Journal of Neuro-Oncology

, Volume 126, Issue 1, pp 27–36 | Cite as

Detecting the H3F3A mutant allele found in high-grade pediatric glioma by real-time PCR

  • Ray Zhang
  • Jing Han
  • David Daniels
  • Haojie Huang
  • Zhiguo Zhang
Laboratory Investigation

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.

Keywords

Histone H3 variant Diffuse intrinsic pontine glioma Real-time PCR Pediatric glioblastoma 

Supplementary material

11060_2015_1936_MOESM1_ESM.docx (177 kb)
Supplementary material 1 (DOCX 176 kb)

References

  1. 1.
    Jones C, Baker SJ (2014) Unique genetic and epigenetic mechanisms driving paediatric diffuse high-grade glioma. Nat Rev Cancer. doi:10.1038/nrc3811 PubMedCentralGoogle Scholar
  2. 2.
    Warren KE (2012) Diffuse intrinsic pontine glioma: poised for progress. Front Oncol 2:205PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Grimm SA, Chamberlain MC (2013) Brainstem glioma: a review. Curr Neurol Neurosci Rep 13:346PubMedCrossRefGoogle Scholar
  4. 4.
    Kebudi R, Cakir FB, Agaoglu FY, Gorgun O, Ayan I, Darendeliler E (2013) Pediatric diffuse intrinsic pontine glioma patients from a single center. Child’s Nerv Syst 29:583–588CrossRefGoogle Scholar
  5. 5.
    Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff E, Jacob K, Sturm D, Fontebasso AM, Quang DA, Tonjes M, Hovestadt V, Albrecht S, Kool M, Nantel A, Konermann C, Lindroth A, Jager N, Rausch T, Ryzhova M, Korbel JO, Hielscher T, Hauser P, Garami M, Klekner A, Bognar L, Ebinger M, Schuhmann MU, Scheurlen W, Pekrun A, Fruhwald MC, Roggendorf W, Kramm C, Durken M, Atkinson J, Lepage P, Montpetit A, Zakrzewska M, Zakrzewski K, Liberski PP, Dong Z, Siegel P, Kulozik AE, Zapatka M, Guha A, Malkin D, Felsberg J, Reifenberger G, von Deimling A, Ichimura K, Collins VP, Witt H, Milde T, Witt O, Zhang C, Castelo-Branco P, Lichter P, Faury D, Tabori U, Plass C, Majewski J, Pfister SM, Jabado N (2012) Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482:226–231PubMedCrossRefGoogle Scholar
  6. 6.
    Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, Qu C, Ding L, Huether R, Parker M, Zhang J, Gajjar A, Dyer MA, Mullighan CG, Gilbertson RJ, Mardis ER, Wilson RK, Downing JR, Ellison DW, Baker SJ (2012) Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 44:251–253PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Goldberg AD, Banaszynski LA, Noh KM, Lewis PW, Elsaesser SJ, Stadler S, Dewell S, Law M, Guo X, Li X, Wen D, Chapgier A, DeKelver RC, Miller JC, Lee YL, Boydston EA, Holmes MC, Gregory PD, Greally JM, Rafii S, Yang C, Scambler PJ, Garrick D, Gibbons RJ, Higgs DR, Cristea IM, Urnov FD, Zheng D, Allis CD (2010) Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140:678–691PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Szenker E, Ray-Gallet D, Almouzni G (2011) The double face of the histone variant H3.3. Cell Res 21:421–434PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Burgess RJ, Zhang Z (2013) Histone chaperones in nucleosome assembly and human disease. Nat Struct Mol Biol 20:14–22PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Buczkowicz P, Hoeman C, Rakopoulos P, Pajovic S, Letourneau L, Dzamba M, Morrison A, Lewis P, Bouffet E, Bartels U, Zuccaro J, Agnihotri S, Ryall S, Barszczyk M, Chornenkyy Y, Bourgey M, Bourque G, Montpetit A, Cordero F, Castelo-Branco P, Mangerel J, Tabori U, Ho KC, Huang A, Taylor KR, Mackay A, Bendel AE, Nazarian J, Fangusaro JR, Karajannis MA, Zagzag D, Foreman NK, Donson A, Hegert JV, Smith A, Chan J, Lafay-Cousin L, Dunn S, Hukin J, Dunham C, Scheinemann K, Michaud J, Zelcer S, Ramsay D, Cain J, Brennan C, Souweidane MM, Jones C, Allis CD, Brudno M, Becher O, Hawkins C (2014) Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations. Nat Genet 46:451–456PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Fontebasso AM, Papillon-Cavanagh S, Schwartzentruber J, Nikbakht H, Gerges N, Fiset PO, Bechet D, Faury D, De Jay N, Ramkissoon LA, Corcoran A, Jones DT, Sturm D, Johann P, Tomita T, Goldman S, Nagib M, Bendel A, Goumnerova L, Bowers DC, Leonard JR, Rubin JB, Alden T, Browd S, Geyer JR, Leary S, Jallo G, Cohen K, Gupta N, Prados MD, Carret AS, Ellezam B, Crevier L, Klekner A, Bognar L, Hauser P, Garami M, Myseros J, Dong Z, Siegel PM, Malkin H, Ligon AH, Albrecht S, Pfister SM, Ligon KL, Majewski J, Jabado N, Kieran MW (2014) Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nat Genet 46:462–466PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Taylor KR, Mackay A, Truffaux N, Butterfield YS, Morozova O, Philippe C, Castel D, Grasso CS, Vinci M, Carvalho D, Carcaboso AM, de Torres C, Cruz O, Mora J, Entz-Werle N, Ingram WJ, Monje M, Hargrave D, Bullock AN, Puget S, Yip S, Jones C, Grill J (2014) Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nat Genet 46:457–461PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Zadeh G, Aldape K (2014) ACVR1 mutations and the genomic landscape of pediatric diffuse glioma. Nat Genet 46:421–422PubMedCrossRefGoogle Scholar
  14. 14.
    Burgess R, Jenkins R, Zhang Z (2008) Epigenetic changes in gliomas. Cancer Biol Ther 7:1326–1334PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, de la Cruz CC, Otte AP, Panning B, Zhang Y (2003) Role of histone H3 lysine 27 methylation in X inactivation. Science 300:131–135PubMedCrossRefGoogle Scholar
  16. 16.
    Zhang Y, Reinberg D (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 15:2343–2360PubMedCrossRefGoogle Scholar
  17. 17.
    Chan KM, Fang D, Gan H, Hashizume R, Yu C, Schroeder M, Gupta N, Mueller S, James CD, Jenkins R, Sarkaria J, Zhang Z (2013) The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes Dev 27:985–990PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Chan KM, Han J, Fang D, Gan H, Zhang Z (2013) A lesson learned from the H3.3K27M mutation found in pediatric glioma: a new approach to the study of the function of histone modifications in vivo? Cell Cycle 12:2546–2552PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Lewis PW, Muller MM, Koletsky MS, Cordero F, Lin S, Banaszynski LA, Garcia BA, Muir TW, Becher OJ, Allis CD (2013) Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science 340:857–861PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Bender S, Tang Y, Lindroth AM, Hovestadt V, Jones DT, Kool M, Zapatka M, Northcott PA, Sturm D, Wang W, Radlwimmer B, Hojfeldt JW, Truffaux N, Castel D, Schubert S, Ryzhova M, Seker-Cin H, Gronych J, Johann PD, Stark S, Meyer J, Milde T, Schuhmann M, Ebinger M, Monoranu CM, Ponnuswami A, Chen S, Jones C, Witt O, Collins VP, von Deimling A, Jabado N, Puget S, Grill J, Helin K, Korshunov A, Lichter P, Monje M, Plass C, Cho YJ, Pfister SM (2013) Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas. Cancer Cell 24:660–672PubMedCrossRefGoogle Scholar
  21. 21.
    Venneti S, Garimella MT, Sullivan LM, Martinez D, Huse JT, Heguy A, Santi M, Thompson CB, Judkins AR (2013) Evaluation of histone 3 lysine 27 trimethylation (H3K27me3) and enhancer of Zest 2 (EZH2) in pediatric glial and glioneuronal tumors shows decreased H3K27me3 in H3F3A K27M mutant glioblastomas. Brain Pathol 23:558–564PubMedCrossRefGoogle Scholar
  22. 22.
    Morlan J, Baker J, Sinicropi D (2009) Mutation detection by real-time PCR: a simple, robust and highly selective method. PLoS One 4:e4584PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Bechet D, Gielen GG, Korshunov A, Pfister SM, Rousso C, Faury D, Fiset PO, Benlimane N, Lewis PW, Lu C, David Allis C, Kieran MW, Ligon KL, Pietsch T, Ellezam B, Albrecht S, Jabado N (2014) Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas. Acta Neuropathol 128:733–741PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Venneti S, Santi M, Felicella MM, Yarilin D, Phillips JJ, Sullivan LM, Martinez D, Perry A, Lewis PW, Thompson CB, Judkins AR (2014) A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas. Acta Neuropathol 128:743–753PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Behjati S, Tarpey PS, Presneau N, Scheipl S, Pillay N, Van Loo P, Wedge DC, Cooke SL, Gundem G, Davies H, Nik-Zainal S, Martin S, McLaren S, Goodie V, Robinson B, Butler A, Teague JW, Halai D, Khatri B, Myklebost O, Baumhoer D, Jundt G, Hamoudi R, Tirabosco R, Amary MF, Futreal PA, Stratton MR, Campbell PJ, Flanagan AM (2013) Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone. Nat Genet 45:1479–1482PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ray Zhang
    • 1
  • Jing Han
    • 1
  • David Daniels
    • 1
  • Haojie Huang
    • 1
  • Zhiguo Zhang
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMayo ClinicRochesterUSA

Personalised recommendations