NIHRDataInsights/HRCSData huggingface dataset:
Title: Understanding granule disorders of myeloid cells by unravelling the interactome and function of the Nbeal2 protein
Funder: Medical Research Council

Abstract: The regulation of myeloid cell secretory granules has a key role in atherogenesis and thrombosis. Understanding granule function could lead to new treatments for haemostatic and cardiovascular diseases. I will focus on NBEAL2 variants, which cause Gray Platelet Syndrome (GPS), a rare bleeding disorder. Molecular studies on cells from patients and knockout mice show Nbeal2 is key to PLT alpha-granule and NEU specific granule regulation and function. I will use iPSC technology and CRISPR genome editing to elucidate the mechanism by which NBEAL2 variants cause granule pathology. Preliminary proteomics studies of iMKs have identified 64 proteins interacting with Nbeal2. Hypothesis 1: GPS variants cause loss of function of one/several interactions with Nbeal2 I will introduce by CRISPR-editing the codons for GPS-causing BEACH-domain variants M2080K, P2100L and G2290W to generate mutants of the line IPSC-S4_NBEAL2_TAP for forward programming to iMKs in biological triplicates (Ghevaert lab protocol; transcription factors GATA-1, FLI-1, TAL-1; tetracycline inducible expression). I will characterise iMKs by ATAC- and RNA- seq, confocal and electron microscopy, platelet formation/function assays and perform pulldowns. Nbeal2's interactions will be defined through proteomics analysis by mass spectrometry (Lamond laboratory) and loss-of-interaction(s) will be confirmed by bi-directional immunoblots, co-localisation and proximity ligation assays. Hypothesis 2: Proteins in the Nbeal2 interactome have increased likelihood of being candidates for inherited blood cell disorders I will apply a new Bayesian algorithm to analyse genome sequencing data for ~750 unexplained BPD cases to determine if the 64 genes encoding Nbeal2's interactors are enriched for rare, possibly causal variants. I will verify putative candidate variants using control databases, detailed clinical phenotyping and co-segregation of pedigrees and functional studies using a CRISPR-knockout iPSC line.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: IMPC: Analysis of the role of Cx57 in the regulation of platelet function, haemostasis and thrombosis
Funder: Medical Research Council

Abstract: Platelets are small cells that control blood clotting. When they encounter cuts or damage to blood vessels they rapidly become sticky and stick to each other. This forms a plug, the first stage of clot formation that stops the bleeding as quickly as possible. Blood vessels become abnormal in cardiovascular disease and platelets can mistake these diseased vessels for cut vessels and consequently trigger the blood to form clots inside blood vessels (this is also known as thrombosis). This can clog up arteries and is the main trigger for heart attacks and strokes, which are frequently fatal. Scientists' successes in identifying how platelets sense healthy and damaged blood vessels, and also communicate with each other, has been critical in developing more effective drugs to prevent heart attacks and strokes, although such cardiovascular diseases still represent the principal causes of death in the UK. We are therefore focussing on increasing our understanding of the molecules and processes that enable platelets to be controlled in order to develop more effective medicines. We have recently found a new protein called Cx57 on platelets and have produced considerable data to show that this is likely to contribute to thrombosis. In order to confirm this we need to study platelets that do not have this protein. In this project we will use mice that have been genetically engineered to lack Cx57. We will test what effect this has on platelet function, bleeding and thrombosis. This will form an important step to determine whether drugs that target this protein may be successful in preventing thrombosis and the other diseases in which platelets are involved.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.2 PSYCHOLOGICAL AND SOCIOECONOMIC PROCESSES
Health categories: BLOOD
Title: IMPC: Phenotyping Cardiovascular Development of Rbms1-null mice
Funder: Medical Research Council

Abstract: The therapeutic stimulation of new blood vessel growth is considered a promising treatment for diseases in which organs become oxygen starved, for example after a heart attack or stroke and in diabetic vascular eye disease. Moreover, blood vessel function is thought to contribute to neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. However, we still have large gaps in our knowledge of how blood vessels normally grow to support the function of the brain, heart and other organs. Moreover, the first therapeutic attempts to stimulate vessel growth to alleviate oxygen starvation were ineffective, because the new vessels were not properly embedded into the organs they should supply. Based on prior work, we now know that two molecules called NRP1 and ITGB1 are essential to guide newly formed blood vessels to the right places in many organs. However, both molecules can also promote the formation of abnormal blood vessels and thereby exacerbate vascular problems in some diseases. Moreover, it is difficult to manipulate these molecules therapeutically, because they also participate in several other essential processes in the body. It is therefore important to identify additional molecules that function together with NRP1 and ITGB1 in blood vessel growth and may provide more selective targets for therapy. Our pilot studies suggest that a molecule termed RBMS1 functions together with NRP1 and ITGB1 in signalling pathways that support blood vessel growth. Moreover, mice lacking RBMS1 die before birth, demonstrating that its normal function is very important. However, very little is still known on the role of RBMS1 in normal cells, how it may support embryonic development and whether it plays a role in blood vessel growth by interacting with NRP1 and ITGB1 or in other pathways. We will therefore establish when during development RBMS1 deficiency causes death, determine which organ systems are affected and examine whether blood vessel growth is defective when RBMS1 is missing.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: BLOOD
Title: Role of regulatory elements in the genetic determination of VWF level in Von Willebrand disease and healthy individuals
Funder: Medical Research Council

Abstract: In the UK there are ~10,000 VWD patients and they mainly present with mucocutaneous bleeding. The ThromboGenomics (TG) high throughput sequencing (HTS) test, introduced as a diagnostic test in the NHS in 2015 identifies causal variants in VWF exons. There is an assumption, partially supported by TG test results that a fraction of VWF variants, labelled in reference databases as LPV and PV have been erroneously ascribed their label. Therefore, the fraction of type 1 VWD patients in whom a conclusive molecular diagnosis can be reached may be lower than the estimated 75%. I will use HTS to analyse the 0.5 MB comprising the VWF-CD9 genes in 1,000 VWD patients to identify LPVs in non-coding elements of the locus through comparison with data from 10,000 WGS-analysed controls from the INTERVAL RCT. I will perform a fine-mapping analysis to associate variants in the 0.5 MB and VWF levels. I will then use a new Bayesian algorithm to search for rare variants enriched in the 1,000 VWD patients using 42,000 INTERVAL and 100KGP controls. Erroneous assignment of pathogenicity status to variants will be minimised by having access to NHS record data of the WGS controls, in contrast to the anonymised whole exome sequenced (WES) samples from the ExAC Consortium. There is a considerable chance that a DNA variant present in a regulatory element of VWF and observed in a few or a single VWD patient(s) but absent from the 42,000 controls is not causal of VWD. So putative causal DNA variants will be verified for co-segregation in pedigrees. Those that co-segregate will be selected for functional validation. The variant will be introduced in iPSCs by CRISPR-Cas9. Wild-type and edited iPSCs will be forward programmed into ECs and MKs, in which I will measure VWF transcript and VWF protein levels. The genome-editing is relatively simple for insertions/deletions but challenging for single nucleotide variants. So as a back-up approach iPSC lines will be generated from the relevant patients.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: Development of highly sensitive methods for defining off target mutations to enable safe gene editing of haematopoietic cells for transplantation
Funder: Medical Research Council

Abstract: Sites of off target CRISPR-Cas9 nuclease activity will initially be defined in purified genomic DNA. Digenome-seq shows that RGEN cleavage in vitro is a good predictor of off target sites but it uses whole genome sequencing to identify cut sites, making it prohibitively expensive and insensitive. The novel method involves ligating a customized sequencing adaptor to RGEN cut sites to allow them to be specifically sequenced. I have previously developed oligonucleotide capture technology for extremely deep sequencing (>100,000 fold coverage). Biotinylated oligonucleotides will be designed to pull down all of the potential sites of Cas9 cleavage identified in vitro and these will then be used to define with high sensitivity whether off target mutations occur during in vivo editing of primary cells. In addition oligonucleotides will be designed to the viral vector and the sequences to be inserted by HDR so that off target sits of integration can be identified. I plan to investigate whether a chromosome conformation capture based method (Capture-C) I pioneered, can improve the sensitivity of the assay detect to sites of off target integration. Off target effects will be studied in two models of genome editing for the treatment of thalassaemia and sickle cell disease. The first model has been developed in the host laboratory to treat HbE beta thalassemia and uses a single CRISPR-Cas9 cut and the non homologous end joining pathway to delete key transcription factor binding sites at the main enhancer of the alpha globin gene. A previously published method will also be set up, which uses homologous recombination to correct the beta globin gene in situ and simultaneously insert a cell surface marker for purification of corrected cells. This uses an adeno-associated virus (AAV) 6 vector to deliver the template for HDR and I plan to use a second AAV-6 vector to deliver Cas9 controlled by an inducible promoter to study how Cas9 expression alters off target effects.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: BLOOD
Title: Molecular mechanism by which the E325K mutation of human KLF1 causes a severe dyserythropoietic anemia, utilising a novel model system of RBC disease
Funder: Medical Research Council

Abstract: KLF1 is an erythroid specific transcription factor essential for erythropoiesis. In recent years the number of individuals identified with disease phenotypes mapped to KLF1 mutations has increased rapidly. Of these the most severe, a form of dyserythropoietic anemia, is causally linked with the monoallelic mutation E325K; the effect of this mutation more severe than monoallelic loss of function KLF1 mutations. However, how the mutation affects the function of KLF1 in vivo resulting in a dominant phenotype is not known. Studying the molecular defects behind many RBC diseases is severely impeded by paucity of suitable and adequate quantities of material from anemic patients. We have recently developed technology and generated the first human immortalized adult erythroid lines, which recapitulate erythropoiesis, and provide a sustainable supply of cells. We have also developed a platform for CRISPR-Cas9 genome editing of these lines. We will use these technologies to create model disease cellular systems, generating a line from a patient with the E325K mutation, and introducing the mutation into an existing line. These systems will be used to (i) obtain a comprehensive map of the complete repertoire of proteins aberrantly expressed in cells expressing E325K KLF1 by comparative proteomics (ii) delineate molecular mechanisms by which the E325K mutation results in disrupted gene regulation, specifically determining if E325K KLF1 exhibits altered DNA binding specificity in vivo and if it interferes with the occupancy rate of wild type KLF1 at gene regulatory regions by ChIP-seq, and how E325K KLF1 then distorts the genetic readout of cells via analysis of chromatin configuration at gene loci required for interaction of regulatory elements and thus regulated gene expression using Capture-C 3C (iii) whether the mutation perturbs KLF1-co-factor binding required for such interactions. The data will also provide further insight into KLF1-directed transcriptional control.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: How do mutations in non-muscle myosin 2A cause bleeding disorders and other defects?
Funder: Medical Research Council

Abstract: Over 80 mutations in MYH9 have been described (Human Genome Mutation Database) that collectively cause an autosomal-dominant disorder known as MYH9 disease. MYH9 encodes non-muscle myosin 2A (NM2A). In humans, the outcomes of this disease range from mild to life-threatening. Symptoms include clotting disorders in which platelets are enlarged and bleeding time is prolonged (thrombocytopaenias), aggregation of NM2A in neutrophils, cataracts, deafness and glomerulosclerosis (which can be lethal). In platelets and other non-muscle cells, NM2A is found both as an inactive compact folded molecule, known as '10S' (based on its sedimentation coefficient), and as an active extended molecule (6S) which forms filaments of 20-30 molecules. In the 10S form, the coiled coil tail wraps around one of the motor domains, and this motor-tail interaction is important in stabilising the inactive state. Plotting the mutations onto a model of the folded molecule, together with our preliminary data, suggest that a significant number of mutations are likely to interfere with this interaction, destabilising the inactive state, which would result in premature filament formation, affecting cell contractility, and thus explain why these mutations result in MYH9 disorders. Our goal is to test this hypothesis by a combination of approaches, from investigating the effects of mutations on filament formation in cells, to investigating the effects of mutations on the secondary structure of the coiled coil, and, by using cryo-EM to obtain a detailed high resolution structure of the folded molecule, to precisely define how the tail interacts with the motor, and thus understand how mutations could disrupt this interaction.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD
Title: Evaluation of the role of ARAP1 in platelet function and thrombosis
Funder: Medical Research Council

Abstract: Platelets are small cells in the blood that are essential for hemostasis, but can also contribute to thrombosis and cardiovascular disease when activated inappropriately. We have previously shown that platelet hyperactivity can contribute to resistance to anti-platelet therapy, potentially increasing the risk of stroke and myocardial infarction in patients with cardiovascular disease receiving anti platelet therapy. One of the main intracellular signalling kinases that contributes to platelet hyperactivity is the lipid kinase PI3kinase, which phosphorylates lipids such as PI(4,5)P2 in the membrane resulting in the generation of PI(3,4,5)P3. The mechanism by which PI3 kinase increases platelet function is still largely unknown but it likely to be mediated though binding of proteins to PI(3,4,5)P3. We recently showed that one of the proteins that binds to PI(3,4,5)P3, and therefore may contribute to platelet hyperactivity, is the Arf and Rho GTPase activating protein ARAP1. Human platelets express relatively high levels of ARAP1 compared to other tissues and ARAP1 is the only ARAP expressed in platelets. Indeed, we confirmed ARAP1 expression in human and mouse platelets, and show that ARAP1 is tyrosine phosphorylated upon platelet activation in a manner that is highly dependent on secondary signalling. At present the regulation and function of ARAP1 in platelets is completely unknown and this application therefore aims to obtain preliminary data using platelets that lack ARAP1 to demonstrate a role for ARAP1 in platelet function and thrombosis. This study will provide more insight in the underlying cause of platelet hyperactivity and potentially identify a novel target to prevent heart attacks and strokes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: BLOOD
Title: IDENTIFICATION OF A NOVEL ANGIOPOIETIN 2 SIGNALLING CENTRE IN THE EMBRYONIC HAEMATOPOIETIC STEM CELL NICHE
Funder: Medical Research Council

Abstract: Transplantations of haematopoietic stem cells (HSC) have been used in the clinic for several decades to treat various blood disorders. However, a significant shortage of donors for some patient groups means there is urgent need to find alternative sources of HSCs. A key goal of current haematology is the search for methods to efficiently produce HSCs in the laboratory. Pluripotent stem cells hold great hope for regenerative medicine, but generating HSCs from them remains a significant challenge, mainly since our current understanding of the mechanisms driving HSC development, particularly in human, is still extremely limited. Understanding how HSCs form in the embryo will be crucial in defining efficient protocols to generate HSCs in vitro. Our recent global transcriptome analysis revealed a novel signaling centre, Ang2, in the mouse aorta-gonad-mesonephros (AGM) region which we found is a strong stimulator of HSC development. We also observed a similar expression pattern in the human AGM region, suggestive of a previously undescribed important HSC regulatory centre. We propose a detailed investigation into the role of Ang2 signaling in human and mouse HSC development, using a combination of cutting-edge techniques including gene expression profiling of sorted cells, confocal immunophenotyping and loss/gain-of-function experiments followed by functional assays. To explore potential links with intra-aortic clusters in which HSCs form, we will use novel image-based spatial correlation analysis to create the first 3D models of the Ang2 signalling centre in the mouse and human AGM region. These and associated datasets will be a valuable resource for the research field to inform further functional studies. We will test whether Angpt2 can enhance haematopoietic differentiation of mouse and human ES cells. In the long term this could help development of a protocol for generating bone fide HSCs from pluripotent cell sources to meet clinical demands.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: BLOOD
Title: Non-invasive assessment and management of coronary heart disease - a translational, data driven approach
Funder: Medical Research Council

Abstract: The aim of the proposed research is to test and improve computational FFR software which can, using non-invasive patient measurements, determine the severity of a coronary artery stenosis. The improvements to the software would include the introduction of more automated, or semi-automated processes. By the end of the project, non-computational fluid dynamic specialists (possibly clinicians) would be able to use the software. Furthermore, machine learning will be performed on a data set of approximately 500 patients. The software, which has been developed in Swansea University, involves the segmentation of patient CT scans, after which reduced-order computational fluid dynamics is performed; this can be used to determine the pressure drop (FFR value) caused by the stenosis in the coronary arteries; and will be compared with the clinically measured FFR value for the patient to test the diagnostic accuracy of the software. Anonymised patient data, which includes CT scans and basic patient information (pressures, body mass index), can be collected through an existing agreement with Derriford hospital in Plymouth. Approximately 500 data sets will be collected for the project. Machine learning will be applied to the data set to determine if any underlying patterns can be found, which could lead to improved calculation of a patient's FFR value. In addition to valuable patient data, the opportunities of such a software could be widespread. The software could improve the patient treatment planning pathway. The methodology used only requires non-invasive patient measurements and imaging techniques. This could potentially reduce the cost of patient diagnosis; it would also improve patient experience, by avoiding invasive catheterisation procedures, and hence would not require patient recovery time. There is also a significant commercial opportunity of such a software.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: BLOOD
Title: A protein-transcriptome atlas of haematopoiesis across the human life span
Funder: Medical Research Council

Abstract: The recent development of Cite-Seq technology coupled with the commercialization of dedicated antibody and droplet-based sequencing reagents represent a golden opportunity to generate a comprehensive Cite-Seq dataset to complement ongoing projects across the Human Cell Atlas Initiative. We have assembled a team of PIs with expert knowledge of human fetal and adult haematopoiesis as well as single cell molecular profiling techniques, complemented with expert collaborators with specialist knowledge of experimental and computational aspects of Cite-Seq technology. Cite-Seq technology enables the joint profiling of mRNA and protein from the same single cells using high-throughput next generation sequencing technology. The specific aims of our proposal are to (i) use Cite-Seq to generate combined protein and transcriptome profiles for over 550,000 haematopoietic progenitor and mature cells across the human lifespan, and (ii) to integrate the resulting datasets into the Human Cell Atlas data platform and establish pipelines for single cell multi-omics analysis. Subfractionation based on CD34+ and CD34- cell subsets will provide the progenitor and mature populations respectively. Samples will be obtained from fetal liver, yolk sac, and bone marrow, newborn cord blood, adult bone marrow and spleen and aged adult bone marrow. Sample acquisition will occur under already existing ethics permissions, thus ensuring that the project can start without any delay. The proposed Cite-Seq datasets for haematopoietic progenitor and mature cells across the human lifespan will provide a multiomic resource highly complementary to currently ongoing HCA projects. Development of data analysis and data integration pipelines will furthermore provide important guidance for future adaptation of the Cite-Seq method not just to projects mapping other tissues, but also to investigators studying blood and/or immune cell disorders.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD