Detection of Recurrent Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MDS/MPD-Derived Secondary AML

Abstract The WHO classification distinguishes MDS/MPD as a distinct entity. The JAK2 V617F mutation is present in a minority of these patients (pts). UPD9p characterizes pts homozygous for the V617F mutation. Other chromosomal abnormalities can also be detected in pts with typical MPD and MDS/MPD, and it is likely that cytogenetic methods with higher resolution could detect additional defects. We applied 250K SNP-arrays to examine genomic composition and identify previously cryptic defects and molecular abnormalities in pts with MDS/MPD-U and secondary AML (sAML) arising from MDS/MPD-U both in pts wild-type for V617F and those with the mutation. Any deletions, duplications, and/or UPD found by SNP-A in 76 controls or on available internet databases were considered copy number variants (CNV) and non-pathogenic. First, we used pts with typical MPD to assess the ability of SNP-A to detect UPD. All pts homozygous for V617F showed UPD9p by SNP-A. In pts with MDS/MPD, several additional cryptic lesions were detected, including segmental micro-deletions on chr 1, 5, 9, and 12. UPD was common, occurring on chromosomes other than 9 in 9/28 patients (32%, i.e. on chr 1,11,12). Shared/overlapping lesions (in >2 pts) included small segmental lesions on chr 7 (N=3), and a small cytoband (q14.1) of chr 11 (N=3). Overall, clonal lesions including segmental UPD were found in 23/28 (82%) pts by SNP-A in comparison to 17/28 (61%) by metaphase cytogenetics (MC). Pts with a history of MDS/MPD-U with (N=14) and without the JAK2 V617F mutation (N=14) were also analyzed. MC revealed chr aberrations in 10/14 (71%) of V617F+ pts, including common lesions such as +8 and del5q. With SNP-A, 1 additional pt with normal MC was found to have an abnormal karyotype (UPD 7 and 9), and 9/10 pts with abnormal MC had additional lesions previously undetected, including UPD on chrs other than 9 in 3/14 pts (21%). Examples of deleted regions include segmental losses within chrs 2, 7, 8 and 13, and UPD on chrs 1p, 7q, and 11. Likewise, additional lesions were identified in MDS/MPD-U pts negative for V617F. 8/14 pts (57%) showed abnormal MC; however SNP-A showed lesions in 12/14. In addition, 6/8 pts with abnormal MC had lesions in addition to those detected by MC. UPD was also common in V617F- pts, occurring in 6/14 (43%), predominantly on chr 11 (in 3/6 pts). No significant difference was found between the number or type of lesions found in pts with and without V617F mutation. SNP-A can also be used to identify lesions acquired during AML evolution. In 1 MPD pt at diagnosis, SNP-A showed UPD9p as a sole abnormality consistent with a homozygous V617F mutation. Upon transformation, repeated SNP-A showed a V617F- leukemic clone (normal chr 9) possessing microdeletions on chr 4 and 19. Similar evolution of a V617 negative leukemic clone was also observed in an MDS/MPD pt in whom a new UPD6 was detected. However, in 3 other MDS/MPD patients, SNP-A showed the presence of a V617+ leukemic clone (showing UPD9) in AML blasts. In summary, SNP-A-based karyotyping complements MC and allows for precise definition of chr aberrations in pts with MDS/MPD, including copy-neutral LOH. UPD is common in both JAK2 V617F+ and V617F- disorders, and is not restricted only to chr 9p, indicating other potential causative genes.

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Tissue Factor and Soluble Markers of Platelet and Endothelial Activation in Essential Thrombocythemia: Relationship with Thrombosis and JAK2 V617F Mutation Status.

Blood ◽

10.1182/blood.v108.11.2707.2707 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2707-2707

Author(s):

Francisco Cervantes ◽

Eduardo Arellano-Rodrigo ◽

Alberto Alvarez-Larran ◽

Juan-Carlos Reverter ◽

Neus Villamor ◽

...

Keyword(s):

Tissue Factor ◽

Essential Thrombocythemia ◽

Plasma Concentrations ◽

Jak2 V617f ◽

Endothelial Activation ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Mutation Status ◽

Significant Difference ◽

V617f Mutation

Abstract There is increasing evidence that platelet and leukocyte activation plays an important role in the thrombotic complications of patients with essential thrombocythemia (ET), but the relationship of both thrombosis occurrence and JAK2 V617F mutation status with the levels of circulating tissue factor (TF) and of soluble markers of platelet and endothelial activation is not known. In 53 ET patients (26 of whom had a previous history of thrombosis), platelet TF expression and plasma levels of TF, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer, and prothrombin fragment 1+2 (F1+2), measured by ELISA, were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 V617F mutation status. ET patients with thrombosis had significantly higher levels of sP-selectin than patients without thrombosis and the controls, whereas ET patients without thrombosis had significantly higher levels than the controls (99.8 ± 47.1 ng/mL versus 70.6 ± 37.8 ng/mL versus 32.4 ± 11.9 ng/mL; p= 0.0001 for all comparisons). The same applied to sCD40L levels (226.7 ± 104.7 pg/mL in patients with thrombosis, 186.4 ± 92.1 pg/mL in patients without thrombosis, and 81.3 ± 22.0 pg/mL in controls; p= 0.0001 for all comparisons). Circulating VWF:Ag and F1+2 levels were higher in ET patients than in controls, but no significant difference was observed between patients with and without thrombosis. No differences in TF platelet expression, TF and sTM plasma concentrations were found between patients and controls. A positive correlation was observed between sP-selectin and F1+2, a marker of thrombin generation (r= 0.378, p= 0.01). Patients with the JAK2 mutation (22 out of 52 assessable patients), as compared with those with the wild-type allele, had significantly higher levels of sP-selectin (p= 0.002), sCD40L (p= 0.03), TF (p= 0.016), VWF:Ag (p= 0.0001), and sTM (p= 0.032). These results support a role for soluble markers of platelet activation in the thrombosis of ET as well as their potential to identify ET patients at greater risk of thrombosis. The association between JAK2 mutation and increased levels of TF and soluble markers of platelet and endothelial activation would suggest that the mutation could promote an enhanced prethrombotic state in ET.

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Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T.

Blood ◽

10.1182/blood.v112.11.1643.1643 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1643-1643

Author(s):

Hadrian Szpurka ◽

Lukasz P Gondek ◽

Sanjay R Mohan ◽

Eric D Hsi ◽

Karl S Theil ◽

...

Keyword(s):

Clonal Selection ◽

Neutral Loss ◽

Significant Proportion ◽

Uniparental Disomy ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Refractory Anemia ◽

Molecular Defect ◽

Ringed Sideroblasts ◽

V617f Mutation

Abstract Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (>600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

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The Relationship Between Wild Type and Mutant-Positive Platelets in Essential Thrombocythemia Patients with a JAK2 V617F Mutation

Blood ◽

10.1182/blood.v112.11.3728.3728 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3728-3728

Author(s):

Jonathan R Lambert ◽

Rosemary Gale ◽

David C. Linch

Keyword(s):

Platelet Count ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Absolute Number ◽

Jak2 V617f Mutation ◽

Secondary Process ◽

Wild Type ◽

Significant Difference ◽

V617f Mutation ◽

The Absolute

Abstract The myeloproliferative condition essential thrombocythemia (ET) is characterized by a persistent thrombocytosis in the absence of a recognizable cause. Approximately 50% of patients carry the acquired mutation JAK2 V617F, but it is unclear whether this is the initiating event in the overproduction of platelets or a secondary process arising in a situation where the thrombopoietic drive is already increased. In vitro studies have shown that mutant-positive erythroid cells have a proliferative advantage compared to wild-type (WT) cells. However, in JAK2 V617F-positive ET patients, the mutation is only found in a proportion of neutrophils, with the mutant level remaining stable over many years, and the JAK2 WT neutrophils are polyclonal by X-chromosome inactivation analysis. This may not be true of platelets as expansion of the mutant-positive cells may be restricted to the megakaryocytic lineage. We therefore quantified the mutant level in neutrophils and platelets purified from 10 JAK2 V617F-positive ET patients prior to the initiation of cytoreductive therapy using PCR with a fluorescently-labeled reverse primer and a mismatch forward primer that allowed discrimination between WT and JAK2 V617F alleles following AflIII digestion. There was no significant difference in the mutant levels determined using neutrophil DNA and RNA (median 15% [range, 11%–27%] and 21% [12%–31%] respectively). Mutant levels in platelet RNA were significantly higher than those in neutrophil RNA (median 27% [range, 20%–39%] versus 21% [12%–31%] respectively; P = 0.002), but still indicated that the JAK2 mutant was present in only a subpopulation of platelets. Assuming that all cells were heterozygous for the mutation, the data indicate that a median of only 54% (range 40%–78%) of the platelets were mutant-positive. We then calculated the absolute number of JAK2 WT and mutant-positive platelets for each patient from the quantified proportion of mutant alleles in platelet RNA and the total platelet count at the time of testing. The absolute number of JAK2 mutant-positive platelets in the patients varied between 263 and 798 × 109/L, and strongly correlated with the percentage of JAK2 mutant alleles (r2 = 0.81, P = 0.0004). The WT platelet count varied between 225 and 426 × 109/L, and there was a significant negative correlation between the absolute number of WT and mutant-positive platelets (r2 = 0.70, P = 0.002). However, when the absolute number of WT platelets was plotted against the total platelet count, there was no relationship between them (r2 = 0.003, P = 0.87). These data suggest that the negative feedback from the total platelet mass on normal (JAK2 WT) thrombopoiesis was incomplete; in no case was the WT platelet count below the lower limit of normal. This may relate to the observation that in ET, levels of thrombopoietin, the lineage-specific cytokine which drives platelet production, are often normal or even increased, unlike the situation in polycythemia vera where erythropoietin levels are reduced and normal red cell production is suppressed. Furthermore, extrapolation of the data from the WT platelet counts and JAK2 V617F mutant levels raises the possibility that when the mutation was first acquired and mutant levels were very low, the WT platelet count was at the upper limit of normality or elevated. This suggests that the JAK2 V617F mutation could have arisen on a background of increased thrombopoiesis, and was not the initiating event in the development of the disorder.

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Evaluation of JAK2 (V617F) Mutation in Iranian Veterans with Delayed Complications of Sulphur Mustard Poisoning

Iranian Jornal of Toxicology ◽

10.32598/ijt.10.4.355.1 ◽

2016 ◽

Vol 10 (3) ◽

pp. 21-24

Author(s):

Mohammad Reza Keramati ◽

◽

Mohammad Hadi Sadeghian ◽

Hossein Ayatollahi ◽

Mohammad Hosein Basharati ◽

...

Keyword(s):

Sulfur Mustard ◽

Preventive Measure ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Control Group ◽

Case Group ◽

Jak2 Mutation ◽

Significant Difference ◽

V617f Mutation

Background: Sulfur mustard was the most widely applied chemical warfare agent by the Iraqi army in Iran–Iraq war (1983-1988). Considering the role of sulfur mustard toxicity in hematopoietic neoplasms and also new role of JAK2 mutation in these neoplasms, we assessed this mutation and delayed hematologic complications in veterans exposed to sulfur mustard. Methods: This case control study was performed in Mashhad University of Medical Sciences, Mashhad, Iran in collaboration with Janbasan Foundation of Khorasan Razavi, Iran in 2012. The case group consists of 42 patients who exposed to sulfur mustard about 30 yr ago and the control group includes 30 healthy persons. For all subjects complete blood counts and ARMSpolymerase chain reaction for JAK2 (V617F) mutation was carried out. Data were analyzed by statistical software using independent sample t-testand Mann- Whitney U test. Results: JAK2 (V617F) mutation was detected, neither in the sulfur mustardveterans nor in the control group. Moreover no significant difference was detected in hematologic parameters between the two groups. Conclusion: Despite sulfur mustard can increase risk of tumor genesis especially hematologic neoplasms but this is probablyas result of other genetic mechanism apart from JAK2 mutation. Considering the health and importance of preventive measure for the sulfur mustard victims, we suggest other genetic aspects of tumor genesis to be assessed in these patients.

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JAK2 V617F Mutation Is Infrequent in “5q-Syndrome” and 5q-Associated MDS.

Blood ◽

10.1182/blood.v108.11.4833.4833 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4833-4833

Author(s):

Ling Zhang ◽

Saskia Gueller ◽

Sophie Raynaud ◽

Phillip H. Koeffler ◽

Stephen Lee

Keyword(s):

Bone Marrow ◽

Chromosomal Abnormalities ◽

Bone Marrow Aspiration ◽

K562 Cells ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Jak2 Mutation ◽

V617f Mutation

Abstract Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”. Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed. Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control. Results: PCR results showed clear wild type PCR patterns in all 21 cases. Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.

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Deletions of Chromosome 13q in Myeloproliferative Neoplasms: Mapping, Relation to the JAK2-V617F Mutation and Evaluation of Potential Tumor Suppressor Candidates

Blood ◽

10.1182/blood.v112.11.3724.3724 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3724-3724 ◽

Cited By ~ 1

Author(s):

Damla Olcaydu ◽

Tiina Berg ◽

Bettina Gisslinger ◽

Heinz Gisslinger ◽

Robert Kralovics

Keyword(s):

Tumor Suppressor ◽

Chromosomal Aberrations ◽

Myeloproliferative Neoplasms ◽

Myeloid Cells ◽

Jak2 V617f ◽

Complete Loss ◽

Jak2 V617f Mutation ◽

Significant Difference ◽

V617f Mutation ◽

Chromosome 13Q

Abstract Chronic myeloproliferative neoplasms (MPN) are a heterogeneous group of disorders characterized by clonal hematopoiesis, excessive production of myeloid cells and an inherent tendency for thrombosis, bleeding, secondary fibrosis and leukemic transformation. Chromosomal aberrations are present at diagnosis in 34% of patients with polycythemia vera (PV), 40% of primary myelofibrosis (PMF) and 5% of essential thrombocythemia (ET) patients. Deletions of the long arm of chromosome 13 (del13q) are among the recurrent cytogenetic abnormalities found not only in MPN but also in other hematological malignancies. The frequency of del13q is highest in PMF and post-polycythemic myelofibrosis (13–20% of all aberrations). A common deleted region of 16 mega base pairs (Mb) has been previously defined for del13q in MPN and the tumor suppressor RB1 was proposed to be the likely target of the deletion. In this study, we aimed to investigate the role of del13q in the clonal evolution and pathogenesis of MPN. We determined the frequency of del13q in a cohort of 367 MPN patients using microsatellite PCR with a series of markers covering a 10 Mb chromosomal region. We identified 8 patients with loss of heterozygosity (LOH) in at least one microsatellite marker (8/367, 2.18%). Three of 8 del13q patients were diagnosed with PMF, 4 with PV and one with ET. To map the minimal deleted region of del13q we performed microarray-based karyotype analysis and defined a common deleted region (CDR) of 9.8 Mb. As this newly defined del13q CDR included the RB1 tumor suppressor gene, we investigated whether haploinsufficiency or complete loss of RB1 function could be involved in MPN pathogenesis. Gene expression analysis of RB1 in del13q-positive MPN patient granulocytes did not show any significant difference in mRNA level compared to del13q-negative patients (P=0.6857) and healthy controls. No point mutations were found by sequence analysis of the remaining RB1 allele in del13q patients. We did not observe any effect of RB1 shRNA knock-down on cytokine-dependent proliferation of UT7/TPO cells. Our data suggest that complete loss or haploinsufficiency of RB1 is an unlikely pathogenetic mechanism associated with del13q in MPN. In addition to complete loss of chromosome 13q, we observed partial allelic loss (partial LOH) in 27 additional patients (7.36%), consistent with the presence of del13q in only a proportion of myeloid cells. To confirm that del13q represents a minor clone in these patients we genotyped BFU-E and CFU-GM progenitor colonies for del13q. In concordance with the microsatellite PCR data patients with partial LOH exhibited del13q only in a proportion of progenitor colonies. Therefore, the overall frequency of del13q in our MPN cohort was 9.54% (35/367). Of these 35 del13q-positive patients, 24 were positive for the JAK2-V617F mutation whereas 11 patients tested negative. Thus, del13q is not acquired preferentially in patients positive for JAK2-V617F (P=0.3642). Furthermore, when progenitor colonies of del13q positive patients were genotyped for both del13q and JAK2-V617F we observed that del13q can occur before or after the acquisition of JAK2-V617F or as a sole chromosomal lesion. In conclusion, del13q is one of the most frequent chromosomal aberrations in MPN and is acquired independently from the JAK2-V617F mutation. Since recent studies of PMF and post-PV myelofibrosis patients with or without del13q did not reveal any significant differences in clinical phenotype, del13q provides only clonal advantage to hematopoietic cells without effecting the disease phenotype. The molecular pathway involved in del13q-dependent clonal expansion remains to be identified.

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JAK2 V617F Analysis in Indonesian Myeloproliferative Neoplasms Patients

Journal of Biomedicine and Translational Research ◽

10.14710/jbtr.v1i2.63 ◽

2015 ◽

Vol 1 (2) ◽

pp. 27

Author(s):

Fanti Saktini ◽

Santosa Santosa ◽

Sultana MH Faradz

Keyword(s):

Cytogenetic Analysis ◽

Chromosomal Abnormalities ◽

Myeloproliferative Neoplasms ◽

Secondary Data ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Amplification Refractory Mutation System ◽

Cross Sectional ◽

V617f Mutation

Background : Three subtypes of myeloproliferative neoplasms (MPNs): Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) showed overlapping phenotype. There has been no specific cytogenetic marker identified in these subtypes. JAK2 V617F mutation prevalence in Caucasian MPNs was first reported as 97% in PV, 57% in ET, and 50% in PMF.Objective: This study was done to define the prevalence of JAK2 V617F mutation and to identify cytogenetic markers in MPNs.Methods : The study design was cross-sectional. Patients who were admitted to Dr. Kariadi Hospital with clinical diagnosis of MPNs were referred for bone marrow cytogenetic analysis in Telogorejo Hospital. JAK2 V617F mutation was tested for using Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) from peripheral blood vein. Clinical data were secondary data retrieved from hospital medical records.Results : There was no cytogenetic abnormality found in all MPNs patients. The prevalence of JAK2 V617F mutation in MPNs patients was 73,68%. Mutation prevalence distribution in each subtypes were 100% in PV, 63,6% in ET and 100% in PMF. Conclusion : Chromosomal abnormality was not found using conventional cytogenetic analysis. More sensitive methods might elucidate submicroscopic chromosomal abnormalities in these patients. The prevalence of JAK2 V617F mutation was comparable with studies in Caucasian. It is recommended that JAK2 V617F testing should be incorporated in the management therapy of MPNs in Indonesia.

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Is JAK2 V617F mutation more than a diagnostic index?

Leukemia Research ◽

10.1016/j.leukres.2008.06.006 ◽

2009 ◽

Vol 33 (1) ◽

pp. 67-73 ◽

Cited By ~ 78

Author(s):

Issa J. Dahabreh ◽

Katerina Zoi ◽

Stavroula Giannouli ◽

Christine Zoi ◽

Dimitrios Loukopoulos ◽

...

Keyword(s):

Jak2 V617f ◽

Jak2 V617f Mutation ◽

V617f Mutation ◽

Diagnostic Index

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CRISPR/Cas12a-Based Ultrasensitive and Rapid Detection of JAK2 V617F Somatic Mutation in Myeloproliferative Neoplasms

Biosensors ◽

10.3390/bios11080247 ◽

2021 ◽

Vol 11 (8) ◽

pp. 247

Author(s):

Miaomiao Chen ◽

Chunhua Zhang ◽

Zhiqing Hu ◽

Zhuo Li ◽

Menglin Li ◽

...

Keyword(s):

Detection System ◽

Myeloproliferative Neoplasms ◽

Cost Effective ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Minimum Detectable Concentration ◽

Lateral Flow Strip ◽

Whole Process ◽

Detectable Concentration ◽

V617f Mutation

The JAK2 V617F mutation is a major diagnostic, therapeutic, and monitoring molecular target of Philadelphia-negative myeloproliferative neoplasms (MPNs). To date, numerous methods of detecting the JAK2 V617F mutation have been reported, but there is no gold-standard diagnostic method for clinical applications. Here, we developed and validated an efficient Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 12a (Cas12a)-based assay to detect the JAK2 V617F mutation. Our results showed that the sensitivity of the JAK2 V617F/Cas12a fluorescence detection system was as high as 0.01%, and the JAK2 V617F/Cas12a lateral flow strip assay could unambiguously detect as low as 0.5% of the JAK2 V617F mutation, which was much higher than the sensitivity required for clinical application. The minimum detectable concentration of genomic DNA achieved was 0.01 ng/μL (~5 aM, ~3 copies/μL). In addition, the whole process only took about 1.5 h, and the cost of an individual test was much lower than that of the current assays. Thus, our methods can be applied to detect the JAK2 V617F mutation, and they are highly sensitive, rapid, cost-effective, and convenient.

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