An acquired gain-of-function mutation in the Janus kinase 2 (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPDs). To test the hypothesis that JAK2-V617F is the disease-initiating mutation, we examined whether all cells of clonal origin carry the JAK2-V617F mutation. Using allele-specific polymerase chain reaction (PCR) assays for the JAK2 mutation and for the X-chromosomal clonality markers IDS and MPP1, we found that the percentage of granulocytes and platelets with JAK2-V617F was often markedly lower than the percentage of clonal granulocytes determined by IDS or MPP1 clonality assays in female patients. Using deletions of chromosome 20q (del20q) as an autosomal, X-chromosome-independent clonality marker, we found a similar discrepancy
IntroductionClonal hematopoiesis has been recognized as a key pathogenetic feature of myeloproliferative disorders (MPDs). 1 Recently, an acquired somatic mutation in the JAK2 gene resulting in a valine-to-phenylalanine substitution at position 617 (JAK2-V617F) was described in patients with MPDs. [2][3][4][5][6] This discovery implied that the presence of the JAK2-V617F mutation could represent the primary causative lesion in MPDs. However, some of the clinical and genetic data suggested that the role of the JAK2-V617F mutation in the MPD pathogenesis could be more complex and led us to propose 2 alternative models for the role of JAK2-V617F in the clonal evolution of MPDs. 5 The first model assumed that JAK2-V617F alone is sufficient to cause MPDs. The second model predicted that MPDs are caused by a mutation in an as-yetunknown gene that precedes the acquisition of the JAK2-V617F mutation and that JAK2-V617F represents a later event in the disease progression. 5 One of the predictions of the first model is that the JAK2-V617F mutation should be present in all cells that represent the MPD clone, whereas according to the second model, only a proportion of clonal cells carry the JAK2-V617F mutation. Here we present data in support of the second model in at least a proportion of patients with MPDs. Cell separations, RNA and DNA isolation, microsatellite PCR All blood samples were processed within 4 hours after collection. Isolation of granulocytes, T lymphocytes, platelets, buccal mucosa, RNA, and DNA, as well as cDNA synthesis, were performed as described. 5,8,9 Loss of heterozygosity on chromosome 20q was determined by microsatellite polymerase chain reaction (PCR) with the markers D20S96 and D20S119 derived from the minimal deleted region. 10
Study design
Detection of JAK2-V617FThe allele-specific PCR for JAK2 genotyping was carried out using 20 ng genomic DNA, 45 nM forward primer JAK2-F, and 22.5 nM each of the allele-specific reverse primers JAK2-R-T and JAK2-R-G (Table S1, available at the Blood website; see the Supplemental Materials link at the top of the online article) in a buffer containing 50 mM KCl, 10 mM Tris pH 8.0, and 1.5 mM MgCl 2 . Thirty PCR cycles with denaturing at 94°C for 30 seconds, annealing at 61°C for 30 seconds, and ...