Jesus Mates

Genetic Analysis of Arrhythmogenic Diseases in the Era of NGS: The Complexity of Clinical Decision-Making in Brugada Syndrome

Background The use of next-generation sequencing enables a rapid analysis of many genes associated with sudden cardiac death in diseases like Brugada Syndrome. Genetic variation is identified and associated with 30–35% of cases of Brugada Syndrome, with nearly 20–25% attributable to variants in SCN5A, meaning many cases remain undiagnosed genetically. To evaluate the role of genetic variants in arrhythmogenic diseases and the utility of next-generation sequencing, we applied this technology to resequence 28 main genes associated with arrhythmogenic disorders. Materials and Methods A cohort of 45 clinically diagnosed Brugada Syndrome patients classified as SCN5A-negative was analyzed using next generation sequencing. Twenty-eight genes were resequenced: AKAP9, ANK2, CACNA1C, CACNB2, CASQ2, CAV3, DSC2, DSG2, DSP, GPD1L, HCN4, JUP, KCNE1, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNQ1, NOS1AP, PKP2, RYR2, SCN1B, SCN3B, SCN4B, SCN5A, SNTA1, and TMEM43. A total of 85 clinically evaluated relatives were also genetically analyzed to ascertain familial segregation. Results and Discussion Twenty-two patients carried 30 rare genetic variants in 12 genes, only 4 of which were previously associated with Brugada Syndrome. Neither insertion/deletion nor copy number variation were detected. We identified genetic variants in novel candidate genes potentially associated to Brugada Syndrome. These include: 4 genetic variations in AKAP9 including a de novo genetic variation in 3 positive cases; 5 genetic variations in ANK2 detected in 4 cases; variations in KCNJ2 together with CASQ2 in 1 case; genetic variations in RYR2, including a de novo genetic variation and desmosomal proteins encoding genes including DSG2, DSP and JUP, detected in 3 of the cases. Larger gene panels or whole exome sequencing should be considered to identify novel genes associated to Brugada Syndrome. However, application of approaches such as whole exome sequencing would difficult the interpretation for clinical purposes due to the large amount of data generated. The identification of these genetic variants opens new perspectives on the implications of genetic background in the arrhythmogenic substrate for research purposes. Conclusions As a paradigm for other arrhythmogenic diseases and for unexplained sudden death, our data show that clinical genetic diagnosis is justified in a family perspective for confirmation of genetic causality. In the era of personalized medicine using high-throughput tools, clinical decision-making is increasingly complex.

Natural and Undetermined Sudden Death: Value of Post-Mortem Genetic Investigation

Background Sudden unexplained death may be the first manifestation of an unknown inherited cardiac disease. Current genetic technologies may enable the unraveling of an etiology and the identification of relatives at risk. The aim of our study was to define the etiology of natural deaths, younger than 50 years of age, and to investigate whether genetic defects associated with cardiac diseases could provide a potential etiology for the unexplained cases. Methods and Findings Our cohort included a total of 789 consecutive cases (77.19% males) <50 years old (average 38.6±12.2 years old) who died suddenly from non-violent causes. A comprehensive autopsy was performed according to current forensic guidelines. During autopsy a cause of death was identified in most cases (81.1%), mainly due to cardiac alterations (56.87%). In unexplained cases, genetic analysis of the main genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. Genetic analysis was performed in suspected inherited diseases (cardiomyopathy) and in unexplained death, with identification of potentially pathogenic variants in nearly 50% and 40% of samples, respectively. Conclusions Cardiac disease is the most important cause of sudden death, especially after the age of 40. Close to 10% of cases may remain unexplained after a complete autopsy investigation. Molecular autopsy may provide an explanation for a significant part of these unexplained cases. Identification of genetic variations enables genetic counseling and undertaking of preventive measures in relatives at risk.

Post-mortem genetic analysis in juvenile cases of sudden cardiac death

BACKGROUND The reason behind a sudden death of a young individual remains unknown in up to 50% of postmortem cases. Pathogenic mutations in genes encoding heart proteins are known to cause sudden cardiac death. OBJECTIVE The aim of our study was to ascertain whether genetic alterations could provide an explanation for sudden cardiac death in a juvenile cohort with no-conclusive cause of death after comprehensive autopsy. METHODS Twenty-nine cases <15 years showing no-conclusive cause of death after a complete autopsy were studied. Genetic analysis of 7 main genes associated with sudden cardiac death was performed using Sanger technology in low quality DNA cases, while in good quality cases the analysis of 55 genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. RESULTS Thirty-five genetic variants were identified in 12 cases (41.37%). Ten genetic/variants in genes encoding cardiac ion channels were identified in 8 cases (27.58%). We also identified 9 cases (31.03%) carrying 25 genetic variants in genes encoding structural cardiac proteins. Nine cases carried more than one genetic variation, 5 of them combining structural and non-structural genes. CONCLUSIONS Our study supports the inclusion of molecular autopsy in forensic routine protocols when no conclusive cause of death is identified. Around 40% of sudden cardiac death young cases carry a genetic variant that could provide an explanation for the cause of death. Because relatives could be at risk of sudden cardiac death, our data reinforce their need of clinical assessment and, if indicated, of genetic analysis.

The Role of clinical, genetic and segregation evaluation in sudden infant death

Sudden infant death syndrome (SIDS) is the leading cause of death in the first year of life. Several arrhythmogenic genes have been associated with cardiac pathologies leading to infant sudden cardiac death (SCD). Our aim was to take advantage of next generation sequencing (NGS) technology to perform a thorough genetic analysis of a SIDS case. A SIDS case was referred to our institution after negative autopsy. We performed a genetic analysis of 104 SCD-related genes using a custom panel. Confirmed variants in index case were also analyzed in relatives. Clinical evaluation of first-degree family members was performed. Relatives did not show pathology. NGS identified seven variants. Two previously described as pathogenic. Four previously catalogued without clinical significance. The seventh variation was novel. Familial segregation showed that the index cases mother carried all same genetic variations except one, which was inherited from the father. The sister of the index case carried three variants. We believe that molecular autopsy should be included in current forensic protocols after negative autopsy. In addition to NGS technologies, familial genetic testing should be also performed to clarify potential pathogenic role of new variants and to identify genetic carriers at risk of SCD.

Large Genomic Imbalances in Brugada Syndrome

Purpose Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. Methods 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). Results The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. Conclusion CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes.

Rare Titin (TTN) Variants in Diseases Associated with Sudden Cardiac Death

A leading cause of death in western countries is sudden cardiac death, and can be associated with genetic disease. Next-generation sequencing has allowed thorough analysis of genes associated with this entity, including, most recently, titin. We aimed to identify potentially pathogenic genetic variants in titin. A total of 1126 samples were analyzed using a custom sequencing panel including major genes related to sudden cardiac death. Our cohort was divided into three groups: 432 cases from patients with cardiomyopathies, 130 cases from patients with channelopathies, and 564 post-mortem samples from individuals showing anatomical healthy hearts and non-conclusive causes of death after comprehensive autopsy. None of the patients included had definite pathogenic variants in the genes analyzed by our custom cardio-panel. Retrospective analysis comparing the in-house database and available public databases also was performed. We identified 554 rare variants in titin, 282 of which were novel. Seven were previously reported as pathogenic. Of these 554 variants, 493 were missense variants, 233 of which were novel. Of all variants identified, 399 were unique and 155 were identified at least twice. No definite pathogenic variants were identified in any of genes analyzed. We identified rare, mostly novel, titin variants that seem to play a potentially pathogenic role in sudden cardiac death. Additional studies should be performed to clarify the role of these variants in sudden cardiac death.

A Genetically Vulnerable Myocardium May Predispose to Myocarditis.

Death from myocarditis requires the presence of myocardial inflammation, without any other structural heart disease, and is frequently associated with a viral infection [(1)][1]. We postulate that genetic defects in structural proteins cause the myocardium to become vulnerable and predisposed to

Role of copy number variants in sudden cardiac death and related diseases: genetic analysis and translation into clinical practice

Several studies have identified copy number variants (CNVs) as responsible for cardiac diseases associated with sudden cardiac death (SCD), but very few exhaustive analyses in large cohorts of patients have been performed, and they have been generally focused on a specific SCD-related disease. The aim of the present study was to screen for CNVs the most prevalent genes associated with SCD in a large cohort of patients who suffered sudden unexplained death or had an inherited cardiac disease (cardiomyopathy or channelopathy). A total of 1765 European patients were analyzed with a homemade algorithm for the assessment of CNVs using high-throughput sequencing data. Thirty-six CNVs were identified (2%), and most of them appeared to have a pathogenic role. The frequency of CNVs among cases of sudden unexplained death, patients with a cardiomyopathy or a channelopathy was 1.4% (8/587), 2.3% (20/874), and 2.6% (8/304), respectively. Detection rates were particularly high for arrhythmogenic cardiomyopathy (5.1%), long QT syndrome (4.7%), and dilated cardiomyopathy (4.4%). As such large genomic rearrangements underlie a non-neglectable portion of cases, we consider that their analysis should be performed as part of the routine genetic testing of sudden unexpected death cases and patients with SCD-related diseases.

Correction: Natural and Undetermined Sudden Death: Value of Post-Mortem Genetic Investigation

[This corrects the article DOI: 10.1371/journal.pone.0167358.].

A novel variant in RyR2 causes familiar catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia is a rare familial arrhythmogenic disease. It usually occurs in juvenile patients with a structurally normal heart and causes exercise-emotion triggered syncope and sudden cardiac death. The main gene associated with catecholaminergic polymorphic ventricular tachycardia is RyR2, encoding the cardiac ryanodine receptor protein which is involved in calcium homeostasis. After the identification of a 16 year-old man presenting with exercise-induced sudden cardiac death, clinically diagnosed as catecholaminergic polymorphic ventricular tachycardia, we collected the family information and performed a comprehensive genetic analysis using Next Generation Sequencing technology. The initial electrocardiogram in the emergency department revealed ventricular fibrillation. On electrocardiogram monitoring, sinus tachycardia degenerated into bidirectional ventricular and into ventricular fibrillation. Catecholaminergic polymorphic ventricular tachycardia was clinically diagnosed in 5 of the 14 family members evaluated. There were no additional reports of seizures, pregnancy loss, neonatal death, or sudden cardiac death in family members. Genetic analysis of the index case identified only one rare novel variant p.Ile11Ser (c.32T>G) in the RyR2 gene. Subsequent familial analysis identified segregation of the genetic variant with the disease. All current evidence supports that novel p.Ile11Ser variant in the RyR2 gene is a potential disease-causing variant in catecholaminergic polymorphic ventricular tachycardia. To our knowledge, there has been no previous case report of catecholaminergic polymorphic ventricular tachycardia associated to this missense variant.