Haematologica 2002; 87:(05)ELT27
[Medline] [prev] [index] [next]Autosomal dominant macrothrombocytopenia with ineffective thrombopoiesis
Fabrizio Fabris, Franca Fagioli,** Giuseppe Basso,* Antonio Girolami
Department of Medical and Surgical Sciences and * Department of Pediatrics, University of Padua Medical School; **Department of Pediatrics and the Laboratory of cell Biology , University of Torino, Medical School, Turin, Italy
Correspondence:Fabrizio Fabris MD, Istituto di Semeiotica Medica,via Ospedale 105, 35128 Padova Italy. Phone +39-049-8212668. Fax number: +39-049-657391. E-mail: fabrizio.fabris@unipd.it
Hereditary forms represent 5% of all thrombocytopenias and an early diagnosis is necessary to avoid inappropriate treatments such as steroids or splenectomy. Congenital thrombocytopenias are heterogeneous and their classification can be made from the inheritance pattern, platelet function, platelet morphology and the presence of additional phenotypic abnormalities. The most frequent form is that characterized by an autosomal dominant trait, mild thrombocytopenia and presence of large platelets.1 Within this group, the defect of MYH9 gene2 and the Bernard-Soulier type Bolzano variant3 have been recently identified in Italy. We recently described 47 subjects belonging to 13 unrelated families characterized by chronic isolated macrothrombocytopenia (CHMT) transmitted as an autosomal dominant trait , mild hemorrhages, large platelets with normal survival, increased expression of surface glycoproteins and absence of associated anomalies;4 this disorder can be considered the "true type" of non-syndromic autosomal dominant macrothrombocytopenia . The pathogenesis of CHMT is still unknown but there is indirect evidence that it is due to ineffective thrombopoiesis. In fact the patients have a normal number of megakaryocytes, a normal percentage of reticulated platelets and only a mild increase of thrombopoietin levels5 as reported in another family with an autosomal dominant form of thrombocytopenia.6 We performed an in vitro study of megakaryocytopoiesis, in 5 patients from 4 families with CHMT to verify the hypothesis of a defective platelet production.
Five milliliters of bone marrow aspirates were obtained from patients and adult volunteers after informed consent. Cultures were performed using 1x105 low-density bone marrow cells (LDBMC) isolated by density centrifugation (Lymphoprep, 1077) and enriched by separation of mature cells by phagocytosis.7 After immunomagnetic purification of LDBMC, CFU-MK were determined, after 14 days, in a plasma clot medium supplemented with a combination of thrombopoietin (TPO) 1 IU/mL and interleukin-3 (IL3) 10 ng/mL. BFU-E colonies were determined in IMDM containing 30% FBS and 1% methylcellulose medium supplemented with 10 ng/mL of IL3, 50 ng/mL of SCF and 3 IU/mL of erythropoietin (EPO) and CFU-GM colonies were determined in semisolid IMDM cultures containing 15% FBS and 0.3% agar supplemented with 10 ng/mL of IL3, 20 ng/mL of G-CSF and 50 ng/mL of GM-CSF. The study of apoptosis was performed by the TUNEL method as previously described.8 CD34+ bone marrow cells were obtained and isolated from LDBMC by an immunoabsorption technique based on the use of polystyrene tissue-flasks coated with anti-CD34 antigen; 4x104 CD34+ cells, were cultured for 14 days in serum-free liquid medium supplemented with a combination of thrombopoietin (TPO) 1 IU/mL and interleukin-3 (IL3) 10 ng/mL. Apoptosis of CD41+ cells was evaluated after 14 days of culture. The results are summarized in Table 1. Compared to normal volunteers, we found a great proliferation of CFU-MK and BFU-E in all five patients with CHMT that we studied while CFU-GM were normal in all but one. This indicates a great number of erythrocyte and platelet progenitors in bone marrow. Furthermore, The MK colonies from the affected patients were confluent and larger than those from controls. MK colonies of two affected patients showed, on day 14, apoptosis of 36% and 32% which was markedly increased in comparison to that of the controls. The study suggests that in CHMT, the peripheral macrothrombocytopenia with normal platelet survival resulted from a sudden death of an increased number of immature MK. Drachman et al.6 Recently showed, a considerable increase of BFU-E and a dramatic proliferation of CFU-MK from two patients with non-syndromic autosomal dominant thrombocytopenia, normal platelet volume and linkage to human chromosome 10. On the other hand, patients with Paris-Trousseau congenital thrombocytopenia Ref 9 associated with a partial deletion of the long arm of chromosome 11, had increased MK, numerous microMK with massive cell death. In conclusion, in spite of the heterogeneous phenotype of autosomal dominant thrombocytopenias, our study supports a common mechanism characterized by increased proliferation of hematopoietic precursors, decreased maturation and excessive apoptosis of MK.6,9 Further genetic and molecular studies should yield new insights into the pathogenesis of the disease.References