To the editor:
Transformation of hematopoietic cells depends on the acquisition of genetic events leading to cytokine independence, typically associated with acquisition of an autocrine cytokine loop or/and increased expression or/and mutation of JAK genes.1 Rearrangement of the JAK2 gene, which presumably alters JAK2 transcription, is reported in hematopoietic cells.2 Murine models of myeloproliferative neoplasms (MPN) demonstrated that the polycythemia vera (PV) phenotype requires the combination of high expression and activation of Jak2.3 Indeed, expression of both wild-type (WT) and mutant JAK2 transcripts can be high in PV.4 PV is characterized by a high frequency of the JAK2 46/1 (GGCC) haplotype (represented in Figure 1A) predisposing to the JAK2V617F mutation.5,6 The JAK2V617F mutation facilitates the acquisition of homozygous status for the JAK2V617F by mitotic homologous recombination (HR) occurring between the JAK2WT and JAK2V617F alleles, resulting in chromosome 9p uniparental disomy (9pUPD).7,8 Here we report 2 cases where high JAK2 mRNA expression was associated with a novel early step in MPN development, HR preceding JAK2 mutation.
Patients Na1061 and Na1253 presented with a high hematocrit, slightly elevated leukocyte counts, normal (Na1061) or elevated (Na1253) platelet counts, aquagenic pruritus, absence of splenomegaly, and presence of JAK2V617F (20.7% for Na1061, 30.0% for Na1253), and were diagnosed with PV (see supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Sequencing and allele-specific qPCR analysis in granulocyte DNA of marker rs12343867 (C/T) in intron 14 of JAK2, characteristic of the 46/1 haplotype, revealed rs12343867 ratios sharply different from JAK2V617F ratios: 80% C-alleles for Na1061 and 100% T-alleles for Na1253 (Figure 1B-C). For both patients, CD3+ lymphocytes were unambiguously heterozygous for rs12343867 (Figure 1C). This indicated granulocyte acquisition of homozygosity for rs12343867 but not for the V617F mutation. In other words, the acquisition of homozygosity for rs12343867 must have preceded JAK2 mutation in these patients. This was confirmed by further analysis of JAK2 in granulocytes and CD3+ lymphocytes (Figure 1C), and of chromosome 9p using SNP arrays (Figure 1D). These studies showed that the DNA regions recombined involved JAK2 exons 6-25 for Na1061, and the complete 46/1 haplotype for Na1253. Moreover, SNP array studies revealed the presence of 1 subclone for Na1253 (28.24 Mb) or 2 subclones for Na1061 (5.7 and 24.54 Mb) with partial 9pUPD (supplemental Figures 2-3 and Figure 1E). Sequencing of the complete JAK2 cDNA excluded any mutation other than V617F.
These first cases of HR of JAK2WT led us to propose a new model for MPN: the 46/1 haplotype may predispose carriers to diverse alteration of JAK2 including early HR of wild-type JAK2, associated or not with mutation in JAK2 or other genes important for myelopoiesis, the V617F mutation facilitating additional HR involving the JAK2V617F-mutated allele, leading to 9pUPD and JAK2V617F homozygosity (Figure 1E-F). The new model allows that a nonidentified somatic genetic event may facilitate JAK2 recombination and subsequent genetic alterations eventually leading to PV phenotype (Figure 1F).
In the context of inherited gene mutations, meiotic HR can increase expression of the gene involved.9 In the case of JAK2, mitotic HR could result in a configuration that amplifies JAK2 expression and subsequently cell growth after activation of Jak2 by cytokine receptors. This is of importance because MPN progenitors produce Jak2-activating cytokines.10 For both patients, cDNA quantitative analysis revealed high JAK2 mRNA levels with > 96% JAK2V617F (see supplemental Table 2), implying an mRNA expression almost 100-fold higher for recombined alleles in V617F/V617F cells than for alleles in WT/WT cells. Finally, finding recurrent JAK2 recombination associated with high mRNA expression suggests that residual JAK2V617F disease may be best assessed in cDNA.
Authorship
The online version of this article contains a data supplement.
Acknowledgments: The authors thank Dr Ariane Plet (Nantes, France), Dr Eric Lippert (Bordeaux, France), and Dr Richard Redon (Nantes, France) for reading the manuscript.
This study was performed thanks to grants from the Association pour la Recherche contre le Cancer (ARC) and the Comités Morbihan and Ille-et-Vilaine of the Ligue Nationale contre le Cancer to S.H. and the MPN Research Foundation to R.K. M.V. is recipient of a scholarship from the French Ministry of Research (2009-2012) and benefited from a scholarship for short term scientific missions (November 2010) from MPN & MPNr-EuroNet (COST Action BM0902). M.V., J.B., and S.H. are members of MPN & MPNr-EuroNet.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Contribution: S.H. designed the research, analyzed data, and wrote the paper; R.K. designed the research and analyzed data; M.V. performed research, analyzed data, and wrote the paper; D.O., A.H., and J.B. performed research and analyzed data; M.T. and J.-F.R. contributed patient samples and clinical data; and J.-M.C. contributed with scientific and technical advice and helped write the paper.
Correspondence: Sylvie Hermouet, Inserm U892, Institut de Recherche Thérapeutique, Université de Nantes, 8 quai Moncousu, 44007 Nantes cedex 1, France; e-mail: sylvie.hermouet@univ-nantes.fr.
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