The Study On The Mutation Burden And Clinical Relationship Of JAK2 V617F In 415 Patients With Myeloproliferative Neoplasm Using MGB Taqman Quantitative Detection

[Background]According to the molecular biological characteristics, myeloproliferative neoplasm (MPN) are divided into BCR-ABL positive and BCR-ABL negative two diseases. BCR-ABL positive MPN mainly for chronic myeloid leukemia (CML).The classical BCR-ABL negative MPN mainly include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), etc. JAK2 V617F mutation in the diagnosis and differential diagnosis of MPN is of great significance. Since 2005, many research groups at home and abroad have researched on JAK2 V617F mutation, but most of the qualitative research, only a handful of the team on the transcript level, mutation load and mutation ratio. But most of such studies and diseases of the single sample size, quantitative methods sensitivity is not high, so a greater difference between the results [1-3]. [Objective]In order to better reveal the the relationship between JAK2 V617F mutation mechanism and its laboratory and MPN patients’clinical features, this study of 415 patients with MPN, adopt more sensitive MGB (Minor groove binder) Taqman double fluorescent probe method to detecte the JAK2 V617F mutation quantitatively and attempt to provide more help for the diagnosis of MPN classification, clinical treatment, disease progression, conversion and prognosis judgement through this research.[Methods]We selected patients with MPN enrolled from September 2011 to May 2014 in department of Hematology, Qilu Hospital, Shandong University and healthy volunteers (JAK2 V617F mutation load of 0%) as subjects, and venous peripheral blood or bone marrow was collected from all the subjects. Cracking of red blood cells, extracting of genomic DNA, PCR amplification,50℃ for 2 minutes,95℃ for 10 minutes beforehand degeneration;95℃ for 15 seconds,62℃ for 1 minute,45 PCR circulation, we collected the fluorescent signals in the 60℃ of the second step PCR cycle.The case group and control group was detected the JAK2V617F mutation in all cases, and according to the formula to calculate the mutation load of all the research objects.Then, according to the JAK2V617F mutation load conditions, the BCR/ABL negative MPN can be divided into JAK2 V617F mutation high group, low mutation group and negative group, comparising the ralationships between groups of patients with clinical features, laboratory indexes and treatment effect with the JAK2 V617F mutation load.[Results]JAK2 V617F was found in 56.9% of all patients (83.5% in PV,55.9% in ET, 41.9% in PMF and 64.7% in MPN-U). The majority of patients carried JAK2 V617F mutation were heterozygous, and homozygote was found only in 12 cases. Most of mutated patients (72.1%) was lower mutation burden (mutation burden <50%) with PV>PMF>ET. The patient’s age and white blood cell count were significantly correlated with higher mutation burden in PV. White blood cell count was significantly related to higher mutation burden in ET. White blood cell count, hemoglobin level and the platelet count were significantly related to higher mutation burden in PMF.[Conclusions]The mutation burden of JAK2 V617F from high to low was PV, ET and PMF. The majority of JAK2 V617F mutation was heterozygous. JAK2 V617F mutation burden was elevated with the increase of age, white blood cell count, Hb and platelet count.