Paola Berne

Gender Differences in Clinical Manifestations of Brugada Syndrome

OBJECTIVES We sought to assess differences in phenotype and prognosis between men and women in a large population of patients with Brugada syndrome. BACKGROUND A male predominance has been reported in the Brugada syndrome. No specific data are available, however, concerning gender differences in the clinical manifestations and their role in prognosis. METHODS Patients with Brugada syndrome were prospectively included in the study. Data on baseline characteristics, electrocardiogram parameters before and after pharmacological test, and events in follow-up were recorded for all patients. RESULTS Among 384 patients, 272 (70.8%) were men and 112 (29.2%) women. At inclusion, men had experienced syncope more frequently (18%) or aborted sudden cardiac death (6%) than women (14% and 1%, respectively, p = 0.04). Men also had greater rates of spontaneous type-1 electrocardiogram, greater ST-segment elevation, and greater inducibility of ventricular fibrillation (p < 0.001 for all). Conversely, conduction parameters and corrected QT intervals significantly increased more in women in response to sodium blockers (p = 0.03 and p = 0.001, respectively). During a mean follow-up of 58 +/- 48 months, sudden cardiac death or documented ventricular fibrillation occurred in 31 men (11.6%) and 3 women (2.8%; p = 0.003). The presence of previous symptoms was the most important predictor for cardiac events in men, whereas a longer PR interval was identified among those women with a greater risk in this series. CONCLUSIONS Men with Brugada syndrome present with a greater risk clinical profile than women and have a worse prognosis. Although classical risk factors identify male patients with worse outcome, conduction disturbances could be a marker of risk in the female population.

Preparation for pacemaker or implantable cardiac defibrillator implants in patients with high risk of thrombo-embolic events: oral anticoagulation or bridging with intravenous heparin? A prospective randomized trial.

Aims Current guidelines recommend stopping oral anticoagulation (OAC) and starting heparin infusion before implanting/replacing a pacemaker/implantable cardioverter-defibrillator (ICD) in patients with high risk for thrombo-embolic events. The aim of this study was to demonstrate that the maintenance of OAC during device implantation/replacement is as safe as bridging to intravenous heparin and shortens in-hospital stay. Methods and results A cohort of 101 consecutive patients with high risk for embolic events and indication for implant/replacement of a pacemaker/ICD were randomized to two anticoagulant strategies: bridging from OAC to heparin infusion (n = 51) vs. maintenance of OAC to reach an INR = 2 ± 0.3 at the day of the procedure (n = 50). Haemorrhagic and thrombo-embolic complications were evaluated at discharge, 15 and 45 days after the procedure. A total of 4/51 patients (7.8%) from heparin group and 4/50 (8.0%) from the OAC group developed pocket haematoma following the implant (P = 1.00). One haematoma in each group required evacuation (1.9 vs. 2%, P = 1.00). No other haemorrhagic events or embolic complications developed during the follow-up. Duration of the hospital stay was longer in the heparin group [median of 5 (4–7) vs. 2 (1–4) days; P < 0.001]. Conclusion Implant of devices maintaining OAC is as safe as bridging to heparin infusion and allows a significant reduction of in-hospital stay.

Genetics of arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy is a rare clinical entity characterised by fibro-fatty replacement of myocardium, mainly involving right ventricular free wall, leading to malignant electrical instability and sudden cardiac death. The disease is inherited in up to 50% of cases, with incomplete penetrance and variable phenotypic expression. To date, more than 300 pathogenic mutations have been identified in 12 genes, mainly with autosomal dominant inheritance. Here, we focus on recent advances in the genetics of arrhythmogenic right ventricular cardiomyopathy. Despite continuous improvements, current genotype–phenotype studies have not contributed yet to establish a genetic risk stratification of the disease.

Analysis of mRNA from human heart tissue and putative applications in forensic molecular pathology

The usefulness of post-mortem mRNA analysis and its potential applications in forensic casework is currently of interest, especially because of several factors affecting the quality of RNA samples that are not practically predictable. In fact, post-mortem RNA degradation is a complex process that has not been studied systematically. The purpose of this work is to establish whether RNA analysis from post-mortem heart tissue could be used as a forensic tool to investigate the cause of death, with special regard to those cases where a cardiac disease is suspected as the manner of death. We analysed heart tissue from 16 individuals with normal cardiac function, 9 with long post-mortem intervals (L-PMI) and 7 from organ donors with very short PMIs (S-PMIs). Right ventricle tissue was homogenised, and the RNA was isolated and reverse transcribed. The resulting cDNA was used in real-time PCR reactions to quantify the gene expression of beta-glucuronidase (GUSB), Nitric Oxide Synthase 3 (NOS3), Collagen 1 (COL1A1) and Collagen 3 (COL3A1). The percentage of samples with high-quality RNA was higher in samples with S-PMI (7 out of 7) than in samples with L-PMI (4 out of 9, p<0.05). No differences in PMI time or cause of exitus were found between samples with degraded or non-degraded RNA in the L-PMI group. When comparing mRNA levels in samples with non-degraded RNA, we found similar values between the L-PMI and S-PMI groups for GUSB, COL1A1 and COL3A1. The NOS3 gene expression in the L-PMI subgroup was less than half that in the S-PMI. These results suggest that high-quality mRNA can be extracted from post-mortem human hearts only in some cases. Moreover, our data show that mRNA levels are independent from the PMI, even though there are mRNAs in which the expression levels are very susceptible to ischemia times. Clear knowledge about the relationship between mRNA integrity and expression and PMI could allow the use of several mRNAs as forensic tools to contribute to the determination of the cause of death with special regard to cardiovascular diseases.

Sinus rhythm detection of conducting channels and ventricular tachycardia isthmus in arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND The identification of conducting channels (CCs) based on its relative high voltage or the presence of electrograms with delayed components has been proposed for substrate-guided scar-related ventricular tachycardia (VT) ablation. The relationship of these channels with the VT isthmuses remains unclear. OBJECTIVE To assess the link between CCs identified during sinus rhythm (SR) and VT isthmuses in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). METHODS Twenty-two consecutive patients with ARVC undergoing substrate-guided VT ablation (scar dechanneling technique) were analyzed. High-density endocardial and epicardial electroanatomic maps were obtained during SR. Standard bipolar cutoff values (0.5-1.5 and <0.5 mV) were used to define border zone and dense scar. The CCs were identified by voltage threshold adjustment (voltage channels) or by tagging the electrograms with delayed components that are sequentially activated (late potential channels). RESULTS A total of 87 CCs were identified; 65 (74.7%) of them on the epicardial surface. Twenty-four (27.6%) CCs were voltage channels, and compared with late potential CCs, these had a higher bipolar voltage (0.96 [0.48-1.29] mV vs 0.39 [0.26-0.50] mV; P < .001] and required more radiofrequency applications (5 [4-7] vs 3 [2-5]; P = .048]. Eighteen (90%) of 20 identified VT isthmuses were located on the epicardium. Only 8 (40%) VT isthmuses were related to a voltage CC. The remaining 12 (60%) VT isthmuses were linked to a late potential CC. CONCLUSION Late potential CCs more frequently act as the VT substrate in ARVC and therefore should also be considered to guide SR substrate-guided ablation.

Clinical and molecular characterization of a cardiac ryanodine receptor founder mutation causing catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a difficult-to-diagnose cause of sudden cardiac death (SCD). We identified a family of 1400 individuals with multiple cases of CPVT, including 36 SCDs during youth. OBJECTIVES We sought to identify the genetic cause of CPVT in this family, to preventively treat and clinically characterize the mutation-positive individuals, and to functionally characterize the pathogenic mechanisms of the mutation. METHODS Genetic testing was performed for 1404 relatives. Mutation-positive individuals were preventively treated with β-blockers and clinically characterized with a serial exercise treadmill test (ETT) and Holter monitoring. In vitro functional studies included caffeine sensitivity and store overload-induced calcium release activity of the mutant channel in HEK293 cells. RESULTS We identified the p.G357S_RyR2 mutation, in the cardiac ryanodine receptor, in 179 family members and in 6 SCD cases. No SCD was observed among treated mutation-positive individuals over a median follow-up of 37 months; however, 3 relatives who had refused genetic testing (confirmed mutation-positive individuals) experienced SCD. Holter monitoring did not provide relevant information for CPVT diagnosis. One single ETT was unable to detect complex cardiac arrhythmias in 72% of mutation-positive individuals, though the serial ETT improved the accuracy. Functional studies showed that the G357S mutation increased caffeine sensitivity and store overload-induced calcium release activity under conditions that mimic catecholaminergic stress. CONCLUSION Our study supports the use of genetic testing to identify individuals at risk of SCD to undertake prophylactic interventions. We also show that the pathogenic mechanisms of p.G357S_RyR2 appear to depend on β-adrenergic stimulation.

A Novel Mutation in Lamin A/C Causing Familial Dilated Cardiomyopathy Associated With Sudden Cardiac Death

BACKGROUND Dilated cardiomyopathy (DCM), a cardiac heterogeneous pathology characterized by left ventricular or biventricular dilatation, is a leading cause of heart failure and heart transplantation. The genetic origin of DCM remains unknown in most cases, but >50 genes have been associated with DCM. We sought to identify the genetic implication and perform a genetic analysis in a Spanish family affected by DCM and sudden cardiac death. METHODS AND RESULTS Clinical assessment and genetic screening were performed in the index case as well as family members. Of all relatives clinically assessed, nine patients showed clinical symptoms related to the pathology. Genetic screening identified 20 family members who carried a novel mutation in LMNA (c.871 G>A, p.E291K). Family segregation analysis indicated that all clinically affected patients carried this novel mutation. Clinical assessment of genetic carriers showed that electrical dysfunction was present previous to mechanical and structural abnormalities. CONCLUSIONS Our results report a novel pathogenic mutation associated with DCM, supporting the benefits of comprehensive genetic studies of families affected by this pathology.

Genetic testing of candidate genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a rare cardiac genetic disease characterized by the presence of structural alterations in the right ventricle which may cause ventricular arrhythmias and may induce sudden cardiac death. ARVC/D has been associated with mutations in genes encoding myocyte adhesion proteins. However, only 30%-50% of patients have mutations in these genes. Genetic testing is useful in obtaining a diagnosis, particularly in individuals who do not completely fulfill clinical criteria, thereby also enabling the undertaking of preventive strategies in family members. The main goal of this study was to identify mutations in candidate genes associated with intercalate disks that could be potentially involved in ARVC/D pathogenesis. We analyze a cohort of 14 Spanish unrelated patients clinically diagnosed with ARVC/D without any genetic alteration in all previously known responsible genes. Thus, a genetic screening has been performed in 7 additional potential candidate genes (ACTC1 -actin alpha cardiac muscle 1-, CDHN -cadherin 2 type 1 or N-cadherin-, CTNNA1 -catenin alpha 1-, Cx43 or GJA1 -gap junction protein alpha 1-, MVCL -Metavinculin-, MYL2 -myosin light chain 2- and MYL3 -myosin light chain 3-) by direct sequencing analysis. Our genetic analysis did not identify any disease-causing mutation. Thirty single nucleotides polymorphisms were found, six of them novel. In conclusion, our ARVC/D Spanish cohort has not shown any mutations in the analyzed candidate genes despite their involvement in formation and maintenance of the intercalated disk.

Genetic analysis, in silico prediction, and family segregation in long QT syndrome

The heritable cardiovascular disorder long QT syndrome (LQTS), characterized by prolongation of the QT interval on electrocardiogram, carries a high risk of sudden cardiac death. We sought to add new data to the existing knowledge of genetic mutations contributing to LQTS to both expand our understanding of its genetic basis and assess the value of genetic testing in clinical decision-making. Direct sequencing of the five major contributing genes, KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2, was performed in a cohort of 115 non-related LQTS patients. Pathogenicity of the variants was analyzed using family segregation, allele frequency from public databases, conservation analysis, and Condel and Provean in silico predictors. Phenotype-genotype correlations were analyzed statistically. Sequencing identified 36 previously described and 18 novel mutations. In 51.3% of the index cases, mutations were found, mostly in KCNQ1, KCNH2, and SCN5A; 5.2% of cases had multiple mutations. Pathogenicity analysis revealed 39 mutations as likely pathogenic, 12 as VUS, and 3 as non-pathogenic. Clinical analysis revealed that 75.6% of patients with QTc≥500 ms were genetically confirmed. Our results support the use of genetic testing of KCNQ1, KCNH2, and SCN5A as part of the diagnosis of LQTS and to help identify relatives at risk of SCD. Further, the genetic tools appear more valuable as disease severity increases. However, the identification of genetic variations in the clinical investigation of single patients using bioinformatic tools can produce erroneous conclusions regarding pathogenicity. Therefore segregation studies are key to determining causality.

Stop-gain mutations in PKP2 are associated with a later age of onset of arrhythmogenic right ventricular cardiomyopathy.

Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a cardiac disease characterized by the presence of fibrofatty replacement of the right ventricular myocardium, which may cause ventricular arrhythmias and sudden cardiac death. Pathogenic mutations in several genes encoding mainly desmosomal proteins have been reported. Our aim is to perform genotype-phenotype correlations to establish the diagnostic value of genetics and to assess the role of mutation type in age-related penetrance in ARVC. Methods and Results Thirty unrelated Spanish patients underwent a complete clinical evaluation. They all were screened for PKP2, DSG2, DSC2, DSP, JUP and TMEM43 genes. A total of 70 relatives of four families were also studied. The 30 patients fulfilled definite disease diagnostic criteria. Genetic analysis revealed a pathogenic mutation in 19 patients (13 in PKP2, 3 in DSG2, 2 in DSP, and 1 in DSC2). Nine of these mutations created a truncated protein due to the generation of a stop codon. Familial assessment revealed 28 genetic carriers among family members. Stop-gain mutations were associated to a later age of onset of ARVC, without differences in the severity of the pathology. Conclusions Familial genetic analysis helps to identify the cause responsible for the pathology. In discrepancy with previous studies, the presence of a truncating protein does not confer a worse severity. This information could suggest that truncating proteins may be compensated by the normal allele and that missense mutations may act as poison peptides.