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

Finol-Urdaneta, R. K.; Tan, C.-Y.; Maksemous, N.; Ma, J. G.; Lockhart, P.; Snell, P.; Malhotra, A.; Thompson, B. A.; Garg, G.; Goel, H.; Griffiths, L. R.; Adams, D. J.; Vandenberg, J. I.; Ng, C. A.

doi:10.64898/2026.05.10.724145

bioRxiv · 10.64898/2026.05.10.724145

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

Finol-Urdaneta, R. K.·Tan, C.-Y.·Maksemous, N.·Ma, J. G.·Lockhart, P.·Snell, P.·Malhotra, A.·Thompson, B. A.·Garg, G.·Goel, H.·Griffiths, L. R.·Adams, D. J.·Vandenberg, J. I.·Ng, C. A.

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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.

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Finol-Urdaneta, R. K., Tan, C.-Y., Maksemous, N., Ma, J. G., Lockhart, P., Snell, P., Malhotra, A., Thompson, B. A., Garg, G., Goel, H., Griffiths, L. R., Adams, D. J., Vandenberg, J. I., Ng, C. A.. 2026-05-18. Calibrated high-throughput electrophysiology enables clinical interpretation of CACNA1G missense variants. https://doi.org/10.64898/2026.05.10.724145

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