Epilepsy-associatedSCN2A(Na_V1.2) Variants Exhibit Diverse and Complex Functional Properties

Abstract

Pathogenic variants in neuronal voltage-gated sodium (NaV) channel genes including SCN2A, which encodes NaV1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and non-syndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A 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 SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A 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 NaV1.2 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 NaV1.2 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 NaV channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.

Biophysics of sodium channels during subthreshold depolarizationin vitroandin silico

“…When reviewing raw data, several of these outliers appeared to result of measurement artifacts, so that it might be worthwhile to amend an outlier analysis. To compensate for outliers destabilizing statistical results, usage of high-throughput, automated patch-clamp systems could also be considered, as they offer a time-efficient method to gather large data sets (Kriegeskorte et al 2023, Thompson et al 2023).…”

“…Larger studies which compare VGSC gating in the same experimental setting were focusing either on CNS VGSCs and their mutations leading e.g. to epilepsy (Thompson et al 2023), or on sensory neuron sodium channels and their temperature dependence (Kriegeskorte et al 2023).…”

Electroencephalographic insights into variant function and clinical outcomes inSCN2Aencephalopathy

“…Conversely, a variant located in a domain known to be enriched for population variants 34 or situated in flexible regions of the protein provides benign evidence. Lastly, in alignment with the ACMG guidelines, significant functional changes of a variant were assessed in an established electrophysiological readout (for details see 34,35 ) as evidence for pathogenicity. A lack of functional change was considered evidence that the variant is benign.…”

“…As a fourth criterion, information on Na V 1.2 domain function identified in previous structure-to-function analyses was included as additional classification evidence. 34 Fifth, electrophysiological readouts of previously tested variants 34,35 were mapped to the set of missense variants from this SCN2A cohort. If these readouts were interpreted by the authors as definitive GoF or LoF effects across all tested splice forms, it was considered strong evidence for the functional effect.…”

No Pain, No Gain of Function: Epilepsy-Associated Variants in SCN2A Defy Classification

“…It is also possible that such variants impact more than biophysical properties, perhaps altering subcellular localization or interaction with other associated proteins in the Naþ channel macromolecular complex (including potential Nav-Nav interactions). 6 Limitations of the Thompson et al 3 work are otherwise minor. First, certain more subtle types of channel dysfunction might be missed using the automated approach, such as changes in resurgent current or induction of gating pore currents.…”