Identification and characterization of two novel KCNH2 mutations contributing to long QT syndrome.

We identified two different inherited mutations in KCNH2 gene, or human ether-a-go-go related gene (hERG), which are linked to Long QT Syndrome. The first mutation was in a 1-day-old infant, whereas the second was in a 14-year-old girl. The two KCNH2 mutations were transiently transfected into either human embryonic kidney (HEK) cells or human induced pluripotent stem-cell derived cardiomyocytes. We performed associated multiscale computer simulations to elucidate the arrhythmogenic potentials of the KCNH2 mutations. Genetic screening of the first and second index patients revealed a heterozygous missense mutation in KCNH2, resulting in an amino acid change (P632L) in the outer loop of the channel and substitution at position 428 from serine to proline (S428P), respectively. Heterologous expression of P632L and S428P into HEK cells produced no hERG current compared to the wild type (WT). Moreover, the co-transfection of WT and P632L yielded no hERG current; however, the co-transfection of WT and S428P yielded partial hERG current. Action potentials were prolonged in a complete or partial blockade of hERG current from computer simulations which was more severe in Purkinje than ventricular myocytes. Three dimensional simulations revealed a higher susceptibility to reentry in the presence of hERG current blockade. Our experimental findings suggest that both P632L and S428P mutations may impair the KCNH2 gene. The Purkinje cells exhibit a more severe phenotype than ventricular myocytes, and the hERG current blockade renders the ventricles an arrhythmogenic substrate from computer modeling.

Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Clinico-histopathologic and single-nuclei RNA-sequencing insights into cardiac injury and microthrombi in critical COVID-19.

Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes - myocarditis and cardiac necrosis - have proved uncommon. To elucidate the pathophysiology of COVID-19-associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at CUIMC in New York City. Of 6 acute cardiac histopathologic features, presence of microthrombi was the most commonly detected among our cohort. We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and C-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single-nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of prothrombotic/antifibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling among cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non-COVID-19, nonfailing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19-associated cardiac microthrombi.

Molecular Pathophysiology of Cardiac Injury and Cardiac Microthrombi in Fatal COVID-19: Insights from Clinico-histopathologic and Single Nuclei RNA Sequencing Analyses.

Cardiac injury is associated with critical COVID-19, yet its etiology remains debated. To elucidate the pathogenic mechanisms of COVID-19-associated cardiac injury, we conducted a single-center prospective cohort study of 69 COVID-19 decedents. Of six cardiac histopathologic features, microthrombi was the most commonly detected (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak ESR and CRP during hospitalization were independently associated with higher odds of microthrombi. Using single nuclei RNA-sequence analysis, we discovered an enrichment of pro-thrombotic/anti-fibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling amongst cardiac fibroblasts in microthrombi-positive COVID-19 hearts relative to microthrombi-negative COVID-19. Non-COVID-19 non-failing hearts were used as reference controls. Our cumulative findings identify the specific transcriptomic changes in cardiac fibroblasts as salient features of COVID-19-associated cardiac microthrombi.

Overlap Arrhythmia Syndromes Resulting from Multiple Genetic Variations Studied in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a "pseudo" QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.

Susceptibility to Ventricular Arrhythmias Resulting from Mutations in FKBP1B, PXDNL, and SCN9A Evaluated in hiPSC Cardiomyocytes.

BACKGROUND: We report an inherited cardiac arrhythmia syndrome consisting of Brugada and Early Repolarization Syndrome associated with variants in SCN9A, PXDNL, and FKBP1B. The proband inherited the 3 mutations and exhibited palpitations and arrhythmia-mediated syncope, whereas the parents and sister, who carried one or two of the mutations, were asymptomatic. METHODS AND RESULTS: We assessed the functional impact of these mutations in induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) derived from the proband and an unaffected family member. Current and voltage clamp recordings, as well as confocal microscopy analysis of Ca2+ transients, were evaluated in hiPSC-CMs from the proband and compared these results with hiPSC-CMs from undiseased controls. Genetic analysis using next-generation DNA sequencing revealed heterozygous mutations in SCN9A, PXDNL, and FKBP1B in the proband. The proband displayed right bundle branch block and exhibited episodes of syncope. The father carried a mutation in FKBP1B, whereas the mother and sister carried the SCN9A mutation. None of the 3 family members screened developed cardiac events. Action potential recordings from control hiPSC-CM showed spontaneous activity and a low upstroke velocity. In contrast, the hiPSC-CM from the proband showed irregular spontaneous activity. Confocal microscopy of the hiPSC-CM of the proband revealed low fluorescence intensity Ca2+ transients that were episodic in nature. Patch-clamp measurements in hiPSC-CM showed no difference in I Na but reduced I Ca in the proband compared with control. Coexpression of PXDNL-R391Q with SCN5A-WT displayed lower I Na density compared to PXDNL-WT. In addition, coexpression of PXDNL-R391Q with KCND3-WT displayed significantly higher I to density compared to PXDNL-WT. CONCLUSION: SCN9A, PXDNL, and FKBP1B variants appeared to alter spontaneous activity in hiPSC-CM. Only the proband carrying all 3 mutations displayed the ERS/BrS phenotype, whereas one nor two mutations alone did not produce the clinical phenotype. Our results suggest a polygenic cause of the BrS/ERS arrhythmic phenotype due to mutations in these three gene variants caused a very significant loss of function of I Na and I Ca and gain of function of I to.

Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome.

BACKGROUND: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. METHODS: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. RESULTS: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P<5×10-8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1(p.Asp85Asn) at the suggestive threshold (P<10-6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (rg=0.40; P=3.2×10-3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P<10-13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P<0.005). CONCLUSIONS: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.

The Phenotypic Spectrum of a Mutation Hotspot Responsible for the Short QT Syndrome.

OBJECTIVES: This study sought to evaluate the phenotypic and functional expression of an apparent hotspot mutation associated with short QT syndrome (SQTS). BACKGROUND: SQTS is a rare channelopathy associated with a high risk of life-threatening arrhythmias and sudden cardiac death (SCD). METHODS: Probands diagnosed with SQTS and their family members were evaluated clinically and genetically. KCNH2 wild-type (WT) and mutant genes were transiently expressed in HEK293 cells, and currents were recorded using whole-cell patch clamp and action potential (AP) clamp techniques. RESULTS: KCNH2-T618I was identified in 18 members of 7 unrelated families (10 men; median age: 24.0 years). All carriers showed 100% penetrance with variable expressivity. Eighteen members in 7 families had SCD. The average QTc intervals of probands and all carriers was 294.1 ± 23.8 ms and 313.2 ± 23.8 ms, respectively. Seven carriers received an implantable cardioverter-defibrillator. Quinidine with adequate plasma levels was effective in prolonging QTc intervals among 5 cases, but 3 cases still had premature ventricular contraction or nonsustained ventricular tachycardia. Bepridil successfully prevented drug-refractory ventricular fibrillation in 1 case with 19-ms prolongation of the QTc interval. Functional studies with KCNE2 revealed a significant increase of IKr (rapidly activating delayed rectifier potassium channel) tail-current density in homozygous (119.0%) and heterozygous (74.6%) expression compared with WT. AP clamp recordings showed IKr was larger, and peak repolarizing current occurred earlier in mutant versus WT channels. CONCLUSIONS: We reported the clinical characteristics and biophysical properties of the highly frequent mutation that contributes to genetically identified SQTS probands. These findings extend our understanding of the spectrum of KCNH2 channel defects in SQTS.

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.

A CACNA1C variant associated with reduced voltage-dependent inactivation, increased CaV1.2 channel window current, and arrhythmogenesis.

Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of CaV1.2 can contribute to QT prolongation.

Mutations in SCN10A are responsible for a large fraction of cases of Brugada syndrome.

BACKGROUND: BrS is an inherited sudden cardiac death syndrome. Less than 35% of BrS probands have genetically identified pathogenic variants. Recent evidence has implicated SCN10A, a neuronal sodium channel gene encoding Nav1.8, in the electrical function of the heart. OBJECTIVES: The purpose of this study was to test the hypothesis that SCN10A variants contribute to the development of Brugada syndrome (BrS). METHODS: Clinical analysis and direct sequencing of BrS susceptibility genes were performed for 150 probands and family members as well as >200 healthy controls. Expression and coimmunoprecipitation studies were performed to functionally characterize the putative pathogenic mutations. RESULTS: We identified 17 SCN10A mutations in 25 probands (20 male and 5 female); 23 of the 25 probands (92.0%) displayed overlapping phenotypes. SCN10A mutations were found in 16.7% of BrS probands, approaching our yield for SCN5A mutations (20.1%). Patients with BrS who had SCN10A mutations were more symptomatic and displayed significantly longer PR and QRS intervals compared with SCN10A-negative BrS probands. The majority of mutations localized to the transmembrane-spanning regions. Heterologous coexpression of wild-type (WT) SCN10A with WT-SCN5A in HEK cells caused a near doubling of sodium channel current compared with WT-SCN5A alone. In contrast, coexpression of SCN10A mutants (R14L and R1268Q) with WT-SCN5A caused a 79.4% and 84.4% reduction in sodium channel current, respectively. The coimmunoprecipitation studies provided evidence for the coassociation of Nav1.8 and Nav1.5 in the plasma membrane. CONCLUSIONS: Our study identified SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our ability to genotype and risk stratify probands and family members.

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.

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.

Brugada-like syndrome in infancy presenting with rapid ventricular tachycardia and intraventricular conduction delay.

BACKGROUND: Brugada syndrome is a potentially serious channelopathy that usually presents in adulthood and has only rarely been described in infancy. In the absence of metabolic or structural cardiac disease, rapid ventricular tachycardia (>200 bpm) and primary cardiac conduction disease are uncommon in infancy. We hypothesized that infants having rapid ventricular tachycardia and conduction abnormalities and not having structural or metabolic pathogeneses were likely to have mutations in depolarizing current channels. METHODS AND RESULTS: A retrospective review of all clinical materials from a single institution over a 9-year period from all infants <2 years old and having a discharge diagnosis of ventricular tachycardia or ventricular fibrillation was performed. Among 32 infants fulfilling inclusion criteria, 12 had a structurally normal heart, and 9 of them had either prolonged QRS duration or Brugada pattern while in sinus rhythm. Of those 5 infants not having a definitive pathogenesis, electrophysiological testing had been performed in 4, and genetic testing had been performed in all 5 of those infants. During electrophysiological testing, a prolonged HV interval was present in 2 of 4, inducible ventricular tachycardia was present in 1 of 4, and a type 1 Brugada pattern was induced by intravenous procainamide in 3 of 4. Genetic testing revealed disease-causing mutations in depolarizing sodium (SCN5A) or calcium (CaCNB2b) channels in all 5 infants. CONCLUSIONS: Infants having rapid ventricular tachycardia and conduction abnormalities in the absence of structural or metabolic abnormalities are likely to have disease-causing mutations in cardiac depolarizing channels.

Multiple arrhythmic syndromes in a newborn, owing to a novel mutation in SCN5A.

BACKGROUND: Mutations in the SCN5A gene have been linked to Brugada syndrome (BrS), conduction disease, Long QT syndrome (LQT3), atrial fibrillation (AF), and to pre- and neonatal ventricular arrhythmias. OBJECTIVE: The objective of this study is to characterize a novel mutation in Na(v)1.5 found in a newborn with fetal chaotic atrial tachycardia, post-partum intraventricular conduction delay, and QT interval prolongation. METHODS: Genomic DNA was isolated and all exons and intron borders of 15 ion-channel genes were sequenced, revealing a novel missense mutation (Q270K) in SCN5A. Na(v)1.5 wild type (WT) and Q270K were expressed in CHO-K1 with and without the Na(v)ß1 subunit. Results. Patch-clamp analysis showed ∼40% reduction in peak sodium channel current (I(Na)) density for Q270K compared with WT. Fast and slow decay of I(Na) were significantly slower in Q270K. Steady-state activation and inactivation of Q270K channels were shifted to positive potentials, and window current was increased. The tetrodotoxin-sensitive late I(Na) was increased almost 3-fold compared with WT channels. Ranolazine reduced late I(Na) in WT and Q270K channels, while exerting minimal effects on peak I(Na). CONCLUSION: The Q270K mutation in SCN5A reduces peak I(Na) while augmenting late I(Na), and may thus underlie the development of atrial tachycardia, intraventricular conduction delay, and QT interval prolongation in an infant.

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.