Aberrant regulation of a poison exon caused by a non-coding variant in Scn1a -associated epileptic encephalopathy

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Na1.1 sodium channel encoded by . Most known DS-causing mutations are in coding regions of , but we recently identified several disease-associated mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved “poison” exon, 20N, whose inclusion leads to transcript degradation. However, it is not clear how these intron 20 variants alter transcript processing or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. heterozygous knock-in () mice exhibited an ~50% reduction in brain mRNA and Na1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult +/+ animals, quantitative RT-PCR assays indicated that ~1% of mRNA included exon 20N, while brain tissue from mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Na1.6, encoded by . For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by and is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.