COLD-PCR and Innovative Microarray Substrates for Detecting and Genotyping MPL Exon 10 W515 Substitutions

BACKGROUND Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. METHODS We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. RESULTS Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. CONCLUSIONS These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.

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TET2 Mutations in a Cohort of Patients with Myeloproliferative Neoplasms Lacking a Molecular Marker.

Blood ◽

10.1182/blood.v114.22.3912.3912 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3912-3912

Author(s):

Luz Maria Martinez-Aviles ◽

Carlos Besses ◽

Alberto Alvarez-Larran ◽

Aina Pons ◽

Sergi Serrano ◽

...

Keyword(s):

Mutational Analysis ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Receptor Gene ◽

Biological Significance ◽

Jak2v617f Mutation ◽

Missense Mutations ◽

Coding Sequence ◽

Exon 10

Abstract Abstract 3912 Poster Board III-848 Background The myeloproliferative neoplasms (MPNs) include different diseases presenting several mutations in variable frequency. The JAK2V617F mutation is present in 90-95% Polycythemia Vera (PV), 50-60% Essential Thrombocythemia (ET) and 50-60% Primary Myelofibrosis (PMF) patients. In addition, JAK2 exon 12 mutations are observed in 1-3% of PV patients and mutations in the thrombopoietin receptor gene (c-MPL) (S505N, W515K/L) are present in a 5-9% of PMF and in a 1-4% of ET patients. Recently, acquired mutations of the Ten-Eleven Translocation (TET) 2 gene, have been reported in about 14% of sporadic MPNs. TET2 mutations may precede the acquisition of JAK2V617F predisposing to a MPN development. However, the incidence of TET2 mutations in patients lacking the JAK2V617F mutation has not been extensively studied. Aim To analyze the incidence of TET2 mutations in a cohort of MPN patients negative for JAK2V617F, JAK2 exon 12 mutations and c-MPL exon 10 mutations (S505N or W515K/L). Patients and methods From a whole cohort of 241 patients with MPN (93 PV, 16 PMF and 132 ET) we excluded those patients positive for JAK2V617F (determined by quantitative allele-specific PCR), JAK2 exon 12 mutations or c-MPL exon 10 mutations (S505N or W515K/L) (analyzed by direct sequencing). We analyzed the TET2 gene in 5 PV, 5 PMF and 53 ET patients lacking any of the aforementioned molecular markers. The mutational analysis of the coding sequence of TET2 was performed by direct sequencing using cDNA from granulocytes. In 13 patients, DNA from T lymphocytes was obtained to indentify the presence of single nucleotide polymorphisms (SNPs) in germline DNA. Results Sixty-three patients were screened for mutations in the whole coding sequence of the TET2 gene. Only 3 ET patients (4.7%) presented deleterious mutations in the TET2 gene. The three distinct TET2 mutations were: Q706X, S1848X and V1395_R1400delinsR. In 48 out of 63 (76.1%) patients we observed a total of 13 different missense mutations and 2 silent mutations in the coding sequence of the gene. The most frequent missense mutation was the I1762V which was detected in 27 patients. In 13 patients whose matched normal DNA was available, we analyzed the presence of missense mutations being all of them present in the control DNA suggesting that they were SNPs and not acquired mutations. The two nonsense mutations (Q706X and S1848X), were not present in matched normal tissue indicating that these mutations were somatically acquired in myeloid cells. Conclusions TET2 pathogenic mutations are infrequent (<5%) in myeloproliferative neoplasms negative for JAKV617F, JAK2 exon 12 and c-MPL (S505, W515K/L) gene mutations. The role and the biological significance of missense mutations in the coding sequence of TET2 has to be elucidated. Acknowledgments Fellowship FI2008 (AGAUR) to LMM-A, FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

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Screening for MPL mutations in Essential Thrombocythemia and Primary Myelofibrosis: Normal Mpl Expression and Absence of Constitutive STAT3 and STAT5 Activation in MPLW515L-Positive Platelets.

Blood ◽

10.1182/blood.v114.22.3910.3910 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3910-3910

Author(s):

Laura Korin ◽

Ana C Glembotsky ◽

Paola R Lev ◽

Carlos D Chazarreta ◽

Rosana F Marta ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Low Frequency ◽

Primary Myelofibrosis ◽

Receptor Activation ◽

Experimental Models ◽

Platelet Surface ◽

Specific Pcr ◽

Exon 10

Abstract Abstract 3910 Poster Board III-846 The identification of exon 10 MPL mutations has begun to unravel the pathogenesis of JAK2V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). Overall frequency of MPL mutations ranges from 1 to 4% for ET and 5 to 11% for PMF patients. Overexpression of these mutant alleles in cell lines and animal models lead to constitutive receptor activation, activation of downstream signaling pathways and hypersensitivity to thrombopoietin (TPO). However, the precise functional effects of these mutations in signaling and TPO-response in patient samples have not been investigated. Decreased Mpl expression is a molecular hallmark of myeloproliferative neoplasms (MPN). This defect is more frequent in patients positive for JAK2V617F, suggesting a biologic relationship between Mpl expression and the underlying molecular pathogenesis. The pattern of Mpl expression in patients with exon 10 MPL mutations has not been explored. The aim of this study was to analyze the frequency of MPLW515L, MPLW515K and MPLS505N mutations in a cohort of patients with ET and PMF and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO) in patient samples. One in one hundred (1%) patients with ET and 1 in 11 with PMF were positive for MPLW515L by allele-specific PCR and sequencing in platelet and/or leukocyte samples, while none harboured the MPLW515K and MPLS505N mutations; both MPL515L-positive patients were JAK2V617F-negative. Platelet surface Mpl expression in the MPLW515L-positive ET patient by flow cytometry did not differ from a normal control, Mpl/isotype ratio was 3.6 vs 3.2, respectively, and normal total Mpl content was found by Western blot, Mpl/B3 integrin ratio was 99% of controls (n=5), while plasma TPO levels were mildly elevated by ELISA, 45.8 pg/mL vs 0 (0-32) pg/mL in controls (n=20). MPL transcripts by real-time RT-PCR in platelets from both MPLW515L-positive patients were similar to values found for this ET cohort (n=20), which did not differ significantly from normal controls (n=10), MPL/GAPDH ratio was 0.25 and 0.26, for MPL-positive patients, 0.24 (0.12-0.97) for MPL-negative patients, and 0.39 (0.21-0.78) for controls, p= 0.1. Constitutive STAT3 and STAT5 phosphorylation was not detected by immunoblotting and phosphorylation in response to increasing concentrations of TPO did not differ from controls. The low frequency of MPL mutations in this cohort is in agreement with previous studies, highlighting the need for identifying additional molecular defects in JAK2V617F-negative patients. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for MPN patients. Therefore, although impaired Mpl expression can arise from a molecular mechanism different from JAK2V617F, this phenotypic abnormality seems not to be linked to MPLW515L. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models. Disclosures: No relevant conflicts of interest to declare.

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From Janus kinase 2 to calreticulin: the clinically relevant genomic landscape of myeloproliferative neoplasms

Blood ◽

10.1182/blood-2014-03-530865 ◽

2014 ◽

Vol 123 (24) ◽

pp. 3714-3719 ◽

Cited By ~ 116

Author(s):

Mario Cazzola ◽

Robert Kralovics

Keyword(s):

Essential Thrombocythemia ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Janus Kinase ◽

Driver Mutations ◽

Mutation Status ◽

Genomic Landscape ◽

Exon 10 ◽

Stat Pathway

Abstract Our understanding of the genetic basis of myeloproliferative neoplasms began in 2005, when the JAK2 (V617F) mutation was identified in polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2 exon 12 and MPL exon 10 mutations were then detected in subsets of patients, and subclonal driver mutations in other genes were found to be associated with disease progression. Recently, somatic mutations in the gene CALR, encoding calreticulin, have been found in most patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. The JAK-STAT pathway appears to be activated in all myeloproliferative neoplasms, regardless of founding driver mutations. These latter, however, have different effects on clinical course and outcomes. Thus, evaluation of JAK2, MPL, and CALR mutation status is important not only for diagnosis but also for prognostication. These genetic data should now also be considered in designing clinical trials.

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Analysis of JAK2 V617F mutation in Tunisian patients with myeloproliferative neoplasms

European Journal of Inflammation ◽

10.1177/20587392211006538 ◽

2021 ◽

Vol 19 ◽

pp. 205873922110065

Author(s):

Soumaya Chadi ◽

Tarak Dhaouadi ◽

Imen Sfar ◽

Hela Baccouche ◽

Rym Nabli ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Pcr Rflp ◽

Tunisian Patients ◽

Healthy Control ◽

V617f Mutation

We aimed to investigate the prevalence of the JAK2 V617F mutation in Tunisian patients with myeloproliferative neoplasms (MPN) and to look for possible associations with diseases’ presentation. In this context, JAK2 V617F polymorphism was detected by PCR-RFLP and direct sequencing in 213 MPN patients (109 with polycythemia vera (PV), 93 with essential thrombocythemia (ET) and 11 with primary myelofibrosis (PMF)), 77 unclassified patients with thrombosis (UPT) and 95 healthy control subjects. The JAK2 V617F mutant allele was present by either PCR-RFLP or direct sequencing in 158 (74.17%) MPN patients while all UPT and controls were negative. Besides, the JAK2 V617F mutation was significantly more frequent in patients with PV 98 (89.9%) than in ET 54 (58.1%) and PMF 6 (54.5%) groups, p < 0.001. Analytic results in MPN patients showed significant associations between the JAK2 SNP and both hemoglobin levels (16.29 ± 3 vs 13.01 ± 3.65) and hematocrit (52.99 ± 8.34 vs 45.37 ± 10.94), p < 0.001 and p < 0.001, respectively. In addition, in the ET subgroup thrombosis was significantly more frequent in patients carrying the V617F mutation (16, (29.6%) vs 3, (7.7%)), p = 0.01. In ET patients, the V617F mutation seems to be predictive of thrombosis occurrence.

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Calreticulin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasms’ in Jeddah Region, Saudi Arabia

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i47a33029 ◽

2021 ◽

pp. 420-428

Author(s):

Heba Alkhatabi ◽

Heyam Abdulqayoom ◽

Raed Alserihi ◽

Raed Felimban ◽

Aisha Elaimi ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Mutation Screening ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Pcr Analysis ◽

Allele Specific ◽

Pcr Method ◽

Calr Mutations ◽

The Impact

Background: The JAK2 V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms’(MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with JAK2V617F negative primary myelofibrosis (PMF), and essential thrombocythemia (ET). Methods: This study analyzed the impact of JAK 2 Exon 12 and CALR common mutations in 65 patients with JAK2V617F negative MPN from the Jeddah region. An allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12 and direct sequencing and PCR analysis were utilized to screen all patients for CALR. Results: None of the patients were positive for the Exon 12 mutation and eight patients were positive for CALR mutations. Conclusions: This is the first Saudi Arabian research that focused on screening CALR hotspot mutations and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. Further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

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Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms

Blood ◽

10.1182/blood-2016-10-695940 ◽

2017 ◽

Vol 129 (6) ◽

pp. 667-679 ◽

Cited By ~ 173

Author(s):

William Vainchenker ◽

Robert Kralovics

Keyword(s):

Disease Progression ◽

Germ Line ◽

Myeloproliferative Neoplasms ◽

Leukemia Virus ◽

Myeloproliferative Neoplasm ◽

Primary Myelofibrosis ◽

Erythropoietin Receptor ◽

Driver Mutations ◽

Disease Initiation ◽

Epigenetic Regulators

Abstract The genetic landscape of classical myeloproliferative neoplasm (MPN) is in large part elucidated. The MPN-restricted driver mutations, including those in JAK2, calreticulin (CALR), and myeloproliferative leukemia virus (MPL), abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors, more particularly the STATs. The most frequent mutation, JAK2V617F, activates the 3 main myeloid cytokine receptors (erythropoietin receptor, granulocyte colony-stimulating factor receptor, and MPL) whereas CALR or MPL mutants are restricted to MPL activation. This explains why JAK2V617F is associated with polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis (PMF) whereas CALR and MPL mutants are found in ET and PMF. Other mutations in genes involved in epigenetic regulation, splicing, and signaling cooperate with the 3 MPN drivers and play a key role in the PMF pathogenesis. Mutations in epigenetic regulators TET2 and DNMT3A are involved in disease initiation and may precede the acquisition of JAK2V617F. Other mutations in epigenetic regulators such as EZH2 and ASXL1 also play a role in disease initiation and disease progression. Mutations in the splicing machinery are predominantly found in PMF and are implicated in the development of anemia or pancytopenia. Both heterogeneity of classical MPNs and prognosis are determined by a specific genomic landscape, that is, type of MPN driver mutations, association with other mutations, and their order of acquisition. However, factors other than somatic mutations play an important role in disease initiation as well as disease progression such as germ line predisposition, inflammation, and aging. Delineation of these environmental factors will be important to better understand the precise pathogenesis of MPN.

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Interlaboratory Quality Control Round of MPL Mutation Detection in Fourteen European Laboratories: A MPN&MPNr-EuroNet Study,

Blood ◽

10.1182/blood.v118.21.3859.3859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3859-3859 ◽

Cited By ~ 1

Author(s):

Naomi A. Porret ◽

Elisabeth Oppliger Leibundgut ◽

Serge Carillo ◽

Bruno Cassinat ◽

Francois Girodon ◽

...

Keyword(s):

Quality Control ◽

Mutation Detection ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Allelic Discrimination ◽

Program Cost ◽

Growth And Survival ◽

Thrombopoietin Receptor

Abstract Abstract 3859 The MPL gene is located on chromosome 1p34 and encodes the thrombopoietin receptor. It includes 12 exons and is a key factor for growth and survival of megacaryocytes. Acquired mutations in this gene activate the thrombopoietin receptor constitutively. MPL515 somatic mutations are stem cell-derived events that involve both myeloid and lymphoid progenitors. Two distinct exon 10 mutations are found in 15% of JAK2-V617F negative myeloproliferative neoplasms (MPN),), i.e. 3% of essential thrombocythemia (ET) and 10% of primary myelofibrosis (PMF).: W515L, W515K and the rare W515A variant. A hereditary mutation, S505N, is associated with familial thrombocytosis. MPL mutation detection is a helpful new tool to detect clonality in JAK2 V617F negative MPN and to establish the diagnosis of MPN. As many laboratories use very different methods and interpretations, standardization is highly warranted. Particularly the methodology is diverse and the results need to be comparable, requiring comprehensive testing. This quality control project was established within workgroup 2 of the MPN&MPNr-EuroNet network, (www.mpneuronet.eu, supported by the European program COST (CoOperation in Science and Technology)). The lab from the ‘Hopital H. Mondor AP-HP Paris' provided 29 samples containing randomized concentrations (between 100% and 1%) of the four mutations MPL W515L, W515K, W515A and S505N. The plasmids used for this quality control experiment spanned exons 9, intron 9 and exon10 of the MPL with S505N, W515L, W515K and W515A mutants diluted first with wild-type plasmid gene and then diluted in human genomic DNA. Thirteen European laboratories tested these 29 samples, each using their own chosen methods (14 altogether). The following methods were used: Mutascreen W515L/K Kit (Ipsogen, France):(n=4), allelic discrimination real-time PCR (n=2), high resolution melting (HRM) (n=7) and sequencing (n=2, 1 Sanger, 1 pyrosequencing)). There were no false positive results in any of the labs. All labs using the Mutascreen W515L/K Kit detected all W515L and W515K mutations, from 100% mutated down to 1% mutated plasmids. The allelic discrimination assays which were also designed for W515L and W515K only, detected the mutations down to 2%. The HRM methods were all designed differently. All except one (which did not recognize S505N) detected all 4 mutations with a sensitivity, down to 5% mutated plasmids, with few exceptions detecting either lower or higher amounts. The Sanger sequencing and pyrosequencing assays had a detection limit of 5–10%, with the pyrosequencing assay not being designed for the S505N mutation. All participating labs detected the most frequent MPL mutations in MPN W515L and W515K, with many designs not including W515A and S505N. Achieved sensitivities differed between methods with cutoffs of 1% to 10% (1.5% for the Ipsogen kit). Most laboratories reported the results as either positive or negative. However, the percentages of mutated alleles reported by a few labs differed greatly from each other and from the original dilutions (range 2–50 times) In conclusion, these results show that the diverse methods for MPL mutation detection used by different European labs yielded comparable specificity with varying sensitivity. Smaller clones might be missed by the less sensitive methods, and quantification of mutated alleles should be interpreted very carefully until standardised reference material for MPL mutation testing will be available. More extensive interlaboratory testing including patient samples is needed to identify the most robust assays suitable for diagnostic mutation detection and particularly for quantification of mutated alleles. Disclosures: No relevant conflicts of interest to declare.

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Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning

Blood ◽

10.1182/blood.2019002561 ◽

2020 ◽

Vol 135 (4) ◽

pp. 287-292 ◽

Cited By ~ 6

Author(s):

Jessica L. Bridgford ◽

Su Min Lee ◽

Christine M. M. Lee ◽

Paola Guglielmelli ◽

Elisa Rumi ◽

...

Keyword(s):

Transmembrane Domain ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Single Amino Acid ◽

Driver Mutations ◽

Myeloproliferative Disease ◽

Sequencing Data ◽

Almost All ◽

Exon 10

Abstract The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F− essential thrombocythemia and primary myelofibrosis patients, respectively, have “canonical” MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other “noncanonical” MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

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Carlituculin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasm’s in Jeddah Region, Saudi Arabia

10.21203/rs.3.rs-849146/v1 ◽

2021 ◽

Author(s):

Heba Alkhatabi ◽

Heyam Abdulqayoom ◽

Raed Alserihi ◽

Raed Felimban ◽

Aisha Elaimi ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Mutation Screening ◽

Gene Mutations ◽

Primary Myelofibrosis ◽

Pcr Analysis ◽

Negative Results ◽

Allele Specific ◽

Pcr Method ◽

The Impact

Abstract PurposeThe JAK2V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms (MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with negative MPNs for JAK2V617F. The CALR gene mutations have been reported in patients with negative JAK2V617F, primary myelofibrosis (PMF), and essential thrombocythemia (ET). This study analyzed the impact of JAK2 Exon 12 and CALR common mutations on MPN patients from the Jeddah region.MethodsAn allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12. Consequently, the results showed that none of the patients were positive for the Exon 12 mutation. Direct sequencing and PCR analysis were utilized to screen all patients for CALR. Results The impact of CALR and JAK2 Exon 12 in 65 patients with a variety of MPN symptoms was investigated. This study included patients who had negative results, when previously screened for JAK2V617F. CALR mutations were identified in eight patients out of 65 JAK2V617F and JAK2 Exon 12 negative patients in this study’s cohort. Conclusions This is the first Saudi Arabian research that focused on screening CALR hotspot mutations, and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. In addition, further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

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AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2

Blood ◽

10.1182/blood-2009-11-254664 ◽

2010 ◽

Vol 116 (2) ◽

pp. 254-266 ◽

Cited By ~ 32

Author(s):

Wei Wang ◽

Sven Schwemmers ◽

Elizabeth O. Hexner ◽

Heike L. Pahl

Keyword(s):

Essential Thrombocythemia ◽

Chromatin Immunoprecipitation ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Jak2v617f Mutation ◽

Binding Partner ◽

Protein Levels ◽

Highly Correlated

Abstract The transcription factor NF-E2 is overexpressed in the majority of patients with polycythemia vera (PV). Concomitantly, 95% of these patients carry the JAK2V617F mutation. Although NF-E2 levels correlate with JAK2V671F allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression has not been described. Here we show that NF-E2 expression is also increased in patients with essential thrombocythemia and primary myelofibrosis independent of the presence of the JAK2V617F mutation. Characterization of the NF-E2 promoter revealed multiple functional binding sites for AML1/RUNX-1. Chromatin immunoprecipitation demonstrated AML1 binding to the NF-E2 promoter in vivo. Moreover, AML1 binding to the NF-E2 promoter was significantly increased in granulocytes from PV patients compared with healthy controls. AML1 mRNA expression was elevated in patients with PV, essential thrombocythemia, and primary myelofibrosis both in the presence and absence of JAK2V617F. In addition, AML1 and NF-E2 expression were highly correlated. RNAi-mediated suppression of either AML1 or of its binding partner CBF-β significantly decreased NF-E2 expression. Moreover, expression of the leukemic fusion protein AML/ETO drastically decreased NF-E2 protein levels. Our data identify NF-E2 as a novel AML1 target gene and delineate a role for aberrant AML1 expression in mediating elevated NF-E2 expression in MPN patients.

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