Incidence and Clinical Profile of JAK2 V617F Mutation in Myelofibrosis with Myeloid Metaplasia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 256-256
Author(s):  
Giovanni Barosi ◽  
Monia Marchetti ◽  
Margherita Massa ◽  
Vittorio Rosti ◽  
Alessandro Vannucchi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Severity Score ◽  
Jak2 V617f ◽  
Mutant Gene ◽  
Jak2 V617f Mutation ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Myeloid Metaplasia ◽  
V617f Mutation ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract An aberrant tyrosine kinase signalling due to non-receptor tyrosine kinase JAK2 V617F mutation has been highlighted in the pathogenesis of polycythemia vera (PV). In myelofibrosis with myeloid metaplasia (MMM) this mutation has been reported to be present in approximately 50% of the patients and its pathogenetic role is not elucidated. Here we report the results of JAK2 V617F mutation in a retrospective analysis of blood samples from 170 patients with MMM. Search for JAK2 mutation was performed by an allele specific PCR from DNA purified from granulocytes. To evaluate whether the mutation was carried in the homozygous or heterozygous state, digestion of PCR products with BsaXI restriction enzyme was performed. The overall frequency of JAK2 V617F mutation was 60% and homozygosity for the mutation was found in 39.2% of mutant samples. Disease duration was similar in JAK2 mutated and wild type patients. Patients who harboured an homozygous mutation had an higher myeloproliferative severity score (that indexed leukocytosis, thrombocytosis and splenomegaly) than patients who had a wild type or heterozygous genotype. In post-PV MMM, the mutant gene was present in 22/22 (100%) of patients, and the frequency of homozygosity was 59% of the mutated cases. In post-ET MMM (n=13), the mutant gene was present in 46% of the patients, and the frequency of homozygosity was 16% of the mutant samples. In idiopathic MMM (n=135), the incidence of the mutational state was 54.8%, with 35.1% of homozygote mutation. Patients who had received a diagnosis of prefibrotic myelofibrosis (WHO classification) had an incidence of the mutation significantly lower than patients who were diagnosed in the fibrotic stage (6/18, 33% vs 68/112, 60.7%; P=0.001). By considering only patients not receiving cytoreductive or disease modifying agents, patients who had an heterozygote mutation, had a mean Hb value higher than wild type patients (9.4 g/dL vs. 11.4 g/dL; P=0.004). Moreover, patients who had an homozygote mutation had the myeloproliferative severity score higher than both heterozygote and wild type patients (2.26 vs 1.59, P=0.008, and 2.26 vs.1.52, P=0.001, respectively). We conclude that JAK2 V617F mutation is significantly represented in MMM patients. It is a necessary event in the transformation from PV to MMM while not in the transformation from ET to MMM. Patients who harboured an heterozygote state maintained higher Hb values than patients with a wild type genotype, while the homozygote mutation was associated with leukocytosis, thrombocytosis and splenomegaly.

JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3503-3503
Author(s):  
Ruben A. Mesa ◽  
Ayalew Tefferi ◽  
Heather Powell ◽  
Terra Lasho ◽  
David Loegering ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Neutrophil Apoptosis ◽  
Wild Type ◽  
Jak2 Mutation ◽  
Myeloid Metaplasia ◽  
Mutation Status ◽  
Stat3 Phosphorylation ◽  
V617f Mutation ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

scholarly journals Eradication of JAK2 V617F mutation after allogeneic transplantation in a patient with myelofibrosis with myeloid metaplasia

Leukemia ◽  
2006 ◽  
Vol 20 (12) ◽  
pp. 2198-2199 ◽  
Author(s):  
A Fiorini ◽  
G Reddiconto ◽  
G Farina ◽  
S Marietti ◽  
M Palladino ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Allogeneic Transplantation ◽  
Jak2 V617f Mutation ◽  
Myeloid Metaplasia ◽  
V617f Mutation ◽  
Myelofibrosis With Myeloid Metaplasia

In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3506-3506 ◽  
Author(s):  
Josef T. Prchal ◽  
Ko-Tung Chang ◽  
Jaroslav Jelinek ◽  
Yongli Guan ◽  
Amos Gaikwad ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Polycythemia Vera ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Erythroid Progenitors ◽  
Erythroid Precursors ◽  
In Vitro Expansion ◽  
V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G>T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G>T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G>T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G>T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

Correlation of JAK2 V617F Mutation Status and Cytogenetic Findings in Myeloproliferative Disorders.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3633-3633
Author(s):  
Guoxian Sun ◽  
Frank Buccini ◽  
Elizabeth Fuentes ◽  
James Weisberger
Keyword(s):  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Chromosome Abnormalities ◽  
Jak2 V617f Mutation ◽  
Homozygous Mutation ◽  
Jak2 Mutation ◽  
Mutation Status ◽  
Trisomy 9 ◽  
V617f Mutation ◽  
Trisomy 9P

Abstract Detection of JAK2 V617F mutation is quickly becoming a front-line screening test for suspected myeloproliferative disorders (MPDs), as the mutation shows high frequency and specificity in non-CML MPDs, PV, ET or CIMF. Routine cytogenetics can detect chromosome abnormalities in approximately 20% of MPDs and is very helpful in establishing or confirming the presence of aberrant clonality, although chromosome changes are often numerical gains and losses, deemed non-specific. To see if there is correlation between JAK2 mutation and karyotypes, we studied 57 consecutive patients with clinically and morphologically confirmed diagnosis of non-CML MPDs. JAK2 V617F mutation performed using allele-specific PCR (sensitive to 10% using pyrosequencing) was found in 72% of patients (41/57), whereas clonal chromosome abnormalities were observed in 15.8% (9/57). There was no correlation between JAK2 mutational status and karyotypes. In 41 patients positive for the JAK2 mutation, 6 were cytogenetically abnormal and 35 normal. In 16 patients negative for the mutation, 3 showed abnormal karyotypes and 13 had normal karyotypes (X2 test, p>0.5). Among 6 patients with both JAK2 mutation and an abnormal karyotype, JAK2 mutation was seen in >50% of each sample in 4 patients, consistent with a homozygous mutation. Interestingly, in two cases, one with PV and trisomy 9 and another with MPD unclassifiable and trisomy 9p resulting from an unbalanced translocation between chromosomes 9p and 13, JAK2 mutation was present in >65% of each sample. Trisomy 9 and trisomy 9p are common abnormalities in MPDs, particularly in PV, seen in over 20% of cytogenetically abnormal cases. JAK2 gene is located on 9p24. Mitotic recombination is considered the most likely cause of loss of heterozygosity (LOH) and thus mutant homozygosity which is undetectable at the cytogenetic level. However, in cases with trisomy 9 or 9p, the JAK2 allele genotypes may be G/T/T and/or T/T/T as well as the usual G/T and/or T/T. Our observations suggest that trisomy 9 or 9p should be taken into consideration when interpreting JAK2 mutation status and that further molecular studies are needed to delineate the implication of trisomy 9 or 9p in potential mutant allele selective advantage and clonal evolution in JAK2 mutation positive MPDs.

Detection of the JAK2 V617F Mutation by Real Time PCR in Granulocytes and Platelets of ET Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4877-4877
Author(s):  
Beatriz Bellosillo ◽  
Eva Gimeno ◽  
Raquel Longaron ◽  
Lourdes Florensa ◽  
Antonio Salar ◽  
...  
Keyword(s):  
Real Time ◽  
Direct Sequencing ◽  
Specific Treatment ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Wild Type ◽  
Rt Pcr ◽  
Jak2 Mutation ◽  
Allele Specific ◽  
V617f Mutation

Abstract Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients. Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR. Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form. Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032). Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.

Presence of JAK2 V617F Mutation in Peripheral Blood of Blood Donors with Upper-Limit Hematocrit and Platelet Values.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2533-2533
Author(s):  
Paola Bianchi ◽  
Elisa Fermo ◽  
Fulvio Mozzi ◽  
Maurizio Marconi ◽  
Alberto Zanella
Keyword(s):  
Polycythemia Vera ◽  
Blood Donors ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Myeloid Metaplasia ◽  
Specific Pcr ◽  
Upper Limit ◽  
Allele Specific ◽  
V617f Mutation ◽  
Allele Specific Pcr

Abstract The somatic mutation V617F of JAK2 gene has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPDs), in particular polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. Recently, two studies showed the presence of this mutation also in 37/3935 subjects with non haematological diseases (Xu et al, 2006) and 5/52 healthy donors (Sidon et al, 2006), suggesting that V617F mutation may occur in the absence of MPD phenotype and that probably is not sufficient per se to induce MPDs. The aim of this study was to search for the presence of JAK2 V617F mutation in healthy blood donors with confirmed upper-limit Hct and/or Plts values. Actually, previous studies indicated that some subjects with upper-limit Hct levels have early stages of polycythemia vera (Zanella et al, 1987). We studied 177 consecutive repeat blood donors (92 M, 85 F; median age 45 years, range 19–66) displaying Hct and/or Plts values higher than the 75° percentile of the normal reference distribution (Hct > 0.47 for M and > 0.42 for F; Plts > 300×109/L), confirmed on at least two different occasions in the last 12 months. All subjects had been accepted for blood donation on the basis of negative clinical history and normal results on both physical examination and routine laboratory testing. 83 of them (55 M and 28 F) had upper-limit Hct levels (median 0.48, range 0.47-0.51 for M; 0.43, range 0.42-0.47 for F); 85 had Plts > 300×109/L (median 338×109/L, range 300–454), and 9 donors had both upper-limit Hct and Plts. DNA was extracted from whole blood; all samples were analyzed by allele-specific polymerase chain reaction (PCR) according to Baxter et al (2005), and by fluorescent allele specific PCR (McClure et al, 2006) on ABI PRISM 310 Genetic Analyzer. Ten subjects were found to be positive for V617F mutation by fluorescent PCR, showing a positive signal when compared to a positive control corresponding to 2% of V617F mutated allele. Six of them showed a positive band also on agarose gel when analyzed with allele specific PCR. The presence of mutation was confirmed by enzymatic digestion with BsaXI. Hematological data of mutated subject are reported in the table. No statistically significant differences of hematological parameters were present between V617F positive and negative subjects. In conclusion, the presence of a V617F positive clone (albeit in a small amount), was found in 4% (3 F and 1 M) donors with upper-limit Hct and in 6% (2 F, 4 M) donors with Plts > 300×109/L. The follow up of these subjects will ascertain whether V617F mutation is a prelude to a myeloproliferative disease. Sex Age (years) Hb (g/dl) Hct Plts (×109/L) WBC (x109/L) Upper-limit Hct 1 F 66 15.1 0.45 202 4.85 2 F 51 14.4 0.43 235 6.40 3 F 64 15.7 0.45 198 7.75 4 M 58 15.9 0.48 220 7.30 Plts > 300×109/L 5 F 53 13.7 0.40 360 6.97 6 F 63 13.5 0.40 301 9.2 7 M 47 15.2 0.45 334 8.64 8 M 47 13.8 0.41 316 6.35 9 M 19 15.2 0.44 321 8 10 M 37 16.1 0.45 379 7.9

Analysis of JAK2 V167F Mutation in Myelodysplastic Syndromes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4591-4591 ◽  
Author(s):  
Elisa Fermo ◽  
Anna Zaninoni ◽  
Francesca G. Imperiali ◽  
Paola Bianchi ◽  
Mariangela Colombi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Myelodysplastic Syndromes ◽  
Prognostic Significance ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Myeloid Metaplasia ◽  
Mutational Status ◽  
V617f Mutation ◽  
Wbc Count

Abstract The somatic mutation JAK2 V617F has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMF). In typical forms of myelodysplastic syndromes (MDS), JAK2 V617F mutation is rarely present (2–5%); on the contrary, it has been found with higher prevalence in patients with RARS-T (i.e. MDS/MPD-U with platelet count >600×109/L and ringed sideroblasts more than 15%) and in a subgroup of MDS patients with isolated 5q deletion and a proliferative bone marrow. In this study we analysed the JAK2 V617F mutational status in 53 MDS patients (26 males, 27 females; median age at the time of the study 76 years, range 45–91). Patients were classified as follows: 4 cases 5q- syndrome, 3 RCMD, 5 MDS/MPD, 1 MDS-U, 23 RA, 12 RARS, 5 RAEB. DNA was extracted from purified granulocytes; all samples were analyzed by allele-specific polymerase chain reaction (PCR), according to Baxter el al (2005). DNA samples were further subjected to direct sequencing for confirmatory testing. The JAK2 V617F mutation was present in 3 cases, with an overall frequency of 5%. With respect to MDS subtype, 1 patient had RA and 2 RARS. Among the 12 RARS patients, the two V617F postive displayed thrombocytosis (680×109/L and 649×109/L), whereas none of the 10 RARS V617F negative patients showed high platelet counts (median Plt 157×109/L, range 5–422×109/L). In one JAK2 mutant case, thrombocytosis required treatment with hydroxyurea. Moreover, the two V617F positive RARS patients displayed higher WBC count (6.2×109/L and 8.5×109/L) than the V617F negatives (median WBC 4.05×109/L); no difference was observed in Hb levels. The JAK2 positive RA patient had 10% of sideroblasts in bone marrow, normal platelet and WBC count and no proliferative characteristics; since the occurrence of the mutation may be an early event and preceed the classical manifestations of MDS/MPD, a longer follow-up is necessary to determine its possible prognostic significance. Considering the V617F negative MDS cases, only one patient, diagnosed as MDS/MPD, showed a platelet count >600×109/L. In conclusion, we confirmed recent reports showing that JAK2 V617F is present with low prevalence (about 5%) in MDS; in particular, the JAK2 mutation identifies a subset of MDS patients with and “overlap” syndrome, characterised by proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis, who may benefit from JAK2 specifically targeted therapies.

Ratio of Mutant JAK2-V617F to Wild Type Jak2 Determines the MPD Phenotypes in Transgenic Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 674-674
Author(s):  
Ralph Tiedt ◽  
Hui Hao-Shen ◽  
Marta A. Sobas ◽  
Renate Looser ◽  
Stephan Dirnhofer ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mice ◽  
Bone Marrow Cells ◽  
Jak2 V617f ◽  
Artificial Chromosome ◽  
Cre Recombinase ◽  
Jak2 V617f Mutation ◽  
Wild Type ◽  
Retroviral Transduction ◽  
V617f Mutation

Abstract The reason why the JAK2-V617F mutation is associated with several phenotypic manifestations of human myeloproliferative disorders (MPD), i.e. polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), is currently unknown. We established an inducible transgenic mouse model for MPD using a bacterial artificial chromosome (BAC) containing the human JAK2-V617F gene under the control of the JAK2 promoter. The sequences encoding the kinase domain were placed in the inverse orientation and flanked with antiparallel loxP sites to make the construct inducible by Cre-recombinase. A transgenic strain (FF1) containing 9 copies of the JAK2-V617F transgene was analyzed in detail. In this strain, Cre activity can lead to activation and/or excision of the multiple transgene copies. Depending on the number of actively rearranged transgene copies, we observed graded levels of expression of the JAK2-V617F mRNA and different MPD phenotypes. Crossing FF1 mice with transgenic mice expressing Cre-recombinase under the control of the hematopoiesis specific Vav promoter (VavCre) led to reduction of the FF1 copy number and low levels of JAK2-V617F expression (approximately 40% of endogenous wild type Jak2). These FF1;VavCre mice developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia (Figure 1A). In contrast, induction of the JAK2-V617F transgene with the interferon-inducible MxCre resulted in less excision and higher JAK2-V617F transgene expression (approximately equal to wild type Jak2). These MxCre;FF1 mice displayed a PV phenotype with increased hemoglobin, thrombocytosis and neutrophilia (Figure 1B). The highest expression levels of JAK2-V617F were achieved by retroviral transduction (approximately 300% of wild type Jak2). Transplantation of these bone marrow cells into irradiated recipients caused a PV-like phenotype without thrombocytosis. Thus, the phenotype correlated with the ratio of mutant to wild type JAK2 mRNA. In patients with MPD, we found a similar correlation between the ratio of mutant to wild type JAK2 mRNA and the ET, PV and PMF phenotypes. In contrast to our transgenic mice, which display graded levels of JAK2-V617F with wild type JAK2 being present in every cell, each individual blood cell from patients with MPD can only be homozygous or heterozygous for the mutation, or normal. Therefore, the molecular mechanism determining the phenotype in humans may be more complex than in our mouse model and appears to be linked to the transition of the JAK2-V617F mutation to homozygosity. Figure Figure

Absence of FLT3 and JAK2 (V617F) Mutations in Langerhans Cell Histiocytosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4496-4496
Author(s):  
Malak Abedalthagafi ◽  
Huimin Guo ◽  
Dan-Paul Hartmann ◽  
Metin Ozdemirli
Keyword(s):  
Tyrosine Kinase ◽  
Soft Tissues ◽  
Jak2 V617f ◽  
Single Strand Conformation Polymorphism ◽  
Langerhans Cell ◽  
Jak2 V617f Mutation ◽  
Pcr Analysis ◽  
Tissue Samples ◽  
Hematopoietic Stem ◽  
V617f Mutation

Abstract Langerhans cell histocytosis (LCH) is a neoplasm of histiocytic cells believed to be derived from cells of the dendritic system, but the disease pathogenesis is unknown. FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that is expressed on the surface of hematopoietic stem cells and plays an important role in normal hematopoiesis. Recently, FLT3 has been shown to play a role in regulating dendritic cell development and elevated levels FLT3-ligand was found in the serum of LCH patients. Therefore we evaluated 14 cases of LCH for FLT3 mutations by PCR analysis of genomic DNA, obtained from formalin-fixed paraffin-embedded tissue samples, followed by PAGE for deletions or internal tandem duplications using a commercially available kit (InVivoScrib Technologies, San Diego, CA). There were 7 males and 7 females with a mean age of 25.1. The tissues involved included bones, lymph nodes, bone marrow, breast, lungs and various soft tissues. No mutations in FLT3 were identified in any of the 14 patient samples. In the same patient population we also looked at the JAK2 (V617F) mutation, which has been found in patients with myeloproliferative disorders other than chronic myeloid leukemia and which has not yet been evaluated in LCH by a single strand conformation polymorphism assay using a commercially available kit (Ipsogen, New Haven, CT). There was no JAK2 V617F mutation in any of the 14 cases. These results suggest that FLT3 and JAK2 V617F mutations do not play a role in the pathogenesis of LCH.

Homologous Recombination of Wild TYPE JAK2, A NOVEL EARLY STEP In the DEVELOPMENT of Myeloproliferative Neoplasm

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2805-2805
Author(s):  
Mathias Vilaine ◽  
Damla Olcaydu ◽  
Ashot Harutyunyan ◽  
Jonathan Bergeman ◽  
Tiab Mourad ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Snp Array ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Wild Type ◽  
Jak2 Mutation ◽  
Chromosome 9P ◽  
V617f Mutation ◽  
Array Analyses

Abstract Abstract 2805 Background: Adequate expression and function of Jak2 in hematopoietic progenitors is critical for normal myelopoiesis. The JAK2 46/1 (GGCC) haplotype, a congenital particularity, predisposes to myeloproliferative neoplasm (MPN) both independently and through mutation of the JAK2 gene. The JAK2 V617F mutation and acquired homozygous status for JAK2 V617F are frequent in MPN. JAK2 V617F homozygosity is currently explained acquisition of the JAK2 V617F mutation followed by mitotic homologous recombination (HR) of JAK2 occurred between wild-type and mutant alleles, leading to uniparental disomy (UPD) of chromosome 9p (9pUPD). Here we report the cases of 2 PV patients (Na1061 and Na1253) with acquired homozygous status for the JAK2 46/1 haplotype yet their granulocytes carried <20% JAK2 V617F. Aim: To determine whether HR of JAK2 can precede the V617F mutation in MPN. Methods: Granulocyte DNA and CD3+ lymphocyte DNA were examined in parallel with qPCR assays specific for the wild type and 46/1 haplotypes using rs12343867, a JAK2 intron 14 marker, as well as 4 other single nucleotide polymorphisms (SNP) on chromosome 9p. 9pUPD clonality and length were determined using SNP array studies. Results: For both patients, lymphocytes were heterozygous for the 46/1 haplotype, confirming that granulocyte 46/1 homozygosity was acquired. Direct sequencing of the JAK2 and GNE genes and SNP array analyses revealed homologous recombination of part of the JAK2 gene (exons 6–19, patient Na1061) and of the complete 46/1 JAK2 haplotype (patient Na1253). Furthermore, for both patients, full length sequencing of JAK2 cDNA revealed no additional mutation. In both cases, HR of wild-type JAK2 was associated with growth advantage and high expression of recombined JAK2. For both patients, further SNP array analyses revealed partial 9pUPD concerning <30% cells, which correlated with %JAK2 V617F and was consistent with 9pUPD having occurred after JAK2 V617F (Figure 1). The distortion of SNP allelic differences was higher at the telomeric end than in the centromeric region of chr. 9p. This indicated 2 distinct partial 9pUPDs for Na1061 and 1 partial 9pUPD for Na1253. Conclusion: Homologous recombination involving wild type JAK2 can precede JAK2 mutation and 9pUPD in MPN. Thus multiple paths and diverse alterations of the JAK2 gene can lead to MPN in individuals carrying the JAK2 GGCC haplotype. We propose a new model with JAK2 HR as early event, followed or not by JAK2 mutation, or/and JAK2 mutation(s) facilitating subsequent recombination resulting in 9pUPD and JAK2 V617F homozygosity. Disclosures: No relevant conflicts of interest to declare.

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