NEW YORK (GenomeWeb) – A National Institutes of Health-led team has uncovered epigenetic changes in a subset of gastrointestinal stromal tumors that appear to silence a key gene to promote tumor formation.
As they reported in Science Translational Medicine today, the National Cancer Institute's Paul Meltzer and his colleagues performed targeted exome sequencing on nearly 60 samples from GIST patients who are deficient in succinate dehydrogenase (SDH). However, the researchers found that slightly more than a quarter of the patients lacked an inactivating mutation in any of the genes encoding SDH subunits. But through methylome profiling, they found that 15 of those SDH-deficient, or dSDH, patients, instead, had hypermethylated promoter regions specific to the SDHC gene.
"The discovery of SDHC epimutation provides a unifying explanation for the pathogenesis of dSDH GIST, whereby loss of SDH function stems from either SDHx mutation or SDHC epimutation." Meltzer and his colleagues wrote in their paper.
SDH-inactivating mutations are thought to give rise to GIST by turning on an angiogenesis, glycolysis, and cell proliferation pathway that is typically induced through hypoxia. The mutations are also thought to lead to nuclear epigenomic remodeling by disrupting the Krebs cycle and thus also contribute to tumorigenesis.
To examine the molecular mechanism behind the disease, the researchers sequenced the genomic regions encoding the SDH subunits from 59 patients with dSDH GIST using a custom-capture, single-molecule next-generation DNA sequencing assay. SDH inactivation was determined through both immunohistochemistry and genomic hypermethylation, a hallmark of the disease.
Of these tumors, 40 had a coding sequence mutation in one of the SDH subunit genes. Copy number and genotyping microarray analysis found that a further three tumors had intragenic deletion. However, 16 dSDH GIST cases had wild-type SDH genes.
By analyzing the DNA methylome of the 59 dSDH GIST cases using the Illumina 450K Infinium methylation BeadArray, Meltzer and his colleagues found that the dSDH GIST cases with wild-type SDH genes had significantly higher methylation levels at 11 CpG targets, compared to the dSDH GIST cases with mutant SDH genes.
Those targets, the researchers noted, mapped to a 656 basepair region that includes the promoter for the gene encoding the SDHC subunit.
Outside this region, they noted no differences in methylation levels between the two groups, including the regions encoding other SDH subunits or their promoters. Additionally, deep sequencing of the dozen dSDH GISTs that were fully methylated was unable to uncover any recurrent genomic aberrations in a 130 kilobase window surrounding SDHC.
"Thus, on the basis of the absence of identified genomic aberration, SDHC promoter methylation in GIST is consistent with primary epimutation, although future discovery of a molecular mechanism may favor reclassification as secondary epimutation," Meltzer and his colleagues said.
Overall, the researchers said that 58 of their 59 dSDH GIST cases had either a mutation in an SDH subunit encoding gene, SDHC promoter hypermethylation, or both a mutation in the SDHC subunit gene and hypermethylation of that gene's promoter.
Meltzer and his colleagues also analyzed the methylation profiles of a reference panel of more than 2,300 normal and cancer tissues, as well as data from The Cancer Genome Atlas and the Gene Expression Omnibus, and found that this hypermethylation epigenotype was largely specific to GIST.
Using an Affymetrix array and 20 GIST samples, the researchers reported that SDHC is silenced in the dSDH GIST cases with wild-type SDH genes that have the SDHC epimutation.
They further examined whether this epimutation was a constitutional change by testing blood and saliva from dSDH GIST cases with patients with wild-type SDH genes. Both whole blood and saliva showed "a small, though statistically significant uptick in SDHC promoter methylation, as compared to the cases with SDH mutations.
"Overall, we interpret SDHC hypermethylation in the blood and saliva of SDHx-WT/SDHC-epimutant GIST patients to be biologically relevant and consistent with a mosaic constitutional epimutation that is clonally expanded in tumor cells; moreover, this somatic mosaicism is consistent with postzygotic SDHC promoter methylation reprogramming," the researchers said.
The researchers also suggested that the SDHC epimutation could present a treatment target, as demethylating agents like decitabine could possibly restore SDHC expression and SDH function