Mutations in SCN3A cause early infantile epileptic encephalopathy

Abstract

Objective: Voltage-gated sodium (Na⁺ ) channels underlie action potential generation and propagation and hence are central to the regulation of excitability in the nervous system. Mutations in the genes SCN1A, SCN2A, and SCN8A, encoding the Na⁺ channel pore-forming (α) subunits Nav1.1, 1.2, and 1.6, respectively, and SCN1B, encoding the accessory subunit β₁ , are established causes of genetic epilepsies. SCN3A, encoding Nav1.3, is known to be highly expressed in brain, but has not previously been linked to early infantile epileptic encephalopathy. Here, we describe a cohort of 4 patients with epileptic encephalopathy and heterozygous de novo missense variants in SCN3A (p.Ile875Thr in 2 cases, p.Pro1333Leu, and p.Val1769Ala).

Methods: All patients presented with treatment-resistant epilepsy in the first year of life, severe to profound intellectual disability, and in 2 cases (both with the variant p.Ile875Thr), diffuse polymicrogyria.

Results: Electrophysiological recordings of mutant channels revealed prominent gain of channel function, with a markedly increased amplitude of the slowly inactivating current component, and for 2 of 3 mutants (p.Ile875Thr and p.Pro1333Leu), a leftward shift in the voltage dependence of activation to more hyperpolarized potentials. Gain of function was not observed for Nav1.3 variants known or presumed to be inherited (p.Arg1642Cys and p.Lys1799Gln). The antiseizure medications phenytoin and lacosamide selectively blocked slowly inactivating over transient current in wild-type and mutant Nav1.3 channels.

Interpretation: These findings establish SCN3A as a new gene for infantile epileptic encephalopathy and suggest a potential pharmacologic intervention. These findings also reinforce the role of Nav1.3 as an important regulator of neuronal excitability in the developing brain, while providing additional insight into mechanisms of slow inactivation of Nav1.3. Ann Neurol 2018;83:703-717.

Figure 1. Locations of epilepsy-associated Nav1.3 variants

(A) Shown is a schematic of the Nav1.3 voltage-gated Na⁺ channel α subunit, with domains (D) 1-4, and transmembrane segments (S) 1-6 (shown for D1). Epilepsy-associated variants previously reported in the literature are shown as closed squares, and those described in the present report are shown as open circles. Variants in bold are recurrent. Amino acid location is based on Reference Sequence NP_008853.3 (hNav1.3 isoform 1). (B) Nav1.3 mutations associated with epileptic encephalopathy occur at highly conserved amino acid residues, as shown using ClustalX multiple sequence alignment of human Nav1.3 isoforms 1, 2, and 3, human Nav1.1 homolog, and various species orthologs. The Nav1.3 variants described in this report are indicated in bold above the corresponding amino acid residue. The highly conserved arginine residues of the S4 helix of the voltage sensor of Nav1.3 are indicated by the boxed regions. Notation indicates conservation across human Na⁺ channel genes and between species: period (.) indicates weakly similar; colon (:), strongly similar; asterisk (*), fully conserved.

Figure 2. Magnetic resonance imaging scans of patients with SCN3A-p.Ile875Thr mutation

(A) MRI for Patient 1. Axial T2 MRI images (Ai-ii) at two rostrocaudal levels demonstrating extensive, bilaterally symmetric, predominantly frontoparietal polymicrogyria. There is incomplete opercularization of the insula with slightly dysplastic sylvian fissures. There is extensive foci of frontal subcortical and periventricular T1 shortening representing calcifications (Aiii). (B) MRI for Patient 2. Axial T2 MRI images (Bi-ii) show diffuse polymicrogyria. T1 images show apparent subcortical calcifications in the frontal lobes (Biii).

Figure 3. Epilepsy-associated mutations alter function of the Nav1.3 channel

(A) Representative single leak-subtracted traces showing families of Na⁺ currents elicited by 20 ms depolarizing voltage steps from -80 to +50 mV in 5 mV increments from a holding potential of -120 mV with a 10 second intersweep interval for wild-type and each Nav1.3 variant co-expressed with β₁ and β₂. (B) Normalized I-V curves showing peak I_NaT current density (in pA/pF) vs. voltage for wild-type (n = 27), p.Ile875Thr (n = 14), p.Pro1333Leu (n = 10), p.Arg1642Cys (n = 8), p.Val1769Ala (n = 27), and p.Lys1799Gln (n = 13), presented as mean ± SEM. (C) Prepulse voltage-dependence of channel inactivation and conductance-voltage relationships for wild-type (n = 27), p.Ile875Thr (n = 14), p.Pro1333Leu (n = 10) and p.Val1769Ala (n = 19 respectively) were fit by a Boltzmann function. (D) Representative individual traces showing I_NaT and I_NaP in response to a 200 ms voltage step from -120 to -10 mV for wild-type Nav1.3 and p.Ile875Thr, p.Pro1333Leu, p.Arg1642Cys, p.Val1769Ala, and p.Lys1799Gln. (E) Bar graph showing ratio of I_NaP/I_NaT for wild-type (n = 27), p.Ile875Thr (n =12), p.Pro1333Leu (n = 14), p.Arg1642Cys (n = 12), p.Val1769Ala (n = 25), and p.Lys1799Gln (n = 13), presented as mean ± SEM. (F) I-V curves of I_NaP in wild-type (n = 27) and Nav1.3 variants p.Ile875Thr (n = 14), p.Pro1333Leu (n = 10), and p.Val1769Ala (n = 13). Note that I_NaP at each voltage is normalized to the maximum value. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. wild-type via one-way ANOVA with Bonferroni correction for multiple comparisons.

Figure 4. Anti-seizure medications partially normalize pathological persistent current in Nav1.3 mutant channels

(A) Traces showing I_NaT and I_NaP of wild-type and mutant NaV1.3 channels before (black) and after (red traces) application of 100 μM lacosamide (A1) and phenytoin (A2). (B) Bar graph showing % blockade of I_NaP by lacosamide (wild-type, n = 10; p.Ile875Thr, n = 6; p.Pro1333Leu, n = 6; p.Val1769Ala, n = 10) and phenytoin (wild-type, n = 3; p.Val1769Ala, n = 7). (C) Representative examples of Nav1.3 currents evoked by a voltage ramp from -120 to +40 mV at 0.8 mV/ms in wild-type Nav1.3 (top left) and p.Val1769Ala mutant channels (top right), with block by phenytoin (bottom). (D) Quantification of charge (in nanocoulombs; nC) between wild-type Nav1.3 (n = 5) and p.Val1769Ala (n = 5) before and after drug application. All data are presented as means ± SEM. * indicates p < 0.05 and ** indicates p < 0.01 via paired two-tailed t-test.