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Makrigiorgos Lab at Dana-Farber Publishes Proof of Concept for Methylation-specific COLD-PCR

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Researchers affiliated with the laboratory of Mike Makrigiorgos at Dana Farber Cancer Institute and Harvard Medical School have demonstrated how their COLD-PCR method can be applied to epigenetics, allowing the enrichment and sequencing of rare unmethylated DNA regions in a background of highly methylated DNA.

Their method — called COLD-MS-PCR for "coamplification at lower denaturation temperature, methylation-specific PCR — adds to the growing stable of specialized COLD-PCR techniques, and could prove useful for detecting loss of methylation or imprinting in cancer, diabetes, or diet-related conditions, the researchers said.

The Makrigiorgos lab developed the original COLD-PCR technique, which takes advantage of the fact that mutant DNA strands denature at lower temperatures in a PCR reaction than normal DNA strands, enabling selective amplification of the mutant DNA with minimal amplification of normal DNA. The technique can be coupled with downstream sequencing or other analysis methods to detect mutant alleles with extremely high sensitivity.

Over the past few years Makrigiorgos and colleagues created several variations of COLD-PCR to either up the sensitivity of the original method or use it in specialized applications. For instance, improved and complete enrichment, or ICE-COLD-PCR enhances sensitivity by using locked nucleic acids and a reference strand that binds PCR amplicons to form duplexes that are preferentially denatured and amplified at a certain temperature.

The lab also previously developed a more efficient version of COLD-PCR called Fast COLD-PCR, which skips the denaturation and intermediate annealing stages of normal COLD-PCR, taking advantage of the fact that in some cases preferential amplification of mutant DNA is so great that it obviates the need to form a mutant-wildtype heteroduplex DNA.

Makrigiorgos told PCR Insider this week that his lab developed this latest methylation-specific version of COLD-PCR — which is described in a paper published this week in PLOS One — to address situations where a researcher would want to detect traces of hypo-methylation.

"We of course are interested in this for cancer purposes, but it also has applications in many other situations like diabetes, or the effect of lifestyle – diet, alcohol, smoking – on [the development of] diabetes, obesity, and cancer," Makrigiorgos said.

For instance, studies have shown that 5-methylcytosine levels in cancer tissues decrease compared to surrounding normal tissue in colon adenocarcinomas, Wilms' tumors, and ovarian epithelial carcinomas.

Furthermore, loss of imprinting — that is, unmethylation of a previously methylated allele — may result in placental defects or a series of genetic conditions that could increase the risk of cancer; and detection of unmethylated pro-insulin DNA in serum has been associated with pancreatic beta-cell death, opening the door to the idea that detecting DNA unmethylation at early stages of disease progression could help guide treatment.

Even more specifically, Makrigiorgos' group developed the method for use in a research collaboration with Boston Medical Center examining the effect of infant diet — breast feeding versus formula feeding — on systemic levels of methylation or unmethylation.

"This can have implications for growth, obesity, and diabetes in particular," Makrigiorgos said. "There have been many links to formula feeding versus breast feeding and subsequent development of diabetes and obesity. But the mechanism is not well-known, and how to inhibit that remains a question. We have a hypothesis that epigenetic regulation can happen … depending on [the way the infant is fed], and that's actually one of the main reasons we developed this methodology."

Many methods for detecting methylated or unmethylated DNA exist, most notably bisulfite sequencing and real-time methylation-specific PCR. Each of these has their advantages, but in general, ease of workflow is not chief among them.

As with all COLD-PCR methods, COLD-MS-PCR works by decreasing the typical 95°-to-98° C denaturation temperature range to enable preferential amplification of sequences with a lower melting temperature — in this case trace unmethylated regions in a sea of methylated DNA. Subsequent detection, however, is relatively streamlined, performed using an intercalating dye and melting analysis followed by direct sequencing of resultant amplicons without the need for specific reagents or special instrumentation, the authors noted in their paper.

As proof of principle for their method, Makrigiorgos and colleagues examined methylation of the gene encoding for O6 methylguanine methyl transferase (MGMT) in DNA from glioblastoma or infant blood samples. They chose to use the Fast COLD-PCR version due to its relative simplicity — their method involved first performing a PCR reaction specific for bisulfite-converted DNA to amplify a region of MGMT, then performing a second, nested PCR in the Fast-COLD-PCR format to favor the amplification of unmethylated DNA.

Immediately following the extension step of the nested reaction, PCR amplicons were subjected to melt curve analysis on a Cepheid SmartCycler II, and resultant amplicons were Sanger sequenced.

Using Fast COLD-MS-PCR, the researchers were able to amplify and sequence 0.05 percent of unmethylated MGMT DNA in an excess of 99.95 percent methylated MGMT DNA. Comparatively, conventional methlylation-specific PCR revealed fractions of unmethylated-to-methylated DNA of 20 to 80 percent, and did not clearly reveal fractions below 5 percent.

Another potential advantage of the new method, the researchers noted, is that it uses primers "that bind sequence positions that do not contain CpG sites, i.e. they are neutral in regards to CpG methylation. This is a potential advantage over approaches that utilize methylation-specific primers, such as [methylation-specific] PCR … as the methylation status of the primer binding sites ideally should not pre-determine which fraction of the target amplicons can be studied."

Although the PLOS One study demonstrated the detection of trace unmethylated regions in a background of methylated DNA, "if one applies other versions, like ICE-COLD-PCR … you can also reverse the process and detect minority alleles that are methylated as opposed to unmethylated," Makrigiorgos said. This, he added, is "very relevant to cancer — methylated tumor suppressor genes, for example," and as such will be a future area of work for his group.

Transgenomic has an exclusive license from Dana-Farber to use standard ICE-COLD-PCR in combination with pyrosequencing and Sanger sequencing for downstream analysis, and has been developing blood-based cancer mutation detection assays using this approach, regularly achieving 10,000-fold enrichment of mutant targets.

It is unclear whether the company has an interest in methylation analysis. In an email to PCR Insider, Transgenomic CEO Paul Kinnon noted that "sensitivity is a critical attribute for any assay that aims at cancer detection and prognosis. The addition of this type of assay/capability would increase sensitivity for detection of rare methylation changes, and could benefit our repertoire of assays that rely on COLD-PCR technology (i.e. our ICE-COLD-PCR and multiplexed ICE-COLD-PCR assays for mutation detection)." In addition, Kinnon said that the technology could "expand our current capabilities for monitoring and surveillance of genetic changes to include epigenetic changes associated with cancer prognosis and therapy and overall bring us one step closer to improved patient-outcomes management."

Makrigiorgos noted that his lab at Dana-Farber has a "long-standing collaboration" with Transgenomic and that the company would be a "natural commercial partner" for COLD-MS-PCR.

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