title: Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data.
abstract: 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 IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E 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 IDH2R172 and BRAFV600E 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.
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Ryusuke
authors.initials: R
authors.lastname: Hatae
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.|Department of Neurosurgery, Clinical Research Institute, National Hospital Organization, Kyushu Medical Center, Fukuoka, Japan.
authors.forename: Nobuhiro
authors.identifier: http://orcid.org/0000-0002-5523-7166
authors.initials: N
authors.lastname: Hata
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Yoshimoto
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kuga
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Akagi
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Murata
authors.affiliation: Department of Neuropathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Satoshi O
authors.initials: SO
authors.lastname: Suzuki
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.|Department of Neurosurgery, Kitakyushu Municipal Medical Center, Kitakyushu, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Mizoguchi
authors.affiliation: Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Iihara
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005910:Glioma / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D013223:Statistics as Topic / Q000379:methods*; D044366:Transition Temperature
chemical_list: C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D048493:Proto-Oncogene Proteins B-raf