Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic ...malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34
cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34
cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.
Glanzmann thrombasthenia (GT) is a genetic bleeding disorder characterised by severely reduced/absent platelet aggregation in response to multiple physiological agonists. The severity of bleeding in ...GT varies markedly, as does the emergency situations and complications encountered in patients. A number of emergency situations may occur in the context of GT, including spontaneous or provoked bleeding, such as surgery or childbirth. While general management principles apply in each of these settings, specific considerations are essential for the management of GT to avoid escalating minor bleeding events. These recommendations have been developed from a literature review and consensus from experts of the French Network for Inherited Platelet Disorders, the French Society of Emergency Medicine, representatives of patients' associations, and Orphanet to aid decision making and optimise clinical care by non-GT expert health professionals who encounter emergency situations in patients with GT.
Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 ...individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel
variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The
variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25-29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7-9%) and autophagosome-like structures (0-3%).
study of megakaryocytes derived from circulating CD34
cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the
variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in
variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of
variant-associated thrombocytopenia.
Introduction
Data on failure to identify the molecular mechanism underlying FXI deficiency by Sanger analysis and the contribution of gene segment deletions are almost inexistent.
Aims and methods
...Prospective and retrospective analysis was conducted on FXI‐deficient patients’ DNA via Next Generation Sequencing (NGS), or Sanger sequencing and Multiplex Probe Ligation‐dependent Assay (MLPA) to detect cryptic causative gene variants or gene segment deletions.
Results
Sanger analysis or NGS enabled us to identify six severe and one partial (median activity 41 IU/dl) FXI deficient index cases with deletions encompassing exons 11–15, the whole gene, or both. After Sanger sequencing, retrospective evaluation using MLPA detected seven additional deletion cases in apparently homozygous cases in non‐consanguineous families, or in previously unsolved FXI‐deficiency cases. Among the 504 index cases with a complete genetic investigation (Sanger/MLPA, or NGS), 23 remained unsolved (no abnormality found n = 14 or rare intronic variants currently under investigation, n = 9). In the 481 solved cases (95% efficiency), we identified F11 gene‐deleted patients (14 cases; 2.9%). Among these, whole gene deletion accounted for four heterozygous cases, exons 11–15 deletion for five heterozygous and three homozygous ones, while compound heterozygous deletion and isolated exon 12 deletion accounted for one case each.
Conclusion
Given the high incidence of deletions in our population (2.9%), MLPA (or NGS with a reliable bioinformatic pipeline) should be systematically performed for unsolved FXI deficiencies or apparently homozygous cases in non‐consanguineous families.
Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor ...(TFPI) level.
To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency.
Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT).
Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was
, AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR.
In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.
GATA1 is an essential transcription factor for both polyploidization and megakaryocyte (MK) differentiation. The polyploidization defect observed in GATA1 variant carriers is not well understood.
To ...extensively phenotype two pedigrees displaying different variants in the GATA1 gene and determine if GATA1 controls MYH10 expression levels, a key modulator of MK polyploidization.
A total of 146 unrelated propositi with constitutional thrombocytopenia were screened on a multigene panel. We described the genotype-phenotype correlation in GATA1 variant carriers and investigated the effect of these novel variants on MYH10 transcription using luciferase constructs.
The clinical profile associated with the p.L268M variant localized in the C terminal zinc finger was unusual in that the patient displayed bleeding and severe platelet aggregation defects without early-onset thrombocytopenia. p.N206I localized in the N terminal zinc finger was associated, on the other hand, with severe thrombocytopenia (15G/L) in early life. High MYH10 levels were evidenced in platelets of GATA1 variant carriers. Analysis of MKs anti-GATA1 chromatin immunoprecipitation-sequencing data revealed two GATA1 binding sites, located in the 3' untranslated region and in intron 8 of the MYH10 gene. Luciferase reporter assays showed their respective role in the regulation of MYH10 gene expression. Both GATA1 variants significantly alter intron 8 driven MYH10 transcription.
The discovery of an association between MYH10 and GATA1 is a novel one. Overall, this study suggests that impaired MYH10 silencing via an intronic regulatory element is the most likely cause of GATA1-related polyploidization defect.
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