SCN1A is the most recurrently mutated gene in patients with epilepsy, accounting for 90% of Dravet syndrome, and ~2% of severe early-onset epilepsies. Despite extensive studies, including exome sequencing,10% of patients with Dravet syndrome do not have a definitive molecular diagnosis. We performed targeted resequencing outside of the SCN1A coding regions and identified five individuals, four of whom had Dravet syndrome, with likely pathogenic variants in an intronic SCN1A region. This highly conserved intronic region contains an exon (20N) that results in a truncated SCN1A protein when included in the transcript. Normally, 20N is only included in non-neuronal cells, where SCN1A is not expressed, but we have no knowledge of the mechanisms or proteins that control splicing of this ‘poison’ exon. We hypothesized that the patient-specific variants disrupt the binding of proteins that control normal splicing of this exon in neurons, leading to aberrant 20N inclusion and a truncated SCN1A protein in these patients.Supported by a Junior Investigator Award from the American Epilepsy Society we established induced pluripotent stem (iPS) cell lines from 3/5 patients and differentiated these iPSCs to neurons. Moreover, using a minigene splicing assay we show that the presence of three of the patient variants lead to aberrant inclusion of exon 20N, while two do not. We hypothesize that this lack of 20N inclusion for two patients-specifc variants may be due to cell-specific splicing factors. In this study, our goal is to build on these results to further characterize the splicing mechanisms that may underlie the aberrant inclusion of exon 20N and investigate functional and physiological properties in neuronal cells.
|Effective start/end date||1/1/19 → 12/31/20|
- Dravet Syndrome Foundation, Inc. (AGMT 1/16/19)