Exome sequencing in 116 patients with inherited thrombocytopenia that remained of unknown origin after systematic phenotype-driven diagnostic workup.

Inherited thrombocytopenias (IT) are genetic diseases characterized by low platelet count, sometimes associated with congenital defects or a predisposition to develop additional conditions. Next-generation sequencing has substantially improved our knowledge of IT, with more than 40 genes identified so far, but obtaining a molecular diagnosis remains a challenge especially for patients with non-syndromic forms, having no clinical or functional phenotypes that raise suspicion about specific genes. We performed exome sequencing (ES) in a cohort of 116 IT patients (89 families), still undiagnosed after a previously validated phenotype-driven diagnostic algorithm including a targeted analysis of suspected genes. ES achieved a diagnostic yield of 36%, with a gain of 16% over the diagnostic algorithm. This can be explained by genetic heterogeneity and unspecific genotype-phenotype relationships that make the simultaneous analysis of all the genes, enabled by ES, the most reasonable strategy. Furthermore, ES disentangled situations that had been puzzling because of atypical inheritance, sex-related effects or false negative laboratory results. Finally, ES-based copy number variant analysis disclosed an unexpectedly high prevalence of RUNX1 deletions, predisposing to hematologic malignancies. Our findings demonstrate that ES, including copy number variant analysis, can substantially contribute to the diagnosis of IT and can solve diagnostic problems that would otherwise remain a challenge.

Expert opinion on the use of platelet secretion assay for the diagnosis of inherited platelet function disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis. We surveyed 26 experts from 10 different countries using the RAND/UCLA methodology, to attain a consensus on sensitivity, specificity, feasibility, time to readout, and cost of most PSAs. Answers were then graded in three categories: appropriate, uncertain, and inappropriate. Equivocal or misinterpretable statements required a second and third round survey involving 14 of the original 26 experts. We report here the consolidated results of the entire procedure. There was uniform agreement on several general statements, including that PSAs should be performed in hemostasis laboratories as first line diagnostic tests even in patients with normal platelet aggregation, and should include a δ-granule secretion marker. Among the specific assays examined, lumiaggregometry, other luciferin/luciferase-based assays, high-performance liquid chromatography methods, radiolabeled-serotonin based assays, and whole-mount transmission electron microscopy were rated as appropriate for the measurement of δ-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for α-granule release. For most of the other PSAs, the expert opinions were widely dispersed. Lack of expert consensus on many PSAs clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of PSAs for clinical laboratory practice.

Dysregulation of oncogenic factors by GFI1B p32: investigation of a novel GFI1B germline mutation.

GFI1B is a transcription factor essential for the regulation of erythropoiesis and megakaryopoiesis, and pathogenic variants have been associated with thrombocytopenia and bleeding. Analysing thrombocytopenic families by whole exome sequencing, we identified a novel GFI1B variant (c.648+5G>A), which causes exon 9 skipping and overexpression of a shorter p32 isoform. We report the clinical data of our patients and critically review the phenotype observed in individuals with different GFI1B variants leading to the same effect on the p32 expression. Since p32 is increased in acute and chronic leukemia cells, we tested the expression level of genes playing a role in various type of cancers, including hematological tumors and found that they are significantly dysregulated, suggesting a potential role for GFI1B in carcinogenesis regulation. Increasing the detection of individuals with GFI1B variants will allow us to better characterize this rare disease and determine whether it is associated with an increased risk of developing malignancies.

A Novel Mutation in GP1BB Reveals the Role of the Cytoplasmic Domain of GPIbß in the Pathophysiology of Bernard-Soulier Syndrome and GPIb-IX Complex Assembly.

Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbß, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbß. The loss of the intracytoplasmic tail of GPIbß results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbß; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbß is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.

The ISTH bleeding assessment tool as predictor of bleeding events in inherited platelet disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: The ISTH Bleeding Assessment Tool (ISTH-BAT) has been validated for clinical screening of suspected von Willebrand disease (VWD) and for bleeding prediction. Recently it has been validated for subjects with inherited platelet disorders (IPD) (BAT-VAL study). OBJECTIVES: To determine whether the ISTH-BAT bleeding score (BS) predicts subsequent bleeding events requiring treatment in IPD patients. METHODS: Patients with IPD, type 1 VWD (VWD-1) and age- and sex-matched healthy controls enrolled in the BAT-VAL study were prospectively followed-up for 2 years and bleeding episodes requiring treatment were recorded. RESULTS: Of the 1098 subjects initially enrolled, 955 were followed-up and 124 suffered hemorrhages during follow-up, 60% of whom had inherited platelet function disorders (IPFD). Total number of events was significantly higher in IPFD (n = 235) than VWD-1 (n = 52) or inherited thrombocytopenia (IT; n = 20). Events requiring transfusions were 66% in IPFD, 5.7% in VWD-1, and 3% in IT. Baseline BS was significantly higher in IPFD patients with a bleeding event at follow-up than in those without (p < .01) and the percentage of subjects suffering a bleeding event increased proportionally to baseline BS quartile. A significant association between the BS and the chance of suffering severe bleeding was found in the overall, IPFD, and VWD-1 populations. Similar results were obtained for the pediatric population. CONCLUSIONS: Inherited platelet function disorder patients with high BS at enrollment are more likely to suffer from bleeding events requiring treatment at follow-up. Moreover, the higher the baseline BS quartile the greater the incidence of subsequent events, suggesting that independently from diagnosis a high BS is associated with a greater risk of subsequent hemorrhage.

Venous thromboembolism and COVID-19: a single center experience from an academic tertiary referral hospital of Northern Italy.

Preliminary evidence supports the notion that COVID-19 patients may have an increased susceptibility to develop venous thromboembolism (VTE). However, the magnitude of this association still needs to be defined. Furthermore, clinical predictors of thrombogenesis, and the relationship with the inflammatory status are currently unknown. On this basis, we conducted a retrospective, observational study on 259 consecutive COVID-19 patients admitted to an academic tertiary referral hospital in Northern Italy between March 19th and April 6th, 2020. Records of COVID-19 patients with a definite VTE event were reviewed for demographic information, co-morbidities, risk factors for VTE, laboratory tests, and anticoagulation treatment. Twenty-five cases among 259 COVID-19 patients developed VTE (9.6%), all of them having a Padua score > 4, although being under standard anticoagulation prophylaxis since hospital admission. In the VTE subcohort, we found a significant positive correlation between platelet count (PLT) and either C reactive protein (CRP) (p < 0.0001) or lactate dehydrogenase (LDH) (p = 0.0013), while a significant inverse correlation was observed between PLT and mean platelet volume (p < 0.0001). Platelet-to-lymphocyte ratio significantly correlated with CRP (p < 0.0001). The majority of VTE patients was male and younger compared to non-VTE patients (p = 0.002 and p = 0.005, respectively). No significant difference was found in D-dimer levels between VTE and non VTE patients, while significantly higher levels of LDH (p = 0.04) and IL-6 (p = 0.04) were observed in VTE patients in comparison to non-VTE patients. In conclusion, our findings showed a quite high prevalence of VTE in COVID-19 patients. Raised inflammatory indexes and increased serum levels of pro-inflammatory cytokines should raise the clinical suspicion of VTE.

Clinical management, ethics and informed consent related to multi-gene panel-based high throughput sequencing testing for platelet disorders: Communication from the SSC of the ISTH.

Molecular diagnostics of inherited platelet disorders (IPD) has been revolutionized by the implementation of high-throughput sequencing (HTS) approaches. A conclusive diagnosis using HTS tests can be obtained quickly and cost-effectively in many, but not all patients. The expanding use of HTS tests has raised concerns regarding complex variant interpretation and the ethical implications of detecting unsolicited findings such as variants in IPD genes RUNX1, ETV6, and ANKRD26, which are associated with increased leukemic risk. This guidance document has been developed and written by a multidisciplinary team of researchers and clinicians, with expertise in hematology, clinical and molecular genetics, and bioethics, alongside a RUNX1 patient advocacy representative. We recommend that for clinical diagnostics, HTS for IPD should use a multigene panel of curated diagnostic-grade genes. Critically, we advise that an HTS test for clinical diagnostics should only be ordered by a clinical expert that is: (a) fully aware of the complexity of genotype-phenotype correlations for IPD; (b) able to discuss these complexities with a patient and family members before the test is initiated; and (c) able to interpret and appropriately communicate the results of a HTS diagnostic report, including the implication of variants of uncertain clinical significance. Each patient should know what an HTS test could mean for his or her clinical management before initiating a test. We hereby propose an exemplified informed consent document that includes information on these ethical concerns and can be used by the community for implementation of HTS of IPD in a clinical diagnostic setting. This paper does not include recommendations for HTS of IPD in a research setting.

Antithrombotic prophylaxis for surgery-associated venous thromboembolism risk in patients with inherited platelet disorders. The SPATA-DVT Study.

Major surgery is associated with an increased risk of venous thromboembolism (VTE), thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of VTE in patients with inherited platelet disorders (IPD) undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in IPD patients undergoing surgery at VTE-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of IPD (VTE-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to VTE-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest VTE-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions use. Two thromboembolic events were registered, both occurring after high VTE-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that VTE incidence is low in patients with IPD undergoing surgery at VTE-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with IPD undergoing invasive procedures at VTE-risk and low-molecular-weight-heparin should be considered for major surgery.

Eltrombopag for the treatment of inherited thrombocytopenias: a phase II clinical trial.

Patients with inherited thrombocytopenias often require platelet transfusions to raise their platelet count before surgery or other invasive procedures; moreover, subjects with clinically significant spontaneous bleeding may benefit from an enduring improvement of thrombocytopenia. The hypothesis that thrombopoietin-mimetics can increase platelet count in inherited thrombocytopenias is appealing, but evidence is scarce. We conducted a prospective, phase II clinical trial to investigate the efficacy of the oral thrombopoietin-mimetic eltrombopag in different forms of inherited thrombocytopenia. We enrolled 24 patients affected by MYH9-related disease, ANKRD26-related thrombocytopenia, X-linked thrombocytopenia/ Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. The average pre-treatment platelet count was 40.4 ×109/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalated manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 ×109/L), ten (43.5%) had a minor response (platelet count at least twice the baseline value), and two patients (8.7%) did not respond. The average increase of platelet count compared to baseline was 64.5 ×109/L (P<0.001). Four patients with clinically significant spontaneous bleeding entered a program of long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages, with the remission persisting throughout the treatment period. Treatment was globally well tolerated: five patients reported mild adverse events and one patient a moderate adverse event. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of inherited thrombocytopenia. Despite these encouraging results, caution is recommended when using thrombopoietinmimetics in inherited thrombocytopenias predisposing to leukemia. ClinicalTrials.gov identifier: NCT02422394.

Validation of the ISTH/SSC bleeding assessment tool for inherited platelet disorders: A communication from the Platelet Physiology SSC.

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 < area under the curve [AUC] < 1), moderate (0.7 < AUC < 0.9) between IPFD and VWD-1 and between IPFD and inherited thrombocytopenia (IT), while it was inaccurate (AUC ≤ 0.7) in discriminating IT from HC. CONCLUSIONS: The ISTH-BAT allows to efficiently discriminate IPFD from HC, while it has lower accuracy in distinguishing IPFD from VWD-1. Therefore, the ISTH-BAT appears useful for identifying subjects requiring laboratory evaluation for a suspected IPFD once VWD is preliminarily excluded.

Fundamentals for a Systematic Approach to Mild and Moderate Inherited Bleeding Disorders: An EHA Consensus Report.

Healthy subjects frequently report minor bleedings that are frequently 'background noise' of normality rather than a true disorder. Nevertheless, unexpected or unusual bleeding may be alarming. Thus, the distinction between normal and pathologic bleeding is critical. Understanding the underlying pathologic mechanism in patients with an excessive bleeding is essential for their counseling and treatment. Most of these patients with significant bleeding will result affected by non-severe inherited bleeding disorders (BD), collectively denominated mild or moderate BD for their relatively benign course. Unfortunately, practical recommendations for the management of these disorders are still lacking due to the current state of fragmented knowledge of pathophysiology and lack of a systematic diagnostic approach. To address this gap, an International Working Group (IWG) was established by the European Hematology Association (EHA) to develop consensus-based guidelines on these disorders. The IWG agreed that grouping these disorders by their clinical phenotype under the single category of mild-to-moderate bleeding disorders (MBD) reflects current clinical practice and will facilitate a systematic diagnostic approach. Based on standardized and harmonized definitions a conceptual unified framework is proposed to distinguish normal subjects from affected patients. The IWG proposes a provisional comprehensive patient-centered initial diagnostic approach that will result in classification of MBD into distinct clinical-pathological entities under the overarching principle of clinical utility for the individual patient. While we will present here a general overview of the global management of patients with MBD, this conceptual framework will be adopted and validated in the evidence-based, disease-specific guidelines under development by the IWG.

MYH9-Related Thrombocytopenia: Four Novel Variants Affecting the Tail Domain of the Non-Muscle Myosin Heavy Chain IIA Associated with a Mild Clinical Evolution of the Disorder.

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). Patients present congenital macrothrombocytopenia and inclusions of NMMHC-IIA in leukocytes, and have a variable risk of developing kidney damage, sensorineural deafness, presenile cataracts and/or liver enzymes abnormalities. The spectrum of mutations found in MYH9-RD patients is limited and the incidence and severity of the non-congenital features are predicted by the causative MYH9 variant. In particular, different alterations of the C-terminal tail domain of NMMHC-IIA associate with remarkably different disease evolution. We report four novel MYH9 mutations affecting the tail domain of NMMHC-IIA and responsible for MYH9-RD in four families. Two variants cause amino acid substitutions in the coiled-coil region of NMMHC-IIA, while the other two are a splicing variant and a single nucleotide deletion both resulting in frameshift alterations of the short non-helical tailpiece. Characterization of phenotypes of affected individuals shows that all of these novel variants are associated with a mild clinical evolution of the disease.

Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ . This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

ACTN1 mutations lead to a benign form of platelet macrocytosis not always associated with thrombocytopenia.

The inherited thrombocytopenias (IT) are a heterogeneous group of diseases resulting from mutations in more than 30 different genes. Among them, ACTN1-related thrombocytopenia (ACTN1-RT; Online Mendelian Inheritance in Man: 615193) is one of the most recently identified forms. It has been described as a mild autosomal dominant macrothrombocytopenia caused by mutations in ACTN1, a gene encoding for one of the two non-muscle isoforms of α-actinin. We recently identified seven new unrelated families with ACTN1-RT caused by different mutations. Two of them are novel missense variants (p.Trp128Cys and p.Pro233Leu), whose pathogenic role has been confirmed by in vitro studies. Together with the 10 families we have previously described, our cohort of ACTN1-RT now consists of 49 individuals carrying ACTN1 mutations. This is the largest case series ever collected and enabled a critical evaluation of the clinical aspects of the disease. We concluded that ACTN1-RT is the fourth most frequent form of IT worldwide and it is characterized by platelet macrocytosis in all affected subjects and mild thrombocytopenia in less than 80% of cases. The risk of bleeding, either spontaneous or upon haemostatic challenge, is negligible and there are no other associated defects, either congenital or acquired. Therefore, ACTN1-RT is a benign form of IT, whose diagnosis provides affected individuals and their families with a good prognosis.