Analysis of JAK2 V617F mutation in Tunisian patients with myeloproliferative neoplasms

Abstract

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.

Keywords

JAK2 mutations myeloproliferative neoplasms Tunisia V617F

Introduction

Dysregulated proliferation and expansion arising from a transformed pluripotent hematopoietic stem cell could lead to clonal hematopoietic malignancies, called myeloproliferative neoplasms (MPNs).¹ In Philadelphia-negative MPN group there are three entities characterized by different clinical features: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary MyeloFibrosis (PMF).¹

The molecular characterization of MPNs was revolutionized in 2005 by the detection of a somatic mutation, the JAK2 V617F.¹ In fact, this peculiar mutation has been detected mostly in patients with PV (65%–97%) and more rarely in patients with ET (23%–57%) and PMF (35%–57%).^2–6 Few years later, in 2008, a revised edition for the classification and diagnosis criteria of MPNs including the JAK2 V617F mutation as major criteria have been published by the World Health Organization (WHO).⁷

Janus kinases (JAK) are a group of tyrosine kinases enzymes which play crucial roles in cytokines signal transduction controlling cell proliferation, survival, and apoptosis.⁸ In this group, JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) are the major kinases.⁸ JAK2, a cytoplasmic kinase, plays a critical role in multiple cytokines and hematopoietic growth-factors receptors signal transduction.¹ JAK2 structure includes four domains: N-terminal FERM domain which mediates receptor interaction, a Src homology-2 domain (SH2) which interacts with several signaling proteins, a C-terminal tyrosine kinase domain JH1 (Jak homology-1) which is negatively regulated by a pseudokinase domain (JH2).¹ The JAK2 V617F mutation consists in a G to T substitution at nucleotide 1849 of exon14.³ This gain of function mutation results in a substitution in codon 617 from valine to phenylalanine of the protein.³ The mutation occurs in the auto-inhibitory domain (JH2) and induces a constant hyperactivation of the JAK2 protein which enhances cytokine-independent growth of hematopoietic progenitors and eventually increases cell proliferation and resistance to apoptosis.¹

The discovery of the JAK2 V617F mutation has made a significant contribution to the understanding of the pathogenesis of MPNs and its high frequency makes it an important laboratory tool for both the diagnosis and the management of these diseases. Moreover, several clinical studies have quantified the V617F mutant allele, known as ‘allele burden’. The allele burden was disparately estimated within MPN, in fact, Allele burden was the lowest in ET patients, intermediate in those with PV and PMF, and the highest in post-PV myelofibrosis patients.⁹

Therefore, we aimed in the present study to determine for the first time in Tunisia the frequency of the JAK2 V617F mutation in Tunisian patients with myeloproliferative neoplasms by performing PCR-RFLP and direct sequencing methods. We intended to compare these two methods because of the reported discrepancies in the threshold of the mutation’s detection. In fact, the direct sequencing is able to detect the mutation at an allelic ratio between 10% and 20% while the threshold for PCR-RFLP was estimated at 2%–10%.¹⁰

Material and methods

Subjects

In this work we intended to perform a transversal comparative study between PCR-RFLF and direct sequencing for JAK2 V617F mutation. This study included 213 MPN patients, 77 unclassified patients with thrombosis (UPT) and 95 healthy blood donors who served as controls. Theoretical percentage (64.4%) of the JAK2 mutation in Tunisian MPN patients was obtained from the study of Mahjoub et al.¹¹ We used the following online calculator to determine the sample size: https://biostatgv.sentiweb.fr/>:?module=etudes/sujets#. For a Beta risk of 0.2, the estimated required sample size is 182 subjects.

The 213 MPN patients were followed-up at the departments of hematology and internal medicine between 2016 and 2019. MPN patients were classified into three subgroups: 109 with PV, 93 with ET, and 11 with PMF. Inclusion conditions were patients meeting the 2008 WHO classification criteria for MPN.⁷ Unfortunately, and because of limited resources we were not able to investigate the JAK2 exon 12, Calreticulin (CALR), and MPL mutations to apply the 2016 WHO classification criteria for MPN.¹² Exclusion criteria were: BCR-ABL1 positive karyotype and bone marrow non-conclusive findings.

We recruited an UPT group because thrombosis is a frequent and major complication in MPN. In the UPT group, thrombotic events included acute limb ischemia, stroke, splanchnic thrombosis, etc.

Healthy controls were used to verify the specificity of the JAK2 V617F mutation detection in MPN diagnosis as the mutation has been noted in the general population in some studies. In fact, Cordua et al.¹³ reported a prevalence of 3.1% in a danish general population which included 19958 healthy participants.

In order to be included in this study, a written informed consent was obtained from all patients and controls. This study was approved by the local Ethics’ committee of Charles Nicolle Hospital.

Methods

A standard salting-out procedure¹⁴ was used to extract genomic DNA for patients and controls from EDTA peripheral blood samples. The detection of the JAK2 V617F mutation was performed by both RFLP-PCR and direct sequencing procedures.

The PCR-RFLP procedure: The PCR-RFLP was performed according to the described procedure reported in the article of Shetty et al.¹⁵ The Figure 1 highlights the results of the products digestion.

Figure 1.

Gel analysis for the JAK2 V617F mutation detection in genomic DNA by PCR-RFLP.

The direct sequencing procedure: The sequencing reaction was performed on the same amplicons as used for PCR-RFLP analysis. The amplified products were sequenced using the BigDye Terminator Sequencing Kit (Applied Biosystems). For a final volume of 20 μl we used 0.5 μl of the PCR product and 0.32 pmol of the JAK2 reverse primer. Excess of Dye Deoxy terminator was removed from the reaction by cleaning the sequencing products using the Dye EXTm 2.0 Spin purification kit (Qiagen). Finally, the samples were sequenced on an ABI/HITACHI 3500 DNA sequencer (Applied Biosystems) and the results were analyzed using Chromas software (Figure 2).

Figure 2.

DNA sequence chromatograms of the JAK2V617F mutation.

Statistical analysis

Statistical evaluation was carried out using the Statistical Package for the Social Sciences (SPSS) version 11 (IBM^®, Armonk, USA). The threshold of significance was fixed at 0.05.

Chi-square or Fisher exact tests were used to test the association between categorical variables. Odds ratio (OR) together with 95% confidence intervals (95%CI) were calculated to estimate the strength of the association.

ANOVA, Mann–Whitney U and Kruskal–Wallis tests were used to analyze quantitative variables as appropriate.

Results

In total, 213 patients with MPN, 77 unclassified patients with thrombosis and 95 controls were included in this study. Mean age was at 57.28 ± 13.9 years for MPN patients, 47.82 ± 15.01 years for the UPT group and 53.19 ± 14.51 years for the controls. The sex ratio (Men/Women) was 0.95 (104/109) for the MPN group, 1.48 (46/31) for the UPT group and 0.93 (41/44) for the controls group (Table 1). Twenty-five (11.7%) MPN patients had a portal vein thrombosis which caused a Budd-Chiari Syndrome.

Table 1.

Characteristics of the study population.

Characteristics	MPN, n = 213	Unclassified patients, n = 77	Controls, n = 95	p
Mean age ± SD (years)	56.91 ± 15.03	47.82 ± 15.01	53.19 ± 14.51	ns
Sex-ratio (male/female)	0.95 (104/109)	1.48 (46/31)	0.93 (41/44)	ns
Thrombocytosis (PLT > 450 × 10⁹/L)	103 (48.5%)	0	0	<10⁻³
Leukocytosis (WBC > 10 × 10⁹/L)	61.8%	0	0	<10⁻³
Thrombosis, n (%)	25 (11.7%)	77 (100%)	0	<10⁻³

SD: standard deviation; ns: not significant; MPN: myeloproliferative neoplasms; WBC: white blood cells; PLT: platelets.

As shown in Table 2, in the MPN group, the mean age was similar in PV, ET, and PMF patients. Inversely, the sex-ratio (Male/Female) was significantly higher in PV group (1.53) than in ET and PMF groups (0.63 and 0.22), p < 0.001. Besides, hemoglobin and hematocrit were significantly higher in patients with PV comparatively to ET and PMF groups; p < 0.001 and p < 0.001, respectively (Table 2). In reverse, platelets count was significantly higher in ET patients than in PV and PMF groups, p < 0.001.

Table 2.

Hematological features of the MPN patients.

Characteristics	PV, n = 109	ET, n = 93	PMF, n = 11	p
Mean age ± SD (years)	57.28 ± 13.9	56.88 ± 16.3	53.55 ± 15.22	ns
Sex-ratio (male/female)	1.53 (66/43)	0.63 (36/57)	0.22 (2/9)	<0.001
Hb (g/dl)	17.8 ± 1.1	12.05 ± 2.35	9.87 ± 0.69	<0.001
HCT (%)	56.19 ± 4.83	37.94 ± 4	33.83 ± 2.65	<0.001
WBC (×10⁹/L)	12.35 ± 6	10.9 ± 4.1	13.53 ± 2.6	ns
PLT (×10⁹/L) (median)	344.5	793	190	<0.001
Thrombocytosis (PLT > 450 × 10⁹/L)	10 (9.2%)	93 (100%)	0	<0.001
Leukocytosis (WBC > 10 × 10⁹/L)	52%	58.5%	100%	0.018
Thrombosis, n (%)	4 (3.7%)	19 (20.4%)	2 (18.2%)	0.001

PV: polycythemia vera; ET: essential thrombocythemia; PMF: primary myelofibrosis; Hb: hemoglobin; HCT: hematocrit; WBC: white blood cell; PLT: platelets; ns: not significant.

Results of the JAK2 genotyping

The JAK2 V617F mutant allele was positive in 154 (40%) subjects by PCR-RFLP and in 150 (38.96%) subjects by direct sequencing. The concordance rate between the two procedures was at 96.88% (373/385), κ = 0.935, p < 0.001 (Table 3).

Table 3.

Comparative analysis between PCR-RFLP and direct sequencing in study population.

		Direct sequencing		Total
		V617F positive	V617F negative	Total
PCR-RFLP	V617F positive	146	8	154
PCR-RFLP	V617F negative	4	227	231
Total		150	235	385

κ = 0.935, p < 0.001.

Considering positive for the JAK2 V617F SNP any detected mutation by either PCR-RFLP or direct sequencing, 158 (74.17%) MPN patients were positive for V617F mutation while all unclassified patients with thrombosis and controls were negative (Table 4). Moreover, 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 (Table 4).

Table 4.

Jak2 V617F mutant allele frequencies by PCR-RFLF and direct sequencing.

Group	Jak2 V617F positive (PCR-RFLP)	Jak2 V617F positive (sequencing)	Jak2 V617F positive
PV (n = 109)	98 (89.9%)	94 (86.2%)	98 (89.9%)
ET (n = 93)	52 (55.9%)	51 (54.8%)	54 (58.1%)
PMF (n = 11)	4 (36.4%)	5 (45.5%)	6 (54.5%)
Unclassified patients (n = 77)	0	0	0
Controls (n = 95)	0	0	0

Analytic results of the JAK2 V617F mutation in MPN patients

In MPN patients, the V617F mutant allele was significantly correlated to patients’ age (59.35 ± 13.64 vs 49.91 ± 16.69), p < 0.001, while no association with gender was noted (Table 5). Biologically, there were significant associations between the JAK2 V617F mutation 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 (Table 5). Inversely, the JAK2 V617F SNP was not associated with either white blood cell or platelet counts.

Table 5.

Analysis of the Jak2 V617F mutation with clinical and biological features of MPN.

Feature		V617F positive, n = 154	V617F negative, n = 59	p	OR [95% CI]
Gender	Male	78	26	ns	–
Gender	Female	80	29	ns	–
Age		59.35 ± 13.64	49.91 ± 16.69	<0.001	–
Hb		16.29 ± 3	13.01 ± 3.65	<0.001	–
HTC		52.99 ± 8.34	45.37 ± 10.94	<0.001	–
WBC		12.14 ± 5	10.53 ± 3.6	ns	–
PLT		711.9 ± 408.8	776.1 ± 275.4	ns	–
Thrombosis		20 (12.7%)	5 (9.1%)	ns	–

In PV and PMF subgroups, the JAK2 V617F polymorphism was not associated to either hemoglobin level or hematocrit or leukocytes and platelets counts or the occurrence of thrombosis (Table 6). Inversely, in the ET subgroup the V617F mutant allele was associated to a higher hematocrit level (39.3 ± 3.34 vs 35.78 ± 4.1) and platelet count (872 vs 719); p = 0.014 and p = 0.033, respectively (Table 6). Moreover, and in ET subgroup, thrombosis occurrence was significantly more frequent in patients carrying the V617F mutation (16, 29.6% vs 3, 7.7%); p = 0.01, OR [95% CI] = 5.05 [1.35–18.81] (Table 6).

Table 6.

Analysis of the Jak2 V617F mutation with clinical and biological features of PV, ET, and PMF.

MPN	PV		ET		PMF
Features	V617F+, n = 98	V617F−, n = 11	V617F+, n = 54	V617F−, n = 39	V617F+, n = 6	V617F−, n = 5
Hb	17.9 ± 1.12	17.8 ± 1	12.5 ± 2.37	11.35 ± 2.2	10.18 ± 0.5	9.5 ± 0.76
p	ns		ns		ns
HTC	56.3 ± 4.9	55.8 ± 1.7	39.3 ± 3.34	35.78 ± 4.1	33.9 ± 2.5	32.1 ± 1.9
p	ns		0.014		ns
WBC	12.76	11.98	11.5 ± 4.3	10 ± 3.8	14.3 ± 3.3	12.6 ± 1.1
p	ns		ns		ns
PLT	389.232	378.215	872	719	170	212
p	ns		0.033		ns
Thrombosis	4 (4.1%)	0	16 (29.6%)	3 (7.7%)	0	2 (40%)
p	ns		0.01		ns

Discussion

The discovery of a gain-of-function somatic mutation in the JAK2 gene, baptized JAK2 V617F, has opened a new era in the understanding of Philadelphia-negative (PN) MPNs.⁹ Currently, the JAK2 V617F mutation screening has become a routine diagnostic workup for patients suspected to have PN-MPN. Thus, we aimed to study the prevalence of the JAK2 V617F mutation in MPN patients (n = 213) comparatively to a group of unclassified patients with thrombosis (n = 77), and a group of healthy subjects (n = 95).

The V617F mutation in the exon 14 of the JAK2 gene was detected by two molecular procedures: PCR-RFLP and direct sequencing. The PCR-RFLP was positive in 154 samples, while the direct sequencing detected only 150 positive patients for the V617F mutation. The concordance rate between the two tests was 96.88% (373/385), κ = 0.935. This, slightly higher sensitivity of PCR-RFLP comparatively to direct sequencing in JAK2 V617F mutation detecting corroborates previous reports. In fact, it has been reported that PCR-RFLP is able to detect the mutation with an allele burden of 2%–10% while direct sequencing with a burden varying between 10% and 20%.¹⁰

In the present study, the JAK2 V617F mutation was detected in 158 (74.17%) MPN patients, while all the UPT and controls were negative. This finding corroborates the results of a Portuguese study¹⁶ in which 75% of MPN patients and none of the controls were positive for JAK2 V617F mutation. Interestingly, the JAK2 V617F have been detected in cohorts of healthy subjects. Using qPCR, Nielsen et al.¹⁷ found the JAK2 V617F prevalence to be 0.1% in a Danish general population. Another study in a Danish general population reported the V617F mutation 613 (3.1%) out of 19958 healthy subjects.¹³ The authors explained their finding by MPN underdiagnosis in the general population.¹³ Besides, in our study, the V617F mutation was present in 98 (89.9%) PV patients, 54 (58.1%) ET patients, and 6 (54.5%) PMF patients, which are higher frequencies comparatively to those reported by the first five JAK2 V617F-discovering studies in 2005.^2–6 In fact, pooled data from the five studies indicated that the mutation was found in 77% (335/434) of PV, 35% (96/280) of ET, and 43% (40/93) of PMF patients.¹ These discrepancies could be explained by different diagnostic criteria which have been updated in 2008⁷ and 2016.¹² Inversely, recent studies performed in patients diagnosed with the updated criteria have reported higher frequencies of the JAK2 V617F mutation in MPN patients.^16,18–21 In fact, the frequency of the JAK2 V617F mutation in PV was 100% in Lebanese¹⁹ and Iranian,²⁰ 87.2% in Portuguese,¹⁶ 82% in Indian,²¹ and 81% in Egyptian¹⁸ populations. For the ET patients, the prevalence of the mutant allele was 73.4% in Portuguese,¹⁶ 70% in Indian,²¹ 68.29% in Lebanese,¹⁹ 50% in Egyptian,¹⁸ and 45.66% in Iranian²⁰ studies. Besides, the JAK2 V617F mutation frequencies in PMF patients were 52% in Indian,²¹ 50% in Portuguese¹⁶ and Egyptian,¹⁸ and 39.7% in Iranian²⁰ reports. The differences between the reported mutation frequencies in PV, ET, and PMF could be due to diverse study designs and different genetic backgrounds.

In this study, we found that MPN JAK2 V617F-positive patients had significantly higher hemoglobin levels and hematocrit which corroborates the results of previous reports.^18,20,21 Inversely, in our PV and PMF subgroups, the JAK2 V617F mutation did not influence either hemoglobin levels, or hematocrit, or leukocytes, and platelets count. These results agree with those of the Iranian²⁰ and Indian²¹ studies. Inversely, the Egyptian report¹⁸ noted significant associations with higher leucocytes and platelets counts in V617F positive PV and PMF patients, but the differences were not relevant (leukocytes: 13.15 vs 14.34 and platelets: 508 vs 591.2). Besides, we noted in the present study that ET JAK2 V617F-positive patients had significantly higher hematocrit and platelets count which has not been reported before in previous studies. In fact, while most of the reports^16,20,21 had not found any associations, the Egyptian report noted higher hemoglobin levels but lower platelets count in ET patients carrying the JAK2 V617F mutation¹⁸ The Egyptian unique finding¹⁸ led the authors to suggest that JAK2 V617F-positive ET are more closely resembling to PV.

Besides, in the present study the ET JAK2 V617F-positive patients had significantly a higher risk (≈5.05) for developing thrombosis. This significant association between the JAK2 V617F mutation and thrombosis has also been reported in previous studies^22,23 and confirmed by a meta-analysis of 17 studies²⁴ in which the estimated OR [95% CI] was 1.84 [1.4–2.43]. The higher risk of thrombosis in our ET JAK2 V617F-positive patients could be explained by the noted significantly higher hematocrit. Indeed, and in contrast to hematocrit, even extreme elevations in platelet counts do not contribute to whole blood viscosity.²⁵ Despite of the intuitive consideration that thrombocytosis increases the risk of thrombosis, most of previous reports failed to confirm it.²⁵ Moreover, the increased risk of thrombosis is not only the consequence of quantitative cellular variations but also qualitative changes. In fact, platelets from patients carrying the JAK2 V617F mutation exhibit biological differences such as increased P-selectin expression.²⁶

In summary, our findings corroborate those of previous reports with a majority of PV patients carrying the JAK2 V617F mutation, while it was present in approximately half of ET and PMF cases. Therefore, screening for the JAK2 V617F has to become a routine approach for MPN diagnosis in the Tunisian population. Nonetheless, the association with thrombosis events in ET patients needs to be replicated on independent cohorts.

In this study, there are some limitations. For instance, the PMF group was too small (n = 11) to carry a statistically proper analysis and needs further patients’ recruitment. Also, we were not able to carry out JAK2 exon 12, MPL exon 10, and CALR exon 9 mutations analysis because of limited resources. Likewise, in the future it would be valuable to quantify the JAK2 allele by qPCR as the threshold of activation by the V617F mutant allele might be disparate for each receptor leading to completely different behaviors for a certain level of JAK2 mutational load.¹

Conclusion

Based on these findings, the frequency of the JAK2 V617F mutation in Tunisian MPN patients corroborate those of previous studies. In ET patients, the V617F mutation seems to be predictive of thrombosis occurrence.

Footnotes

Acknowledgements

This study was granted and supported by the Research Laboratory in Immunology of Renal Transplantation and Immunopathology (LR03SP01), Charles Nicolle Hospital, Tunis El Manar University, Tunisia.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

Ethical approval for this study was obtained from the local Ethics’ committee of Charles Nicolle Hospital (Approval number: HCN_2016_21; January 07, 2016).

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Informed consent

Written informed consent was obtained from all subjects before the study.

ORCID iD

Tarak Dhaouadi

References

James

Ugo

Casadevall

(2005) A JAK2 mutation in myeloproliferative disorders: Pathogenesis and therapeutic and scientific prospects. Trends in Molecular Medicine 11: 546–554.

James

Ugo

Le Couédic

J-P

, et al. (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434(7037): 1144–1148.

Kralovics

Passamonti

Buser

, et al. (2005) A gain-of-function mutation of JAK2 in myeloproliferative disorders. The New England Journal of Medicine 352(17): 1779–1790.

Levine

Wadleigh

Cools

, et al. (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7(4): 387–397.

Baxter

Scott

Campbell

, et al. (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365(9464): 1054–1061.

Zhao

Xing

, et al. (2005) Identification of an acquired JAK2 mutation in polycythemia vera. The Journal of Biological Chemistry 280(24): 22788–22792.

Vardiman

Thiele

Arber

, et al. (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: Rationale and important changes. Blood 114: 937–951.

Ghoreschi

Laurence

O’Shea

(2009) Janus kinases in immune cell signaling. Immunological Reviews 228(1): 273–287.

Passamonti

Rumi

(2009) Clinical relevance of JAK2 (V617F) mutant allele burden. Haematologica 94(1): 7–10.

10.

Bench

White

Foroni

, et al. (2013) Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations. British Journal of Haematology 160: 25–34.

11.

Mahjoub

Baccouche

Sahnoun

, et al. (2015) La mutation JAK2 dans les syndromes myéloprolifératifs BCR-ABL négatifs: facteur prédictif de thrombose [The JAK2 mutation in myeloproliferative disorders: A predictive factor of thrombosis]. La Tunisie Médicale 93(7): 474–477.

12.

Arber

Orazi

Hasserjian

, et al. (2016) The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 127: 2391–2405.

13.

Cordua

Kjaer

Skov

, et al. (2019) Prevalence and phenotypes of JAK2 V617F and calreticulin mutations in a Danish general population. Blood 134(5): 469–479.

14.

Miller

Dykes

Polesky

(1988) A sampling salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research 16: 1218.

15.

Shetty

Kulkarni

Pai

, et al. (2010) JAK2 mutations across a spectrum of venous thrombosis cases. American Journal of Clinical Pathology 134(1): 82–85.

16.

Azevedo

Silva

Reichert

, et al. (2017) Prevalence of the Janus kinase 2 V617F mutation in Philadelphia-negative myeloproliferative neoplasms in a Portuguese population. Biomedical Reports 7: 370–376.

17.

Nielsen

Birgens

Nordestgaard

, et al. (2013) Diagnostic value of JAK2 V617F somatic mutation for myeloproliferative cancer in 49 488 individuals from the general population. British Journal of Haematology 160(1): 70–79.

18.

Ayad

Nafea

(2011) Acquired mutation of the tyrosine kinase JAK2V617F in Egyptian patients with myeloid disorders. Genetic Testing and Molecular Biomarkers 15: 17–21.

19.

Mahfouz

RAR

Hoteit

Salem

, et al. (2011) JAK2 V617F gene mutation in the laboratory work-up of myeloproliferative disorders: Experience of a major referral center in Lebanon. Genetic Testing and Molecular Biomarkers 15: 263–265.

20.

Poopak

Farshdousti Hagh

Saki

, et al. (2013) JAK2 V617F mutation in Iranian patients with myeloproliferative neoplasms: clinical and laboratory findings. Turkish Journal of Medical Sciences 43: 347–353.

21.

Sazawal

Bajaj

Chikkara

, et al. (2010) Prevalence of JAK2 V617F mutation in Indian patients with chronic myeloproliferative disorders. The Indian Journal of Medical Research 132: 423–427.

22.

Finazzi

Rambaldi

Guerini

, et al. (2007) Risk of thrombosis in patients with essential thrombocythemia and polycythemia vera according to JAK2 V617F mutation status. Haematologica 92: 135–136.

23.

Takata

Seki

Kanajii

, et al. (2014) Association between thromboembolic events and the JAK2 V617F mutation in myeloproliferative neoplasms. The Kurume Medical Journal 60: 89–97.

24.

Ziakas

(2008) Effect of Jak2 V617F on thrombotic risk in patients with essential thrombocythemia: measuring the uncertain. Haematologica 93: 1412–1414.

25.

Austin

Lambert

(2008) The JAK2V617F mutation and thrombosis. British Journal of Haematolgy 143: 307–320.

26.

Arellano-Rodrigo

Alvarez-Larran

Reverter

, et al. (2006) Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocythemia and correlation with the JAK2 mutational status. Haematologica 91: 169–175.