Abstracts

Rationale: Dravet Syndrome (DS) is understood to be a disorder driven primarily by loss-of-function mutations in SCN1A, a gene encoding the NaV1.1 sodium channel. However, in 20% of cases, no mutation can be found in the SCN1A coding sequence. We hypothesize that mutations to non-coding DNA regulatory elements (REs) that control SCN1A regulation represent a secondary causal mechanism. Changes to REs can produce strong phenotypes, and there are instances of large genomic deletions affecting the Scn1a locus in mice, yet the role of gene regulation in DS is not well understood. Methods: We used the Cas9/CRISPR system to generate a mouse model harboring a deletion of the h1b non-coding RE of Scn1a. Three genotypes were investigated at all developmental time points: wild type (WT), deletion carrier (Scn1a-REdel+/-), and homozygous deletion (Scn1a-REdel-/-). We performed quantitative real-time PCR (qPCR) and RNA sequencing (RNA-seq) on P7 forebrain of all three genotypes to examine Scn1a transcription across coding and non-coding regions as well as to test for changes in expression of other genes. Gross expression levels of NaV1.1 in forebrain at postnatal days P14, P21, and P28 were characterized using western blot (WB) and immunohistochemistry (IHC). Regional distribution and expression of NaV1.1 was characterized by IHC of vibratome sections using antibodies directed against NaV1.1 and cell-specific markers. The seizure phenotype of young adult deletion carrier mice will be validated by electroencephalogram (EEG), and cognitive effects of reduced Scn1a expression will be described with first pass behavioral testing. Results: Mice expressing a homozygous deletion of RE demonstrated 0% survival after P28, indicating that homozygous deletion of the h1b RE is lethal. No early lethality effects have been noted in deletion carrier mice. Scn1a was the most significant differentially expressed gene, with dosage-sensitive down-regulation in h1b deletion brain. We did not identify significant transcription from the h1a or h1b Scn1a non-coding regions, leading to questions of how these regions function as promoter or enhancer elements. We did not identify a strong global signature of differential expression at P7, but did find suggestive evidence of pathology-related changes. We are currently evaluating Scn1a transcription and global expression changes via RNA-seq at later ages. WB analysis and IHC indicates NaV1.1 expression follows a dose-dependent relationship, with expression differences most pronounced at the later developmental time points (P21, P28). Behavioral and EEG experiments are ongoing. Conclusions: These data suggest we have created a novel alternative mouse model of DS that relies upon deletion of a non-coding regulatory element associated with SCN1A. Our model captures a possible mechanism for the 20% of DS cases in which no mutations in the coding regions of SCN1A are found. This work and extension of the approach to characterize other Scn1a REs has the potential to generate new insights about pathology and guide diagnosis and treatment of DS and Scn1a-related disorders in the future. Funding: Supported by the Dravet Syndrome Foundation (Award Number 201600552) and institutional startup funding provided by UC Davis Center for Neuroscience.