JAK2-V617F mutation in Moroccan patients with myeloproliferative disorders: Contribution diagnosis and therapeutic prospects

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Original article

JAK2-V617F mutation in Moroccan patients with myeloproliferative disorders:

Contribution, diagnosis and therapeutic prospects

La mutation V617F du ge`ne JAK2 chez les malades de syndromes mye´loprolife´ratifs au Maroc : contribution au diagnostic et perspectives the´rapeutiques

A. Benmoussa

^a,

*, H. Dehbi

^a

, S. Fehri

^b

, A. Quessar

^b

, S. Nadiﬁ

^a

aLaboratory of Genetics and Molecular Pathologies, Faculty of Medicine, Casablanca, Morocco

bService d’he´matologie et d’oncologie pe´diatrique, hoˆpital 20-Aouˆt-1953, Casablanca, Morocco

Pathologie Biologie 59 (2011) e89–e92

A R T I C L E I N F O

Article history:

Received 21 May 2009 Accepted 26 June 2009

Available online 24 November 2009

Keywords:

JAK2 V617F

Myeloproliferative disorders in Morocco Polycythemia vera

Essential thrombocythemia Idiopathic myeloﬁbrosis AS-PCR

Mots cle´s:

JAK2 V617F

Syndromes mye´loprolife´ratifs au Maroc Polyglobulie de vaquez

Thrombocyte´mie essentielle Mye´loﬁborse idiopathique AS-PCR

A B S T R A C T

Aims. – The JAK2 V617F is a recent discovery. The implication of this mutation in the pathogenesis of the myeloproliferative disorders (MPDs) is currently conﬁrmed. Our study is the ﬁrst to be interested in the status of the JAK2 V617F mutation among myeloproliferative disorders patients in Morocco.

Patients and methods. – Our study focused on 70 non-CML MPD patients, attending several departments of hematology and internal medicine across Morocco. The mutation was detected by allele-speciﬁc PCR (AS-PCR).

Results. – The V617F JAK2 mutation incidence in polycythemia vera, essential thrombocythemia and idiopathic myeloﬁbrosis are respectively 89.47%, 62.5% and 33.33%. The V617F JAK2 mutation was absent within the patients with secondary erythrocytosis or thrombocytosis. We also found that the patients carrying the mutation displayed a leucocytosis and higher levels of haemoglobin and hematocrit than mutation-negative patients.

Conclusion. – Our study is the ﬁrst to assess the status of the JAK2 V617F mutation in patients with MPDs in Morocco. However, our data seem to conﬁrm that the JAK2 V617F mutation is rather uncommon in myeloid malignancies other than the classical BCR/ABL MPD negative.

R E´ S U M E´

Objectifs. –La mutation JAK2 V617F est de dećouverte rećente. L’implication de cette mutation dans la pathogeńie des syndromes mye´loprolife´ratifs (SMP) est actuellement confirmeé. Notre e´tude est la premie`re a` s’inte´resser au statut de la mutation JAK2 V617F chez les patients souffrant d’un syndrome mye´loprolife´ratif au Maroc.

Patients et me´thodes. –L’e´tude a porte´ sur 70 patients. La mutation a e´te´ de´tecteé par PCR alle`le spećifique (AS-PCR).

Re´sultats. – L’incidence de la mutation V617F JAK2 dans la polyglobulie de vaquez, thrombocyte´mie essentielle et la mye´loﬁbrose idiopathique sont respectivement de 89,47 %, 62,5 % et 33,33 %. La mutation V617F JAK2 est absente chez les patients avec polyglobulie secondaire ou une thrombocytose secondaire. Nous avons e´galement constate´ que les patients porteurs de cette mutation pre´sentent des niveaux plus e´leve´s de l’he´moglobine et l’he´matocrite par rapport aux malades ne portant pas cette mutation.

Conclusion. – Notre e´tude est la premie`re a` e´valuer la mutation JAK2 V617F chez des patients avec syndromes mye´loprolife´ratifs au Maroc. Nos donneés semblent confirmer que la mutation JAK2 V617F est spećifique aux syndromes mye´loprolife´ratifs.

ß2009 Elsevier Masson SAS. Tous droits re´serve´s.

* Corresponding author.

E-mail addresses:[email protected],[email protected](A. Benmoussa).

doi:10.1016/j.patbio.2009.06.005

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1. Introduction

Myeloproliferative disorders (MPDs) are clonal proliferations affecting pluripotent hematopoietic stem cells. The pathogenesis of these diseases remains unclear, except for chronic myeloid leukaemia (CML) where the fusion protein Bcr– Abl exhibits constitutive kinase activity

[1]. A similar mechanism is thought be

involved for the other MPDs. JAK2 protein is a key mediator of intracellular signal transduction of cytokine receptors, erythro- poietin receptor (EPO-R) and the thrombopoietin receptor (TPO-R) which are involved in hematopoiesis. It has been shown recently that the mutation on the gene coding for JAK2 protein at the position 617 (V617F) impaired its tyrosine kinase activity

[2–5].

The mutation of JAK2 protein at the position V617F is a genetic somatic abnormality where the substitution of a Thymine at position 1849 by a Guanine on the exon 12 of JAK2 protein coding gene. This mutation is located in the pseudokinase domain of JAK2 (JH2). A deﬁcient JH2 results in the suppression of the negative regulation of JAK2 protein kinase activity

[6–8]. Therefore, JAK2

kinase is in constitutive activation state and the cells presenting this mutation develop a hypersensitivity to a variety of cytokines and growth factors such as IL3, TPO, IGF1 and EPO, inducing a high level of proliferation and survival.

[4]. The JAK2 V617F mutation is

mostly found in polycythemia vera (PV) patients (65% to 97%) and also frequently found in idiopathic myeloﬁbrosis (IMF) and essential thrombocythemia (TE) cases (25% to 50%). However, this mutation is occasionally detected in hypereosinophilic syndrome patients

[2,5]. Studying JAK2 mutation represents an

important step in the understanding of its function in the molecular pathogenesis of the (MPDs). In addition, this would allow in future to establish a new classiﬁcation of these diseases and to develop new therapeutic approaches.

We have screened a group of Moroccan patients with myelo- proliferative disorders (MPDs) to investigate whether the JAK2 V617F mutation can be routinely used in the diagnostic evaluation.

2. Patients and methods

Our study focused on 70 non-CML MPD patients, attending several departments of hematology and internal medicine across Morocco (Table 1). All the patients gave informed written consent, and the study was approved by the local Ethics Committee of the Faculty of Medicine in Casablanca.

The extraction of genomic DNA from whole blood samples was carried out using the salt method. The quality and amount of DNA extracted was measured by

spectrophotometry. We detected the JAK2 V617F mutation by allele-specific polymerization chain reaction(AS-PCR). A double amplification was performed on the JAK2 exon 12 with two different pairs of primers: one pair of primers (M) amplified the mutant allele (F617) JAK2 gene, and the other pair of primers (S) amplified the wild allele (V617)[10–11]. The AS-PCR products were separated by electrophresis in a 2% agarose gel.

The correlations between the presence of the JAK2 V617F mutation, the clinical and biological characteristics of the patients were statistically analyzed using the Chi-square test, and the Student test[12]; this allow us to compare between the frequencies and the means, respectively.

3. Results and discussion

Among the 70 patients studied, 50 exhibited a myeloprolifera- tive disorder (MPD) while the remaining 20 suffered from secondary polycythemia or thrombocytosis. In the MPDs patients, 58% (29/50) have both wild type and mutated (V617F) alleles of JAK2 gene. The frequencies of JAK2 V617F mutation in the PV, TE and IMF patients are 89.47%, 62.5%, and 33.33%, respectively. This mutation was detected only in one out of ﬁve patients with idiopathic hypereosinophilic syndrome. Whereas, the only case of CMML examined for the presence of the JAK2 V617F mutation, was positive. On the other hand, the mutation was absent in 20 patients with secondary erythrocytosis or thrombocytosis (Table 2).

The mean age of the 29 patients presenting JAK2 V617F mutation is higher than that of the patients with only the wild type allele, 60.78 years and 51.84 years, respectively. This difference is not signiﬁcant for a threshold of signiﬁcance a = 5% (tc

<

tt) (Table 3). The difference in the risk of presenting a leukocytosis or high levels of hematocrite and hemoglobin are signiﬁcant between patients with mutation and patients without the V617F JAK2 mutation to the threshold of signiﬁcance a = 5% (C2c

>

C2t) (Table 3).

The implication of the JAK2 V617F mutation in the pathogenesis of the MPDs is conﬁrmed. But recent studies report higher rates of the presence of this mutation. This is mainly related to the choice of the technique used, the AS-PCR technique being known to be the most sensitive (2 to 3%) for the detection of point mutations

[2–4,13]. In our series the frequency of the JAK2 V617F mutation

amongst cases of PV is 89.47%, which corresponds to previous reports

[3,4]. We found the mutation in ﬁve out of 12 cases of

essential thrombocythemia (62.5%). This result is similar to frequencies reported by others

[13]. However, we believe that

our frequency is slightly high, given the small size of our sample,

Table 1 Patients.

Pathology Number Men/women Average diagnostic age by years (age interval)

Polycythemia vera(PV) 19 7/12 56.52 (36–72)

Essential thrombocythemia(TE) 8 4/4 54.87 (39–76)

Idiopathic myeloﬁbrosis(IMF) 12 7/5 56.83 (44–70)

Hypereosinophilic syndrome 5 5/0 39.6 (13–60)

Chronic Myelomonocytic Leukemia (LMMC) 1 1/0 76

MPDs unclassiﬁable 5 3/2 53.8 (36–77)

Secondary erythrocytosis or thrombocytosis 20 11/9 53.5 (24–70)

Table 2

Frequency of the JAK2 V617F mutation.

Pathology Number of patients Number of mutant patients (frequency [%])

Polycythemia vera (PV) 19 17 (89.47%)

Essential thrombocythemia (TE) 8 5 (62.5%)

Idiopathic myeloﬁbrosis (IMF) 12 4 (33.33%)

Chronic Myelomonocytic Leukemia (LMMC) 1 1

Hypereosinophilic syndrome 5 1

Secondary erythrocytosis or thrombocytosis. 20 0

MPDs unclassiﬁable 5 1

A. Benmoussa et al. / Pathologie Biologie 59 (2011) e89–e92 e90

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probably because some cases of PV that were included and considered as cases of essential thrombocythemia. In our series the frequency of the mutation in the case of IMF corresponds to 33.33%.

This is relatively low value compared to the results reported elsewhere

[2,3,14]. This difference could be explained by the small

size of our sample (12 cases of IMF), or by a possible inaccurate diagnosis.

In has been shown that the JAK2 V617F mutation is occasionally found in other MPDs

[14,15]. In our study, we detected the

mutation in one case among ﬁve of hypereosinophilic syndrome and in the only case of chronic myelomonocytic leukemia.

The small size of the sample does not allow us to conclude on the frequency of the mutation in these pathologies; however, our results strongly suggest the presence of this mutation not only in the PV, the essential thrombocythemia and idiopathic myeloﬁ- brosis but also in other MPDs.

The MPDs are a group of very heterogeneous disorders often difficult to diagnose. In our sample, one of the five MPDs cases is unclassifiable but was positive for the presence of the mutation, suggesting that this could help the clinicians to make a more precise diagnosis of this disease. On the other hand, mutated JAK2 allele is absent amongst the 20 cases of secondary polycythemia and thrombocytosis, confirming that this mutation is associated with MPDs.

The V617F JAK2 mutation is mainly heterozygous, but it can also be found in a homozygous state, especially in the case of PV

[3–4,16]. In our study, we found both wild type and mutated alleles

in all the patients presenting a mutated JAK2 V617F, but we cannot conclude that these patients present heterozygote alleles of the mutation.

In fact, the DNA of patients was extracted from leucocytes including lymphocytes that rarely present the mutation

[4]. The

wild type allele is always detectable in all the patients; for this reason it could be used as a control for the DNA ampliﬁcations.

On the other hand, 42% of the patients did not have the JAK2 V617F mutation, but they may present other mutations on the exon 12. Furthermore, other mutations in other genes may also be responsible for these diseases such as MPL W515L and MPL W515K mutations identiﬁed recently in 5% of the IMF and in 1% of TE

[17,18]. The positive correlation between the age of the late onset

of the disease and the presence of the JAK2 V617F mutation was also reported by the others

[3,13,19]. This could be due to an

overestimation of the age onset of the disease, because of the difﬁculty to determine the exact age of the emergence of the disease especially for patients diagnosed at a late stage.

We did not ﬁnd a signiﬁcant difference between patients with the JAK2 V617F mutation and patients without this mutation in the presentation of splenomegaly, hepatomegaly and thrombocytosis.

By contrast, hemoglobin, hematocrite and white blood cells are signiﬁcantly higher in patients with the mutation, in agreement with the ﬁnding reported elsewhere

[13]. We have not registered

cases of thrombosis in our series. However, a positive correlation

between the presence of the mutation and the risk of developing thrombosis has been reported

[3,4,19]. Finally, the duration of our

patients follow-up, is short, we could not correlate the presence of the JAK2 V617F mutation and the acute transformation and the response to different treatments. It has been shown that ET patients positive for the presence of the JAK2 V617F mutation are more sensitive to Hydroxyurea than negative patients

[19]. Also it

has been reported that the rate of the mutant allele decreased after treatment with INF alpha in 89% of the MPDs patients presenting JAK2 V617F the mutation

[20].

4. Conclusion

At our knowledge this study is the ﬁrst to assess the status of the JAK2 V617F mutation in patients with MPDs in Morocco. However our data seem to conﬁrm that the JAK2 V617F mutation is rather uncommon in myeloid malignancies other than the classical BCR/

ABL MPD negative.

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Table 3

Correlations between the presence of the JAK2 V617F mutation and some clinical and biological characteristics of patients.

Biological and clincals characteristics JAK2 V617F mutation status Statistical analysis

positive negative used Test Calculated critical value Table critical value

the age of the late onset of the MPD (years) 60.78 51.84 Student tc= 0.180 tt= 2

Frequency of splenomegaly (%) 58.62 61.9 Khi 2 C²c= 0.054 C²t= 3.84

Frequency of hepatomegaly (%) 13.79 33.33 Khi 2 C²c= 2.71 C²t= 3.84

Frequency of leukocytosis (%) 89.65 57.14 Khi 2 C²c= 7.05 C²t= 3.84

Frequency of elevated haematocrit (%) 55.17 9.52 Khi 2 C²c= 11.01 C²t= 3.84

Frequency of high haemoglobin (%) 65.51 28.57 Khi 2 C²c= 6.65 C²t= 3.84

Frequency of thrombocytosis (%) 62.06 38.09 Khi 2 C²c= 2.79 C²t= 3.84

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