Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

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 SureSelectQXT 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.

Recent years have seen increasing recognition of a subgroup of inherited platelet function disorders which are due to defects in transcription factors that are required to regulate megakaryopoiesis and platelet production. Thus, germline mutations in the genes encoding the haematopoietic transcription factors RUNX1, GATA-1, FLI1, GFI1b and ETV6 have been associated with both quantitative and qualitative platelet abnormalities, and variable bleeding symptoms in the affected patients. Some of the transcription factor defects are also associated with an increased predisposition to haematologic malignancies (RUNX1, ETV6), abnormal erythropoiesis (GATA-1, GFI1b, ETV6) and immune dysfunction (FLI1). The persistence of MYH10 expression in platelets is a surrogate marker for FLI1 and RUNX1 defects. Characterisation of the transcription factor defects that give rise to platelet function disorders, and of the genes that are differentially regulated as a result, are yielding insights into the roles of these genes in platelet formation and function.

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) (OMIM #601399) is an autosomal dominant disorder characterized by quantitative and qualitative platelet defects and an increased risk of AML. FPD/AML shares phenotypic similarities with Jacobsen syndrome; platelet counts show mild to moderate reductions but are variable between individuals with the same genetic etiology of disease, and a reduction in dense granule secretion is often observed as a secondary qualitative abnormality [1]. The major clinical complication of this disorder, however, is not the bleeding tendency experienced by some patients, but the propensity for a proportion of patients to develop myelodysplasia or leukemia [2]. The molecular genetic cause of FPD/AML was first elucidated by linkage studies which mapped the underlying genetic defect to a region on human chromosome 21q [3]. Contained within this region is the gene encoding the master regulator of hematopoiesis, Runt-related transcription factor 1 (RUNX1). Variants have been identified throughout the coding region of RUNX1 but those clustered within the region encoding the Runt homology domain (RHD), which mediates DNA binding and heterodimerization with core binding factor beta (CBF-β) [4], and are most likely to be detrimental [5]. RUNX1 mutation can result in haploinsufficiency of RUNX1, or reduced RUNX1 function as a result of a dominant-negative effect, that disrupts the formation of complexes with CBF-β, thereby disturbing the regulation of genes necessary for hematopoietic stem cell (HSC) maintenance, maturation, and differentiation [6,7]. Over 40 RUNX1 mutations associated with FPD/AML have been reported in patients to date (Table I, Figure 1). However, the prevalence of RUNX1 defects is believed to be underestimated and as sequencing technologies improve an increasing number of patients are being reported [8,9]. The mutations reported are predominantly missense and phenotypically platelets from patients present with dense granule secretion defects and persistence of MYH10 expression which can be used as a biomarker of genetic variation [1,10]. It has been suggested that the risk of malignancy is reduced in those cases having RUNX1 defects that cause haploinsufficiency when compared to those patients with dominant-negative RUNX1 defects. Due to the associated predisposition to myeloid malignancy with some variants in RUNX1, it is critical to establish diagnosis as early as possible to aid in patient management and guidance.

https://doi.org/10.1160/TH16-10-0791 Thromb Haemost 2017; 117: 642–643 Dear Sirs, We were interested to read the paper by Navarro-Fernández et al. regarding antithrombin Dublin (p.Val30Glu) and its capacity to cause transient antithrombin deficiency (1). Antithrombin Dublin was first described by one of us in 1987 when it was identified as part of a study that aimed to determine the effects of asparaginase therapy on the different plasma isoforms of antithrombin that can be distinguished by isoelectric focusing. The Dublin variant was found to be more negatively charged when compared with normal antithrombin (2). Sequencing of SERPINC1 encoding antithrombin subsequently revealed a single nucleotide variation predicting a p.Val30Glu substitution in the signal peptide of antithrombin which redirects signal peptidase cleavage to a site two amino acids into the mature protein and results in removal of the N-terminal dipeptide from antithrombin (3). Of note, while none of the heterozygous carriers of the Dublin variant identified in the original study had a history of thrombosis at the time of investigation, in our subsequent study we reported the Dublin variant in a patient with a history of spontaneous deep vein thrombosis and pulmonary embolus at age 41 and the presence of anti-IIa and anti-Xa activity levels at the lower end of the normal range (2, 3). Its variable association with thrombosis and the observation that the Dublin variant, denoted rs2227624, is relatively prevalent in the European population (MAF 0.0017 in the ExAC database (http://exac.broadinstitute.org/variant/1–173884010-A-T) have meant that its clinical significance has remained elusive. Navarro-Fernández et al. (1) observed that recombinant antithrombin Dublin adopted a hyperstable conformation of reduced activity when it was expressed in HEK cells. This led them to speculate that the p.Val30Glu mutation facilitates the formation of disulphide-linked polymers of antithrombin, similar to those reported for antithrombins Wibble and Rouen VI, two variants caused by mutations that facilitate the conversion of antithrombin to its latent form and the development of antithrombin deficiency under conformational stress conditions such as pyrexia (4, 5). However, they did not identify the factors that might have triggered the conformation stress conditions in the symptomatic carriers of the Dublin variant in their study. We recently identified antithrombin Dublin in a patient who developed a pulmonary embolism during pregnancy. Her thrombin-based antithrombin levels (Berichrom, Sysmex UK) on the ACL TOP analyser (Werfen UK) were initially reduced (0.72, 0.69 and 0.73 IU/ml) but increased and indeed normalised after delivery (1.04, 0.92 and 0.86 IU/ml). Antithrombin antigen (ELISA) agreed well with antithrombin activity levels during pregnancy (0.75, 0.76, 0.74 IU/ml) and after delivery (0.98 IU/ml). Our adult reference ranges for antithrombin activity and antigen are 0.85–1.31 IU/ml and 0.83–1.24 IU/ml, respectively, and our antithrombin activity inter-assay coefficient of variation (CV) is 2.5 % at an antithrombin level of 0.92 IU/ml (95 % limits 0.87–0.96 IU/ml, n=83). The patient did not have an increased body temperature or fever at the time of her thrombotic event, leading us to speculate that the classical triggering factor of pregnancy may have predisposed her to the development of transient antithrombin deficiency that may be associated with the Dublin variant, though other possible explanations for variations in antithrombin level, including pre-analytical variables, cannot be excluded. Interestingly, we noticed that the patient had specimens with poor anti-Xa duplicate assay levels (Coamatic assay, Quadratech UK, on Sysmex CS-5100 analyser) in response to low-molecular-weight heparin (LMWH) treatment, in two samples during the pregnancy, duplicates were 0.29 and 0.65 IU/ml and in another sample, 0.33 and 0.59 IU/ml. These two pregnancy samples were assayed for factor Xa-based antithrombin level (Innovance, Sysmex) and results were borderline at 0.85 and 0.81 IU/ml (adult reference range 0.85–1.15 IU/ml) and higher than the thrombin-based assays (0.72 and 0.69 IU/ml). Her samples, when tested repeatedly, yielded a CV of approximately 20 % in comparison to a control sample with CV of 10 %. The reasons for the unexpected poor precision in the patient’s anti-Xa levels, and the higher Xa-based antithrombin are unexplained, but it would be interesting to know if LMWHs affect antithrombin levels in carriers of the antithrombin Dublin variant, and if so, to what extent. Whilst our observations above are consistent with the findings of NavarroFernández et al. (1), we would like to add a word of caution regarding measurement of antithrombin levels. A number of different assays for antithrombin activity are in use by different laboratories, and this can contribute to variation in results. The majority of laboratories will only employ one assay for antithrombin activity, and some defects in the antithrombin gene have been shown to give markedly different results with different assays (6). In the authors’ laboratory, at an antithrombin level of 90 IU/dl, the interassay coefficient of variation for bovinethrombin-based and FXa-based chromogenic antithrombin assays is approximately 3 %, and at a level of 50 IU/dl it is approximately 4 %. Recent EQA data (UK NEQAS, unpublished) demonstrate generally good agreement between antithrombin assay results in different laboratories, with CVs of between 5 and 8 %, and comparable results for different assays using either bovine

Introduction: Inherited macrothrombocytopenia represents a heterogeneous group of disorders which are characterized by the presence of a reduced number of abnormally large platelets in the circulation, which may or may not be associated with a bleeding tendency. In spite of several causative genes having been identified, the underlying genetic defects remain to be identified in approximately half of the cases. Aims: To understand the molecular pathology of isolated giant platelet disorder from India. Materials and methods: We studied 112 cases that were referred for investigation of macrothrombocytopenia. Agonist induced platelet aggregation and platelet GP1b/IX/V receptor expression were investigated to assess GP1b/IX/V receptor expression and the GP1BA, GP1BB, GP9, ABCG5, ABCG8, TUBB1 and MYH9 genes were analysed to identify candidate gene defects. Results: Twenty-three candidate gene defects were identified in 48 of 112 cases, 20 of which were novel. Of the candidate defects identified, 91% were missense and 9% were nonsense variations. The missense variations were in GP9 (9), ABCG5 (4), GP1BB (3), GP1BA (3) and MYH9 (2), while the nonsense defects occurred in MYH9 (1) and GP1BA (1). Conclusions: This study increases the understanding of the molecular basis of an isolated giant platelet disorder, a common heterogeneous condition prevalent in north and eastern India.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK, Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK, Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK, Haemophilia Comprehensive Care Centre, University Hospital Birmingham, Edgbaston, Birmingham, UK, Program in Genetics & Genomic Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada, Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia 30003, Spain, and The William Harvey Research Institute, Barts & The London School of Medicine & Dentistry, Queen Mary University of London, London, UK

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified “pathogenic” or “likely pathogenic” variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

BackgroundInherited platelet function disorders (PFDs) are heterogeneous, and identification of the underlying genetic defects is difficult when based solely on phenotypic and clinical features of the patient.ObjectiveTo analyze 329 genes regulating platelet function, number, and size in order to identify candidate gene defects in patients with PFDs.Patients/methodsTargeted analysis of candidate PFD genes was undertaken after next-generation sequencing of exomic DNA from 18 unrelated index cases with PFDs who were recruited into the UK Genotyping and Phenotyping of Platelets (GAPP) study and diagnosed with platelet abnormalities affecting either Gi signaling (n=12) or secretion (n=6). The potential pathogenicity of candidate gene defects was assessed using computational predictive algorithms.ResultsAnalysis of the 329 candidate PFD genes identified 63 candidate defects, affecting 40 genes, among index cases with Gi signaling abnormalities, while 53 defects, within 49 genes, were identified among patients with secretion abnormalities. Homozygous gene defects were more commonly associated with secretion abnormalities. Functional annotation analysis identified distinct gene clusters in the two patient subgroups. Thirteen genes with significant annotation enrichment for intracellular signaling' harbored 16 of the candidate gene defects identified in nine index cases with Gi signaling abnormalities. Four gene clusters, representing 14 genes, with significantly associated gene ontology annotations were identified among the cases with secretion abnormalities, the most significant association being with establishment of protein localization.'ConclusionOur findings demonstrate the genetic complexity of PFDs and highlight plausible candidate genes for targeted analysis in patients with platelet secretion and Gi signaling abnormalities.

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

SummaryPlatelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Inherited thrombocytopenias are a group of disorders that are characterized by a low platelet count and are sometimes associated with excessive bleeding that ranges from mild to severe. We evaluated 36 unrelated patients and 17 family members displaying thrombocytopenia that were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study. All patients had a history of excessive bleeding of unknown etiology. We performed platelet phenotyping and whole-exome sequencing (WES) on all patients and identified mutations in schlafen 14 (SLFN14) in 12 patients from 3 unrelated families. Patients harboring SLFN14 mutations displayed an analogous phenotype that consisted of moderate thrombocytopenia, enlarged platelets, decreased ATP secretion, and a dominant inheritance pattern. Three heterozygous missense mutations were identified in affected family members and predicted to encode substitutions (K218E, K219N, and V220D) within an ATPase-AAA-4, GTP/ATP-binding region of SLFN14. Endogenous SLFN14 expression was reduced in platelets from all patients, and mutant SLFN14 expression was markedly decreased compared with that of WT SLFN14 when overexpressed in transfected cells. Electron microscopy revealed a reduced number of dense granules in affected patients platelets, correlating with a decreased ATP secretion observed in lumiaggregometry studies. These results identify SLFN14 mutations as cause for an inherited thrombocytopenia with excessive bleeding, outlining a fundamental role for SLFN14 in platelet formation and function.

Background The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY12 mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y12 receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. Objectives To determine the functional consequences of the R122C substitution for P2Y12 function. Patient/methods We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y12 stably expressed in cells was also performed. Results ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y12 activity in the patient and family members. Cell surface R122C P2Y12 expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Conclusions Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait.

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

Inherited platelet function disorders (PFDs), associated with normal or reduced platelet counts, account for a significant proportion of bleeding diatheses. Identification of the underlying genetic defects is difficult in the majority of cases due to the variable clinical expression of the bleeding symptoms and the redundancy of platelet receptor and signalling pathways, which add to the complexity of diagnosis. The gold standard method for phenotyping platelets, light transmission aggregometry (LTA), has allowed classification of functional defects in the majority of patients referred for investigation of suspected PFDs, while DNA-based analysis has primarily played a confirmatory role and been restricted mainly to analysis of candidate genes. Recent advances in next generation sequencing have facilitated the identification of gene defects in patients with PFDs where the underlying genetic defect was previously unknown, especially when combined with genome-wide linkage analysis. These studies have provided new insights into the mechanisms controlling platelet formation and function, and it is likely that, as understanding of the relationships between platelet phenotype and genotype increases and pipelines for the interpretation of genetic variations identified in patients are developed, DNA-based analysis will play an increasingly important role in the first-line investigation of patients with PFDs.