Publications by authors named "Neil V Morgan"

77 Publications

GoldVariants, a resource for sharing rare genetic variants detected in bleeding, thrombotic, and platelet disorders: Communication from the ISTH SSC Subcommittee on Genomics in Thrombosis and Hemostasis.

J Thromb Haemost 2021 10 5;19(10):2612-2617. Epub 2021 Aug 5.

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium.

The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15459DOI Listing
October 2021

Evidence that autosomal recessive spastic cerebral palsy-1 (CPSQ1) is caused by a missense variant in .

Brain Commun 2021 28;3(1):fcab002. Epub 2021 Jan 28.

Department of Medical Genetics, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge CB2 0QQ, UK.

A subset of individuals diagnosed with cerebral palsy will have an underlying genetic diagnosis. Previously, a missense variant in was described as a candidate mutation in a single family diagnosed with autosomal recessive spastic cerebral palsy-1 (CPSQ1; OMIM 603513). Following the ascertainment of a further branch of the CPSQ1 kindred, we found that the previously reported variant did not segregate with the neurological disease phenotype in the recently ascertained branch of the kindred. Following genetic linkage studies to map autozygous regions and whole-exome sequencing, a missense variant (c.527 T > C; p. Leu176Pro, rs773333490) in the gene was detected and found to segregate with disease status in both branches of the kindred. encodes a 371-amino acid protein (4-Hydroxyphenylpyruvate Dioxygenase Like) that localizes to mitochondria but whose function is uncertain. Recently, biallelic loss of function variants and missense substitution-causing variants in were reported to cause a childhood onset progressive spastic movement disorder with a variable presentation. These findings suggest that related neurological disease may mimic spastic cerebral palsy and that should not be included in diagnostic gene panels for inherited cerebral palsy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/braincomms/fcab002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892364PMC
January 2021

A novel RUNX1 exon 3 - 7 deletion causing a familial platelet disorder.

Platelets 2021 Feb 22:1-4. Epub 2021 Feb 22.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3-7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2021.1887470DOI Listing
February 2021

Heterozygous mutation SLFN14 K208N in mice mediates species-specific differences in platelet and erythroid lineage commitment.

Blood Adv 2021 01;5(2):377-390

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences.

Schlafen 14 (SLFN14) has recently been identified as an endoribonuclease responsible for cleaving RNA to regulate and inhibit protein synthesis. Early studies revealed that members of the SLFN family are capable of altering lineage commitment during T-cell differentiation by using cell-cycle arrest as a means of translational control by RNase activity. SLFN14 has been reported as a novel gene causing an inherited macrothrombocytopenia and bleeding in human patients; however, the role of this endoribonuclease in megakaryopoiesis and thrombopoiesis remains unknown. To investigate this, we report a CRISPR knock-in mouse model of SLFN14 K208N homologous to the K219N mutation observed in our previous patient studies. We used hematological analysis, in vitro and in vivo studies of platelet and erythrocyte function, and analysis of spleen and bone marrow progenitors. Mice homozygous for this mutation do not survive to weaning age, whereas heterozygotes exhibit microcytic erythrocytosis, hemolytic anemia, splenomegaly, and abnormal thrombus formation, as revealed by intravital microscopy, although platelet function and morphology remain unchanged. We also show that there are differences in erythroid progenitors in the spleens and bone marrow of these mice, indicative of an upregulation of erythropoiesis. This SLFN14 mutation presents distinct species-specific phenotypes, with a platelet defect reported in humans and a severe microcytic erythrocytosis in mice. Thus, we conclude that SLFN14 is a key regulator in mammalian hematopoiesis and a species-specific mediator of platelet and erythroid lineage commitment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020002404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839357PMC
January 2021

Post-translational polymodification of β1-tubulin regulates motor protein localisation in platelet production and function.

Haematologica 2020 12 17;Online ahead of print. Epub 2020 Dec 17.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK, B15 2TT.

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which β1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2020.270793DOI Listing
December 2020

Cell-Free DNA in the Investigation of Miscarriage.

J Clin Med 2020 Oct 26;9(11). Epub 2020 Oct 26.

West Midlands Regional Genetics Laboratory, Birmingham Women's and Children's Hospital, Birmingham B15 2TG, UK.

Approximately one in four pregnancies result in pregnancy loss, and ~50% of these miscarriages are caused by chromosomal abnormalities. Genetic investigations are recommended after three consecutive miscarriages on products of conception (POC) tissue. Cell-free DNA (cfDNA) has been utilised for prenatal screening, but very little work has been carried out in nonviable pregnancies. We investigated the use of cfDNA from maternal blood to identify chromosomal abnormalities in miscarriage. One hundred and two blood samples from women experiencing a first trimester miscarriage were collected and stored. The mean gestational age was 7.1 weeks (range: 5-11 weeks). In this research, samples without a genetic test result from POC were not analysed. CfDNA was extracted and analysed using a modified commercial genome-wide non-invasive prenatal test. No results were provided to the patient. In 57 samples, cytogenetic results from POC analysis were available. Chromosomal abnormalities were identified in 47% (27/57) of POC analyses, and cfDNA analysis correctly identified 59% (16/27) of these. In total, 75% (43/57) of results were correctly identified. The average cfDNA fetal fraction was 6% (2-19%). In conclusion, cfDNA can be used to detect chromosomal abnormalities in miscarriages where the 'fetal fraction' is high enough; however, more studies are required to identify variables that can affect the overall results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/jcm9113428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693881PMC
October 2020

Novel gene variants in patients with platelet-based bleeding using combined exome sequencing and RNAseq murine expression data.

J Thromb Haemost 2021 01 28;19(1):262-268. Epub 2020 Oct 28.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Essentials Identifying genetic variants in platelet disorders is challenging due to its heterogenous nature. We combine WES, RNAseq, and python-based bioinformatics to identify novel gene variants. We find novel candidates in patient data by cross-referencing against a murine RNAseq model of thrombopoiesis. This innovative combined bioinformatic approach provides novel data for future research in the field. ABSTRACT: Background The UK Genotyping and Phenotyping of Platelets study has recruited and analyzed 129 patients with suspected heritable bleeding. Previously, 55 individuals had a definitive genetic diagnosis based on whole exome sequencing (WES) and platelet morphological and functional testing. A significant challenge in this field is defining filtering criteria to identify the most likely candidate mutations for diagnosis and further study. Objective Identify candidate gene mutations for the remaining 74 patients with platelet-based bleeding with unknown genetic cause, forming the basis of future re-recruitment and further functional testing and assessment. Methods Using python-based data frame indexing, we first identify and filter all novel and rare variants using a panel of 116 genes known to cause bleeding across the full cohort of WES data. This identified new variants not previously reported in this cohort. We then index the remaining patients, with rare or novel variants in known bleeding genes against a murine RNA sequencing dataset that models proplatelet-forming megakaryocytes. Results Filtering against known genes identified candidate variants in 59 individuals, including novel variants in several known genes. In the remaining cohort of "unknown" patients, indexing against differentially expressed genes revealed candidate gene variants in several novel unreported genes, focusing on 14 patients with a severe clinical presentation. Conclusions We identified candidate mutations in a cohort of patients with no previous genetic diagnosis. This work involves innovative coupling of RNA sequencing and WES to identify candidate variants forming the basis of future study in a significant number of undiagnosed patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jth.15119DOI Listing
January 2021

A comprehensive bioinformatic analysis of 126 patients with an inherited platelet disorder to identify both sequence and copy number genetic variants.

Hum Mutat 2020 11 8;41(11):1848-1865. Epub 2020 Oct 8.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Inherited bleeding disorders (IBDs) comprise an extremely heterogeneous group of diseases that reflect abnormalities of blood vessels, coagulation proteins, and platelets. Previously the UK-GAPP study has used whole-exome sequencing in combination with deep platelet phenotyping to identify pathogenic genetic variants in both known and novel genes in approximately 40% of the patients. To interrogate the remaining "unknown" cohort and improve this detection rate, we employed an IBD-specific gene panel of 119 genes using the Congenica Clinical Interpretation Platform to detect both single-nucleotide variants and copy number variants in 126 patients. In total, 135 different heterozygous variants in genes implicated in bleeding disorders were identified. Of which, 22 were classified pathogenic, 26 likely pathogenic, and the remaining were of uncertain significance. There were marked differences in the number of reported variants in individuals between the four patient groups: platelet count (35), platelet function (43), combined platelet count and function (59), and normal count (17). Additionally, we report three novel copy number variations (CNVs) not previously detected. We show that a combined single-nucleotide variation (SNV)/CNV analysis using the Congenica platform not only improves detection rates for IBDs, suggesting that such an approach can be applied to other genetic disorders where there is a high degree of heterogeneity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.24114DOI Listing
November 2020

Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions.

Blood Adv 2020 07;4(13):2953-2961

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.

The role of glycoprotein VI (GPVI) in platelets was investigated in 3 families bearing an insertion within the GP6 gene that introduces a premature stop codon prior to the transmembrane domain, leading to expression of a truncated protein in the cytoplasm devoid of the transmembrane region. Western blotting and flow cytometry of GP6hom (homozygous) platelets confirmed loss of the full protein. The level of the Fc receptor γ-chain, which associates with GPVI in the membrane, was partially reduced, but expression of other receptors and signaling proteins was not altered. Spreading of platelets on collagen and von Willebrand factor (which supports partial spreading) was abolished in GP6hom platelets, and spreading on uncoated glass was reduced. Anticoagulated whole blood flowed over immobilized collagen or a mixture of von Willebrand factor, laminin, and rhodocytin (noncollagen surface) generated stable platelet aggregates that express phosphatidylserine (PS). Both responses were blocked on the 2 surfaces in GP6hom individuals, but adhesion was not altered. Thrombin generation was partially reduced in GP6hom blood. The frequency of the GP6het (heterozygous) variant in a representative sample of the Chilean population (1212 donors) is 2.9%, indicating that there are ∼4000 GP6hom individuals in Chile. These results demonstrate that GPVI supports aggregation and PS exposure under flow on collagen and noncollagen surfaces, but not adhesion. The retention of adhesion may contribute to the mild bleeding diathesis of GP6hom patients and account for why so few of the estimated 4000 GP6hom individuals in Chile have been identified.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020001761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362345PMC
July 2020

Germline TET2 loss of function causes childhood immunodeficiency and lymphoma.

Blood 2020 08;136(9):1055-1066

Primary Immunodeficiency Group, Newcastle University Translational and Clinical Research Institute, Newcastle upon Tyne, United Kingdom.

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020005844DOI Listing
August 2020

New insights into glycoprotein Ibα desialylation-mediated platelet clearance.

Platelets 2020 07 4;31(5):621-623. Epub 2020 Jun 4.

Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham , Midlands, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2020.1764922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446028PMC
July 2020

An adaptable analysis workflow for characterization of platelet spreading and morphology.

Platelets 2021 Jan 23;32(1):54-58. Epub 2020 Apr 23.

Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham , Midlands, UK.

The assessment of platelet spreading through light microscopy, and the subsequent quantification of parameters such as surface area and circularity, is a key assay for many platelet biologists. Here we present an analysis workflow which robustly segments individual platelets to facilitate the analysis of large numbers of cells while minimizing user bias. Image segmentation is performed by interactive learning and touching platelets are separated with an efficient semi-automated protocol. We also use machine learning methods to robustly automate the classification of platelets into different subtypes. These adaptable and reproducible workflows are made freely available and are implemented using the open-source software KNIME and ilastik.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2020.1748588DOI Listing
January 2021

Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in.

Sci Rep 2019 10 2;9(1):14219. Epub 2019 Oct 2.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK.

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-50733-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775134PMC
October 2019

gene mutations associated with bleeding.

Platelets 2020 4;31(3):407-410. Epub 2019 Aug 4.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2019.1648781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055508PMC
November 2020

Inherited Thrombocytopenia: Update on Genes and Genetic Variants Which may be Associated With Bleeding.

Front Cardiovasc Med 2019 19;6:80. Epub 2019 Jun 19.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Inherited thrombocytopenia (IT) is comprised of a group of hereditary disorders characterized by a reduced platelet count as the main feature, and often with abnormal platelet function, which can subsequently lead to impaired haemostasis. Inherited thrombocytopenia results from genetic mutations in genes implicated in megakaryocyte differentiation and/or platelet formation and clearance. The identification of the underlying causative gene of IT is challenging given the high degree of heterogeneity, but important due to the presence of various clinical presentations and prognosis, where some defects can lead to hematological malignancies. Traditional platelet function tests, clinical manifestations, and hematological parameters allow for an initial diagnosis. However, employing Next-Generation Sequencing (NGS), such as Whole Genome and Whole Exome Sequencing (WES) can be an efficient method for discovering causal genetic variants in both known and novel genes not previously implicated in IT. To date, 40 genes and their mutations have been implicated to cause many different forms of inherited thrombocytopenia. Nevertheless, despite this advancement in the diagnosis of IT, the molecular mechanism underlying IT in some patients remains unexplained. In this review, we will discuss the genetics of thrombocytopenia summarizing the recent advancement in investigation and diagnosis of IT using phenotypic approaches, high-throughput sequencing, targeted gene panels, and bioinformatics tools.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcvm.2019.00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593073PMC
June 2019

Potential genetic causes of miscarriage in euploid pregnancies: a systematic review.

Hum Reprod Update 2019 07;25(4):452-472

Tommy's National Centre for Miscarriage Research, Birmingham Women's and Children's Hospital, Birmingham, UK.

Background: Approximately 50% of pregnancy losses are caused by chromosomal abnormalities, such as aneuploidy. The remainder has an apparent euploid karyotype, but it is plausible that there are cases of pregnancy loss with other genetic aberrations that are not currently routinely detected. Studies investigating the use of exome sequencing and chromosomal microarrays in structurally abnormal pregnancies and developmental disorders have demonstrated their clinical application and/or potential utility in these groups of patients. Similarly, there have been several studies that have sought to identify genes that are potentially causative of, or associated with, spontaneous pregnancy loss, but the evidence has not yet been synthesized.

Objective And Rationale: The objective was to identify studies that have recorded monogenic genetic contributions to pregnancy loss in euploid pregnancies, establish evidence for genetic causes of pregnancy loss, identify the limitations of current evidence, and make recommendations for future studies. This evidence is important in considering additional research into Mendelian causes of pregnancy loss and appropriate genetic investigations for couples experiencing recurrent pregnancy loss.

Search Methods: A systematic review was conducted in MEDLINE (1946 to May 2018) and Embase (1974 to May 2018). The search terms 'spontaneous abortion', 'miscarriage', 'pregnancy loss', or 'lethal' were used to identify pregnancy loss terms. These were combined with search terms to identify the genetic contribution including 'exome', 'human genome', 'sequencing analysis', 'sequencing', 'copy number variation', 'single-nucleotide polymorphism', 'microarray analysis', and 'comparative genomic hybridization'. Studies were limited to pregnancy loss up to 20 weeks in humans and excluded if the genetic content included genes that are not lethal in utero, PGD studies, infertility studies, expression studies, aneuploidy with no recurrence risk, methodologies where there is no clinical relevance, and complex genetic studies. The quality of the studies was assessed using a modified version of the Newcastle-Ottawa scale.

Outcomes: A total of 50 studies were identified and categorized into three themes: whole-exome sequencing studies; copy number variation studies; and other studies related to pregnancy loss including recurrent molar pregnancies, epigenetics, and mitochondrial DNA aberrations. Putatively causative variants were found in a range of genes, including CHRNA1 (cholinergic receptor, nicotinic, alpha polypeptide 1), DYNC2H1 (dynein, cytoplasmic 2, heavy chain 1), and RYR1 (ryanodine receptor 1), which were identified in multiple studies. Copy number variants were also identified to have a causal or associated link with recurrent miscarriage.

Wider Implications: Identification of genes that are causative of or predisposing to pregnancy loss will be of significant individual patient impact with respect to counselling and treatment. In addition, knowledge of specific genes that contribute to pregnancy loss could also be of importance in designing a diagnostic sequencing panel for patients with recurrent pregnancy loss and also in understanding the biological pathways that can cause pregnancy loss.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/humupd/dmz015DOI Listing
July 2019

Investigation of the contribution of an underlying platelet defect in women with unexplained heavy menstrual bleeding.

Platelets 2019 6;30(1):56-65. Epub 2018 Dec 6.

b Institute of Cardiovascular Sciences, College of Medical and Dental Sciences , University of Birmingham , Birmingham , UK.

Heavy menstrual bleeding (HMB) is often undiagnosed in women and can cause discomfort and distress. A haemostatic cause for excessive bleeding is often not routinely investigated and can lead to hysterectomy at an early age. A prospective cohort study was carried out to determine whether certain patients with unexplained HMB have an underlying platelet function defect (PFD). The Genotyping and Phenotyping of Platelets (GAPP) study recruited 175 women with HMB and 44 unrelated volunteers from 25 Haemophilia Centres across the UK, and a tertiary gynaecology service. Bleeding history was assessed using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT). Platelet count, platelet size, haemoglobin and mean corpuscular volume were measured in whole blood using the Sysmex XN-1000 Haematology Analyzer. Platelet function testing using lumiaggregometry and flow cytometry was performed in patients included in this study. A PFD was identified in 47% (82/175) of patients with HMB. Cutaneous bleeding was the most frequent additional bleeding symptom (89% in PFD and 83% with no PFD). Whole blood platelet count was significantly lower (P < 0.0001) between the PFD group and no PFD group. The prevalence of anaemia did not differ between patients and healthy volunteers. Clinical evaluation alone is insufficient to determine presence of an underlying PFD in patients with HMB. Platelet function tests may be considered and clinical guidelines may include them in their algorithms. An appropriate diagnosis and subsequent tailored management of HMB may prevent unnecessary surgery and help manage future haemostatic challenges.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2018.1543865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406209PMC
March 2019

Evaluation of the Total Thrombus-Formation System (T-TAS): application to human and mouse blood analysis.

Platelets 2019 26;30(7):893-900. Epub 2018 Oct 26.

Institute of Inflammation and Ageing, University of Birmingham , Birmingham , UK.

The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyze platelet (PL) or fibrin-rich thrombus formation, respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N = 22) increased with shear within PL (4:40 ± 1.11, 3:25 ± 0.43 and 3:12 ± 0.48 mins [1000, 1500 and 2000s]) and AR chips (3:55 ± 0.42 and 1:49 ± 0.19 [240s and 600s]). The area under the curve (AUC) on the PL chip was also reduced at 1000s compared to 1500/2000s (260 ± 51.7, 317 ± 55.4 and 301 ± 66.2, respectively). In contrast, no differences in the AUC between 240s and 600s were observed in the AR chip (1593 ± 122 and 1591 ± 158). The intra-assay coefficient of variation (CV) (n = 10) in the PL chip (1000s) and AR chip (240s) were T14.1%, T16.7%, T22.8% and AUC24.4% or T 9.03%, T8.64%, T23.8% and AUC5.1%. AR chip thrombus formation was inhibited by rivaroxaban (1 µM), but not with ticagrelor (10 µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n = 30) with normal PL counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a PL function defect. The onset (T) of thrombus formation in WT mice (N = 4) was shorter when compared to humans e.g. PL chip (1000s) T were 02:02 ± 00:23 and 03:30 ± 0:45, respectively). T-TAS measures thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected PL function defects and monitoring PL function within mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2018.1535704DOI Listing
January 2020

A comprehensive targeted next-generation sequencing panel for genetic diagnosis of patients with suspected inherited thrombocytopenia.

Res Pract Thromb Haemost 2018 Oct 8;2(4):640-652. Epub 2018 Oct 8.

Institute of Cardiovascular Sciences College of Medical and Dental Sciences University of Birmingham Birmingham UK.

Background: Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet counts and often disproportionate bleeding with over 30 genes currently implicated. Previously the UK-GAPP study using whole exome sequencing (WES) identified a pathogenic variant in 19 of 47 (40%) patients of which 71% had variants in genes known to cause IT.

Aims: To employ a targeted next-generation sequencing platform to improve efficiency of diagnostic testing and reduce overall costs.

Methods: We have developed an IT-specific gene panel as a pre-screen for patients prior to WES using the Agilent SureSelect transposon-based enrichment system.

Results: Thirty-one patients were analyzed using the panel-based sequencing, of which; 10% (3/31) were identified with a classified pathogenic variant, 16% (5/31) were identified with a likely pathogenic variant, 51% (16/31) were identified with variants of unknown significance, and 23% (7/31) were identified with either no variant or a benign variant.

Discussion And Conclusion: Although requiring further clarification of the impact of the genetic variations, the application of an IT-specific next generation sequencing panel is an viable method of pre-screening patients for variants in known IT-causing genes prior to WES. With an added benefit of distinguishing IT from idiopathic thrombocytopenic purpura (ITP) and the potential to identify variants in genes known to have a predisposition to hematological malignancies, it could become a critical step in improving patient clinical management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rth2.12151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178765PMC
October 2018

Mutation in is associated with severe congenital thrombocytopenia.

Blood 2018 10 25;132(17):1855-1858. Epub 2018 Jun 25.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2018-04-847798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238157PMC
October 2018

Role of the novel endoribonuclease SLFN14 and its disease-causing mutations in ribosomal degradation.

RNA 2018 07 20;24(7):939-949. Epub 2018 Apr 20.

Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, New York 11203, USA.

Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by a low platelet count and are frequently associated with excessive bleeding. is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all variants colocalize with ribosomes and mediate rRNA endonucleolytic degradation. Compared to SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients' platelets. Overall, we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.066415.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004054PMC
July 2018

Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders.

Haematologica 2018 01 5;103(1):148-162. Epub 2017 Oct 5.

Servicio de Hematología, Hospital Universitario de Salamanca-IBSAL-USAL, Spain

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in (n=2) and (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2017.171132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777202PMC
January 2018

CRISPR-Cas9 Mediated Labelling Allows for Single Molecule Imaging and Resolution.

Sci Rep 2017 08 16;7(1):8450. Epub 2017 Aug 16.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Single molecule imaging approaches like dSTORM and PALM resolve structures at 10-20 nm, and allow for unique insights into protein stoichiometry and spatial relationships. However, key obstacles remain in developing highly accurate quantitative single molecule approaches. The genomic tagging of PALM fluorophores through CRISPR-Cas9 offers an excellent opportunity for generating stable cell lines expressing a defined single molecule probe at endogenous levels, without the biological disruption and variability inherent to transfection. A fundamental question is whether these comparatively low levels of expression can successfully satisfy the stringent labelling demands of super-resolution SMLM. Here we apply CRISPR-Cas9 gene editing to tag a cytoskeletal protein (α-tubulin) and demonstrate a relationship between expression level and the subsequent quality of PALM imaging, and that spatial resolutions comparable to dSTORM can be achieved with CRISPR-PALM. Our approach shows a relationship between choice of tag and the total expression of labelled protein, which has important implications for the development of future PALM tags. CRISPR-PALM allows for nanoscopic spatial resolution and the unique quantitative benefits of single molecule localization microscopy through endogenous expression, as well as the capacity for super-resolved live cell imaging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-08493-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559501PMC
August 2017

Schlafen 14 (SLFN14) is a novel antiviral factor involved in the control of viral replication.

Immunobiology 2017 11 11;222(11):979-988. Epub 2017 Jul 11.

Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea. Electronic address:

Schlafen (SLFN) proteins have been suggested to play important functions in cell proliferation and immune cell development. In this study, we determined the antiviral activities of putative RNA-helicase domain-containing SLFN14. Murine SLFN14 expression was specifically induced by TLR3-mediated pathways and type I interferon (IFN) in RAW264.7 mouse macrophages. To examine the role of SLFN during viral infection, cells were infected with either wild-type PR8 or delNS1/PR8 virus. SLFN14 expression was specifically induced following influenza virus infection. Overexpression of SLFN14 in A549 cells reduced viral replication, whereas knockdown of SLFN14 in RAW264.7 cells enhanced viral titers. Furthermore, SLFN14 promoted the delay in viral NP translocation from cytoplasm to nucleus and enhanced RIG-I-mediated IFN-β signaling. In addition, SLFN14 overexpression promoted antiviral activity against varicella zoster virus (VZV), a DNA virus. In conclusion, our data suggest that SLFN14 is a novel antiviral factor for both DNA and RNA viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.imbio.2017.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990420PMC
November 2017

Gene of the issue: RUNX1 mutations and inherited bleeding.

Platelets 2017 03 17;28(2):208-210. Epub 2017 Feb 17.

b Department of Infection , Immunity and Cardiovascular Disease, University of Sheffield , Sheffield , UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2017.1280151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359732PMC
March 2017

Whole exome sequencing identifies a mutation in thrombomodulin as the genetic cause of a suspected platelet disorder in a family with normal platelet function.

Platelets 2017 Sep 7;28(6):611-613. Epub 2017 Mar 7.

a Institute of Cardiovascular Sciences, College of Medical and Dental Sciences , University of Birmingham , Birmingham , UK.

Here, we describe a mother and son with a lifelong bleeding tendency and posttraumatic bleeding who were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study with a suspected platelet function disorder. However, despite a clinically significant bleeding score, both had normal platelet counts and normal platelet function. The patients' blood was analyzed by light transmission aggregometry and genotyping by whole exome sequencing, as outlined by the GAPP study. Approximately 25 000 genetic variants were found for each patient as a result of sequencing and were filtered using a specialized bioinformatics pipeline. A heterozygous variant displaying autosomal dominant inheritance (c.1611 C>A) was found in the gene THBD which encodes the glycoprotein thrombomodulin. This sequence change results in a stop codon (p.Cys537Stop) and truncation of the protein and has been previously described in two other families with bleeding events which suggests it may be a recurrent mutation. In summary, this study shows that patients with a suspected platelet disorder but who present with a normal pattern of platelet aggregation should be investigated for defects in nonplatelet genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2017.1283011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646180PMC
September 2017

ISTH Advanced Training Course on platelet bleeding disorders: How should they be investigated?.

Platelets 2016 12 14;27(8):719-721. Epub 2016 Nov 14.

a Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham , Birmingham , UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09537104.2016.1256726DOI Listing
December 2016
-->