The Profile Of Mutation R882and Gene Expression For Dnmt3A Gene In Acute Leukemia

BackgroundAcute leukemia (AL) is a group of heterogeneous malignancies that jeopardizes human health of AL patients. DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression by controlling gene methylation. Part of acute myeloid leukemia (AML) patients were found DNMT3A mutations and deletions which were shown enriched in M4and M5subtypes, according to the French-American-British (FAB) classification system. Another research suggested that DNMT3A mutations were independent factors predicting poor prognosis, such as the shorter overall survival (OS) and the lower complete remission (CR) rate. So far. the relationship among DNMT3A mutations, DNMT3A expression and the prognosis is still not very clear. In addition, DNMT3A mutations and expression in acute lymphoblastic leukemia (ALL) are rarely reported. Therefore, DNMT3A mutations and DNMT3A expression status in AL need a further study.ObjectivesIn the present study, we examine DNMT3A mutations and expression in bone marrow mononuclear cells (BN4MCs) of AL patients to investigate the relationship among DNMT3A mutations, DNMT3A expression and prognosis. We discuss the possible mechanism of DNMT3A in the occurrence and development of AL to provide a new marker for the risk stratification of AL.Materials and Methods 1. Patients and controls:We selected99cases of patients who were newly diagnosed as acute leukemia from July2011to July2012accepted their first treatment in Qilu Hospital (research targets). Of the99cases,52are male and47are female. The median age is41(range from12to83). There are57AML patients,41ALL patients, and one biphenotypic acute leukemia(BAL)patient in all these99cases, with their laboratory datas and the informations of clinical diagnosis and treatment collected. We also selected16healthy volunteers (normal control),6are male,10are female. The median age is46(range from34to65).2. All people’s BMMCs were collected, then we isolated DNA and RNA from BMMCs. We established polymerase chain reaction (PCR) method to amplify the DNA fragment of379bp covering the R882site in the exon23of DNMT3A gene. Then the PCR products were sequenced bidirectionally using ABI3730XL DNA Analyzer in Shandong Province Academy of Agricultural Sciences Sequencing Center. Then used real-time quantitative RT-PCR (Q-RT-PCR) to test the expression of DNMT3A in all of people and β-actin was selected as internal control.3. Statistical analysis SPSS Statistics Base17.0statistical software was used to compare the DNMT3A expression and CR rate after induction therapy in different groups. P<0.05was considered statistically significant.Results1. We found10patients harbored DNMT3A mutations in AML, six AML samples had the R882H variant, and two had R882C variant, one had R882P variant and one had M880V variant. The frequency of DNMT3A mutations was17.5%. None of ALL was found DNMT3A mutations. AML patients with DNMT3A mutations showed lower CR rate than those with wild-type DNMT3A.2. DNMT3A expression in AML patients:DNMT3A expression in AML patients showed significantly higher than that in ALL patients or normal controls. DNMT3A expression in AML with DNMT3A mutations was decreased significantly compared to AML with wild-type, and showed no difference from normal controls. AML patients with wild-type DNMT3A showed significantly higher DNMT3A expression compared to normal controls. There was significant difference for DNMT3A expression between different AML subtypes (P=0.006). M5subtype AML showed significantly lower DNMT3A expression level than the other subtypes AML3. There was no statistical difference in DNMT3A expression between ALL patients and normal controls. And no difference was found between T-ALL patients and B-ALL patients.4. The lower expression levels of DNMT3A were associated with a statistically significant lower complete remission (CR) rate in AML (T=0.002). However, in ALL. there was no statistical significance.Conclusion1. Part of AML patients harbored DNMT3A mutations. AML patients with DNMT3A mutations showed lower CR rate than those with wild-type DNMT3A. On the contrary. DNMT3A mutations in ALL were not observed.2. In AML, DNMT3A expression with DNMT3A mutations was significantly lower than that without mutation.3. In AML, The lower DNMT3A expression group showed lower CR rate than the higher DNMT3A expression group. In ALL, the difference of CR rate between the two groups did not reach statistical significance.