Whole-exome sequencing to identify causative variants in juvenile sudden cardiac death.

BACKGROUND: Juvenile sudden cardiac death (SCD) remains unexplained in approximately 40% of cases, leading to a significant emotional burden for the victims' families and society. Comprehensive investigations are essential to uncover its elusive causes and enable cascade family screening. This study aimed to enhance the identification of likely causative variants in juvenile SCD cases (age ≤ 50 years), particularly when autopsy findings are inconclusive. RESULTS: Autopsy revealed diagnostic structural abnormalities in 46%, non-diagnostic findings in 23%, and structurally normal hearts in 31% of cases. Whole-exome sequencing (WES), refined through a customized virtual gene panel was used to identify variants. These variants were then evaluated using a multidisciplinary approach and a structured variant prioritization scheme. Our extended approach identified likely causative variants in 69% of cases, outperforming the diagnostic yields of both the cardio panel and standard susceptibility gene analysis (50% and 16%, respectively). The extended cardio panel achieved an 80% diagnostic yield in cases with structurally normal hearts, demonstrating its efficacy in challenging scenarios. Notably, half of the positive cases harboured a single variant, while the remainder had two or more variants. CONCLUSION: This study highlights the efficacy of a multidisciplinary approach employing WES and a tailored virtual gene panel to elucidate the aetiology of juvenile SCD. The findings support the expansion of genetic testing using tailored gene panels and prioritization schemes as part of routine autopsy evaluations to improve the identification of causative variants and potentially facilitate early diagnosis in first-degree relatives.

A novel TGFßR2 splice variant in patient with aortic aneurysm and family history for aortic dissection: a case report.

We report the clinical presentation and genetic screening of a 31-year-old man with dilatation of the aortic root and ascending aorta and a positive family history for aortic dissection and sudden death. A novel heterozygous variant in a splice acceptor site (c.1600-1G>T) of TGFßR2 gene was identified by using a targeted multi-gene panel analysis. Bioinformatics tools predicted that the c.1600-1G>T variant is pathogenic by altering acceptor splice site at - 1 position affecting pre-mRNA splicing. These data confirm that the diverging splicing in the TGF-ß pathway genes may be an important process in aneurismal disease and emphasize the utility of genetic sequencing in the identification of high-risk patients for a more patient's management able to improve outcomes and minimize costs for the care of patients with heritable thoracic aortic aneurysm and dissection.

[Box: see text].

Low HDL cholesterol and the eNOS Glu298Asp polymorphism are associated with inducible myocardial ischemia in patients with suspected stable coronary artery disease.

BACKGROUND: The endothelial nitric oxide synthase (eNOS) gene deficiency is known to cause impaired coronary vasodilating capability in animal models. In the general clinical population, the eNOS gene polymorphisms, able to affect eNOS activity, were associated with cardiometabolic risk features and prevalence of coronary artery disease (CAD). AIM: To investigate the association of eNOS Glu298Asp gene polymorphism, cardiometabolic profile, obstructive CAD and inducible myocardial ischemia in patients with suspected stable CAD. METHODS: A total of 506 patients (314 males; mean age 62 ± 9 years) referred for suspected CAD was enrolled. Among these, 325 patients underwent stress ECG or cardiac imaging to assess the presence of inducible myocardial ischemia and 436 patients underwent non-invasive computerized tomography or invasive coronary angiography to assess the presence of obstructive CAD. Clinical characteristics and blood samples were collected for each patient. RESULTS: In the whole population, 49.6% of patients were homozygous for the Glu298 genotype (Glu/Glu), 40.9% heterozygotes (Glu/Asp) and 9.5% homozygous for the 298Asp genotype (Asp/Asp). Obstructive CAD was documented in 178/436 (40.8%) patients undergoing coronary angiography while myocardial ischemia in 160/325 (49.2%) patients undergoing stress testing. Patients with eNOS Asp genotype (Glu/Asp + Asp/Asp) had no significant differences in clinical risk factors and in circulating markers. Independent predictors of obstructive CAD were age, gender, obesity, and low HDL-C. Independent predictors of myocardial ischemia were gender, obesity, low HDL-C and Asp genotype. In the subpopulation in which both stress tests and coronary angiography were performed, the Asp genotype remained associated with increased myocardial ischemia risk after adjustment for obstructive CAD. CONCLUSION: In this population, low-HDL cholesterol was the only cardiometabolic risk determinant of obstructive CAD. The eNOS Glu298Asp gene polymorphism was significantly associated with inducible myocardial ischemia independently of other risk factors and presence of obstructive CAD.

Molecular Autopsy of Sudden Cardiac Death in the Genomics Era.

Molecular autopsy is the process of investigating sudden death through genetic analysis. It is particularly useful in cases where traditional autopsy is negative or only shows non-diagnostic features, i.e., in sudden unexplained deaths (SUDs), which are often due to an underlying inherited arrhythmogenic cardiac disease. The final goal of molecular autopsy in SUD cases is to aid medico-legal inquiries and to guide cascade genetic screening of the victim's relatives. Early attempts of molecular autopsy relied on Sanger sequencing, which, despite being accurate and easy to use, has a low throughput and can only be employed to analyse a small panel of genes. Conversely, the recent adoption of next-generation sequencing (NGS) technologies has allowed exome/genome wide examination, providing an increase in detection of pathogenic variants and the discovery of newer genotype-phenotype associations. NGS has nonetheless brought new challenges to molecular autopsy, especially regarding the clinical interpretation of the large number of variants of unknown significance detected in each individual.

The potential and limitations of plasma BNP measurement in the diagnosis, prognosis, and management of children with heart failure due to congenital cardiac disease: an update.

The aim of this article is to review the diagnostic and prognostic relevance of measurement of brain natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in pediatric patients with heart failure caused by various acquired and congenital heart diseases (CHD). In January 2013, we performed a computerized literature search in the National Library of Medicine (PubMed access to MEDLINE citations; http://www.ncbi.nlm.nih.gov/PubMed/ ). The search strategy included a mix of Medical Subject Headings and free-text terms for the key concepts, starting from BNP assay and 'NT-proBNP assay', children, CHD. The search was further refined by adding the keywords neonate/s, newborn/s, heart failure, cardiomyopathy, screening, prognosis, follow-up, and management. BNP values are age and method dependent, even in pediatric populations. Regardless of age, there is great variability in BNP/NT-proBNP values within CHD characterized by different hemodynamic and clinical conditions. There is enough evidence to support the use of BNP/NT-proBNP as an adjunctive marker in the integrated evaluation of patients with congenital and acquired heart disease to help define severity and progression of heart failure as well in the monitoring of response to treatment. BNP/NT-proBNP can also be used for the screening of heart failure and as a prognostic marker in children undergoing cardiac surgery; however, to date, there are studies with heterogeneous patient groups, and diverse outcome measures selected are still few. BNP/NT-proBNP can be used as adjunctive markers in the integrated screening, diagnosis, management, and follow-up of children with heart failure caused by various acquired and congenital heart disease.

Circulating forms of the b-type natriuretic peptide prohormone: pathophysiologic and clinical considerations.

Recent studies reported that many different biochemical forms of B-type-related peptides circulate in human blood. In particular, a significant amount of the prohormone peptide (i.e., proBNP108) can be detected in plasma of patients with heart failure. These data indicate that the posttranslational maturation processing of the B-type natriuretic peptide (BNP) precursor may not be efficient in heart failure. The aim of this chapter is to describe the biochemical pathways of proBNP108 maturation and to discuss the pathophysiological relevance of alteration of the posttranslational maturation mechanisms in heart failure. An impaired cardiac endocrine function was proposed to explain the altered electrolyte and fluid homeostasis occurring in chronic heart failure. Recent studies demonstrated that a great part of BNPs assayed by immunoassay methods in healthy subjects and in patients with cardiovascular disease is devoid of biological activity. These findings suggest that an alteration in posttranslational maturation of BNP precursor may promote the resistance to biological action of BNP in patients with heart failure at a prereceptor level. These studies also open a new and more complex scenario regarding the circulating BNPs. The active hormone (i.e., BNP1-32) may be produced even in vivo from the circulating precursor proBNP108 by plasma enzyme degradation, such as the soluble form of corin, possibly able to process the circulating intact precursor of natriuretic hormones. As a future perspective, the simultaneous measurement of the proBNP1-108 and the active peptide BNP1-32 with more specific methods could allow a more accurate estimation of both production/secretion of B-type-related peptides from cardiomyocytes and the true activity of the cardiac endocrine function.

The paradox of low BNP levels in obesity.

The aim of this review is to analyze in detail some possible pathophysiological mechanisms linking obesity and cardiac endocrine function, in order to try to explain the negative association previously observed between BMI and BNP values in both healthy subjects and patients with cardiovascular diseases. In particular, we discuss the hypothesis that the response of the cardiac endocrine system is the integrated resultant of several and contrasting physiological and pathological interactions, including the effects of peptide and steroid hormones, cytokines, cardiovascular hemodynamics, clinical conditions, and pharmacological treatment. Several studies suggested that gonadal function regulates both body fat distribution and cardiac endocrine function. Visceral fat expansion can increase the clearance of active natriuretic peptides by means of an increased expression of clearance receptors on adipocytes, and in this way, it may contribute to decrease the activity of the cardiac endocrine system. Moreover, obesity is associated with ectopic lipid deposition even in the heart, which may directly exert a lipotoxic effect on the myocardium by secreting in loco several cytokines and adipokines. Obese subjects are frequently treated for hypertension and coronary artery disease. Pharmacological treatment reduces plasma level of cardiac natriuretic peptides, and this effect may explain almost in part the lower BNP levels of some asymptomatic subjects with increased BMI values. At present time, it is not possible to give a unique and definitive answer to the crucial question concerning the inverse relationship between the amount of visceral fat distribution and BNP levels. Our explanation for these unsatisfactory results is that the cardiac endocrine response is always the integrated resultant of several pathophysiological interactions. However, only few variables can be studied together; as a result, it is not possible to perform a complete evaluation of pathophysiological mechanisms under study. We are still not able to well integrate these multiple information together; therefore, we should learn to do it.

Measurement of the pro-hormone of brain type natriuretic peptide (proBNP): methodological considerations and pathophysiological relevance.

Recent studies demonstrated that large amounts of the pro-hormone peptide of brain natriuretic peptide (proBNP) can be detected in plasma of healthy subjects and in particular of patients with heart failure. As a result, a great part of B-type natriuretic peptides measured in patients with cardio-vascular disease may be devoid of biological activity. These findings stimulated the set up of specific immunoassay methods for the measurement of the intact proBNP peptide. The aim of this review article is to discuss the methodological characteristics and the possible clinical relevance of specific immunoassay methods for the measurement of the proBNP peptide. From an analytical point of view, a fully automated immunoassay of proBNP has some theoretical advantages (e.g., a more stable molecule with higher molecular weight than the derived peptides) compared to the active hormone BNP. Recent studies supported the concept that the precursor proBNP might be actually considered a circulating prohormone, which can be cleaved by specific plasma proteases in BNP, the active hormone, and NT-proBNP, an inactive peptide. The peripheral processing of circulating proBNP could likely be submitted to regulatory rules, which might be impaired in patients with heart failure, opening new perspectives in the treatment of heart failure (e.g., by studying drugs inducing the cleavage of the prohormone into active BNP). Furthermore, as a future perspective, the specific assay in the same plasma sample of the intact precursor proBNP and of the biologically active peptide BNP, could allow a more accurate estimation of the production/secretion of B-type related peptides from cardiomyocytes and of the global cardiac endocrine function.

[Genetic screening of Gata4 and Nkx2.5 mutations in hereditary congenital heart defects: 5 familial cases]. / Screening genetico di Gata4 e Nkx2,5 nelle cardiopatie congenite: cinque casi familiari.

Single gene mutations in Gata4 and Nkx2.5 genes have been identified as a causative factor for various clinical forms of hereditary congenital heart diseases (CHDs), especially for cardiac septal defects. However, the role of Gata4 and Nkx2.5 mutations in familial CHD is not clear yet. We report 5 cases of familial CHD with a positive history of cardiac septal defects. Our data suggest that mutations of either the Gata4 or Nkx2.5 genes are very uncommonly found in familial cases of CHD, supporting the genetic heterogeneity of cardiac congenital defects and the limitation of genetic testing in clinical setting.

Thirty years of the heart as an endocrine organ: physiological role and clinical utility of cardiac natriuretic hormones.

Thirty years ago, De Bold et al. (20) reported that atrial extracts contain some biologically active peptides, which promote a rapid and massive diuresis and natriuresis when injected in rats. It is now clear that the heart also exerts an endocrine function and in this way plays a key role in the regulation of cardiovascular and renal systems. The aim of this review is to discuss some recent insights and still-debated findings regarding the cardiac natriuretic hormones (CNHs) produced and secreted by cardiomyocytes (i.e., atrial natriuretic peptide and B-type natriuretic peptide). The functional status of the CNH system depends not only on the production/secretion of CNHs by cardiomyocytes but also on both the peripheral activation of circulating inactive precursor of natriuretic hormones and the transduction of the hormone signal by specific receptors. In this review, we will discuss the data supporting the hypothesis that the production and secretion of CNHs is the result of a complex integration among mechanical, chemical, hemodynamic, humoral, ischemic, and inflammatory inputs. The cross talk among endocrine function, adipose tissue, and sex steroid hormones will be discussed more in detail, considering the clinically relevant relationships linking together cardiovascular risk, sex, and body fat development and distribution. Finally, we will review the pathophysiological role and the clinical relevance of both peripheral maturation of the precursor of B-type natriuretic peptides and hormone signal transduction.

[A novel lamin A/C mutation in a family with dilated cardiomyopathy and a strong history of sudden cardiac death]. / Identificazione di una nuova mutazione nel gene della lamina A/C in una famiglia con cardiomiopatia dilatativa e storia di morte improvvisa.

Diseases related to lamin A/C mutations (laminopathies) are extremely heterogeneous. The common cardiac phenotype is idiopathic dilated cardiomyopathy with atrioventricular block and/or arrhythmias. Moreover, patients with lamin A/C gene mutations are at increased risk for sudden cardiac death. Here we present a family with a strong positive history of sudden cardiac death in presence of idiopathic dilated cardiomyopathy and cardiac conduction abnormalities, related to a novel lamin A/C mutation in exon 3.

A novel LMNA mutation (R189W) in familial dilated cardiomyopathy: evidence for a 'hot spot' region at exon 3: a case report.

We describe a case of a patient with idiopathic dilated cardiomyopathy and cardiac conduction abnormalities who presented a strong family history of sudden cardiac death. Genetic screening of lamin A/C gene revealed in proband the presence of a novel missense mutation (R189W), near the most prevalent lamin A/C mutation (R190W), suggesting a "hot spot" region at exon 3.

New and emerging biomarkers of heart failure.

Heart failure (HF) may be considered as the fatal finishing line of all cardiovascular disorders. There is not a single diagnostic test for HF, and its diagnosis relies on clinical judgment based on a combination of history, physical examination, and appropriate investigations. For these reasons, the accuracy of diagnosis by clinical means alone is often inadequate. Despite the enormous advances in understanding and treatment that have taken place during the last 50 years, HF continues to have a poor prognosis. Diagnosis and risk stratification of patients with HF depend on the availability of specific, accurate, and effective disease or risk markers. Thus, there is an increasing interest in the development of new cardiovascular biomarkers, and, consequently, a great number of laboratory tests have recently been proposed for their assay. In this review, we briefly discuss the characteristics of an ideal HF biomarker and describe the analytical performance and clinical relevance of available biomarker assay methods, comparing their performances with that of an ideal biomarker for HF. Finally, we present a scheme to search for more efficient diagnostic and prognostic biomarkers for HF.

Diagnostic accuracy and clinical relevance of brain natriuretic peptide assay in pediatric patients with congenital heart diseases.

BACKGROUND: The clinical usefulness of B-type natriuretic peptide (BNP) assay in congenital heart diseases is still controversial. We evaluated the diagnostic accuracy and clinical relevance of this assay in different cardiovascular hemodynamic conditions in pediatric patients with congenital heart diseases. MATERIALS AND METHODS: BNP was measured in 173 healthy individuals and in 382 pediatric patients with congenital heart diseases. We identified five subgroups of hemodynamic conditions: left ventricular volume overload, right ventricular volume overload, left ventricular pressure overload, right ventricular pressure overload, and biventricular volume overload. RESULTS: BNP was higher (P<0.0001) in patients (median 49.0 ng/l, range 0.45-14363 ng/l) than in the reference population (median 6.1 ng/l, range 1.0-29.7 ng/l). BNP assay showed a good diagnostic accuracy in discriminating between healthy individuals and patients (area under the receiver operating characteristic curve 0.95, SE 0.009). Lower BNP values were found in right ventricular pressure overload than in left ventricular pressure overload, left ventricular volume overload, or biventricular volume overload. In the left ventricular volume overload subgroup, BNP significantly correlated with peak systolic gradients (rho=0.622, P<0.001) and left ventricular dilatation (rho=0.35, P=0.03). In the right ventricular volume overload subgroup, BNP correlated with right ventricular pressure (rho=0.622, P<0.001) and right ventricular dilatation (rho=0.377, P=0.0077). Moreover, in cyanotic defects, BNP showed a significant correlation with O2 saturation (rho=0.204 P=0.0128). CONCLUSION: BNP assay showed a good diagnostic accuracy in discriminating between healthy individuals and patients. Congenital heart disease with left ventricular pressure overload and biventricular volume overload has higher BNP values than that with right ventricular pressure overload.

The search for a pathophysiological link between gender, cardiac endocrine function, body mass regulation and cardiac mortality: proposal for a working hypothesis.

BACKGROUND: The discovery of cardiac natriuretic hormones determined a radical revision of the concept of heart function. It is now clear that the heart is not merely a pump but, through its endocrine function, exerts a nodal role in a complex information network. As a matter of fact the heart plays a key role in the regulation of circulation, salt-water homeostasis, and can exchange physiologically relevant information with other organs and systems. CONTENT: Highlighting the most important recent literature observations, this review discusses the inter-relationship between endocrine function of the heart and gonadal function. We have first considered the cross-talk between cardiac endocrine system and sex steroid hormones, examining the different actions of female sex steroid hormones and androgens on cardiac endocrine function, and then the action of cardiac natriuretic hormones on female and male gonadal function. Then, we have highlighted the clinical relevance of the relationships between cardiac endocrine function and sex steroid hormones in several clinical conditions associated with cardiovascular risk, focusing on mechanisms linking adipose tissue to natriuretic peptide and sex steroid hormone actions. CONCLUSION: The knowledge of the relation between cardiac endocrine function and other neurohormonal systems, including gonadal function, is crucial to explain the increased cardiovascular risk in some clinical conditions, such as obesity, arterial hypertension and metabolic syndrome.

Nitric oxide treatment reduces neo-intimal formation and modulates osteopontin expression in an ex-vivo human model of intimal hyperplasia.

In this study the effects of nitric oxide (NO) on intimal hyperplasia (IH) were evaluated in an ex-vivo model of human saphenous vein (SV). SV segments were cultured in conditions able to reproduce IH (FCS), or in medium alone (RPMI), or in presence of a NO donor (NO). Osteopontin (OPN) and Interleukin (IL)-6 were determined in the medium at different culture times and in the tissue, at the end of experiment. OPN and IL-6 release in medium was increased in FCS with respect to RPMI (OPN: 13.9+/-2.9 vs. 2.3+/-0.8 microg/ml, p=0.0011; IL-6: 304.2+/-64.7 vs. 42.0+/-10.1 ng/ml, p<0.0006) as well as intima thickness, that positively correlated with OPN production (r=0.81). In tissue OPN was higher in FCS (82.0+/-30.3 ng/mg protein) than in RPMI (13.8+/-4.2, p=0.0051) and at baseline (3.7+/-0.7, p=0.018). NO reduces IH progression (25%) and both OPN and IL-6 expression (OPN/GAPDH: undetectable baseline; 0.27+/-0.06 RPMI; 0.89+/-0.28 FCS; 0.09+/-0.05 NO; p=0.026 FCS vs. baseline, p=0.018 vs. RPMI, p=0.005 vs. NO). The beneficial NO effect on IH reduction appears to be mediated by the indirect inhibition of OPN production. NO could modulates the initial inflammatory signals that induces the OPN over-production with the related cascade of events leading to IH.

Old and new biomarkers of heart failure.

Heart failure (HF) may be considered as the fatal finishing line of all cardiovascular disorders. Despite advances in the understanding and treatment, it still has a poor prognosis. Heart failure is a syndrome, rather than a primary diagnosis, which results from any structural or functional cardiac disorder that impairs the ability of the heart to support the physiological circulation. This is sustained by a chronic imbalance in the neurohormonal control of circulation. Unfortunately, there is no single diagnostic test for HF, and the accuracy of diagnosis by clinical means only (i.e. a combination of history, physical examination and appropriate investigations) is often inadequate. Diagnosis and risk stratification depend on the availability of accurate, and effective markers of either risk or disease. There is an increasing interest in the development of new biomarkers, and a great number of laboratory tests have recently been proposed. The goals of this 'commentary' are to (i) briefly discuss the characteristics of an ideal HF biomarker; (ii) describe the analytical performance and clinical relevance of currently available biomarker assay methods, (iii) evaluate newer biomarkers and finally, (iv) design a scheme to optimize the search for efficient diagnostic and prognostic biomarkers for HF.

Comparison between analytical performances of polyclonal and monoclonal electrochemiluminescence immunoassays for NT-proBNP.

BACKGROUND: We evaluated the analytical characteristics of electrochemiluminescence (ECLIA) immunoassay for NT-proBNP (proBNP II, Roche Diagnostics, Germany) and compared its analytical performance to that of the previous polyclonal method. METHODS: We measured NT-proBNP in EDTA plasma samples of 177 consecutive cardiac patients (69 females and 108 males; mean age 62.2+/-16.4 years, range 13 to 96 years) with monoclonal and polyclonal ECLIA methods following manufacturer's instructions using an Elecsys 2010 analyzer. RESULTS: Monoclonal ECLIA method for NT-proBNP assay showed an imprecision (CV%) lower than 3% at the cut-off value (i.e., 150 ng/L). No significant interference was found in plasma samples containing high levels of hemoglobin, triglycerides or bilirubin. EDTA plasma showed slightly, but significantly lower NT-proBNP values than serum (on average -6.3%) and lithium-heparinized plasma (on average -3.9%) samples. Finally, a very close linear regression was found between the NT-proBNP values found by either monoclonal or polyclonal ECLIA method (monoclonal ECLIA=-72.17+1.04 polyclonal ECLIA, n=177, R=0.993). CONCLUSIONS: The monoclonal ECLIA method showed very similar analytical characteristics with slightly lower NT-proBNP results (on average -2.5%) than the polyclonal ECLIA method. The difference between monoclonal and polyclonal methods seems to be too slight to change the reference range and decisional values for NT-proBNP assay, as previously assessed by the polyclonal ECLIA method.

Clinical utility of genetic tests for inherited hypertrophic and dilated cardiomyopathies.

Genetic testing has become an increasingly important part of medical practice for heritable form of cardiomyopathies. Hypertrophic cardiomyopathy and about 50% of idiopathic dilatative cardiomyopathy are familial diseases, with an autosomal dominant pattern of inheritance.Some genotype-phenotype correlations can provide important information to target DNA analyses in specific genes. Genetic testing may clarify diagnosis and help the optimal treatment strategies for more malignant phenotypes. In addition, genetic screening of first-degree relatives can help early identification and diagnosis of individuals at greatest risk for developing cardiomyopathy, allowing to focus clinical resources on high-risk family members.This paper provides a concise overview of the genetic etiology as well as the clinical utilities and limitations of genetic testing for the heritable cardiomyopathies.