Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

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.

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

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.

Transcription factor defects causing platelet disorders.

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.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects.

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.

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

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.

SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects.

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.

Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

Platelet 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.

What is the role of genetic testing in the investigation of patients with suspected platelet function disorders?

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.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

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.

Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects.

We analyzed candidate platelet function disorder genes in 13 index cases with a history of excessive bleeding in association with a significant reduction in dense granule secretion and impaired aggregation to a panel of platelet agonists. Five of the index cases also had mild thrombocytopenia. Heterozygous alterations in FLI1 and RUNX1, encoding Friend leukemia integration 1 and RUNT-related transcription factor 1, respectively, which have a fundamental role in megakaryocytopoeisis, were identified in 6 patients, 4 of whom had mild thrombocytopenia. Two FLI1 alterations predicting p.Arg337Trp and p.Tyr343Cys substitutions in the FLI1 DNA-binding domain abolished transcriptional activity of FLI1. A 4-bp deletion in FLI1, and 2 splicing alterations and a nonsense variation in RUNX1, which were predicted to cause haploinsufficiency of either FLI1 or RUNX1, were also identified. Our findings suggest that alterations in FLI1 and RUNX1 may be common in patients with platelet dense granule secretion defects and mild thrombocytopenia.

Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel.

Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

Lack of association between polymorphisms in the interleukin-1 gene cluster and familial thrombophilia.

INTRODUCTION: Inflammation and venous thrombosis are intimately linked, and there is evidence that levels of inflammatory cytokines influence risk of venous thrombosis. We investigated the hypothesis that allelic variation within the IL-1 gene cluster, which encompasses the genes encoding the inflammatory cytokines IL-1α and IL-1ß and the competitive IL-1 receptor antagonist, is associated with venous thrombosis among patients with heritable thrombophilia. SUBJECTS AND METHODS: Genomic DNA samples from 181 index cases with heritable thrombophilia and 323 control subjects were genotyped for four SNPs, and four microsatellite markers located within the IL-1 gene cluster. The distributions of SNP genotypes and of microsatellite marker alleles were then compared between the patient and control groups. RESULTS: There was no significant difference in the distribution of alleles between the patients and control subjects for any of the four microsatellite loci studied. Likewise, the distribution of genotypes for each of the four SNPs investigated was similar among the cases and control subjects. Haplotype analysis showed no difference in the estimated frequencies of any of the IL-1 gene cluster haplotypes between the patients and control subjects. CONCLUSIONS: Our findings in this study suggest that inherited variation within the IL-1 gene cluster is not associated with thrombosis among patients with heritable thrombophilia and that alterations in inflammatory cytokines encoded by loci in the IL-1 gene cluster are more likely to occur as a result, rather than a cause, of venous thrombosis.

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets.

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.

A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA(2)R, U46619 did not increase cytosolic free Ca(2+) concentration, indicating loss of receptor function. The TxA(2)R antagonist [(3)H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA(2)R. This is the second naturally occurring TxA(2)R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA(2)R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA(2)R function in vivo.

Identification and characterization of a novel P2Y 12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study.

We investigated whether defects in the P2Y(12) ADP receptor gene (P2RY12) contribute to the bleeding tendency in 92 index cases enrolled in the European MCMDM-1VWD study. A heterozygous mutation, predicting a lysine to glutamate (K174E) substitution in P2Y(12), was identified in one case with mild type 1 von Willebrand disease (VWD) and a VWF defect. Platelets from the index case and relatives carrying the K174E defect changed shape in response to ADP, but showed reduced and reversible aggregation in response to 10 muM ADP, unlike the maximal, sustained aggregation observed in controls. The reduced response was associated with an approximate 50% reduction in binding of [(3)H]2MeS-ADP to P2Y(12), whereas binding to the P2Y(1) receptor was normal. A hemagglutinin-tagged K174E P2Y(12) variant showed surface expression in CHO cells, markedly reduced binding to [(3)H]2MeS-ADP, and minimal ADP-mediated inhibition of forskolin-induced adenylyl cyclase activity. Our results provide further evidence for locus heterogeneity in type 1 VWD.

Common sequence variations in the P2Y12 and CYP3A5 genes do not explain the variability in the inhibitory effects of clopidogrel therapy.

The efficacy of the platelet P2Y12 receptor antagonist clopidogrel, which undergoes cytochrome-mediated metabolism to its active form, shows marked inter-individual variability. We investigated whether polymorphic variations in the P2Y12 gene, which have been linked to platelet aggregation phenotypes, or the cytochrome P450 3A5 gene 6986G > A polymorphism, which largely determines CYP3A5 expression, influence the response to clopidogrel therapy. Fifty-four patients listed for elective percutaneous coronary intervention were studied using ADP-induced optical aggregometry, whole-blood single platelet counting (WBSPC) aggregometry, and flow-cytometric analysis of platelet P-selectin expression and platelet-monocyte conjugate formation. Platelet reactivity was measured at baseline, 4 h post clopidogrel 300 mg, and 10 and 28 days following clopidogrel 75 mg daily. A further 55 patients were studied using ADP-induced WBSPC at baseline and 4 h post clopidogrel 600 mg. Patients were genotyped for P2Y12 haplotype and the CYP3A5 6986G > A single nucleotide polymorphism. Neither genotype was found to significantly influence the inhibition of platelet responses by either clopidogrel regimen. In conclusion, common sequence variations within the P2Y12 and CYP3A5 genes do not contribute any major effect to the inter-patient variability in clopidogrel efficacy.