Germ-line heterozygous mutations in the hematopoietic transcription factor AML1 (RUNX1) have been identified in patients with familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), which is characterized by thrombocytopenia, abnormal platelet function, and propensity to myeloid malignancies. We identified a novel mutation in the AML1 gene in an FPD/AML pedigree characterized by a single nucleotide deletion that generates a frameshift and premature chain termination (Pro218fs-Ter225). Both wild-type and mutant transcripts were expressed in affected individuals by allele-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Thrombopoietin (TPO) binds to the Mpl receptor and is the major regulator of megakaryopoiesis. To explore the mechanisms underlying thrombocytopenia, we studied the TPO/Mpl pathway in this newly identified pedigree. TPO levels were mildly to moderately elevated. On flow cytometry and immunoblotting, Mpl receptor expression was decreased and TPOinduced signaling was impaired. While no mutations were identified in the MPL gene by sequence analysis, low MPL mRNA levels were found, suggesting decreased gene expression. Of particular interest, several AML1-binding motifs are present in the MPL promoter, suggesting MPL is an AML1 target. In conclusion, we identified a C-terminal AML1 mutation that leads to a decrease in Mpl receptor expression, providing a potential explanation for thrombocytopenia in this FPD/AML pedigree. IntroductionFamilial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder characterized by moderate thrombocytopenia, a defect in platelet function, and propensity to develop myeloid malignancy. 1 Germ-line heterozygous mutations in the hematopoietic transcription factor AML1, also known as RUNX1 and CBFA2, have been identified in 12 pedigrees reported so far, including missense, frameshift, and nonsense mutations and a large intragenic deletion. [2][3][4][5][6] AML1 is the DNA-binding subunit of the core binding factor (CBF) transcription complex. Heterodimerization with the non-DNA-binding subunit, CBF, increases the affinity of AML1 to DNA and protects it from proteolytic degradation. 7,8 AML1 includes a runt homology domain (RHD), which mediates DNA binding and heterodimerization with CBF, and a carboxy (C)-terminal domain responsible for transcriptional activation. 9 The AML1/CBF complex regulates the expression of genes specific to hematopoiesis, including several cytokines and their receptors, such as granulocyte macrophage colony-stimulating factor, interleukin-3, and macrophage colony-stimulating factor receptor. 7 This transcription factor is essential for the establishment of definitive hematopoiesis, as demonstrated in AML1 Ϫ/Ϫ mice, which show a complete absence of fetal liver hematopoiesis and die during embryonic development, precluding analysis of the effects of AML1 in later stages of blood-cell development. 10 The role of this transcription factor in plate...
The mechanisms underlying increased thrombotic risk in chronic myeloproliferative neoplasms (MPN) are incompletely understood. We assessed whether neutrophil extracellular traps (NETs), which promote thrombosis, contribute to the procoagulant state in essential thrombocythemia, polycythemia vera and myelofibrosis (MF) patients. Although MPN neutrophils showed increased basal reactive oxygen species (ROS), enhanced NETosis by unstimulated neutrophils was an infrequent finding, whereas PMA-triggered NETosis was impaired, particularly in MF, due to decreased PMA-triggered ROS production. Elevated circulating nucleosomes were a prominent finding and were higher in patients with advanced disease, which may have potential prognostic implication. Histone-MPO complexes, proposed as specific NET biomarker, were seldomly detected, suggesting NETs may not be the main source of nucleosomes in most patients, whereas their correlation with high LDH points to increased cell turn-over as a plausible origin. Lack of association of nucleosomes or NETs with thrombosis or activation markers does not support their use as predictors of thrombosis although prospective studies in a larger cohort may help define their potential contribution to MPN thrombosis. These results do not provide evidence for relevant in vivo NETosis in MPN patients under steady state conditions, although availability of standardized NET biomarkers may contribute to further research in this field.
The frequency of the JAK2V617F mutation in platelets was similar to that reported in granulocytes in the literature, suggesting this mutation does not occur as an isolated event in the megakaryocyte lineage. If confirmed in a larger study, the observed higher frequency of thrombosis in patients younger than 60 might be a useful predictive marker for thrombosis in this subset of patients. Even though this mutation has been predicted to constitutively activate the JAK2 kinase, spontaneous phosphorylation of STAT5 does not seem to be a frequent finding in platelets from ET patients.
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