NEW YORK (GenomeWeb) – By relying on cell type-specific methylation patterns, an international team of researchers has developed a way to trace the origin of circulating cell-free DNA, and thus in which tissues cell death is occurring.
In a series of proof-of-principle studies appearing in the Proceedings of the National Academy of Sciences this week, the Hebrew University-Hadassah Medical School's Yuval Dor and his colleagues showed that they could identify methylation patterns specific to pancreatic and brain tissues, and uncover cell-free DNAs harboring those patterns in patients with diseases affecting those tissues, like type I diabetes and multiple sclerosis.
"[W]e present a method for the detection of cell death in specific tissues, based on tissue-specific methylation patterns in circulating DNA," the researchers wrote in their paper. "The approach may have multiple applications, including assessment of tissue damage after injury, evaluation of both targeted and off-target (toxicity) cell death in response to therapy, and early diagnosis of diseases such as T1D, neurodegenerative disease, and cancer."
Current approaches that analyze circulating cell-free DNA from fetuses, for example, rely on differences between the fetus and mother. However, Dor and his colleagues reasoned that conserved differences in methylation patterns between tissue types could be used to tell the difference between circulating DNA that's otherwise identical.
Further, as cell death — a hallmark of pathogenesis — accounts for a portion of cell-free circulating DNA, those methylation patterns could reveal sites in the body that may be diseased.
The team combed through the Cancer Genome Atlas, the Gene Expression Omnibus, and methylomes they'd generated locally to uncover CpG dinucleotides with differential methylation patterns between tissue types. And rather than depend on a single CpG dinucleotide, the researchers focused on a region of four to nine CpG sites near the marker site, as DNA methylation status is often regional. This way, they said, any random methylation or demethylation events would be unlikely to affect the entire region and influence the assay.
To test this idea, Dor and his colleagues used the INS promoter, which contains a number of CpG sites, as a β-cell specific marker. They amplified and then sequenced a 160-basepair stretch of the promoter from bisulfite-treated DNA obtained from a variety of tissues.
Each individual CpG site was unmethylated in 90 percent to 95 percent of the DNA molecules from the β cells, they reported, and in 5 percent to 15 percent of DNA molecules from other tissues. But when they analyzed the six sites in combination, they were all unmethylated in about 80 percent of DNA molecules from β cells and in less that 0.01 percent of DNA molecules from other tissues, indicating a high specificity for β cells.
Then to test the sensitivity, they spiked human β-cell DNA into human lymphocyte DNA at varying levels to determine the portion of INS promoter sites that were unmethylated. They found that β-cell DNA could be detected even when highly diluted.
Meanwhile, only a small portion of circulating DNA from a cohort of 31 healthy human volunteers had a fully unmethylated INS promoter — 0.12 percent of circulating fragments were totally unmethylated — but tests on 11 patients recently diagnosed with T1D exhibited a clear signal of an unmethylated INS promoter in their circulating cell-free DNA, according to the researchers.
The team likewise uncovered a methylation pattern specific to oligodendrocytes based on the methylation state of CpG sites near the 3' UTR of myelin basic protein (MBP3) and an unannotated site they dubbed WM1. Healthy people, they noted, had only low levels of unmethylated MBP3 or WM1 in circulation.
In comparison, multiple sclerosis patients during disease relapse, as determined clinically and through MRI, had unmethylated MBP3, WM1, or both in their serum, the researchers reported. Stable MS patients had minimal or no signal in their serum.
The team similarly uncovered a cluster of nine CpG sites near the locus G09787504, which they called Brain1, that act as a brain-wide tissue marker. Forty-seven healthy people had low levels of unmethylated Brain1 in their serum or plasma, the researchers noted, but patients who'd had had a heart attack with ischemic brain damage and patients with traumatic brain injury both had high levels of unmethylated Brain1 in their serum.
Dor and his colleagues further reported that their approach could also be applied to identify exocrine pancreas DNA in patients with pancreatic cancer or pancreatitis.
All together, they said that this series of studies suggests that their approach could be developed into a blood test to detect tissue damage before disease is even suspected.
"We believe that such a tool will have broad utility in diagnostic medicine and in the study of human biology," the researchers added.