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 Suzuki, Masahiro Mizoguchi, Koji Iihara

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Article numbere0160489
JournalPloS one
Volume11
Issue number8
DOIs
Publication statusPublished - Aug 1 2016

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Differentiation (calculus)
calculi
Calculi
melting
Glioma
Freezing
Melting
Tissue
mutation
Mutation
tissues
Molecular Pathology
Tumors
Brain
Screening
rapid methods
Brain Neoplasms
sampling
analytical methods
Derivatives

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

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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.",
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AU - Yoshimoto, Koji

AU - Kuga, Daisuke

AU - Akagi, Yojiro

AU - Murata, Hideki

AU - Suzuki, Satoshi

AU - Mizoguchi, Masahiro

AU - Iihara, Koji

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