Publications by authors named "Mattia Frontini"

56 Publications

Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions.

Sci Rep 2021 06 11;11(1):12358. Epub 2021 Jun 11.

Department of Biochemistry, CARIM, Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.

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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http://dx.doi.org/10.1038/s41598-021-91661-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196183PMC
June 2021

Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease.

Nat Commun 2021 04 16;12(1):2298. Epub 2021 Apr 16.

Human Genetics, Wellcome Sanger Institute, Genome Campus, Hinxton, UK.

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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http://dx.doi.org/10.1038/s41467-021-22548-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052402PMC
April 2021

Transcriptional characterization of human megakaryocyte polyploidization and lineage commitment.

J Thromb Haemost 2021 05 29;19(5):1236-1249. Epub 2021 Mar 29.

Department of Hematology, University of Cambridge, Cambridge, UK.

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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http://dx.doi.org/10.1111/jth.15271DOI Listing
May 2021

A rare coding mutation in the MAST2 gene causes venous thrombosis in a French family with unexplained thrombophilia: The Breizh MAST2 Arg89Gln variant.

PLoS Genet 2021 01 19;17(1):e1009284. Epub 2021 Jan 19.

EA3878-GETBO, Univ Brest, Department of internal medicine and chest diseases, FCRIN_INNOVTE, CHU Brest, Brest, France.

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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http://dx.doi.org/10.1371/journal.pgen.1009284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846112PMC
January 2021

Breaking barriers: Quebec platelet disorder.

Authors:
Mattia Frontini

Blood 2020 12;136(23):2603-2604

University of Exeter Medical School.

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http://dx.doi.org/10.1182/blood.2020008213DOI Listing
December 2020

Paired rRNA-depleted and polyA-selected RNA sequencing data and supporting multi-omics data from human T cells.

Sci Data 2020 11 9;7(1):376. Epub 2020 Nov 9.

Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Laboratory Medicine, State Key Laboratory of Biotherapy, West China Second Hospital, Sichuan University, Chengdu, Sichuan, 610041, China.

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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http://dx.doi.org/10.1038/s41597-020-00719-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652884PMC
November 2020

Cell type-specific novel long non-coding RNA and circular RNA in the BLUEPRINT hematopoietic transcriptomes atlas.

Haematologica 2021 10 1;106(10):2613-2623. Epub 2021 Oct 1.

Department of Haematology, University of Cambridge, School of Clinical Medicine.

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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http://dx.doi.org/10.3324/haematol.2019.238147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485671PMC
October 2021

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Blood 2020 10;136(17):1956-1967

Service d'Hématologie Biologique, Hospices Civils de Lyon, Lyon, France.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.
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http://dx.doi.org/10.1182/blood.2019004776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582559PMC
October 2020

Whole-genome sequencing of patients with rare diseases in a national health system.

Nature 2020 07 24;583(7814):96-102. Epub 2020 Jun 24.

Department of Medical Genetics, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK.

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 discovered. 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 participants, 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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http://dx.doi.org/10.1038/s41586-020-2434-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610553PMC
July 2020

Activated αβ on platelets mediates flow-dependent NETosis via SLC44A2.

Elife 2020 04 21;9. Epub 2020 Apr 21.

Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom.

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 αβ activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet αβ to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in (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 αβ and VWF-primed platelets. This was confirmed using neutrophils homozygous for the 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 rs2288904-A polymorphism in venous thrombosis.
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http://dx.doi.org/10.7554/eLife.53353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253179PMC
April 2020

Dynamic Transcriptome-Proteome Correlation Networks Reveal Human Myeloid Differentiation and Neutrophil-Specific Programming.

Cell Rep 2019 11;29(8):2505-2519.e4

Department of Blood Cell Research, Sanquin Research, Amsterdam University Medical Center (AUMC), University of Amsterdam, Amsterdam, the Netherlands; Department of Paediatric Immunology and Infectious Diseases, Emma Children's Hospital, AUMC, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.10.082DOI Listing
November 2019

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

Blood 2019 12;134(23):2070-2081

National Institute for Health Research (NIHR) BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge, United Kingdom.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.
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http://dx.doi.org/10.1182/blood.2019000782DOI Listing
December 2019

High-throughput elucidation of thrombus formation reveals sources of platelet function variability.

Haematologica 2019 06 13;104(6):1256-1267. Epub 2018 Dec 13.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands

In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced αβ activation and secretion. Common sequence variation of and , associated with GPVI-induced αβ activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.
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http://dx.doi.org/10.3324/haematol.2018.198853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545858PMC
June 2019

Sphingolipid dysregulation due to lack of functional KDSR impairs proplatelet formation causing thrombocytopenia.

Haematologica 2019 05 22;104(5):1036-1045. Epub 2018 Nov 22.

NIHR BioResource, Cambridge University Hospitals, Cambridge Biomedical Campus, UK

Sphingolipids are fundamental to membrane trafficking, apoptosis, and cell differentiation and proliferation. KDSR or 3-keto-dihydrosphingosine reductase is an essential enzyme for sphingolipid synthesis, and pathogenic mutations in result in the severe skin disorder Four of the eight reported cases also had thrombocytopenia but the underlying mechanism has remained unexplored. Here we expand upon the phenotypic spectrum of KDSR deficiency with studies in two siblings with novel compound heterozygous variants associated with thrombocytopenia, anemia, and minimal skin involvement. We report a novel phenotype of progressive juvenile myelofibrosis in the propositus, with spontaneous recovery of anemia and thrombocytopenia in the first decade of life. Examination of bone marrow biopsies showed megakaryocyte hyperproliferation and dysplasia. Megakaryocytes obtained by culture of CD34 stem cells confirmed hyperproliferation and showed reduced proplatelet formation. The effect of KDSR insufficiency on the sphingolipid profile was unknown, and was explored and by a broad metabolomics screen that indicated activation of an compensatory pathway that leads to normalization of downstream metabolites such as ceramide. Differentiation of propositus-derived induced pluripotent stem cells to megakaryocytes followed by expression of functional KDSR showed correction of the aberrant cellular and biochemical phenotypes, corroborating the critical role of in proplatelet formation. Finally, Kdsr depletion in zebrafish recapitulated the thrombocytopenia and showed biochemical changes similar to those observed in the affected siblings. These studies support an important role for sphingolipids as regulators of cytoskeletal organization during megakaryopoiesis and proplatelet formation.
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http://dx.doi.org/10.3324/haematol.2018.204784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518879PMC
May 2019

Histone Acetyltransferase KAT2A Stabilizes Pluripotency with Control of Transcriptional Heterogeneity.

Stem Cells 2018 12 17;36(12):1828-1838. Epub 2018 Oct 17.

Department of Haematology, University of Cambridge, Cambridge, United Kingdom.

Cell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity; however, existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin-associated factors that modulate levels of transcriptional noise. Herein, we show destabilization of pluripotency-associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl-transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5), have been inhibited. Functionally, network destabilization associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we show evidence of a relationship between transcriptional heterogeneity and cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells, suggesting a general principle that could be exploited in other normal and malignant stem cell fate transitions. Stem Cells 2018;36:1828-11.
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http://dx.doi.org/10.1002/stem.2919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334525PMC
December 2018

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Cell Rep 2018 09;24(10):2784-2794

National Center for Genomic Analysis (CNAG), Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, Carrer Baldiri i Reixac 4, 08028 Barcelona, Spain.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.
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http://dx.doi.org/10.1016/j.celrep.2018.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326331PMC
September 2018

Chromosome contacts in activated T cells identify autoimmune disease candidate genes.

Genome Biol 2017 09 4;18(1):165. Epub 2017 Sep 4.

Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 0SP, UK.

Background: Autoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4 T cells. Linking such regulatory regions to gene promoters in disease-relevant cell contexts facilitates identification of candidate disease genes.

Results: Within 4 h, activation of CD4 T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases, we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach.

Conclusions: Our systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes.
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http://dx.doi.org/10.1186/s13059-017-1285-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584004PMC
September 2017

Platelet function is modified by common sequence variation in megakaryocyte super enhancers.

Nat Commun 2017 07 13;8:16058. Epub 2017 Jul 13.

Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.
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http://dx.doi.org/10.1038/ncomms16058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511350PMC
July 2017

SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume.

PLoS One 2017 23;12(5):e0178095. Epub 2017 May 23.

Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut, United States of America.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178095PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441597PMC
September 2017

Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations.

Am J Pathol 2017 Jul 11;187(7):1512-1522. Epub 2017 May 11.

School of Biomedical Sciences, University of Western Australia, Crawley, Western Australia, Australia; PathWest Laboratory Medicine, Nedlands, Western Australia, Australia. Electronic address:

Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.
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http://dx.doi.org/10.1016/j.ajpath.2017.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500825PMC
July 2017

Detection of Atherosclerotic Inflammation by Ga-DOTATATE PET Compared to [F]FDG PET Imaging.

J Am Coll Cardiol 2017 Apr;69(14):1774-1791

Division of Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom. Electronic address:

Background: Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([F]FDG PET), [F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover.

Objectives: This study tested the efficacy of gallium-68-labeled DOTATATE (Ga-DOTATATE), a somatostatin receptor subtype-2 (SST)-binding PET tracer, for imaging atherosclerotic inflammation.

Methods: We confirmed Ga-DOTATATE binding in macrophages and excised carotid plaques. Ga-DOTATATE PET imaging was compared to [F]FDG PET imaging in 42 patients with atherosclerosis.

Results: Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific Ga-DOTATATE ligand binding to SST receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBR) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). Ga-DOTATATE mTBR predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [F]FDG mTBR differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [F]FDG spillover rendered coronary [F]FDG scans uninterpretable in 27 patients (64%). Coronary Ga-DOTATATE PET scans were readable in all patients.

Conclusions: We validated Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188).
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http://dx.doi.org/10.1016/j.jacc.2017.01.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381358PMC
April 2017

CSB ablation induced apoptosis is mediated by increased endoplasmic reticulum stress response.

PLoS One 2017 2;12(3):e0172399. Epub 2017 Mar 2.

Unit of Molecular Genetics of Aging-Department of Ecology and Biology-University of Tuscia, Viterbo, Italy.

The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stress-mediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172399PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333825PMC
August 2017

Potency of Human Cardiosphere-Derived Cells from Patients with Ischemic Heart Disease Is Associated with Robust Vascular Supportive Ability.

Stem Cells Transl Med 2017 05 16;6(5):1399-1411. Epub 2017 Feb 16.

Radcliffe Department of Medicine.

Cardiosphere-derived cell (CDC) infusion into damaged myocardium has shown some reparative effect; this could be improved by better selection of patients and cell subtype. CDCs isolated from patients with ischemic heart disease are able to support vessel formation in vitro but this ability varies between patients. The primary aim of our study was to investigate whether the vascular supportive function of CDCs impacts on their therapeutic potential, with the goal of improving patient stratification. A subgroup of patients produced CDCs which did not efficiently support vessel formation (poor supporter CDCs), had reduced levels of proliferation and increased senescence, despite them being isolated in the same manner and having a similar immunophenotype to CDCs able to support vessel formation. In a rodent model of myocardial infarction, poor supporter CDCs had a limited reparative effect when compared to CDCs which had efficiently supported vessel formation in vitro. This work suggests that not all patients provide cells which are suitable for cell therapy. Assessing the vascular supportive function of cells could be used to stratify which patients will truly benefit from cell therapy and those who would be better suited to an allogeneic transplant or regenerative preconditioning of their cells in a precision medicine fashion. This could reduce costs, culture times and improve clinical outcomes and patient prognosis. Stem Cells Translational Medicine 2017;6:1399-1411.
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http://dx.doi.org/10.1002/sctm.16-0229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442720PMC
May 2017

Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types.

Genome Biol 2017 01 26;18(1):18. Epub 2017 Jan 26.

UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6BT, UK.

Background: A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability.

Results: We apply a novel analytical approach to measure and compare transcriptional and epigenetic variability genome-wide across CD14CD16 monocytes, CD66bCD16 neutrophils, and CD4CD45RA naïve T cells from the same 125 healthy individuals. We discover substantially increased variability in neutrophils compared to monocytes and T cells. In neutrophils, genes with hypervariable expression are found to be implicated in key immune pathways and are associated with cellular properties and environmental exposure. We also observe increased sex-specific gene expression differences in neutrophils. Neutrophil-specific DNA methylation hypervariable sites are enriched at dynamic chromatin regions and active enhancers.

Conclusions: Our data highlight the importance of transcriptional and epigenetic variability for the key role of neutrophils as the first responders to inflammatory stimuli. We provide a resource to enable further functional studies into the plasticity of immune cells, which can be accessed from: http://blueprint-dev.bioinfo.cnio.es/WP10/hypervariability .
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http://dx.doi.org/10.1186/s13059-017-1156-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270224PMC
January 2017

Increased DNA methylation variability in type 1 diabetes across three immune effector cell types.

Nat Commun 2016 11 29;7:13555. Epub 2016 Nov 29.

CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 4, 08028 Barcelona, Spain.

The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.
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http://dx.doi.org/10.1038/ncomms13555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141286PMC
November 2016

DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation.

Cell Stem Cell 2016 12 17;19(6):808-822. Epub 2016 Nov 17.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria; Max Planck Institute for Informatics, Saarland Informatics Campus, 66123 Saarbrücken, Germany; Department of Laboratory Medicine, Medical University of Vienna, 1090 Vienna, Austria; Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, 1090 Vienna, Austria. Electronic address:

Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell methylome sequencing to assess cell-to-cell heterogeneity. The resulting dataset identified characteristic differences between HSCs derived from fetal liver, cord blood, bone marrow, and peripheral blood. We also observed lineage-specific DNA methylation between myeloid and lymphoid progenitors, characterized immature multi-lymphoid progenitors, and detected progressive DNA methylation differences in maturing megakaryocytes. We linked these patterns to gene expression, histone modifications, and chromatin accessibility, and we used machine learning to derive a model of human hematopoietic differentiation directly from DNA methylation data. Our results contribute to a better understanding of human hematopoietic stem cell differentiation and provide a framework for studying blood-linked diseases.
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http://dx.doi.org/10.1016/j.stem.2016.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5145815PMC
December 2016

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

Cell 2016 11;167(5):1415-1429.e19

Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK; Department of Statistics, University of Oxford, 1 South Parks Road, Oxford OX1 3TG, UK.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.
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http://dx.doi.org/10.1016/j.cell.2016.10.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300907PMC
November 2016

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Cell 2016 11;167(5):1398-1414.e24

Human Genetics, McGill University, 740 Dr. Penfield, Montreal, QC H3A 0G1, Canada.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14 monocytes, CD16 neutrophils, and naive CD4 T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.
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http://dx.doi.org/10.1016/j.cell.2016.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119954PMC
November 2016

Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters.

Cell 2016 11;167(5):1369-1384.e19

Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK. Electronic address:

Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.
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http://dx.doi.org/10.1016/j.cell.2016.09.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123897PMC
November 2016
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