Mutations in NaV1.5 Reveal Calcium-Calmodulin Regulation of Sodium Channel.

Mutations in the SCN5A gene, encoding the cardiac voltage-gated sodium channel NaV1.5, are associated with inherited cardiac arrhythmia and conduction disease. Ca2+-dependent mechanisms and the involvement of ß-subunit (NaVß) in NaV1.5 regulation are not fully understood. A patient with severe sinus-bradycardia and cardiac conduction-disease was genetically evaluated and compound heterozygosity in the SCN5A gene was found. Mutations were identified in the cytoplasmic DIII-IV linker (K1493del) and the C-terminus (A1924T) of NaV1.5, both are putative CaM-binding domains. These mutants were functionally studied in human embryonic kidney (HEK) cells and HL-1 cells using whole-cell patch clamp technique. Calmodulin (CaM) interaction and cell-surface expression of heterologously expressed NaV1.5 mutants were studied by pull-down and biotinylation assays. The mutation K1493del rendered NaV1.5 non-conductive. NaV1.5K1493del altered the gating properties of co-expressed functional NaV1.5, in a Ca2+ and NaVß1-dependent manner. NaV1.5A1924T impaired NaVß1-dependent gating regulation. Ca2+-dependent CaM-interaction with NaV1.5 was blunted in NaV1.5K1493del. Electrical charge substitution at position 1493 did not affect CaM-interaction and channel functionality. Arrhythmia and conduction-disease -associated mutations revealed Ca2+-dependent gating regulation of NaV1.5 channels. Our results highlight the role of NaV1.5 DIII-IV linker in the CaM-binding complex and channel function, and suggest that the Ca2+-sensing machinery of NaV1.5 involves NaVß1.

Coexistence of atrioventricular accessory pathways and drug-induced type 1 Brugada pattern.

BACKGROUND: Atrial arrhythmias, particularly atrioventricular nodal reentrant tachycardia, can coexist with drug-induced type 1 Brugada electrocardiogram (ECG) pattern (DI-Type1-BrP). The present study was designed to determine the prevalence of DI-Type1-BrP in patients with atrioventricular accessory pathways (AV-APs) and to investigate the clinical, electrocardiographic, electrophysiologic, and genetic characteristics of these patients. METHODS: One-hundred twenty-four consecutive cases of AV-APs and 84 controls underwent an ajmaline challenge test to unmask DI-Type1-BrP. Genetic screening and analysis was performed in 55 of the cases (19 with and 36 without DI-Type1-BrP). RESULTS: Patients with AV-APs were significantly more likely than controls to have a Type1-BrP unmasked (16.1 vs 4.8%, P = 0.012). At baseline, patients with DI-Type1-BrP had higher prevalence of chest pain, QR/rSr' pattern in V1 and QRS notching/slurring in V2 and aVL during preexcitation, rSr' pattern in V1 -V2 , and QRS notching/slurring in aVL during orthodromic atrioventricular reentrant tachycardia (AVRT) compared to patients without DI-Type1-BrP. Abnormal QRS configuration (QRS notching/slurring and/or fragmentation) in V2 during preexcitation was present in all patients with DI-Type1 BrP. The prevalence of spontaneous preexcited atrial fibrillation (AF) and history of AF were similar (15% vs 18.3%, P = 0.726) in patients with and without DI-Type1-BrP, respectively. The prevalence of mutations in Brugada-susceptibility genes was higher (36.8% vs 8.3%, P = 0.02) in patients with DI-Type1-BrP compared to patients without DI-Type1-BrP. CONCLUSIONS: DI-Type1-BrP is relatively common in patients with AV-APs. We identify 12-lead ECG characteristics during preexcitation and orthodromic AVRT that point to an underlying type1-BrP, portending an increased probability for development of malignant arrhythmias.

Further Insights in the Most Common SCN5A Mutation Causing Overlapping Phenotype of Long QT Syndrome, Brugada Syndrome, and Conduction Defect.

BACKGROUND: Phenotypic overlap of type 3 long QT syndrome (LQT3), Brugada syndrome (BrS), cardiac conduction disease (CCD), and sinus node dysfunction (SND) is observed with SCN5A mutations. SCN5A-E1784K is the most common mutation associated with BrS and LQTS3. The present study examines the genotype-phenotype relationship in a large family carrying SCN5A-E1784K and SCN5A-H558R polymorphism. METHODS AND RESULTS: Clinical work-up, follow-up, and genetic analysis were performed in 35 family members. Seventeen were SCN5A-E1784K positive. They also displayed QTc prolongation, and either BrS, CCD, or both. One carrier exhibited SND. The presence of SCN5A-H558R did not significantly alter the phenotype of SCN5A-E1784K carriers. Fourteen SCN5A-E1784K patients underwent implantable cardioverter-defibrillator (ICD) implantation; 4 developed VF and received appropriate ICD shocks after 8±3 months of follow-up. One patient without ICD also developed VF after 6.7 years. These 5 cases carried both SCN5A-E1784K and SCN5A-H558R. Functional characterization was achieved by expressing SCN5A variants in TSA201 cells. Peak (INa,P) or late (INa,L) sodium currents were recorded using whole-cell patch-clamp techniques. Co-expression of SCN5A-E1784K and SCN5A-WT reduced INa,P to 70.03% of WT, shifted steady-state inactivation by -11.03 mV, and increased INa,L from 0.14% to 1.86% of INa,P. Similar changes were observed when SCN5A-E1784K was co-expressed with SCN5A-H558R. CONCLUSIONS: We demonstrate a strong genotype-phenotype correlation with complete penetrance for BrS, LQTS, or CCD in the largest family harboring SCN5A-E1784K mutation described so far. Phenotype of LQTS is present during all decades of life, whereas CCD develops with increasing age. Phenotypic overlap may explain the high event rate in carriers.

High prevalence of concealed Brugada syndrome in patients with atrioventricular nodal reentrant tachycardia.

BACKGROUND: Atrioventricular nodal reentrant tachycardia (AVNRT) may coexist with Brugada syndrome (BrS). OBJECTIVES: The present study was designed to determine the prevalence of drug-induced type 1 Brugada ECG pattern (concealed BrS) in patients presenting with clinical spontaneous AVNRT and to investigate their electrocardiographic, electrophysiological, and genetic characteristics. METHODS: Ninety-six consecutive patients without any sign of BrS on baseline electrocardiogram undergoing electrophysiological study and ablation for symptomatic, drug-resistant AVNRT and 66 control subjects underwent an ajmaline challenge to unmask BrS. Genetic screening was performed in 17 patients displaying both AVNRT and BrS. RESULTS: A concealed BrS electrocardiogram was uncovered in 26 of 96 patients with AVNRT (27.1%) and in 3 of 66 control subjects (4.5%) (P ≤ .001). Patients with concealed BrS were predominantly female patients (n=23 [88.5%] vs n=44 [62.9%], P = .015), had higher prevalence of chest pain (n=10 [38.5%] vs n=13 [18.6%], p=0.042), migraine headaches (n=10 [38.5%] vs n=10 [14.2%], p=0.008), and drug-induced initiation and/or worsening of duration and/or frequency of AVNRT (n=4 [15.4%] vs n=1 [1.4%], p=0.006) as compared to patients with AVNRT without BrS. Genetic screening identified 19 mutations or rare variants in 13 genes in 13 of 17 patients with both AVNRT and BrS (yield = 76.5%). Ten of these 13 genotype-positive patients (76.9%) harbored genetic variants known or suspected to cause a loss of function of cardiac sodium channel current (SCN5A, SCN10A, SCN1B, GPD1L, PKP2, and HEY2). CONCLUSION: Our results suggest that spontaneous AVNRT and concealed BrS co-occur, particularly in female patients, and that genetic variants that reduce sodium channel current may provide a mechanistic link between AVNRT and BrS and predispose to expression of both phenotypes.

ABCC9 is a novel Brugada and early repolarization syndrome susceptibility gene.

BACKGROUND: Genetic defects in KCNJ8, encoding the Kir6.1 subunit of the ATP-sensitive K(+) channel (I(K-ATP)), have previously been associated with early repolarization (ERS) and Brugada (BrS) syndromes. Here we test the hypothesis that 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 -18.0 mV (p<0.01) and increased late I(Na) from 0.14% to 2.01% of peak I(Na) (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 I(K-ATP) when coupled with a loss-of-function in SCN5A may underlie type 3 ERS, which is associated with a severe arrhythmic phenotype.

Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in KCNJ8.

BACKGROUND: Adenosine triphosphate (ATP)-sensitive potassium cardiac channels consist of inward-rectifying channel subunits Kir6.1 or Kir6.2 (encoded by KCNJ8 or KCNJ11) and the sulfonylurea receptor subunits SUR2A (encoded by ABCC9). OBJECTIVE: To examine the association of mutations in KCNJ8 with Brugada syndrome (BrS) and early repolarization syndrome (ERS) and to elucidate the mechanism underlying the gain of function of ATP-sensitive potassium channel current. METHODS: Direct sequencing of KCNJ8 and other candidate genes was performed on 204 BrS and ERS probands and family members. Whole-cell and inside-out patch-clamp methods were used to study mutated channels expressed in TSA201 cells. RESULTS: The same missense mutation, p.Ser422Leu (c.1265C>T) in KCNJ8, was identified in 3 BrS and 1 ERS probands but was absent in 430 alleles from ethnically matched healthy controls. Additional genetic variants included CACNB2b-D601E. Whole-cell patch-clamp studies showed a 2-fold gain of function of glibenclamide-sensitive ATP-sensitive potassium channel current when KCNJ8-S422L was coexpressed with SUR2A-wild type. Inside-out patch-clamp evaluation yielded a significantly greater half maximal inhibitory concentration for ATP in the mutant channels (785.5 ± 2 vs 38.4 ± 3 µM; n = 5; P <.01), pointing to incomplete closing of the ATP-sensitive potassium channels under normoxic conditions. Patients with a CACNB2b-D601E polymorphism displayed longer QT/corrected QT intervals, likely owing to their effect to induce an increase in L-type calcium channel current (I(Ca-L)). CONCLUSIONS: Our results support the hypothesis that KCNJ8 is a susceptibility gene for BrS and ERS and point to S422L as a possible hotspot mutation. Our findings suggest that the S422L-induced gain of function in ATP-sensitive potassium channel current is due to reduced sensitivity to intracellular ATP.

Long QT, syndactyly, joint contractures, stroke and novel CACNA1C mutation: expanding the spectrum of Timothy syndrome.

Timothy syndrome (TS) is an autosomal dominant condition with the constellation of features including prolonged QT interval, hand and foot abnormalities, and mental retardation or autism. Splawski et al. [2004] previously described two phenotypes associated with TS distinguished by two unique and different mutations within the CACNA1C gene. We report on a newborn who presented with prolonged QT interval and associated polymorphic ventricular tachycardia, dysmorphic facial features, syndactyly of the hands and feet, and joint contractures, suggestive of TS. He developed a stroke, subsequent intractable seizures, and was found to have cortical blindness and later profound developmental delay. Initial targeted mutation analysis did not identify either of the previously described TS associated mutations; however, full gene sequencing detected a novel CACNA1C gene mutation (p.Ala1473Gly). The clinical and genetic findings in our case expand both the clinical and molecular knowledge of TS.

A novel rare variant in SCN1Bb linked to Brugada syndrome and SIDS by combined modulation of Na(v)1.5 and K(v)4.3 channel currents.

BACKGROUND: Cardiac sodium channel ß-subunit mutations have been associated with several inherited cardiac arrhythmia syndromes. OBJECTIVE: To identify and characterize variations in SCN1Bb associated with Brugada syndrome (BrS) and sudden infant death syndrome (SIDS). METHODS: All known exons and intron borders of the BrS-susceptibility genes were amplified and sequenced in both directions. Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using co-immunoprecipitation and whole-cell patch-clamp techniques. RESULTS: Patient 1 was a 44-year-old man with an ajmaline-induced type 1 ST-segment elevation in V1 and V2 supporting the diagnosis of BrS. Patient 2 was a 62-year-old woman displaying a coved-type BrS electrocardiogram who developed cardiac arrest during fever. Patient 3 was a 4-month-old female SIDS case. A R214Q variant was detected in exon 3A of SCN1Bb (Na(v)1B) 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%). Co-expression of SCN5A/WT + SCN1Bb/R214Q resulted in peak sodium channel current (I(Na)) 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 (transient outward potassium current, I(to)) 70.6% greater compared with KCND3/WT + SCN1Bb/WT (n = 10-11, P<0.01). Co-immunoprecipitation indicated structural association between Na(v)ß1B and Na(v)1.5 and K(v)4.3. CONCLUSION: Our results suggest that R214Q variation in SCN1Bb is a functional polymorphism that may serve as a modifier of the substrate responsible for BrS or SIDS phenotypes via a combined loss of function of sodium channel current and gain of function of transient outward potassium current.

LQT5 masquerading as LQT2: a dominant negative effect of KCNE1-D85N rare polymorphism on KCNH2 current.

AIMS: KCNE1 encodes an auxiliary subunit of cardiac potassium channels. Loss-of-function variations in this gene have been associated with the LQT5 form of the long QT syndrome (LQTS), secondary to reduction of I(Ks) current. We present a case in which a D85N rare polymorphism in KCNE1 is associated with an LQT2 phenotype. METHODS AND RESULTS: An 11-year old competitive athlete presented with mild bradycardia and a QTc interval of 470 ms. An LQT2 phenotype, consisting of low-voltage bifid T waves, was evident in the right precordial electrocardiogram leads. During the tachycardia phase following adenosine, QTc increased to 620 ms. Genetic analysis revealed a rare heterozygous polymorphism in KCNE1 predicting the substitution of asparagine for aspartic acid at position 85 of minK (D85N). Patch clamp experiments showed that KCNE1-D85N, when co-expressed with KCNH2 in TSA201 cells, significantly reduced I(Kr). Homozygous co-expression of the mutant with KCNH2 reduced I(Kr) tail current by 85%, whereas heterozygous co-expression reduced the current by 52%, demonstrating for the first time a dominant-negative effect of D85N to reduce I(Kr). Co-expression of the mutant with KCNQ1, either homozygously or heterozygously, produced no change in I(Ks). CONCLUSIONS: Our results suggest that a rare polymorphism KCNE1-D85N underlies the development of an LQT2 phenotype in this young athlete by interacting with KCNH2 to cause a dominant-negative effect to reduce I(Kr). Our data provide further evidence in support of the promiscuity of potassium channel ß subunits in modulating the function of multiple potassium channels leading to a diversity of clinical phenotypes.

Biophysical and molecular characterization of a novel de novo KCNJ2 mutation associated with Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia mimicry.

BACKGROUND: Mutations in KCNJ2, the gene encoding the human inward rectifier potassium channel Kir2.1 (IK1 or IKir2.1), have been identified in Andersen-Tawil syndrome. Andersen-Tawil syndrome is a multisystem inherited disease exhibiting periodic paralysis, cardiac arrhythmias, and dysmorphic features at times mimicking catecholaminergic polymorphic ventricular tachycardia. METHODS AND RESULTS: Our proband displayed dysmorphic features including micrognathia, clinodactyly, and syndactyly and exhibited multiform extrasystoles and bidirectional ventricular tachycardia both at rest and during exercise testing. The patient's symptoms continued after administration of nadolol but subsided after treatment with flecainide. Molecular genetic screening revealed a novel heterozygous mutation (c.779G>C/p.R260P) in KCNJ2. Whole-cell patch-clamp studies conducted in TSA201 cells transfected with wild-type human KCNJ2 cDNA (WT-KCNJ2) yielded robust IKir2.1 but no measurable current in cells expressing the R260P mutant. Coexpression of WT and R260P-KCNJ2 (heterozygous expression) yielded a markedly reduced inward IKir2.1 compared with WT alone (-36.5±9.8 pA/pF versus -143.5±11.4 pA/pF, n=8 for both, P<0.001, respectively, at -90 mV), indicating a strong dominant negative effect of the mutant. The outward component of IKir2.1 measured at -50 mV was also markedly reduced with the heterozygous expression versus WT (0.52±5.5 pA/pF versus 23.4±6.7 pA/pF, n=8 for both, P<0.001, respectively). Immunocytochemical analysis indicates that impaired trafficking of R260P-KCNJ2 channels. CONCLUSIONS: We report a novel de novo KCNJ2 mutation associated with classic phenotypic features of Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia mimicry. The R260P mutation produces a strong dominant negative effect leading to marked suppression of IK1 secondary to a trafficking defect.

Overlapping LQT1 and LQT2 phenotype in a patient with long QT syndrome associated with loss-of-function variations in KCNQ1 and KCNH2.

Long QT syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and potentially life-threatening arrhythmias. Mutations in 12 different genes have been associated with LQTS. Here we describe a patient with LQTS who has a mutation in KCNQ1 as well as a polymorphism in KCNH2. The proband (MMRL0362), a 32-year-old female, exhibited multiple ventricular extrasystoles and one syncope. Her ECG (QT interval corrected for heart rate (QTc) = 518ms) showed an LQT2 morphology in leads V4-V6 and LQT1 morphology in leads V1-V2. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 7 LQTS susceptibility genes were amplified and sequenced. Variations were detected predicting a novel missense mutation (V110I) in KCNQ1, as well as a common polymorphism in KCNH2 (K897T). We expressed wild-type (WT) or V110I Kv7.1 channels in CHO-K1 cells cotransfected with KCNE1 and performed patch-clamp analysis. In addition, WT or K897T Kv11.1 were also studied by patch clamp. Current-voltage (I-V) relations for V110I showed a significant reduction in both developing and tail current densities compared with WT at potentials >+20 mV (p < 0.05; n = 8 cells, each group), suggesting a reduction in IKs currents. K897T- Kv11.1 channels displayed a significantly reduced tail current density compared with WT-Kv11.1 at potentials >+10 mV. Interestingly, channel availability assessed using a triple-pulse protocol was slightly greater for K897T compared with WT (V0.5 = -53.1 ± 1.13 mV and -60.7 ± 1.15 mV for K897T and WT, respectively; p < 0.05). Comparison of the fully activated I-V revealed no difference in the rectification properties between WT and K897T channels. We report a patient with a loss-of-function mutation in KCNQ1 and a loss-of-function polymorphism in KCNH2. Our results suggest that a reduction of both IKr and IKs underlies the combined LQT1 and LQT2 phenotype observed in this patient.

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

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, ß2, 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, ß2, 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.

A common single nucleotide polymorphism can exacerbate long-QT type 2 syndrome leading to sudden infant death.

BACKGROUND: Identification of infants at risk for sudden arrhythmic death remains one of the leading challenges of modern medicine. We present a family in which a common polymorphism (single nucleotide polymorphism) inherited from the father, combined with a stop codon mutation inherited from the mother (both asymptomatic), led to 2 cases of sudden infant death. METHODS AND RESULTS: KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, CACNA1c, CACNB2b, and KCNJ2 genes were amplified and analyzed by direct sequencing. Functional electrophysiological studies were performed with the single nucleotide polymorphism and mutation expressed singly and in combination in Chinese ovary (CHO-K1) and COS-1 cells. An asymptomatic woman presenting after the death of her 2-day-old infant and spontaneous abortion of a second baby in the first trimester was referred for genetic analysis. The newborn infant had nearly incessant ventricular tachycardia while in utero and a prolonged QTc (560 ms). The mother was asymptomatic but displayed a prolonged QTc. Genetic screening of the mother revealed a heterozygous nonsense mutation (P926AfsX14) in KCNH2, predicting a stop codon. The father was asymptomatic with a normal QTc but had a heterozygous polymorphism (K897T) in KCNH2. The baby who died at 2 days of age and the aborted fetus inherited both K897T and P926AfsX14. Heterologous coexpression of K897T and P926AfsX14 led to loss of function of HERG current much greater than expression of K897T or P926AfsX14 alone. CONCLUSIONS: Our data suggest that a common polymorphism (K897T) can markedly accentuate the loss of function of mildly defective HERG channels, leading to long-QT syndrome-mediated arrhythmias and sudden infant death.

Dual variation in SCN5A and CACNB2b underlies the development of cardiac conduction disease without Brugada syndrome.

BACKGROUND: Inherited loss of function mutations in SCN5A have been linked to overlapping syndromes including cardiac conduction disease and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood. METHODS: Direct sequencing was performed in a family with cardiac conduction disease. Wild-type (WT) and mutant channels were expressed in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)-fused WT or mutant genes were used to assess channel trafficking. RESULTS: A novel SCN5A mutation, P1008S, was identified in all family members displaying first-degree atrioventricular block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (P < 0.001). Confocal microscopy showed that WT channels tagged with GFP were localized on the cell surface, whereas GFP-tagged P1008S channels remained trapped in intracellular organelles. Trafficking could be rescued by incubation at room temperature, but not by incubation with mexiletine (300 muM) at 37 degrees C. We also identified a novel polymorphism (D601E) in CACNB2b that slowed inactivation of L-type calcium current (I(Ca,L)), significantly increased total charge. Using the Luo-Rudy action potential (AP) model, we show that the reduction in sodium current (I(Na)) can cause loss of the right ventricular epicardial AP dome in the absence but not in the presence of the slowed inactivation of I(Ca,L). Slowed conduction was present in both cases. CONCLUSIONS: Our results suggest genetic variations leading to a loss-of-function in I(Na) coupled with a gain of function in I(Ca,L) may underlie the development of cardiac conduction disease without BrS.

A mutation in the beta 3 subunit of the cardiac sodium channel associated with Brugada ECG phenotype.

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 beta 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/WT+SCN1B/WT+SCN3B/L10P resulted in an 82.6% decrease in peak sodium current density, accelerated inactivation, slowed reactivation, and a -9.6-mV shift of half-inactivation voltage compared with SCN5A/WT+SCN1B/WT+SCN3B/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)beta 3 in human ventricular myocardium. CONCLUSIONS: Our results provide support for the hypothesis that mutations in SCN3B can lead to loss of transport and functional expression of the hNa(v)1.5 protein, leading to reduction in sodium channel current and clinical manifestation of a Brugada phenotype.

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

SCN5A mutation associated with acute myocardial infarction.

Ventricular tachycardia and fibrillation (VT/VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) have both been linked to phase 2 reentry. Because of these mechanistic similarities in arrhythmogenesis, we examined the contribution of SCN5A mutations to VT/VF complicating AMI. Nineteen consecutive patients developing VF during AMI were enrolled. Wild-type (WT) and mutant SCN5A genes were co-expressed with SCN1B in TSA201 cells and studied using whole-cell patch-clamp techniques. One missense mutation (G400A) in SCN5A was detected in a conserved region among the cohort of 19 patients. A H558R polymorphism was detected on the same allele. Unlike the other 18 patients who each developed 1-2 VF episodes during acute MI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST segment changes and were initiated by short-coupled extrasystoles. We describe the first sodium channel mutation to be associated with the development of an arrhythmic storm during acute ischemia. These findings suggest that a loss of function in SCN5A may predispose to ischemia induced arrhythmic storm. These results could be very useful for forensic implications regarding genetic screening in relatives.

Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 beta-subunit, has been shown to reduce IKs. OBJECTIVE: The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF. METHODS: One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5. RESULTS: A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to -40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT. CONCLUSION: The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs.

Functional effects of KCNE3 mutation and its role in the development of Brugada syndrome.

INTRODUCTION: The Brugada Syndrome (BrS), an inherited syndrome associated with a high incidence of sudden cardiac arrest, has been linked to mutations in four different genes leading to a loss of function in sodium and calcium channel activity. Although the transient outward current (I(to)) is thought to play a prominent role in the expression of the syndrome, mutations in I(to)-related genes have not been identified as yet. METHODS AND RESULTS: One hundred and five probands with BrS were screened for ion channel gene mutations using single strand conformation polymorphism (SSCP) electrophoresis and direct sequencing. A missense mutation (R99H) in KCNE3 (MiRP2) was detected in one proband. The R99H mutation was found 4/4 phenotype positive and 0/3 phenotype-negative family members. Chinese hamster ovary (CHO)-K1 cells were co-transfected using wild-type (WT) or mutant KCNE3 and either WT KCND3 or KCNQ1. Whole-cell patch clamp studies were performed after 48 hours. Interactions between Kv4.3 and KCNE3 were analyzed in co-immunoprecipitation experiments in human atrial samples. Co-transfection of R99H-KCNE3 with KCNQ1 produced no alteration in current magnitude or kinetics. However, co-transfection of R99H KCNE3 with KCND3 resulted in a significant increase in the I(to) intensity compared to WT KCNE3+KCND3. Using tissues isolated from left atrial appendages of human hearts, we also demonstrate that K(v)4.3 and KCNE3 can be co-immunoprecipitated. CONCLUSIONS: These results provide definitive evidence for a functional role of KCNE3 in the modulation of I(to) in the human heart and suggest that mutations in KCNE3 can underlie the development of BrS.

Novel mutation in the SCN5A gene associated with arrhythmic storm development during acute myocardial infarction.

BACKGROUND: Ventricular tachycardia (VT) and ventricular fibrillation (VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) both have been linked to phase 2 reentry. OBJECTIVE: Given the mechanistic similarities in arrhythmogenesis, the purpose of this study was to examine the contribution of SCN5A mutations to VT/VF complicating AMI. METHODS: Nineteen consecutive patients developing VF during AMI were enrolled in the study. Wild-type (WT) and mutant SCN5A genes were coexpressed with SCN1B in TSA201 cells and studied using whole-cell patch clamp techniques. RESULTS: Among the cohort of 19 patients, one missense mutation (G400A) in SCN5A was detected in a conserved region. An H558R polymorphism was detected on the same allele. Unlike the other 18 patients, who each developed 1-2 VF episodes during AMI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST-segment changes and were initiated by short-coupled extrasystoles. Flecainide and adenosine challenge performed to unmask Brugada and long QT syndromes both were negative. Peak G400A and G400A+H558R current were 70.7% and 88.4% less than WT current at -35 mV (P