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Background  Mutations in epigenetic modifiers were reported in patients with acute myeloid leukaemia (AML) including mutations in DNA methyltransferase 3A gene ( DNMT3A)  in 20%-30% patients and mutations in isocitrate dehydrogenase 1/2 gene ( IDH1/2 ) in 5%-15% patients. Novel studies have shown that mutations in  DNMT3A  and  IDH1/2  influence prognosis, indicating an increasing need to detect these mutations during routine laboratory analysis. DNA sequencing for the identification of these mutations is time-consuming and cost-intensive. This study aimed to establish rapid screening tests to identify mutations in  DNMT3A  and  IDH1/2  that could be applied in routine laboratory procedures and that could influence initial patient management.    Methods  In this study we developed an endonuclease restriction method to identify the most common  DNMT3A  mutation (R882H) and an amplification-refractory mutation system (ARMS) to analyse  IDH2  R140Q mutations. Furthermore, we compared these methods with HRM analysis and evaluated the latter for the detection of  IDH1  mutations.    Results  Of 230 samples from patients with AML 30 (13%) samples had  DNMT3A  mutations, 16 (7%) samples had  IDH2  R140Q mutations and 36 (16%) samples had  IDH1  mutations. Sensitivity assays performed using serial dilutions of mutated DNA showed that ARMS analysis had a sensitivity of 4.5%, endonuclease restriction had a sensitivity of 0.05% and HRM analysis had a sensitivity of 5.9%–7.8% for detecting different mutations. HRM analysis was the best screening method to determine the heterogeneity of  IDH1  mutations. Furthermore, for the identification of mutations in  IDH2  and  DNMT3A,  endonuclease restriction and ARMS methods showed a perfect concordance (100%) with Sanger sequencing while HRM analysis showed a near-perfect concordance (approximately 98%).    Conclusion  Our study suggested that all the developed methods were rapid, specific and easy to use and interpret. HRM analysis is the most timesaving and cost-efficient method to rapidly screen all the 3 genes at diagnosis in samples obtained from patients with AML. Endonuclease restriction and ARMS assays can be used separately or in combination with HRM analysis to obtain more reliable results. We propose that early screening of mutations in patients with AML having normal karyotype could facilitate risk stratification and improve treatment options.

Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia