Clinical Study On The Intragenic Methylation Pattern Of DNMT3A In Myeloid Malignancies

Objective This study was aimed to investigate the methylation status of intragenic differentially methylated region 2(DMR2) in DNA methyltransferase 3A(DNMT3A) gene, then further to analyze its clinical significance in patients with acute myeloid leukemia(AML), chronic myeloid leukemia(CML) and myelodysplastic syndrome(MDS).Methods Real-time quantitative PCR(RQ-PCR) was carried out to detect the level of four DNMT3 A isoforms in 56 AML patients and 18 controls with available mRNA. Real-time quantitative methylation-specific PCR(RQ-MSP) was carried out to detect the methylation status and level of DNMT3 A DMR2 in the bone marrow mononuclear cells(BMMNCs) from AML patients(n=152), CML(n=82), MDS(n=90) and controls(n=18). Bisulfite-sequencing PCR was carried out to analyze the methylation density of DNMT3 A DMR2.Results The DNMT3 A DMR2 hypomethylation was identified in AML, CML and MDS patients:(1) DNMT3 A DMR2 methylation status in AML patients 84(55%) cases of 152 AML patients were observed with DNMT3 A DMR2 hypomethylation. BSP was performed in two controls, two AML patients with DNMT3 A DMR2 hypomethylation and two patients with DNMT3 A DMR2 hypermethylation. DNMT3 A DMR2 hypomethylated patients had a quite lower density of the region(1.72% and 3.88%) than the DNMT3 A DMR2 hypermethylated patients(93.97% and 72.41%). The density of two normal controls was 60.34% and 70.49%, respectively. There was no differentiation between DNMT3 A DMR2 hypermethylation and DNMT3 A DMR2 hypomethylation in age, sex, white blood cell count(WBC), hemoglobin level(HB) and platelet count(PLT)(P>0.05). Moreover, no significant differences were observed in the distribution of French-American-British(FAB), World Health Organization(WHO) and karyotype classifications between DNMT3 A hypomethylated and hypermethylated patients(P>0.05). There were no differences in the status of DNMT3 A DMR2 methylation between AML patients with and without DNMT3 A gene, NPM1 gene, FLT3 gene, IDH1/2 gene, C/EBPA gene, C/KIT gene, U2AF1 gene or RAS gene mutation(P>0.05). Patients with DNMT3 A DMR2 hypomethylation and hypermethylation had similar complete remission(CR) rates after induction therapy(48.3% vs 52.5%). Survival analyses showed that the patients with DNMT3 A DMR2 hypomethylation had significantly shorter overall survival(OS) than those with DNMT3 A hypermethylation(median 7 months vs 11 months, P=0.034). Moreover, DNMT3A-hypomethylated cases also showed significantly shorter OS than DNMT3A-methylated cases in both non-M3(median 7 months vs 9 months, P=0.019) and cytogenetically normal AML(CN-AML)(median 7 months vs 25 months, P=0.011). Multivariate analysis confirmed that DNMT3 A DMR2 hypomethylation was an independent adverse prognostic factor in CN-AML. However, no correlations were observed between four transcripts expression and DNMT3 A unmethylation.(2) DNMT3 A DMR2 methylation status in CML patients60(73%) cases of 82 CML patients were observed with DNMT3 A DMR2 hypomethylation. DNMT3 A DMR2 hypomethylated patients had a quite lower density of the region(2.07% and 3.02%) than the DNMT3 A DMR2 hypermethylated patients(50.34% and 37.16%). There was no differentiation between DNMT3 A DMR2 hypermethylation and DNMT3 A DMR2 hypomethylation in age, sex, WBC, HB, PLT and chromosomal abnormalities of patients(P>0.05). DNMT3 A DMR2 hypomethylation in patients in chronic phase, in accelerated phase and in blast crisis were 72%(46/64), 100%(5/5) and 69%(9/13), respectively, but the difference was not statistically significant(P>0.05).(3) DNMT3 A DMR2 methylation status in MDS patients64(71%) cases of 90 MDS patients were observed with DNMT3 A DMR2 hypomethylation. DNMT3 A DMR2 hypomethylated patients had a quite lower density of the region(0.49% and 1.72%) than the DNMT3 A DMR2 hypermethylated patients(45.3% and 51.7%). There was no differentiation between DNMT3 A DMR2 hypermethylation and DNMT3 A DMR2 hypomethylation in age, sex, WBC, HB and PLT(P>0.05). And there was no significant difference in hypomethylation incidence among FAB, karyotypic classifications or the International Prognostic Scoring System(IPSS) subtypes(P>0.05). Furthermore, the association between DNMT3 A DMR2 hypomethylation and DNMT3 A, IDH1/2, U2AF1 or SF3B1 mutations was also not observed(P>0.05). Moreover, although the overall survival(OS) time of the DNMT3 A DMR2 hypomethylated group(median 20 months) was shorter than that of the DNMT3 A DMR2 hypermethylated group(median 33 months), the difference was not statistically significant(P>0.05).Conclusions(1) DNMT3 A DMR2 hypomethylation is a common molecular event in patients with myeloid malignancies including AML, CML and MDS;(2) DNMT3 A DMR2 hypomethylation is an independent adverse prognostic factor in CNAML;(3) DNMT3 A DMR2 hypomethylation is unlikely to provide helpful prognostic in MDS patients.