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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. ispartof: JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol:19 issue:10 pages:2612-2617 ispartof: location:England status: published

International audience; Inherited disorders of platelet function (IPFD) and/or number (IPND) are heterogeneous conditions that result in variable mucocutaneous bleeding symptoms as a result of deranged primary haemostasis caused by platelet dysfunction or thrombocytopenia. Diagnosis is important to guide post-operative bleeding prophylactic strategies, to avoid treatment with inappropriate medications, and inform prognosis. Achieving an accurate diagnosis has traditionally been hampered by the requirement of multiple, often complex, laboratory tests that are not always available at single centres. To improve the diagnosis of these disorders a research collaborative was established, the Sydney Platelet Group, that explored an integrated approach combining traditional and contemporary platelet phenotypic and genetic diagnostic platforms available at four Sydney tertiary hospitals. Herein we report the outcomes of the first 50 patients evaluated using this approach. The cohort included 22 individuals with suspected IPFD and 28 with thrombocytopenia. Bleeding scores were higher in individuals with IPFD (mean 5.75; SD 4.83) than those with IPNDs (mean 2.14; SD 2.45). In cases with suspected IPFD, diagnosis to the level of the defective pathway was achieved in 71% and four individuals were found not to have a definitive platelet function defect. Dense granule secretion disorders were the most common platelet pathway abnormality detected (n=5). Mean bleeding scores in these individuals were not significantly different to individuals with defects in other commonly detected platelet pathways (dense granules, signal transduction and 'undetermined'). A molecular diagnosis was achieved in 52% of individuals with IPNDs and 5% with IPFD. Likely pathogenic and pathogenic variants detected included variants associated with extra-haematological complications (DIAPH1, MYH9) and potential for malignancy (ANKRD26 and RUNX1). The level of platelet investigation undertaken by this initiative is currently not available elsewhere in Australia and initial results confirm the utility of this integrated phenotypic-genetic approach.

Essentials Intracellular calcium pathways regulate megakaryopoiesis but details are unclear.We examined effects of NMDAR‐mediated calcium influx on normal and leukemic cells in culture.NMDARs facilitated differentiation of normal but proliferation of leukemic megakaryocytes.NMDAR inhibitors induced differentiation of leukemic Meg‐01 cells. Background N‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts. Objectives The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. Methods Meg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls. Results The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. Conclusions NMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

Essentials Platelet function defects may cause atypical bleeding symptoms in immune thrombocytopenia (ITP).An isolated platelet defect of collagen‐induced aggregation was explored in a patient with ITP.ITP mediated by antibodies to glycoprotein (GP) VI curtail receptor function.Inclusion of GPVI in diagnostic antibody detection assays may improve their diagnostic utility. Idiopathic immune thrombocytopenia (ITP) is an autoimmune disorder characterized by relapsing/ remitting thrombocytopenia. Bleeding complications are infrequent with platelet counts above 30×109/L, and this level is commonly used as a threshold for treatment. The question of another/ co‐existent diagnosis or an alternate mechanism of platelet destruction arises when bleeding is experienced with platelet counts above this threshold. We report a case of anti‐GPVI mediated ITP that was diagnosed following investigations performed to address this key clinical question. A patient with ITP experienced exaggerated bruising symptoms despite a platelet count of 91×109/L. Platelet functional testing showed an isolated platelet defect of collagen‐induced aggregation. Next generation sequencing excluded a pathogenic variant of GP6, and anti‐GPVI antibodies that curtailed GPVI function were confirmed by extended platelet phenotyping. We propose that anti‐GPVI mediated ITP may be under‐recognized, and that inclusion of GPVI in antibody detection assays may improve their diagnostic utility and in turn, facilitate a better understanding of ITP pathophysiology and aid individualized treatment approaches.

Background: Careful assessment of bleeding history is the first step in the evaluation of patients with mild/moderate bleeding disorders, and the use of a bleeding assessment tool (BAT) is strongly encouraged. Although a few studies have assessed the utility of the ISTH-BAT in patients with inherited platelet function disorders (IPFD) none of them was sufficiently large to draw conclusions and/or included appropriate control groups. Objectives: The aim of the present study was to test the utility of the ISTH-BAT in a large cohort of patients with a well-defined diagnosis of inherited platelets disorder in comparison with two parallel cohorts, one of patients with type-1 von Willebrand disease (VWD-1) and one of healthy controls (HC). Patients/Methods: We enrolled 1098 subjects, 482 of whom had inherited platelet disorders (196 IPFD and 286 inherited platelet number disorders [IT]) from 17 countries. Results: IPFD patients had significantly higher bleeding score (BS; median 9) than VWD-1 patients (median 5), a higher number of hemorrhagic symptoms (4 versus 3), and higher percentage of patients with clinically relevant symptoms (score > 2). The ISTH-BAT showed excellent discrimination power between IPFD and HC (0.9

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SummaryBackground Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.