Brugada Syndrome and Pulmonary Atresia with Intact Interventricular Septum: Fortuitous Finding or New Genetic Connection?

Brugada syndrome is a rare arrhythmogenic syndrome associated mainly with pathogenic variants in the SCN5A gene. Right ventricle outflow tract fibrosis has been reported in some cases of patients diagnosed with Brugada syndrome. Pulmonary atresia with an intact ventricular septum is characterized by the lack of a functional pulmonary valve, due to the underdevelopment of the right ventricle outflow tract. We report, for the first time, a 4-year-old boy with pulmonary atresia with an intact ventricular septum who harbored a pathogenic de novo variant in SCN5A, and the ajmaline test unmasked a type-1 Brugada pattern. We suggest that deleterious variants in the SCN5A gene could be implicated in pulmonary atresia with an intact ventricular septum embryogenesis, leading to overlapping phenotypes.

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.

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.

Role of microRNAs in arrhythmogenic cardiomyopathy: translation as biomarkers into clinical practice.

Arrhythmogenic cardiomyopathy is a rare inherited entity, characterized by a progressive fibro-fatty replacement of the myocardium. It leads to malignant arrhythmias and a high risk of sudden cardiac death. Incomplete penetrance and variable expressivity are hallmarks of this arrhythmogenic cardiac disease, where the first manifestation may be syncope and sudden cardiac death, often triggered by physical exercise. Early identification of individuals at risk is crucial to adopt protective and ideally personalized measures to prevent lethal episodes. The genetic analysis identifies deleterious rare variants in nearly 70% of cases, mostly in genes encoding proteins of the desmosome. However, other factors may modulate the phenotype onset and outcome of disease, such as microRNAs. These small noncoding RNAs play a key role in gene expression regulation and the network of cellular processes. In recent years, data focused on the role of microRNAs as potential biomarkers in arrhythmogenic cardiomyopathy have progressively increased. A better understanding of the functions and interactions of microRNAs will likely have clinical implications. Herein, we propose an exhaustive review of the literature regarding these noncoding RNAs, their versatile mechanisms of gene regulation and present novel targets in arrhythmogenic cardiomyopathy.

Sex differences in long QT syndrome.

Long QT Syndrome (LQTS) is a rare, inherited channelopathy characterized by cardiac repolarization dysfunction, leading to a prolonged rate-corrected QT interval in patients who are at risk for malignant ventricular tachyarrhythmias, syncope, and even sudden cardiac death. A complex genetic origin, variable expressivity as well as incomplete penetrance make the diagnosis a clinical challenge. In the last 10 years, there has been a continuous improvement in diagnostic and personalized treatment options. Therefore, several factors such as sex, age diagnosis, QTc interval, and genetic background may contribute to risk stratification of patients, but it still currently remains as a main challenge in LQTS. It is widely accepted that sex is a risk factor itself for some arrhythmias. Female sex has been suggested as a risk factor in the development of malignant arrhythmias associated with LQTS. The existing differences between the sexes are only manifested after puberty, being the hormones the main inducers of arrhythmias. Despite the increased risk in females, no more than 10% of the available publications on LQTS include sex-related data concerning the risk of malignant arrhythmias in females. Therein, the relevance of our review data update concerning women and LQTS.

Characterization of cardiac involvement in children with LMNA-related muscular dystrophy.

Introduction: LMNA-related muscular dystrophy is a rare entity that produce "laminopathies" such as Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B (LGMD1B), and LMNA-related congenital muscular dystrophy (L-CMD). Heart failure, malignant arrhythmias, and sudden death may occur. No consensus exists on cardiovascular management in pediatric laminopathies. The aim was to perform an exhaustive cardiologic follow-up in pediatric patients diagnosed with LMNA-related muscular dystrophy. Methods: Baseline cardiac work-up consisted of clinical assessment, transthoracic Doppler echocardiography, 12-lead electrocardiogram, electrophysiological study, and implantation of a long-term implantable cardiac loop recorder (ILR). Results: We enrolled twenty-eight pediatric patients diagnosed with EDMD (13 patients), L-CMD (11 patients), LGMD1B (2 patients), and LMNA-related mild weakness (2 patients). Follow-up showed dilated cardiomyopathy (DCM) in six patients and malignant arrhythmias in five (four concomitant with DCM) detected by the ILR that required implantable cardioverter defibrillator (ICD) implantation. Malignant arrhythmias were detected in 20% of our cohort and early-onset EDMD showed worse cardiac prognosis. Discussion: Patients diagnosed with early-onset EDMD are at higher risk of DCM, while potentially life-threatening arrhythmias without DCM appear earlier in L-CMD patients. Early onset neurologic symptoms could be related with worse cardiac prognosis. Specific clinical guidelines for children are needed to prevent sudden death.

Molecular autopsy: Twenty years of post-mortem diagnosis in sudden cardiac death.

In the forensic medicine field, molecular autopsy is the post-mortem genetic analysis performed to attempt to unravel the cause of decease in cases remaining unexplained after a comprehensive forensic autopsy. This negative autopsy, classified as negative or non-conclusive, usually occurs in young population. In these cases, in which the cause of death is unascertained after a thorough autopsy, an underlying inherited arrhythmogenic syndrome is the main suspected cause of death. Next-generation sequencing allows a rapid and cost-effectives genetic analysis, identifying a rare variant classified as potentially pathogenic in up to 25% of sudden death cases in young population. The first symptom of an inherited arrhythmogenic disease may be a malignant arrhythmia, and even sudden death. Early identification of a pathogenic genetic alteration associated with an inherited arrhythmogenic syndrome may help to adopt preventive personalized measures to reduce risk of malignant arrhythmias and sudden death in the victim's relatives, at risk despite being asymptomatic. The current main challenge is a proper genetic interpretation of variants identified and useful clinical translation. The implications of this personalized translational medicine are multifaceted, requiring the dedication of a specialized team, including forensic scientists, pathologists, cardiologists, pediatric cardiologists, and geneticists.

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.

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.

Evaluation of FMR4, FMR5 and FMR6 Expression Levels as Non-Invasive Biomarkers for the Diagnosis of Fragile X-Associated Primary Ovarian Insufficiency (FXPOI).

Female FMR1 (Fragile X mental retardation 1) premutation carriers are at risk for developing fragile X-associated primary ovarian insufficiency (FXPOI), a condition characterized by amenorrhea before age 40 years. Not all women with a FMR1 premutation suffer from primary ovarian insufficiency and nowadays there are no molecular or other biomarkers that can help predict the occurrence of FXPOI. Long non-coding RNAs (lncRNAs) comprise a group of regulatory transcripts which have versatile molecular functions, making them important regulators in all aspects of gene expression. In recent medical studies, lncRNAs have been described as potential diagnostic biomarkers in many diseases. The present study was designed to determine the expression profile of three lncRNAs derived from the FMR1 locus, FMR4, FMR5 and FMR6, in female FMR1 premutation carriers in order: (i) to determine a possible role in the pathogenesis of FXPOI and (ii) to investigate whether they could serve as a biomarker for the diagnosis of FXPOI. FMR4, FMR5 and FMR6 transcripts levels were evaluated in total RNA extracted from peripheral blood by digital droplet PCR and compared between FMR1 premutation carriers with FXPOI and without FXPOI. The diagnostic value of lncRNAs was evaluated by receiver operating characteristic (ROC) analysis. Results revealed a significant association between FXPOI and high expression levels of FMR4. No association was obtained for FMR5 or FMR6. ROC curve analysis revealed that FMR4 can distinguish FMR1 premutation carrier with FXPOI with a diagnostic power of 0.67. These findings suggest a potential role of FMR4 as a possible biomarker for FXPOI.

Brugada Syndrome in Women: What Do We Know After 30 Years?

Brugada syndrome (BrS) was initially described in 1992 by Josep and Pedro Brugada as an arrhythmogenic disease characterized by ST segment elevation in the right precordial leads and increased risk of sudden cardiac death (SCD). Alterations in the SCN5A gene are responsible for approximately 30% of cases of BrS, following an autosomal dominant pattern of inheritance. However, despite its autosomal transmission, sex-related differences are widely accepted. BrS is more prevalent in males than in females (8-10 times), with males having a 5.5-fold higher risk of SCD. There are also differences in clinical presentation, with females being more frequently asymptomatic and older than males at the time of diagnosis. Some factors have been identified that could explain these differences, among which testosterone seems to play an important role. However, only 30% of the available publications on the syndrome include sex-related information. Therefore, current findings on BrS are based on studies conducted mainly in male population, despite the wide acceptance of gender differences. The inclusion of complete clinical and demographic information in future publications would allow a better understanding of the phenotypic variability of BrS in different age and sex groups helping to improve the diagnosis, management and risk management of SCD.

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.

Clinical Genetics of Inherited Arrhythmogenic Disease in the Pediatric Population.

Sudden death is a rare event in the pediatric population but with a social shock due to its presentation as the first symptom in previously healthy children. Comprehensive autopsy in pediatric cases identify an inconclusive cause in 40-50% of cases. In such cases, a diagnosis of sudden arrhythmic death syndrome is suggested as the main potential cause of death. Molecular autopsy identifies nearly 30% of cases under 16 years of age carrying a pathogenic/potentially pathogenic alteration in genes associated with any inherited arrhythmogenic disease. In the last few years, despite the increasing rate of post-mortem genetic diagnosis, many families still remain without a conclusive genetic cause of the unexpected death. Current challenges in genetic diagnosis are the establishment of a correct genotype-phenotype association between genes and inherited arrhythmogenic disease, as well as the classification of variants of uncertain significance. In this review, we provide an update on the state of the art in the genetic diagnosis of inherited arrhythmogenic disease in the pediatric population. We focus on emerging publications on gene curation for genotype-phenotype associations, cases of genetic overlap and advances in the classification of variants of uncertain significance. Our goal is to facilitate the translation of genetic diagnosis to the clinical area, helping risk stratification, treatment and the genetic counselling of families.

Genetic Abnormalities in Large to Giant Congenital Nevi: Beyond NRAS Mutations.

Large and giant congenital melanocytic nevi (CMN) are rare melanocytic lesions mostly caused by postzygotic NRAS alteration. Molecular characterization is usually focused on NRAS and BRAF genes in a unique biopsy sample of the CMN. However, large/giant CMN may exhibit phenotypic differences among distinct areas, and patients differ in features such as presence of multiple CMN or spilus-like lesions. Herein, we have characterized a series of 21 large/giant CMN including patients with spilus-type nevi (9/21 patients, 42.8%). Overall, 53 fresh frozen biopsy samples corresponding to 40 phenotypically characterized areas of large/giant CMNs and 13 satellite lesions were analyzed with a multigene panel and RNA sequencing. Mutational screening showed mutations in 76.2% (16/21) of large/giant CMNs. A NRAS mutation was found in 57.1% (12/21) of patients, and mutations in other genes such as BRAF, KRAS, APC, and MET were detected in 14.3% (3/21) of patients. RNA sequencing showed the fusion transcript ZEB2-ALK and SOX5-RAF1 in large/giant CMN from two patients without missense mutations. Both alterations were not detected in unaffected skin and were detected in different areas of affected skin. These findings suggest that large/giant CMN may result from distinct molecular events in addition to NRAS mutations, including point mutations and fusion transcripts.

Aplicación de la secuenciación masiva de nueva generación al diagnóstico molecular de la hipercolesterolemia familiar / Applicability of next generation sequencing to the molecular diagnosis of familial hypercholesterolemia

Introducción: La hipercolesterolemia familiar es una enfermedad autosómica dominante que cursa con niveles plasmáticos elevados de colesterol unido a lipoproteínas de baja densidad. La presencia de esta alteración en el metabolismo lipídico se asocia a un aumento del riesgo cardiovascular en los pacientes que la padecen, siendo de gran importancia la realización de un diagnóstico genético para ofrecer un tratamiento adecuado y disminuir la morbimortalidad. El objetivo de este trabajo es describir la aplicación de la secuenciación de nueva generación al diagnóstico genético de la hipercolesterolemia familiar, en comparación con la secuenciación Sanger, la técnica convencional usada hasta el momento. Material y métodos: Se analizaron 110 muestras de sangre venosa periférica procedente de pacientes que presentaban cuadro clínico de hipercolesterolemia familiar mediante secuenciación de nueva generación, utilizando un panel comercial que permite la identificación de mutaciones en los genes LDLR, APOB, PCSK9 yLDLRAP1 (SEQPRO Lipo, Progenika) con la tecnología GS JUNIOR 454 (Roche). Resultados: Aplicando esta tecnología fue posible secuenciar los genes asociados a la hipercolesterolemia familiar descritos hasta el momento en grupos de hasta 20 pacientes simultáneamente. Se detectaron un total de 35 mutaciones en las 110 muestras analizadas, localizándose el 94,29% en el gen LDLR. Todas las mutaciones identificadas fueron confirmadas mediante el método de secuenciación Sanger. Conclusiones: La utilización de la secuenciación masiva de nueva generación permite la realización de un diagnóstico genético más rápido y un análisis molecular más eficiente de los genes implicados en la hipercolesterolemia familiar con una fiabilidad similar a la técnica convencional Sanger (AU)

Introduction: Familial hypercholesterolemia is an autosomal dominant disorder that causes increased levels of cholesterol associated with low density lipoproteins in plasma. The presence of altered lipid metabolism in these patients increases their level of risk of suffering from a cardiovascular disease. The certainty of having this genetic disorder by making a timely and precise molecular diagnostic is crucial for the appropriate treatment and the reduction of the disease morbidity-mortality. The aim of this work is to describe the applicability of next generation sequencing technology to the genetic diagnosis of familial hypercholesterolemia and compare this novel method with the conventional Sanger sequencing method. Material and methods: A next generation sequencing commercial panel (SEQPRO Lipo, Progenika) was used to analyze 110 peripheral venous blood samples from patients with familial hypercholesterolemia. This enables the assessment of mutations in genes associated with the disease (e.g. LDLR, APOB, PCSK9 and LDLRAP1)with the GS JUNIOR 454 technology (Roche). Results: Application of next generation sequencing enables the sequencing of the genes involved in the familial hypercholesterolemias, described so far, in groups of 20 patients simultaneously. Using this novel technology, a total of 35 mutations were detected in the 110 analysed samples, with 94.29% being located in the LDLR gene. Mutations were confirmed by Sanger sequencing. Conclusion: Next generation sequencing enables a quick genetic diagnosis and a more efficient molecular analysis of all genes described so far to be involved in familial hypercholesterolemia, with similar reliability to that of conventional Sanger sequencing (AU)

MLPA: a prenatal diagnostic tool for the study of congenital heart defects?

Congenital heart defects (CHD) represent the most common birth defects, so they are not a rare finding when performing routine ultrasound examinations during pregnancy. Once chromosome abnormalities have been excluded in a fetus with a CHD, chromosome 22q11.2 deletion is usually investigated by FISH, as it is the most frequent microdeletion syndrome and is generally associated with cardiac malformations. If 22q11.2 microdeletion is ruled out, the etiology of the CHD remains generally unexplained, making familial genetic counseling difficult. To evaluate the usefulness of Multiplex Ligation-dependent Probe Amplification (MLPA) kits designed for the study of 22q11.2 and other genomic regions previously associated with syndromic CHD, we performed MLPA in 55 pregnancies with fetuses presenting CHD, normal karyotype and negative FISH results for 22q11.2 microdeletion, which constitutes the largest prenatal series reported. Definitive MLPA results were obtained in 50 pregnancies, and in this setting such MLPA kits did not detect any imbalance. On the other hand, to compare FISH and MLPA techniques for the study of 22q11.2 microdeletions, we performed MLPA in 4 pregnancies known to have 22q11.2 deletions (by FISH). All four 22q11.2 microdeletions were also detected by MLPA, which corroborates that it is a reliable technique for the diagnosis and characterization of 22q11.2 deletions. Finally, we evaluated the possibility of replacing conventional FISH by MLPA for the prenatal diagnosis of CHD, comparing the diagnostic potential, results delivery times, repetition and failure rates and cost of both techniques, and concluded that FISH should still be the technique of choice for the prenatal diagnosis of fetuses with CHD.