Analysis Of TET2 Gene Mutation And DNA Dioxygenase In Myeloid Tumor Cells

Objective: Myeloid tumor is a kind of bone marrow clonal diseases derived from hematopoietic stem cells, and is a general term for a class of malignant hematological diseases. It includes acute myelocytic leukemia(AML), myelodysplastic syndromes(MDS), chronic myeloproliferative neoplasms(cMPN), etc. cMPN also includes essential thrombocythemia(ET), polycythemiavera(PV) and primary myelofibrosis(PMF), etc. Recent studies demonstrated that TET2 gene mutations existed frequent mutations in AML, MDS, cMPN and other hematopoietic malignancies. In these patients, the hypermethylation status of gene promoter region caused the transcriptional silence of tumor suppressor gene, which was the most common cause leading to the inactivation of malignant tumor suppressor gene. DNA methyltransferase(DNMT) catalyzed promoter region methylation and TET2 protein catalyzed active demethylation process,which lead to a dynamic equilibrium. A large number of data indicated that the presence of DNA methylation disorders in the hematopoietic malignancies, in addition to the role of DNMT related genes, TET2 gene mutation and function loss were also closely related to the incidence and development of the hematopoietic malignancies.In this study, we detected the rate of TET2 gene mutation and analysised the mRNA and protein levels of TET2 gene, as well as 5-hydroxymethyl cytosine(5-hmC) levels in patients with myeloid tumor. TET2 gene mutation incidence rates of patients with myeloid tumor were investigated, the relationship and mechanism of the gene mutation and gene expression, and their effects on hematopoietic function regulation and tumorigenesis were analyzed.Methods:De novo AML(non-APL), MDS and MPN patients who received initial treatment in the admission office and outpatient of internal medicine of hematology were selected from February 2015 to January 2016 in the Second Hospital of Hebei Medical University. There were 40 cases of initial treatment AML patients with 23 cases of male patients and 17 cases of female patients, an age ranging from 16 to 70 years, and a median age of 40 years. There were 16 cases of AML patients who received complete response(CR) with 9 cases of male patients and 7 cases of female patients, an age ranging from 18 to 67 years, and a median age of 39 years. There were 15 cases of MDS patients with 8 cases of male patients and 7 cases of female patients, an age ranging from 20 to 64 years, and a median age of 56 years. There were 15 cases of MPN patients with 9 cases of male patients and 6 cases of female patients, an age ranging from 35 to 69 years, and a median age of 48 years. At the same time, 17 cases of patients with non-malignant diseases in blood system were screened as a control, including 9 cases of males and 8 cases of females with an age ranging from 16 to 59 years and a median age of 37 years. The TET2 gene mutations in bone marrow mononuclear cells(BM-MNCs), the mRNA and protein levels of TET2 expressions, and 5-hmC content levels of sample genome were detected.Results:1 TET2 mutationIn 40 cases of de novoAML patients, 7 patients existed TET2 missense mutation, with a 17.5 % mutation rate. Mutation occurs mainly in the exon 3?9?11. The median age of patients in the missense mutation group was 34(20-69)years old, while the median age of patients in the non-mutation group/nonsense mutation group was 44(16-70)years old,(P=0.807). There was no statistically significant difference in gender, classification, karyotype and complete remission rate after treatment.In 15 cases of MDS patients, 2 patients existed TET2 missense mutation, with a 13.33 % mutation rate. one of the two was diagnosed with MDS-RAEB2, the other is MDS-RCMD. Mutation occurs mainly in the exon 3,7,9,11.In 15 cases of cMPN patients, the patient is made up of three parts(3 MF patient?7 ET patient and 5 PV patient),only a patient with PV was found the TET2 mutation.In myeloid tumor, TET2 mutation rates of AML, MDS and cMPN were 17.5%, 13.33% and 6.67% respectively.2 The TET2 mRNA expression levelThe level of TET2 mRNA expression in de novo AML?CR AML?cMPN?MDS and control group were 0.414±0.069?0.469±0.073?0.320±0.028?0.769±0.082 and 0.817±0.09 respectively.The level of TET2 mRNA expression in de novo AML patients was significantly lower than that in the normal control(P < 0.01), and was no significant difference(P >0.05) in the mutation group than the non-mutation group(0.461±0.035 vs. 0.405±0.071,P>0.05). The level of TET2 mRNA expression in AML-CR patients is higer than the de novo AML patients(P<0.01).TET2 gene expression levels showed no differences in de novo AML patients with AML-CR(P>0.05).3 The TET2 protein expression levelThe levels of TET2 protein in de novo AML?MPN?MDS and control group were 0.414±0.069;0.469±0.073;0.320±0.028 and 0.817±0.09, respectively. The TET2 protein levels in de novo AML?MPN and MDS patients was significantly lower than that in the normal control(P < 0.01).The AML-CR group was significantly higer than de novo AML group( 0.788±0.079 vs. 0.329±0.034;P<0.01), and no significant difference were found in the control group(0.788±0.079 vs. 0.837±0.078;P>0.05).There was no significant difference in the mutation group than the non-mutation group(0.310±0.030 vs. 0.334±0.034,P>0.05) of the de novo AML patients.4 5-hmC content in the genomic DNAThe 5-hmC contentof initial treatment AML?CR AML?cMPN?MDS and control group were 0.063±0.011%, 0.081±0.021%, 0.013±0.003%, 0.128±0.026%, 0.094±0.04% respectively in the mononuclear cells. The 5-hmC content of initial treatment AML patients was significantly lower than that in the normal control bone marrow cells(P = 0.005) and was no significant difference in the TET2 mutation group(0.054±0.013)% than the non-mutation group(0.065±0.025)%(P=0.137). Initial treatment AML patients5-hmC contentwere higer than AML-CR patients(0.094±0.04)%,(P=0.04). The 5-hmC content of cMPN and MDS patients were lower than that in the normal control bone marrow cells respectively(P <0.001, P <0.001).Conclusions:1 TET2 gene mutations were detected in AML(non APL) patients, MDS patients and cMPN patients, and the highest incidence rate was AML. But most of mutations were found in patients with MDS.2 In patients with myeloid neoplasms, TET2 gene expression, TET2 protein content, concentration of 5-hmC have lower than normal control group. TET2 may be play a key role in the regulation of hematopoietic differentiation and balance, the lack of TET2 can induce the transformation of myeloid neoplasms.3 There is no significant statistical difference in remission rate of patients with TET2 mutations and no mutations patients.4 The TET2 mRNA expression level and 5-hmC content of AML-CR patients were elevated compared to de novo AML patients. It may be associated with reduced tumor load in patients.