Epilepsy-associated SCN2A (NaV1.2) variants exhibit diverse and complex functional properties

Abstract

Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A 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 SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method 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 variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.

Optimizing clinical interpretability of functional evidence in epilepsy-related ion channel variants

“…Moreover, as a result of our cross-channel approach, we were able to apply our calibration beyond SCN1A , which accounts for our benign variant dataset, to an additional 117 variants in SCN2A/3A/8A , which are the causative genes in up to an additional 11% of all genetic epilepsies in large exome studies. 1,2 Given the applicability of some functional insight for a majority of reported individuals with disease-causing SCN2A variants, as well as the rise of automated electrophysiological approaches in the research space, 15-18 we expect that systematic functional evaluations will facilitate diagnosis and expand clinical trial readiness.…”

“…Voltage clamp experiments are the standard in research for the functional study of voltage-gated ion channel variants, and emerging technologies are rapidly increasing scale and throughput of these assays. 15-18 Rigorously leveraging these data within the ACMG/AMP framework would increase available evidence for variant interpretation. However, reporting of results is sparse and heterogeneous.…”

The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data

“…These days, risk stratification of ion channelopathies is largely based on changes in the electrophysiological properties of ion channel variants. So far, such risk stratification has been performed not only for a selection of cardiac ion channelopathies, as already outlined in the Introduction section, but also for neuromuscular diseases with Na V 1.4 variants [ 87 ], hearing loss related to KCNQ4 variants [ 88 ], and encephalopathies, including schizophrenia, with CACNA1I variants [ 89 ], and epilepsy with SCN1A [ 90 ], KCNB1 [ 91 ], HCN1 [ 92 ], KCNQ2 [ 93 ], and SCN2A [ 94 ] variants. In the present study, we tested whether risk stratification for sinus bradycardia can be based on AP clamp experiments on transfected cells to compute the charge carried by (mutant) I f during the diastolic depolarization phase of a prerecorded human SAN AP, using this AP as command potential.…”

Clinical characteristics and genetic analysis of pediatric patients with sodium channel gene mutation-related childhood epilepsy: a review of 94 patients

“…It is obvious, then, that Nav1.2 electrophysiologic changes noted in SCN2A mutation-related EE are more complex. Studies have shown that many patients with epilepsy-related SCN2A mutations present with complex GoF or LoF characteristics, and some rare mutations share both characteristics ( 19 , 20 ). Hence, it is difficult to apply a simple scheme with respect to classification.…”