title: Development of a robust and sensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas.
abstract: Assessment of the mutational status of the isocitrate dehydrogenase 1/2 (IDH1/2) gene has become an integral part of the standard diagnostic procedure and, therefore, needs to be accurate. This may, however, be compromised by various factors including the method of analysis and a low tumor cell content. We have developed a rapid, sensitive and robust assay to detect all types of mutation in either IDH1 or IDH2 using pyrosequencing. The efficacy of detecting mutation was evaluated using a panel of control plasmids representing all the different types of IDH1/2 mutation and a set of 160 tumor specimens. The sensitivity of the assays was examined by a serial dilution analysis performed on samples containing various ratios of wild-type and mutant alleles. The pyrosequencing assay detected as little as 5 % of mutant alleles for most mutation types, while conventional Sanger sequencing required the presence of at least 20 % of mutant alleles for identifying mutations. The pyrosequencing assay detected IDH1/2 mutations in three samples which were missed by Sanger sequencing due to their low tumor cell contents. Our assay is particularly useful for the analysis of a large number of specimens as in a retrospective clinical study for example.
authors.affiliation: Department of Neurosurgery and Neuro-Oncology, National Cancer Center Hospital, Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Arita
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Narita
authors.forename: Yuko
authors.initials: Y
authors.lastname: Matsushita
authors.forename: Shintaro
authors.initials: S
authors.lastname: Fukushima
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yoshida
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Takami
authors.forename: Yasuji
authors.initials: Y
authors.lastname: Miyakita
authors.forename: Makoto
authors.initials: M
authors.lastname: Ohno
authors.forename: Soichiro
authors.initials: S
authors.lastname: Shibui
authors.forename: Koichi
authors.initials: K
authors.lastname: Ichimura
mesh_terms: D001932:Brain Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D005910:Glioma / Q000235:genetics*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics*; D009154:Mutation; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004273:DNA, Neoplasm; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human