High-resolution melting analysis for a reliable and two-step scanning of mutations in the tyrosine kinase domain of the chimerical bcr-abl gene.
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.
Doi
Yuko
Y
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Sasaki
Daisuke
D
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Terada
Chiharu
C
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Mori
Sayaka
S
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Tsuruda
Kazuto
K
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Matsuo
Emi
E
Department of Hematology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Miyazaki
Yasushi
Y
Department of Hematology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Nagai
Kazuhiro
K
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Hasegawa
Hiroo
H
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Yanagihara
Katsunori
K
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Yamada
Yasuaki
Y
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan.
Kamihira
Shimeru
S
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan. kamihira@nagasaki-u.ac.jp.
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
D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl