An African loss-of-function CACNA1C variant p.T1787M associated with a risk of ventricular fibrillation

Abstract

Calcium regulation plays a central role in cardiac function. Several variants in the calcium channel Ca_v1.2 have been implicated in arrhythmic syndromes. We screened patients with Brugada syndrome, short QT syndrome, early repolarisation syndrome, and idiopathic ventricular fibrillation to determine the frequency and pathogenicity of Ca_v1.2 variants. Ca_v1.2 related genes, CACNA1C, CACNB2 and CACNA2D1, were screened in 65 probands. Missense variants were introduced in the Ca_v1.2 alpha subunit plasmid by mutagenesis to assess their pathogenicity using patch clamp approaches. Six missense variants were identified in CACNA1C in five individuals. Five of them, A1648T, A1689T, G1795R, R1973Q, C1992F, showed no major alterations of the channel function. The sixth C-terminal variant, Ca_vα_1c-T1787M, present mostly in the African population, was identified in two patients with resuscitated cardiac arrest. The first patient originated from Cameroon and the second was an inhabitant of La Reunion Island with idiopathic ventricular fibrillation originating from Purkinje tissues. Patch-clamp analysis revealed that Ca_vα_1c-T1787M reduces the calcium and barium currents by increasing the auto-inhibition mediated by the C-terminal part and increases the voltage-dependent inhibition. We identified a loss-of-function variant, Ca_vα_1c-T1787M, present in 0.8% of the African population, as a new risk factor for ventricular arrhythmia.

Figure 1

Family pedigrees of Cases 1 to 5 (A–E) with the members carrying CACNA1C variants. The probands are indicated by an arrow. Males are represented by squares, females by circles, affected subjects by filled symbols, and healthy subjects by open symbols. Ca_vα_1c missense variants or their absence (no) are indicated below each subject that has been genotyped.

Figure 2

Electrocardiograms of Cases 1 and 2. Case 1, (A) just after reanimation showing early repolarisation pattern, (B) 5 years later. Case 2, (C) short-coupled torsades de pointes, (D) short-coupled premature ventricular beats.

Figure 3

Loss-of-function of Ca_vα_1c-T1787M variant: calcium currents (I_Ca). (A) Representative whole-cell I_Ca traces showing the decrease of current density with the Ca_vα_1c-T1787M variant compare to WT. (B) Current-voltage relationships in cells transfected with either WT (○) or Ca_vα_1c-T1787M variants (●) of calcium channels (*p < 0.05). (C) Steady-state inactivation and activation curves of either WT (white symbol) or Ca_vα_1c-T1787M variants (back symbol) of calcium channels showing no major alterations. (D) Calcium-dependent inactivation (CDI) with either WT (○) or Ca_vα_1c-T1787M variants (●) of calcium channels showing no alterations. The number of cells is indicated in parentheses.

Figure 4

Loss-of function of Ca_vα_1c-T1787M variant: barium currents (I_Ba). (A) Representative whole-cell I_Ba traces showing the decrease of current density with the Ca_vα_1c-T1787M variant compare to WT. (B) Current-voltage relationships in cells transfected with either WT (○) or Ca_vα_1c-T1787M variants (●) of calcium channels (*p < 0.05). (C) Steady-state inactivation and activation curves of either WT (white symbol) or Ca_vα_1c-T1787M variant (back symbol) of calcium channels showing no major alterations. (D) Voltage-dependent inactivation (VDI) with either WT (○) or Ca_vα_1c-T1787M variant (●) of calcium channels showing an increase of the VDI (*p < 0.05 and **p < 0.01). The number of cells is indicated in parentheses.

Figure 5

Scheme of the C-terminal part of the voltage-gated calcium channel. Schematic representation modified from Lyu et al. 2017 showing the C-terminal domain of Ca_vα_1c with EF-hand like region (EF), pre-IQ, IQ, PCRD, DCRD, and CaM-competitive domain (CCD). The interaction between the PCRD and the DCRD forming the auto-inhibitory complex and the different sites of interaction with CaM (pre-IQ, IQ, and CCD) are depicted with dashed arrows. Asterisks correspond to human loss-of-function variants (present data, red). The in vivo cleavage site by proteolysis correspond to 1764-NANINNANN-1772 in human. The scissors show the position of the truncation of Ca_vα_1c subunit performed in this study (A1770, 1800 in rabbit).

Figure 6

The I_Ba inhibition mediated by the cleaved CT part on the CT-cleaved Ca_vα_1c construct is stronger when the Ca_vα_1c-T1787M variant is present. (A) Representative raw traces of I_Ba density recorded in presence of CT-cleaved Ca_vα_1c alone or with either WT or T1787M-cleaved CT part. (B) I-V relationships showing the effect mediated by T1787M variant on the CT-cleaved Ca_vα_1c when introduced in the cleaved CT part (*p < 0.05: CT-cleaved Ca_vα_1c + WT-cleaved CT vs CT-cleaved Ca_vα_1c + T1787M-cleaved CT). The number of cells is indicated in parentheses.