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Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.
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BACKGROUND: Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments. METHODS: We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1-/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan. FINDINGS: We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure. CONCLUSION: This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them. FUNDING: This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant.
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Blood cells contain functionally important intracellular structures, such as granules, critical to immunity and thrombosis. Quantitative variation in these structures has not been subjected previously to large-scale genetic analysis. We perform genome-wide association studies of 63 flow-cytometry derived cellular phenotypes-including cell-type specific measures of granularity, nucleic acid content and reactivity-in 41,515 participants in the INTERVAL study. We identify 2172 distinct variant-trait associations, including associations near genes coding for proteins in organelles implicated in inflammatory and thrombotic diseases. By integrating with epigenetic data we show that many intracellular structures are likely to be determined in immature precursor cells. By integrating with proteomic data we identify the transcription factor FOG2 as an early regulator of platelet formation and α-granularity. Finally, we show that colocalisation of our associations with disease risk signals can suggest aetiological cell-types-variants in IL2RA and ITGA4 respectively mirror the known effects of daclizumab in multiple sclerosis and vedolizumab in inflammatory bowel disease.
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Estudo de Associação Genômica Ampla , Proteômica , Microscopia , Fatores de Transcrição , CausalidadeRESUMO
Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140 214 unrelated UK Biobank (UKB) participants found that each of them carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade gene (DGG) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12 367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18 410 nodes and 571 917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1 or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants.
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Estudo de Associação Genômica Ampla , Trombose , Humanos , Bancos de Espécimes Biológicos , Hemostasia , Hemorragia/genética , Doenças RarasRESUMO
An altered thrombo-hemorrhagic profile has long been observed in patients with myeloproliferative neoplasms (MPNs). We hypothesized that this observed clinical phenotype may result from altered expression of genes known to harbor genetic variants in bleeding, thrombotic, or platelet disorders. Here, we identify 32 genes from a clinically validated gene panel that were also significantly differentially expressed in platelets from MPN patients as opposed to healthy donors. This work begins to unravel previously unclear mechanisms underlying an important clinical reality in MPNs. Knowledge of altered platelet gene expression in MPN thrombosis/bleeding diathesis opens opportunities to advance clinical care by: (1) enabling risk stratification, in particular, for patients undergoing invasive procedures, and (2) facilitating tailoring of treatment strategies for those at highest risk, for example, in the form of antifibrinolytics, desmopressin or platelet transfusions (not current routine practice). Marker genes identified in this work may also enable prioritization of candidates in future MPN mechanistic as well as outcome studies.
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BACKGROUND AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) develops due to impaired hepatic lipid fluxes and is a risk factor for chronic liver disease and atherosclerosis. Lipidomic studies consistently reported characteristic hepatic/VLDL "lipid signatures" in NAFLD; whole plasma traits are more debated. Surprisingly, the HDL lipid composition by mass spectrometry has not been characterised across the NAFLD spectrum, despite HDL being a possible source of hepatic lipids delivered from peripheral tissues alongside free fatty acids (FFA). This study characterises the HDL lipidomic signature in NAFLD, and its correlation with metabolic and liver disease markers. METHODS: We used liquid chromatography-mass spectrometry to determine the whole serum and HDL lipidomic profile in 89 biopsy-proven NAFLD patients and 20 sex and age-matched controls. RESULTS: In the whole serum of NAFLD versus controls, we report a depletion in polyunsaturated (PUFA) phospholipids (PL) and FFA; with PUFA PL being also lower in HDL, and negatively correlated with BMI, insulin resistance, triglycerides, and hepatocyte ballooning. In the HDL of the NAFLD group we also describe higher saturated ceramides, which positively correlate with insulin resistance and transaminases. CONCLUSION: NAFLD features lower serum lipid species containing polyunsaturated fatty acids; the most affected lipid fractions are FFA and (HDL) phospholipids; our data suggest a possible defect in the transfer of PUFA from peripheral tissues to the liver in NAFLD. Mechanistic studies are required to explore the biological implications of our findings addressing if HDL composition can influence liver metabolism and damage, thus contributing to NAFLD pathophysiology.
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Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácidos Graxos não Esterificados , Lipoproteínas HDL , Ácidos Graxos Insaturados , FosfolipídeosRESUMO
BACKGROUND: Assessing inflammatory disease activity in large vessel vasculitis (LVV) can be challenging by conventional measures. OBJECTIVES: We aimed to investigate somatostatin receptor 2 (SST2) as a novel inflammation-specific molecular imaging target in LVV. METHODS: In a prospective, observational cohort study, in vivo arterial SST2 expression was assessed by positron emission tomography/magnetic resonance imaging (PET/MRI) using 68Ga-DOTATATE and 18F-FET-ßAG-TOCA. Ex vivo mapping of the imaging target was performed using immunofluorescence microscopy; imaging mass cytometry; and bulk, single-cell, and single-nucleus RNA sequencing. RESULTS: Sixty-one participants (LVV: n = 27; recent atherosclerotic myocardial infarction of ≤2 weeks: n = 25; control subjects with an oncologic indication for imaging: n = 9) were included. Index vessel SST2 maximum tissue-to-blood ratio was 61.8% (P < 0.0001) higher in active/grumbling LVV than inactive LVV and 34.6% (P = 0.0002) higher than myocardial infarction, with good diagnostic accuracy (area under the curve: ≥0.86; P < 0.001 for both). Arterial SST2 signal was not elevated in any of the control subjects. SST2 PET/MRI was generally consistent with 18F-fluorodeoxyglucose PET/computed tomography imaging in LVV patients with contemporaneous clinical scans but with very low background signal in the brain and heart, allowing for unimpeded assessment of nearby coronary, myocardial, and intracranial artery involvement. Clinically effective treatment for LVV was associated with a 0.49 ± 0.24 (standard error of the mean [SEM]) (P = 0.04; 22.3%) reduction in the SST2 maximum tissue-to-blood ratio after 9.3 ± 3.2 months. SST2 expression was localized to macrophages, pericytes, and perivascular adipocytes in vasculitis specimens, with specific receptor binding confirmed by autoradiography. SSTR2-expressing macrophages coexpressed proinflammatory markers. CONCLUSIONS: SST2 PET/MRI holds major promise for diagnosis and therapeutic monitoring in LVV. (PET Imaging of Giant Cell and Takayasu Arteritis [PITA], NCT04071691; Residual Inflammation and Plaque Progression Long-Term Evaluation [RIPPLE], NCT04073810).
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Aterosclerose , Arterite de Células Gigantes , Infarto do Miocárdio , Arterite de Takayasu , Humanos , Receptores de Somatostatina , Estudos Prospectivos , Fluordesoxiglucose F18 , Inflamação/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Imageamento por Ressonância Magnética , Vasos Coronários/patologia , Aterosclerose/diagnóstico por imagem , Compostos Radiofarmacêuticos/farmacologiaRESUMO
Since the early inception of genome-wide association studies (GWAS), it became clear that, in all diseases or traits studied, most genetic variants are likely to exert their effect on gene expression mainly by altering the function of regulatory elements. At the same time, the regulation of the gene expression field broadened its boundaries, from the univocal relationship between regulatory elements and genes to include genome organization, long-range DNA interactions, and epigenetics. Next-generation sequencing has introduced genome-wide approaches that have greatly improved our understanding of the general principles of gene expression. However, elucidating how these apply in every single genomic locus still requires painstaking experimental work, in which several independent lines of evidence are required, and often this is helped by rare genetic variants in individuals with rare diseases. This review will focus on the non-coding features of the genome involved in transcriptional regulation, that when altered, leads to known cases of inherited (familial) thrombotic and hemostatic phenotypes, emphasizing the role of enhancers and super-enhancers.
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Estudo de Associação Genômica Ampla , Trombose , Elementos Facilitadores Genéticos , Variação Genética , Hemostasia/genética , Humanos , Fenótipo , Trombose/genéticaRESUMO
BACKGROUND: This work is aimed at improving the understanding of cardiometabolic syndrome pathophysiology and its relationship with thrombosis by generating a multi-omic disease signature. METHODS/RESULTS: We combined classic plasma biochemistry and plasma biomarkers with the transcriptional and epigenetic characterisation of cell types involved in thrombosis, obtained from two extreme phenotype groups (morbidly obese and lipodystrophy) and lean individuals to identify the molecular mechanisms at play, highlighting patterns of abnormal activation in innate immune phagocytic cells. Our analyses showed that extreme phenotype groups could be distinguished from lean individuals, and from each other, across all data layers. The characterisation of the same obese group, 6 months after bariatric surgery, revealed the loss of the abnormal activation of innate immune cells previously observed. However, rather than reverting to the gene expression landscape of lean individuals, this occurred via the establishment of novel gene expression landscapes. NETosis and its control mechanisms emerge amongst the pathways that show an improvement after surgical intervention. CONCLUSIONS: We showed that the morbidly obese and lipodystrophy groups, despite some differences, shared a common cardiometabolic syndrome signature. We also showed that this could be used to discriminate, amongst the normal population, those individuals with a higher likelihood of presenting with the disease, even when not displaying the classic features.
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Lipodistrofia , Síndrome Metabólica , Obesidade Mórbida , Metilação de DNA , Epigênese Genética , Humanos , Síndrome Metabólica/genética , Obesidade Mórbida/cirurgia , FenótipoRESUMO
Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.
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Hematopoese , Células-Tronco Hematopoéticas , Adulto , Medula Óssea , Células da Medula Óssea/fisiologia , Eritropoese , Humanos , MegacariócitosRESUMO
Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43-0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.
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Plaquetas/metabolismo , Doenças Hematológicas/genética , Megacariócitos/metabolismo , Proteoma/genética , Transcriptoma , Humanos , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/metabolismoRESUMO
Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.
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Doenças Autoimunes/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/imunologia , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Adulto , Idoso , Doenças Autoimunes/imunologia , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Regiões Promotoras Genéticas/genética , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/imunologia , Adulto JovemRESUMO
BACKGROUND: Megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs. OBJECTIVES: To characterize MKs and HSCs from human bone marrow using single-cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis. RESULTS: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet-specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK-biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state. CONCLUSIONS: Here, we use single-cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC.
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Megacariócitos , Trombopoese , Plaquetas , Medula Óssea , Diferenciação Celular , Humanos , Trombopoese/genéticaRESUMO
Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.
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Proteínas Associadas aos Microtúbulos/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Serina-Treonina Quinases/genética , Trombofilia/genética , Trombose Venosa/genética , Adulto , Células Endoteliais/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Lipoproteínas/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Fatores de Risco , Trombofilia/patologia , Tromboembolia Venosa/genética , Tromboembolia Venosa/patologia , Trombose Venosa/patologia , Sequenciamento do ExomaRESUMO
Transcriptional profiling of hematopoietic cell subpopulations has helped to characterize the developmental stages of the hematopoietic system and the molecular bases of malignant and non-malignant blood diseases. Previously, only the genes targeted by expression microarrays could be profiled genome-wide. High-throughput RNA sequencing, however, encompasses a broader repertoire of RNA molecules, without restriction to previously annotated genes. We analyzed the BLUEPRINT consortium RNA-sequencing data for mature hematopoietic cell types. The data comprised 90 total RNA-sequencing samples, each composed of one of 27 cell types, and 32 small RNA-sequencing samples, each composed of one of 11 cell types. We estimated gene and isoform expression levels for each cell type using existing annotations from Ensembl. We then used guided transcriptome assembly to discover unannotated transcripts. We identified hundreds of novel non-coding RNA genes and showed that the majority have cell type-dependent expression. We also characterized the expression of circular RNA and found that these are also cell type-specific. These analyses refine the active transcriptional landscape of mature hematopoietic cells, highlight abundant genes and transcriptional isoforms for each blood cell type, and provide a valuable resource for researchers of hematologic development and diseases. Finally, we made the data accessible via a web-based interface: https://blueprint.haem.cam.ac.uk/bloodatlas/.
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RNA Longo não Codificante , Transcriptoma , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA Circular , RNA Longo não Codificante/genética , Análise de Sequência de RNARESUMO
Both poly(A) enrichment and ribosomal RNA depletion are commonly used for RNA sequencing. Either has its advantages and disadvantages that may lead to biases in the downstream analyses. To better access these effects, we carried out both ribosomal RNA-depleted and poly(A)-selected RNA-seq for CD4+ T naive cells isolated from 40 healthy individuals from the Blueprint Project. For these 40 individuals, the genomic and epigenetic data were also available. This dataset offers a unique opportunity to understand how library construction influences differential gene expression, alternative splicing and molecular QTL (quantitative loci) analyses for human primary cells.
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Poli A/genética , RNA Ribossômico/genética , Análise de Sequência de RNA , Linfócitos T/metabolismo , Processamento Alternativo , Epigenômica , Expressão Gênica , Biblioteca Gênica , Genômica , Humanos , Locos de Características QuantitativasRESUMO
Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.
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Internacionalidade , Programas Nacionais de Saúde , Doenças Raras/diagnóstico , Doenças Raras/genética , Sequenciamento Completo do Genoma , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Bases de Dados Factuais , Eritrócitos/metabolismo , Fator de Transcrição GATA1/genética , Humanos , Fenótipo , Locos de Características Quantitativas , Receptores de Trombopoetina/genética , Medicina Estatal , Reino UnidoRESUMO
Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Ibα-dependent platelet 'priming' induces integrin αIIbß3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet αIIbß3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated αIIbß3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil crosstalk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.
Platelets in our blood form clots over sites of injury to stop us from bleeding. Blood clots can also occur in places where they are not needed, such as deep veins in our legs or other regions of the body. Developing such clots also known as deep vein thrombosis (or DVT for short) is one of the most common cardiovascular diseases and a major cause of death. Although certain inherited factors have been linked to DVT, the underlying mechanisms of the disease remain poorly understood. In addition to platelets, the pathological (or dangerous) clots that cause DVT also contain immune cells called neutrophils which fight off bacterial infections. Platelets are recruited to the wall of the vein by a protein called "von Willebrand Factor" (or VWF for short). However, it remained unclear how these recruited platelets interact with neutrophils and whether this promotes the onset of DVT. To answer this question, Constantinescu-Bercu et al. used a device that mimics the flow of blood to study how human platelets change when they are exposed to VWF. This revealed that VWF 'primes' the platelets to interact with neutrophils via a protein called integrin αIIbß3. Further experiments showed that integrin αIIbß3 binds to a protein on the surface of neutrophils called SLC44A2. Once the neutrophils interacted with the 'primed' platelets, they started making traps which increased the size of the blood clot by capturing other blood cells and proteins. Finally, Constantinescu-Bercu et al. studied a genetic variant of the SLC44A2 protein which is found in 22% of people and is associated with a lower risk of developing DVT. This genetic mutation caused SLC44A2 to interact with 'primed' platelets more weakly, which may explain why people with this genetic variant are protected from getting DVT. These findings suggest that blocking the interaction between 'primed' platelets and neutrophils could reduce the risk of DVT. Although current treatments for DVT can prevent patients from forming dangerous blood clots, they can also cause severe bleeding. Since neutrophils are not crucial for normal blood clots to form at the site of injury, drugs targeting SLC44A2 could inhibit inappropriate clotting without causing excess bleeding.
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Armadilhas Extracelulares/fisiologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Neutrófilos/metabolismo , Ativação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose Venosa , Plaquetas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Polimorfismo Genético , Trombose Venosa/genética , Trombose Venosa/metabolismoRESUMO
Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data, we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development, which can be linked to functional maturation of typical end-stage blood neutrophil killing activities.