According to the British Society for Cell Biology: “In its modern sense, epigenetics is the term used to describe inheritance by mechanisms other than through the DNA sequence of genes. It can apply to characteristics passed from a cell to its daughter cells in cell division and to traits of a whole organism. It works through chemical tags added to chromosomes that in effect switch genes on or off.” To study epigenetics, scientists can select from various tools.
“The most interesting applications in epigenetic diagnostics are related to changes in DNA methylation,” says Robert Philibert, CEO at Behavioral Diagnostics. “In brief, although there are a number of methods through which the genetic ‘hardware’ is modified by the epigenetic software, they tend to be highly intercorrelated, and this makes DNA methylation a particularly tractable method for monitoring epigenetic status, because several scalable methods for measuring the quantitative nature of DNA methylation already exist.” The same cannot be said for other epigenetic changes.
In some cases, scientists can track illuminating pathways to disease from lifestyle decisions that trigger methylation and then disease.
As an example, Michael Mendelson, pediatric cardiologist at Boston University School of Medicine, along with his colleagues, used microarrays to compare body mass index (BMI) and changes in the methylation of more than 400,000 cytosine-phosphate-guanine structures (CpGs) in human DNA [1]. The results revealed BMI-related differences in methylation at 83 CpGs, and changes at one CpG have been linked to heart disease. The research team concluded: “BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases.”
As scientists develop simpler and more complete methods of tracking DNA methylation, medical researchers will find more ways for clinicians to use the results. The applications will involve both diagnostic methods and treatments.
Measuring methylation
Specific sites of methylation in DNA can be detected with various techniques, including PCR. Although Andrea Fuso, psychology faculty member and an epigenetics expert at Sapienza University of Rome, has used various approaches to analyzing methylation, he now uses strand-specific PCR followed by Sanger sequencing. “This technology is still my preferred approach,” he says.
As Fuso points out, “This technique allows me to profile DNA methylation at the single-cytosine level in quite large promoter sequences—400 to 800 base pairs.” He adds, “Although more complex and time-consuming than pyrosequencing, Sanger sequencing results in longer sequences and gives me the opportunity to cover a larger portion of the 5¢-flanking region when I don’t have any indication of the possible relevant sites of methylation.”
Most significantly, this technique can detect and profile non-CpG methylation, Fuso points out. This refers to methylation that is not in an area of DNA where a cytosine nucleotide is followed by a guanine nucleotide.
By tracking methylation, Fuso and his team have discovered several important medical connections. For example, he says, “We demonstrated that non-CpG methylation is modulated in the presenilin1 gene in an Alzheimer’s Disease murine experimental model.”
Analyzing substance use
DNA methylation can also reveal if someone consumes alcohol or uses tobacco products.
“DNA methylation measures cannot only be used to establish diagnostic status but to monitor change in substance use/consumption in response to treatment,” says Philibert. “It is ‘precision medicine’ for the treatment of addiction.”
An epigenetic tool to test for smoking already exists. In collaboration with IBI Scientific, Behavioral Diagnostics released Smoke Signature, which Philibert describes as “the first blood-based epigenetic test.” He adds, “This easy-to-perform test is capable of quantitatively assessing smoking consumption.”
Without such a technique, clinicians rely on self-reporting from patients about smoking, and that method is typically very inaccurate. Instead, Smoke Signature measures methylation at an aryl hydrocarbon receptor repressor gene—cg05575921—which, Philibert says, “sensitively and quantitatively determines smoking status.”
Regarding the training required to use this technology, Philibert says: “The assay runs off the Bio-Rad QX200™ Droplet Digital™ PCR system, and anyone familiar with traditional fluorescent qPCR or genotyping techniques will be readily capable of running this test on this Droplet Digital PCR platform.”
Revving up RNA
In tracking epigenetic changes, scientists can look beyond DNA. “Over the past few years, there has been an interest in studying not only methylation in DNA, but more recently also methylation in RNA,” says Blanca Valle, scientific support specialist at Epigentek. “6-methyladenosine, or m6A, is the most common modification in RNA in eukaryotes.” In fact, m6A makes up about 80% of base methylations in RNA, and it arises in messenger (mRNA), transfer (tRNA), ribosomal (rRNA) and noncoding RNA (ncRNA).
“M6A controls several aspects of RNA metabolism, including splicing, mRNA translation and RNA stability,” Valle says. “Altered levels of m6A may also contribute to obesity, brain developmental abnormalities and other physiological conditions.”
To study RNA methylation, scientists target m6A with antibodies, collect the methylated adenosine nucleotides by immunoprecipitation and analyze the results with next-generation sequencing. These techniques require expertise and time. “To address these challenges,” Valle explains, “we offer the EpiQuik m6A RNA Methylation Quantification Kit to measure global levels of m6A in RNA, starting from as low as 100 nanograms.” This ELISA-based kit uses an m6A-specific antibody and colorimetric reagents. “The absorbance, which is proportional to the levels of m6A, can be measured in a microplate reader, which is readily available in most labs,” Valle says. “Therefore, it is a fast and easy way to measure m6A in a large number of samples without the need for costly equipment or much expertise.”
Making it easier to track this epigenetic event in RNA could provide various clinical applications. “The study of m6A in mRNAs and ncRNAs in different tissues may aid in having a better understanding of disease and developing therapeutic approaches,” Valle explains.
The range of diseases and other physical concerns created or controlled by epigenetics is only beginning to be known. The expanding availability of techniques and technology will allow scientists to study more aspects of epigenetic impacts on health and how the mechanisms could be controlled or even prevented.
References
[1] Mendelson, MM, et al. “Association of body mass index with DNA methylation and gene expression in blood cells and relations to cardiometabolic disease: a Mendelian randomization approach,” PLoS Medicine, 14: e1002215, 2017.
[PMID: 28095459]
Image: Shutterstock Images