RESUMO
The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.
Assuntos
Microvasos , Fenótipo , Placa Aterosclerótica , Espectrina , Peixe-Zebra , Humanos , Placa Aterosclerótica/patologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Peixe-Zebra/genética , Animais , Microvasos/patologia , Microvasos/metabolismo , Espectrina/genética , Espectrina/metabolismo , Transcriptoma/genética , Permeabilidade Capilar/genética , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, here we perform an unbiased survey of C-degrons in budding yeast. We identify over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast Cterminome. Combining machine learning, high-throughput mutagenesis and genetic screens reveals that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.
Assuntos
Degrons , Proteínas Ligases SKP Culina F-Box , Animais , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Domínios Proteicos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Mamíferos/metabolismoRESUMO
The Anopheles mosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved1. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation (SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.
Assuntos
Processamento Alternativo , Anopheles , Mecanismo Genético de Compensação de Dose , Proteínas de Insetos , Caracteres Sexuais , Diferenciação Sexual , Cromossomo X , Animais , Feminino , Masculino , Anopheles/genética , Anopheles/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Diferenciação Sexual/genética , Cromossomo X/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic as well as systemic insulin resistance even in the absence of type 2 diabetes. The extent and pathways through which hepatic inflammation modulates insulin sensitivity in NAFLD are only partially understood. We explored the contribution of hepatic interleukin (IL)-1 signalling in a novel conditional knockout mouse model and expand the knowledge on this signalling pathway with regard to its liver-specific functions. METHODS: A high-fat, high-carbohydrate diet (HFD) over 12 weeks was used in male hepatocyte-specific IL-1 receptor type 1 (IL-1R1) knockout mice (Il1r1Hep-/- ) and wild-type (WT) littermates. RESULTS: Both genotypes developed an obese phenotype and accompanying macrovesicular hepatic steatosis. In contrast to WT mice, microvesicular steatosis and ballooning injury was less pronounced in HFD-fed Il1r1Hep-/- mice, and alanine aminotransferase remained in the normal range. This was paralleled by the suppression of injurious and proinflammatory hepatic c-Jun N-terminal kinases and extracellular signal-regulated kinases signalling, stable peroxisome proliferator activated receptor gamma coactivator-1alpha and farnesoid X receptor-alpha expression and preservation of mitochondrial function. Strikingly, despite HFD-feeding Il1r1Hep-/- mice remained highly insulin sensitive as indicated by lower insulin levels, homeostatic model assessment for insulin resistance, higher glucose tolerance, more stable hepatic insulin signalling cascade, and less adipose tissue inflammation compared to the WT. CONCLUSIONS: The current data highlights that hepatocyte IL-1R1 contributes to hepatic and extrahepatic insulin resistance. Future liver-directed therapies in NAFLD could have effects on insulin sensitivity when improving hepatic inflammation and IL-1R1 signalling.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Animais , Inflamação , Insulina , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
As mediators of intercellular communication, extracellular vesicles containing molecular cargo, such as microRNAs, are secreted by cells and taken up by recipient cells to influence their cellular phenotype and function. Here we report that cardiac stress-induced differential microRNA content, with miR-200c-3p being one of the most enriched, in cardiomyocyte-derived extracellular vesicles mediates functional cross-talk with endothelial cells. Silencing of miR-200c-3p in mice subjected to chronic increased cardiac pressure overload resulted in attenuated hypertrophy, smaller fibrotic areas, higher capillary density, and preserved cardiac ejection fraction. We were able to maximally rescue microvascular and cardiac function with very low doses of antagomir, which specifically silences miR-200c-3p expression in non-myocyte cells. Our results reveal vesicle transfer of miR-200c-3p from cardiomyocytes to cardiac endothelial cells, underlining the importance of cardiac intercellular communication in the pathophysiology of heart failure.
Assuntos
Vesículas Extracelulares , MicroRNAs , Animais , Comunicação Celular , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Genomic instability, the unresolved accumulation of DNA variants, is hypothesized as one of the contributors to the natural aging process. We assessed the frequency of unresolved DNA damage reaching the transcriptome of the murine myocardium during the course of natural aging and in hearts from four distinct mouse models of premature aging with established aging-related cardiac dysfunctions. RNA sequencing and variant calling based on total RNA sequencing was compared between hearts from naturally aging mice, mice with cardiomyocyte-specific deficiency of Ercc1, a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity, Tert-deficient mice with reduced telomere length, and a mouse model of human Hutchinson-Gilford progeria syndrome (HGPS). Our results demonstrate that no enrichment in variants is evident in the naturally aging murine hearts until 2 y of age from the HGPS mouse model or mice with reduced telomere lengths. In contrast, a dramatic accumulation of variants was evident in Ercc1 cardiomyocyte-specific knockout mice with deficient DNA repair machinery, in mice with reduced mitochondrial antioxidant capacity, and in the intestine, liver, and lung of naturally aging mice. Our data demonstrate that genomic instability does not evidently contribute to naturally aging of the mouse heart in contrast to other organs and support the contention that the endogenous DNA repair machinery is remarkably active to maintain genomic integrity in cardiac cells throughout life.
Assuntos
Senilidade Prematura/genética , Senescência Celular/genética , Instabilidade Genômica/genética , Envelhecimento/genética , Animais , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Endonucleases/genética , Endonucleases/metabolismo , Feminino , Coração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Miocárdio/metabolismoRESUMO
lncRNAs are at the core of many regulatory processes and have also been recognized to be involved in various complex diseases. They affect gene regulation through direct interactions with RNA, DNA or proteins. Accordingly, lncRNA structure is likely to be essential for their regulatory function. Point mutations, which manifest as SNPs (single nucleotide polymorphisms) in genome screens, can substantially alter their function and, subsequently, the expression of their downstream regulated genes. To test the effect of SNPs on structure, we investigated lncRNAs associated with dilated cardiomyopathy. Among 322 human candidate lncRNAs, we demonstrate first the significant association of an SNP located in lncRNA H19 using data from 1084 diseased and 751 control patients. H19 is generally highly expressed in the heart, with a complex expression pattern during heart development. Next, we used MFE (minimum free energy) folding to demonstrate a significant refolding in the secondary structure of this 861 nt long lncRNA. Since MFE folding may overlook the importance of sub-optimal structures, we showed that this refolding also manifests in the overall Boltzmann structure ensemble. There, the composition of structures is tremendously affected in their thermodynamic probabilities through the genetic variant. Finally, we confirmed these results experimentally, using SHAPE-Seq, corroborating that SNPs affecting such structures may explain hidden genetic variance not accounted for through genome wide association studies. Our results suggest that structural changes in lncRNAs, and lncRNA H19 in particular, affect regulatory processes and represent optimal targets for further in-depth studies probing their molecular interactions.
Assuntos
Cardiomiopatia Dilatada/patologia , Predisposição Genética para Doença , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Pareamento de Bases , Sequência de Bases , Cardiomiopatia Dilatada/genética , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
Assuntos
Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína Vermelha FluorescenteRESUMO
Previous genome-wide association studies (GWASs) have established several susceptibility genes for venous thromboembolism (VTE) and suggested many others. However, a large proportion of the genetic variance in VTE remains unexplained. Here, we report genome-wide single- and multimarker as well as gene-level associations with VTE in 964 cases and 899 healthy controls of European ancestry. We report 19 loci at the genome-wide level of association (p ≤ 5 × 10-8). Our results add to the strong support for the association of genetic variants in F5, NME7, ABO, and FGA with VTE, and identify several loci that have not been previously associated with VTE. Altogether, our novel findings suggest that 20 susceptibility genes for VTE were newly discovered by our study. These genes may impact the production and prothrombotic functions of platelets, endothelial cells, and white and red blood cells. Moreover, the majority of these genes have been previously associated with cardiovascular diseases and/or risk factors for VTE. Future studies are warranted to validate our findings and to investigate the shared genetic architecture with susceptibility factors for other cardiovascular diseases impacting VTE risk.
Assuntos
Loci Gênicos , Polimorfismo de Nucleotídeo Único , Tromboembolia Venosa/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Medição de Risco , Fatores de Risco , Tromboembolia Venosa/diagnóstico , Adulto JovemRESUMO
The role of the A>G polymorphism at position 19911 in the prothrombin gene (factor [F] 2 at rs3136516) as a risk factor for venous thromboembolism [VTE] is still unclear. To evaluate the presence of the F2 polymorphism in VTE patients compared to healthy blood donors and to adjust the results for common inherited thrombophilias [IT], age at onset and blood group [BG], and to calculate the risk of VTE recurrence. We investigated 1012 Caucasian patients with a diagnosis of VTE for the presence of the F2 rs3136516 polymorphism and compared these with 902 healthy blood donors. Odds ratios [OR] together with their 95% confidence intervals were calculated adjusted for F5 at rs6025, F2 at rs1799963, blood group, age and gender. In addition, we evaluated the risk of recurrent VTE during patient follow-up calculating hazard ratios [HR] together with their 95% CI. Compared with the AA wildtype, the F2 GG and AG genotypes (rs3136516) were associated with VTE (OR 1.48 and 1.45). The OR in F5 carriers compared to controls was 5.68 and 2.38 in patients with F2 (rs1799963). BG "non-O" was significantly more often diagnosed in patients compared to BG "O" (OR 2.74). VTE recurrence more often occurred in males (HR 2.3) and in carriers with combined thrombophilia (HR 2.11). Noteworthy, the rs3136516 polymorphism alone was not associated significantly with recurrence. In Caucasian patients with VTE the F2 GG/GA genotypes (rs3136516) were moderate risk factors for VTE. Recurrence was associated with male gender and combined thrombophilia.
Assuntos
Antígenos de Grupos Sanguíneos , Polimorfismo de Nucleotídeo Único , Protrombina/genética , Tromboembolia Venosa/genética , Adulto , Antígenos de Grupos Sanguíneos/sangue , Feminino , Predisposição Genética para Doença , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fatores Sexuais , Tromboembolia Venosa/sangue , Tromboembolia Venosa/etiologia , Adulto JovemRESUMO
We recently reported a family-based genome wide association study (GWAS) for pediatric stroke pointing our attention to two significantly associated genes of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family ADAMTS2 (rs469568, p = 8x10-6) and ADAMTS12 (rs1364044, p = 2.9x10-6). To further investigate these candidate genes, we applied a targeted resequencing approach on 48 discordant sib-pairs for pediatric stroke followed by genotyping of the detected non-synonymous variants in the full cohort of 270 offspring trios and subsequent fine mapping analysis. We identified eight non-synonymous SNPs in ADAMTS2 and six in ADAMTS12 potentially influencing the respective protein function. These variants were genotyped within a cohort of 270 affected offspring trios, association analysis revealed the ADAMTS12 variant rs77581578 to be significantly under-transmitted (p = 6.26x10-3) to pediatric stroke patients. The finding was validated in a pediatric venous thromboembolism (VTE) cohort of 189 affected trios. Subsequent haplotype analysis of ADAMTS12 detected a significantly associated haplotype comprising the originally identified GWAS variant. Several ADAMTS genes such as ADAMTS13 are involved in thromboembolic disease process. Here, we provide further evidence for ADAMTS12 to likely play a role in pediatric stroke. Further functional studies are warranted to assess the functional role of ADAMTS12 in the pathogenesis of stroke.
Assuntos
Proteínas ADAMTS/genética , Predisposição Genética para Doença , Acidente Vascular Cerebral/genética , Criança , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos/genética , Humanos , Masculino , Pediatria , Polimorfismo de Nucleotídeo Único/genética , Acidente Vascular Cerebral/patologiaRESUMO
We investigated the transcriptomic landscape of the murine myocardium along the course of natural aging and in three distinct mouse models of premature aging with established aging-related cardiac dysfunction. Genome-wide total RNA-seq was performed and the expression patterns of protein-coding genes and non-coding RNAs were compared between hearts from naturally aging mice, mice with cardiac-specific deficiency of a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity and mice with reduced telomere length. Our results demonstrate that no dramatic changes are evident in the transcriptomes of naturally senescent murine hearts until two years of age, in contrast to the transcriptome of accelerated aged mice. Additionally, these mice displayed model-specific alterations of the expression levels of protein-coding and non-coding genes with hardly any overlap with age-related signatures. Our data demonstrate very limited similarities between the transcriptomes of all our murine aging models and question their reliability to study human cardiovascular senescence.
Assuntos
Senilidade Prematura/genética , Envelhecimento/genética , Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Proteínas/genética , Envelhecimento/metabolismo , Senilidade Prematura/metabolismo , Senilidade Prematura/fisiopatologia , Animais , Feminino , Humanos , Ictiose Lamelar/genética , Ictiose Lamelar/metabolismo , Ictiose Lamelar/fisiopatologia , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas/metabolismo , Telômero/genética , Telômero/metabolismo , Encurtamento do Telômero , TranscriptomaRESUMO
Unraveling the genetic susceptibility of complex diseases such as chronic kidney disease remains challenging. Here, we used inbred rat models of kidney damage associated with elevated blood pressure for the comprehensive analysis of a major albuminuria susceptibility locus detected in these models. We characterized its genomic architecture by congenic substitution mapping, targeted next-generation sequencing, and compartment-specific RNA sequencing analysis in isolated glomeruli. This led to prioritization of transmembrane protein Tmem63c as a novel potential target. Tmem63c is differentially expressed in glomeruli of allele-specific rat models during onset of albuminuria. Patients with focal segmental glomerulosclerosis exhibited specific TMEM63C loss in podocytes. Functional analysis in zebrafish revealed a role for tmem63c in mediating the glomerular filtration barrier function. Our data demonstrate that integrative analysis of the genomic architecture of a complex trait locus is a powerful tool for identification of new targets such as Tmem63c for further translational investigation.
Assuntos
Loci Gênicos , Predisposição Genética para Doença , Hipertensão Renal/fisiopatologia , Hipertensão/complicações , Herança Multifatorial , Nefrite/fisiopatologia , Albuminúria/patologia , Animais , Modelos Animais de Doenças , Humanos , Hipertensão Renal/patologia , Nefrite/patologia , Ratos , Peixe-ZebraRESUMO
BACKGROUND/AIMS: Inflammatory processes are controlled by the fine-tuned balance of monocyte subsets. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C that is highly expressed on inflammatory monocytes (Ly6Chigh) and to a lesser extent on patrolling monocytes (Ly6Clow). Our previous study revealed an accumulation of Ly6Chigh monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (SOCS)-1 leading to an increased atherosclerotic burden. To decipher the underlying mechanisms, we performed a genome-wide analysis of SOCS-1-dependent gene regulation in Ly6Chigh and Ly6Clow monocytes. METHODS: In monocyte subsets from SOCS-1competent and -deficient mice differentially regulated genes were identified using an Illumina mRNA microarray (45,200 transcripts), which were randomly validated by qPCR. Principal component analysis was performed to further characterize mRNA profiles in monocyte subsets. To unravel potential regulatory mechanisms behind the differential mRNA expression, in silico analysis of a transcription factor (TF) network correlating with SOCS-1-dependent mRNA expression was carried out and combined with a weighted correlation network analysis (WGCNA). RESULTS: mRNA analysis in monocyte subsets revealed 46 differentially regulated genes by 2-fold or more. Principal component analysis illustrated a distinct separation of mRNA profiles in monocyte subsets from SOCS-1-deficient mice. Notably, two cell surface receptors crucially involved in the determination of monocyte differentiation and survival, C-X3-C chemokine receptor 1 (CX3CR1) and colony stimulating factor 1 receptor (CSF1R), were identified to be regulated by SOCS-1. Moreover, in silico analysis of a TF network in combination with the WGCNA revealed genes coding for PPAR-γ, NUR77 and several ETSdomain proteins that act as pivotal inflammatory regulators. CONCLUSION: Our study reveals that SOCS-1 is implicated in a TF network regulating the expression of central transcription factors like PPAR-γ and NUR77 thereby influencing the expression of CX3CR1 and CSF1R that are known to be pivotal for the survival of Ly6Clow monocytes.
Assuntos
Antígenos Ly , Aterosclerose/metabolismo , Regulação da Expressão Gênica , Monócitos/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Sobrevivência Celular , Camundongos , Camundongos Knockout , Monócitos/patologia , Proteína 1 Supressora da Sinalização de Citocina/genéticaRESUMO
Immune-mediated heparin-induced thrombocytopenia (HIT) is the clinically most important adverse drug reaction (ADR) in response to heparin therapy characterized by a prothrombotic state despite a decrease in platelet count. We conducted a genome-wide association study in 96 suspected HIT cases and 96 controls to explore the genetic predisposition for HIT within a case-control pharmacovigilance study followed by replication in additional 86 cases and 86 controls from the same study. One single nucleotide polymorphism (SNP, rs1433265, P = 6.5 × 10-5, odds ratio (OR) 2.79) from 16 identified SNPs was successfully replicated (P = 1.5 × 10-4, OR 2.77; combined data set P = 2.7 × 10-8, OR 2.77) and remained the most strongly associated SNP after imputing locus genotypes. Fine mapping revealed a significantly associated risk-conferring haplotype (P = 4.9 × 10-6, OR 2.41). In order to find rare variants contributing to the association signals, we applied a targeted resequencing approach in a subgroup of 73 HIT patients and 23 controls for the regions with the 16 most strongly HIT-associated SNPs. C-alpha testing was applied to test for the impact of rare variants and we detected two candidate genes, the discoidin domain receptor tyrosine kinase 1 (DDR1, P = 3.6 × 10-2) and the multiple C2 and transmembrane domain containing 2 (MCTP2, P = 4.5 × 10-2). For the genes interactor of little elongation complex ELL subunit 1 (ICE1) and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, 16 (ADAMTS16) nearby rs1433265, we identified several missense variants. Although replication in an independent population is warranted, these findings provide a basis for future studies aiming to identify and characterize genetic susceptibility factors for HIT. KEY MESSAGES: We identified and validated a HIT-associated locus on chromosome 5. Targeted NGS analysis for rare variants identifies DDR1 and MCTP2 as novel candidates. In addition, missense variants for ADAMTS16 and ICE1 were identified in the locus.
Assuntos
Anticoagulantes/efeitos adversos , Cromossomos Humanos Par 5 , Predisposição Genética para Doença , Heparina/efeitos adversos , Locos de Características Quantitativas , Trombocitopenia/etiologia , Idoso , Alelos , Biomarcadores , Estudos de Casos e Controles , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Testes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Trombocitopenia/sangue , Trombocitopenia/diagnósticoRESUMO
BACKGROUND: Inhibition of PKC-α (protein kinase C-α) enhances contractility and cardioprotection in animal models, but effects in humans are unknown. Genotypes at rs9912468 strongly associate with PRKCA expression in the left ventricle, enabling genetic approaches to measure effects of reduced PKC-α in human populations. METHODS AND RESULTS: We analyzed the cis expression quantitative trait locus for PRKCA marked by rs9912468 using 313 left ventricular specimens from European Ancestry patients. The forward strand minor allele (G) at rs9912468 is associated with reduced PKC-α transcript abundance (1.7-fold reduction in minor allele homozygotes, P=1×10-41). This association was cardiac specific in expression quantitative trait locus data sets that span 16 human tissues. Cardiac epigenomic data revealed a predicted enhancer in complete (R2=1.0) linkage disequilibrium with rs9912468 within intron 2 of PRKCA. We cloned this region and used reporter constructs to verify cardiac-specific enhancer activity in vitro in cardiac and noncardiac cells and in vivo in zebrafish. The PRKCA enhancer contains 2 common genetic variants and 4 haplotypes; the haplotype correlated with the rs9912468 PKC-α-lowering allele (G) showed lowest activity. In contrast to previous reports in animal models, the PKC-α-lowering allele is associated with adverse left ventricular remodeling (higher mass, larger diastolic dimension), reduced fractional shortening, and higher risk of dilated cardiomyopathy in human populations. CONCLUSIONS: These findings support PKC-α as a regulator of the human heart but suggest that PKC-α inhibition may adversely affect the left ventricle depending on timing and duration. Pharmacological studies in human subjects are required to discern potential benefits and harms of PKC-α inhibitors as an approach to treat heart disease.
Assuntos
Ventrículos do Coração/metabolismo , Proteína Quinase C-alfa/genética , Remodelação Ventricular/genética , Adulto , Idoso , Alelos , Animais , Feminino , Genes Reporter , Predisposição Genética para Doença , Genótipo , Haplótipos , Homozigoto , Humanos , Íntrons , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Proteína Quinase C-alfa/metabolismo , Locos de Características Quantitativas , Peixe-ZebraRESUMO
The transcription factor ISL1 is thought to be key for conveying the multipotent and proliferative properties of cardiac precursor cells. Here, we investigate its function upon cardiac induction of human embryonic stem cells. We find that ISL1 does not stabilize the transient cardiac precursor cell state but rather serves to accelerate cardiomyocyte differentiation. Conversely, ISL1 depletion delays cardiac differentiation and respecifies nascent cardiomyocytes from a ventricular to an atrial identity. Mechanistic analyses integrate this unrecognized anti-atrial function of ISL1 with known and newly identified atrial inducers. In this revised view, ISL1 is antagonized by retinoic acid signaling via a novel player, MEIS2. Conversely, ISL1 competes with the retinoic acid pathway for prospective cardiomyocyte fate, which converges on the atrial specifier NR2F1. This study reveals a core regulatory network putatively controlling human heart chamber formation and also bears implications for the subtype-specific production of human cardiomyocytes with enhanced functional properties.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/fisiologia , Proteínas com Homeodomínio LIM/metabolismo , Miócitos Cardíacos/fisiologia , Fatores de Transcrição/metabolismo , Fator I de Transcrição COUP/metabolismo , HumanosRESUMO
Paediatric venous thromboembolism (VTE) is a rare disorder but a rising incidence has been observed in recent years, due to improved VTE diagnosis and increased use of central venous catheters in the treatment of severe diseases. Risk assessment strategies are well established for adult patients, however, similar guidelines for paediatric patients are largely lacking. Several risk prediction tools have been reported in recent literature, which make use of established risk factors to assess VTE risk in paediatric subgroups, such as hospitalised children, cancer-diagnosed children and paediatric trauma patients. Although these models suffer several limitations regarding their study size and heterogeneous selection of predictor variables, they offer potential for improving the thromboprophylaxis management in these children. Here, we give an overview on recently reported risk prediction models for paediatric VTE.
Assuntos
Tromboembolia Venosa/prevenção & controle , Coagulação Sanguínea/fisiologia , Criança , Pré-Escolar , Previsões , Hospitalização , Humanos , Lactente , Recém-Nascido , Neoplasias/complicações , Medição de Risco/tendências , Fatores de Risco , Trombofilia/genética , Tromboembolia Venosa/sangue , Tromboembolia Venosa/etiologia , Ferimentos e Lesões/complicaçõesRESUMO
BACKGROUND: Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases. RESULTS: Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts. CONCLUSIONS: RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.