Impact of JAK2 V617F Mutation on Hemogram Variation in Patients with Non-Reactive Elevated Platelet Counts

Background

Non-reactive platelet counts elevation occurs mainly in myeloproliferative disorders (MPDs), which have been reported to be closely associated with JAK2 V617F mutation. Complete blood cell count (CBC) is essential in diagnosis of MPDs, however, the impact of JAK2 V617F mutation on the patients’ hemogram variation remains not clear.

Methods

JAK2 V617F mutation was detected by allele specific real-time quantitative fluorescence PCR (AS-qPCR).

Results

Of the 402 non-reactive platelet elevating patients, JAK2 V617F mutation was detected in 222 (55.2%) patients. RBC counts, WBC counts, platelet-large contrast ratio (P-LCR), platelet distribution width (PDW) and mean platelet volume (MPV) were much higher in JAK2 V617F mutated patients, except platelet counts. In addition, when the patients were classified into subgroups by blood cell counts, it was found that JAK2 V617F mutation rate increased progressively with the increase of RBC counts and WBC counts, other than platelet counts. Furthermore, trilineage hyperplasia group showed highest JAK2 V617F mutation rate (93.26%), followed by the bilineage hyperplasia groups. Lastly, JAK2 V617F mutant allele burden was found much higher in polycythemia vera (PV) patients [median(P₂₅–P₇₅): 45.02%(35.12%–54.22%)] than in essential thrombocythemia (ET) patients [median(P₂₅–P₇₅): 28.23%(17.77%–41.66%)], and that it increased with WBC counts (r = 0.393, p = 0.000) and RBC counts(r = 0.215, p = 0.001), other than platelet counts (r = −0.051, p = 0.452). Further analysis revealed that in ET patients, JAK2 V617F mutant allele burden correlated with WBC counts and platelet counts positively, other than RBC counts, while in PV patients, it correlated with WBC counts and RBC counts positively, but not platelet counts.

Conclusions

JAK2 V617F mutation occurs frequently in patients with non-reactive elevated platelet counts. The presence of JAK2 V617F mutation has great impact on hemogram variation, including RBC counts, WBC counts, platelet parameters and lineage hyperplasia, but not on platelet counts. Besides, JAK2 V617F mutant allele burden affects the blood cell proliferation pattern.     

JAK2 V617F Genotype Is a Strong Determinant of Blast Transformation in Primary Myelofibrosis

Purpose

The influence of JAK2 V617F mutation on blast transformation (BT) and overall survival (OS) in primary myelofibrosis (PMF) is controversial. In a large cohort of patients we applied competing risks analysis for studying the influence of JAK2V617F mutation on BT in PMF.

Patients and Methods

In 462 PMF–fibrotic type patients (bone marrow [BM] fibrosis grade >0) we computed the incidence of BT and death in the framework of Cox regression analysis and of Fine and Gray competing risks analysis for BT.

Results

At the Cox regression analysis, having either a wild-type (wt) or a homozygous JAK2V617F genotype were factors for BT (HR, 1.98 and 2.04, respectively, with respect to the heterozygous genotype), but not for OS. At the competing risks regression analysis, the risk for BT in wt and homozygous V617F patients increased with respect to Cox analysis, giving a sHR of 2.17 and 2.12, respectively. Correcting the results for the variables that could have influence on BT, JAK2V617F wt and homozygous genotypes remained independently associated with BT. In a validation cohort of 133 independent cases with PMF-prefibrotic type (BM fibrosis grade  = 0), the BT predictive model including JAK2V617F genotype and older age retained high discriminant capacity (C statistics, 0.70; 95% CI, 0.47 to 0.92).

Conclusion

The accumulation of mutated alleles in the JAK2V617F clone or the selective acquisition of a proliferative advantage in the wt clone are two relevant routes to BT in PMF. The influence of these results on treatment decisions with anti-JAK2 agents should be tested.     

Absence of JAK2 V617F mutation in thalassemia intermedia patients

JAK2 is a cytoplasmic tyrosine kinase that has a vital role in signal transduction from several hemopoietic growth factor receptors. The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia Vera, essential thrombocythemia, and idiopathic Myelofibrosis but has not been previously described in Thalassemia patients. We studied 36 Lebanese patients diagnosed with thalassemia intermedia and assessed the presence or absence of the JAK2 V617F mutation using JAK2 activating mutation assay (In VivoScribe Technologies) and Polymerase Chain Reaction (PCR). None of the thalassemia intermedia patients were positive for this mutation. To our knowledge, this study is the first to determine the status of JAK2 V617F mutation in thalassemia intermedia patients and expands the international published literature on JAK2. The latter’s V617F mutation does not seem to play a role in this hematologically important clinical entity.     

Recognizing and managing the expanded risk of tumor lysis syndrome in hematologic and solid malignancies

Background

Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34⁺ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters.

Results

By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34⁺ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34⁺ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-X _L and BCLW. In contrast, pro-apoptotic BID and BIM _EL expression were downregulated in ET leukocytes. Increased BCL-X_L protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly.

Conclusions

Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.     

Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor

Background

The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.

Methodology

In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.

Conclusions

Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.     

Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

Background

Myeloproliferative disorders are characterized by clonal expansion of normal mature blood cells. Acquired mutations giving rise to constitutive activation of the JAK2 tyrosine kinase has been shown to be present in the majority of patients. Since the demonstration that the V617F mutation in the exon 14 of the JAK2 gene is present in about 90% of patients with Polycythemia Vera (PV), the detection of this mutation has become a key tool for the diagnosis of these patients. More recently, additional mutations in the exon 12 of the JAK2 gene have been described in 5 to 10% of the patients with erythrocytosis. According to the updated WHO criteria the presence of these mutations should be looked for in PV patients with no JAK2 V617F mutation. Reliable and accurate methods dedicated to the detection of these highly variable mutations are therefore necessary.

Methods/Findings

For these reasons we have defined the conditions of a High Resolution DNA Melting curve analysis (HRM) method able to detect JAK2 exon 12 mutations. After having validated that the method was able to detect mutated patients, we have verified that it gave reproducible results in repeated experiments, on DNA extracted from either total blood or purified granulocytes. This HRM assay was further validated using 8 samples bearing different mutant sequences in 4 different laboratories, on 3 different instruments.

Conclusion

The assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.     

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera

Background

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.

Methodology/Principal Findings

Asymmetric PCR for detection of JAK2 V617F with a 3′-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.

Conclusions

With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.     

Improved Diagnosis of the Transition to JAK2 V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 ^V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2 ^V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 ^V617F, is highly desirable. In this study, we present an approach to assess the JAK2 ^V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 ^V617F spaced from one template for JAK2^{Wild Type} were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2^V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53±4.2% and 51.46±4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 ^V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 ^V617F from heterozygosity to homozygosity.     

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga,a halophytic species

Key message

QTL mapping in F ₂ population [ V. luteola × V. marina subsp. oblonga ] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL.

Abstract

The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F₂ population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F_2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50 % of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

Absence of <Emphasis Type="Italic">JAK2V617F</Emphasis> mutation in patients with beta-thalassemia major and thrombocytosis due to splenectomy

The report of Janus Kinase 2 (JAK2) mutations in myeloid malignancies with high frequency in myeloproliferative neoplasms has been well known since 2005. By monitoring allele burden, it is found that the expression of JAK2V617F mutation is increasing significantly from essential thrombocytosis to polycythemia vera. Furthermore, JAK2 abnormalities are reported in the majority of unexplained thrombotic episodes. Thalassemic syndromes are characterized by ineffective erythropoiesis and thrombocytosis, mainly due to splenectomy. The high incidence of thromboembolic events has led to the identification of a prothrombotic state in these patients. The contribution of JAK2 mutations to the hypercoagulable state of thalassemic patients is still unknown. Furthermore, the potential role of Janus Kinase mutations in hepcidin expression and consequently in ineffective erythropoiesis is still under investigation. This study was scheduled to determine whether the presence of JAK2V617F mutation in thalassemic patients is associated with thrombocytosis. We studied 20 patients DNA with beta-thalassemia for JAK2V617F mutation by using RG-PCR method. None of the patients were positive for this particular mutation. More studies are needed to prove the role of JAK2 in ineffective erythropoiesis, iron metabolism and thrombocytosis and to determine if using JAK2 inhibitors in thalassemic patients can be a potential therapeutic option.     

Evaluation of clinical and laboratory findings with JAK2 V617F mutation as an independent variable in essential thrombocytosis

Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia and thrombocytes are increased with abnormal functions. Discovery of the protein tyrosine kinase JAK2 V617F allele contributed to better understanding of the pathogenetic mechanisms of MPNs. Acquired single point mutation in the JAK2 V617F was determined approximately 50–60 % of patients with ET. In this study we aimed to investigate the relationship between JAK2 V617F gene mutation, hematologic, biochemical markers and the complications in the ET patients. A total of 268 patients diagnosed with ET and 219 of those studied for JAK2 gene mutation were followed at the hematology clinics of three major hospitals between 2008 and 2013 were screened retrospectively. Laboratory, clinical and hematologic parameters were compared for JAK2 V617F positive and JAK2 V617F negative patients with ET. 102 (46 %) patients were positive with the JAK2 V617F mutation. The complications were observed in 61 (28 %) patients and 38 (62 %) of them had JAK2 V617F mutation. The levels of white blood cells, neutrophil, basophil, red blood cells, hemoglobin, hematocrit, mean platelet volume, thrombocytes, eosinophil; urea, creatinine were significantly different in patients with the JAK2 V617F mutation (P < 0.05). Presence of the JAK2 V617F mutation supports the diagnosis of ET. It would be useful to investigate the JAK2 V617F mutation and the hematologic and biochemical markers at diagnosis with respect to consider the risk of developing complications and to take the precautions against these complications.     

A novel 9-bp insertion detected in steroid 21-hydroxylase gene (CYP21A2): prediction of its structural and functional implications by computational methods

Background

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods.

Methods

A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation.

Results

Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed.

Conclusion

The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.