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.
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Acknowledgments
We thank Dr. Dominick Sinicropi for providing advice to RZ during the primer and blocker oligonucleotide designs; Dr. Chan Kui-Ming for providing H3F3A wild type and K27M mutant cDNA libraries; and Dr. Yu Chao and Mrs. Hui Zhou for assistance with qPCR reactions. We also thank Dr. Suzanne Baker for cDNA from DIPG1, DIPG2 and DIPG4.
Funding
This work is supported by a NIH grant (CA157489) to ZZ. This paper is subject to the NIH Public Access Policy.
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Zhang, R., Han, J., Daniels, D. et al. Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR. J Neurooncol 126, 27–36 (2016). https://doi.org/10.1007/s11060-015-1936-5
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DOI: https://doi.org/10.1007/s11060-015-1936-5