A De Novo , Intragenic Modifier in Cis Rescues Scn8a -N1768D Epilepsy and Defines a Therapeutic Window Bounded by Gain- and Loss-of-Function

Gain-of-function variants in SCN8A , encoding Na _V 1.6, cause developmental and epileptic encephalopathy with early-onset, drug-resistant seizures. SCN8A is a “Goldilocks” gene: both excess and deficit of Na _V 1.6 are deleterious, so therapies that lower Na _V 1.6 must operate within a narrow window. Here we characterize a congenic C3H/HeJ.C57BL/6J colony carrying Scn8a -N1768D and identify a tightly linked, cis -acting modifier (L) that converts a severe phenotype into graded outcomes. Among N1768D homozygotes on a 98.4% C3H/HeJ.C57BL/6J background, three phenotypes segregate: short-lived (SL) mice with early lethal tonic-clonic seizures (TCS), long-lived (LL) mice with delayed onset and reduced seizure burden, and hindlimb-paralysis (HP) mice with early death. Heterozygotes separate into D/+ SL mice, which develop spontaneous TCS and die of epilepsy, and D/+ LL mice, which lack spontaneous TCS and epilepsy-related death. Genetic analysis places a second locus (L) tightly linked to Scn8a and co-segregating with phenotype. Hippocampal RNA-seq shows dose-dependent Scn8a mRNA reduction: none in SL (D+/D+), ∼30% in LL (DL/D+), and ∼70% in HP (DL/DL). Long-read trio DNA sequencing identifies a de novo 2-bp frameshift in coding exon 15 of Scn8a , in cis with N1768D, that introduces a premature stop codon ∼26 kb upstream of the 3′-most exon–exon junction, predicted to trigger nonsense-mediated decay. Allele-specific read counting confirms selective, cis -acting loss of DL transcripts with preserved D+ output. Thus, a de novo , intragenic frameshift variant titrates Na _V 1.6 through a Goldilocks window in which intermediate reduction rescues Gain-of-function epilepsy, whereas insufficient or excessive reduction remains lethal, validating Scn8a -lowering and its therapeutic dose window.