A Novel De Novo Gain-of-Function CACNA1D Variant in Neurodevelopmental Disease With Congenital Tremor, Seizures, and Hypotonia

Abstract

Background and objectives: De novo gain-of-function variants in the CACNA1D gene, encoding the L-type voltage-gated Ca²⁺ channel Ca_V1.3, cause a multifaceted syndrome. Patients show variable degrees of autism spectrum disorder, developmental delay, epilepsy, and other neurologic and endocrine abnormalities (primary aldosteronism and/or hyperinsulinemic hypoglycemia). We study here a novel variant [c.3506G>A, NM_000720.4, p.(G1169D)] in 2 children with the same CACNA1D mutation but different disease severity.

Methods: The clinical data of the study patients were collected. After molecular analysis and cloning by site-directed mutagenesis, patch-clamp recordings of transfected tsA201 cells were conducted in whole-cell configuration. The functional effects of wild-type and mutated channels were analyzed.

Results: One child is a severely affected boy with a novel de novo CACNA1D variant with additional clinical symptoms including prenatal-onset tremor, congenital respiratory insufficiency requiring continuous positive airway pressure ventilation, and sensorineural deafness. Despite episodes of hypoglycemia, insulin levels were normal. Aldosterone:renin ratios as a screening parameter for primary aldosteronism were variable. In the second patient, putative mosaicism of the p.(G1169D) variant was associated with a less severe phenotype. Patch-clamp electrophysiology of the p.(G1169D) variant in a heterologous expression system revealed pronounced activity-enhancing gating changes, including a shift of channel activation and inactivation to more hyperpolarized potentials, as well as impaired channel inactivation and deactivation. Despite retained sensitivity to the Ca²⁺ channel blocker isradipine in vitro, no beneficial effects of isradipine or nifedipine treatment were observed in the index case.

Discussion: Through this report, we expand the knowledge about the disease presentation in patients with CACNA1D variants and show the novel variant's modulatory effects on Ca_V1.3 gating.

Figure 1. Ca_v1.3 Structure and Position of Identified De Novo Mutations

CACNA1D encodes the pore-forming Ca_V1.3 α1 subunit. Each of its 4 homologous repeats comprises 6 transmembrane helices. Helices S5 and S6 and their connecting loops of each repeat form the Ca²⁺-conducting ion pore, and helices S1-S4 the voltage-sensing domain. The auxiliary subunits α2δ and β support channel function and membrane targeting. p.(G1169D) found in the patients presented here is highlighted (G1169D) along with previously identified de novo pathogenic CACNA1D germline variants implicated in neurodevelopmental disease.

Figure 2. Biophysical and Pharmacologic Characterization of p.(G1169D)-Containing Human Ca_V1.3 Ca²⁺ Channels

Whole-cell patch-clamp recordings in tsA201 cells of transiently transfected wild-type (WT, black) or p.(G1169D) (grey/G1169D) Ca_V1.3 Ca²⁺ channel complexes (coexpressed with β3 and α2δ1, 15 mM Ca²⁺). Data were measured using a holding potential of −109.3 mV (A–E) and are given as mean ± SEM for the indicated number of recordings. Parameters and statistics are provided in eTables 1–3. (A) The p.(G1169D) variant shifted the voltage dependence of activation (G-V curve, circles) and steady-state inactivation (squares) by ∼35 mV toward more negative potentials. (B) This enabled a significantly increased constant background Ca²⁺ influx (“window current”) at subthreshold potentials. Two-way ANOVA (interaction, potential, genotype: p < 0.001) with the Šídák multiple comparison post hoc test as indicated. (C) During a prolonged (5 s) depolarization to the voltage of maximal activation (V_max), the p.(G1169D) variant displayed slower inactivation kinetics. Averaged current traces (mean ± SEM) are shown. (D–E) Deactivation kinetics of the p.(G1169D) variant were significantly slower, as shown by representative traces of tail currents elicited by 40-ms long repolarization from the reversal potential (V_rev) to −59.3 mV or −39.3 mV (D) and quantification of the normalized tail current area for all tested repolarization potentials (details on normalization in Methods). Two-way ANOVA (interaction, repolarization potential, genotype: p < 0.001) with the Šídák multiple comparison post hoc test as indicated. (F) Sensitivity to the L-type Ca²⁺ channel inhibitor isradipine (30 and 100 nM) was evaluated during 100-ms long depolarizations to the V_max (holding potential −89.3 mV, 0.1 Hz, 3 independent transfections). Normalized representative current traces for 30 nM and subsequent full block with 3-µM isradipine are shown (left, tail currents were cut). Inhibition was corrected for current run-down (details in Methods) and is presented as means ± SEM (right). No statistically significant difference in isradipine inhibition was observed between WT and p.(G1169D) Ca_V1.3 Ca²⁺ channels (two-way ANOVA). This provides class IV evidence. It is an observational study without controls.