The JAK2V617F Mutation In Non-MPD Individuals Occurs at a Low Frequency During Erythroid Differentiation and Not In the Stem Cells.

Abstract 1583

A somatic point mutation in the JAK2 gene results in a constitutive activation of the JAK2 kinase in the erythropoietin (epo) signal transduction pathway. The mutation was found in Polycythemia Vera (PV) patients (>95%) as well as in other myeloproliferative disorders (MPD). In MPD the mutation was shown to occur in pluripotential hematopoietic stem cells and to be present in all blood cell lineages. We analyzed DNA of blood donors for the JAK2V617F mutation by allele specific Real Time PCR. The mutation was detected in 9% of 114 samples with a very low mutation frequency of < 0.002% per sample (mutant/wild-type DNA allelic ratio) in comparison to frequency of 50–100% in PV.

We speculated that accelerated erythropoiesis with increased erythroid differentiation will result in higher mutation rate. We therefore studied the presence of V617F mutation in two conditions associated with increased erythropoiesis, thalassemia and smoking.

In beta-thalassemia major patients, the mutation was found in 22.2% (12/54), a significant 2.4-fold increase in mutation incidence compared to healthy individuals (p=0.016). The mutation frequency was 0.035% per positive sample.

The V617F mutation was studied in a case – control study of hospitalized smokers (n=81) and non-smokers (n=61). Smoking was defined as 10 or more cigarettes per day, everyday of the week, for at least 10 consecutive years. Smoking status was assessed by a questionnaire and validated with measurement of exhaled carbon monoxide. Smokers suffered more chronic lung disease than non-smokers (45.7% vs. 6.6%, respectively, p<0.0001) and had a higher hematocrit, RBC and WBC (47.8% vs. 41.7% p<0.0001, 5.2 vs. 4.4×10¹² cells/liter, p<0.0001 and 9.4 vs. 7.6×10⁹ cells/liter, p=0.003, respectively). In all subjects epo was within the normal range or higher. The proportion of subjects with higher than normal epo was similar in smokers and non-smokers.

The prevalence of the mutation was significantly higher among the smokers (36%) than among the controls (15%) (p= 0.005) The median V617F mutation frequency in smokers was 10 fold higher than in controls (0.032% and 0.0032% respectively; p=0.027).

Very low V617F mutation frequency in DNA of non-MPD blood donors led us to question whether in these non-MPD individuals V617F mutation occurs in stem sells as was shown for PV patients. We found that the mutation was more frequent in peripheral blood mononuclear cells than in CD34⁺ cells purified from these mononuclear cells. Following 3 weeks culturing of CD34⁺ cells with epo mutation frequency increased by 9-folds (range 6–14) in hemoglobin-containing normoblasts. We next grew individual erythroid colonies from 8 healthy donors in semi-solid culture in the presence of epo. Out of 201 single colonies analyzed 13% contained the JAK2V617F mutation in a very small proportion of the cells (< 0.02%). These results demonstrate that in non-MPD individuals V617F mutation arises in very low frequency during cell differentiation.

We established a cell line model to study the occurrence of JAK2V617F mutation during erythroid differentiation. Mouse erythroleukemia (MEL) cells were transfected with the 1kb fragment of human JAK2 gene containing exon 14 sub-cloned into a mammalian expression vector. MEL cells were stimulated to differentiate by hexamethylene bisacetamide and the frequency of sequence changes in the JAK2 exon 14 was investigated. We found that changes in the JAK2 exon 14 sequence occurred at late stages of differentiation.

We hypothesize that maintenance of DNA fidelity is down graded with cell differentiation, rendering the cells susceptible for mutations in general and in JAK2 in particular.

In summary, accelerated erythropoiesis results in increased V617F mutation frequency such as in beta-thalassemia major patients and in heavy smokers. We demonstrate that unlike in MPD where the JAK2V617F mutation occurs in pluripotential stem cells and expands extensively, in non-MPD individuals it arises at very low frequency during cell differentiation. These differences determine the lack of clinical significance of the JAK2V617F mutation in non-MPD individuals.