GEN Exclusives

More »

Assay Tutorials

More »
Oct 1, 2010 (Vol. 30, No. 17)

Detection of Key Epigenetic Biomarkers

Approach Strives to Make Hydroxymethylated and Methylated Cytosine Quantification Reproducible

  • Epigenetics refers to the study of heritable changes in genome function that occur without a change in the primary DNA sequence of an organism. One such change is DNA methylation, most frequently characterized by an enzymatic modification at the fifth position of cytosine (5-mC), present abundantly in the context of CpG dinucleotides. The formation and maintenance of 5-mC is catalyzed by cellular DNA (cytosine-5) methyltransferases (DNMTs).

    The biology of 5-mC and its role in epigenetic inheritance and gene expression are well known. In addition to 5-mC, mammalian DNA also contains a variety of other modified nucleobases at low levels, which arise by DNA damage, normal metabolism, and other environmental factors. These modifications are generally removed during the onset of a new cell cycle.

    Recent observations involving mouse Purkinje and granule neuron cells identified an additional modified cytosine, 5-hydroxymethylcytosine (5-hmC). This modified base was also found in the genome of undifferentiated embryonic stem (ES) cells. A family of genes encoding Tet (ten eleven translocation) proteins (Tet1, Tet2, and Tet3) catalyze the oxidation reaction of 5-mC to 5-hmC.

    In addition to this mechanism, the transfer of formaldehyde to cytosine by DNMTs may be another cause of 5-hmC formation. The biological role of 5-hmC is the subject of much current speculation. A popular hypothesis is that 5-hmC may be an intermediate between 5-mC and cytosine in the mammalian genome. For example, conversion of 5-mC to 5-hmC and subsequent deamination of 5-hmC to 5-hydroxymethyluracil (5-hmU) would generate a mismatch between two opposite bases (5-hmU:G), resulting in activation of mismatch repair pathways.

    In another study, a reversible enzymatic reaction catalyzed by DNA (cytosine-5) methyltransferases led to the release of formaldehyde from 5-hmC, producing unmodified cytosine. Therefore, through this mechanism, 5-hmC may participate directly in DNA demethylation.

    Furthermore, disruption of the Tet1 and Tet2 genetic loci has been reported to associate with hematologic malignancies. A fusion of human Tet1 with the histone methyltransferase MLL has been identified in several cases of acute myeloid leukemia (AML) associated with the t(10;11)(q22;q23) translocation.

    Homozygous null mutations and chromosomal deletions of the Tet2 locus have been discovered in myeloproliferative disorders, suggesting that Tet2 may function as a tumor-suppressor protein.

    The cytosine nucleobase can reside as either unmethylated or methylated, including hydroxymethylated (5-mC and 5-hmC) forms. Methylated cytosine is generally associated with gene repression.

    Several biomarkers rely on methylation status as a disease prognostic and/or diagnostic marker (e.g., methylation of Sept9 as a biomarker for colon cancer). However, neither gene expression nor biomarker studies have taken the presence of 5-hmC into account as, currently, there are no routinely used technologies that can distinguish 5-mC from 5-hmC.

    The gold-standard approach to detect methylated cytosine is bisulphite conversion followed by DNA sequencing.  Although this chemical method can reliably identify cytosine versus 5-methylcytosine, it does not distinguish between 5-mC and 5-hmC.

Related content


GEN Jobs powered by connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
More »

Be sure to take the GEN Poll

Patient Access to Genetic Information

Do you think patients have the absolute right to gain access to their own genetic information from medical or clinical laboratories?

More »