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. 2020 Dec;61(12):2847-2856.
doi: 10.1111/epi.16741. Epub 2020 Nov 2.

Gabra2 is a genetic modifier of Scn8a encephalopathy in the mouse

Wenxi Yu  1 , Sophie F Hill  1   2 , James G Xenakis  3 , Fernando Pardo-Manuel de Villena  3 , Jacy L Wagnon  1 , Miriam H Meisler  1   2
Affiliations

Gabra2 is a genetic modifier of Scn8a encephalopathy in the mouse

Wenxi Yu et al. Epilepsia. 2020 Dec.

Abstract

Objective: SCN8A encephalopathy is a developmental epileptic encephalopathy typically caused by de novo gain-of-function mutations in Nav 1.6. Severely affected individuals exhibit refractory seizures, developmental delay, cognitive disabilities, movement disorders, and elevated risk of sudden death. Patients with the identical SCN8A variant can differ in clinical course, suggesting a role for modifier genes in determining disease severity. The identification of genetic modifiers contributes to understanding disease pathogenesis and suggesting therapeutic interventions.

Methods: We generated F1 and F2 crosses between inbred mouse strains and mice carrying the human pathogenic variants SCN8A-R1872W and SCN8A-N1768D. Quantitative trait locus (QTL) analysis of seizure-related phenotypes was used for chromosomal mapping of modifier loci.

Results: In an F2 cross between strain SJL/J and C57BL/6J mice carrying the patient mutation R1872W, we identified a major QTL on chromosome 5 containing the Gabra2 gene. Strain C57BL/6J carries a splice site mutation that reduces expression of Gabra2, encoding the α2 subunit of the aminobutyric acid type A receptor. The protective wild-type allele of Gabra2 from strain SJL/J delays the age at seizure onset and extends life span of the Scn8a mutant mice. Additional Scn8a modifiers were observed in the F2 cross and in an F1 cross with strain C3HeB/FeJ.

Significance: These studies demonstrate that the SJL/J strain carries multiple modifiers with protective effects against seizures induced by gain-of-function mutations in Scn8a. Homozygosity for the hypomorphic variant of Gabra2 in strain C57BL/6J is associated with early seizure onset and short life span. GABRA2 is a potential therapeutic target for SCN8A encephalopathy.

Keywords: Gabra2; Scn8a; epilepsy; epileptic encephalopathy; genetic modifier; voltage-gated sodium channel.

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Conflict of interest statement

None of the authors has any conflict of interest to disclose.

Figures

FIGURE 1
FIGURE 1
Strain background modifies survival of Scn8aR1872W/+, Emx1Cre/+ mice. The Scn8a‐R1872W patient mutation was generated on the (C57BL/6J × SJL/J)F2 background and then backcrossed to strain C57BL/6J. A, Longer survival is correlated with a greater contribution of strain SJL/J. Approximate percentage SJL is 50% in the F2 mice, 6% in N3, and 0.2% in N8. B, Breeding scheme for double heterozygous F2 mice with genotype Scn8a R1972W/+ , Emx1Cre/+. (N3 data are from previously published work 10 )
FIGURE 2
FIGURE 2
Seizure phenotypes in (B6 × SJL)F2 and C57BL/6J.N8 mutant mice. A, B, Distribution of age at seizure onset and length of survival in N8 mice. C, D, Distribution of age of onset and length of survival in F2 mice. See also Figure S1. E, Direct correlation between age at seizure onset and length of survival (r 2 = 0.7, n = 60; 1 outlier excluded from the calculation). F, In vivo activation of the conditional Scn8aR1872W allele in Scn8aR1872W/+, Emx1Cre/+ F2 mice. Scn8a transcripts were counted after deep sequencing of a reverse transcriptase polymerase chain reaction product containing codon1872. The proportion of mutant transcripts was 26 ± 2% (mean ± SD, n = 16) and did not differ between mice with early and late seizure onset
FIGURE 3
FIGURE 3
Disease duration is not correlated with age at onset of first seizure. Duration is defined as the time interval between first observed seizure and death. A, Narrow distribution of disease duration in (B6 × SJL)F2 mutant mice, from 20 to 40 days. B, Age at first seizure is not correlated with disease duration (r 2 = 0.006)
FIGURE 4
FIGURE 4
Quantitative trait locus mapping of survival in (B6 × SJL) F2 mice. One hundred eleven F2 mice were genotyped with the MiniMUGA genotyping array. Lod scores were calculated for 2792 informative loci. Chromosomes 1‐19 are represented numerically on the x‐axis. The relative width of the space allotted for each chromosome reflects the size of each chromosome. The y‐axis represents the lod score
FIGURE 5
FIGURE 5
The Gabra2 splice site variant cosegregates with seizure onset and survival in Scn8aR1872W/+, Emx1Cre F2 mice. A, SJL/J mice lack the splice site variant. A polymerase chain reaction product containing exon 5 was amplified from genomic DNA and sequenced. The T nucleotide in the wild‐type allele at position −3 upstream of exon 5 is present in strain SJL/J (arrow). The single‐bp deletion at position −3 (Mulligan et al 23 ) is present in our C57BL/6J mice. B, The splice site variant reduces Gabra2 transcripts. RNA was isolated from hippocampus of strains C57BL/6J, SJL/J, and C3Heb/FeJ, and Gabra2 transcripts were quantitated with TaqMan assay. Gabra2 transcripts are fourfold more abundant in strain SJL/J than in strain C57BL/6J. C, Gabra2 genotype and length of survival in F2 mice. D, Gabra2 genotype and age at first seizure in F2 mice. The dotted lines mark 35 days of surviva. See also Figure S2.
FIGURE 6
FIGURE 6
Survival of C3H Scn8aN1768D/+ F1 mice. N20 mice from the congenic strain C3H Scn8aN1768D/+ were crossed with inbred mice from strains SJL/J, FVB/NJ, and DBA/2J. Survival of F1 mice carrying the N1768D allele was monitored for 6 months. Survival was completely rescued in the (C3H × SJL)F1 mice
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Comment in

  • More Genes, Better Outcome?
    Goldman AM. Goldman AM. Epilepsy Curr. 2021 Jul 31;21(4):300-302. doi: 10.1177/15357597211025127. eCollection 2021 Jul-Aug. Epilepsy Curr. 2021. PMID: 34690574 Free PMC article. No abstract available.

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