DNMT3A expression Analysis in AML

Introduction

DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for the regulation of gene expression. In the last years, DNMT3A and its potentially pathway partner have been extensively studied. Recently, it was shown that PRMT5 (protein arginine methyltransferase 5) and DNMT3A function cooperatively in gene silencing. Furthermore, GFI1 (Growth Factor Independent 1) is a one of the targets of PRMT5 in complex processes of epigenetic regulation. The aim of this study was to analyze expression level of DNMT3A, DNMT3B, PRMT5, andGFI1 genes in AML patients in context of mutation status.

Methods

Using RT-qPCR and 2^−ΔCT method with GAPDH as an endogenous control, we analyzed gene expression in 152 AML patients at the time of diagnosis. PCR and direct sequencing were performed to investigate mutations of DNMT3A, NPM1, FLT3, and IDH1/2genes. Statistical analysis was performed using the GraphPad Prism 7.0 and SPSS 17.0 statistical software.

Results

Expression levels of all four genes were significantly higher in the AML patients compared with those of healthy bone marrow MNC controls (p=0.02 for DNMT3A, p=0.002 for DNMT3B, p=0.01 for PRMT5, and p=0.04 for GFI1). DNMT3A mutation were found in 13% of patients, FLT3-ITD and FLT3-TKD mutations were observed in 20% and 9%, respectively. The incidence of NPM1 mutations was 29%. IDH1 and IDH2 mutations were seen in 13% and 8% of patients. Interestingly, expression levels of all studied genes were significantly lower in the AML patients with DNMT3A mutations compared with patients without mutations (p=0.0009 for DNMT3A, p=0.0001 for DNMT3B, p=0.001 for PRMT5, and p=0.0001 for GFI1). There was no correlation between expression level and type of DNMT3A mutation. Lower expression levels of DNMT3A and DNMT3B were also seen in AML patients with NPM1 mutation (p=0.03 and 0.007, respectively). Up-regulation of PRMT5 (p=0.01) and down-regulation of GFI1 (p=0.0007) were found in AML patients with FLT3-ITD mutation. Finally, we found a negative effect of lower DNMT3A expression levels on the outcome of AML, especially in the context of relapse rate (p=0.009).

Conclusion

We can conclude that AML patients are characterized by upregulation of DNMT3A expression and its partners such as DNMT3B, PRMT5 and GFI1. However, within the AML cohort patients with DNMT3A mutation presented with lower expression of these genes compared to other AML cases. This could be caused by the inactivating mutation. Moreover, our data demonstrate that not only mutations in DNMT3A but its expression level correlates with outcome of AML.