title: Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR.
abstract: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed. Here, we describe a real-time PCR-based procedure involving a mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can differentiate samples with H3F3A K27M mutation from those that do not. We first tested four different mutant-specific primers for their ability to selectively amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant allele more efficiently than the rest. We then determined the optimal concentration of blocker oligo that significantly improved amplification of the H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed eleven samples, two of which containing H3F3A K27M mutation, and found that these two samples were differentially amplified from the nine others. In addition, we were able to discern the H3F3A K27M mutation in a newly obtained pediatric brainstem glioblastoma sample whose H3.3 status was not known previously, and in three other DIPG samples as well as paraffin embedded samples. These results demonstrate that we have developed a new reliable procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma patient samples.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1ST SW, Rochester, MN, 55905, USA.
authors.forename: Ray
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1ST SW, Rochester, MN, 55905, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1ST SW, Rochester, MN, 55905, USA.
authors.forename: David
authors.initials: D
authors.lastname: Daniels
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1ST SW, Rochester, MN, 55905, USA.
authors.forename: Haojie
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1ST SW, Rochester, MN, 55905, USA. zhang.zhiguo@mayo.edu.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000235:genetics*; D002648:Child; D004252:DNA Mutational Analysis; D015723:Gene Library; D005910:Glioma / Q000235:genetics*; D006657:Histones / Q000235:genetics*; D006801:Humans; D008239:Lysine / Q000235:genetics; D008297:Male; D008715:Methionine / Q000235:genetics; D051379:Mice; D009154:Mutation / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: C000609950:H3-3A protein, human; D006657:Histones; D008715:Methionine; D008239:Lysine
keywords: Diffuse intrinsic pontine glioma; Histone H3 variant; Pediatric glioblastoma; Real-time PCR