CACNA1C Mutations Research Articles

Novel SCN10A variants associated with Brugada syndrome.

The expression of sodium channel Nav1.8 in cardiac nervous systems has been identified, and variants of SCN10A that encodes Nav1.8 contribute to the development of Brugada syndrome (BrS) by modifying the function of Nav1.5 or directly reducing the sodium current. The aim of this study was to identify the frequency of SCN10A mutations in Japanese patients with BrS and to compare the phenotypical differences between patients with BrS and those who have other BrS-causative genes. This study involved 240 Japanese probands who were clinically suspected with BrS and were negative for mutations in major BrS-related genes. We screened for the SCN10A gene using a high-resolution melting method and direct sequencing. In addition, we compared the clinical characteristics among the probands with gene mutations in SCN10A, 6 probands with CACNA1C and 17 probands with SCN5A. We identified six SCN10A variant carriers (2.5%): W189R, R844H (in two unrelated probands), N1328K, R1380Q, and R1863Q. Five were male. Four were symptomatic: one died following sudden cardiopulmonary arrest at age 35, one suffered ventricular fibrillation, and two had recurrent syncope. Compared with BrS patients carrying SCN5A or CACNA1C mutations, although there were no significant differences among them, symptomatic patients in the SCN10A group tended to be older than those in the other gene groups. In six BrS probands who carried SCN10A variants, most experienced severe arrhythmic attacks. It is of clinical importance to screen SCN10A mutations in BrS, although the functional significance of these variants remains unclear.

Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome

Gain-of-function mutations in CACNA1C, encoding the L-type Ca2+ channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.

Novel Timothy syndrome mutation leading to increase in CACNA1C window current

Timothy syndrome (TS) is a rare multisystem genetic disorder characterized by a myriad of abnormalities, including QT prolongation, syndactyly, and neurologic symptoms. The predominant genetic causes are recurrent de novo missense mutations in exon 8/8A of the CACNA1C-encoded L-type calcium channel; however, some cases remain genetically elusive. The purpose of this study was to identify the genetic cause of TS in a patient who did not harbor a CACNA1C mutation in exon 8/A, and was negative for all other plausible genetic substrates. Diagnostic exome sequencing was used to identify the genetic substrate responsible for our case of TS. The identified mutation was characterized using whole-cell patch-clamp technique, and the results of these analyses were modeled using a modified Luo-Rudy dynamic model to determine the effects on the cardiac action potential. Whole exome sequencing revealed a novel CACNA1C mutation, p.Ile1166Thr, in a young male with diagnosed TS. Functional electrophysiologic analysis identified a novel mechanism of TS-mediated disease, with an overall loss of current density and a gain-of-function shift in activation, leading to an increased window current. Modeling studies of this variant predicted prolongation of the action potential as well as the development of spontaneous early afterdepolarizations. Through expanded whole exome sequencing, we identified a novel genetic substrate for TS, p.Ile1166Thr-CACNA1C. Electrophysiologic experiments combined with modeling studies have identified a novel TS mechanism through increased window current. Therefore, expanded genetic testing in cases of TS to the entire CACNA1C coding region, if initial targeted testing is negative, may be warranted.

Rare mutations of CACNB2 found in autism spectrum disease-affected families alter calcium channel function.

Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism.

Long QT syndrome type 8: novel CACNA1C mutations causing QT prolongation and variant phenotypes.

CACNA1C mutations have been reported to cause LQTS type 8 (LQT8; Timothy syndrome), which exhibits severe phenotypes, although the frequency of patients with LQT8 exhibiting only QT prolongation is unknown. This study aimed to elucidate the frequency of CACNA1C mutations in patients with long QT syndrome (LQTS), except those with Timothy syndrome and investigate phenotypic variants. CACNA1C gene screening was performed in 278 probands negative for LQTS-related gene mutations. Functional analysis of mutant channels using a whole-cell patch-clamp technique was also performed. Using genetic screening, we identified five novel CACNA1C mutations: P381S, M456I, A582D, R858H, and G1783C in seven (2.5%) unrelated probands. Seven mutation carriers showed alternative clinical phenotypes. Biophysical assay of CACNA1C mutations revealed that the peak calcium currents were significantly larger in R858H mutant channels than those of wild-type (WT). In contrast, A582D mutant channels displayed significantly slower inactivation compared with WT. The two mutant channels exerted different gain-of-function effects on calcium currents. In patients with LQTS, the frequency of CACNA1C mutations was higher than reported. Even without typical phenotypes of Timothy syndrome, CACNA1C mutations may cause QT prolongation and/or fatal arrhythmia attacks.

Gain‐of‐Function KCNH2 Mutations in Patients with Brugada Syndrome

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST segment elevation on electrocardiograms (ECGs) that predisposes patients to sudden cardiac death as a result of polymorphic ventricular tachyarrhythmia or ventricular fibrillation (VF). In BrS patients, except for SCN5A, mutations in other responsible genes are poorly elucidated. We identified 4 KCNH2 mutations, T152I, R164C, W927G, and R1135H, in 236 consecutive probands with BrS or Brugada-like ECG. Three of these mutation carriers showed QTc intervals shorter than 360 milliseconds and 1 experienced VF. We performed patch-clamp analyses on I(Kr) reconstituted with the KCNH2 mutations in Chinese hamster ovary cells and compared the phenotypes of the patients with different genotypes. Three mutations, R164C, W927G, and R1135H, increased I(Kr) densities. Three mutations, T152I, R164C, and W927G, caused a negative shift in voltage-dependent activation curves. Only the R1135H mutant channel prolonged the deactivation time constants. We also identified 20 SCN5A and 5 CACNA1C mutation carriers in our cohort. Comparison of probands' phenotypes with 3 different genotypes revealed that KCNH2 mutation carriers showed shorter QTc intervals and SCN5A mutation carriers had longer QRS durations. All KCNH2 mutations that we identified in probands with BrS exerted gain-of-function effects on I(Kr) channels, which may partially explain the ECG findings in our patients.

Illumina TruSight Cardiomyopathy Sequencing Panel: Application to hypertrophic cardiomyopathy

exon 8 mutations has been described, to date. In contrast to multiorgan disease caused by G406R either in exon 8A or 8, the G402S carrier manifested only an isolated cardiac phenotype with LQT and cardiac arrest. This isolated phenotype was suggested to be a cause of somatic mosaicism. We describe a teenage patient resuscitated from ventricular fibrillation and treated with ICD. She has no other organ manifestations, no syndactyly, her QTc is <480ms, and she has normal neurodevelopment. There is no family history of arrhythmias or sudden deaths. Targeted oligonucleotideselective sequencing of channelopathy genes revealed a de novo G402S in the exon 8 of CACNA1C. Direct sequencing of blood and oral mucosa derived DNA revealed an identical mutation peak suggesting the mutation is not expressed as mosaicism. The phenotype of our patient and the previously described patient show an isolated arrhythmia disease with no clinical manifestations of TS. Based on current evidence, we suggest the G402S mutation in CACNA1C should not be considered as causative for TS.

A CACNA1C Mutation that Causes a Subset of Timothy Syndrome Phenotypes Correlates

Background Timothy syndrome (TS) is a rare congenital long QT syndrome (LQTS) associated with extracardiac manifestations including craniofacial dysmorphia and dental abnormalities. The locus for TS is CACNA1C , which encodes the Ca V 1.2 L-type Ca 2+ channel, for which canonical mutations lead to a decrease in voltage-dependent inactivation (VDI). However, a recent report of a patient with LQTS in isolation and a CACNA1C mutation that did not affect VDI raised the question whether altered VDI is necessary for extracardiac phenotypes. In a patient with a maternally inherited microdeletion with a chromosomal translocation who presented with LQTS and associated ventricular tachyarrhythmias (Figure A), a subset of TS phenotypes, and a skeletal myopathy not readily explained by the translocation, we sought to identify a causative mutation for the TS phenotypes. Methods A candidate gene approach identified a mutation in CACNA1C that was absent in the mother. We performed electrophysiologic studies on the mutant and characterized CACNA1C expression in skeletal muscle with a mouse CACNA1C reporter line. Results We identified a glycine to arginine mutation at position 1911 (G1911R) in Ca V 1.2. Functional studies revealed that G1911R increased Ca V 1.2 channel availability (Figures B and C) and decreased VDI (Figure D). The CACNA1C reporter mouse showed no Ca V 1.2 expression in skeletal muscle. Conclusions We describe a CACNA1C mutation that leads to a subset of TS phenotypes. In the context of a recently described CACNA1C mutation that does not affect VDI in an LQTS patient without extracardiac phenotypes, these data suggest that the extracardiac phenotypes seen in TS require effects on VDI.

Missense Mutations in Plakophilin-2 Can Cause Brugada Syndrome Phenotype By Decreasing Sodium Current and Nav1.5 Membrane Localization

Background Brugada syndrome (BrS) is associated with loss of sodium channel function. Previous studies showed features consistent with sodium current (I Na ) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy ([AC]; or arrhythmogenic right ventricular cardiomyopathy [ARVC]). Experimental models showed correlation between loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced I Na . We hypothesized that PKP2 variants that reduce I Na could yield a BrS phenotype, even without cardiomyopathic features of AC. Methods and Results We searched for PKP2 variants in genomic DNA of 200 patients with BrS diagnosis, no signs of AC, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L, and MOG1 . We identified 5 cases of single amino acid substitutions. One (Q62K) was previously described in AC patients as a variant of unknown significance; 4 were unreported. In a family with multiple cases of syncope and/or suspect ECG, novel variant R635Q cosegregated with the phenotype in all affected relatives and was absent in the nonaffected ones. Mutations were tested in HL-1–derived cells endogenously expressing Na v 1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased I Na and Na v 1.5 at site of cell contact. These deficits were restored by transfection of wild-type PKP2 (PKP2-WT) but not of BrS-related PKP2 mutants. Similar results were obtained when cells were cotransfected with PKP2-WT and the BrS-related PKP2 variants, to mimic heterozygosity. Human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from a patient with PKP2 deficit showed drastically reduced I Na . The deficit was restored by transfection of WT but not BrS-related PKP2 variant R635Q. Superresolution microscopy in murine PKP2-deficient cardiomyocytes related I Na deficiency to reduced number of channels at the intercalated disk and increased separation of microtubules from the cell end. Conclusions This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

FGF12 is a candidate Brugada syndrome locus

Less than 30% of the cases of Brugada syndrome (BrS) have an identified genetic cause. Of the known BrS-susceptibility genes, loss-of-function mutations in SCN5A or CACNA1C and their auxiliary subunits are most common. On the basis of the recent demonstration that fibroblast growth factor (FGF) homologous factors (FHFs; FGF11-FGF14) regulate cardiac Na(+) and Ca(2+) channel currents, we hypothesized that FHFs are candidate BrS loci. The goal of this study was to test whether FGF12 is a candidate BrS locus. We used quantitative polymerase chain reaction to identify the major FHF expressed in the human ventricle and then queried a phenotype-positive, genotype-negative BrS biorepository for FHF mutations associated with BrS. We queried the effects of an identified mutant with biochemical analyses combined with electrophysiological assessment. We designed a novel rat ventricular cardiomyocyte system in which we swapped the endogenous FHF with the identified mutant and defined its effects on multiple ionic currents in their native milieu and on the cardiac action potential. We identified FGF12 as the major FHF expressed in the human ventricle. In 102 individuals in the biorepository, we identified a single missense mutation in FGF12-B (Q7R-FGF12). The mutant reduced binding to the NaV1.5 C terminus, but not to junctophilin-2. In adult rat cardiac myocytes, Q7R-FGF12, but not wild-type FGF12, reduced Na(+) channel current density and availability without affecting Ca(2+) channel function. Furthermore, the mutant, but not wild-type FGF12, reduced action potential amplitude, which is consistent with a mutant-induced loss of Na(+) channel function. These multilevel investigations strongly suggest that Q7R-FGF12 is a disease-associated BrS mutation. Moreover, these data suggest for the first time that FHF effects on Na(+) and Ca(2+) channels are separable. Most significantly, this study establishes a new method to analyze effects of human arrhythmogenic mutations on cardiac ionic currents.

Inhibition of Late Sodium Current by Mexiletine

Background— Timothy syndrome (TS) is a rare long-QT syndrome caused by CACNA1C mutations G406R in exon 8A (TS1) and G402S/G406R in exon 8 (TS2). Management of TS is a challenge and prognosis is poor. This study aimed to explore the inheritance pattern and mechanism of an I Na blocker, mexiletine, to improve clinical manifestations in TS. Methods and Results— A 2-year-old Chinese girl with a typical TS1 phenotype underwent candidate gene screening. Qualitative and quantitative cloning sequence and analyses for mosaicism were performed on family members. Therapeutic effects of mexiletine were evaluated using ECG and Holter monitoring. The electrophysiological effect of mexiletine was evaluated in a TS model using rabbit ventricular wedges. The proband with severe syndactyly and delayed language skills was identified harboring a G406R mutation in CACNA1C . Her baseline ECG showed markedly prolonged QTc, 2:1 AV block and macro-T wave alternans. G406R was absent in her mother but expressed in her father’s oral mucosa, sperm, and white blood cells, indicating a mosaic carrier. Although asymptomatic, he exhibited mild QTc prolongation (470–490 ms) and syndactyly. Mexiletine shortened QTc from 584 to 515 ms, blunted QT–RR relationship, and abolished 2:1 AV block and T wave alternans in the girl. In in vitro studies, mexiletine inhibited late I Na with IC 50 of 17.6±1.9 µmol/L and attenuated brady-dependent QT prolongation and reduced QT–RR slope in the TS model using BayK 8644. Conclusions— Mexiletine shortened QTc, attenuated QT–RR slope, abolished 2:1 AV block and T wave alternans in a TS1 patient and TS model via inhibition of late I Na .

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca2+ channel CaV1.2 in nonexcitable cells. How abnormal Ca2+ influx through CaV1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established CaV1.2 roles. Here, we show that CaV1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca2+ influx through CaV1.2 regulates jaw development. Cranial neural crest migration was unaffected by CaV1.2 knockdown, suggesting a role for CaV1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca2+ signals through CaV1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca2+ channels in nonexcitable cells during development.

L-Type Calcium Channel Mutations in Japanese Patients With Inherited Arrhythmias

Mutations in genes encoding the L-type cardiac calcium channel (LTCC) are associated with various types of inherited arrhythmias, including Brugada syndrome (BrS). However, the frequency in Asian populations remains unknown. This study aimed to elucidate disease-causing mutations in LTCC-related genes in Japanese patients diagnosed as BrS or idiopathic ventricular fibrillation (IVF), early repolarization syndrome, short QT syndrome, and compare them with those carrying SCN5A mutations. We screened CACNA1C and CACNB2b in 312 probands and compared the clinical characteristics between probands with gene mutations in CACNA1C or SCN5A. In results, we identified 6 CACNA1C mutations in 7 unrelated probands and SCN5A mutations in 20 probands. There were no CACNB2b mutation carriers. In topology, half of the mutations were located in the C-terminus. Among 7 CACNA1C mutation carriers, 2 were female and 3 were symptomatic; 2 patients were resuscitated from ventricular fibrillation, and 1 patient had syncope. Compared with SCN5A mutation carriers, there were no significant differences in the ECG characteristics. 2 of 3 symptomatic CACNA1C patients were female, but all female SCN5A mutation carriers remained asymptomatic. We identified 6 CACNA1C mutations in BrS and IVF patients and their phenotypes were varied. Although mutation frequency was not high, screening of LTCC channel genes may be clinically important to prevent unexpected sudden death.

Molecular and Functional Characterization of a Novel Pathogenic Substrate for Autosomal Dominant Long QT Syndrome Discovered by Whole Exome Sequencing, Genomic Triangulation, and Systems Biology

<h3>Background</h3> Long QT syndrome (LQTS) is the most common cardiac channelopathy with 13 elucidated long QT syndrome (LQTS) susceptibility genes. An estimated 25% of LQTS cases remain elusive genetically. We combined whole exome sequencing (WES), systems biology, and genomic triangulation to identify the pathogenic substrate responsible for nonsyndromic, LQT1-13 genotype negative, autosomal dominant LQTS in a multigenerational pedigree. <h3>Methods</h3> Genomic DNA was isolated from the symptomatic index case (QTc = 506 ms), unaffected father (QTc = 383 ms), and affected maternal aunt (QTc = 479 ms). DNA libraries were sequenced with 35× coverage, and the Mayo Clinic Bioinformatics Core aligned the samples. Common variants in dbSNP, 1000 genomes, or 200 BGI exomes were subtracted. Familial genomic triangulation and the published LQTS interactome were used to filter the remaining variants, which were then ranked using 3 disease network analysis algorithms (ToppGene, Endeavor, and SUSPECTS). The top ranking mutation was characterized functionally using whole-cell patch-clamp technique in a heterologous HEK293 expression system. <h3>Results</h3> After filtering, 6 missense mutations and 2 insertions/deletions remained. Direct DNA sequencing identified 1 variant, P857R-CACNA1C, which segregated with the disease phenotype within the pedigree. This variant was also ranked by all 3 disease network algorithms as the most probable LQTS susceptibility gene. This L-type calcium channel (LTCC) α-subunit mutation involves a highly conserved residue localizing to a critical PEST domain in the II-III linker, a region thought to signal for LTCC protein degradation. Functional expression of P857R-CACNA1C in HEK293 cells resulted in a gain of function with a 2.1-fold (–34 ± 7.5 pA/pF vs 16 ± 1.7 pA/pF, <i>P</i> <.05) increase in peak I_Ca,L at +10 mV compared with wild type, consistent with the prolonged QT interval phenotype. <h3>Conclusions</h3> The coupling of WES, genomic triangulation, and systems biology is a novel approach for the identification of potential disease-causing mutations. The identification of a non-Timothy syndrome, gain-of-function mutation in CACNA1C suggests that perturbations in the LTCC may be a bona fide pathogenic substrate for classic, autosomal dominant LQTS.

THE MOSAICISM GENETIC PATTERN FOR TIMOTHY SYNDROME WITH CACNA1C MUTATION G406R

ObjectivesTimothy syndrome (TS), also known as LQT8, is a rare and severe form of Long QT syndrome (LQTS) and most patients are caused by the CACNA1C mutation, G406R. So far...

Hallmarks of the channelopathies associated with L-type calcium channels : A focus on the Timothy mutations in Ca v1.2 channels

Within the voltage-gated calcium channels (Cav channels) family, there are four genes coding for the L-type Cav channels (Cav1). The Cav1 channels underly many important physiological functions like excitation–contraction coupling, hormone secretion, neuronal excitability and gene transcription. Mutations found in the genes encoding the Cav channels define a wide variety of diseases called calcium channelopathies and all four genes coding the Cav1 channels are carrying such mutations. L-type calcium channelopathies include muscular, neurological, cardiac and vision syndromes. Among them, the Timothy syndrome (TS) is linked to missense mutations in CACNA1C, the gene that encodes the Ca v1.2 subunit. Here we review the important features of the Cav1 channelopathies. We also report on the specific properties of TS–Ca v1.2 channels, which display non-inactivating calcium current as well as higher plasma membrane expression. Overall, we conclude that both electrophysiological and surface expression properties must be investigated to better account for the functional consequences of mutations linked to calcium channelopathies.

Screening for Copy Number Variation in Genes Associated With the Long QT Syndrome: Clinical Relevance

The aim of this study was to investigate, in a set of 93 mutation-negative long QT syndrome (LQTS) probands, the frequency of copy number variants (CNVs) in LQTS genes. LQTS is an inherited cardiac arrhythmia characterized by a prolonged heart rate-corrected QT (QTc) interval associated with sudden cardiac death. Recent studies suggested the involvement of duplications or deletions in the occurrence of LQTS. However, their frequency remains unknown in LQTS patients. Point mutations in KCNQ1, KCNH2, and SCN5A genes were excluded by denaturing high-performance liquid chromatography or direct sequencing. We applied Multiplex Ligation-dependent Probe Amplification (MLPA) to detect CNVs in exons of these 3 genes. Abnormal exon copy numbers were confirmed by quantitative multiplex PCR of short fluorescent fragment (QMPSF). Array-based comparative genomic hybridization (array CGH) analysis was performed using Agilent Human Genome 244K Microarrays to further map the genomic rearrangements. We identified 3 different deletions in 3 unrelated families: 1 in KCNQ1 and 2 involving KCNH2. We showed in the largest family that the deletion involving KCNH2 is fully penetrant and segregates with the long QT phenotype in 7 affected members. Our study demonstrates that CNVs in KCNQ1 and KCNH2 explain around 3% of LQTS in patients with no point mutation in these genes. This percentage is likely higher than the frequency of point mutations in ANKB, KCNE1, KCNE2, KCNJ2, CACNA1C, CAV3, SCN4B, AKAP9, and SNTA1 together. Thus, we propose that CNV screening in KCNQ1 and KCNH2 may be performed routinely in LQTS patients.

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

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. 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. 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. 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.

Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome

Recent studies have demonstrated an association between mutations in CACNA1c or CACNB2b and Brugada syndrome (BrS). Previously described mutations all caused a loss of function secondary to a reduction of peak calcium current ( I Ca). We describe a novel CACNB2b mutation associated with BrS in which loss of function is caused by accelerated inactivation of I Ca. The proband, a 32 year old male, displayed a Type I ST segment elevation in two right precordial ECG leads following a procainamide challenge. EP study was positive with induction of polymorphic VT/VF. Interrogation of implanted ICD revealed brief episodes of very rapid ventricular tachycardia. He was also diagnosed with vasovagal syncope. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 15 ion channel genes were amplified and sequenced. The only mutation uncovered was a missense mutation (T11I) in CACNB2b. We expressed WT or T11I CACNB2b in TSA201 cells co-transfected with WT CACNA1c and CACNA2d. Patch clamp analysis showed no significant difference between WT and T11I in peak I Ca density, steady-state inactivation or recovery from inactivation. However, both fast and slow decays of I Ca were significantly faster in mutant channels between 0 and + 20 mV. Action potential voltage clamp experiments showed that total charge was reduced by almost half compared to WT. We report the first BrS mutation in CaCNB2b resulting in accelerated inactivation of L-type calcium channel current. Our results suggest that the faster current decay results in a loss-of-function responsible for the Brugada phenotype.

Abstract 4413: Accelerated Inactivation of the L-type Calcium due to a Mutation in CACNB2b Underlies the Development of a Brugada ECG Phenotype

Background: Ion channelopathies are responsible for a number of genetic cardiac arrhythmia syndromes. Recent work demonstrated an association between mutations in CACNA1c and CACNB2b, the genes that encode the α and β subunits of the L-type calcium channel, and the Brugada syndrome (BrS). The mutations previously described all caused a loss of function secondary to a major reduction in peak calcium channel current (I Ca ). In the present study we describe a novel CACNB2b mutation associated with BrS in which loss of function was caused by accelerated inactivation of I Ca . Methods and Results: The proband, a 32 yo male, displayed a saddleback ST segment elevation in the right precordial leads that converted to a coved-type ECG following a procainamide challenge. His EP study was positive with double extrastimuli inducing polymorphic VT/VF. He was also diagnosed with vasovagal syncope. Genomic DNA was isolated from blood lymphocytes. All exons and intron borders of 12 ion channel genes were amplified and sequenced. The only mutation uncovered was a missense mutation (T11I) in CACNB2b. The effect of this mutation was studied by expression of WT or T11I CACNB2b in TSA201 cells co-transfected with WT CANCA1c and CACNA2d. Patch clamp analysis showed no difference in I Ca density between WT and T11I (17.9±1.8 vs 22.5±4.3 pA/pF, respectively at +20mV). Similarly, steady-state inactivation and channel recovery was not different between WT and T11I mutant channels. However, both the fast and slow decay of I Ca produced by T11I mutant were significantly faster compared to WT at potentials between −10 to +30 mV, suggesting a reduction in depolarizing current during the course of an action potential. Application of action potential voltage clamp pulses confirmed that T11I total charge was reduced by 42±2.3% compared to WT (p&lt;0.05). Conclusion: We report the first Brugada syndrome mutation in CaCNB2b resulting in accelerated inactivation of the L-type calcium channel. The T11I mutation caused a faster decay of cardiac L-type calcium current but did not significantly alter the magnitude of the peak current. Our results suggest that a reduced total charge carried by I Ca during the plateau of the action potential predisposes to the Brugada phenotype.