scholarly journals PCR-HRM for Detecting JAK2V617F Gene Mutation: Is It a Sensitive Assay?

2021 ◽  
Author(s):  
Mitra Rezaei ◽  
Mihan PourAbdollah Toutkaboni ◽  
Babak Salimi ◽  
Sharareh Seifi ◽  
Fatemeh Maryam Sheikholeslami
Keyword(s):  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Sensitive Assay ◽  
Chain Reaction ◽  
Jak2 Mutation ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Neoplastic Disorders ◽  
V617f Mutation ◽  
Sensitivity Specificity

Background: A substitution of G to T at nucleotide 1849 in exon 14 of the Janus kinase2 (JAK2) gene is well recognized in myeloproliferative neoplastic disorders (MPNs). Based on WHO guidelines, detection of the mutation is very important to confirm the disease in suspected patients. Methods: Eighty-seven patients with different background diseases were tested for JAK2 V617F mutation by four different methods, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), polymerase chain reaction-high resolution melting (PCR-HRM), and two different commercial kits. Results: The mean age of patients was 53.38±17.43 years, 72.4% were males, and 37.6% were females. JAK2 mutation was detected in 16 patients (18.3%). Of those, 7 (43.75%) suffered from PV, 5 (31.25%) from ET, 3 (18.75%) from PMF, and 1 (6.15%) from unclassified neoplastic disorders. The frequency of JAK2 mutation was 71.4% (5/7) in PV, 80% (4/5) in ET, and 66.7% (2/3) in PMF patients. The sensi- tivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and GE of PCR-HRM for detection of the JAK2 mutation was 86.7%, 100%, 100%, 97.3%, and 97.7%, respectively. While the sensitivity, specificity, PPV, NPV, and GE of PCR-RFLP were 93.3%, 80.5%, 50%, 98.3%, and 82.7%, respectively. On the other hand, the sensitivity, specificity, PPV, NPV, and GE of ARMS assays were evaluated by about 80%, 96%, 100%, 96%, and 96.5%, respectively. Conclusion: This study showed that PCR-HRM was a more sensitive assay to detect the JAK2 V617F mutation than the other assays. So, it can be used as a quick, easy, and effective method for screening the JAK2 V617F mutation in patients with MPNs disorders. PCR-RFLP must accompany it as a gold standard method for confirmation of the mutation of JAK2 V617F.

scholarly journals Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2173-2181 ◽  
Author(s):  
Hadrian Szpurka ◽  
Ramon Tiu ◽  
Gurunathan Murugesan ◽  
Samer Aboudola ◽  
Eric D. Hsi ◽  
...  
Keyword(s):  
Melting Curve ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Melting Curve Analysis ◽  
Jak2 Mutation ◽  
Ringed Sideroblasts ◽  
Polymerase Chain ◽  
V617f Mutation ◽  
Reaction Sequencing

Abstract JAK2 V617F mutation recently was identified as a pathogenic factor in typical chronic myeloproliferative diseases (CMPD). Some forms of myelodysplastic syndromes (MDS) show a significant overlap with CMPD (classified as MDS/MPD), but the diagnostic assignment may be challenging. We studied blood or bone marrow from 270 patients with MDS, MDS/MPD, and CMPD for the presence of JAK2 V617F mutation using polymerase chain reaction, sequencing, and melting curve analysis. The detection rate of JAK2 V617F mutants for polycythemia vera, chronic idiopathic myelofibrosis, and essential thrombocythemia (n = 103) was similar to the previously reported results. In typical forms of MDS (n = 89) JAK2 V617F mutation was very rare (n = 2). However, a higher prevalence of this mutation was found in patients with MDS/MPD-U (9 of 35). Within this group, most of the patients harboring JAK2 V617F mutation showed features consistent with the provisional MDS/MPD-U entity refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T). Among 9 RARS-T patients, 6 showed the presence of JAK2 V617F mutation, and in 1 patient without mutation, aberrant, positive phospho-STAT5 staining was seen that is typically present in association with JAK2 V617F mutation. In summary, we found that RARS-T reveals a high frequency of JAK2 V617F mutation and likely constitutes another JAK2 mutation-associated form of CMPD.

scholarly journals Development of a Quantitative Real-Time Polymerase Chain Reaction Assay for the Detection of the JAK2 V617F Mutation

2007 ◽  
Vol 9 (1) ◽  
pp. 42-46 ◽  
Author(s):  
Elizabeth C. Wolstencroft ◽  
Katy Hanlon ◽  
Lorna W. Harries ◽  
Graham R. Standen ◽  
Alexander Sternberg ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
V617f Mutation

scholarly journals Detection of the Janus kinase 2 V617F mutation using a locked nucleic-acid, real-time polymerase chain reaction assay

2018 ◽  
Vol 7 (1) ◽  
Author(s):  
Tshiphiri Senamela ◽  
Marleen Kock ◽  
Piet Becker ◽  
Joachim J.C. Potgieter
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Jak2 V617f ◽  
Locked Nucleic Acid ◽  
Jak2 V617f Mutation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
V617f Mutation

The purpose of this study was to develop a real time polymerase chain reaction (PCR) assay for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Sanger sequencing and a newly developed locked nucleic-acid, real-time PCR assay were used to detect the JAK2 V617F mutation. There was 100% agreement between the sequencing and PCR analysis. Both assays were able to detect the mutation in all 24 of the 60 test specimens harbouring the mutation.

Markers of Myeloproliferative Diseases in Childhood Polycythemia Vera and Essential Thrombocythemia

2007 ◽  
Vol 25 (9) ◽  
pp. 1048-1053 ◽  
Author(s):  
Luciana Teofili ◽  
Fiorina Giona ◽  
Maurizio Martini ◽  
Tonia Cenci ◽  
Francesco Guidi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Similar Proportion ◽  
Control Group ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
V617f Mutation

Purpose Polycythemia vera (PV) and essential thrombocythemia (ET) can present in pediatric age as sporadic or familial diseases. To define the biologic profile of childhood PV and ET, we evaluated specific markers in a cohort of pediatric patients affected by PV and ET, including cases with familial occurrence. Patients and Methods Thirty-eight children with PV and ET were investigated. The control group included 58 adults with PV and ET. Endogenous erythroid colonies, qualitative reverse transcriptase polymerase chain reaction for polycythemia rubra vera-1 (PRV-1) RNA expression, human androgen receptor assay and allele specific polymerase chain reaction for JAK2 V617F mutation were undertaken in all patients. Thrombopoietin, thrombopoietin receptor (c-mpl), and erythropoietin receptor mutation analysis was performed by direct sequencing in familial cases. Results The JAK2 V617F mutation in children with PV was significantly less frequent than in adult PV. The most common myeloproliferative marker found in these patients was PRV-1 RNA overexpression. Children and adults with sporadic ET showed a similar proportion of patients with PRV-1 RNA overexpression, JAK2 V617F mutation, and clonality, while none of the familial ET showed JAK2 V617F mutation and clonality. Also, PRV-1 RNA overexpression was significantly less common. Furthermore, most patients with familial ET exhibited the dominant-positive activating mutation of c-mpl. Finally, children with PV and ET had a significant lower incidence of thrombosis than adults. Conclusion This study demonstrates that familial and sporadic ET recognize different pathogenetic mechanisms. Myeloproliferative markers are specific tests for the diagnosis of ET in children with sporadic forms, while a significant proportion of children with PV can prove negative.

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.

Myeloid Blasts in Transformed JAK2-V617F Positive Myeloproliferative Disorders Are Frequently Negative for the JAK2-V617F Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 375-375 ◽  
Author(s):  
Alexandre Theocharides ◽  
Marjorie Boissinot ◽  
Richard Garand ◽  
François Girodon ◽  
Soon-Siong Teo ◽  
...  
Keyword(s):  
De Novo ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Allelic Ratio ◽  
Pcr Products ◽  
Blast Cells ◽  
V617f Mutation ◽  
De Novo Aml

Abstract Acute myeloid leukemia (AML) is a common complication of myeloproliferative disorders (MPDs). The role of the JAK2-V617F mutation in this process is unknown. We performed a retrospective analysis of DNA samples from MPD patients with secondary AML. We analysed DNA samples taken at the time of transformation to AML from 54 MPD patients (24 PV, 21 ET, 9 IMF). In addition, DNA samples taken at diagnosis of MPD were obtained in 21 of these patients. DNA was extracted from bone marrow or peripheral blood films, purified granulocytes or frozen cells. FACS sorting of blast cells, T cells and neutrophils was performed in some of the samples. The allelic ratio of JAK2-V617F was determined by allele-specific quantitative PCR (AS-PCR). We obtained AS-PCR data on 52/54 samples taken at the time of transformation (96%), whereas 2 samples did not yield PCR products: 24/52 samples were negative for JAK2-V617F (46%) and 28/52 were positive (54%). For 14/24 negative patients (58%) we had additional DNA samples taken at the time of MPD diagnosis and interestingly, 5 of these 14 patients (36%) were positive for JAK2-V617F at this earlier time point before AML transformation. This suggests that in these patients the JAK2-V617F positive clone was lost during the evolution to AML. Furthermore, comparison of the JAK2-V617F allelic ratios with the percentage of blast cells in patient samples positive at transformation revealed 8/28 cases where the JAK2-V617F allelic ratio was markedly lower than the percentage of blasts, e.g. 8%T-allele and 52% myeloid blast cells. In these patients a JAK2-V617F negative AML clone most likely co-exists with a JAK2-V617F positive MPD clone. To address the question whether the AML clone arose independently from the JAK2-V617F clone, we analyzed loss of heterozygosity on chromosome 9p (9pLOH) in one informative patient who displayed a high allelic ratio of mutant JAK2 at diagnosis (94%T). The CD15+ cells from this patient showed 9pLOH at diagnosis, as demonstrated with two independent microsatellite markers. In contrast, the FACS sorted blast cells at the time of transformation contained both parental alleles in the 9p region and were JAK2-V617F negative by AS-PCR. This excludes the possibility that the AML clone lost the JAK2V617F in the process of undergoing mitotic recombination at a stage heterozygous for JAK2-V617F. Analysis of additional patients is under way. In summary, we found in a cohort of 54 MPD patients, 13 patients initially positive for JAK2-V617F that transformed into JAK2-V617F negative AML. Although not confirmed in the one patient analyzed, we cannot exclude that other patients the JAK2-V617F positive MPD clone lost the JAK2 mutation during the process of transformation. Alternatively, the AML clone could have developed de novo from a JAK2-V617F negative progenitor or stem cell. The latter model has difficulties explaining the high incidence of de novo AML (8/54 patients), unless the JAK2-V617F negative progenitor already carried an as yet unknown mutation and was part of the MPD clone.

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.

Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4687-4687
Author(s):  
Yue Xu ◽  
Changxin Yin ◽  
Han He ◽  
Lingling Shu ◽  
Fuqun Wu ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Western Countries ◽  
Arms Pcr ◽  
V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

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