Mutation, Expression And Enzyme Activity Of DNMT3A Gene In Myeloid Tumor

Objective: Myeloid tumor is a kind of malignant clonal disease derived from myeloid hematopoietic system,including acute myeloid leukemia(AML),myelodysplastic syndrome(MDS),chronic myeloproliferative neoplasms(cMPN)etc.With the completion of the human genome project,the regulation of gene expression,namely epigenetic changes causes more and more people's attention.Recently studies have shown that epigenetic abnormalities can be found in some patients with myeloid tumor.Epigenetic regulation drugs have been designed to reverse the epigenetic abnormalities,so as to achieve the purpose of treating diseases.For instance,by inhibiting the activity of DNMTs,demethylating drugs can reactivate the silenced genes due to from hypermethylation to hypomethylation In addition,the activity of tumor suppressor genes can be restored by lowering the level of methylation,in order to achieve the purpose of treatment of malignant tumors[1],while reducing the side effects and the risk of recurrence,and even realizing the "clinical cure" for these patients.DNA methylation is one of the most common epigenetic abnormalities.As an important molecular biological marker,DNMT3 A plays a very important role in the occurrence,development and prognosis of myeloid tumors[2].In this study,the incidence and distribution of DNMT3 A gene mutation were detected in patients with myeloid tumor.In addition,the mRNA level of DNMT3 A and its enzyme activity was investigated in patients with myeloid tumor in this study.Moreover,the inherent relations among gene mutation,mRNA expression level and DNA methyltransferase activity,plus their implications with the prognosis and clinical features of the disease,were explored,laying the foundation for its future use as the molecular marker and treatment target that guide the prognosis of myeloid tumor.Methods:1 The mononuclear cells were separated from the bone marrow sample,and the genomic DNA was extracted using the test kit(Tiangen Biotech).The partial fragment of DNMT3 A gene containing R882 locus in the 23 rd exon was amplified by using PCR,and then the sample was sent to Beijing for sequencing.2 To reflect the expression of DNMT3 A gene in the bone marrow sample cells,the mRNA level of DNMT3 A gene was detected by using RQ-PCR method.3 The protein activity of DNMTs in the bone marrow sample was determined by using isotope labeling,and HL-60 cell was set as positive control and negative control(without methyl acceptor Poly[ d(I-C)d(I-C)]).The relative activity of DNMTs was represented by the value of sample isotope liquid scintillation count/HL-60 isotope liquid scintillation count.Results: 1 Analysis of DNMT3 A gene mutation in patients with myeloid tumor 1.1 In the detected 40 cases of AML patients,the total incidence rate of gene mutation in patients with AML was(6/40)15%.Among the 6 mutation positive patients with AML,1 was M2 mutation rate was(1/15)6.7%,2 were M4 mutation rate was(2/13)15.4%,and 3 were M5 mutation rate was(3/12)25%(according to WHO classification).Among the 6 mutation positive patients with AML,4(66.7%)were R882 H heterozygous mutation,1(16.7%)was R882 C heterozygous mutation,and 1(16.7%)was R882 P heterozygous mutation.1.2 In the detected 15 cases of MDS patients,the incidence rate of gene mutation in patients with MDS was(0/15)0%.1.3 In the detected 15 cases of cMPN patients,the incidence rate of gene mutation in patients with c MPN was(0/15)0%.2 Analysis of mRNA level of DNMT3 A gene in AML patients 2.1 The mRNA level of DNMT3 A gene in the mutation-positive AML patients is 0.9441±0.1309 ? 2.2 The mRNA level of DNMT3 A gene in the mutation-negative AML patients is 1.0014±0.1146 ? 2.3 The mRNA level of DNMT3 A gene in the control group is 0.9822±0.1021 ? 2.4 Between the mutation-positive and mutation-negative AML patients,the difference in the mRNA level of DNMT3 A was not statistically significant(P>0.05);between the mutation-positive patients and the control group,the difference was not statistically significant(P>0.05);the difference was not statistically significant between the mutation-negative patients and the control group(P>0.05).3 Comparison of DNMTs relative activity among AML patients 3.1 The relative enzyme activity of DNMTs in 6 DNMT3 A mutation-positive AML patients was(20.3±2.3)%.3.2 The relative enzyme activity of DNMTs in 34 DNMT3 A mutation-negative AML patients was(4.3±2.0)%.3.3 The relative enzyme activity of DNMTs in the control group was(3.9±1.6)%.3.4 Pairwise comparisons of the three groups: The relative enzyme activity of DNMTs was significantly increased in DNMT3 A mutation-positive AML patients,as compared to the DNMT3 A mutation-negative AML patients and the control group,with all P<0.01,the difference was statistically significant;the difference in the relative activity of DNMTs was not statistically significant between the DNMT3 A mutation-negative AML patients and the control group(P>0.05).4 Analysis of clinical features and prognosis of the DNMT3 A mutationpositive AML patients and the DNMT3 A mutation-negative AML patientsAmong the 6 DNMT3 A mutation-positive AML patients,with a mean age of 32.5 years old;white blood cell count was(×109/L)16.09±5.65;the proportion of male patients was 66.7%;marrow blast count was(53±13.14)%;platelet count was(×109/L)M=69;hemoglobin was(g/L)74±16.91.CR was(4/6)66.7% after a course of standard chemotherapy.Among the 34 DNMT3 A mutation-negative AML patients,with a mean age of 46.5 years old;white blood cell count was(×109/L)M=12.13;the proportion of male patients was 58.8%;marrow blast count was(57.9±13.46)%;platelet count was(×109/L)M=56.5;hemoglobin was(g/L)69±25.42.CR was(28/34)82.4% after a course of standard chemotherapy.The gene mutations of FLT3-ITD and NPM1 in the AML patients were also inspected,and among the six DNMT3 A mutation-positive AML patients,there was 1 with FLT3-ITD(+)and NPM1(-);1 with FLT3-ITD(+)and NPM1(+);4 with FLT3-ITD(-)and NPM1(+),while the 2 with FLT3-ITD(+)and NPM1(-),and FLT3-ITD(+)and NPM1(+),respectively,were AML patients who failed to achieve complete remission after a course of standard chemotherapy.Conclusions:1 In myeloid tumors,the incidence of DNMT3 A gene mutation was highest in AML.In the 3 types of DNMT3 A gene mutations detected,the mutation rate of R882 H was the highest.2 In the AML patients,DNMT3 A gene mutation rate was not significantly correlated with the gender,age,proportion of bone marrow original cells,initial white blood cell count and other clinical features.3 In AML patients,whether the DNMT3 A gene mutation or not does not change the expression level of mRNA.4 DNMTs enzyme activity was increased in the AML patients with DNMT3 A gene mutation.5 The rate of CR showed no difference between the DNMT3 A gene mutation positive and mutation negative AML patients after one standard chemotherapy.6 Mutations of DNMT3 A gene were frequently associated with NPM1 gene mutations in AML patients,so the prognosis of AML is controlled by multiple genes.