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Updated: Apr 28, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid
Rimma Berenstein1, Igor Wolfgang Blau, Asiye Kar
1Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. rimma.berenstein@charite.de.
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.
Insights
Rapid screening tests for DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1/2 (IDH1/2) mutations in acute myeloid leukaemia (AML) were developed. High-resolution melting (HRM) analysis proved most efficient for screening these critical AML mutations.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Mutations in epigenetic modifiers like DNMT3A and IDH1/2 are common in acute myeloid leukaemia (AML), impacting patient prognosis.
- Current DNA sequencing methods for detecting these mutations are time-consuming and costly.
- There is a clinical need for rapid and accessible diagnostic tools for these genetic alterations in AML.
Purpose of the Study:
- To establish rapid and reliable screening methods for common mutations in DNMT3A, IDH1, and IDH2 genes in AML patients.
- To evaluate the efficiency and accuracy of novel endonuclease restriction, amplification-refractory mutation system (ARMS), and high-resolution melting (HRM) analyses.
- To provide a basis for improved risk stratification and treatment strategies in AML.
Main Methods:
- Development of an endonuclease restriction assay for the common DNMT3A R882H mutation.
- Implementation of an ARMS assay for the IDH2 R140Q mutation.
- Evaluation of HRM analysis for detecting mutations in IDH1 and comparison with other methods.
Main Results:
- The study analyzed 230 AML samples, identifying DNMT3A mutations in 13%, IDH2 R140Q in 7%, and IDH1 mutations in 16%.
- Sensitivity assays demonstrated high performance: endonuclease restriction (0.05%), ARMS (4.5%), and HRM (5.9%-7.8%).
- Endonuclease restriction and ARMS showed 100% concordance with Sanger sequencing, while HRM achieved ~98% concordance, proving effective for IDH1 heterogeneity.
Conclusions:
- The developed endonuclease restriction, ARMS, and HRM methods are rapid, specific, and user-friendly for AML mutation screening.
- HRM analysis is the most time-saving and cost-efficient method for simultaneously screening DNMT3A, IDH1, and IDH2 mutations at diagnosis.
- Combining HRM with endonuclease restriction and ARMS assays enhances diagnostic reliability, supporting early risk stratification and optimized treatment for AML patients.
