Epilepsy-associated (Na 1.2) Variants Exhibit Diverse and Complex Functional Properties.

Pathogenic variants in neuronal voltage-gated sodium (Na ) channel genes including , which encodes Na 1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated variants have not been functionally annotated. We determined the functional properties of more than 30 variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of Na 1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of Na 1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of Na channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.