BACKGROUND:GNE-related thrombocytopenia (GNE-RT) is a very rare disorder caused by biallelic variants in GNE, encoding a key enzyme for the sialic acids biosynthesis. Patients usually present severe thrombocytopenia and excessive bleeding. Knowledge about this condition is still poor. There are no recognized tools for diagnosis. Previous study of one patient showed reduced platelet half-life as a mechanism of thrombocytopenia; however, it is still unclear if the sialylation defect also impairs platelet biogenesis. OBJECTIVES:To gain insights into clinical aspects and platelet biogenesis in GNE-RT. METHODS:We investigated 4 novel GNE-RT patients (3 families). A recently standardized flow cytometry assay was applied to characterize platelet sialylation. Patients' megakaryocytes were cultured to study megakaryopoiesis and proplatelet formation. A validated three-dimensional bone marrow model was exploited to investigate platelet production. RESULTS:We characterized 3 novel GNE variants, demonstrating the pathogenicity of 2 VUS. In all individuals, platelet flow cytometry detected an obvious increase in RCA-1 and ECL lectins binding and decreased MAL-II binding. Sialylation of serum transferrin did not show straightforward alterations. Although presenting the sialylation defect, patients' megakaryocytes showed preserved differentiation, maturation, and proplatelet formation. Megakaryocytes generated ex-vivo a normal number of normal-sized platelets. Two patients received Eltrombopag, achieving a durable clinical response (38- and 72-month follow-up). CONCLUSIONS:The profound megakaryocyte sialylation defect induced by GNE variants does not affect platelet biogenesis. Platelet flow cytometry for RCA-1 and MAL-II binding is a reliable and simple assay for diagnosis of GNE-RT. Based on literature review, 55% of GNE-RT patients responded to thrombopoietin-mimetics.

Immunofluorescence-based platelet phenotyping using peripheral blood smears has recently emerged as a promising method for characterizing a subgroup of inherited platelet disorders (IPD). A single-center study demonstrated its potential for accurate diagnosis of 9 disorders with characteristic platelet structural changes. The aim of this study was to evaluate the reproducibility of this approach through an interlaboratory validation study. Native, air-dried blood smears from healthy controls and patients with confirmed IPD were shipped to 7 participating laboratories, blinded for the sample origin. Samples were fixed and stained using a shared panel of 13 commercially available primary antibodies and 2 fluorescence-labelled secondary antibodies. Laboratories formulated diagnostic predictions based solely on immunofluorescence findings. The pre-workshop method establishment involved other samples and feedback with the coordinating laboratory to address technical issues before blinded sample validation. All 7 laboratories (Brisbane, Greifswald, Murcia, Paris, Pavia, Perugia, and Tübingen) correctly diagnosed MYH9-related disease, Bernard-Soulier syndrome, Glanzmann thrombasthenia, and GFI1B-related thrombocytopenia. Six of 7 laboratories accurately identified TUBB1-related disorder and quantitative δ-storage pool disorder, while 5 of 7 correctly diagnosed GATA1-related thrombocytopenia. Immunofluorescence-based platelet phenotyping on peripheral blood smears demonstrated high sensitivity for diagnosing MYH9-related disease, Bernard-Soulier syndrome, Glanzmann thrombasthenia, and GFI1B-related thrombocytopenia, and reasonable sensitivity for TUBB1-related disorder, quantitative δ-storage pool disorder, and GATA1-related thrombocytopenia. Immunofluorescence analysis of blood smears may be of help in the diagnostic work-up of IPD.

Purpose Lemierre syndrome is a rare condition traditionally defined by bacterial infection of the head/neck region, local thrombophlebitis, and septic embolism. Although in most cases Fusobacterium necrophorum is isolated, it is questionable whether the presence of this microbe is mandatory for diagnosis. In this study, we investigated microorganisms isolated in cases of Lemierre syndrome and their association with demographical and clinical features. Methods We conducted an analysis of individual patient data from 712 patients diagnosed with Lemierre syndrome. Demographics, clinical presentation, treatment strategies, and outcomes according to different pathogens were evaluated. Results Among a total of 712 patients, in 574 cases bacterial growth was detected. In 415 patients Fusobacterium spp. was isolated, in 108 either Streptococcus spp. or Staphylococcus spp., and in 51 other bacteria. Patients with different bacteria differed markedly in age, site of preceding infections, clinical presentation, and treatment. Fusobacterium spp. was typically isolated in younger patients (69% of patients aged 16 to 30 years) while Streptococcus spp. and Staphylococcus spp. were more prevalent in older subjects (30% of patients aged over 45 years). Of all cases with Fusobacterium spp., 63% had a thrombosis of the internal jugular vein and 91% septic embolism, compared with 94% and 69%, respectively, in cases with Streptococcus spp. or Staphylococcus spp. Conclusion In contrast to the available literature, our study suggests that Lemierre syndrome may be caused by multiple bacterial species, and that the clinical presentation and course may vary according to the specific bacterial species involved.

ACTN1-related thrombocytopenia is a rare disorder caused by heterozygous variants in the ACTN1 gene characterized by macrothrombocytopenia and mild bleeding tendency. We describe for the first time two patients affected with ACTN1-RT caused by a homozygous variant in ACTN1 (c.982G>A) with mild heart valve defects unexplained by any other genetic variants investigated by WES. Within the reported family, the homozygous sisters have moderate thrombocytopenia and marked platelet macrocytosis with giant platelets, revealing a more severe haematological phenotype compared to their heterozygous relatives and highlighting a significant effect of allelic burden on platelet size. Moreover, we hypothesize that some ACTN1 variants, especially when present in the homozygous state, may also contribute to the cardiac abnormalities.

Correct interpretation of the pathogenicity of germline RUNX1 variants is essential for FPD/AML diagnosis, clinical management and leukaemia surveillance. We report two families with clear FPD/AML phenotypic features harbouring missense variants at RHD critical residue Gly168. Although classified as of unknown significance (VUS) by RUNX1-specific curation guidelines, these variants should rather be considered likely pathogenic, as supported by computational tools, structural modelling and dysregulated platelet expression of RUNX1-targets, adding Gly168 among amino acids currently recognised as mutational hotspots. Our data could help reduce the number of variants classified as VUS, providing evidence for updating RUNX1 guidelines, thus improving FPD/AML diagnosis.

Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants.

Hematopoietic stem and progenitor cells (HSPCs) continuously generate platelets throughout one's life. Inherited Platelet Disorders affect ≈ 3 million individuals worldwide and are characterized by defects in platelet formation or function. A critical challenge in the identification of these diseases lies in the absence of models that facilitate the study of hematopoiesis ex vivo. Here, a silk fibroin-based bioink is developed and designed for 3D bioprinting. This bioink replicates a soft and biomimetic environment, enabling the controlled differentiation of HSPCs into platelets. The formulation consisting of silk fibroin, gelatin, and alginate is fine-tuned to obtain a viscoelastic, shear-thinning, thixotropic bioink with the remarkable ability to rapidly recover after bioprinting and provide structural integrity and mechanical stability over long-term culture. Optical transparency allowed for high-resolution imaging of platelet generation, while the incorporation of enzymatic sensors allowed quantitative analysis of glycolytic metabolism during differentiation that is represented through measurable color changes. Bioprinting patient samples revealed a decrease in metabolic activity and platelet production in Inherited Platelet Disorders. These discoveries are instrumental in establishing reference ranges for classification and automating the assessment of treatment responses. This model has far-reaching implications for application in the research of blood-related diseases, prioritizing drug development strategies, and tailoring personalized therapies.

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.

INTRODUCTION:Lemierre syndrome is a thromboembolic complication following an acute bacterial infection of the head/neck area, often due to anaerobes. Data on the prognostic role of laboratory parameters is lacking. METHODS:We analyzed individual-patient level data from a multinational cohort of patients with Lemierre-syndrome. Patients had an infection in the head/neck area, and contiguous vein thrombosis or septic embolism, irrespective of the causal pathogen. We studied the patterns of white blood cell count, platelet count, and C-reactive protein concentration investigating their association with baseline characteristics and in-hospital clinical outcomes (septic embolism, major bleeding, all-cause death). RESULTS:A total of 447 (63%) patients had complete data for analysis. White blood cells were elevated across all subgroups (median 17 × 103/μL; Q1-Q3:12-21). Median platelet count was 61 × 103/μL (Q1-Q3:30-108) with decreasing levels with increasing age. Males, patients with renal failure or cardiopulmonary impairment, and those with typical Lemierre syndrome (tonsillitis, septic thromboembolism, positivity for Fusobacterium spp.) had the lowest platelet count. Median C-reactive protein was 122 (Q1-Q3:27-248) mg/L with higher values in patients who also had more severe thrombocytopenia. The overall risk of complications was similar across subgroups of patients stratified according to white blood cell and C-reactive protein levels. Patients in the lowest third of platelet count (<42 × 103/μL) had the highest rate of complications (26%), as opposed to those in the highest third (11%), notably septic embolic events. CONCLUSIONS:Common laboratory tests correlate with the clinical presentation of Lemierre syndrome. However, extreme values did not appear to be prognostically relevant for in-hospital complications and potentially able to improve clinical management.

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a recessive disorder characterized by severe reduction of megakaryocytes and platelets at birth, which evolves toward bone marrow aplasia in childhood. CAMT is mostly caused by mutations in MPL (CAMT-MPL), the gene encoding the receptor of thrombopoietin (THPO), a crucial cytokine regulating hematopoiesis. CAMT can be also due to mutations affecting the THPO coding region (CAMT-THPO). In a child with the clinical picture of CAMT, we identified the homozygous c.-323C>T substitution, affecting a potential regulatory region of THPO. Although mechanisms controlling THPO transcription are not characterized, bioinformatics and in vitro analysis showed that c.-323C>T prevents the binding of transcription factors ETS1 and STAT4 to the putative THPO promoter, impairing THPO expression. Accordingly, in the proband the serum THPO concentration indicates defective THPO production. Based on these findings, the patient was treated with the THPO-mimetic agent eltrombopag, which induced a significant increase in platelet count and stable remission of bleeding symptoms. Herein, we report a novel pathogenic variant responsible for CAMT and provide new insights into the mechanisms regulating transcription of the THPO gene.

Background  Lemierre syndrome is a potentially life-threatening disease, which affects otherwise healthy young adults and adolescents. It is characterized by acute neck vein thrombosis and septic embolism, usually complicating a bacterial infection. Data on the syndrome are sparse, particularly concerning arterial complications. Methods  We evaluated the frequency and patterns of cerebral arterial and cardiac involvement (“arterial complications”) in an individual patient level cohort of 712 patients, representing all cases described over the past 20 years in the medical literature who fulfilled the criteria: (1) bacterial infection in the neck/head site and (2) objectively confirmed thrombotic complication or septic embolism. The study outcomes were defined as all-cause in-hospital deaths and the occurrence of clinical sequelae at discharge or in the postdischarge period. Results  A total of 55 (7.7%) patients had an arterial complication. The most frequent arterial complications were carotid involvement (52.7%), stroke (38.2%), and pericardial complications (20%). Patients with an arterial involvement were more likely to be treated with a greater number of antibiotics (23 vs. 10%) and to receive anticoagulation. In addition, patients with arterial complications had a greater risk of all-cause death ( n  = 20/600, 3.3% vs. n  = 6/52, 12%; odds ratio [OR]: 3.8; 95% confidence interval [CI]: 1.5–9.9) and late clinical sequelae ( n  = 49/580, 9.0% vs. n  = 15/46, 35%; OR: 5.2; 95% CI: 2.65–10.37). Conclusions  While Lemierre syndrome is known to be primarily characterized by venous thromboembolic events, our results suggest that local or distant arterial complications may occur in approximately one-tenth of patients and may be associated with a greater risk of long-term sequelae and death.