Calibrated high-throughput electrophysiology enables clinical interpretation of CACNA1G missense variants

Abstract

ObjectiveAccurate classification of ion channel variants of uncertain significance (VUS) remains a persistent challenge in clinical genomics, limiting diagnostic resolution in neurological disorders. MethodsWe developed a calibrated electrophysiological framework to generate functional evidence for clinical interpretation of CACNA1G variants encoding the low-voltage-activated calcium channel Cav3.1. Functional metrics derived from automated patchclamp recordings were calibrated against benign/likely benign (B/LB) and pathogenic/likely pathogenic (P/LP) reference variants to enable conservative application of ACMG/AMP functional criteria within clinical variant interpretation workflows. ResultsCalibration using 25 B/LB and 16 P/LP CACNA1G variants showed that more than 80% of P/LP variants exhibited reduced current density (CD). Deactivation kinetics ({tau}Deact) provided complementary discriminatory information by identifying gating abnormalities in variants with preserved CD. Application of this dual-metric framework to five VUS identified in Australian patients revealed two variants (Cav3.1-R186Q and R1394Q) with abnormal functional profiles consistent with voltage-sensor perturbation, supporting reassessment as likely pathogenic under ACMG/AMP guidelines. The remaining VUS displayed functional properties overlapping the benign reference distribution. ConclusionThese findings establish a calibrated functional framework for generating electrophysiological evidence that supports clinical interpretation of CACNA1G missense variants under ACMG/AMP guidelines. When applied as external functional evidence, this approach improves resolution of CACNA1G-associated VUS while maintaining conservative standards for variant classification.