Epilepsy-associated SCN2A (Na V 1.2) Variants Exhibit Diverse and Complex Functional Properties

Abstract

Pathogenic variants in neuronal voltage-gated sodium (Na _V ) channel genes including SCN2A , which encodes Na _V 1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic 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 Na _V 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 _V 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 _V channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.

Fig. 1.. Functional validation of a training set of Na_V1.2 variants.

Average normalized whole-cell sodium currents of (A) WT Na_V1.2 (left), untransfected cells (right), (B) a set of population variants, and (C) known pathogenic variants representing loss-of-function (LOF, red) -and gain-of-function (GOF, blue) phenotypes. Average traces are from 5 to 65 cells.

Fig. 2.. Na_V1.2 variants alter whole-cell current density.

(A) Average deviation of whole-cell sodium current density from WT Na_V1.2 for population and disease-associated variants. (B) Average traces for WT Na_V1.2, a gain-of-function (GOF) variant Na_V1.2A-D1050V, and two loss-of-function (LOF) variants Na_V1.2A-A880S, and Na_V1.2A-F978L. (C) Volcano plot highlighting variants significantly different from WT. Red symbols denote loss-of-function and blue symbols denote gain-of-function with p < 0.05 (n = 5–79).

Fig. 3.. Na_V1.2 variants alter voltage-dependence of activation.

(A) Average deviation from WT Na_V1.2 for V_1/2 of activation (in mV). (B) Conductance-voltage curves showing a gain-of-function (Na_V1.2-K1260Q) and a loss-of-function (R1882L) variant. (C) Volcano plot highlighting variants significantly different from WT. Red symbols denote loss-of-function and blue symbols denote gain-of-function with p < 0.05 (n = 5–65).

Fig. 4.. Na_V1.2 variants alter voltage-dependence of inactivation.

(A) Average deviation from WT Na_V1.2 for V_1/2 of inactivation (in mV). (B) Steady-state inactivation curves showing a gain-of-function (Na_V1.2-R1626Q) and a loss-of-function (Na_V1.2-A1773T) variant. (C) Volcano plot highlighting variants significantly different from WT. Red symbols denote loss-of-function and blue symbols denote gain-of-function with p < 0.05 (n = 8–122).

Fig. 5.. Na_V1.2 variants alter inactivation time constants.

(A) Average deviation of inactivation time constant (tau) measured at 0 mV from WT Na_V1.2 for disease-associated variants. (B) Average traces for WT Nav1.2 and a GOF variant Na_V1.2-M1879T recorded at 0 mV. (C) Volcano plot highlighting variants significantly different from WT. Red symbols denote loss-of-function and blue symbols denote gain-of-function with p < 0.05 (n = 9–120).

Fig. 6.. Na_V1.2 variants alter inactivation ramp currents.

(A) Average deviation of ramp currents from WT Na_V1.2 for disease-associated variants. (B) Average traces for WT Na_V1.2 and a GOF variant Na_V1.2-M1879T. (C) Volcano plot highlighting variants significantly different from WT. Red symbols denote loss-of-function and blue symbols denote gain-of-function with p < 0.05 (n = 5–45).

Fig. 7.. Validation of difficult to record Na_V1.2 variants using manual patch clamp.

(A) Average whole-cell current traces of WT and three difficult to characterize variants (G882E, F978L, and Q1479P) recorded using manual patch-clamp. (B) Summary current voltage relationship (left), voltage-dependence of activation and inactivation (middle), and persistent current (right) of Na_V1.2 variants recorded using manual patch clamp. All data were from 9 to 19 cells.

Fig. 8.. Comparison of epilepsy associated variants in the adult and neonatal isoforms of Nav1.2.

Heat maps showing GOF (blue) and LOF (red) phenotypes measured for epilepsy associated Na_V1.2 variants in the (A) adult and (B) neonatal splice isoforms. Only properties which reached the threshold for statistical significance are highlighted.