Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage-gated sodium channel Scn2a

Abstract

More than 1,200 mutations in neuronal voltage-gated sodium channel (VGSC) genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2a(Q54) transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2a(Q54) phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2a(Q54) mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2a(Q54) mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2a(Q54) phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2a(Q54) seizure phenotype. Scn2a(Q54) mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.

Keywords: Genetics; Mouse model; Seizures; Voltage-gated calcium channels; Voltage-gated ion channels.

Figure 1. Characterization of Cacna1g transgenic mice

(A,B) To assess expression at the level of mRNA, a quantitative digital droplet PCR (ddPCR) Taqman assay was performed on hemizygous transgenic (SJL.1GL or B6.1GH) male offspring and controls (SJL.WT or B6.WT). The expression of Cacna1g in six week old whole-brain was normalized to Tbp expression. P-value was determined by unpaired Student’s T-test. Error bars represent upper and lower limits derived from SEM. (A) Normalized Cacna1g expression was observed to be: SJL.WT = 1.0 (n=7) and hemizygous SJL.1GL = 1.27 (n=4). p<0.005. (B) Normalized Cacna1g expression was observed to be: wildtype control males = 1.0 (n=6) and hemizygous B6.1GH males = 2.66 (n=6). p<0.0001. (C & D) Whole-brain membrane fractions prepared from six week old B6.1GH (C) or B6.1G^KO/+ and B6.1G^KO/KO (D) mice were assayed using mouse monoclonal Cav3.1 (Neuromab, N178A/9) (upper panels) and β-tubulin antibodies (Sigma, TUB 2.1) (lower panels). Expression of Cav3.1 was determined by densitometry using ImageJ. (C) Cav3.1 expression was significantly elevated in B6.1GH hemizygous males (2.69; n=5) relative to wildtype controls (1; n=4) (p < 0.001; Student’s T-test). (D) Cav3.1 expression was decreased by approximately half in 1G^KO/+ heterozygotes and absent in 1G^KO/KO homozygotes.

Figure 2. Effect of genetic modulation of Cacna1g on Scn2a^Q54 seizure frequency

(A–C) Average number of focal motor seizures (FMS) for each genotype is shown. Error bars represent SEM. (A) Average seizure frequencies in 60 minutes were compared between F1.Q54 (22.3 ± 1.76) and F1.Q54;1GL (29.6 ± 1.68) using Student’s T-test (p<0.004). (B) Average seizure frequencies in 60 minutes were compared between B6.Q54 (0.45 ± 0.15) and B6.Q54;1GH (6.68 ± 1.37) using Mann-Whitney rank-sum test (p<0.0001). (C) Average seizure frequencies in 30 minutes were compared between F1.Q54 (11.47 ± 1.9) and F1.Q54;1G^KO/+ (6.93 ± 1.0) using Student’s T-test (p<0.05).