Functional Annotation of a Comprehensive Set of SCN2A Variants

We propose to develop and test a strategy to predetermine which amino acid substitutions in SCN2A lead to altered function in a high-throughput pooled screen. This strategy will couple saturation mutagenesis of genetic ‘hot spots’ and next-generation sequencing with an assay to determine the ability of cells transfected with a missense variant of SCN2A to survive when exposed to a potent NaV channel activator. The goal of this pilot study is to demonstrate proof-of-principle that we can create a comprehensive database for all possible substitutions in SCN2A that cause altered function. If successful, this approach will generate an allele characterization framework that can be scaled to accomplish genome-guided genotype-phenotype mapping.