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Investigation of screening method for DNMT3A mutations by high-resolution melting analysis in acute myeloid leukemia.

Authors
  • Mizuta, Shumpei1, 2
  • Yamane, Noriko1
  • Komai, Takao1
  • Koba, Yusuke3
  • Kawata, Takahito3, 4
  • Ukyo, Naoya3
  • Tamekane, Akira3
  • Watanabe, Mitsumasa3
  • 1 Department of Clinical Laboratory, Hyogo Prefectural Amagasaki General Medical Center, Hyogo, Japan. , (Japan)
  • 2 Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, Hyogo, Japan. , (Japan)
  • 3 Department of Hematology, Hyogo Prefectural Amagasaki General Medical Center, Hyogo, Japan. , (Japan)
  • 4 Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. , (Japan)
Type
Published Article
Journal
International journal of laboratory hematology
Publication Date
Oct 01, 2019
Volume
41
Issue
5
Pages
593–600
Identifiers
DOI: 10.1111/ijlh.13056
PMID: 31149783
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Acute myeloid leukemia (AML) is a heterogeneous disease associated with various genetic abnormalities. Somatic mutations in nucleophosmin 1 (NPM1), fms-related tyrosine kinase 3 (FLT3), and DNA methyltransferase 3 alpha (DNMT3A) are the most frequent mutations associated with AML. However, because DNMT3A mutations are broadly distributed, they are challenging to analyze in routine laboratory tests. Hence, we developed a rapid screening method for DNMT3A mutations by high-resolution melting (HRM) analysis for clinical use at the point of AML diagnosis. The detection limit for DNMT3A mutations from exons 8-23 by an HRM analysis was investigated using plasmid mixtures. In 69 patients with AML, somatic mutations in NPM1, FLT3-internal tandem duplication (ITD), FLT3-tyrosine kinase domain (TKD), DNMT3A, and isocitrate dehydrogenase 1/2 were screened using HRM analysis, and direct sequencing was performed for positive samples. High-resolution melting analysis enabled complete mutation detection in samples with 20% mutant alleles in all regions. In a clinical laboratory test, DNMT3A mutations were detected in 12 cases (17.3%), and we identified five novel mutations. Simultaneous NPM1, FLT3-ITD, and DNMT3A mutations constituted the most common pattern (30%) in de novo cytogenetically normal AML. High-resolution melting analysis has sufficient performance for the detection of DNMT3A mutations in AML. This approach can facilitate rapid AML genotyping at diagnosis in clinical settings. © 2019 John Wiley & Sons Ltd.

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