A benign control-calibrated multiparametric functional assay supports CACNA1A variant assessment

CACNA1A-related disorders arise from altered function of the P/Q-type calcium channel Cav2.1 and encompass a broad spectrum of neurological phenotypes, including developmental and epileptic encephalopathy, familial hemiplegic migraine type 1 and episodic ataxia type 2. Although loss-of-function and gain-of-function mechanisms have both been implicated, interpretation of rare CACNA1A missense variants remains challenging because clinical presentation is heterogeneous and many variants identified through diagnostic sequencing remain classified as variants of uncertain significance. To generate clinically interpretable functional evidence, we developed a calibrated high-throughput automated patch-clamp assay to assess the CACNA1A variants. Twenty-six curated benign/likely benign and 30 curated pathogenic/likely pathogenic missense variants were expressed in Flp-In HEK293 cells with CACNB4 and CACNA2D1. We quantified current density, {Delta}V0.5 activation, {Delta}V0.5 inactivation and recovery from inactivation (T0.5 RFI), and used benign controls to define parameter-specific normal ranges with {+/-}3 Z-score thresholds. This multi-parameter framework classified all benign controls as functionally normal and 29 of 30 pathogenic controls as abnormal, yielding 100% specificity and 96.7% sensitivity. Current density identified 19 pathogenic controls with loss of function, while gating and recovery from inactivation identified additional abnormal variants with preserved or partially preserved current density. The assay generated likelihood ratios of 0.033 for normal function and 25.13 for abnormal function, supporting BS3 and PS3 functional evidence, respectively. Application to 23 CACNA1A variants identified in Australian patients showed abnormal function in all four variants already classified as pathogenic or likely pathogenic. Among 19 variants of uncertain significance, 11 were functionally abnormal and ten were reclassified as likely pathogenic after integration of PS3 evidence with other ACMG/AMP criteria. These data show that CACNA1A missense variants can produce loss-of-function, gain-of-function or mixed effects, and that multi-parameter functional assessment is required for accurate interpretation. This calibrated automated patch-clamp framework provides clinically interpretable evidence for refining CACNA1A variant classification in diagnostic practice.