Open AccessPrecise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data
Author(s) -
Ryusuke Hatae,
Nobuhiro Hata,
Koji Yoshimoto,
Daisuke Kuga,
Yojiro Akagi,
Hideki Murata,
Satoshi Suzuki,
Masahiro Mizoguchi,
Koji Iihara
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0160489
Subject(s) - glioma , high resolution melt , heteroduplex , mutation , mutation testing , biology , computational biology , pathology , genetics , gene , medicine , polymerase chain reaction
High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1 R132 and IDH2 R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAF V600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2 R172 and BRAF V600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments.