Background-Cardiac ion channelopathies are responsible for an ever-increasing number and diversity of familial cardiac arrhythmia syndromes. We describe a new clinical entity that consists of an ST-segment elevation in the right precordial ECG leads, a shorter-than-normal QT interval, and a history of sudden cardiac death. Methods and Results-Eighty-two consecutive probands with Brugada syndrome were screened for ion channel gene mutations with direct sequencing. Site-directed mutagenesis was performed, and CHO-K1 cells were cotransfected with cDNAs encoding wild-type or mutant CACNB2b (Ca v␤2b ), CACNA2D1 (Ca v␣2␦1 ), and CACNA1C tagged with enhanced yellow fluorescent protein (Ca v 1.2). Whole-cell patch-clamp studies were performed after 48 to 72 hours. Three probands displaying ST-segment elevation and corrected QT intervals Յ360 ms had mutations in genes encoding the cardiac L-type calcium channel. Corrected QT ranged from 330 to 370 ms among probands and clinically affected family members. Rate adaptation of QT interval was reduced. Quinidine normalized the QT interval and prevented stimulation-induced ventricular tachycardia. Genetic and heterologous expression studies revealed loss-of-function missense mutations in CACNA1C (A39V and G490R) and CACNB2 (S481L) encoding the ␣ 1 -and ␤ 2b -subunits of the L-type calcium channel. Confocal microscopy revealed a defect in trafficking of A39V Ca v 1.2 channels but normal trafficking of channels containing G490R Ca v 1.2 or S481L Ca v␤2b -subunits. Conclusions-This is the first report of loss-of-function mutations in genes encoding the cardiac L-type calcium channel to be associated with a familial sudden cardiac death syndrome in which a Brugada syndrome phenotype is combined with shorter-than-normal QT intervals. Key Words: arrhythmia Ⅲ genetics Ⅲ electrophysiology Ⅲ tachycardia Ⅲ fibrillation C ardiac arrhythmias are responsible for an estimated 1 million cases of syncope and sudden cardiac death (SCD) among Europeans and Americans each year. 1 Cardiac arrhythmias can be acquired as a consequence of coronary heart disease or may be secondary to familial or inherited syndromes. The past decade has witnessed an explosion of information linking cardiac ion channel mutations with a wide variety of inherited arrhythmia syndromes. 2 The long-QT syndrome has been associated with 10 different Clinical Perspective p 449genes, in large part owing to the pioneering studies of Keating and coworkers. The LQT8 form of long-QT syndrome, also known as Timothy syndrome, is associated with gain-offunction mutations in cardiac calcium channel activity. 3,4 The cardiac L-type calcium channel is a protein complex formed by at least 3 subunits, ␣ 1 , ␤, and ␣ 2␦ . The pore-forming Ca v 1. subunit, encoded by CACNB2b, modulates calcium channel activity in the human heart and enables trafficking by suppressing an endoplasmic reticulum retention signal in the I-II loop of the ␣ 1 -subunit. 5 The short-QT syndrome (SQTS), a clinical entity first described in 2000,6 has been associated ...

BACKGROUND-Brugada syndrome (BrS) is a common heritable channelopathy. Mutations in the SCN5A-encoded sodium channel (BrS1) culminate in the most common genotype.

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.

Background-Brugada syndrome, characterized by ST-segment elevation in the right precordial ECG leads and the development of life-threatening ventricular arrhythmias, has been associated with mutations in 6 different genes. We identify and characterize a mutation in a new gene. Methods and Results-A 64-year-old white male displayed a type 1 ST-segment elevation in V1 and V2 during procainamide challenge. Polymerase chain reaction-based direct sequencing was performed using a candidate gene approach. A missense mutation (L10P) was detected in exon 1 of SCN3B, the ␤3 subunit of the cardiac sodium channel, but not in any other gene known to be associated with Brugada syndrome or in 296 controls. Wild-type (WT) and mutant genes were expressed in TSA201 cells and studied using whole-cell patch-clamp techniques. Coexpression of SCN5A/WTϩSCN1B/WTϩSCN3B/L10P resulted in an 82.6% decrease in peak sodium current density, accelerated inactivation, slowed reactivation, and a Ϫ9.6-mV shift of half-inactivation voltage compared with SCN5A/WTϩSCN1B/ WTϩSCN3B/WT. Confocal microscopy revealed that SCN5A/WT channels tagged with green fluorescent protein are localized to the cell surface when coexpressed with WT SCN1B and SCN3B but remain trapped in intracellular organelles when coexpressed with SCN1B/WT and SCN3B/L10P. Western blot analysis confirmed the presence of Na V ␤3 in human ventricular myocardium. Conclusions-Our

BACKGROUND Cardiac sodium channel β-subunit mutations have been associated with several inherited cardiac arrhythmia syndromes. OBJECTIVE To identify and characterize variations in SCN1Bb associated with Brugada (BrS) and sudden infant death syndromes (SIDS). METHODS AND RESULTS Patient 1 was a 44-y/o male with an ajmaline-induced Type-1 ST-segment elevation in V1 and V2 supporting the diagnosis of BrS. Patient 2 was a 62-y/o female displaying a coved-type BrS ECG who developed cardiac arrest during fever. Patient 3 was a 4-m/o female SIDS case. All known exons and intron borders of BrS and SIDS susceptibility genes were amplified and sequenced in both directions. A R214Q variant was detected in exon 3A of SCN1Bb (Navβ1B) in all three probands, but not in any other gene previously associated with BrS or SIDS. R214Q was identified in 4 of 807 ethnically-matched healthy controls (0.50%). Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using whole-cell patch-clamp and co-immunoprecipitation techniques. Co-expression of SCN5A/WT+SCN1Bb/R214Q resulted in peak sodium channel current (INa) 56.5% smaller compared to SCN5A/WT+SCN1Bb/WT ( n=11–12, p<0.05 ). Co-expression of KCND3/WT+SCN1Bb/R214Q induced a Kv4.3 current (Ito) 70.6% greater compared with KCND3/WT+SCN1Bb/WT(n=10–11, p<0.01). Co-immunoprecipitation indicated structural association between Navβ1B and Nav1.5 and Kv4.3. CONCLUSION Our results suggest that R214Q variation in SCN1Bb is a functional polymorphism that may serve as a modifier of the substrate responsible for Brugada or SIDS phenotypes via a combined loss of function of INa and gain of function of Ito.

Background Genetic defects in KCNJ8, encoding the Kir6.1 subunit of the ATP-sensitive K+ channel (IK-ATP), have previously been associated with early repolarization (ERS) and Brugada (BrS) syndromes. Here we test the hypothesis genetic variants in ABCC9, encoding the ATP-binding cassette transporter of IK-ATP (SUR2A), are also associated with both BrS and ERS. Methods and Results Direct sequencing of all ERS/BrS susceptibility genes was performed on 150 probands and family members. Whole-cell and inside-out patch-clamp methods were used to characterize mutant channels expressed in TSA201-cells. Eight ABCC9 mutations were uncovered in 11 male BrS probands. Four probands, diagnosed with ERS, carried a highly-conserved mutation, V734I-ABCC9. Functional expression of the V734I variant yielded a Mg-ATP IC50 that was 5-fold that of wild-type (WT). An 18-y/o male with global ERS, inherited an SCN5A-E1784K mutation from his mother, who displayed long QT intervals, and S1402C-ABCC9 mutation from his father, who displayed an ER pattern. ABCC9-S1402C likewise caused a gain of function of IK-ATP with a shift of ATP IC50 from 8.5±2 mM to 13.4±5 μM (p<0.05). The SCN5A mutation reduced peak INa to 39% of WT (p<0.01), shifted steady-state inactivation by −18.0mV (p<0.01) and increased late INa from 0.14% to 2.01% of peak INa (p<0.01). Conclusion Our study is the first to identify ABCC9 as a susceptibility gene for ERS and BrS. Our findings also suggest that a gain-of-function in IK-ATP when coupled with a loss-of-function in SCN5A may underlie type 3 ERS, which is associated with a severe arrhythmic phenotype.

Background-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 .

Background-Loss-of-function mutations in SCN5A have been associated with the Brugada syndrome. We report the first Brugada syndrome family with compound heterozygous mutations in SCN5A. The proband inherited 1 mutation from each parent and transmitted 1 to each daughter. Methods and Results-The effects of the mutations on the function of the sodium channel were evaluated with heterologous expression in TSA201 cells, patch-clamp study, and confocal microscopy. Genetic analysis revealed that the proband carried 2 heterozygous missense mutations (P336L and I1660V) on separate alleles. He displayed a coved-type ST-segment elevation and a prolonged PR interval (280 ms). One daughter inherited P336L and exhibited a prolonged PR (210 ms). The other daughter inherited mutation I1660V and displayed a normal PR interval. Both daughters had a slightly elevated, upsloping ST-segment elevation. The parents had normal ECGs. Patch-clamp analysis showed that the P336L mutation reduced I Na by 85% relative to wild type. The I1660V mutation produced little measurable current, which was rescued by room temperature incubation for 48 hours. Sodium channel blockers also rescued the I1660V current, with mexiletine proving to be the most effective. Confocal immunofluorescence showed that I1660V channels conjugated to green fluorescent protein remained trapped in intracellular organelles. Conclusions-Mutation P336L produced a reduction in cardiac I Na , whereas I1660V abolished it. Only the proband carrying both mutations displayed the Brugada syndrome phenotype, whereas neither mutation alone produced the clinical phenotype. I1660V channels could be rescued pharmacologically and by incubation at room temperature. The present data highlight the role of compound heterozygosity in modulating the phenotypic expression and penetrance of Brugada syndrome.