RESUMEN

AIM: Numerous genes are known to cause dilated cardiomyopathy (DCM). However, until now technological limitations have hindered elucidation of the contribution of all clinically relevant disease genes to DCM phenotypes in larger cohorts. We now utilized next-generation sequencing to overcome these limitations and screened all DCM disease genes in a large cohort. METHODS AND RESULTS: In this multi-centre, multi-national study, we have enrolled 639 patients with sporadic or familial DCM. To all samples, we applied a standardized protocol for ultra-high coverage next-generation sequencing of 84 genes, leading to 99.1% coverage of the target region with at least 50-fold and a mean read depth of 2415. In this well characterized cohort, we find the highest number of known cardiomyopathy mutations in plakophilin-2, myosin-binding protein C-3, and desmoplakin. When we include yet unknown but predicted disease variants, we find titin, plakophilin-2, myosin-binding protein-C 3, desmoplakin, ryanodine receptor 2, desmocollin-2, desmoglein-2, and SCN5A variants among the most commonly mutated genes. The overlap between DCM, hypertrophic cardiomyopathy (HCM), and channelopathy causing mutations is considerably high. Of note, we find that >38% of patients have compound or combined mutations and 12.8% have three or even more mutations. When comparing patients recruited in the eight participating European countries we find remarkably little differences in mutation frequencies and affected genes. CONCLUSION: This is to our knowledge, the first study that comprehensively investigated the genetics of DCM in a large-scale cohort and across a broad gene panel of the known DCM genes. Our results underline the high analytical quality and feasibility of Next-Generation Sequencing in clinical genetic diagnostics and provide a sound database of the genetic causes of DCM.

Asunto(s)

Cardiomiopatía Dilatada/genética , Análisis de Secuencia de ADN/métodos , Cardiomiopatía Dilatada/diagnóstico , Europa (Continente) , Estudios de Factibilidad , Femenino , Marcadores Genéticos/genética , Genotipo , Heterocigoto , Humanos , Masculino , Mutación/genética , Fenotipo , Características de la Residencia

RESUMEN

BACKGROUND: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists. METHODS: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR. RESULTS: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 (95% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait. CONCLUSIONS: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , MicroARNs/genética , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/genética , Neoplasias/patología , Fenotipo , Pronóstico

RESUMEN

Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome-wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A). Mass-spectrometric analysis and bisulphite-sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13. In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure.

Asunto(s)

Cardiomiopatía Dilatada/genética , Metilación de ADN , Epigénesis Genética , Miocardio/metabolismo , Adulto , Anciano , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Biopsia , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/fisiopatología , Estudios de Casos y Controles , Análisis por Conglomerados , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/metabolismo , Ratas , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de Proteína , Transfección , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

BACKGROUND: Alterations in microRNA (miRNA) expression patterns in whole blood may be useful biomarkers of diverse cardiovascular disorders. We previously reported that miRNAs are significantly dysregulated in acute myocardial infarction (AMI) and applied machine-learning techniques to define miRNA subsets with high diagnostic power for AMI diagnosis. However, the kinetics of the time-dependent sensitivity of these novel miRNA biomarkers remained unknown. METHODS: To characterize temporal changes in the expressed human miRNAs (miRNome), we performed here the first whole-genome miRNA kinetic study in AMI patients. We measured miRNA expression levels at multiple time points (0, 2, 4, 12, 24 h after initial presentation) in patients with acute ST-elevation myocardial infarction by using microfluidic primer extension arrays and quantitative real-time PCR. As a prerequisite, all patients enrolled had to have cardiac troponin T concentrations <50 ng/L on admission as measured with a high-sensitivity assay. RESULTS: We found a subset of miRNAs to be significantly dysregulated both at initial presentation and during the course of AMI. Additionally, we identified novel miRNAs that are dysregulated early during myocardial infarction, such as miR-1915 and miR-181c*. CONCLUSIONS: The present proof-of-concept study provides novel insights into the dynamic changes of the human miRNome during AMI.

Asunto(s)

MicroARNs/sangre , Infarto del Miocardio/sangre , Infarto del Miocardio/diagnóstico , Biomarcadores/sangre , Femenino , Genómica , Humanos , Cinética , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Troponina I/sangre

RESUMEN

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

Asunto(s)

N-Metiltransferasa de Histona-Lisina/metabolismo , Músculo Esquelético/enzimología , Miocardio/enzimología , Miosinas/metabolismo , Sarcómeros/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Clonación Molecular , Citoesqueleto/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , Análisis por Micromatrices , Contracción Muscular/fisiología , Músculo Esquelético/ultraestructura , Mutación/genética , Miocardio/ultraestructura , Unión Proteica , Sarcómeros/genética , Transgenes/genética , Pez Cebra , Proteínas de Pez Cebra/genética

RESUMEN

BACKGROUND: The molecular mechanisms that guide heart valve formation are not well understood. However, elucidation of the genetic basis of congenital heart disease is one of the prerequisites for the development of tissue-engineered heart valves. METHODS AND RESULTS: We isolated here a mutation in zebrafish, bungee (bng(jh177)), which selectively perturbs valve formation in the embryonic heart by abrogating endocardial Notch signaling in cardiac cushions. We found by positional cloning that the bng phenotype is caused by a missense mutation (Y849N) in zebrafish protein kinase D2 (pkd2). The bng mutation selectively impairs PKD2 kinase activity and hence Histone deacetylase 5 phosphorylation, nuclear export, and inactivation. As a result, the expression of Histone deacetylase 5 target genes Krüppel-like factor 2a and 4a, transcription factors known to be pivotal for heart valve formation and to act upstream of Notch signaling, is severely downregulated in bungee (bng) mutant embryos. Accordingly, the expression of Notch target genes, such as Hey1, Hey2, and HeyL, is severely decreased in bng mutant embryos. Remarkably, downregulation of Histone deacetylase 5 activity in homozygous bng mutant embryos can rescue the mutant phenotype and reconstitutes notch1b expression in atrioventricular endocardial cells. CONCLUSIONS: We demonstrate for the first time that proper heart valve formation critically depends on Protein kinase D2-Histone deacetylase 5-Krüppel-like factor signaling.

Asunto(s)

Desarrollo Embrionario/fisiología , Válvulas Cardíacas/embriología , Histona Desacetilasas/fisiología , Proteínas Quinasas/fisiología , Pez Cebra/embriología , Animales , Embrión no Mamífero/fisiología , Desarrollo Embrionario/genética , Histona Desacetilasas/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Modelos Animales , Mutación Missense/genética , Proteína Quinasa D2 , Proteínas Quinasas/genética , Receptor Notch1/fisiología , Transducción de Señal/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología

RESUMEN

Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or ß-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

Asunto(s)

Proteínas Musculares/fisiología , Contracción Miocárdica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Insuficiencia Cardíaca/etiología , Músculo Esquelético , Miocardio/química , Miocardio/enzimología , Fosforilación , Sarcómeros/química , Pez Cebra

RESUMEN

Although it is well known that mutations in the cardiac essential myosin light chain-1 (cmlc-1) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan (laz), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195-deficient cMLC-1 (cMLC-1(S195A)) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1(S195D)) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility.

Asunto(s)

Corazón/embriología , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Codón sin Sentido , Etilnitrosourea/toxicidad , Regulación del Desarrollo de la Expresión Génica , Genotipo , Corazón/efectos de los fármacos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Fuerza Muscular , Mutágenos/toxicidad , Contracción Miocárdica/genética , Miocitos Cardíacos/efectos de los fármacos , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Fenotipo , Fosforilación , Estabilidad Proteica , Estructura Terciaria de Proteína , Sarcómeros/metabolismo , Homología de Secuencia de Aminoácido , Serina , Factores de Tiempo , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética

RESUMEN

BACKGROUND: Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking. METHODS AND RESULTS: In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go-related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish. CONCLUSIONS: With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.

Asunto(s)

Arritmias Cardíacas/genética , Modelos Animales de Enfermedad , Canales de Potasio Éter-A-Go-Go/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/genética , Potenciales de Acción/genética , Sustitución de Aminoácidos , Animales , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/embriología , Arritmias Cardíacas/fisiopatología , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Canales de Potasio Éter-A-Go-Go/deficiencia , Canales de Potasio Éter-A-Go-Go/genética , Genotipo , Corazón/embriología , Activación del Canal Iónico/genética , Mutación Missense , Contracción Miocárdica , Oocitos , Técnicas de Placa-Clamp , Potasio/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Bloqueo Sinoatrial/tratamiento farmacológico , Bloqueo Sinoatrial/genética , Bloqueo Sinoatrial/fisiopatología , Síndrome , Terfenadina/uso terapéutico , Xenopus laevis , Pez Cebra/embriología , Pez Cebra/fisiología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética