Increased Expression of the PRV-1 Gene in Thalassemia Reflects the Rate of the Underlying Erythropoietic Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2687-2687
Author(s):  
Katerina Zoi ◽  
Christine Zoi ◽  
Ersi Voskaridou ◽  
Evangelos Terpos ◽  
Dimitris Loukopoulos
Keyword(s):  
Polycythemia Vera ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Close Relation ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia ◽  
Ineffective Erythropoiesis ◽  
Elevated Expression ◽  
Normal Controls

Abstract Over the last years, PRV-1 expression has been considered to be closely connected with polycythemia vera (PV). However, the precise function of the PRV-1 protein has never been clarified while the search for the pathogenetic mechanism of PV has shifted to the association between the PRV-1 gene and the JAK2 V617F mutation rather than the activity of PRV-1 alone. Our present knowledge regarding the PRV-1 protein is summarized in that it is a GPI-anchored membrane protein and that its amount in the surface of the mononuclear cells of PV patients is almost normal in contrast to its high mRNA expression. The PRV-1 gene is also overexpressed in some ET and IMF cases, but not in CML, AML and secondary erythrocytosis and thrombocytosis. Whilst studying PRV-1 expression in PV patients, we noticed elevated expression of this gene in a number of controls. Further analysis revealed that these controls were not hematologically normal but instead were beta thalassaemia carriers. We therefore went onto to study PRV-1 expression in 20 beta-thalassemia carriers, 20 patients with thalassemia intermedia (hence, intensive ineffective erythtopoiesis), 20 with sickle cell thalassemia (not transfused; hence, moderately intensive ineffective erythropoiesis), 14 heavily transfused patients with thalassemia major (hence, suppressed erythropoiesis) and 34 normal controls. Expression of PRV-1 was determined by RQ-PCR assay using the ABI PRISM 7000SDS. The values obtained were normalized using the ABL gene as endogenous control with the deltadeltaCt method. Furthermore, we examined two more parameters which are considered to reflect the extent of total erythropoiesis, i.e. the serum levels of (a) Erythropoetin (EPO) and (b) Transferrin Receptors, using standard ELISA based assays. Results in TABLE I display an unequivocally elevated expression of the PRV-1 transcripts in all groups of patients with thalassemia, in apparent relation with the intensity of ineffective erythropoiesis occurring in each individual case. The latter statement is supported by the observation that the increase of PRV-1 expression in the mononuclear cells of patients with various types of thalassemia occurs in close relation to their EPO and sTFR levels. We conclude that elevated PRV-1 expression is not confined to the myeloproliferative disorders, but is also seen in genetic disorders with ineffective hematopoiesis. This finding brings up a number of challenging hypotheses regarding the significance of the PRV-1 protein, especially when considering that the high expression of this gene is detected in the mononuclear WBC of the patients and not in their erythroid cells, and opens the way for several additional experiments. TABLE I No PRV-1/ABL (m±SD) EPO (m±SD) sTfR (m±SD) Normal controls 34 12.9±20.05 4.5±10.9 17.9±11.5 beta-thalassemia carriers 20 67.5±98.37 not determined not determined Thalassemia intermedia 20 430.8±308.33 110.0±59.3 104.2±18.6 HbS/beta-thalassemia 20 190.5±352.5 103.0±114.4 94.5±22.7 Thalassemia major 14 76.8±46.7 66.4±52.5 76.2±34.4 Polycythemia vera 52 8921.1± 12861 not determined not determined

scholarly journals Erythrocyte membrane skeleton abnormalities in severe beta-thalassemia

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 158-164 ◽  
Author(s):  
E Shinar ◽  
O Shalev ◽  
EA Rachmilewitz ◽  
SL Schrier
Keyword(s):  
Electron Microscopy ◽  
Protein Composition ◽  
Thalassemia Major ◽  
Membrane Skeleton ◽  
Skeletal Structure ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia ◽  
Transmission Electron ◽  
Membrane Skeletons ◽  
Normal Controls

The protein composition of ghosts, inside-out vesicles (IOV), and membrane skeletons (MS) of erythrocytes (RBC) from splenectomized (spx) and nonsplenectomized (non-spx) patients with beta-thalassemia major and beta-thalassemia intermedia was determined. Ghosts from spx thalassemia intermedia patients had a significant increase in their globin content (which was mostly heme reactive) and contained extra polypeptides in the protein 4.2 to 5 and 6-globin areas. The Triton- extracted MS from all of the thalassemic patients showed two major abnormalities: they retained up to twice the amount of protein 3 when compared with controls; they had a significant increase in their globin content, the concentration of which was independent of their protein 3 content. Analysis of the IOV revealed no differences between those prepared from normal controls and those of the patients. MS from spx thalassemia intermedia patients were grossly abnormal when examined by scanning electron microscopy and they exhibited aggregates of material that on transmission electron microscopy suggested the presence of globin precipitates. We propose that, although the integral protein composition, as reflected in the IOV, from severely affected beta- thalassemics is intact, their MS assembly is deranged. The altered skeletal structure of thalassemic RBC could result from attachment of denatured globin to the skeleton components. These abnormalities may contribute to the premature cell death seen in severe beta-thalassemia.

scholarly journals Erythrocyte membrane skeleton abnormalities in severe beta-thalassemia

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 158-164 ◽  
Author(s):  
E Shinar ◽  
O Shalev ◽  
EA Rachmilewitz ◽  
SL Schrier
Keyword(s):  
Electron Microscopy ◽  
Protein Composition ◽  
Thalassemia Major ◽  
Membrane Skeleton ◽  
Skeletal Structure ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia ◽  
Transmission Electron ◽  
Membrane Skeletons ◽  
Normal Controls

Abstract The protein composition of ghosts, inside-out vesicles (IOV), and membrane skeletons (MS) of erythrocytes (RBC) from splenectomized (spx) and nonsplenectomized (non-spx) patients with beta-thalassemia major and beta-thalassemia intermedia was determined. Ghosts from spx thalassemia intermedia patients had a significant increase in their globin content (which was mostly heme reactive) and contained extra polypeptides in the protein 4.2 to 5 and 6-globin areas. The Triton- extracted MS from all of the thalassemic patients showed two major abnormalities: they retained up to twice the amount of protein 3 when compared with controls; they had a significant increase in their globin content, the concentration of which was independent of their protein 3 content. Analysis of the IOV revealed no differences between those prepared from normal controls and those of the patients. MS from spx thalassemia intermedia patients were grossly abnormal when examined by scanning electron microscopy and they exhibited aggregates of material that on transmission electron microscopy suggested the presence of globin precipitates. We propose that, although the integral protein composition, as reflected in the IOV, from severely affected beta- thalassemics is intact, their MS assembly is deranged. The altered skeletal structure of thalassemic RBC could result from attachment of denatured globin to the skeleton components. These abnormalities may contribute to the premature cell death seen in severe beta-thalassemia.

scholarly journals Unusual combination and existence of hemolytic anemias; Thalassemia and G6PD deficient anemia in female patient

2018 ◽  
Vol 6 (4) ◽  
pp. 308-314
Author(s):  
Abdulhamza Rajooj Hmood ◽  
Rawaa Hatif Abd
Keyword(s):  
Hemolytic Anemia ◽  
Broad Bean ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia ◽  
Ineffective Erythropoiesis ◽  
Acid Therapy ◽  
First Case ◽  
Unusual Combination ◽  
Appropriate Therapy ◽  
Carrier Diagnosis

Beta-thalassemia intermedia exhibits feature of ineffective erythropoiesis and hemolytic anemia. Diagnosis can be made via hemoglobin electrophoresis. G6PD deficiency is an X-linked recessive disorder commonly affecting males while females are carrier. Diagnosis is made by measuring G6PD level. A 19-year old pregnant lady, known case of β-thalassemia intermedia, had been presented with episode of acute hemolytic anemia. Investigations were highly suggestive of G6PD deficient anemia. This was confirmed with low G6PD level. Back to her immediate history, consumption of broad bean was ascertained. After appropriate therapy, the patient felt better and her medical derangement was normalized. She delivered normally and kept on life-long folic acid therapy. The importance of recording such a case report is to expect unusual combination of hemolytic anemia despite the uncommon finding of X-linked recessive disorder in women. This was the first case recorded in Karbala province

scholarly journals Globin Chain Synthesis in the Marrow and Reticulocytes of Beta Thalassemia, Hemoglobin H Disease, and Beta Delta Thalassemia

Blood ◽  
1972 ◽  
Vol 40 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Mordechai Shchory ◽  
Bracha Ramot
Keyword(s):  
Bone Marrow ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Globin Chain ◽  
Thalassemia Intermedia ◽  
Thalassemia Minor ◽  
Order Of Magnitude ◽  
Hb H Disease ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract α, β, and γ globin chain synthesis in bone marrow and peripheral blood reticulocytes were studied in two patients with thalassemia major, two with thalassemia intermedia, one with thalassemia minor, one with Hb H disease, and one with homozygous βδ-thalassemia. Nine nonthalassemic patients served as controls. In thalassemia major, a marked imbalance of α- to β-chain synthesis was found in the bone marrow as well as in reticulocytes. The imbalance, however, was slightly more evident in the latter. In the patients with thalassemia intermedia and minor the α- to β-globin chain ratios in the reticulocytes were of the same order of magnitude, despite the marked clinical differences between thalassemia intermedia and minor. A balanced synthesis was found in the bone marrow of the patient with thalassemia minor. The bone marrow globin synthesis in thalassemia intermedia was not studied. Contrary to that in Hb H disease and βδ-thalassemia, the imbalance was more apparent in the bone marrow. In the latter, no evidence for imbalance was detected in the reticulocytes. These results point out the need for further studies on globin chain synthesis in the bone marrow and reticulocytes of patients With the various thalassemia syndromes and the effect of the free globin chain pool on those results.

scholarly journals Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe beta-thalassemia (Cooley's anemia) [see comments]

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 374-377 ◽  
Author(s):  
J Yuan ◽  
E Angelucci ◽  
G Lucarelli ◽  
M Aljurf ◽  
LM Snyder ◽  
...  
Keyword(s):  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Ineffective Erythropoiesis ◽  
Normal Individuals ◽  
Erythroid Precursors ◽  
Transplantation Program ◽  
Life Threatening ◽  
Thalassemia Trait ◽  
Cooley's Anemia ◽  
Morphologic Data

Abstract The profound and life-threatening anemia in patients with Cooley's anemia is ascribed primarily to intramedullary hemolysis (ineffective erythropoiesis), the cause of which is obscure. Based on prior morphologic data showing nuclear abnormalities, we hypothesized that accelerated apoptosis could occur in these erythroid precursors. The highly successful bone marrow (BM) transplantation program for patients with Cooley's anemia provided us with a unique opportunity to test this hypothesis. We obtained pretransplantation BM aspiration samples from patients undergoing BM transplantation in Pesaro, Italy and from their allogeneic donors. The erythroid precursors were isolated using ficoll sedimentation and then panning selecting fro CD45- cells. Cytospin and Giemsa staining showed that the separation provided greater than 90% erythroblasts. Five million of these erythroblasts were lysed and their DNA was isolated. There were obvious ladder patterns of DNA breakdown products in beta-thalassemia major samples, with less occurring in beta- thalassemia trait. Normal individuals showed only a slight smear of breakdown of DNA. These results indicate there is enhanced apoptosis in the erythroblasts in the BMs of Cooley's anemia patients. This finding might partially explain why most of these erythroblasts never survive to become mature erythrocytes.

scholarly journals Early detection of premature atherosclerosis in β-thalassemia patients by measuring carotid intima-media thickness

QJM ◽  
2020 ◽  
Vol 113 (Supplement_1) ◽  
Author(s):  
W E Ibrahim ◽  
O I Youssef ◽  
H G A Ali ◽  
D M A Alnagar
Keyword(s):  
Carotid Artery ◽  
Early Detection ◽  
Iron Overload ◽  
Lipid Profile ◽  
Intima Media Thickness ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia ◽  
Premature Atherosclerosis ◽  
Media Thickness

Abstract Background Beta-thalassemia patients still suffer from many complications. Transfused patients may develop complications related to iron overload including growth retardation and failure or delay of sexual maturation, cardiac involvement (dilated cardiomyopathy or rarely arrhythmia), liver (fibrosis and cirrhosis), endocrine glands (diabetes mellitus, hypogonadism, insufficiency of parathyroid, thyroid, pituitary and less commonly, adrenal glands). Purpose The present study was undertaken to evaluate the role of Carotid artery intima media thickness (CIMT) measurement as an early detector of premature atherosclerosis in beta-thalassemia children and early adolescents and its relation to biochemical risk factors as iron overload and lipid profile. Patients and Method Twenty-two β-thalassemia major (TM), 8 β-thalassemia intermedia (TI) with confirmed diagnosis of beta-thalassemia (major and intermedia) proved by clinical and laboratory investigations, frequent blood transfusion, chelation therapy with their age ranging from 10 to18 years old and 30 age-and sex matched healthy controls were included. Lipid profile (by colorimetric assay), serum ferritin, and CIMT measurements using high-resolution B-mode ultrasonography were estimated. Results CIMT of thalassemic patients (major and intermedia) was highly significantly increased compared to controls with no significant difference between β-thalassemia major and β thalassemia intermedia groups could be detected. CIMT was positively correlated with serum ferritin, TG, Total cholesterol level in both diseased groups and LDL level in B-TM group only. This provides a good evidence of the presence of premature atherosclerosis in vascular-free TM and TI patients and its relation to increased body iron and dyslipidemia. Conclusion Carotid artery intima media thickness represented a simple, accurate and non-invasive method for early detection of premature atherosclerosis which started early in β- thalassemia patients This study identified a relationship between body iron status, dyslipidemia and increased carotid IMT..

scholarly journals Molecular analysis of beta zero-thalassemia intermedia in Sardinia

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 823-827 ◽  
Author(s):  
R Galanello ◽  
E Dessi ◽  
MA Melis ◽  
M Addis ◽  
MA Sanna ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
Frameshift Mutation ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Thalassemia Intermedia ◽  
Mild Phenotype ◽  
Beta Globin Gene ◽  
Alpha Thalassemia

Abstract In this study we have carried out alpha- and beta-globin gene analysis and defined the beta-globin gene polymorphisms in a group of patients with thalassemia intermedia of Sardinian descent. A group of patients (109) with thalassemia major of the same origin served as control. Characterization of the beta-thalassemia mutation showed either a frameshift mutation at codon 6 or a codon 39 nonsense mutation. We found that homozygotes for the frameshift mutation at codon 6 or compound heterozygotes for this mutation and for the codon 39 nonsense mutation develop thalassemia intermedia more frequently than thalassemia major. The frameshift mutation at codon 6 was associated with haplotype IX that contains the C-T change at position -158 5′ to the G gamma globin gene implicated in high gamma chain production and thus the mild phenotype. In patients' homozygotes for codon 39 nonsense mutation, those with thalassemia intermedia more frequently had the two- gene deletion form of alpha-thalassemia, or functional loss of the alpha 2 gene as compared with those with thalassemia major. In a few siblings with thalassemia major and intermedia, the thalassemia intermedia syndrome correlated with the presence of the -alpha/-alpha genotype. No cause for the mild phenotype was detected in the majority of patients who had not inherited either haplotype IX or alpha- thalassemia.

Relationship between JAK2 V617F Mutation Status, Granulocyte CD177 mRNA Expression and CD177 Soluble Protein Level in Patients with Polycythemia Vera.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2578-2578
Author(s):  
Daniela Pietra ◽  
Alessandra Balduini ◽  
Carmela Marseglia ◽  
Matteo G. Della Porta ◽  
Luca Malcovati ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Polycythemia Vera ◽  
Protein Level ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Serum Samples ◽  
Close Relationship ◽  
V617f Mutation

Abstract A unique gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently described in patients with polycythemia vera (PV), essential thrombocythemia and chronic idiopathic myelofibrosis [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Although the currently available data clearly demonstrate that the JAK2 V617F mutation participates in the pathogenesis of myeloproliferative disorders, the mutation’s precise place in the hierarchical order of pathogenetic events remains to be established. We have recently reported that altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2 (Blood. 2005 Aug 4; Epub ahead of print). Granulocyte CD177 (PRV1) mRNA overexpression has been initially reported as a potential marker of PV but later shown by us to rather be a marker of neutrophil activation [Br J Haematol. 2004 Sep;126(5):650–6]. In this study, we analyzed the relationship between JAK2 V617F mutation status, granulocyte CD177 mRNA expression and CD177 soluble protein level in 72 patients with PV. We also investigated the ontogeny of CD177 expression by hematopoietic cells with the aim of defining the stage of mRNA expression during myeloid, erythroid and megakaryocytic cell differentiation. Finally we studied the effect of soluble CD177 protein on hematopoietic cell proliferation and differentiation. Granulocyte CD177 mRNA expression and percentage of JAK2 V617F alleles were evaluated by quantitative Real Time PCR (qRT-PCR), while serum CD177 protein level was measured by a flow cytometry-based competitive antibody-binding assay. Liquid cultures were performed by culturing peripheral blood mononuclear cells obtained from healthy individuals and PV patients in the presence of high CD177-expressing, low CD177-expressing or CD177-depleted sera. After 12 days of culture, cells were collected, counted and evaluated for colony growth, and for flow cytometry analysis of myeloid, erythroid, megakaryocytic and CD34-positive cell subpopulations. qRT-PCR studies showed a close relationship between CD177 mRNA level and percentage of JAK2 V617F alleles (r=0.412, P<0.001). CD177 mRNA expression was almost undetectable in cell populations other than granulocytes. Studies of CFU-GM growth and differentiation indicated that CD177 mRNA expression is a late event restricted to the neutrophil stage of differentiation. Analysis of serum samples showed variable values for mean fluorescence intensity (MFI), indicating variable levels of the soluble CD177 protein in the patients studied. A very close relationship was found between granulocyte CD177 mRNA expression and soluble CD177 protein level (r=0.56, P=0.02). Incubation of mononuclear cells with serum samples showing high levels of soluble CD177 protein resulted in increased numbers of CD34-positive cells (P<0.02) and of erythroid progenitors (P<0.03). This effect was not detectable when low CD177-expressing or CD177-depleted sera were employed. These observations clearly indicate that the JAK2 V617F mutation is associated with enhanced granulocyte CD177 mRNA expression, and that this latter results in high levels of soluble CD177 protein. These elevated levels might contribute to the increased red cell production that characterizes polycythemia vera.

In Vitro and In Vivo Antioxidant Effect of Fermented Papaya Preparation (FPP) on Blood Cells of Beta-Thalassaemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1766-1766
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Ada Goldfarb ◽  
Eliezer Rachmilewitz
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia

Abstract In sickle cell anemia (SCD) and thalassemia, although the basic lesions are mutations in the globin genes, the pathophysiology involves oxidative stress-mediated cell damage in the bone marrow (ineffective erythropoiesis due to apoptosis of early erythroid precursors) and in the peripheral blood (chronic hemolysis of mature RBC). In addition, some patients develop thromboembolic complications and recurrent bacterial infections, the etiology of which is related at least in part, to documented oxidative stress in platelets and neutrophils (PMN), respectively. To study the presence and the role of oxidative stress in thalassemia and SCD, we adapted flow cytometry techniques for measuring the generation of Reactive Oxygen Species (ROS), the content of reduced glutathione (GSH), membrane lipid peroxidation and externalization of phosphatidylserine (PS) moieties in RBC, platelets and PMN. Cells derived from the peripheral blood of patients with beta-thalassemia major, intermedia or SCD showed increased oxidative status (increased ROS, lipid peroxidation and PS externalization, and decreased GSH) compared with their normal counterparts. Incubating fresh blood samples from patients with thalassemia major and thalassemia intermedia with 10 mg/ml FPP for 16 hours at 37oC reduced the oxidative status of RBC as well as platelets and PMN. Experiments carried out in normal and thalassemic mice (Th3/+, a mouse model of human beta-thalassemia intermedia demonstrated that mice treated for one week with 10 mg/ml FPP (dissolved in the drinking water) had reduced oxidative stress compared to control mice. The in-vivo effect of FPP was tested on 9 patients with beta-thalassemia (6 - major and 3 - intermedia) treated with 3 gr FPP per os three times a day for 12–15 weeks. Following the treatment, the ROS in RBC, platelets and PMN decreased and the GSH increased in all patients (see table). Six of these patients responded by a modest increase in RBC, reticulocytes and hemoglobin levels. These results suggest that FPP may have an important clinical efficacy as an antioxidant in thalassemia and sickle cell anemia. The in vivo effect of FPP treatment of beta-thalassemia patients Baseline After treatment n Mean ± SE Mean ± SE P-value* * Paired samples t-test RBC 9 324.07 ± 29.19 209.55 ± 23.65 0.001 ROS Platelets 9 223.73 ± 26.49 109.11 ± 8.71 0.001 PMN 9 222.72 ± 46.42 117.61 ± 8.98 0.045 RBC 9 55.37 ± 5.37 94.88 ± 3.71 0.001 GSH Platelets 9 59.41 ± 4.98 97.55 ± 5.26 <0.0001 PMN 9 58.29 ± 5.35 90.06 ± 5.87 0.005

JAK2 Mutation Status in Granulocytes, Platelets and Erythrocytes Differs Between Polycythemia Vera and Essential Thrombocythemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5228-5228
Author(s):  
Kohtaro Toyama ◽  
Norifumi Tsukamoto ◽  
Akio Saito ◽  
Hirotaka Nakahashi ◽  
Yoko Hashimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Mononuclear Cells ◽  
Rna Extraction ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Mutation Status ◽  
V617f Mutation

Abstract Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p &lt; 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

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