De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias

Abstract

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α₁-subunit of the voltage-gated Ca_V2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed Ca_V2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Keywords: CACNA1E, ion channel; arthrogryposis; calcium channel; epilepsy.

Figure 1

Secondary Structure of the Ca_v2.3 Channel with the Distribution of Disease-Causing Missense Variants Variants are highly clustered in the cytoplasmic ends of all four S6 transmembrane segments, which line the inner pore of the channel and form the activation gate of Ca_v2.3 and other voltage-gated cation channels. Numbers in parentheses indicate the number of individuals harboring the variant.

Figure 2

Functional Effects of CACNA1E (GenBank: NM_001205293.1) Domain II S6 Variants Introduced into α_1E-1 and Co-expressed with β_2d in tsA-201 Cells using 15 mM Ba²⁺ as Charge Carrier (A) Current traces elicited by depolarizing voltage pulses in 7.5-mV steps from a holding potential of −90 mV. (B) The voltage dependence of activation was similarly and highly significantly shifted for all three variants. (C) The residual current (r400) at +5 mV was determined by dividing the mean current of the last 10 ms of a 400-ms test pulse (I_res) by the peak current (I_Peak) of the same pulse. Numbers in parentheses denote the number of experimental readings per variant. Data are represented as means ± SEM (and in addition all measured values in C). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 3

Functional Effects of the CACNA1E (GenBank: NM_001205293.1) Domain II S4-S5 Variant p.Ile603Leu Introduced into α_1E-3 and Co-expressed with β_1b and α₂δ₁ in tsA-201 Cells (A) Current traces elicited by depolarizing voltage pulses in 5 mV steps from a holding potential of −100 mV as recorded with 2 mM Ca²⁺ as charge carrier. (B) Voltage dependence of activation recorded in 2 mM Ca²⁺ revealing a significant difference between WT and mutant channels. Numbers in parentheses denote the number of experimental readings per variant. (C) Mean peak current densities for WT and p.Ile603Leu channels recorded in 2 mM Ca²⁺ or 5 mM Ba²⁺. Data are represented as mean ± SEM. Numbers in parentheses denote the number of experimental readings per variant. Asterisks denote statistical significance (Student’s t test, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗∗p ≤ 0.0001).