Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice

Abstract

In a chemical mutagenesis screen, we identified the novel Scn8a(8J) allele of the gene encoding the neuronal voltage-gated sodium channel Na(v)1.6. The missense mutation V929F in this allele alters an evolutionarily conserved residue in the pore loop of domain 2 of Na(v)1.6. Electroencephalography (EEG) revealed well-defined spike-wave discharges (SWD), the hallmark of absence epilepsy, in Scn8a(8J) heterozygotes and in heterozygotes for two classical Scn8a alleles, Scn8a(med) (null) and Scn8a(med-jo) (missense). Mouse strain background had a significant effect on SWD, with mutants on the C3HeB/FeJ strain showing a higher incidence than on C57BL/6J. The abnormal EEG patterns in heterozygous mutant mice and the influence of genetic background on SWD make SCN8A an attractive candidate gene for common human absence epilepsy, a genetically complex disorder.

Figure 1.

Genetic mapping and identification of the Scn8a^8J mutation. (A) LOD score plot of locomotor impairment in mice from G₃ and G₃F₁ populations. The markers from left to right are D15Mit171 (90.1 Mb), D15Mit243 (93.1 Mb), D15Jmp29 (96.2 Mb), D15Mit161 (97.0 Mb), D15Mit43 (97.9 Mb), D15Mit14 (99.7 Mb), D15Mit246 (102.1 Mb) and D15Mit79 (103.4 Mb). Scn8a is located at 100.7 Mb (arrow). The maximum LOD score was 36.4 at 99.7 Mb. (B) Identification of the mutation (G>T) in a homozygote from the 596-16 pedigree. (C) Evolutionary conservation of residue valine 929 in eight vertebrate orthologs and five mouse paralogs of Scn8a. (D) Location of V929 in the ion pore region of domain 2. (E) Western blot of membrane protein from mutant and wild-type mouse brain. Lanes contained 75 µg of protein.

Figure 2.

Representative SWD in Scn8a mutants. (A) Traces correspond to six differential recordings from a Scn8a^8J/+ mouse from each of four epidural electrodes; RF–LF (right front–left front), RF–LB (right front–left back), etc. carried out at Jackson Laboratory as described in Materials and Methods. SWD data from individual Scn8a^8J/+ mice is shown in Figure 4. (B) and (C) Representative EEG traces from Scn8a^med/+ and Scn8a^med-jo/+ mutants, respectively, carried out at Emory University as described in Materials and Methods. EEG₁ and EEG₂ represent cortical activity from the right and left hemispheres, respectively. SWD are present in both hemispheres and are associated with lack of muscle movement.

Figure 3.

Effect of genetic background on incidence of SWD in heterozygous Scn8a^8Jmice. Each point shows the number of SWD per hour for an individual mouse, in different genetic backgrounds. Heterozygous (het) genotypes are shown in filled symbols, wild-type (wt) in open symbols. The inset shows a plot of the average seizure frequency (±SE) in the four Scn8a^8J genotypes. The average seizure frequency per hour (±SE) are: G₃× C3HeB/FeJ: 132.1 ± 15.4 (het), 22.3 ± 4.3 (wt); G₃N₂ and G₃N₃: 104.6 ± 14.3 (het); (G₃N₃ X C57BL/6J)F1: 80.2 ± 12 (het), 0.8 ± .5 (wt); F₁ X C57BL/6J: 60.6 ± 7.8 (het); C3HeB/FeJ–Scn8a^med/+ 67.9 ± 2.5.

Figure 4.

Effect of ETX treatment on SWD in Scn8a mutants. ETX treatment decreased the length and incidence of SWD. (A) Scn8a^8J/+ examined at Jackson Laboratory. (B) Scn8a^med/+ tested at Emory University. (C) Effect of the injection procedure on the incidence of SWD. Each diamond represents a single spike-wave discharge, and all of the SWD for a single mouse are shown in each panel. SWD were decreased following ETX administration and increased by the handling associated with saline injection. The average incidence before and after treatment as well as sample sizes are given in Results.

Figure 5.

Relationship between SWD and states of vigilance in Scn8a^med/+ mice. The percentage of time spent in SWD during wakefulness was similar in the light and dark cycles. A greater amount of time was spent in SWD during slow-wave sleep (SWS) and paradoxical sleep (PS) in the dark cycle when compared with the light cycle. The percentage of time spent in SWD during wakefulness, SWS and PS was compared between the light and dark cycles using the Wilcoxon signed-rank test implemented in SAS software. *, statistical difference between percentage time spent in SWD in light and dark cycles.