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1.
Nature ; 608(7922): 353-359, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35922509

RESUMO

Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.


Assuntos
Alelos , Perfilação da Expressão Gênica , Especificidade de Órgãos , RNA-Seq , Transcriptoma , Processamento Alternativo/genética , Linhagem Celular , Conjuntos de Dados como Assunto , Genótipo , Ribonucleoproteínas Nucleares Heterogêneas/deficiência , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Especificidade de Órgãos/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/deficiência , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Reprodutibilidade dos Testes , Transcriptoma/genética
2.
Nature ; 608(7921): 174-180, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35732739

RESUMO

Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function1,2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts3,4, but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFß1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.


Assuntos
Cardiomiopatia Dilatada , Cardiomiopatia Hipertrófica , Núcleo Celular , Perfilação da Expressão Gênica , Insuficiência Cardíaca , Análise de Célula Única , Sistemas CRISPR-Cas , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Estudos de Casos e Controles , Núcleo Celular/genética , Células Cultivadas , Técnicas de Inativação de Genes , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , RNA-Seq , Transcrição Gênica , Fator de Crescimento Transformador beta1
3.
Circ Res ; 132(10): 1290-1301, 2023 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-37167361

RESUMO

From the onset of the pandemic, evidence of cardiac involvement in acute COVID-19 abounded. Cardiac presentations ranged from arrhythmias to ischemia, myopericarditis/myocarditis, ventricular dysfunction to acute heart failure, and even cardiogenic shock. Elevated serum cardiac troponin levels were prevalent among hospitalized patients with COVID-19; the higher the magnitude of troponin elevation, the greater the COVID-19 illness severity and in-hospital death risk. Whether these consequences were due to direct SARS-CoV-2 infection of cardiac cells or secondary to inflammatory responses steered early cardiac autopsy studies. SARS-CoV-2 was reportedly detected in endothelial cells, cardiac myocytes, and within the extracellular space. However, findings were inconsistent and different methodologies had their limitations. Initial autopsy reports suggested that SARS-CoV-2 myocarditis was common, setting off studies to find and phenotype inflammatory infiltrates in the heart. Nonetheless, subsequent studies rarely detected myocarditis. Microthrombi, cardiomyocyte necrosis, and inflammatory infiltrates without cardiomyocyte damage were much more common. In vitro and ex vivo experimental platforms have assessed the cellular tropism of SARS-CoV-2 and elucidated mechanisms of viral entry into and replication within cardiac cells. Data point to pericytes as the primary target of SARS-CoV-2 in the heart. Infection of pericytes can account for the observed pericyte and endothelial cell death, innate immune response, and immunothrombosis commonly observed in COVID-19 hearts. These processes are bidirectional and synergistic, rendering a definitive order of events elusive. Single-cell/nucleus analyses of COVID-19 myocardial tissue and isolated cardiac cells have provided granular data about the cellular composition and cell type-specific transcriptomic signatures of COVID-19 and microthrombi-positive COVID-19 hearts. Still, much remains unknown and more in vivo studies are needed. This review seeks to provide an overview of the current understanding of COVID-19 cardiac pathophysiology. Cell type-specific mechanisms and the studies that provided such insights will be highlighted. Given the unprecedented pace of COVID-19 research, more mechanistic details are sure to emerge since the writing of this review. Importantly, our current knowledge offers significant clues about the cardiac pathophysiology of long COVID-19, the increased postrecovery risk of cardiac events, and thus, the future landscape of cardiovascular disease.


Assuntos
COVID-19 , Cardiopatias , Miocardite , Humanos , COVID-19/complicações , SARS-CoV-2 , Células Endoteliais , Mortalidade Hospitalar , Síndrome de COVID-19 Pós-Aguda , Coração , Troponina , Miócitos Cardíacos
4.
Circ Res ; 133(4): 313-329, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-37449401

RESUMO

BACKGROUND: ZFHX3 (zinc finger homeobox 3), a gene that encodes a large transcription factor, is at the second-most significantly associated locus with atrial fibrillation (AF), but its function in the heart is unknown. This study aims to identify causative genetic variation related to AF at the ZFHX3 locus and examine the impact of Zfhx3 loss on cardiac function in mice. METHODS: CRISPR-Cas9 genome editing, chromatin immunoprecipitation, and luciferase assays in pluripotent stem cell-derived cardiomyocytes were used to identify causative genetic variation related to AF at the ZFHX3 locus. Cardiac function was assessed by echocardiography, magnetic resonance imaging, electrophysiology studies, calcium imaging, and RNA sequencing in mice with heterozygous and homozygous cardiomyocyte-restricted Zfhx3 loss (Zfhx3 Het and knockout, respectively). Human cardiac single-nucleus ATAC (assay for transposase-accessible chromatin)-sequencing data was analyzed to determine which genes in atrial cardiomyocytes are directly regulated by ZFHX3. RESULTS: We found single-nucleotide polymorphism (SNP) rs12931021 modulates an enhancer regulating ZFHX3 expression, and the AF risk allele is associated with decreased ZFHX3 transcription. We observed a gene-dose response in AF susceptibility with Zfhx3 knockout mice having higher incidence, frequency, and burden of AF than Zfhx3 Het and wild-type mice, with alterations in conduction velocity, atrial action potential duration, calcium handling and the development of atrial enlargement and thrombus, and dilated cardiomyopathy. Zfhx3 loss results in atrial-specific differential effects on genes and signaling pathways involved in cardiac pathophysiology and AF. CONCLUSIONS: Our findings implicate ZFHX3 as the causative gene at the 16q22 locus for AF, and cardiac abnormalities caused by loss of cardiac Zfhx3 are due to atrial-specific dysregulation of pathways involved in AF susceptibility. Together, these data reveal a novel and important role for Zfhx3 in the control of cardiac genes and signaling pathways essential for normal atrial function.


Assuntos
Fibrilação Atrial , Proteínas de Homeodomínio , Animais , Humanos , Camundongos , Fibrilação Atrial/genética , Cálcio/metabolismo , Dilatação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética
5.
Circ Res ; 128(8): 1214-1236, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33856918

RESUMO

A pandemic of historic impact, coronavirus disease 2019 (COVID-19) has potential consequences on the cardiovascular health of millions of people who survive infection worldwide. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, can infect the heart, vascular tissues, and circulating cells through ACE2 (angiotensin-converting enzyme 2), the host cell receptor for the viral spike protein. Acute cardiac injury is a common extrapulmonary manifestation of COVID-19 with potential chronic consequences. This update provides a review of the clinical manifestations of cardiovascular involvement, potential direct SARS-CoV-2 and indirect immune response mechanisms impacting the cardiovascular system, and implications for the management of patients after recovery from acute COVID-19 infection.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , Doenças Cardiovasculares/virologia , Miócitos Cardíacos/virologia , SARS-CoV-2/fisiologia , Internalização do Vírus , Biomarcadores/metabolismo , COVID-19/complicações , COVID-19/epidemiologia , COVID-19/terapia , Cardiomiopatias/virologia , Expressão Gênica , Humanos , Sistema Imunitário/fisiologia , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Neuropilina-1/metabolismo , Ativação Plaquetária , RNA Mensageiro/metabolismo , Sistema Renina-Angiotensina/fisiologia , Volta ao Esporte , Fatores de Risco , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Troponina/metabolismo , Remodelação Ventricular , Ligação Viral , Internalização do Vírus/efeitos dos fármacos
6.
Curr Cardiol Rep ; 25(3): 171-184, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36897483

RESUMO

PURPOSE OF REVIEW: Cardiac consequences occur in both acute COVID-19 and post-acute sequelae of COVID-19 (PASC). Here, we highlight the current understanding about COVID-19 cardiac effects, based upon clinical, imaging, autopsy, and molecular studies. RECENT FINDINGS: COVID-19 cardiac effects are heterogeneous. Multiple, concurrent cardiac histopathologic findings have been detected on autopsies of COVID-19 non-survivors. Microthrombi and cardiomyocyte necrosis are commonly detected. Macrophages often infiltrate the heart at high density but without fulfilling histologic criteria for myocarditis. The high prevalences of microthrombi and inflammatory infiltrates in fatal COVID-19 raise the concern that recovered COVID-19 patients may have similar but subclinical cardiac pathology. Molecular studies suggest that SARS-CoV-2 infection of cardiac pericytes, dysregulated immunothrombosis, and pro-inflammatory and anti-fibrinolytic responses underlie COVID-19 cardiac pathology. The extent and nature by which mild COVID-19 affects the heart is unknown. Imaging and epidemiologic studies of recovered COVID-19 patients suggest that even mild illness confers increased risks of cardiac inflammation, cardiovascular disorders, and cardiovascular death. The mechanistic details of COVID-19 cardiac pathophysiology remain under active investigation. The ongoing evolution of SARS-CoV-2 variants and vast numbers of recovered COVID-19 patients portend a burgeoning global cardiovascular disease burden. Our ability to prevent and treat cardiovascular disease in the future will likely depend on comprehensive understanding of COVID-19 cardiac pathophysiologic phenotypes.


Assuntos
COVID-19 , Cardiopatias , Miocardite , Trombose , Humanos , COVID-19/complicações , COVID-19/epidemiologia , SARS-CoV-2/genética , Coração/diagnóstico por imagem , Miocardite/etiologia , Cardiopatias/complicações , Trombose/complicações
7.
J Mol Cell Cardiol ; 166: 23-35, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35114252

RESUMO

Atrial fibrillation (AF) affects over 1% of the population and is a leading cause of stroke and heart failure in the elderly. A feared side effect of sodium channel blocker therapy, ventricular pro-arrhythmia, appears to be relatively rare in patients with AF. The biophysical reasons for this relative safety of sodium blockers are not known. Our data demonstrates intrinsic differences between atrial and ventricular cardiac voltage-gated sodium currents (INa), leading to reduced maximum upstroke velocity of action potential and slower conduction, in left atria compared to ventricle. Reduced atrial INa is only detected at physiological membrane potentials and is driven by alterations in sodium channel biophysical properties and not by NaV1.5 protein expression. Flecainide displayed greater inhibition of atrial INa, greater reduction of maximum upstroke velocity of action potential, and slowed conduction in atrial cells and tissue. Our work highlights differences in biophysical properties of sodium channels in left atria and ventricles and their response to flecainide. These differences can explain the relative safety of sodium channel blocker therapy in patients with atrial fibrillation.


Assuntos
Fibrilação Atrial , Flecainida , Potenciais de Ação , Idoso , Antiarrítmicos/farmacologia , Antiarrítmicos/uso terapêutico , Fibrilação Atrial/metabolismo , Flecainida/metabolismo , Flecainida/farmacologia , Flecainida/uso terapêutico , Átrios do Coração/metabolismo , Humanos , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo
8.
Am J Physiol Heart Circ Physiol ; 323(4): H797-H817, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36053749

RESUMO

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.


Assuntos
Insuficiência Cardíaca , Animais , Gatos , Modelos Animais de Doenças , Feminino , Ventrículos do Coração , Humanos , Masculino , Volume Sistólico/fisiologia , Função Ventricular Esquerda/fisiologia
9.
Circ Res ; 127(2): 229-243, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32248749

RESUMO

RATIONALE: Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in noncoding regions of the genome and likely include variants that modulate the function of transcriptional regulatory elements (REs) such as enhancers. However, the actual REs modulated by variants and the target genes of such REs remain to be identified. Thus, the biological mechanisms by which genetic variation promotes AF has thus far remained largely unexplored. OBJECTIVE: To identify REs in genome-wide association study loci that are influenced by AF-associated variants. METHODS AND RESULTS: We screened 2.45 Mbp of human genomic DNA containing 12 strongly AF-associated loci for RE activity using self-transcribing active regulatory region sequencing and a recently generated monoclonal line of conditionally immortalized rat atrial myocytes. We identified 444 potential REs, 55 of which contain AF-associated variants (P<10-8). Subsequently, using an adaptation of the self-transcribing active regulatory region sequencing approach, we identified 24 variant REs with allele-specific regulatory activity. By mining available chromatin conformation data, the possible target genes of these REs were mapped. To define the physiological function and target genes of such REs, we deleted the orthologue of an RE containing noncoding variants in the Hcn4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction, and selective loss of Hcn4 expression. CONCLUSIONS: We have identified REs at multiple genetic loci for AF and found that loss of an RE at the HCN4 locus results in sinus node dysfunction and reduced gene expression. Our approach can be broadly applied to facilitate the identification of human disease-relevant REs and target genes at cardiovascular genome-wide association studies loci.


Assuntos
Fibrilação Atrial/genética , Elementos Facilitadores Genéticos , Animais , Fibrilação Atrial/metabolismo , Loci Gênicos , Genoma Humano , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo
10.
Circ Res ; 127(1): 34-50, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32717170

RESUMO

Genome-wide association studies have uncovered over a 100 genetic loci associated with atrial fibrillation (AF), the most common arrhythmia. Many of the top AF-associated loci harbor key cardiac transcription factors, including PITX2, TBX5, PRRX1, and ZFHX3. Moreover, the vast majority of the AF-associated variants lie within noncoding regions of the genome where causal variants affect gene expression by altering the activity of transcription factors and the epigenetic state of chromatin. In this review, we discuss a transcriptional regulatory network model for AF defined by effector genes in Genome-wide association studies loci. We describe the current state of the field regarding the identification and function of AF-relevant gene regulatory networks, including variant regulatory elements, dose-sensitive transcription factor functionality, target genes, and epigenetic states. We illustrate how altered transcriptional networks may impact cardiomyocyte function and ionic currents that impact AF risk. Last, we identify the need for improved tools to identify and functionally test transcriptional components to define the links between genetic variation, epigenetic gene regulation, and atrial function.


Assuntos
Fibrilação Atrial/genética , Epigênese Genética , Redes Reguladoras de Genes , Animais , Fibrilação Atrial/metabolismo , Loci Gênicos , Humanos , Transcriptoma
11.
Proc Natl Acad Sci U S A ; 116(45): 22692-22698, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636200

RESUMO

Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to PITX2 Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2 AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2.


Assuntos
Fibrilação Atrial/genética , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas , Mapeamento Cromossômico , Epigênese Genética , Estudo de Associação Genômica Ampla , Camundongos , Camundongos Knockout , Proteína Homeobox PITX2
12.
Circulation ; 142(5): 466-482, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32403949

RESUMO

BACKGROUND: The human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low-input RNA sequencing have allowed definitions of cellular transcriptomes at single-cell resolution at scale, we have applied these approaches to assess the cellular and transcriptional diversity of the nonfailing human heart. METHODS: Microfluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from 7 human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based on transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data. RESULTS: We sequenced the transcriptomes of 287 269 single cardiac nuclei, revealing 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include 2 distinct groups of resident macrophages, 4 endothelial subtypes, and 2 fibroblast subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Intersection of our data set with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity. CONCLUSIONS: Using large-scale single nuclei RNA sequencing, we defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.


Assuntos
Miocárdio/citologia , Transcrição Gênica , Adipócitos/metabolismo , Adulto , Idoso , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Células Endoteliais/classificação , Células Endoteliais/metabolismo , Fibroblastos/classificação , Fibroblastos/metabolismo , Ontologia Genética , Coração/inervação , Átrios do Coração/citologia , Cardiopatias/tratamento farmacológico , Ventrículos do Coração/citologia , Homeostase , Humanos , Subpopulações de Linfócitos/metabolismo , Macrófagos/classificação , Macrófagos/metabolismo , Técnicas Analíticas Microfluídicas , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , RNA-Seq , Caracteres Sexuais , Análise de Célula Única , Transcriptoma
13.
Am J Hum Genet ; 99(6): 1281-1291, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27866707

RESUMO

The most significantly associated genetic locus for atrial fibrillation (AF) is in chromosomal region 4q25, where four independent association signals have been identified. Although model-system studies suggest that altered PITX2c expression might underlie the association, the link between specific variants and the direction of effect on gene expression remains unknown for all four signals. In the present study, we analyzed the AF-associated region most proximal to PITX2 at 4q25. First, we identified candidate regulatory variants that might confer AF risk through a combination of mammalian conservation, DNase hypersensitivity, and histone modification from ENCODE and the Roadmap Epigenomics Project, as well as through in vivo analysis of enhancer activity in embryonic zebrafish. Within candidate regions, we then identified a single associated SNP, rs2595104, which displayed dramatically reduced enhancer activity with the AF risk allele. CRISPR-Cas9-mediated deletion of the rs2595104 region and editing of the rs2595104 risk allele in human stem-cell-derived cardiomyocytes resulted in diminished PITX2c expression in comparison to that of the non-risk allele. This differential activity was mediated by activating enhancer binding protein 2 alpha (TFAP2a), which bound robustly to the non-risk allele at rs2595104, but not to the risk allele, in cardiomyocytes. In sum, we found that the AF-associated SNP rs2595104 altered PITX2c expression via interaction with TFAP2a. Such a pathway could ultimately contribute to AF susceptibility at the PITX2 locus associated with AF.


Assuntos
Fibrilação Atrial/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Polimorfismo de Nucleotídeo Único/genética , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/genética , Alelos , Animais , Cromossomos Humanos Par 4/genética , Sequência Conservada/genética , Desoxirribonucleases/metabolismo , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença , Histonas/química , Histonas/metabolismo , Humanos , Mamíferos/genética , Miócitos Cardíacos/citologia , Peixe-Zebra/genética , Proteína Homeobox PITX2
14.
PLoS Genet ; 12(10): e1006367, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27792790

RESUMO

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10-4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10-10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10-4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.


Assuntos
Displasia Fibromuscular/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas dos Microfilamentos/genética , Animais , Artérias/metabolismo , Artérias/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Espessura Intima-Media Carotídea , Modelos Animais de Doenças , Exoma/genética , Feminino , Displasia Fibromuscular/patologia , Regulação da Expressão Gênica , Genótipo , Humanos , Hipertensão/genética , Hipertensão/patologia , Masculino , Proteínas dos Microfilamentos/biossíntese , Miócitos de Músculo Liso , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Peixe-Zebra/genética
15.
JAMA ; 320(22): 2354-2364, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30535219

RESUMO

Importance: Atrial fibrillation (AF) is the most common arrhythmia affecting 1% of the population. Young individuals with AF have a strong genetic association with the disease, but the mechanisms remain incompletely understood. Objective: To perform large-scale whole-genome sequencing to identify genetic variants related to AF. Design, Setting, and Participants: The National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine Program includes longitudinal and cohort studies that underwent high-depth whole-genome sequencing between 2014 and 2017 in 18 526 individuals from the United States, Mexico, Puerto Rico, Costa Rica, Barbados, and Samoa. This case-control study included 2781 patients with early-onset AF from 9 studies and identified 4959 controls of European ancestry from the remaining participants. Results were replicated in the UK Biobank (346 546 participants) and the MyCode Study (42 782 participants). Exposures: Loss-of-function (LOF) variants in genes at AF loci and common genetic variation across the whole genome. Main Outcomes and Measures: Early-onset AF (defined as AF onset in persons <66 years of age). Due to multiple testing, the significance threshold for the rare variant analysis was P = 4.55 × 10-3. Results: Among 2781 participants with early-onset AF (the case group), 72.1% were men, and the mean (SD) age of AF onset was 48.7 (10.2) years. Participants underwent whole-genome sequencing at a mean depth of 37.8 fold and mean genome coverage of 99.1%. At least 1 LOF variant in TTN, the gene encoding the sarcomeric protein titin, was present in 2.1% of case participants compared with 1.1% in control participants (odds ratio [OR], 1.76 [95% CI, 1.04-2.97]). The proportion of individuals with early-onset AF who carried a LOF variant in TTN increased with an earlier age of AF onset (P value for trend, 4.92 × 10-4), and 6.5% of individuals with AF onset prior to age 30 carried a TTN LOF variant (OR, 5.94 [95% CI, 2.64-13.35]; P = 1.65 × 10-5). The association between TTN LOF variants and AF was replicated in an independent study of 1582 patients with early-onset AF (cases) and 41 200 control participants (OR, 2.16 [95% CI, 1.19-3.92]; P = .01). Conclusions and Relevance: In a case-control study, there was a statistically significant association between an LOF variant in the TTN gene and early-onset AF, with the variant present in a small percentage of participants with early-onset AF (the case group). Further research is necessary to understand whether this is a causal relationship.


Assuntos
Fibrilação Atrial/genética , Conectina/genética , Mutação com Perda de Função , Adulto , Idade de Início , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Controle de Qualidade
17.
BMC Med Genet ; 17(1): 83, 2016 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-27855642

RESUMO

BACKGROUND: The genetic basis for dilated cardiomyopathy (DCM) can be difficult to determine, particularly in familial cases with complex phenotypes. Next generation sequencing may be useful in the management of such cases. METHODS: We report two large families with pleiotropic inherited cardiomyopathy. In addition to DCM, the phenotypes included atrial and ventricular septal defects, cardiac arrhythmia and sudden death. Probands underwent whole exome sequencing to identify potentially causative variants. RESULTS: Each whole exome sequence yielded over 18,000 variants. We identified distinct mutations affecting a common amino acid in NKX2.5. Segregation analysis of the families support the pathogenic role of these variants. CONCLUSION: Our study emphasizes the utility of next generation sequencing in identifying causative mutations in complex inherited cardiac disease. We also report a novel pathogenic NKX2.5 mutation.


Assuntos
Aminoácidos/genética , Cardiomiopatia Dilatada/genética , Proteína Homeobox Nkx-2.5/genética , Aminoácidos/metabolismo , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/patologia , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Análise Mutacional de DNA , Morte Súbita Cardíaca/etiologia , Eletrocardiografia , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único
18.
Circ Res ; 114(9): 1469-82, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24763465

RESUMO

Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity. As the population ages and the prevalence of AF continues to rise, the socioeconomic consequences of AF will become increasingly burdensome. Although there are well-defined clinical risk factors for AF, a significant heritable component is also recognized. To identify the molecular basis for the heritability of AF, investigators have used a combination of classical Mendelian genetics, candidate gene screening, and genome-wide association studies. However, these avenues have, as yet, failed to define the majority of the heritability of AF. The goal of this review is to describe the results from both candidate gene and genome-wide studies, as well as to outline potential future avenues for creating a more complete understanding of AF genetics. Ultimately, a more comprehensive view of the genetic underpinnings for AF will lead to the identification of novel molecular pathways and improved risk prediction of this complex arrhythmia.


Assuntos
Fibrilação Atrial/genética , Variação Genética , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Estudos de Associação Genética , Loci Gênicos , Predisposição Genética para Doença , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Hereditariedade , Humanos , Linhagem , Fenótipo , Medição de Risco , Fatores de Risco
20.
Circulation ; 130(15): 1225-35, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25124494

RESUMO

BACKGROUND: Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. METHODS AND RESULTS: To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13-1.23; P=6.5×10(-16)), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06-1.14; P=2.2×10(-8)), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08-1.16; P=5.7×10(-11)), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06-1.14; P=9.8×10(-9)). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26-1.39; P=2.0×10(-25)) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08-1.16; P=3.9×10(-9)). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10(-19)), GJA1 (P=2.66×10(-6)), and TBX5 (P=1.36×10(-5)). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). CONCLUSIONS: We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.


Assuntos
Fibrilação Atrial/genética , Conexina 43/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteínas com Domínio T/genética , Ubiquitina-Proteína Ligases/genética , Idoso , Animais , Fibrilação Atrial/etnologia , Fibrilação Atrial/fisiopatologia , Mapeamento Cromossômico , Conexina 43/fisiologia , Europa (Continente) , Feminino , Técnicas de Silenciamento de Genes , Loci Gênicos/fisiologia , Predisposição Genética para Doença/etnologia , Genótipo , Proteínas de Homeodomínio/fisiologia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Proteínas Musculares , Proteínas Nucleares/fisiologia , Locos de Características Quantitativas , Proteínas Repressoras/fisiologia , Proteínas com Domínio T/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Proteína Homeobox PITX2
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