JAK2 Mutation in Granulocytes and Platelets and X-Chromosome Gene-Based Clonal Analysis in Chronic Myeloproliferative Disorders.
Authors: Toyama Kohtaro , Tsukamoto Norifumi , Hiroyuki Irisawa , Yokohama Akihiko , Saitoh Takayuki , Handa Hiroshi , Matsushima Takafumi , Murakami Hirokazu , Miyawaki Shuichi , Nojima Yoshihisa , Karasawa Masamitsu 
Published: April 2020

Abstract
BCR/ABL-negative chronic myeloproliferative disorders (CMPDs) are considered to arise in a pluripotent progenitor cell. High prevalence of a point mutation in Janus Kinase 2 (JAK2) exon12 gene (JAK2-V617F) in CMPDs has been observed in many reports. Since most studies, however, are restricted mainly in granulocyte lineage, information about the JAK2 gene status in other cell lineage is limited. Heparinized peripheral blood was obtained from 49 patients with polycythemia vera (PV), 109 with essential thrombocytosis (ET), and 5 with chronic idiopathic myelofibrosis (CIMF). After centrifugation, platelets were collected from the upper plasma layer. The remaining blood layer was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet and T-cells recovered from the interface were further positively selected using either anti-CD3 or anti-CD4 immunoconjugated magnetic beads (Miltenyi Biotec, Germany). After DNA and RNA extraction, PCR amplified JAK2 exon 12 gene was sequenced to check the presence of JAK2-V617F mutation. Furthermore, X-chromosome linked clonal analysis using human androgen receptor (HUMARA) gene was carried out in 58 females. Correlation between the mutation status and clinical data was analyzed using statiscal software R (The R Development Core team). In PV, the frequency of JAK2-V617F mutation in granulocytes and platelets was 72.3 % and 74.3%, respectively (p-value 0.844). In ET, this mutation rate was 51.9% in granulocytes and 73% in platelets and the difference was significant (p-value 0.005). In all CMPDs, the cases showing JAK2-V617F mutation in granulocytes always had the mutation in platelets as well, but in one case of PV and 11 cases of ET, the JAK2-V617F mutation was observed only in platelets. T cell fraction was negative for JAK2-V617F mutation in all cases examined. White blood cell count was significantly higher in cases of JAK2-V617F mutation in their platelets compared to that in cases without this mutation (12.6 × 109/l vs. 9.2 × 109/l), but hemoglobin level and platelet count did not differ between the two groups (14.9g/l vs. 14.6 g/l, 81.4 × 109/l vs. 95.2 × 109/l). In addition, cases showing this mutation in platelets were more likely to be associated with thrombosis than cases showing the wild type gene (p-value 0.018). On clonal analyses using the HUMARA gene, 6/8 patients with PV, 15/27 patients with ET, and 1/1 patient with CIMF demonstrated a monoclonal pattern in their granulocytes. Interestingly, in 23 cases showing the JAK2-V617F mutation, 7 patients showed the polyclonal HUMARA pattern; in 13 cases without mutation, 7 showed the monoclonal HUMARA pattern. The discrepancy of JAK2-V617F mutation rate between platelets and granulocytes in ET suggests that analysis of platelet fraction is advantageous for detecting the mutation. This finding also suggests that the JAK2 mutation could be involved only in megakaryocyte lineage in some ET patients. HUMARA analysis confirmed that some ET patients with monoclonal disease lack the JAK2-V617F mutation, which is consistent with previous reports.