Altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of Jak2

AbstractWe identified 13 new gene expression markers that were elevated and one marker, ANKRD15, that was down-regulated in patients with polycythemia vera (PV). These 14 markers, as well as the previously described PRV1 and NF-E2, exhibited the same gene expression alterations also in patients with exogenously activated granulocytes due to sepsis or granulocyte colony-stimulating factor (G-CSF) treatment. The recently described V617F mutation in the Janus kinase 2 (JAK2) gene allows defining subclasses of patients with myeloproliferative disorders based on the JAK2 genotype. Patients with PV who were homozygous or heterozygous for JAK2-V617F exhibited higher levels of expression of the 13 new markers, PRV1, and NF-E2 than patients without JAK2-V617F, whereas ANKRD15 was down-regulated in these patients. Our results suggest that the alterations in expression of the markers studied are due to the activation of the Jak/signal transducer and activator of transcription (STAT) pathway through exogenous stimuli (sepsis or G-CSF treatment), or endogenously through the JAK2-V617F mutation.

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The Hypoxic Response and Altered Gene Expression in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood.v120.21.3245.3245 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3245-3245

Author(s):

Victor R. Gordeuk ◽

Xu Zhang ◽

Wei Zhang ◽

Shwu-Fan Ma ◽

Galina Miasniakova ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Solute Carrier Family ◽

Cell Disease ◽

Hypoxic Response ◽

Tetratricopeptide Repeats ◽

Altered Gene Expression ◽

Solute Carrier ◽

Altered Gene ◽

Gene Expression Alterations

Abstract Abstract 3245 Sickle cell disease (SCD) and Chuvash polycythemia (CP) are both monogenic hematologic disorders, the first resulting in hemolytic anemia and the second in polycythemia related to an upregulated hypoxic response at normoxia. Specifically, homozygosity for the VHLR200W mutation leads to increased levels of the transcription factors hypoxia inducible factor (HIF)-1 and HIF-2 at normoxia and altered transcription of many genes. Much attention in the pathophysiology of SCD has focused on the adverse effects of chronic inflammation, enhanced cellular adhesion pathways and hemolytic rate. However, the chronic anemia of SCD is associated with an upregulation of the hypoxic response as evidenced by high erythropoietin concentrations. In this prospective study, we compared gene expression alterations in peripheral blood mononuclear cells in SCD and CP. Our pre-specified hypothesis was that we could identify hypoxia-mediated gene expression alterations in SCD through a comparison with altered gene expression in CP and reveal molecular pathways that are potentially shared by these two conditions. We prospectively compared gene expression profiles in two independent cohorts, an SCD cohort comprised of 22 SCD patients and 19 African American controls and a CP cohort comprised of 8 VHLR200W homozygotes and 17 Chuvash controls with wildtype VHL. Because iron deficiency can influence the hypoxic response, we excluded iron-deficient subjects from each cohort. We used the identical Affymetrix exon array platform for both cohorts. Differential gene expression was highly correlated between the two conditions, with Spearman correlation ρ = 0.75 between their expression profiles across 16,642 genes, suggesting that 56% of expression variation triggered by beta hemoglobin mutation in SCD may be explained by hypoxic transcriptional responses. A small portion of differential genes were highly induced in both conditions. Among 54 genes up-regulated >1.5-fold in SCD patients, 24 (44%) overlapped with the 31 genes up-regulated >1.5-fold in VHLR200W homozygotes. The genes highly induced in both conditions included FAM46C (family with sequence similarity 46 member C), SELENBP1 (selenium binding protein 1), IL1B (interleukin 1, beta), MOP-1, SNCA (synuclein, alpha), GMPR (guanosine monophosphate reductase), BPGM (2,3-bisphosphoglycerate mutase), SLC25A37 (solute carrier family 25 member 37), CA1 (carbonic anhydrase I), DCAF12 (DDB1 and CUL4 associated factor 12), EPB42 (erythrocyte membrane protein band 4.2), AHSP (alpha hemoglobin stabilizing protein), SLC4A1 (solute carrier family 4 member 1), HBB (hemoglobin, beta), HBD (hemoglobin, delta), CSDA (cold shock domain protein A), FECH (ferrochelatase), BCL2L1 (BCL2-like 1), OSBP2 (oxysterol binding protein 2), APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A), IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), IFI44L (interferon-induced protein 44-like), and IFI27 (interferon, alpha-inducible protein 27). Three genes were down-regulated >1.5-fold in SCD patients. One of these, GIMAP7 (GTPase, IMAP family member 7), was among the 11 genes down-regulated >1.5-fold in VHLR200Whomozygotes. These results suggest that there is a broad upregulation of the hypoxic response in SCD and that the hypoxic response may underlie or interact with an important proportion of the clinical and pathophysiologic manifestations of SCD. Disclosures: No relevant conflicts of interest to declare.

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Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders

Blood ◽

10.1182/blood-2005-11-009605 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1377-1380 ◽

Cited By ~ 197

Author(s):

Robert Kralovics ◽

Soon-Siong Teo ◽

Sai Li ◽

Alexandre Theocharides ◽

Andreas S. Buser ◽

...

Keyword(s):

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Janus Kinase ◽

Jak2 Mutation ◽

Genetic Event ◽

Allele Specific ◽

Unknown Gene ◽

Clonal Origin ◽

Polymerase Chain ◽

V617f Mutation

AbstractAn acquired gain-of-function mutation in the Janus kinase 2 (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPDs). To test the hypothesis that JAK2-V617F is the disease-initiating mutation, we examined whether all cells of clonal origin carry the JAK2-V617F mutation. Using allele-specific polymerase chain reaction (PCR) assays for the JAK2 mutation and for the X-chromosomal clonality markers IDS and MPP1, we found that the percentage of granulocytes and platelets with JAK2-V617F was often markedly lower than the percentage of clonal granulocytes determined by IDS or MPP1 clonality assays in female patients. Using deletions of chromosome 20q (del20q) as an autosomal, X-chromosome–independent clonality marker, we found a similar discrepancy between the percentage of cells carrying JAK2-V617F and del20q. Our results suggest that in a proportion of patients with MPDs, JAK2-V617F occurs on the background of clonal hematopoiesis caused by a somatic mutation in an as-yet-unknown gene.

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Allelic Expression Imbalance of JAK2 V617F Mutant Contributes to Phenotypic Variation in BCR-ABL Negative Chronic Myeloproliferative Disorders.

Blood ◽

10.1182/blood.v110.11.4651.4651 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4651-4651

Author(s):

Myung-Geun Shin ◽

Hye-Ran Kim ◽

Hyeoung-Joon Kim ◽

Mi-Ji Kim ◽

Hee-Nam Kim ◽

...

Keyword(s):

Gene Expression ◽

Genomic Dna ◽

Mutant Allele ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Allelic Expression ◽

Jak2 V617f Mutation ◽

Allelic Expression Imbalance ◽

Mutation Status ◽

V617f Mutation

Abstract Background: An acquired somatic mutation in the JAK2 gene (V617F) could present the primary causative lesion in BCR-ABL-negative chronic myeloproliferative disorders (CMPD). However, some of the clinical and genetic data implied that the pathophysiological role of JAK2 V617F mutation in the CMPD would be more complex. Quantitative assessment of JAK2 V617F mutation has shown substantial heterogeneity in the genomic DNA. Recently, it has been reported that allelic variation in gene expression is common in the human genome. To test the hypothesis that JAK2 V617F mutant allele could be increased in cDNA, we examined JAK2 V617F mutation status in the genomic DNA and cDNA from a total of 78 patients with BCR-ABL-negative CMPD. Patients and Methods: Enrolled patients with BCR-ABL-negative CMPD in this study comprised 42 cases of essential thrombocythemia (ET), 26 polycythemia vera (PV), 7 idiopathic myelofibrosis (MF) and 3 unclassifiable (UC) CMPD. A 364-bp PCR product containing JAK2 V617F mutation was bidirectionally sequenced from total BM cells. A quantitative real time PCR-based allelic discrimination assay and pyrosequencing (Pyrosequencer PSQ96) were developed for quantitative analysis of JAK2 V617F mutation status. Homozygous JAK2 V617F mutation was defined if the mutant peak was more than 50% of total peak area. Results: The proportion of mutant alleles ranged from 36% to 100% in real-time PCR and pyrosequencing analysis. Patients with MF had higher percentages of JAK2 mutant alleles than patients with ET (MF > PV > ET). The prevalence of homozygous V617F mutations was significantly higher in PV patients (73%) than in patients with ET (17%). Allelic expression imbalance of heterozygous JAK2 mutation was common in patients with PV and ET. Interestingly, allelic expression imbalance in patients with MF was not remarkably impaired, but preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with PV and ET. Conclusion: Allelic imbalance in the gene expression of JAK2 V617F mutant could provide the underlying mechanisms to elucidate phenotypic variation in BCR-ABL negative CMPD.

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The Janus kinase 2 (JAK2) V617F mutation in Chinese patients with chronic myeloproliferative disorders

Haematologica ◽

10.3324/haematol.12337 ◽

2008 ◽

Vol 93 (5) ◽

pp. 787-788 ◽

Cited By ~ 5

Author(s):

Z. Xiao ◽

Y. Zhang ◽

L. Li ◽

L. Nie ◽

L. Yang ◽

...

Keyword(s):

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Chinese Patients ◽

Janus Kinase 2 ◽

Chronic Myeloproliferative Disorders ◽

V617f Mutation

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Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera

Blood ◽

10.1182/blood-2008-12-196535 ◽

2010 ◽

Vol 115 (3) ◽

pp. 667-676 ◽

Cited By ~ 9

Author(s):

Maria Luigia Randi ◽

Anna Maria Brunati ◽

Margherita Scapin ◽

Martina Frasson ◽

Renzo Deana ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Tyrosine Kinases ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Janus Kinase ◽

Resting Cells ◽

Src Tyrosine Kinase ◽

V617f Mutation ◽

Signaling Activation

Abstract Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca2+]c increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

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Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162521 ◽

1996 ◽

Vol 54 ◽

pp. 12-13

Author(s):

W. K. Jones ◽

J. Robbins

Keyword(s):

Gene Expression ◽

Pulmonary Veins ◽

Microscopic Analysis ◽

Mouse Tissue ◽

Adult Heart ◽

Heavy Chains ◽

Altered Gene Expression ◽

Altered Gene ◽

Pulmonary Myocardium

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of altered α-MyHC expression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results of in situ hybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that of α-MyHC (Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding of α-MyHC expression and delimit a third cardiac compartment.

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