Abstracts

This is a Late Breaking abstract

Rationale: The expanding list of potential epilepsy genes comes the problem of genetic variants of uncertain significance (VUS).The number of VUS are growing for many genes as more patients undergo next-generation sequencing (NGS) but we lack in vivo tools to interpret VUS effects within these genes or study pathogenic mechanisms. Here, we use Cre in utero electroporation (IUE) mouse model to investigate its feasibility for VUS functional evaluation.

Methods: Cre-IUE was performed on floxed Stxbp1 transgenic mice to knockout Stxbp1 in forebrain excitatory neurons. These Stxbp1 null-background neurons were then used to validate specific STXBP1 antibodies in vivo and in vitro. Neurodevelopmental assays were performed to investigate the phenotype that can be used as the readout for VUS assays. Human STXBP1 pathogenic and benign variants were then expressed in null background neurons to determine their pathogenicity.   

Results: Cre-mediated Stxbp1 deletion was successfully achieved by Cre-IUE, and the lack of Stxbp1 expression was confirmed by specific antibody staining in vitro and in vivo. Stxbp1 knockout in cortical excitatory neurons caused cell death, which was then used as the readout for VUS function assay. Interestingly, both the pathogenic variant (C180Y) and wildtype STXBP1 successfully rescued cell death. However, rescued neurons with C180Y variant expression were dysmorphic and showed a markedly reduced dendritic complexity and increased soma circularity.   

Conclusions: Cre-IUE could be used as the in vivo platform to study the pathogenicity of VUS. Our data also suggested that the STXBP1 is not only crucial for presynaptic transmission but has a multifaceted function, including survival, dendritic growth, and polarity.  

Funding: U54NS117170