Background:
CACNA1A-related disorders encompass a broad spectrum of neurological, neuropsychiatric, and neuromuscular dysfunctions caused by altered function of the Cav2.1 calcium channel encoded by CACNA1A. Over one thousand CACNA1A variants have been reported in ClinVar; however, approximately 72% of the 1998 missenses variants are classified as variants of uncertain significance (VUS).
Aim:
To establish an automated patch clamp assay for Cav2.1 and then determine the sensitivity and specificity of the assay for differentiating between known benign and pathogenic variants.
Method:
A double Flp-In method was employed to achieve stable expression of human CACNA1A, CACNB4, and CACNA2D1 in HEK293 cells (Jensen et al., 2020). Stable colonies were selected using hygromycin and puromycin. Electrophysiological properties were assessed using automated patch clamping (SyncroPatch 384PE). Measured parameters included current density (pA/pF), steady-state half-activation (V0.5 act) and half-inactivation (V0.5 inact) voltages, and time for 50% (T₅₀) of channels to recover from inactivation (RFI).
Results:
All 27 benign controls fell within the ±3 Z-score range (i.e., functionally normal) across all four parameters, whereas 29 of 30 pathogenic controls fell outside this range for at least one parameter. The odds of pathogenicity (OddsPath) analysis indicated that the assay provides strong level of evidence both for benign (BS3) and pathogenic (PS3) variants. The assay was then tested on an independent set of variants provided by the Victorian Clinical Genetics Services (VCGS). 5/5 VCGS pathogenic variants had abnormal function. In addition of the 10 VUS tested, six had abnormal function, supporting potential reclassification based on functional data.
Conclusion:
We have developed a high throughput functional genomic assay for CACNA1A variants that can provide strong level evidence to assist with classification of clinical variants.