Pathogenic in-Frame Variants in SCN8A: Expanding the Genetic Landscape of SCN8A-Associated Disease

Figures

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  FIGURE 1. Patient SCN8A variants and in-frame variants from gnomAD and ClinVar. (A) SCN8A channel with variants denoted by: red star, R1620L mutation; green star, in-frame deletion in Patients 1 and 2; filled blue circle, in-frame duplication in proband from Stringer et al., 2021; filled green circle, in-frame variants from Johannesen et al., 2021; open circles, in-frame deletions from ClinVar (July 2021); filled black circle, in-frame deletions and duplications from the gnomAD database (v2.1.1 and v3.1.1; July 2021); filled red circle, in-frame duplication in both gnomAD and ClinVar. (B) DNA and protein sequence alignment for Patients 1 and 2 and the proband identified in Stringer et al., 2021. Green indicates amino acids deleted in Patients 1 and 2. Dash (-) indicates a deleted nucleotide or amino acid compared to WT. Blue indicates inserted DNA nucleotides or amino acids. Underline shows close proximity of amino acids deleted in ΔVIR and ΔIRL mouse lines to the variant identified in Stringer et al., 2021.

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  FIGURE 2. Generation and characterization of the ΔIRL and ΔVIR mouse lines. (A) DNA and protein sequence alignment. WT, wild-type mouse sequence, RL, the human R1620L mutation (red) in the RL mouse line, ΔVIR, the 3 amino acid deletion in the ΔVIR mouse line, ΔIRL, the 3 amino acid deletion in the ΔIRL mouse line. Dash (-) indicates a deleted nucleotide or amino acid when compared to the WT sequence. (B) Homozygous ΔIRL/ΔIRL mutants weigh significantly less than same-sex heterozygous ΔIRL/+ mutants and WT littermates. Kruskal-Wallis test followed by Dunn’s multiple comparisons. (C) Approximately 25% of ΔIRL/ΔIRL mutants and 90% of ΔIRL/+ mutants survive to postnatal day 90 (P90). Log-rank Mantel Cox test. (B and C) WT male N = 10; WT female N = 11; ΔIRL/+ male N = 29; ΔIRL/+ female N = 17, ΔIRL/ΔIRL male N = 13; ΔIRL/ΔIRL female N = 11. (D) Scn8a mRNA expression levels were comparable for the three genotypes in the ΔIRL mouse line. Kruskal-Wallis test. N = 3–4/genotype. (E) Heterozygous ΔVIR/+ male mutants weigh significantly less than same-sex WT littermates. Female ΔVIR/+ mutants and WT littermates had comparable weights. Paired Student's t test. (F) Approximately 95% of ΔVIR/+ mutants…

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  FIGURE 3. ΔIRL/+ and ΔVIR/+ mutants exhibit increased seizure susceptibility. (A) ΔIRL/+ mutants are significantly more susceptible to 6 Hz-induced seizures compared to WT littermates. Mann-Whitney test. WT N = 25; ΔIRL/+ N = 21. (B,C) ΔIRL/+ mutants exhibit a lower average latency to the first MJ (B) and GTCS (C) compared to WT littermates. Unpaired student’s t test. WT N = 15; ΔIRL/+ N = 14. (D) ΔVIR/+ mutants are more susceptible to 6 Hz-induced seizures compared to WT littermates. Mann-Whitney test. WT N = 20; ΔVIR/+: N = 23. (E,F) ΔVIR/+ mutants exhibit lower average latencies to the first MJ (E) and GTCS (F) compared to WT littermates. Unpaired Student's t test. WT N = 15; ΔVIR/+ N = 16. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

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  FIGURE 4. ΔIRL/+ and ΔVIR/+ mutants exhibit spontaneous seizures. (A) Representative cortical EEG traces from a ΔVIR/+ mutant before, during, and after a spontaneous seizure. (B) Number of spontaneous seizures observed in 5 male ΔIRL/+ and 5 male ΔVIR/+ mutants. SE denotes status epilepticus.

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  FIGURE 5. Generation and characterization of ΔVIR/RL biallelic heterozygous mice. (A,B) ΔVIR/RL mutants weighed less than same-sex littermates from P12. (C) Male and female ΔVIR/RL mutants exhibited a maximum lifespan of 22 and 31 days, respectively. Only 3/22 ΔVIR/+ mutants died prematurely. Log-rank Mantel Cox test. (A-C) WT male N = 12; WT female N = 10; RL/+ male N = 12; RL/+ female N = 3; ΔVIR/+ male N = 16; ΔVIR/+ female N = 14; ΔVIR/RL male N = 7; ΔVIR/RL female N = 8. (D) Compared to WT littermates, ΔVIR/+ mutants exhibited a significantly lower average latency to the first MJ. (E) ΔVIR/+ mutants exhibit a significantly lower average latency to the first GTCS compared to RL/+ mutants and WT littermates. (D,E) Kruskal-Wallis test followed by Dunn’s multiple comparisons. WT N = 13; RL/+ N = 12; ΔVIR/+ N = 15. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

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  FIGURE 6. In silico analysis of in-frame deletions in the DIVS4 and DIIS4 transmembrane segments. (A–C) Alpha helix wheel diagrams of the WT DIVS4 transmembrane segment (A), the ΔVIR mutation (B), and the ΔIRL mutation (C). (D–F) Alpha helix wheel diagrams of the WT DIIS4 transmembrane segment (D), the Δ9 mutation (E), and the ∇3 mutation (F) from Inglis et al., 2020. The alpha helix wheel diagrams were generated using NetWheels (http://lbqp.unb.br/NetWheels/).