title: Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia.
abstract: 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%).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Hematology, Oncology and Tumourimmunology, Charité Universitätsmedizin Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. rimma.berenstein@charite.de.
authors.forename: Rimma
authors.initials: R
authors.lastname: Berenstein
authors.forename: Igor Wolfgang
authors.initials: IW
authors.lastname: Blau
authors.forename: Asiye
authors.initials: A
authors.lastname: Kar
authors.forename: Ruhiye
authors.initials: R
authors.lastname: Cay
authors.forename: Annette
authors.initials: A
authors.lastname: Sindram
authors.forename: Claudia
authors.initials: C
authors.lastname: Seide
authors.forename: Olga
authors.initials: O
authors.lastname: Blau
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D000090205:DNA Methyltransferase 3A; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: C000717967:DNMT3A protein, human; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A