Precise detection of IDH1/2 and BRAF hotspot mutations in clinical glioma tissues by a differential calculus analysis of high-resolution melting data

Ryusuke Hatae, Nobuhiro Hata, Koji Yoshimoto, Daisuke Kuga, Yojiro Akagi, Hideki Murata, Satoshi O. Suzuki, Masahiro Mizoguchi, Koji Iihara

研究成果: ジャーナルへの寄稿学術誌査読

31 被引用数 (Scopus)

抄録

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.

本文言語英語
論文番号e0160489
ジャーナルPloS one
11
8
DOI
出版ステータス出版済み - 8月 2016

All Science Journal Classification (ASJC) codes

  • 生化学、遺伝学、分子生物学(全般)
  • 農業および生物科学(全般)
  • 一般

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