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, yet the role of gene regulation in DS is not well understood. Methods: Here, we investigate the biochemical and anatomical consequences of loss of an RE associated with SCN1A. We used the Cas9/CRISPR system to generate a mouse model harboring a deletion of this non-coding sequence. We then used quantitative real-time PCR (qPCR) and RNA-sequencing (RNA-seq) to determine the levels of Scn1a transcript in wild type (WT), RE-deletion carrier (Scn1a-REdel+/-), and homozygous RE-deletion (Scn1a-REdel-/-) animals. Distribution and expression levels of Nav1.1 in forebrain at P21 and P60 were characterized using western blot (WB), fluorescent immunohistochemistry (IHC), and laser scanning confocal microscopy (LSCM). Results: Homozygous deletion of the RE is lethal, with 0% survivability in Scn1a-REdel-/- mice after postnatal day 28 (P28), similar to Scn1a gene knockout lines. No early lethality effects have been seen so far in Scn1a-REdel+/- mice. Scn1a-REdel+/- and Scn1a-REdel-/- mice exhibit significant reduction in Scn1a transcript abundance in forebrain in a dose-dependent manner. LSCM analysis of Nav1.1-immunoreactivity in mouse forebrain indicates specific immunoreactivity in cortex, thalamus, and hippocampus. The intensity of antibody fluorescence and relative levels of Nav1.1 protein are consistent with RNA expression analysis. 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. We are currently characterizing and examining neurodevelopmental and behavioral phenotypes specific to this regulatory element. 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.