Mutations in the cardiac L-type calcium channel associated with inherited J-wave syndromes and sudden cardiac death

Abstract

Background: L-type calcium channel (LTCC) mutations have been associated with Brugada syndrome (BrS), short QT (SQT) syndrome, and Timothy syndrome (LQT8). Little is known about the extent to which LTCC mutations contribute to the J-wave syndromes associated with sudden cardiac death.

Objective: The purpose of this study was to identify mutations in the α1, β2, and α2δ subunits of LTCC (Ca(v)1.2) among 205 probands diagnosed with BrS, idiopathic ventricular fibrillation (IVF), and early repolarization syndrome (ERS). CACNA1C, CACNB2b, and CACNA2D1 genes of 162 probands with BrS and BrS+SQT, 19 with IVF, and 24 with ERS were screened by direct sequencing.

Methods/results: Overall, 23 distinct mutations were identified. A total of 12.3%, 5.2%, and 16% of BrS/BrS+SQT, IVF, and ERS probands displayed mutations in α1, β2, and α2δ subunits of LTCC, respectively. When rare polymorphisms were included, the yield increased to 17.9%, 21%, and 29.1% for BrS/BrS+SQT, IVF, and ERS probands, respectively. Functional expression of two CACNA1C mutations associated with BrS and BrS+SQT led to loss of function in calcium channel current. BrS probands displaying a normal QTc had additional variations known to prolong the QT interval.

Conclusion: The study results indicate that mutations in the LTCCs are detected in a high percentage of probands with J-wave syndromes associated with inherited cardiac arrhythmias, suggesting that genetic screening of Ca(v) genes may be a valuable diagnostic tool in identifying individuals at risk. These results are the first to identify CACNA2D1 as a novel BrS susceptibility gene and CACNA1C, CACNB2, and CACNA2D1 as possible novel ERS susceptibility genes.

Figure 1

Representative ECGs of Brugada syndrome (BrS), BrS with shorter than normal QT (BrS/SQT), and early repolarization syndrome (ERS). Arrows denote type I ST-segment elevation in the BrS patients and early repolarization pattern in the ERS patient.

Figure 2

Pedigree of the available families for CACNA1C and CACNB2 mutations. BrS = Brugada syndrome; BrS/SQT = BrS with shorter than normal QT; ER = early repolarization pattern; ERS = early repolarization syndrome; IVF = idiopathic ventricular fibrillation. + = heterozygous for the mutation; ++ = homozygous for the mutation. Arrows indicates proband. Numbers represent the Masonic Medical Research Laboratory ID number. Asterisk denotes previously published mutations.,

Figure 3

Predicted topology of the Cav1.2 (β1c) subunit with associated β2 and α2δ subunits shows the location of the mutations. AID and BID show the position of interaction of α1c and β2 subunits and the position of the α-subunit interaction domain (AID) and β-subunit interaction domain (BID). GK = guanylate kinase–like domain, SH3 = Src homology domain; Src-. Larger symbols with numbers denote multiple probands with the same mutation. Asterisk denotes previously published mutations.,

Figure 4

A: ECG recorded with leads V₁–V₃ of patient #219 before and after procainamide. B: Electropherogram of wild-type (WT) and mutant CACNA1C gene showing heterozygous transition c.6040 G>A predicting replacement of valine by isoleucine at position 2014. C: Amino acid sequence alignment showing that valine at position 2014 is highly conserved among multiple species.

Figure 5

The p.V2014I-CACNA1C mutation causing a loss of function of I_Ca together with a p.D601E-CACNB2b single nucleotide polymorphism causing a gain of function of late I_Ca, result in Brugada syndrome (BrS) with normal QTc (MMRL219). A: Representative calcium current traces recorded in Chinese hamster ovary (CHO) cells transfected with wild-type (WT; left) or p.V2014I (right) CACNA1C subunits in response to the voltage clamp protocol shown at the top. B: I_Ca recorded in response to the inactivation protocol shown. C: Overlapping calcium traces recorded from human embryonic kidney (TSA201) cells expressing WT and p.D601E-CACNB2b rare polymorphism. D: Current–voltage relationship. E: Activation curve showing conductance–voltage. F: Normalized inactivation curves in WT or p.V2014I CACNA1C. G: Bar graph showing I_Ca current density recorded with WT versus p.D601E CACNB2b at different times (100, 200, and 300 ms) into the depolarized testing pulse at 0 mV (protocol inset). *P <.05, **P <.01 vs WT data. Each datapoint/bar represents mean ± SEM of 6–8 experiments.

Figure 6

A: Duplication of five amino acids in Cav1.2 leads to loss of function of I_Ca resulting in Brugada syndrome (BrS) with shorter than normal QT interval (QTc = 346 ms). ECG recorded in leads V₁–V₃ of patient #300 at baseline. B: Electropherogram of wild-type (WT) and mutant CACNA1C showing duplication of five amino acids EETSQ. C: Representative calcium current traces recorded in human embryonic kidney (TSA201) cells transfected with WT (left) and p.E1829_Q1833-dup mutant (right) CACNA1C subunits by applying the protocol shown at the top. D: Current–voltage relationship (I–V curve) p.E1829_Q1833-dup mutant effect in Cav1.2 channels. Data are given as mean ± SEM of at least eight cells. *P <.05.