Identification And Quantitative Assay For JAK2V617F Mutation In Patients With Myeloproliferative Neoplasms

Background and ObjectiveRecent researches suggested that JAK2V617F mutation was identified in most of polycythemia ver(aPV) patients and about half of essential thrombocythemia (ET ) and idiopathic myelofibrosis (IMF) patients. The identification of the JAK2V617F allele greatly improved our understanding of the molecular pathogenesis of myeloproliferative neoplasms(CMPN) and a positive mutation test is highly suggestive of the diagnosis. The discovery has led to revised WHO diagnostic criteria for PV,ET and IMF that incorporate JAK2V617F mutation screening in 2008 and JAK2V617F mutation screening plays the first step in the diagnosis of PV. With the investigation thorough, two kinds of mutation have been identified—homozygotic and heterozygotic status, furthermore, the ratio of mutant/wild-type JAK2 mRNA favor MPN phenotype. This study enlarged sample size and observed the relation between JAK2V617F mutation and clinical practice in CMPN. Because the mutational status and the relative quantitation of JAK2V617F mRNA in a great cohort of Chinese patients with CMPN still have not been reported, amplification- refractory mutation sequencing polymerase chain reaction (ARMS-PCR) were established. Blood samples from 136 CMPN patients were collected and examined, along with capillary electrophoresis, the clinical relevance of mutated JAK2 mRNA was analyzed.MethordsIn the frist part , the study included 114 patients with PV ,229 with ET, 24 with IMF, 42 with myeloproliferative disorders- unclassified(MPD-U), 2 with chronic neutrophilic leukemiaCNL) and 1 with chronic eosinophilic leukemia (CEL), diagnosed according to the 2001 WHO (World Health Organization) criteria. All of these patients were admitted to our hospital between August 2005 and November 2008. Twenty healthy blood donors were used as controls .Genomic DNA was extracted from fresh bone marrow mononuclear cells(BMMCs). Allele-specific PCR and sequencing of JAK2 exon 14 was performed. Amplicons were size fractioned by standard agarose gel electrophoresis. Direct sequencing of PCR products was performed in selected patients.In the second part of the study, A quantitative assay for JAK2V617F mutation in 136 CMPN patients by ARMS-PCR and capillary electrophoresis. This part included 38 patients with PV ,94 with ET and 4 with IMF, diagnosed according to the 2001 WHO criteria. All of these patients were admitted to our hospital between August 2003 and June 2008. Twenty healthy blood donors were used as controls. 10ml of fresh blood was collected from the vein into tubes containing ethylenediamine tetraacetic acid(EDTA). Granulocytes were enriched by density-gradient centrifugation with Ficoll-Paque solution. Total cellar RNA was extracted from granulocytes by using the TRIzol reagent strictly according to manufacturer's instructions. The First-strand complementary DNA synthesis was performed with random hexamers and 300U M-MLV reverse transcriptase. ARMS-PCR of JAK2 exon 14 was performed. Amplicons were size fractioned by agarose gel electrophoresis. Direct sequencing of PCR products was performed in selected patients. The mutated mRNA ratio was evaluated by capillary electrophoresis, the clinical relevance of mutated JAK2 mRNA was analyzed.ResultsJAK2V617F mutation was detected in 277of the 412 patients with MPN, we detected JAK2V617F in 95.6% of PV, 55.9% of ET, 66.7% of IMF and 52.4% of MPD-U,respectively. The mutation was also detected in 2 of 2 cases of CNL, but it was none in 1 CEL and 20 normal samples.7 of 18 MPD-U patients with the burden of JAK2V617F turned out to be typical PV and 4 patients evolved to be ET, 1 patient turned out to be CNL, only 2 out of 17 patients without this mutation evolved to typical ET after follow-up for 1～22 month , which demonstrated a statistically significant difference between these two groups (P<0.05). JAK2V617F mutation was detected in 96 of the 136 patients with MPN by ARMS-PCR, we detected JAK2V617F in 95.6% of PV, 55.9% of ET and 66.7% of IMF. In the 96 CMPD cases carrying JAK2V617F mutation, 18 patients (47.3% 18/38) with PV and 17 (18.1% 17/94) patients with ET and 1(25% 1/4) patient with IMF were homozygotes. ET patients showed a lower frequency of homozygosity than PV patients(P<0.05); The mutated JAK2 mRNA ratio in the JAK2V617F heterozygote and homozygote patients was (89.5±6.0)% and(56.9±6.4)%, respectively, demonstrating a statistically significant difference between these two groups (P<0.05). The levels of mutated JAK2 mRNA were (64.0±6.7)% and (53.5±6.2)% in the heterozygote patients with PV and heterozygote patients with ET, heterozygote ET patients showed significantly lower JAK2 mutated mRNA levels as compared to PV heterozygote patients (P<0.05).The levels of JAK2V617F mRNA was found to be significantly correlative with advanced age at diagnosis, the patients whose age>60 years showed significantly higher JAK2 mutated RNA levels as compared to patients whose age< 60 years[ (82.6±9.4)% versus (60.0±6.7)%, P<0.05]. As compared to patients carrying lower JAK2 mutated RNA levels, higher leukocyte count was observed in patients with higher JAK2 mutated RNA levels [(23.59±10.3)×109/L versus (23.59±10.3)×109/L, P﹤0.05], JAK2V617F-positive patients had a higher median white cell count than wild-type patients(P﹤0.05).Our findings suggest that the JAK2V617F mutation is observed in Chinese patients with a significant percentage; The presence of JAK2V617F in MPD-U was found to be associated with disease development.The ARMS-PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation ,along with capillary electrophoresis, a quantitative assay for mutated JAK2 mRNA and the diagnosis of MPN and the estimate of minimal residual disease become possible.ET patients showed a lower frequency of JAK2V617F homozygosity than PV patients ; homozygote patients showed significantly higher JAK2 mutated RNA levels as compared to patients carrying heterozygote mutation; heterozygote ET patients showed significantly lower JAK2 mutated mRNA levels as compared to PV heterozygote patients.The JAK2V617F mutation was found to be significantly correlative with advanced age and higher leukocyte counts at diagnosis in patients with classic CMPN.The patients whose age>60 years showed significantly higher JAK2 mutated RNA levels as compared to patients whose age< 60 years, higher leukocyte counts were observed in PV and ET patients with higher levels of mutated JAK2 mRNA.