title: Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.
abstract: Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML.
authors.affiliation: Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Patryk
authors.initials: P
authors.lastname: Gorniak
authors.affiliation: Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Ejduk
authors.affiliation: Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Borg
authors.affiliation: Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Hanna
authors.initials: H
authors.lastname: Makuch-Lasica
authors.affiliation: Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Grazyna
authors.initials: G
authors.lastname: Nowak
authors.affiliation: Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.|Centre for Postgraduate Medical Education, Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Lech-Maranda
authors.affiliation: Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Prochorec-Sobieszek
authors.affiliation: Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Warzocha
authors.affiliation: Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland.
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Juszczynski
mesh_terms: D000328:Adult; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D000090205:DNA Methyltransferase 3A; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D008297:Male; D009154:Mutation; D009691:Nucleic Acid Denaturation; D012189:Retrospective Studies
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
keywords: DNA (cytosine-5-)-methyltransferase 3; high-resolution melting; isocitrate dehydrogenase 1/2; mutation screening; sensitivity