Identification of a novel loss-of-function calcium channel gene mutation in short QT syndrome (SQTS6)

Abstract

Aims: Short QT syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death. Thus far, mutations in five different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for an L-type calcium channel subunit.

Methods and results: The electrocardiogram of the affected member of a single family revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and symmetrical T-waves. Invasive electrophysiological testing showed short ventricular refractory periods and increased vulnerability to induce ventricular fibrillation. DNA screening of the patient identified no mutation in previously known SQTS genes; however, a new variant at a heterozygous state was identified in the CACNA2D1 gene (nucleotide c.2264G > C; amino acid p.Ser755Thr), coding for the Ca(v)α(2)δ-1 subunit of the L-type calcium channel. The pathogenic role of the p.Ser755Thr variant of the CACNA2D1 gene was analysed by using co-expression of the two other L-type calcium channel subunits, Ca(v)1.2α1 and Ca(v)β(2b), in HEK-293 cells. Barium currents (I(Ba)) were recorded in these cells under voltage-clamp conditions using the whole-cell configuration. Co-expression of the p.Ser755Thr Ca(v)α(2)δ-1 subunit strongly reduced the I(Ba) by more than 70% when compared with the co-expression of the wild-type (WT) variant. Protein expression of the three subunits was verified by performing western blots of total lysates and cell membrane fractions of HEK-293 cells. The p.Ser755Thr variant of the Ca(v)α(2)δ-1 subunit was expressed at a similar level compared with the WT subunit in both fractions. Since the mutant Ca(v)α(2)δ-1 subunit did not modify the expression of the pore-forming subunit of the L-type calcium channel, Ca(v)1.2α1, it suggests that single channel biophysical properties of the L-type channel are altered by this variant.

Conclusion: In the present study, we report the first pathogenic mutation in the CACNA2D1 gene in humans, which causes a new variant of SQTS. It remains to be determined whether mutations in this gene lead to other manifestations of the J-wave syndrome.

Figure 1

Twelve-lead electrocardiogram of the patient with short QT syndrome. (A) QT interval is 317 ms (QTc 329 ms) on resting surface electrocardiogram at presentation (paper speed 25 mm/s). (B) Surface electrocardiogram prior to and after flecainide challenge test. (1) Twelve-lead electrocardiogram shows incomplete right bundle branch block at baseline. (2) Tenminutes after flecainide (2 mg/kg) administration, a prominent notch in V1 in the early ST-segment (arrow) is depicted. Notably, the ST-segment becomes more convex. (3) Fifteen minutes after flecainide challenge, reduction in the QRS amplitude in V1 is observed. (4) Twenty minutes later, a prominent Q-wave is seen in V1_. Six hours later, the right precordial electrocardiogram changes returned to baseline.

Figure 2

Genetic analysis identified a novel CACNA2D1 mutation. (A) Electropherograms of wild-type (WT) and mutant CACNA2D1 gene showing a heterozygous transition c.2264G > C predicting replacement of serine by threonine at position 755 (p.Ser755Thr). (B) Amino acid sequence alignment showing that serine at position 755 is highly conserved among mammalian species. (C) Predicted topology of the L-type calcium channel Ca_vα₂δ-1 subunit showing the location of the S755T mutation (red circle) at the external carboxyl terminal of CACNA2D1. AID, α-subunit-interacting domain. BID, β-subunit-interacting domain.

Figure 3

(A) Pedigree of the reported family. (B) Right precordial electrocardiographic leads (V1, V2, and V3; 25 mm/s; electrodes were placed in the normal position) and the QTc intervals of the family members. Paternal grandmother had a previous anteroseptal myocardial infarction and paternal grandfather had an implanted pacemaker. The black arrows mark the index patient.

Figure 4

The barium current (I_Ba) is reduced by Ca_vα₂δ-1 p.Ser755Thr. (A) Western blot showing that the expression of all subunits is not modified in the condition where Ca_vα₂δ-1 p.Ser755Thr is expressed compared with the control condition (Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). (B) Representative whole-cell current traces at 0 mV during 200 ms. (C) I–V relationships recorded (protocol in inset) from HEK-293 cells transfected with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 channels (open circle) or with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p.Ser755Thr (filled circle). (D) Activation curves and steady-state inactivation curves recorded (protocol in inset) from HEK-293 cells transfected under the same condition of transfection as in (C) and fitted as mentioned in the Methods section. Activation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open circle) V_1/2= −10.5 ± 0.6 mV, K = 5.9 ± 0.4; Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p_.Ser755Thr (filled circle) V_1/2 = −8.4 ± 0.9 mV (*compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 6.1 ± 0.3 (n.s. compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). Steady-state inactivation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open diamond) V_1/2 = −33.1 ± 0.9 mV, K = 7.9 ± 0.3; Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p.Ser755Thr (filled diamond) V_1/2 = −30.6 ± 0.5 mV (**compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 8.2 ± 0.5 (n.s. compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). (E) I–V relationships recorded (protocol in inset) from HEK-293 cells transfected with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 channels (open circle) or with Ca_v1.2α1/Ca_vβ_2b (filled circle). (F) Activation curves and steady-state inactivation curves recorded (protocol in inset) from HEK-293 cells transfected in the same condition as in (E) and fitted as mentioned in the Methods section. Activation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open circle) V_1/2 = −10.5 ± 0.6 mV, K = 5.9 ± 0.4; Ca_v1.2α1/Ca_vβ_2b (filled circle) V_1/2 = −2.0 ± 0.6 mV (***compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 7.6 ± 0.3 (**compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). Steady-state inactivation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open diamond) V_1/2 = −33.1 ± 0.9 mV, K = 7.9 ± 0.3; Ca_v1.2α1/Ca_vβ_2b (filled diamond) V_1/2 = −26.0 ± 2.8 mV (*compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 12.4 ± 1.3 (**compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). The number of cells recorded is indicated in parentheses. *P < 0.05, **P< 0.01, and ***P< 0.001.

Figure 4

The barium current (I_Ba) is reduced by Ca_vα₂δ-1 p.Ser755Thr. (A) Western blot showing that the expression of all subunits is not modified in the condition where Ca_vα₂δ-1 p.Ser755Thr is expressed compared with the control condition (Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). (B) Representative whole-cell current traces at 0 mV during 200 ms. (C) I–V relationships recorded (protocol in inset) from HEK-293 cells transfected with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 channels (open circle) or with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p.Ser755Thr (filled circle). (D) Activation curves and steady-state inactivation curves recorded (protocol in inset) from HEK-293 cells transfected under the same condition of transfection as in (C) and fitted as mentioned in the Methods section. Activation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open circle) V_1/2= −10.5 ± 0.6 mV, K = 5.9 ± 0.4; Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p_.Ser755Thr (filled circle) V_1/2 = −8.4 ± 0.9 mV (*compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 6.1 ± 0.3 (n.s. compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). Steady-state inactivation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open diamond) V_1/2 = −33.1 ± 0.9 mV, K = 7.9 ± 0.3; Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p.Ser755Thr (filled diamond) V_1/2 = −30.6 ± 0.5 mV (**compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 8.2 ± 0.5 (n.s. compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). (E) I–V relationships recorded (protocol in inset) from HEK-293 cells transfected with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 channels (open circle) or with Ca_v1.2α1/Ca_vβ_2b (filled circle). (F) Activation curves and steady-state inactivation curves recorded (protocol in inset) from HEK-293 cells transfected in the same condition as in (E) and fitted as mentioned in the Methods section. Activation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open circle) V_1/2 = −10.5 ± 0.6 mV, K = 5.9 ± 0.4; Ca_v1.2α1/Ca_vβ_2b (filled circle) V_1/2 = −2.0 ± 0.6 mV (***compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 7.6 ± 0.3 (**compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). Steady-state inactivation curves: Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 (open diamond) V_1/2 = −33.1 ± 0.9 mV, K = 7.9 ± 0.3; Ca_v1.2α1/Ca_vβ_2b (filled diamond) V_1/2 = −26.0 ± 2.8 mV (*compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1), K = 12.4 ± 1.3 (**compared with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1). The number of cells recorded is indicated in parentheses. *P < 0.05, **P< 0.01, and ***P< 0.001.

Figure 5

Surface biotinylation assays were performed using Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 and Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 p.Ser755Thr subunit-transfected HEK-293 cells. (A) Western blots showing Ca_v1.2α1 and Ca_vα₂δ-1 subunits detected in the whole-cell lysates and (C) corresponding biotinylated fraction. (B and D) Bar graphs summarizing the effect of the mutation on the expression of Ca_v1.2α1 and Ca_vα₂δ-1 subunits in whole-cell lysates (B) and in biotinylated fractions (D). The number of independent experiments is indicated in parentheses; n.s., not significant compared with control cells transfected with Ca_v1.2α1/Ca_vβ_2b/Ca_vα₂δ-1 subunits.

Figure 6

Model of the sensory domain (residues 659–889) of CACNA2D1. (A) Helices, sheets, turns, and coil regions are coloured in red, cyan, green, and white, respectively. (B and C) The site of the p.Ser755Thr mutation is shown in space-filled presentation and is shown as an enlargement in the right panels. (B) Interactions of Ser755 and Val799 in the WT protein. The hydrophobic moieties of both side chains pack tightly, but no clashes are observed. Residue 755 is coloured by atom type and Val799 is depicted in yellow. (C) Interactions of Thr755 and Val799 in the mutant protein. The magenta arrow indicates clashes between the methyl groups of Thr755 and Val799. Colour coding as in (B).