Abstracts

Rationale: CACNA1E encodes the pore-forming a_1E-subunit of the high voltage-activated Ca_v2.3 R-type Ca²⁺channel, which is widely expressed at neuronal presynapses and plays an important role in synaptic transmission. While pathogenic variants in several Ca²⁺channels, including CACNA1A, CACNA1C, and CACNA1D, cause neurologic and/or cardiac disorders, CACNA1E has not clearly been linked with human disease. Here, we explore the clinical and functional consequences of CACNA1E variants in patients with Developmental and Epileptic Encephalopathy (DEE). Methods: Patients with de novo likely pathogenic CACNA1E variants identified via Next Generation Sequencing were ascertained and phenotyped via an international collaborative network of research- and diagnostic sequencing laboratories. Functional effects of selected variants were examined using whole cell patch clamp recordings of mammalian tsA201 cells that were transiently transfected with either the pore-forming wild type or mutant a_1E-subunit, along with the auxiliary ß_2d-subunit. Results: We identified 14 unique CACNA1E missense variants in 26 patients with DEE (median age 5 years; range 11 months – 25 years), characterized by refractory infantile-onset epilepsy, prominent hypotonia, and profound global developmental impairment. Seizures were reported in 24/26 (92%) patients. Median age of seizure onset was 5 months (range 1 day – 47 months), and the most common presenting seizure type was epileptic spasms (14/24; 58%) and then focal motor seizures (6/24; 25%). Congenital contractures were observed in 10/26 (38%) patients, ranging from talipes equinovarus to arthrogryposis. Congenital macrocephaly in the setting of normal neuroimaging was reported in 7/16 (44%) patients for whom data were available. In 6/26 patients, disease progression led to premature death (median age at death 2.9 years; range 11 months – 25 years). In 25/26 patients, the variants were confirmed to occur de novo. Four recurrent variants were observed in 17/26 patients. The majority (11/14; 79%) of variants are located in one of the four S6 segments containing the channel’s gate. Functional analysis of three selected variants revealed highly consistent gain-of-function effects including (i) a -10 mV shift of the voltage-dependence of activation decreasing the activation threshold and (ii) a slowing of the inactivation time course increasing the Ca²⁺inward current. Very similar effects have been reported previously for two other variants detected in our cohort, which had been previously studied in the context of functional characterization of Ca²⁺channels including Ca_v2.3. Conclusions: CACNA1E-related encephalopathy can be distinguished from other genetic causes of DEE by profound hypotonia, often in conjunction with congenital arthrogryposis or other isolated contractures and macrocephaly. Functional analysis revealed a clear gain-of-function effect indicating facilitated and increased presynaptic Ca²⁺currents and synaptic transmission. Funding: Supported by the DFG Research Unit FOR 2715 (HL, IH) and by a Sigmund Kiener stipend of the Hertie Foundation to RJL.