Implementing a New Algorithm for Reinterpretation of Ambiguous Variants in Genetic Dilated Cardiomyopathy.

Dilated cardiomyopathy is a heterogeneous entity that leads to heart failure and malignant arrhythmias. Nearly 50% of cases are inherited; therefore, genetic analysis is crucial to unravel the cause and for the early identification of carriers at risk. A large number of variants remain classified as ambiguous, impeding an actionable clinical translation. Our goal was to perform a comprehensive update of variants previously classified with an ambiguous role, applying a new algorithm of already available tools. In a cohort of 65 cases diagnosed with dilated cardiomyopathy, a total of 125 genetic variants were classified as ambiguous. Our reanalysis resulted in the reclassification of 12% of variants from an unknown to likely benign or likely pathogenic role, due to improved population frequencies. For all the remaining ambiguous variants, we used our algorithm; 60.9% showed a potential but not confirmed deleterious role, and 24.5% showed a potential benign role. Periodically updating the population frequencies is a cheap and fast action, making it possible to clarify the role of ambiguous variants. Here, we perform a comprehensive reanalysis to help to clarify the role of most of ambiguous variants. Our specific algorithms facilitate genetic interpretation in dilated cardiomyopathy.

Reevaluation of ambiguous genetic variants in sudden unexplained deaths of a young cohort.

Sudden death cases in the young population remain without a conclusive cause of decease in almost 40% of cases. In these situations, cardiac arrhythmia of genetic origin is suspected as the most plausible cause of death. Molecular autopsy may reveal a genetic defect in up to 20% of families. Most than 80% of rare variants remain classified with an ambiguous role, impeding a useful clinical translation. Our aim was to update rare variants originally classified as of unknown significance to clarify their role. Our cohort included fifty-one post-mortem samples of young cases who died suddenly and without a definite cause of death. Five years ago, molecular autopsy identified at least one rare genetic alteration classified then as ambiguous following the American College of Medical Genetics and Genomics' recommendations. We have reclassified the same rare variants including novel data. About 10% of ambiguous variants change to benign/likely benign mainly because of improved population frequencies. Excluding cases who died before one year of age, almost 21% of rare ambiguous variants change to benign/likely benign. This fact makes it important to discard these rare variants as a cause of sudden unexplained death, avoiding anxiety in relatives' carriers. Twenty-five percent of the remaining variants show a tendency to suspicious deleterious role, highlighting clinical follow-up of carriers. Periodical reclassification of rare variants originally classified as ambiguous is crucial, at least updating frequencies every 5 years. This action aids to increase accuracy to enable and conclude a cause of death as well as translation into the clinic.

Clinical impact of rare variants associated with inherited channelopathies: a 5-year update.

A proper interpretation of the pathogenicity of rare variants is crucial before clinical translation. Ongoing addition of new data may modify previous variant classifications; however, how often a reanalysis is necessary remains undefined. We aimed to extensively reanalyze rare variants associated with inherited channelopathies originally classified 5 years ago and its clinical impact. In 2016, rare variants identified through genetic analysis were classified following the American College of Medical Genetics and Genomics' recommendations. Five years later, we have reclassified the same variants following the same recommendations but including new available data. Potential clinical implications were discussed. Our cohort included 49 cases of inherited channelopathies diagnosed in 2016. Update show that 18.36% of the variants changed classification mainly due to improved global frequency data. Reclassifications mostly occurred in minority genes associated with channelopathies. Similar percentage of variants remain as deleterious nowadays, located in main known genes (SCN5A, KCNH2 and KCNQ1). In 2016, 69.38% of variants were classified as unknown significance, but now, 53.06% of variants are classified as such, remaining the most common group. No management was modified after translation of genetic data into clinics. After 5 years, nearly 20% of rare variants associated with inherited channelopathies were reclassified. This supports performing periodic reanalyses of no more than 5 years since last classification. Use of newly available data is necessary, especially concerning global frequencies and family segregation. Personalized clinical translation of rare variants can be crucial to management if a significant change in classification is identified.

A microRNA Signature for the Diagnosis of Statins Intolerance.

Atherosclerotic cardiovascular diseases (ASCVD) are the leading cause of morbidity and mortality in Western societies. Statins are the first-choice therapy for dislipidemias and are considered the cornerstone of ASCVD. Statin-associated muscle symptoms are the main reason for dropout of this treatment. There is an urgent need to identify new biomarkers with discriminative precision for diagnosing intolerance to statins (SI) in patients. MicroRNAs (miRNAs) have emerged as evolutionarily conserved molecules that serve as reliable biomarkers and regulators of multiple cellular events in cardiovascular diseases. In the current study, we evaluated plasma miRNAs as potential biomarkers to discriminate between the SI vs. non-statin intolerant (NSI) population. It is a multicenter, prospective, case-control study. A total of 179 differentially expressed circulating miRNAs were screened in two cardiovascular risk patient cohorts (high and very high risk): (i) NSI (n = 10); (ii) SI (n = 10). Ten miRNAs were identified as being overexpressed in plasma and validated in the plasma of NSI (n = 45) and SI (n = 39). Let-7c-5p, let-7d-5p, let-7f-5p, miR-376a-3p and miR-376c-3p were overexpressed in the plasma of SI patients. The receiver operating characteristic curve analysis supported the discriminative potential of the diagnosis. We propose a three-miRNA predictive fingerprint (let-7f, miR-376a-3p and miR-376c-3p) and several clinical variables (non-HDLc and years of dyslipidemia) for SI discrimination; this model achieves sensitivity, specificity and area under the receiver operating characteristic curve (AUC) of 83.67%, 88.57 and 89.10, respectively. In clinical practice, this set of miRNAs combined with clinical variables may discriminate between SI vs. NSI subjects. This multiparametric model may arise as a potential diagnostic biomarker with clinical value.

miR-16-5p Suppression Protects Human Cardiomyocytes against Endoplasmic Reticulum and Oxidative Stress-Induced Injury.

Oxidative stress, defined as the excess production of reactive oxygen species (ROS) relative to antioxidant defense, plays a significant role in the development of cardiovascular diseases. Endoplasmic reticulum (ER) stress has emerged as an important source of ROS and its modulation could be cardioprotective. Previously, we demonstrated that miR-16-5p is enriched in the plasma of ischemic dilated cardiomyopathy (ICM) patients and promotes ER stress-induced apoptosis in cardiomyocytes in vitro. Here, we hypothesize that miR-16-5p might contribute to oxidative stress through ER stress induction and that targeting miR-16-5p may exert a cardioprotective role in ER stress-mediated cardiac injury. Analysis of oxidative markers in the plasma of ICM patients demonstrates that oxidative stress is associated with ICM. Moreover, we confirm that miR-16-5p overexpression promotes oxidative stress in AC16 cardiomyoblasts. We also find that, in response to tunicamycin-induced ER stress, miR-16-5p suppression decreases apoptosis, inflammation and cardiac damage via activating the ATF6-mediated cytoprotective pathway. Finally, ATF6 is identified as a direct target gene of miR-16-5p by dual-luciferase reporter assays. Our results indicate that miR-16-5p promotes ER stress and oxidative stress in cardiac cells through regulating ATF6, suggesting that the inhibition of miR-16-5p has potential as a therapeutic approach to protect the heart against ER and oxidative stress-induced injury.

Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples.

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members' follow-up.

Genetic interpretation and clinical translation of minor genes related to Brugada syndrome.

Brugada syndrome (BrS) is an inherited arrhythmogenic disease associated with sudden cardiac death. The main gene is SCN5A. Additional variants in 42 other genes have been reported as deleterious, although these variants have not yet received comprehensive pathogenic analysis. Our aim was to clarify the role of all currently reported variants in minor genes associated with BrS. We performed a comprehensive analysis according to the American College of Medical Genetics and Genomics guidelines of published clinical and basic data on all genes (other than SCN5A) related to BrS. Our results identified 133 rare variants potentially associated with BrS. After applying current recommendations, only six variants (4.51%) show a conclusive pathogenic role. All definitively pathogenic variants were located in four genes encoding sodium channels or related proteins: SLMAP, SEMA3A, SCNN1A, and SCN2B. In total, 33.83% of variants in 19 additional genes were potentially pathogenic. Beyond SCN5A, we conclude definitive pathogenic variants associated with BrS in four minor genes. The current list of genes associated with BrS, therefore, should include SCN5A, SLMAP, SEMA3A, SCNN1A, and SCN2B. Comprehensive genetic interpretation and careful clinical translation should be done for all variants currently classified as potentially deleterious for BrS.

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.

Update about atrial fibrillation genetics.

PURPOSE OF REVIEW: Atrial fibrillation is an important cause of morbidity in the aging population. The mechanisms responsible for the triggering and maintenance of the chaotic atrial rhythm are still poorly understood. In this review, we will focus on the genetic aspects of atrial fibrillation, to understand causality, with special emphasis on recent studies published in the field. RECENT FINDINGS: Diseases such as hypertension, valvular heart disease, and heart failure may induce atrial fibrillation, which increases the risk of stroke and sudden cardiac death. Clinical studies published in these last two decades have provided evidence that genetics play a key role in atrial fibrillation. Thus, a family history of the disease has been identified in up to 30% of clinically diagnosed patients. In those genotyped families, most carry rare genetic variants in genes associated with ionic channels, calcium handling protein, or predisposing to fibrosis, conduction system disease, and inflammatory processes. SUMMARY: Currently, atrial fibrillation is the most common sustained arrhythmia in clinical practice. The pathophysiological mechanisms of atrial fibrillation are complex. A better understanding of the molecular basis will help improve both current risk stratification and clinical management.

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.

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.

Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart.

The α subunit of the cardiac voltage-gated sodium channel, NaV1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of NaV1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any NaV1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of NaV1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore NaV1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure.

Generation of the induced pluripotent stem cell line ESi108-A from a familial atrial fibrillation patient.

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.

LMNA-related muscular dystrophy: Identification of variants in alternative genes and personalized clinical translation.

Background: Laminopathies are caused by rare alterations in LMNA, leading to a wide clinical spectrum. Though muscular dystrophy begins at early ages, disease progression is different in each patient. We investigated variability in laminopathy phenotypes by performing a targeted genetic analysis of patients diagnosed with LMNA-related muscular dystrophy to identify rare variants in alternative genes, thereby explaining phenotypic differences. Methods: We analyzed 105 genes associated with muscular diseases by targeted sequencing in 26 pediatric patients of different countries, diagnosed with any LMNA-related muscular dystrophy. Family members were also clinically assessed and genetically analyzed. Results: All patients carried a pathogenic rare variant in LMNA. Clinical diagnoses included Emery-Dreifuss muscular dystrophy (EDMD, 13 patients), LMNA-related congenital muscular dystrophy (L-CMD, 11 patients), and limb-girdle muscular dystrophy 1B (LGMD1B, 2 patients). In 9 patients, 10 additional rare genetic variants were identified in 8 genes other than LMNA. Genotype-phenotype correlation showed additional deleterious rare variants in five of the nine patients (3 L-CMD and 2 EDMD) with severe phenotypes. Conclusion: Analysis f known genes related to muscular diseases in close correlation with personalized clinical assessments may help identify additional rare variants of LMNA potentially associated with early onset or most severe disease progression.

Generation of an induced pluripotent stem cell line from a healthy Caucasian male.

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.

High-quality RNA improves sensitivity of SARS-CoV-2 detection by colorimetric RT-LAMP.

The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.

Structural Heart Alterations in Brugada Syndrome: Is it Really a Channelopathy? A Systematic Review.

Brugada syndrome (BrS) is classified as an inherited cardiac channelopathy attributed to dysfunctional ion channels and/or associated proteins in cardiomyocytes rather than to structural heart alterations. However, hearts of some BrS patients exhibit slight histologic abnormalities, suggesting that BrS could be a phenotypic variant of arrhythmogenic cardiomyopathy. We performed a systematic review of the literature following Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement (PRISMA) criteria. Our comprehensive analysis of structural findings did not reveal enough definitive evidence for reclassification of BrS as a cardiomyopathy. The collection and comprehensive analysis of new cases with a definitive BrS diagnosis are needed to clarify whether some of these structural features may have key roles in the pathophysiological pathways associated with malignant arrhythmogenic episodes.

Post-mortem toxicology analysis in a young sudden cardiac death cohort.

Risk of sudden cardiac death (SCD) increases with age, and several studies have examined the impact of different drugs on cardiovascular function. However, few studies have integrated epidemiological drug consumption data and genetic background in the context of cardiac death. We performed a retrospective population-based study in forensic sudden death cases from a 9-year period in Catalonia. The young cohort included 924 cases 18-50 years old, 566 of which had a cardiac cause of death. Complete autopsy, toxicological, and histopathological studies were performed. Molecular autopsy using next-generation sequencing was performed in nearly 400 cardiac cases. Cases related with fatal acute intoxication were excluded. Drug consumption prevalence was similar between forensic cases of cardiac and non-cardiac origin (62.5% versus 69.5%), with the exception of alcohol, which was more prevalent in the cardiac group than in the non-cardiac group (23.3% versus 17.1%). Individuals in the toxicology-positive group were carriers of more rare genetic variants and were significantly younger than the toxicology-negative group. Psychopharmacological drugs were identified in 22.3% of cardiac cases, and molecular autopsy identified an association between antiepileptic drugs or caffeine and pathogenic or likely pathogenic variants in arrhythmogenic genes. Specific substances could therefore play an essential role as triggers of SCD in genetically predisposed young people.

Rare variants in genes encoding structural myocyte contribute to a thickened ventricular septum in sudden death population without ventricular alterations.

Unexpected cardiac deaths are a current challenge to healthcare systems. In adults, coronary artery disease and acquired cardiomyopathies are the most frequent causes of sudden cardiac death while in younger than 35 years old, the main cause is represented by non-ischemic diseases, usually inherited. Nowadays, around 10%-15% of unexpected deaths remain without a definite cause of decease after a complete autopsy, then classified as deaths potentially due to an inherited arrhythmia. Discrete abnormalities in some of the heart measures have been considered as potential predictors or risk factors for sudden cardiac death. However, role of non-benign genetic variants in these scattered heart alterations remains to be clarified, especially if variants are classified of ambiguous role. Clinicians usually only take into consideration pathogenic variants for decision-making. It is yet unclear what the role of VUS genetic variants in modifying the anatomical parameters of the heart. We hypothesize that some heart measures might be influenced by polygenic components as some variants may individually confer minor risk but may actually produce additive effects when combined with others. Our aim was to investigate whether carrying non-benign rare variants in genes related to inherited arrhythmias may contribute to scattered cardiac alterations in anatomical normal hearts. The study is composed by 761 samples collected from autopsies of SD suffered by adults from 18 to 50 years of age who occurred in Catalonia (Spain) in a 9-year period. Complete medico-legal autopsy was performed to determine the cause of death. Molecular autopsy was performed as part of our forensic protocol, including genes associated with inherited diseases.To evaluate the effect of genetic rare variants into hearts measures we performed a linear regression model and data were presented as regression. This study showed, for the first time, that rare variants, regardless of significance (pathogenic, probably pathogenic or uncertain significance), may contribute to interventricular septum width in the structurally normal heart. While the cohort is based on sudden death cases, further studies and case-control studies will be necessary to conclude that the genetic determinants of septal thickness contributes to sudden cardiac death. We conclude that non-benign rare variants contribute to modify scattered septum width in structural normal hearts, being a potential risk factor of arrhythmia in genetic harbors. These evidence support the current recommendation in forensic protocols of including histologic analysis of septum when inherited arrhythmogenic disease is suspicious cause of decease.

Discerning the Ambiguous Role of Missense TTN Variants in Inherited Arrhythmogenic Syndromes.

The titin gene (TTN) is associated with several diseases, including inherited arrhythmias. Most of these diagnoses are attributed to rare TTN variants encoding truncated forms, but missense variants represent a diagnostic challenge for clinical genetics. The proper interpretation of genetic data is critical for translation into the clinical setting. Notably, many TTN variants were classified before 2015, when the American College of Medical Genetics and Genomics (ACMG) published recommendations to accurately classify genetic variants. Our aim was to perform an exhaustive reanalysis of rare missense TTN variants that were classified before 2015, and that have ambiguous roles in inherited arrhythmogenic syndromes. Rare missense TTN variants classified before 2015 were updated following the ACMG recommendations and according to all the currently available data. Our cohort included 193 individuals definitively diagnosed with an inherited arrhythmogenic syndrome before 2015. Our analysis resulted in the reclassification of 36.8% of the missense variants from unknown to benign/likely benign. Of all the remaining variants, currently classified as of unknown significance, 38.3% showed a potential, but not confirmed, deleterious role. Most of these rare missense TTN variants with a suspected deleterious role were identified in patients diagnosed with hypertrophic cardiomyopathy. More than 35% of the rare missense TTN variants previously classified as ambiguous were reclassified as not deleterious, mainly because of improved population frequencies. Despite being inconclusive, almost 40% of the variants showed a potentially deleterious role in inherited arrhythmogenic syndromes. Our results highlight the importance of the periodical reclassification of rare missense TTN variants to improve genetic diagnoses and help increase the accuracy of personalized medicine.