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Epigenetic Markers in Glioblastoma Linked to Poor Prognosis

NEW YORK (GenomeWeb) – Low levels of a certain epigenetic mark are correlated with poorer prognosis in glioblastoma patients, according to a team of researchers from the Geisel School of Medicine at Dartmouth.

Researchers led by Dartmouth's Brock Christensen characterized patterns of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) DNA modifications in set of 30 glioblastoma (GBM) samples. As they reported Friday in Nature Communications, the researchers used a combination of sodium bisulfite and oxidative sodium bisulfite treatment followed by hybridization to DNA methylation arrays to find that 5hmc modifications are generally depleted in glioblastomas. Those that are present, though, are typically localized to tissue-specific transcription and super-enhancers and are linked to RNA regulation and immune functions, among other processes.

At the same time, the researchers reported that their clustering analysis found that low 5hmC patterns were associated with poorer overall survival.

"Here, we uncovered that specific DNA 5mC and 5hmC patterns are disrupted in GBM and uniquely characterize the molecular switches of the genome known as 'enhancers,'" Christensen said in a statement. "Importantly, we discovered that 5hmC signatures had a particularly strong association with patient survival."

Since sodium bisulfite treatment alone cannot distinguish 5mC modifications from 5hmC modifications, the researchers relied on that approach plus oxidative sodium bisulfite treatment prior to methylation array analysis to disentangle the two. They applied these approaches to a set of 30 primary GBM tumors that had wild-type IDH1 and IDH2.

Using a novel algorithm dubbed OxyBS, they then determined genome-wide 5mC and 5hmC estimates for these samples.

By comparing those 5hmC estimates against those from the prefrontal cortices from an independent group of healthy patients, the researchers uncovered a total reduction of 5hmC content in GBM as compared to healthy tissue. Such genome-wide loss of 5hmC has been reported in other tumor types, the investigators said, suggesting that the loss of 5hmC regulation is a key part of tumorigenesis.

While all GBM samples had lower 5hmC levels overall, some regions of the genome exhibited elevated 5hmC levels, Christensen and his colleagues reported.

According to the investigators, 5hmC modifications were enriched at glioblastoma-specific enhancer elements, alternative mRNA splicing sites, and near genes that often mutated in glioblastoma. For instance, the researchers noted that high 5hmC CpGs were more likely to be in introns, but depleted in promoter regions. They also noted a small enrichment among 5hmC CpGs for glioblastoma DNase hypersensitivity sites, suggesting a link to open chromatin. The link to enhancers and super-enhancers also indicated that 5hmC might serve as a mark of cellular identity, the researchers said.

At the same time, they found that the intronic regions of genes like EGFR, PTEN, NF1, PIK3R1, RB1, PDGFRA, and QKI — all of which are commonly mutated in glioblastoma — had high 5hmC levels.

They further reported that high 5hmC levels appeared to be linked to active transcription and thus might influence cancerous processes.

When Christensen and his colleagues clustered their cases based on their 5hmC patterns at CpG sites, they uncovered low- and high-5hmC clusters. The patients with low-5hmC tumors were older at diagnosis and had shorter median survival times. A multivariable model indicated that patients in the low-5hmC cluster had a significantly increased death hazard. However, the researchers noted that total 5hmC was not associated with prognosis.

This, they wrote in this paper, indicates "that the genomic location of 5hmC is an important consideration in association with disease progression."