A circle of life: platelet and megakaryocyte cytoskeleton dynamics in health and disease.

Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.

ISTH bleeding assessment tool and platelet function analyzer in children with mild inherited platelet function disorders.

OBJECTIVES: To evaluate the diagnostic performance of platelet function analyzer (PFA) and The International Society on Thrombosis and Hemostasis bleeding-assessment-tool (ISTH-BAT) in detecting mild inherited platelet function disorders (IPFDs) in children with suspected bleeding disorders. METHODS: Prospective single-center diagnostic study including consecutive patients <18 years with suspected bleeding disorder and performing a standardized workup for platelet function defects including ISTH-BAT, PFA, platelet aggregation testing, blood smear-based immunofluorescence, and next-generation sequencing-based genetic screening for IPFDs. RESULTS: We studied 97 patients, of which 34 von Willebrand disease (VWD, 22 type-1, 11 type-2), 29 IPFDs (including delta-/alpha-storage pool disease, Glanzmann thrombasthenia, Hermansky-Pudlak syndrome) and 34 with no diagnosis. In a model combining PFA-adenosine diphosphate (ADP), PFA-epinephrine (EPI), and ISTH-BAT overall performance to diagnose IPFDs was low with area under the curves of 0.56 (95% CI 0.44, 0.69) compared with 0.84 (95% CI 0.76, 0.92) for VWD. Correlation of PFA-EPI/-ADP and ISTH-BAT was low with 0.25/0.39 Spearman's correlation coefficients. PFA were significantly prolonged in patients with VWD and Glanzmann thrombasthenia. ISTH-BAT-scores were only positive in severe bleeding disorders, but not in children with mild IPFDs or VWD. CONCLUSION: Neither ISTH-BAT nor PFA or the combination of both help diagnosing mild IPFDs in children. PFA is suited to exclude severe IPFDs or VWD and is in this regard superior to ISTH-BAT in children.

Efficacy of novel agents against cellular models of familial platelet disorder with myeloid malignancy (FPD-MM).

Germline, mono-allelic mutations in RUNX1 cause familial platelet disorder (RUNX1-FPD) that evolves into myeloid malignancy (FPD-MM): MDS or AML. FPD-MM commonly harbors co-mutations in the second RUNX1 allele and/or other epigenetic regulators. Here we utilized patient-derived (PD) FPD-MM cells and established the first FPD-MM AML cell line (GMR-AML1). GMR-AML1 cells exhibited active super-enhancers of MYB, MYC, BCL2 and CDK6, augmented expressions of c-Myc, c-Myb, EVI1 and PLK1 and surface markers of AML stem cells. In longitudinally studied bone marrow cells from a patient at FPD-MM vs RUNX1-FPD state, we confirmed increased chromatin accessibility and mRNA expressions of MYB, MECOM and BCL2 in FPD-MM cells. GMR-AML1 and PD FPD-MM cells were sensitive to homoharringtonine (HHT or omacetaxine) or mebendazole-induced lethality, associated with repression of c-Myc, EVI1, PLK1, CDK6 and MCL1. Co-treatment with MB and the PLK1 inhibitor volasertib exerted synergistic in vitro lethality in GMR-AML1 cells. In luciferase-expressing GMR-AML1 xenograft model, MB, omacetaxine or volasertib monotherapy, or co-treatment with MB and volasertib, significantly reduced AML burden and improved survival in the immune-depleted mice. These findings highlight the molecular features of FPD-MM progression and demonstrate HHT, MB and/or volasertib as effective agents against cellular models of FPD-MM.

Bleeding phenotype and hemostatic evaluation by thrombin generation in children with Noonan syndrome: A prospective study.

BACKGROUND: This study aimed to evaluate the bleeding phenotype and to conduct a comprehensive hemostatic evaluation in individuals with Noonan syndrome (NS), a dominantly inherited disorder caused by pathogenic variants in genes associated with the Ras/MAPK signaling pathway. METHODS: Children with a genetically confirmed diagnosis of NS underwent clinical evaluation, routine laboratory tests, platelet function testing, and thrombin generation (TG) assessment. RESULTS: The study included 24 children. The most frequently reported bleeding symptoms were easy bruising and epistaxis, while bleeding complications were observed in 15% of surgical procedures. Various hemostatic abnormalities were identified, including platelet dysfunction, von Willebrand disease, and clotting factor deficiencies. Abnormal platelet function was observed in 50% of the patients, and significantly lower TG parameters were found compared to controls. However, no significant correlation was observed between bleeding symptoms and TG results. CONCLUSIONS: The study suggests that the bleeding diathesis in NS is multifactorial, involving both platelet dysfunction and deficiencies of plasma coagulation factors. The potential role of TG assay as an ancillary tool for predicting bleeding tendencies in individuals with NS undergoing surgery warrants further investigation.

Evaluating the clinical validity of genes related to hemostasis and thrombosis using the Clinical Genome Resource gene curation framework.

BACKGROUND: Inherited bleeding, thrombotic, and platelet disorders (BTPDs) are a heterogeneous set of diseases, many of which are very rare globally. Over the past 5 decades, the genetic basis of some of these disorders has been identified, and recently, high-throughput sequencing has become the primary means of identifying disease-causing genetic variants. OBJECTIVES: Knowledge of the clinical validity of a gene-disease relationship is essential to provide an accurate diagnosis based on results of diagnostic gene panel tests and inform the construction of such panels. The Scientific and Standardization Committee for Genetics in Thrombosis and Hemostasis undertook a curation process for selecting 96 TIER1 genes for BTPDs. The purpose of the process was to evaluate the evidence supporting each gene-disease relationship and provide an expert-reviewed classification for the clinical validity of genes associated with BTPDs. METHODS: The Clinical Genome Resource (ClinGen) Hemostasis/Thrombosis Gene Curation Expert Panel assessed the strength of evidence for TIER1 genes using the semiquantitative ClinGen gene-disease clinical validity framework. ClinGen Lumping and Splitting guidelines were used to determine the appropriate disease entity or entities for each gene, and 101 gene-disease relationships were identified for curation. RESULTS: The final outcome included 68 Definitive (67%), 26 Moderate (26%), and 7 Limited (7%) classifications. The summary of each curation is available on the ClinGen website. CONCLUSION: Expert-reviewed assignment of gene-disease relationships by the ClinGen Hemostasis/Thrombosis Gene Curation Expert Panel facilitates accurate molecular diagnoses of BTPDs by clinicians and diagnostic laboratories. These curation efforts can allow genetic testing to focus on genes with a validated role in disease.

Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice.

Germ line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM.

Progress in Hemostasis (Part 1): Improved Management of Inherited Platelet Disorders: Reality or Illusion?

Platelets are key drivers of hemostasis. Low platelet counts, dysfunction in platelet adhesion, and aggregation lead to increased bleeding tendency. Inherited platelet disorders (IPDs) form a highly heterogeneous group of rare diseases with variable bleeding tendency. IPDs may be associated with other signs and symptoms often referred to as "syndromic." The underlying genetic defect may prone patients to develop hematopoietic diseases such as leukemia. Over the last decade, accumulating knowledge in genetics has led to the detection of many "new" platelet disorders. However, still many patients with a well-described platelet dysfunction remain undetected until severe bleeding occurs.

State-of-the-Art Targeted High-Throughput Sequencing for Detecting Inherited Platelet Disorders.

Inherited platelet disorders (IPDs) are a heterogeneous group of rare entities caused by molecular divergence in genes relevant for platelet formation and function. A rational diagnostic approach is necessary to counsel and treat patients with IPDs. With the introduction of high-throughput sequencing at the beginning of this millennium, a more accurate diagnosis of IPDs has become available. We discuss advantages and limitations of genetic testing, technical issues, and ethical aspects. Additionally, we provide information on the clinical significance of different classes of variants and how they are correctly reported.

Treatment of Inherited Platelet Disorders: Current Status and Future Options.

Inherited platelet disorders (IPDs) comprise a heterogeneous group of entities that manifest with variable bleeding tendencies. For successful treatment, the underlying platelet disorder, bleeding severity and location, age, and sex must be considered in the broader clinical context. Previous information from the AWMF S2K guideline #086-004 (www.awmf.org) is evaluated for validity and supplemented by information of new available and future treatment options and clinical scenarios that need specific measures. Special attention is given to the treatment of menorrhagia and risk management during pregnancy in women with IPDs. Established treatment options of IPDs include local hemostatic treatment, tranexamic acid, desmopressin, platelet concentrates, and recombinant activated factor VII. Hematopoietic stem cell therapy is a curative approach for selected patients. We also provide an outlook on promising new therapies. These include autologous hematopoietic stem cell gene therapy, artificial platelets and nanoparticles, and various other procoagulant treatments that are currently tested in clinical trials in the context of hemophilia.

Platelet genetic testing by next-generation sequencing: A practical update.

Inherited platelet disorders (IPDs) are a heterogeneous group of disorders characterized by normal or reduced platelet counts, bleeding diatheses of varying severities, and the presence (syndromic) or absence (non-syndromic) of involvement of other organs. Due to the lack of highly specific platelet function tests and overlapping clinical and laboratory features, diagnosing the underlying cause of IPDs remains challenging. In recent years, genetic testing via next-generation sequencing (NGS) technologies to rapidly analyze multiple genes has gradually emerged as an important part of the laboratory investigation of patients with IPDs. A systemic clinical and laboratory testing approach and thorough phenotype and genotype correlation studies of both patients and their family members are crucial for accurate diagnoses of IPDs.

[A new form of familial platelet disorder caused by germline mutations in RUNX1 in a pedigree].

Objective: To investigate the clinical and biological characteristics of familial platelet disorder (FPD) with germline Runt-related transcription factor (RUNX) 1 mutations. Methods: Patients diagnosed with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with RUNX1 mutations from February 2016 to December 2021 in Wuhan No.1 Hospital underwent pedigree analysis and were screened for gene mutations (somatic and germline). Patients diagnosed with FPD with germline RUNX1 mutations were enrolled and evaluated in terms of clinical characteristics and biological evolution. Bioinformatics analysis was used to assess the pathogenicity of mutations and to analyze the effect of mutated genes on the function of the corresponding protein. Results: Germline RUNX1 mutations were detected in three out of 34 patients suffering from MDS/AML who had RUNX1 mutations. A pedigree of FPD with RUNX1 (RUNX1-FPD) c.562A>C and RUNX1 c.1415T>C mutations was diagnosed, and the mutations were of patrilineal origin. Bioinformatics analysis indicated that the locus at positions 188 and 472 in the AML-1G type of RUNX1 was highly conserved across different species, and that variations might influence functions of the proteins. The mutations were evaluated to be highly pathogenic. Of the nine cases with germline RUNX1 mutations: two patients died due AML progression; one case with AML survived without leukemia after transplantation of hemopoietic stem cells; four patients showed mild-to-moderate thrombocytopenia; two cases had no thrombocytopenia. During the disease course of the proband and her son, mutations in RUNX1, NRAS and/or CEBPA and KIT appeared in succession, and expression of cluster of differentiation-7 on tumor cells was enhanced gradually. None of the gene mutations correlated with the tumor were detected in the four cases not suffering from MDS/AML, and they survived until the end of follow-up. Conclusions: RUNX1-FPD was rare. The mutations c.562A>C and c.1415T>C of RUNX1 could be the disease-causing genes for the family with RUNX1-FPD, and these mutations could promote malignant transformation. Biological monitoring should be carried out regularly to aid early intervention for family members with RUNX1-FPD.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities for mucocutaneous bleeding disorders.

BACKGROUND: Excessive or abnormal mucocutaneous bleeding (MCB) may impact all aspects of the physical and psychosocial wellbeing of those who live with it (PWMCB). The evidence base for the optimal diagnosis and management of disorders such as inherited platelet disorders, hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndromes (EDS), and von Willebrand disease (VWD) remains thin with enormous potential for targeted research. RESEARCH DESIGN AND METHODS: National Hemophilia Foundation and American Thrombosis and Hemostasis Network initiated the development of a National Research Blueprint for Inherited Bleeding Disorders with extensive all-stakeholder consultations to identify the priorities of people with inherited bleeding disorders and those who care for them. They recruited multidisciplinary expert working groups (WG) to distill community-identified priorities into concrete research questions and score their feasibility, impact, and risk. RESULTS: WG2 detailed 38 high priority research questions concerning the biology of MCB, VWD, inherited qualitative platelet function defects, HDS/EDS, HHT, bleeding disorder of unknown cause, novel therapeutics, and aging. CONCLUSIONS: Improving our understanding of the basic biology of MCB, large cohort longitudinal natural history studies, collaboration, and creative approaches to novel therapeutics will be important in maximizing the benefit of future research for the entire MCB community.

More people experience mucocutaneous bleeding (MCB), affecting tissues like skin and gums, than have hemophilia A or B. MCB is not understood as well as hemophilia. Common types of MCB include nosebleeds, bleeding gums, heavy menstrual bleeding, and digestive tract bleeding. Mucocutaneous inherited bleeding disorders include inherited platelet disorders, hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD) and Ehlers-Danlos syndromes (EDS), von Willebrand Disease (VWD), and others. Diagnosing and treating MCB is complicated and sometimes medical providers dismiss the bleeding that patients report when they cannot find a medical explanation for it. Many people with mucocutaneous bleeding (PWMCB) do not receive the care they need; for example, women with VWD live with symptoms for, on average, 16 years before they are diagnosed in the US. This struggle to obtain care has important negative impacts on patients' physical and psychological health and their quality-of-life. The National Hemophilia Foundation (NHF), a large US bleeding disorders patient advocacy organization, set out to develop a National Research Blueprint for Inherited Bleeding Disorders focused on community priorities. They brought together a group of patients, providers, and researchers with MCB expertise to identify the research that would most improve the lives of PWMCB through targeted and accessible diagnostics and therapies. We report in this paper that research is needed to better understand the biology of MCB and to define the mechanisms of disease in these disorders. We also describe high priority research questions for each of the main disorders, novel therapeutics, and aging.

Unrelated hematopoietic stem cell transplantation for familial platelet disorder/acute myeloid leukemia with germline RUNX1 mutations.

Germline mutations in RUNX1 result in rare autosomal-dominant familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). As genetic analysis is becoming increasingly prevalent, the diagnosis rate of FPD/AML is expected to increase. In this report, we present two pedigrees, one diagnosed molecularly and another highly suspected to be FPD/AML, whose members both received allogeneic hematopoietic stem cell transplantation (HSCT). Both pedigrees had a family history of thrombocytopenia, platelet dysfunction, and hematological malignancies. One family inherited a frameshift mutation (p.P240fs) of RUNX1, a known pathogenic variant. Another family inherited a point mutation (p.G168R) in the runt-homology domain, the clinical significance of which is uncertain at this point. As this mutation was completely absent from all population databases and had a relatively high REVEL score of 0.947, we thought that it would be dangerous to ignore its possible pathogenicity. Consequently, we avoided choosing HSCT donors from relatives of both families and performed HSCT from unrelated donors. In conclusion, our experience with two families of FPD/AML highlights the importance of searching for gene mutations associated with germline predisposition and indicates the necessity of developing a donor coordination system for FPD/AML patients, as well as a support system for families.

Validation of immunofluorescence analysis of blood smears in patients with inherited platelet disorders.

BACKGROUND: Inherited platelet disorders (IPDs) are rare diseases characterized by reduced blood platelet counts and/or impaired platelet function. Recognizing IPDs is advisable but often challenging. The diagnostic tools include clinical evaluation, platelet function tests, and molecular analyses. Demonstration of a pathogenic genetic variant confirms IPDs. We established a method to assess the platelet phenotype on blood smears using immunofluorescence microscopy as a diagnostic tool for IPDs. OBJECTIVES: The aim of the present study was to validate immunofluorescence microscopy as a screening tool for IPDs in comparison with genetic screening. METHODS: We performed a blinded comparison between the diagnosis made using immunofluorescence microscopy on blood smears and genetic findings in a cohort of 43 families affected with 20 different genetically confirmed IPDs. In total, 76% of the cases had inherited thrombocytopenia. RESULTS: Immunofluorescence correctly predicted the underlying IPD in the vast majority of patients with 1 of 9 IPDs for which the typical morphologic pattern is known. Thirty of the 43 enrolled families (70%) were affected by 1 of these 9 IPDs. For the other 11 forms of IPD, we describe alterations of platelet structure in 9 disorders and normal findings in 2 disorders. CONCLUSION: Immunofluorescence microscopy on blood smears is an effective screening tool for 9 forms of IPD, which include the most frequent forms of inherited thrombocytopenia. Using this approach, typical changes in the phenotype may also be identified for other rare IPDs.

Frequency and molecular basis of CD36 deficiency among platelet donors in Kunming, China.

CD36 is a multifunctional receptor expressed on the surface of many cell types. Among healthy individuals, CD36 may be absent on platelets and monocytes (type I deficiency) or platelets alone (type II deficiency). However, the exact molecular mechanisms underlying CD36 deficiency remain unclear. In this study, we aimed to identify individuals with CD36 deficiency and investigate the molecular basis underlying it. Blood samples were collected from platelet donors at Kunming Blood Center. Platelets and monocytes were isolated and CD36-expression levels were analyzed using flow cytometry. DNA from whole blood and mRNA isolated from monocytes and platelets of individuals with CD36 deficiency were analyzed using polymerase chain reaction (PCR) testing. The PCR products were cloned and sequenced. Among the 418 blood donors,7 (1.68%) were CD36 deficient: 1 (0.24%) with type I deficiency and 6(1.44%) with type II deficiency. Six heterozygous mutations occurred, including c.268C>T (in type I individuals), c.120 + 1 G>T, c.268C>T, c.329_330del/AC, c.1156 C>T, c.1163A>C, and c.1228_1239del/ATTGTGCCTATT (in type II individuals). Mutations were not detected in one type II individual . At the cDNA level, only mutant, but not wild-type, transcripts were detected in the platelets and monocytes of type I individual. In type II individuals, only mutant transcripts were found in platelets, whereas monocytes possessed wild-type and mutant transcripts. Interestingly, only alternative splicing transcripts were observed in the individual without mutation. We report the incidence rates of type I and II CD36 deficiencies among platelet donors in Kunming. Molecular genetic analyses of DNA and cDNA demonstrated that homozygous mutations on the cDNA level in platelets and monocytes or platelets alone identified type I and II deficiencies, respectively. Furthermore, alternatively spliced products also potentially contribute to the mechanism of CD36 deficiency.

What is the context? Healthy individuals may lack CD36 on platelets and (or) monocytes, which are defined as Type I and Type II CD36 deficiency. These individuals could develop anti-CD36 antibodies associated with immune-mediated disorders. However, the mechanism underlying the CD36 deficiency is still unclear. In this study, we reported the incidence of CD36 deficiency in Kunming platelet donors and found the new molecular basis of CD36 deficiency individuals.What's new? Molecular genetic analysis of cDNA derived from type I subjects showed the presence of mutant transcript only, both in platelets and monocytes. In type II subjects, platelets only carry mutant transcript, whereas monocytes possessed both wild-type and mutant transcripts. Furthermore, we found that alternatively spliced product of CD36 transcript could also contribute to the mechanism of CD36 deficiencies.What's the impact? Our finding indicates that analysis of CD36 at cDNA level is mandatory to verify different forms of CD36 deficiencies. This information may help us to understand the development of anti-CD36 antibodies in CD36 deficient individuals.

Hereditary platelet disorders associated with germ line variants in RUNX1, ETV6, and ANKRD26.

Hereditary platelet disorders (HPDs) are a group of blood disorders with variable severity and clinical impact. Although phenotypically there is much overlap, known genetic causes are many, prompting the curation of multigene panels for clinical use, which are being deployed in increasingly large-scale populations to uncover missing heritability more efficiently. For some of these disorders, in particular RUNX1, ETV6, and ANKRD26, pathogenic germ line variants in these genes also come with a risk of developing hematological malignancy (HM). Although they may initially present as similarly mild-moderate thrombocytopenia, each of these 3 disorders have distinct penetrance of HM and a different range of somatic alterations associated with malignancy development. As our ability to diagnose HPDs has improved, we are now faced with the challenges of integrating these advances into routine clinical practice for patients and how to optimize management and surveillance of patients and carriers who have not developed malignancy. The volume of genetic information now being generated has created new challenges in how to accurately assess and report identified variants. The answers to all these questions involve international initiatives on rare diseases to better understand the biology of these disorders and design appropriate models and therapies for preclinical testing and clinical trials. Partnered with this are continued technological developments, including the rapid sharing of genetic variant information and automated integration with variant classification relevant data, such as high-throughput functional data. Collective progress in this area will drive timely diagnosis and, in time, leukemia preventive therapeutic interventions.

Proteomic landscapes of inherited platelet disorders with different etiologies.

BACKGROUND: Inherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited. OBJECTIVES: The objective of this study was to profile the platelet proteomics signatures of IPDs. METHODS: We performed unbiased label-free quantitative mass spectrometry (MS)-based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development. RESULTS: In line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins. CONCLUSIONS: With this study, we demonstrate a MS-based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function.

Clinical application of multigene panel testing for bleeding, thrombotic, and platelet disorders: a 3-year Belgian experience.

BACKGROUND: The international study ThromboGenomics has evaluated the diagnostic rate using a targeted multigene panel test for the screening of inherited bleeding, thrombotic and platelet disorders. OBJECTIVES: We retrospectively analyzed the results of the implementation of genetic testing for inherited bleeding, thrombotic and platelet disorders in Belgian clinical practice and evaluated possible reclassification of reported variants. PATIENTS/METHODS: We implemented a Thrombosis-Hemostasis multigene panel test using whole exome sequencing to diagnose 487 patients recruited by 27 different Belgian hospitals with the implementation of stringent laboratory accreditation standards and by studying up to 100 diagnostic-grade genes. RESULTS: This Thrombosis-Hemostasis multigene panel test was able to detect at least one genetic variant in 58% of the 487 patients of which 50% were (likely) pathogenic variants and the others were variants of unknown significance. Polygenic variants were detected in 65 patients (13%). A multi-step workflow for results discussion by multidisciplinary team meetings and patients' recalls for segregation studies and additional laboratory testing was set up. Variants were also submitted to the GoldVariants database from the International Society on Thrombosis and Haemostasis (ISTH). The aim of these approaches is to optimize variant interpretation and to (re)classify variants of unknown significance as (likely) pathogenic or (likely) benign. CONCLUSIONS: The growing implementation of multigene panel tests in clinical diagnostics comes with difficulties in interpreting genetic results. Additional efforts are needed to continuously optimize the diagnostic outcome.