title: High-resolution melting analysis for a reliable and two-step scanning of mutations in the tyrosine kinase domain of the chimerical bcr-abl gene.
abstract: For relevant imatinib therapy against Philadelphia (Ph)-positive leukemias, it is essential to monitor mutations in the chimerical bcr-abl tyrosine kinase domain (TKD). However, there is no universally acceptable consensus on how to efficiently identify mutations in the target TKD. Recently, high-resolution melting (HRM) technology was developed, which allows gene scanning using an inexpensive generic heteroduplex-detecting dsDNA-binding dye. This study aimed to validate the introduction of HRM in a practical clinical setting for screening of mutations in sporadic sites of the chimerical bcr-abl TKD. All chimerical and wild-type abl TKD regions selectively amplified were used for HRM assays and direct sequencing. The HRM test had approximately 5-90% detection sensitivity for mutations. In contrast to mixture samples with mutant and wild-type cells, all mutant cell samples had indeterminate melting curves equivalent to those of the wild-type due to formation of only a homodulex. This issue was improved by the addition of exogenous wild-type DNA after PCR. Subsequently, HRM results gave a high accordance rate of 97.8% (44/45 samples) compared to the sequencing data. The discordant results in one appear to be due to unsuccessful amplification. Thus, HRM may be considered to be suitable for reliable scanning of mutations in the chimerical abl TKD in a clinical setting.
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Doi
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sasaki
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Terada
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Mori
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Tsuruda
authors.affiliation: Department of Hematology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Matsuo
authors.affiliation: Department of Hematology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Miyazaki
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nagai
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Hiroo
authors.initials: H
authors.lastname: Hasegawa
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Yanagihara
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Yamada
authors.affiliation: Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan. kamihira@nagasaki-u.ac.jp.
authors.forename: Shimeru
authors.initials: S
authors.lastname: Kamihira
mesh_terms: D001549:Benzamides; D004252:DNA Mutational Analysis; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000235:genetics*; D016313:Genes, abl / Q000235:genetics*; D006801:Humans; D000068877:Imatinib Mesylate; D019169:Jurkat Cells; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D008297:Male; D009154:Mutation; D010677:Philadelphia Chromosome; D010879:Piperazines / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D017434:Protein Structure, Tertiary / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D011743:Pyrimidines / Q000627:therapeutic use; D020298:U937 Cells
chemical_list: D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl