De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP

Abstract

Individuals with severe, sporadic disorders of infantile onset represent an important class of disease for which discovery of the underlying genetic architecture is not amenable to traditional genetic analysis. Full-genome sequencing of affected individuals and their parents provides a powerful alternative strategy for gene discovery. We performed whole-genome sequencing (WGS) on a family quartet containing an affected proband and her unaffected parents and sibling. The 15-year-old female proband had a severe epileptic encephalopathy consisting of early-onset seizures, features of autism, intellectual disability, ataxia, and sudden unexplained death in epilepsy. We discovered a de novo heterozygous missense mutation (c.5302A>G [p.Asn1768Asp]) in the voltage-gated sodium-channel gene SCN8A in the proband. This mutation alters an evolutionarily conserved residue in Nav1.6, one of the most abundant sodium channels in the brain. Analysis of the biophysical properties of the mutant channel demonstrated a dramatic increase in persistent sodium current, incomplete channel inactivation, and a depolarizing shift in the voltage dependence of steady-state fast inactivation. Current-clamp analysis in hippocampal neurons transfected with p.Asn1768Asp channels revealed increased spontaneous firing, paroxysmal-depolarizing-shift-like complexes, and an increased firing frequency, consistent with a dominant gain-of-function phenotype in the heterozygous proband. This work identifies SCN8A as the fifth sodium-channel gene to be mutated in epilepsy and demonstrates the value of WGS for the identification of pathogenic mutations causing severe, sporadic neurological disorders.

Figure 1

The De Novo Proband Substitution p.Asn1768Asp in Sodium-Channel SCN8A The altered amino acid residue is located at the cytoplasmic end of transmembrane segment 6 in domain 4 of the channel (for simplicity of display, we use 1-letter amino acid codes). Residue 1768 is evolutionarily conserved in mammalian and invertebrate sodium channels, as indicated by the examples shown on the right. The polar asparagine residue is altered to the charged residue aspartate in our proband. A substitution in the corresponding residue of SCN1A, Asn1788Lys, was identified as a de novo mutation in an individual with Dravet syndrome, another early-onset epileptic encephalopathy. The following abbreviations are used: h, human; a, anole lizard; f, fish (Fugu); Dm, Drosophila melanogaster; para, fly voltage-gated sodium channel (encoded by paralytic); hSCN5A, human cardiac sodium channel; hSCN1A, human neuronal sodium channel; N, Asn; D, Asp; and K, Lys. Dots represent amino acid identity.

Figure 2

Effect of the De Novo SCN8A Substitution p.Asn1768Asp on Biophysical Properties of the Channel (A) Representative inward currents recorded from ND7/23 cells transiently transfected with Nav1.6_R WT or mutant channels. Cells were held at −120 mV, and a family of step depolarizations (−80 to +60 mV in 5 mV increments) were applied every 5 s. Insets show persistent inward currents (normalized by maximal transient peak currents) from WT and p.Asn1768Asp channels at the end of a 100 ms step depolarization to −80 mV (black) and +20 mV (red). (B) Voltage dependence of persistent current. The amplitude of persistent current was measured as the mean value of currents 93–98 ms after the onset of depolarization and is presented as a percentage of the maximal transient peak current. (C) Voltage dependence of channel activation and steady-state fast inactivation. Channel activation was analyzed as previously described. Steady-state fast inactivation was assessed with a series of 100 ms step depolarizations (−130 to −10 mV in 10 mV increments) and was followed by a test pulse (−10 mV) so the remaining fraction of noninactivated channels could be measured. The p.Asn1768Asp channels do not completely inactivate, which is consistent with the large persistent current. (D) Development of closed-state inactivation at −60 mV. Cells were held at −120 mV, and closed-state inactivation was assessed with a prepulse set to −60 mV with a duration varying from 0 to 500 ms, and remaining available channels were assessed with a test pulse set to 0 mV (20 ms). (E) Mean ramp currents generated by WT (black) and p.Asn1768Asp (red) channels. The response to a slow ramp stimulus was evaluated with a ramp depolarization from −120 to +40 mV over 800 ms. The p.Asn1768Asp mutation increases the amplitude of the ramp current (normalized by transient peak current; p.Asn1768Asp [13.6 ± 1.9%, n = 5, p < 0.05] versus WT [1.2 ± 0.2%, n = 7]).

Figure 3

Effect of the De Novo SCN8A Substitution p.Asn1768Asp on Hippocampal Neuronal Excitability (A) p.Asn1768Asp channels increase excitability of hippocampal neurons. (Ai) An example of spontaneous firing in a neuron transfected with p.Asn1768Asp channels. (Aii) Representative PDS-like complexes recorded from two hippocampal pyramidal neurons transfected with p.Asn1768Asp. The dashed lines indicate −80 mV. (B) Percentage of neurons displaying spontaneous firing. The asterisk indicates p < 0.05. (C) Number of action potentials (APs) evoked by a series of 1 s step depolarizating current injections (from 5 to 40 pA with a 5 pA increment). Neurons transfected with p.Asn1768Asp produce more APs than neurons transfected with WT. An asterisk indicates p < 0.05.