De novo mutations of voltage-gated sodium channel alphaII gene SCN2A in intractable epilepsies

Abstract

Background: Mutations of voltage-gated sodium channel alpha(II) gene, SCN2A, have been described in a wide spectrum of epilepsies. While inherited SCN2A mutations have been identified in multiple mild epilepsy cases, a de novo SCN2A-R102X mutation, which we previously reported in a patient with sporadic intractable childhood localization-related epilepsy, remains unique. To validate the involvement of de novo SCN2A mutations in the etiology of intractable epilepsies, we sought to identify additional instances.

Methods: We performed mutational analyses on SCN2A in 116 patients with severe myoclonic epilepsy in infancy, infantile spasms, and other types of intractable childhood partial and generalized epilepsies and did whole-cell patch-clamp recordings on Na(v)1.2 channels containing identified mutations.

Results: We discovered 2 additional de novo SCN2A mutations. One mutation, SCN2A-E1211K, was identified in a patient with sporadic infantile spasms. SCN2A-E1211K produced channels with altered electrophysiologic properties compatible with both augmented (an approximately 18-mV hyperpolarizing shift in the voltage dependence of activation) and reduced (an approximately 22-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation and a slowed recovery from inactivation) channel activities. The other de novo mutation, SCN2A-I1473M, was identified in a patient with sporadic neonatal epileptic encephalopathy. SCN2A-I1473M caused an approximately 14-mV hyperpolarizing shift in the voltage dependence of activation.

Conclusions: The identified de novo mutations SCN2A-E1211K, -I1473M, and -R102X indicate that SCN2A is an etiologic candidate underlying a variety of intractable childhood epilepsies. The phenotypic variations among patients might be due to the different electrophysiologic properties of mutant channels.

Figure 1 Novel SCN2A nucleotide changes leading to amino acid substitutions identified in patients with intractable childhood epilepsies (A) Pedigree for proband 1 (II-1) with a history of infantile spasms that evolved to symptomatic generalized epilepsy who showed the nucleotide change c.3631C>T (E1211K). The putative haplotypes determined by analyzing microsatellite markers, namely, D2S151, D2S2330, and D2S335, flanking the SCN2A locus are shown. Markers are given in order, from the p telomere to the q telomere. + = Wild-type allele; m = mutated allele; filled square = infantile spasms. (B) Pedigree for proband 2 (II-1) with neonatal epileptic encephalopathy who showed the nucleotide change c.4419A>G (I1473M). The putative haplotypes determined by analyzing microsatellite markers flanking the SCN2A locus are shown. Genetic recombination occurred in the proband’s or her brother’s maternal chromosome within the region between D2S151 and D2S2330. + = Wild-type allele, m = mutated allele; hatched circle = neonatal epileptic encephalopathy; slash = deceased. (C) Pedigree for proband 3 (II-2) with SMEB who showed the nucleotide change c.1724C>T (A575V). The nucleotide change was also detected in her asymptomatic father. Genomic DNA of her sister was not available for the analysis. + = Wild-type allele; m = mutated allele; hatched circle = SMEB; symbol with a dot = asymptomatic mutation carrier.

Figure 2 SCN2A nucleotide changes leading to amino acid substitutions detected in intractable childhood epilepsies An asterisk indicates a nonsense mutation, SCN2A-R102X, described before. SCN2A-A575V is assigned to the intracellular linker between domain I (DI) and DII. The alanine residue A575 (highlighted in black) is conserved among human, mouse, and rat Na_v1.2 but not in other types of mammalian VGSC α subunits. SCN2A-E1211K is localized to transmembrane segment 1 (S1) of DIII. The glutamate residue E1211 (highlighted in black) is significantly conserved through vertebrate and invertebrate VGSC α subunits and human calcium channel α subunits. I1473M is localized to S6 of DIII. The isoleucine residue I1473 (highlighted in black) is perfectly conserved through vertebrate and invertebrate sodium channel α subunits. Filled square = de novo nonsense mutation; filled circle = de novo missense mutation; open circle = possible nonpathogenic variant. Sources of amino acid sequences are as follows (notations refer to accession numbers): Human Na_v1.2, NP_001035232; mouse Na_v1.2, NP_001092768; rat Na_v1.2, NP_036779; human Na_v1.1, NP_008851; human Na_v1.3, NP_008853; human Na_v1.4, NP_000325; human Na_v1.5, NP_932173; human Na_v1.6, NP_055006; human Na_v1.7, NP_002968; human Na_v1.8, NP_006505; human Na_v1.9, NP_054858; cockroach sodium channels, AAC47483 and AAK01090; human Ca_v1.1, NP_000060; human Ca_v1.2, NP_000719; human Ca_v1.3, NP_000711; human Ca_v1.4, NP_005174; human Ca_v2.1, NP_000059; human Ca_v2.2, NP_000709; human Ca_v2.3, NP_000712.

Figure 3 Voltage-gated Na⁺ currents recorded from HEK293 cells expressing human wild-type and mutant (E1211K, I1473M, and A575V) Na_v1.2 channels (A) Peak sodium conductance-voltage relationships for wild-type (WT; closed circle), E1211K (closed triangle), I1473M (open triangle), and A575V (open circle). Na⁺ currents were evoked by 10 msec depolarizations to various test potentials (−80 mV to 20 mV) from a holding potential of −120 mV. Sodium conductance (g_Na) was calculated according to the equation g_Na = I_Na/(V_g − V_r), where I_Na is the peak amplitude of the Na⁺ current, V_g is the test potential, and V_r is the reversal potential for Na⁺. To compare voltage dependence of activation, data were fitted by the least-squares fit of the data to a Boltzmann function, according to the equation g_Na/g_Namax = 1 − 1/{1 − exp[(V_h − V_1/2)/k]}, where g_Namax is the maximum conductance, V_h is the potential of individual step pulses, V_1/2 is the potential at which g_Na is one-half maximal, and k is the slope factor. The data points represent the average of g_Na/g_Namax. Note that E1211K and I1473M opened at significantly lower voltages. (B) Half activation potentials of Na_v1.2 channels. Half activation potentials were calculated for individual cells and averaged. (C) Steady-state voltage dependence of inactivation for WT (closed circle), E1211K (closed triangle), I1473M (open triangle), and A575V (open circle). Cells were pre-pulsed for 2 seconds at various holding potentials (from −120 mV to 10 mV in 10-mV increments), and then Na⁺ current was evoked by a step depolarization to 0 mV. The peak amplitudes of the Na⁺ currents measured at individual test potentials were normalized to the peak amplitude of the Na⁺ current measured at a holding potential of −120 mV. Data were fitted by the least-squares fit of the data to a Boltzmann function, according to the equation I/I_max = 1/{1 + exp[(V_h − V_1/2)/k]}, where I_max is the magnitude of the peak Na⁺ current observed at a holding potential of −120 mV, V_h is the holding potential, V_1/2 is the potential at which the Na⁺ current is one-half maximal, and k is the slope factor. The data points represent the average of I/I_max. Note that the curve for E1211K is significantly shifted to the hyperpolarized direction. (D) Half inactivation potentials of Na_v1.2 channels. Half inactivation potentials were calculated for individual cells and averaged. Values represent means ± SEM, *p < 0.05, **p < 0.01.

Figure 4 Recovery from inactivated state for human wild-type and mutant (E1211K, I1473M, and A575V) Na_v1.2 channels (A) Recovery from inactivated state for WT (closed circle), E1211K (closed triangle), I1473M (open triangle), and A575V (open circle). Two depolarizing pulses (step to 10 mV, 10 msec in duration) with various interpulse intervals (from 0.5 msec to 50 msec) were successively applied to activate Na⁺ currents. The peak amplitude of Na⁺ currents evoked by the second pulse (I₂) was normalized to the peak amplitude of Na⁺ currents evoked by the first pulse (I₁) and I₂/I₁ is expressed as recovery ratios. Data were fitted by the equation I₂/I₁ = 1 − exp(−A_rect), where A_rec is the factor to determine the speed of recovery. The data points represent the average of I₂/I₁. Note that the recovery for E1211K was significantly prolonged. (B) A_rec of Na_v1.2 channels. A_rec was calculated for individual cells and averaged. Values represent means ± SEM, *p < 0.05, **p < 0.01.