Irene Mademont-Soler

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.

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.

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.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort.

Background Brugada syndrome (BrS) is a rare genetic cardiac arrhythmia that can lead to sudden cardiac death in patients with a structurally normal heart. Genetic variations in SCN5A can be identified in approximately 20-25% of BrS cases. The aim of our work was to determine the spectrum and prevalence of genetic variations in a Spanish cohort diagnosed with BrS. Methodology/Principal Findings We directly sequenced fourteen genes reported to be associated with BrS in 55 unrelated patients clinically diagnosed. Our genetic screening allowed the identification of 61 genetic variants. Of them, 20 potentially pathogenic variations were found in 18 of the 55 patients (32.7% of the patients, 83.3% males). Nineteen of them were located in SCN5A, and had either been previously reported as pathogenic variations or had a potentially pathogenic effect. Regarding the sequencing of the minority genes, we discovered a potentially pathogenic variation in SCN2B that was described to alter sodium current, and one nonsense variant of unknown significance in RANGRF. In addition, we also identified 40 single nucleotide variations which were either synonymous variants (four of them had not been reported yet) or common genetic variants. We next performed MLPA analysis of SCN5A for the 37 patients without an identified genetic variation, and no major rearrangements were detected. Additionally, we show that being at the 30-50 years range or exhibiting symptoms are factors for an increased potentially pathogenic variation discovery yield. Conclusions In summary, the present study is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish BrS cohort. The mean pathogenic variation discovery yield is higher than that described for other European BrS cohorts (32.7% vs 20-25%, respectively), and is even higher for patients in the 30-50 years age range.

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.

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.

Molecular autopsy in a cohort of infants died suddenly at rest

Sudden infant death syndrome is the leading cause of death during the first year of life. A large part of cases remains without a conclusive cause of death after complete autopsy. In these situations, cardiac arrhythmia of genetic origin is suspected as the most plausible cause of death. Our aim was to ascertain whether genetic variants associated with sudden cardiac death might be the cause of death in a cohort of infants died suddenly. We analyzed 108 genes associated with sudden cardiac death in 44 post-mortem samples of infants less than 1 year old of age who died at rest. Definite cause of death was not conclusive in any case after a complete autopsy. Genetic analysis identified at least one rare variant in 90.90% of samples. A total of 121 rare genetic variants were identified. Of them, 33.05% were novel and 39.66% were located in genes encoding ion channels or associated proteins. A comprehensive genetic analysis in infants who died suddenly enables the unraveling of potentially causative cardiac variants in 2045% of cases. Molecular autopsy should be included in forensic protocols when no conclusive cause of death is identified. Large part genetic variants remain of uncertain significance, reinforcing the crucial role of genetic interpretation before clinical translation but also in early identification of relatives at risk.

Genetic analysis in post-mortem samples with micro-ischemic alterations

Sudden cardiac arrest is a leading cause of death worldwide. Most cardiac arrests happen in patients who have previously suffered a myocardial infarct. The risk of sudden death after infarction may increase in people who carry a pathogenic genetic alteration in cardiac ion channels. We hypothesized that micro-ischemia could trigger lethal arrhythmogenesis, thus we sought to identify genetic alterations in cardiac ion channels in patients with micro-ischemic disease. We studied a cohort of 56 post-mortem samples. Autopsy studies identified myocardial infarction as the cause of death in each case. We used both Sanger sequencing and next-generation sequencing to screen candidate genes associated with sudden cardiac death. We identified six rare missense genetic variations in five unrelated patients. Two variants have been previously reported; one is associated with atrial fibrillation (SCN5A_p.H445D), and the other is predicted to be benign (ANK2_p.T2059M). The novel variants were predicted in silico as benign, except for one (RyR2_p.M4019T), which was classified as deleterious. Our post-mortem, micro-infarction cohort displayed a rate of nearly 10% non-common genetic variants. However, the clinical significance of most of the identified variants remains unknown due to lack of family assessment. Further analyses should be performed in large cohorts to clarify the role of ion-channel gene analysis in samples showing microscopic ischemic alterations.