Connie R. Bezzina

Sodium channel β1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans

Brugada syndrome is a genetic disease associated with sudden cardiac death that is characterized by ventricular fibrillation and right precordial ST segment elevation on ECG. Loss-of-function mutations in SCN5A, which encodes the predominant cardiac sodium channel alpha subunit NaV1.5, can cause Brugada syndrome and cardiac conduction disease. However, SCN5A mutations are not detected in the majority of patients with these syndromes, suggesting that other genes can cause or modify presentation of these disorders. Here, we investigated SCN1B, which encodes the function-modifying sodium channel beta1 subunit, in 282 probands with Brugada syndrome and in 44 patients with conduction disease, none of whom had SCN5A mutations. We identified 3 mutations segregating with arrhythmia in 3 kindreds. Two of these mutations were located in a newly described alternately processed transcript, beta1B. Both the canonical and alternately processed transcripts were expressed in the human heart and were expressed to a greater degree in Purkinje fibers than in heart muscle, consistent with the clinical presentation of conduction disease. Sodium current was lower when NaV1.5 was coexpressed with mutant beta1 or beta1B subunits than when it was coexpressed with WT subunits. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.

Genotype-phenotype relationship in Brugada syndrome: electrocardiographic features differentiate SCN5A-related patients from non–SCN5A-related patients☆

OBJECTIVES We have tested whether a genotype-phenotype relationship exists in Brugada syndrome (BS) by trying to distinguish BS patients with (carriers) and those without (non-carriers) a mutation in the gene encoding the cardiac sodium channel (SCN5A) using clinical parameters. BACKGROUND Brugada syndrome is an inherited cardiac disease characterized by a varying degree of ST-segment elevation in the right precordial leads and (non)specific conduction disorders. In a minority of patients, SCN5A mutations can be found. Genetic heterogeneity has been demonstrated, but other causally related genes await identification. If a genotype-phenotype relationship exists, this might facilitate screening. METHODS In a multi-center study, we have collected data on demographics, clinical history, family history, electrocardiogram (ECG) parameters, His to ventricle interval (HV), and ECG parameters after pharmacologic challenge with I(Na) blocking drugs for BS patients with (n = 23), or those without (n = 54), an identified SCN5A mutation. RESULTS No differences were found in demographics, clinical history, or family history. Carriers had a significantly longer PQ interval on the baseline ECG and a significantly longer HV time. A PQ interval of > or =210 ms and an HV interval > or =60 ms seem to be predictive for the presence of an SCN5A mutation. After I(Na) blocking drugs, carriers had significantly longer PQ and QRS intervals and more increase in QRS duration. CONCLUSIONS We observed significantly longer conduction intervals on baseline ECG in patients with established SCN5A mutations (PQ and HV interval and, upon class I drugs, more QRS increase). These results concur with the observed loss of function of mutated BS-related sodium channels. Brugada syndrome patients with, and those without, an SCN5A mutation can be differentiated by phenotypical differences.

Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome : a combined electrophysiological, genetic, histopathologic, and computational study

Background— The mechanism of ECG changes and arrhythmogenesis in Brugada syndrome (BS) patients is unknown. Methods and Results— A BS patient without clinically detected cardiac structural abnormalities underwent cardiac transplantation for intolerable numbers of implantable cardioverter/defibrillator discharges. The patient’s explanted heart was studied electrophysiologically and histopathologically. Whole-cell currents were measured in HEK293 cells expressing wild-type or mutated sodium channels from the patient. The right ventricular outflow tract (RVOT) endocardium showed activation slowing and was the origin of ventricular fibrillation without a transmural repolarization gradient. Conduction restitution was abnormal in the RVOT but normal in the left ventricle. Right ventricular hypertrophy and fibrosis with epicardial fatty infiltration were present. HEK293 cells expressing a G1935S mutation in the gene encoding the cardiac sodium channel exhibited enhanced slow inactivation compared with wild-type channels. Computer simulations demonstrated that conduction slowing in the RVOT might have been the cause of the ECG changes. Conclusions— In this patient with BS, conduction slowing based on interstitial fibrosis, but not transmural repolarization differences, caused the ECG signs and was the origin of ventricular fibrillation.

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Familial Sudden Death Is an Important Risk Factor for Primary Ventricular Fibrillation A Case-Control Study in Acute Myocardial Infarction Patients

Background— Primary ventricular fibrillation (VF) accounts for the majority of deaths during the acute phase of myocardial infarction. Identification of patients at risk for primary VF remains very poor. Methods and Results— We performed a case-control study in patients with a first ST-elevation myocardial infarction (STEMI) to identify independent risk factors for primary VF. A total of 330 primary VF survivors (cases) and 372 controls were included; patients with earlier infarcts or signs of structural heart disease were excluded. Baseline characteristics, including age, gender, drug use, and ECG parameters registered well before the index infarction, as well as medical history, were not different. Infarct size and location, culprit coronary artery, and presence of multivessel disease were similar between groups. Analysis of ECGs performed at hospital admission for the index STEMI revealed that cumulative ST deviation was significantly higher among cases (OR per 10-mm ST deviation 1.59, 95% CI 1.25 to 2.02). Analysis of medical histories among parents and siblings showed that the prevalence of cardiovascular disease was similar between cases and controls (73.1% and 73.0%, respectively); however, familial sudden death occurred significantly more frequently among cases than controls (43.1% and 25.1%, respectively; OR 2.72, 95% CI 1.84 to 4.03). Conclusions— In a population of STEMI patients, the risk of primary VF is determined by cumulative ST deviation and family history of sudden death.

Genetic variation in SCN10A influences cardiac conduction

To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 × 10−15) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10−5 to 10−20). SCN10A encodes NaV1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a−/− mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.

Common sodium channel promoter haplotype in asian subjects underlies variability in cardiac conduction.

Background— Reduced cardiac sodium current slows conduction and renders the heart susceptible to ventricular fibrillation. Loss of function mutations in SCN5A, encoding the cardiac sodium channel, are one cause of the Brugada syndrome, associated with slow conduction and a high incidence of ventricular fibrillation, especially in Asians. In this study, we tested the hypothesis that an SCN5A promoter polymorphism common in Asians modulates variability in cardiac conduction. Methods and Results— Resequencing 2.8 kb of SCN5A promoter identified a haplotype variant consisting of 6 polymorphisms in near-complete linkage disequilibrium that occurred at an allele frequency of 22% in Asian subjects and was absent in whites and blacks. Reporter activity of this variant haplotype, designated HapB, in cardiomyocytes was reduced 62% compared with wild-type haplotype (P=0.006). The relationship between SCN5A promoter haplotype and PR and QRS durations, indexes of conduction velocity, was then analyzed in a cohort of 71 Japanese Brugada syndrome subjects without SCN5A mutations and in 102 Japanese control subjects. In both groups, PR and QRS durations were significantly longer in HapB individuals (P≤0.002) with a gene-dose effect. In addition, up to 28% and 48% of variability in PR and QRS durations, respectively, were attributable to this haplotype. The extent of QRS widening during challenge with sodium channel blockers, known to be arrhythmogenic in Brugada syndrome and other settings, was also genotype dependent (P=0.002). Conclusions— These data demonstrate that genetically determined variable sodium channel transcription occurs in the human heart and is associated with variable conduction velocity, an important contributor to arrhythmia susceptibility.

Cardiomyocytes Derived From Pluripotent Stem Cells Recapitulate Electrophysiological Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease

Background— Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na+ current (INa) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na+ channel mutation causing a cardiac Na+ channel overlap syndrome. Method and Results— Induced PSC (iPSC) lines were generated from mice carrying the Scn5a1798insD/+ (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in INa density and a larger persistent INa compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A1795insD/+ mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. Conclusion— Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na+ channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na+ channel disease.

Genetic control of sodium channel function.

Sodium ion (Na) influx through cardiac Na channels triggers the action potential in cells of the working myocardium and the specialized conduction system. Na channels thus act as key molecular determinants of cardiac excitability and impulse propagation. Na channel dysfunction may cause life-threatening arrhythmias. Here, we review the ways in which Na channel function can be aberrant due to genetic changes. We discuss how biophysical studies of mutant Na channels combined with precise clinical phenotyping may improve our understanding of Na channel function in health and disease and may be useful as a model from which to derive improved treatment strategies for common disease.

Type of SCN5A mutation determines clinical severity and degree of conduction slowing in loss-of-function sodium channelopathies

BACKGROUND Patients carrying loss-of-function SCN5A mutations linked to Brugada syndrome (BrS) or progressive cardiac conduction disease (PCCD) are at risk of sudden cardiac death at a young age. The penetrance and expressivity of the disease are highly variable, and new tools for risk stratification are needed. OBJECTIVES We aimed to establish whether the type of SCN5A mutation correlates with the clinical and electrocardiographic phenotype. METHODS We studied BrS or PCCD probands and their relatives who carried a SCN5A mutation. Mutations were divided into 2 main groups: missense mutations (M) or mutations leading to premature truncation of the protein (T). The M group was subdivided according to available biophysical properties: M mutations with <or=90% (M(active)) or >90% (M(inactive)) peak I(Na) reduction were analyzed separately. RESULTS The study group was composed of 147 individuals with 32 different mutations. No differences in age and sex distribution were found between the groups. Subjects carrying a T mutation had significantly more syncopes than those with an M(active) mutation (19 of 75 versus 2 of 35, P = .03). Also, mutations associated with drastic peak I(Na) reduction (T and M(inactive) mutants) had a significantly longer PR interval, compared with M(active) mutations. All other electrocardiographic parameters were comparable. After drug provocation testing, both PR and QRS intervals were significantly longer in the T and M(inactive) groups than in the M(active) group. CONCLUSION In loss-of-function SCN5A channelopathies, patients carrying T and M(inactive) mutations develop a more severe phenotype than those with M(active) mutations. This is associated with more severe conduction disorders. This is the first time that genetic data are proposed for risk stratification in BrS.