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. 2018 Feb 12;9(17):13807-13821.
doi: 10.18632/oncotarget.24480. eCollection 2018 Mar 2.

Integrated DNA methylation analysis identifies topographical and tumoral biomarkers in pilocytic astrocytomas

Manila Antonelli  1 , Antonio Fadda  2 , Eleonora Loi  2 , Loredana Moi  2   3 , Cesare Zavattari  4 , Pia Sulas  5 , Davide Gentilini  6   7 , Cinzia Cameli  8 , Elena Bacchelli  8 , Manuela Badiali  3 , Antonella Arcella  9 , Isabella Morra  10 , Felice Giangaspero  1   9 , Patrizia Zavattari  2
Affiliations

Integrated DNA methylation analysis identifies topographical and tumoral biomarkers in pilocytic astrocytomas

Manila Antonelli et al. Oncotarget. .

Abstract

Pilocytic astrocytoma (PA) is the most common glioma in pediatric patients and occurs in different locations. Chromosomal alterations are mostly located at chromosome 7q34 comprising the BRAF oncogene with consequent activation of the mitogen-activated protein kinase pathway. Although genetic and epigenetic alterations characterizing PA from different localizations have been reported, the role of epigenetic alterations in PA development is still not clear. The aim of this study was to investigate whether distinctive methylation patterns may define biologically relevant groups of PAs. Integrated DNA methylation analysis was performed on 20 PAs and 4 normal brain samples by Illumina Infinium HumanMethylation27 BeadChips. We identified distinct methylation profiles characterizing PAs from different locations (infratentorial vs supratentorial) and tumors with onset before and after 3 years of age. These results suggest that PA may be related to the specific brain site where the tumor arises from region-specific cells of origin. We identified and validated in silico the methylation alterations of some CpG islands. Furthermore, we evaluated the expression levels of selected differentially methylated genes and identified two biomarkers, one, IRX2, related to the tumor localization and the other, TOX2, as tumoral biomarker.

Keywords: gene expression alteration; human methylation beadchips; methylome alteration; pilocytic astrocytomas; topographic and tumor biomarkers.

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Conflict of interest statement

CONFLICTS OF INTEREST None of the authors has conflicts of interest

Figures

Figure 1
Figure 1. Unsupervised hierarchical clustering analysis of significantly differently methylated CpG Islands related to the two subgroups considered in the study: tumor location and age at onset
(A) infratentorial and supratentorial samples (248 CpG Islands); (B) age at onset ≤ 3 or > 3 (360 CpG Islands).
Figure 2
Figure 2. Flowchart describing the selection, annotation, probe enrichment and validation of the methylation alterations relative to the Supratentorial/Infratentorial differential methylation comparison
Figure 3
Figure 3. 27K/450K enrichment and comparison
CpG Islands found significantly altered in PAs analyzed in the present study (using the HumanMethylation27 beadchip), were compared and enriched in silico with PA data analyzed by Lambert and colleagues (HumanMethylation450K beadchip). Here is the example of the EN2 gene.
Figure 4
Figure 4. EN2, IRX2, TOX2 gene expression profile
(A) Differential expression obtained by qRT-PCR on the PA samples collected in the present study, calculated using the ΔΔCT method and expressed as fold change relative to the supratentorial mean expression value; (B) Differential expression obtained by qRT-PCR on commercially available normal brain RNA, calculated using the ΔΔCT method and expressed as fold change relative to the lowest expression value; (C) Gene expression in normal brain tissue, according to GTEx portal (Genotype-Tissue Expression portal). Expression value are shown in log10(RPKM) (Reads Per Kilobase of transcript per Million mapped reads), calculated from a gene model with isoforms collapsed to a single gene. In orange: supratentorial localization and related brain regions; in yellow: infratentorial localization and related brain regions.
Figure 5
Figure 5. 27K/450K enrichment and comparison
CpG Islands found significantly altered in PAs analyzed in the present study (using the HumanMethylation27 beadchip), were compared and enriched in silico with PA data analyzed by Lambert and colleagues (HumanMethylation450K beadchip). Here is the example of the IRX2 gene.
Figure 6
Figure 6. 27K/450K enrichment and comparison
CpG Islands found significantly altered in PAs analyzed in the present study (using the HumanMethylation27 beadchip), were compared and enriched in silico with PA data analyzed by Lambert and colleagues (HumanMethylation450K beadchip). Here is the example of the TOX2 gene.
Figure 7
Figure 7. In silico LGG survival curves of patients classified by
(A) TOX2 and (B) IRX2 expression in the indicated dataset [TCGA-LGG] (x-axis: survival time in days; y-axis: overall survival). Samples were divided into high and low expression groups bifurcating at median expression value for mRNA expression.
See this image and copyright information in PMC

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