Evaluation of a novel antibody to define histone 3.3 G34R mutant brain tumours.
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Date
2017-06-06ICR Author
Author
Haque, F
Varlet, P
Puntonet, J
Storer, L
Bountali, A
Rahman, R
Grill, J
Carcaboso, AM
Jones, C
Layfield, R
Grundy, RG
Type
Journal Article
Metadata
Show full item recordAbstract
Missense somatic mutations affecting histone H3.1 and H3.3 proteins are now accepted as the hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) as well as a subset of adult glioblastoma multiforme (GBM). Different mutations give rise to one of three amino acid substitutions at two critical positions within the histone tails, K27M, G34R/V. Several studies have highlighted gene expression and epigenetic changes associated with histone H3 mutations; however their precise roles in tumourigenesis remain incompletely understood. Determining how such amino acid substitutions in a protein affect its properties can be challenging because of difficulties in detecting and tracking mutant proteins within cells and tissues. Here we describe a strategy for the generation of antibodies to discriminate G34R and G34V mutant histone H3 proteins from their wild-type counterparts. Antibodies were validated by western blotting and immunocytochemistry, using recombinant H3.3 proteins and paediatric GBM cell lines. The H3-G34R antibody demonstrated a high degree of selectivity towards its target sequence. Accordingly, immunostaining on a cohort of 22 formalin-fixed paraffin embedded tumours with a previously known H3.3 G34R mutation status, detected successfully the corresponding mutant protein in 11/11 G34R cases. Since there was a high concordance between genotype and immunohistochemical analysis of G34R mutant tumour samples, we analysed a series of tissue microarrays (TMAs) to assess the specificity of the antibody in a range of paediatric brain tumours, and noted immunoreactivity in 2/634 cases. Importantly, we describe the generation and validation of highly specific antibodies for G34 mutations. Overall our work adds to an extremely valuable portfolio of antibodies, not only for histopathologic detection of tumour-associated mutant histone sequences, but also facilitating the study of spatial/anatomical aspects of tumour formation and the identification of downstream targets and pathways in malignant glioma progression.
Collections
Subject
Cell Line, Tumor
Animals
Rabbits
Humans
Glioma
Brain Neoplasms
Histones
Recombinant Proteins
Antibodies
Blotting, Western
Enzyme-Linked Immunosorbent Assay
Tissue Array Analysis
Immunohistochemistry
Sensitivity and Specificity
Cohort Studies
Mutation
Research team
Glioma Team
Language
eng
Date accepted
2017-05-26
License start date
2017-06-06
Citation
Acta neuropathologica communications, 2017, 5 (1), pp. 45 - ?
Publisher
BMC