Elsevier

Heart Rhythm

Volume 7, Issue 12, December 2010, Pages 1872-1882
Heart Rhythm

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

https://doi.org/10.1016/j.hrthm.2010.08.026Get rights and content

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 (Cav1.2) 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 Cav 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.

Introduction

Sudden cardiac death (SCD) is often associated with inherited cardiac arrhythmia syndromes.1 Twenty-five percent of all unexplained sudden deaths may be due to inherited cardiac diseases such as Brugada syndrome (BrS), idiopathic ventricular fibrillation (IVF), and long QT syndrome (LQTS).2 BrS, early repolarization syndrome (ERS), and some forms of IVF represent a continuous spectrum of phenotypic expression that differ with respect to the magnitude and lead location of abnormal J-wave manifestations, which we and others have proposed be termed J-wave syndromes.3

The past decade has witnessed a veritable explosion of information linking inherited cardiac arrhythmia syndromes to cardiac ion channel mutations. BrS has been associated with mutations in seven genes classified as BrS1 through BrS7.4 Mutations in SCN5A, which encodes the Nav1.5 protein forming the α subunit of the sodium channel, have been associated with 11% to 28% of BrS probands by different groups.5 A genotype has not yet been identified in the majority of BrS probands. ERS has thus far been associated with one mutation in KCNJ8, a gene encoding the pore-forming subunit of the IK-ATP channel.6 Expression studies suggesting a functional effect of this mutation has recently been reported.7

Little is known about the contribution of calcium channel gene variations to the etiology of inherited cardiac arrhythmia syndromes. Splawski et al8, 9 first described gain-of-function mutations in CACNA1C, a gene encoding Cav1.2 protein that forms the α subunit of the L-type calcium channel (LTCC), associated with a multiorgan dysfunction causing long QT intervals, arrhythmias, and autism known as Timothy syndrome (LQT8). Our group first described loss-of-function mutations in the α and β subunits of the cardiac LTCC associated with BrS and shorter than normal QT intervals and SCD.10, 11

The LTCC is composed of four subunits: the main pore-forming α1 (Cav1.2) subunit, which determines the main biophysical and pharmacologic properties of the channel, and three auxiliary subunits, including a cytoplasmic β subunit, encoded by CACNB, α2δ encoded by CACNA2D, and a γ subunit, which is present in skeletal, but not cardiac, muscle.12, 13, 14 Although a number of isoforms for the auxiliary subunits have been identified, in this study we focused on β2 (CACNB2), the dominant isoform known to play an essential role in the voltage dependence of LTCC,15, 16 and the extracellular α2 and transmembrane δ1 (CACNA2D1), which are linked to each other via disulfide bonds.

Few data are available on the extent to which mutations in the various subunits of LTCC contribute to SCD, the extent to which they are associated with ST-segment elevation and QT abbreviation giving rise to the BrS and BrS+SQT phenotypes, and their pathogenicity. The present study sought to identify genetic variations in the α1, β2, and α2δ1 subunits of LTCC among probands diagnosed with BrS, ERS, and IVF and the extent to which they contribute to pathogenesis of these syndromes. We tested the hypothesis that mutations in LTCC genes are relatively common among probands diagnosed with these syndromes. We also examined the hypothesis that LTCC mutation-mediated BrS associated with a normal QTc is attributable to additional genetic variations known to prolong the QT interval.

Section snippets

Diagnosis

The probands and their family members were diagnosed as having BrS, BrS with shorter than normal QT (BrS/SQT), IVF, or ERS based on established criteria.17, 18, 19, 20 Diagnosis was made based on 12-lead ECG, personal history of syncope, seizures, or aborted cardiac death, and family history of SCD or arrhythmic events. BrS patients displayed a coved-type ST-segment elevation in at least one right precordial lead under baseline conditions or after sodium channel block challenge with ajmaline or

Clinical characteristics

A total of 205 unrelated probands enrolled at the Masonic Medical Research Laboratory (MMRL) inherited cardiac arrhythmia registry over the past 5 years diagnosed with BrS, BrS/SQT, IVF, or ERS and their families were included in the study. Figure 1 shows representative 12-lead ECGs from BrS, ERS, and BrS/SQT phenotypes. The cohort consisted of 152 probands diagnosed with BrS, 10 with BrS/SQT, 19 with IVF, and 24 with ERS. Demographic characteristics are given in Table 1. Average age ranged

Discussion

This study is the first comprehensive attempt to associate inherited cardiac arrhythmia syndromes with genetic variations in the cardiac LTCC. We identified 23 mutations in three genes encoding the three subunits of the LTCC in 25 unrelated probands and six rare polymorphisms in 17 additional probands diagnosed with BrS, BrS/SQT, IVF, or ERS. 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. The total

Study limitations

The LTCC subunit genes, especially CACNB, have multiple isoforms. Our focus on CACNB2 in this study may have resulted in an underestimation of linkage of LTCC mutations to inherited cardiac arrhythmia disease. Thus far, a total of seven genes have been identified as associated with BrS.40 Our findings of three BrS probands associated with mutations in highly conserved residues of CACNA2D1 suggest that it may be a new gene for BrS. In support of this hypothesis, our preliminary functional

Acknowledgments

We thank Judy Hefferon for creative work on the figures, Susan Bartkowiak for maintaining the genetics database, and Gabriel Caceres for DNA isolation. We also thank Drs. Nikolai Soldatov and Michael C. Sanguinetti for expression constructs.

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      The SCN5A gene is the most prevalent and is the only gene confirmed by definitive evidence to be associated with BrS, while the clinical utility of systematic screening of the other genes remains under scrutiny due to the lack of information on prevalence and function often leading to an inconclusive interpretation of laboratory findings. Next-generation sequencing has opened a “veritable floodgate of biological data of unknown clinical significance”2 to a point such that some investigators have suggested that, in the absence of functional data, the interpretation of variants is almost impossible.3 The second most frequent gene reported in association with BrS is CACNA1C, which encodes for the α-subunit of the voltage-gated calcium channel (locus BrS3).

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    Supported by Grant HL47678 from the National Heart, Lung, and Blood Institute, New York State and Florida Masonic Grand Lodges to Dr. Antzelevitch, and Grant SAF2008-04903 from the Spanish Ministry of Sciences to Dr. Delpón.

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