RESUMO
Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.
Assuntos
Síndrome da Plaqueta Cinza , Animais , Plaquetas , Proteínas Sanguíneas , Grânulos Citoplasmáticos , Síndrome da Plaqueta Cinza/genética , Humanos , Camundongos , NeutrófilosRESUMO
Gray platelet syndrome (GPS) is an inherited disorder. Patients harboring GPS have thrombocytopenia with large platelets lacking α-granules. A long-term complication is myelofibrosis with pancytopenia. Hematopoietic stem cell transplant (HSCT) could be a curative treatment. We report a male GPS patient with severe pancytopenia, splenomegaly and a secondary myelofibrosis needing red blood cells transfusion. He received an HSCT from a 10/10 matched HLA-unrelated donor after a myeloablative conditioning regimen. Transfusion independence occurred at day+21, with a documented neutrophil engraftment. At day+ 180, we added ruxolitinib to cyclosporine and steroids for a moderate chronic graft versus host disease (GVHD) and persistent splenomegaly. At day+240 GVHD was controlled and splenomegaly reduced. Complete donor chimesrism was documented in blood and marrow and platelets functions and morphology normalized. At day+ 720, the spleen size normalized and there was no evidence of marrow fibrosis on the biopsy. In GPS, HSCT may be a curative treatment in selected patients with pancytopenia and myelofibrosis.
Assuntos
Plaquetas/patologia , Síndrome da Plaqueta Cinza/terapia , Transplante de Células-Tronco Hematopoéticas , Mielofibrose Primária/terapia , Adulto , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Ciclosporina/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Síndrome da Plaqueta Cinza/tratamento farmacológico , Síndrome da Plaqueta Cinza/fisiopatologia , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Nitrilas , Pirazóis/uso terapêutico , Pirimidinas , Esplenomegalia/tratamento farmacológico , Esplenomegalia/etiologia , Fatores de Tempo , Condicionamento Pré-TransplanteRESUMO
Venous thrombosis affecting thrombocytopenic patients is challenging. We report the case of a woman affected by deep vein thrombosis and pulmonary embolism in a thrombocytopenic context leading to the discovery of a heterozygous mutation in the gene encoding ankyrin repeat domain 26 (ANKRD26) associated with a heterozygous factor V (FV) Leiden mutation. This woman was diagnosed with lower-limb deep vein thrombosis complicated by pulmonary embolism. Severe thrombocytopenia was observed. The genetic study evidenced a heterozygous FV Leiden mutation. Molecular study sequencing was performed after learning that her family had a history of thrombocytopenia. Previously described heterozygous mutation c-127C>A in the 5'untranslated region (5'UTR) of the ANKRD26 gene was detected in the patient, her aunt, and her grandmother. ANKRD26-related thrombocytopenia and thrombosis are rare. This is, to our knowledge, the first case reported in the medical literature. This mutation should be screened in patients with a family history of thrombocytopenia.
RESUMO
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 FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 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%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Trombocitopenia/etiologia , Adulto , Plaquetas/patologia , Plaquetas/ultraestrutura , Núcleo Celular/química , Variação Genética , Humanos , Masculino , Megacariócitos/patologia , Pessoa de Meia-Idade , Agregação Plaquetária/genética , Proteína Proto-Oncogênica c-fli-1/genética , Trombocitopenia/congênito , Transcrição Gênica , Adulto JovemRESUMO
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.
Assuntos
Plaquetas/metabolismo , Mutação em Linhagem Germinativa , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Trombopoese/genética , Antígenos CD34/metabolismo , Contagem de Células Sanguíneas , Diferenciação Celular , Família , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Hiperplasia , Masculino , Megacariócitos/citologia , Megacariócitos/metabolismo , Megacariócitos/patologia , Linhagem , Fenótipo , Contagem de Plaquetas , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
MYH9-related disease (MYH9-RD) is one of the most frequent autosomal-dominant forms of inherited macrothrombocytopenias and is caused by mutations in MYH9 (nonmuscle myosin IIA), the gene coding for the heavy chain of the nonmuscle myosin IIA. Affected individuals can present with isolated thrombocytopenia, and whereas only some will have bleeding events requiring intervention, nearly all will require the use of prophylactic platelet transfusions before surgery. Here we report the first prophylactic use of eltrombopag before surgery in a child with MYH9-RD. Our patient was a 13-year-old girl with an MYH9 S96L missense mutation who required a tympanoplasty due to chronic otitis media. Pretreatment microscopic platelet count was 10 × 10(9)/L. The child was treated with eltrombopag starting 4 weeks before her planned surgery. On the day of surgery her platelet count was 70 × 10(9)/L. She required no platelet transfusions and no abnormal bleeding was reported either during surgery or postoperatively. Given these results, the first reported in a child, we suggest that the use of this thrombopoietic agent should be further evaluated as a useful presurgical prophylactic option in this hereditary thrombocytopenia, thus avoiding the use of platelet transfusions and their associated risks, which include alloimmunization and the transmission of infectious agents.
Assuntos
Benzoatos/uso terapêutico , Aberrações Cromossômicas , Hidrazinas/uso terapêutico , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Otite Média/cirurgia , Cuidados Pré-Operatórios , Pirazóis/uso terapêutico , Receptores de Trombopoetina/agonistas , Trombocitopenia/tratamento farmacológico , Trombocitopenia/genética , Perfuração da Membrana Timpânica/cirurgia , Timpanoplastia , Adolescente , Feminino , Humanos , Mutação de Sentido Incorreto , Otite Média/sangue , Contagem de Plaquetas , Trombocitopenia/sangue , Perfuração da Membrana Timpânica/sangueRESUMO
We report a case of lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) in an 11 year old girl initially hospitalized for bleeding. The patient presented with petechia, persisting bleeding after tooth extraction performed two days before, nephritic syndrome (renal failure, proteinuria and macroscopic hematuria), severe anemia, thrombocytopenia, lymphopenia. The association of these abnormalities suggested LAHPS secondary to severe systemic lupus. Immediate treatment with fresh frozen plasma and intravenous immunoglobulins (400 mg/kg/5d) was started and followed by steroid (500 mg/d) and cyclophosphamide (800 mg/m(2)) pulse therapy leading to rapid improvement of bleeding, renal involvement and prothrombin levels within 13 days. Lupus diagnosis was confirmed by immunological investigations and renal biopsy. Two early relapses occurred despite adequate treatment. After a follow-up of two years, no further disease activity is noted while the patient is treated only by mycophenolate mofetil (1 200 mg/m(2)/d). LAHPS did not relapse during this follow-up.