A novel de novo mutation of SCN8A (Nav1.6) with enhanced channel activation in a child with epileptic encephalopathy - PubMed (original) (raw)

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A novel de novo mutation of SCN8A (Nav1.6) with enhanced channel activation in a child with epileptic encephalopathy

Mark Estacion et al. Neurobiol Dis. 2014 Sep.

Abstract

Rare de novo mutations of sodium channels are thought to be an important cause of sporadic epilepsy. The well established role of de novo mutations of sodium channel SCN1A in Dravet Syndrome supports this view, but the etiology of many cases of epileptic encephalopathy remains unknown. We sought to identify the genetic cause in a patient with early onset epileptic encephalopathy by whole exome sequencing of genomic DNA. The heterozygous mutation c. 2003C>T in SCN8A, the gene encoding sodium channel Nav1.6, was detected in the patient but was not present in either parent. The resulting missense substitution, p.Thr767Ile, alters an evolutionarily conserved residue in the first transmembrane segment of channel domain II. The electrophysiological effects of this mutation were assessed in neuronal cells transfected with mutant or wildtype cDNA. The mutation causes enhanced channel activation, with a 10mV depolarizing shift in voltage dependence of activation as well as increased ramp current. In addition, pyramidal hippocampal neurons expressing the mutant channel exhibit increased spontaneous firing with PDS-like complexes as well as increased frequency of evoked action potentials. The identification of this new gain-of-function mutation of Nav1.6 supports the inclusion of SCN8A as a causative gene in infantile epilepsy, demonstrates a novel mechanism for hyperactivity of Nav1.6, and further expands the role of de novo mutations in severe epilepsy.

Keywords: De novo mutation; Epilepsy; Epileptic encephalopathy; Sodium channel.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Location of the p.Thr767Ile mutation in Nav1.6 and evolutionary conservation of the mutated residue

(A) Threonine 767 is located in transmembrane segment 1 of domain II of the voltage-gated sodium channel alpha subunit. (B) This threonine residue is conserved in the vertebrate SCN8A channel from reptile (a, anotole) and fish (f, Fugu) and in the homologous invertebrate channel from drosophila and Ciona. (C) Threonine (T) at position 767 is conserved in the members of the human voltage-gated sodium channel gene family, including muscle and cardiac channels SCN4A and SCN5A, respectively.

Figure 2

Figure 2. Voltage-clamp analysis of Nav1.6 p.Thr767Ile mutant channels

(A) Representative traces recorded from ND7/23 cells expressing mNav1.6R (WT, black lines) and T767I (red lines) in response to the activation stimulation protocol (100 msec duration pulses). (B) The peak currents are normalized for cell capacitance and then are averaged together to obtain the activation current-voltage (I–V) relation for cells expressing WT (black symbols) or T767I (red symbols) as described in the Methods. (C) The G-V curves for both WT (black symbols) and T767I (red symbols) cell are normalized and then averaged to obtain the Boltzmann fit for activation data, which shows hyperpolarizing shift of activation of T767I channels compared to WT channels. (D) The responses to the fast-inactivation protocol are analyzed to obtain the voltage-dependence of fast-inactivation as described in the methods. The normalized and averaged and the Boltzmann’s fit are shown for both WT (black symbols) and T767I (red symbols). The voltage-dependence of fast-inactivation of T767I channels is not different from that of WT channels. Error bars are standard error of the mean (SEM). (E) The averaged currents evoked during a slow ramp stimulus beginning at −100 mV and ending at 20 mV over a duration of 600 msec are shown from WT (black line, n=6) and T767I (red line, n=10) expressing cells. The traces have been normalized to the maximal peak current recorded from that cell. T767I channels produce a larger normalized ramp current which peaks at a more hyperpolarized voltage compared to that of WT channels.

Figure 3

Figure 3. Neurons expressing Nav1.6 - T767I exhibit an increase in spontaneous activity

Continuous segments of current-clamp recording from hippocampal pyramidal neurons illustrate patterns of spontaneous action potential firing seen in a subset of cells expressing T767I channels. (A1) Trace from a representative spontaneously active cell. Scale bar indicates 20 mV vertical, 5 sec horizontal. (A2) A small region of the trace was expanded to illustrate firing behavior riding on top of transient depolarizations of the membrane potential, paroxsysmal depolarizing shift-like complexes, characteristic of epileptic discharges. Scale bar indicates 20mV vertical, 1sec horizontal. (B) Trace from a hippocampal pyramidal cell expressing Nav1.6-WT with the same scaling as panel A1 for comparison. Scale bar indicates 20 mV vertical, 5 sec horizontal.

Figure 4

Figure 4. Neurons expressing Nav1.6 - T767I are hyperexcitable compared to those expressing the WT channel

Selected traces illustrating action potentials elicited near threshold stimulus current injections for Nav1.6-T767I expressing (A) and Nav1.6-WT expressing (B) pyramidal hippocampal neurons. (C) The average number of spikes elicited to one second long current injections of 0 pA to 40 pA in 5 pA increments from hippocampal pyramidal neurons expressing WT (black symbols, N=8) or T767I (red symbols, N=13) channels. The increase of the response for T767I expressing neurons was significant by ANOVA (p<0.05). Data presented as mean ± SEM.

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