-function variants in the pathophysiology of the most severe phenotypes associated with SCN1A.

Keywords: Hodgkin–Huxley model; SCN1A; epileptic encephalopathy; non-inactivating current; sodium-channel gating.

Graphical Abstract

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Figure 1

EEG aged 15 months. (A) (Awake) and (B) (sleep): EEG (HF filter, 70 Hz, sensitivity, 20 mV/mm; timebase, 30 mm/s) showing a slow awake background (mainly theta activity) and infrequent sharp waves. In sleep, epileptiform sharp waves were activated in bilateral temporal regions.

Figure 2

EEG aged 23 months. (A) (Awake) and (B) (sleep): EEG (HF filter, 70 Hz, sensitivity, 30 mV/mm; timebase, 30 mm/s) showing an epileptic encephalopathy with slow-wave and spike 2.5 Hz epileptiform discharges. Activation of continuous generalized epileptiform discharges in sleep.

Figure 3

EEG aged 3 years. (A) (Atypical absence during awake state) and (B) (sleep): EEG (HF filter, 70 Hz, sensitivity, 30 mV/mm; timebase, 30 mm/s). Clinical event characterized by staring and cessation of hyperkinetic movements for 8 s with an EEG correlation of high-amplitude generalized regular 2Hz discharges. The epileptiform discharges become continuous in sleep.

Figure 4

T782I and A1685S mutants in Nav1.1 produce functional channels. (A,B) Macroscopic inward currents during depolarizing steps from a holding potential of −130 mV through WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 32°C and 37°C. (C) Conductance-voltage relationships for WT (black), T782I (red), A1685S (blue) and co-transfected T782I-A1685S (purple) Nav1.1 at 32°C. (D) Conductance-voltage relationships for WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 37°C using the same colour scheme as (C). (E) Average voltage of half-maximal conductance for WT (black; N = 12 at 32°C; N = 13 at 37°C), T782I (red; N = 10 at 32°C; N = 13 at 37°C), A1685S (blue; N = 9 at 32°C; N = 9 at 37°C) and co-transfected T782I-A1685S (purple; N = 10 at 32°C; N = 10 at 37°C) Nav1.1 at 32°C (open symbols) and 37°C (closed symbols). Individual observations in each condition are shown as smaller symbols. (F) Average peak current at 0 mV for WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 32°C and 37°C using the same colour scheme as (F). The number of observations in (F) are the same for each condition as in (F). Individual observations in each condition are shown as smaller symbols. Each midpoint measurement reflects a mean and each error bar reflects a standard error of the mean. Independent replicates are individual cells recorded in the whole-cell patch clamp configuration. * indicates a statistically significant difference between two midpoints. All statistical analysis was performed with a two-factor ANOVA followed by a Dunnett’s post hoc of pairwise comparisons to WT. Exact P-values and details of statistical tests are found in the main text. Number of individual replicates, means and standard error of the mean for all conditions are found in Table 2 and https://doi.org/10.17605/OSF.IO/YS5RG.

Figure 5

T782I and A1685S mutants in Nav1.1 minimally alter fast inactivation. (A) Fast inactivation voltage-dependence for WT (black, N = 11), T782I (red, N = 8), A1685S (blue, N = 8) and co-transfected T782I-A1685S (purple, N = 10) Nav1.1 at 32°C. (B) Fast inactivation voltage-dependence for WT (N = 12), T782I (N = 12), A1685S (N = 9) and co-transfected T782I-A1685S (N = 9) Nav1.1 at 37°C using the same colour scheme as (A). (C) Time course of fast inactivation recovery at −90 mV for WT (N = 11), T782I (N = 8), A1685S (N = 9) and co-transfected T782I-A1685S (N = 7) Nav1.1 at 32°C using the same colour scheme as (A). (D) Time course of fast inactivation recovery at −90 mV for WT (N = 11), T782I (N = 11), A1685S (N = 9) and co-transfected T782I-A1685S (N = 7) Nav1.1 at 37°C using the same colour scheme as (A). (E) Time constants of fast inactivation onset for WT (N = 11), T782I (N = 8), A1685S (N = 7) and co-transfected T782I-A1685S (N = 10) Nav1.1 at 32°C using the same colour scheme as (A). (F) Time constants of fast inactivation onset for WT (N = 13), T782I (N = 12), A1685S (N = 8) and co-transfected T782I-A1685S (N = 9) Nav1.1 at 37°C using the same colour scheme as (A). Each mid-point measurement represents a mean and each error bar represents a standard error of the mean. Independent replicates are individual cells recorded in the whole-cell patch clamp configuration. Number of individual replicates, means and standard error of the mean for all conditions are found in Table 2 and https://doi.org/10.17605/OSF.IO/YS5RG.

Figure 6

(A) Sample currents at −20 mV from WT (black), T782I (red), A1685S (blue) and co-transfected T782I-A1685S (purple) Nav1.1 at 32°C. (B) Sample non-inactivating currents at −20 mV in WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 32°C using the same colour scheme as (A). (C) Sample currents at −20 mV from WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 37°C using the same colour scheme as (A). (D) Sample non-inactivating currents at −20 mV in WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 37°C using the same colour scheme as (A). Note the A1685S cell shown had a larger peak current than the WT cell. E) Average amplitude of non-inactivating current at −20 mV for WT (N = 8 at 32°C; N = 7 at 37°C), T782I (N = 6 at 32°C; N = 7 at 37°C), A1685S (N = 4 at 32°C; N = 5 at 37°C) and co-transfected T782I-A1685S (N = 6 at 32°C; N = 6 at 37°C) Nav1.1 at 32°C (open symbols) and 37°C (closed symbols) using the same colour scheme as (A). Individual observations in each condition are shown as smaller symbols. (F) Average fraction of non-inactivating current at −20 mV normalized to peak current for WT, T782I, A1685S and co-transfected T782I-A1685S Nav1.1 at 32°C and 37°C using the same colour scheme as (E). The number of observations in (F) are the same for each condition as in (E). Individual observations in each condition are shown as smaller symbols. Each midpoint measurement reflects a mean and each error bar reflects a standard error of the mean. Independent replicates are individual cells recorded in the whole-cell patch clamp configuration. * indicates a statistically significant difference between two midpoints. All statistical analysis was performed with a two-factor ANOVA followed by a Dunnett’s post hoc of pairwise comparisons to WT. Exact P-values and details of statistical tests are found in the main text. Number of individual replicates, means and standard error of the mean for all conditions are found in Table 2 and https://doi.org/10.17605/OSF.IO/YS5RG.

Figure 7

Mutant voltage-gated sodium channels confer differences in neuronal response to stimulation. (A) Single compartment neuron simulations for WT and mutant sodium channels with slowly increasing stimulating current I_stim. T782I mutant channels confer hyperexcitability at low input current levels, whilst A1685S mutants allow firing even at much higher levels than WT. In the T782I-A1685S model, the effects of T782I predominate. (B) Gating variables plotting limit cycles during action potentials at different input current levels. The limit cycles appear similar for wild type and A1685S mutants, whilst T782I and T782I-A1685S do not achieve full inactivation at any input current amplitude. (C) Results from repeat simulations at different levels of input currents. Top four plots show maximum and minimum values during the simulated period at steady state, with oscillatory regimes highlighted with shading. The bottom plot shows simulated firing frequency at the different input current levels. Wildtype and A1685S mutants show threshold-like phase transitions at certain I_stim values when neuronal firing is initiated. Even at the lowest input currents tested here, both T782I and T782I-A1685S simulated neurons already show fast action potential firing. At very high input currents, all simulated cells cease firing.