Characterization of the functional and clinical impacts of CACNA1A missense variants found in neurodevelopmental disorders

CACNA1A encodes the P/Q-type CaV2.1 calcium channels whose function underlies neuronal excitability, presynaptic neurotransmitter release, and Ca ²⁺ signaling in neurons. Pathogenic variants in CACNA1A have been found in individuals with various neurological conditions, including hemiplegic migraine, epilepsy, developmental delay, and ataxia. Clinical presentations can vary significantly between patients, with limited information known about the underlying neurobiology of these different clinical patterns. Adding further complication, prior work on pathogenic missense variants has demonstrated variable impacts on CaV2.1 channel function, sometimes in opposite directions. As such, the relationships between specific coding variants, electrophysiological properties, and clinical phenotypes remain elusive. In this study, we determined the biophysical properties of an allelic series of 42 de novo missense CACNA1A variants discovered in a neurodevelopmental disorder cohort of more than 31,000 individuals, together with the most common eight coding variants found in the general population. We found that all but one de novo variant altered at least one aspect of the channel properties examined, and the majority (70%) of the variants reduced the channel current density. In addition, for variants that encode human CaV2.1 channels (hCaV2.1) with detectable currents, nearly 50% altered how channels respond to membrane potential, while common variations did not significantly change any channel biophysical properties. Coupled with our functional analyses and AlphaMissense prediction, we showed that CaV2.1 missense variants significantly underlie the risk of developmental epileptic encephalopathy. Subsequently, we examined the physiological impact of variant hCaV2.1 using NEURON simulations as an omnibus output of neuronal function and found that abnormal biophysical channel properties have a profound impact on Purkinje cell excitability. Most interestingly, we correlated the clinical phenotype with molecular consequences of missense variants provided by our comprehensive functional analyses and found that distinct CaV2.1 channel molecular function is significantly associated with different clinical outcomes. By analyzing an entire allelic series of CACNA1A de novo changes in a large cohort of individuals with neurodevelopmental disorders, we provide a powerful approach to dissecting the role of missense variants in CACNA1A channelopathy, which in turn may help pave the way for future precision medicine initiatives.