Molecular Epidemiology

Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. Epigenetic regulation utilizes DNA methylation, various post-translational modifications at histones and small, non-coding RNAs. 

DNA methylation is a biochemical process that involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring. DNA methylation is the most frequent and stable form of epigenetic modification.

Epigenetic mechanisms control modifications in chromatin, regulating its accessibility to transcription factors, and thus contributing to determine the level of expression of different genes. Direct DNA methylation prevents transcription factor binding and at the same time favours the binding of transcription inhibiting proteins. The degree of activation of a given gene is generally dependent upon its degree of methylation. An important feature of DNA methylation is that it can be faithfully copied during the process of DNA replication. The fact, that the methylation state is potentially reversible, makes these epigenetic modifications an attractive potential target in therapy.

Epidemiological evidence increasingly suggests that environmental exposures early in development, but also during the whole lifetime, have a role in susceptibility to disease in later life. Epigenetic modifications provide a plausible link between the environment and alterations in gene expression that might lead to disease phenotypes.

Many studies suggest that epigenetics may in part mediate the complex gene-by-environment interactions that can lead to various complex diseases such as cancer or asthma and allergies.


Illumina 450K: Genome-wide DNA methylation analysis


The Infinium 450K Methylation Assay (Illumina) detects cytosine methylation at CpG sites based on highly multiplexed genotyping of bisulfite-converted genomic DNA and enables genome-wide DNA methylation profiles and large scale measurement. The panel of methylation sites includes about 485.000 CpG loci consisting of:

 ·           All designable human RefSeq genes, including promoter, 5' and 3’ regions, and miRNA

·           CpG islands and island shores

·           Sites outside of CpG islands

·           non-CpG methylated sites identified in human stem cells

·           Differentially methylated sites identified in tumor versus normal (multiple forms of cancer) and across several tissue types

·           CpG islands outside of coding regions

·           miRNA promoter regions

·           Disease-associated regions identified through GWAS

A single BeadChip accommodates 12 samples. A small amount of genomic DNA (500 - 1000 ng) is required for the initial bisulfite conversion step prior to performing the automated Infinium Assay. Unmethylated cytosines are chemically deaminated to uracil in the presence of bisulfite, while methylated cytosines are refractory to the effects of bisulfite and remain cytosine.

After bisulfite conversion, each sample will be whole-genome amplified (WGA), enzymatically fragmented and applied to the BeadChips. During hybridization, the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The 450K Methylation BeadChip applies both Infinium I & II assay chemistry technology. Infinium I assay design employs two bead types per CpG locus, one each for the methylated (C) and unmethylated (T) states. Allele-specific primer annealing is followed by single-base extension using DNP- and Biotin-labeled ddNTPs. Both bead types for the same CpG locus will incorporate the same type of labeled nucleotide, determined by the base preceding the interrogated “C” in the CpG locus, and therefore will be detected in the same color channel.

Infinium II design uses only one bead type with a unique type of probe allowing detection of both alleles. The methylated and unmethylated signals are generated respectively in the green and the red channels.

In both cases, the percentage of methylation of a given cytosine is reported as a ß‑value, which is a continuous variable between 0 and 1, corresponding to the ratio of the methylated signal over the sum of the methylated and unmethylated signals.

 At the GAC, equipment for Illumina includes the BeadArray platform, a scanner, an autoloader and the BeadStudio Software.


Sequenom EpiTYPER: Region-specific DNA methylation analysis

The EpiTYPER method (Sequenom), described by Ehrich and colleagues (Ehrich et al., 2007), is a tool for the detection and quantitative analysis of DNA methylation using base-specific cleavage of bisulfite-converted genomic DNA and Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

The method starts with bisulfite treatment of genomic DNA, followed by PCR amplification in which a T7- promoter tag is introduced. The PCR primers should be designed to yield a product within a 200-600 bp range. The significant advantage of this method is that the PCR primers are independent of the methylation state of the genomic DNA, meaning they bind to both methylated and non-methylated template. Only two primers are needed to screen for methylation changes within a region of several hundred bases in a single experiment. Next, in vitro RNA transcription is performed on the reverse strand, followed by base specific cleavage. In the cleavage reaction, the reverse strand is cleaved by RNase A at specific bases (U or C). MALDI-TOF MS analyzes the cleavage products, and a distinct signal pair pattern results from the methylated and non-methylated template DNA.

This assay offers methylation profiles of selected regions/genes; so the intention is fine mapping of specific regions. Up to 85% of CpG sites within a target region are represented in the cleavage reactions.

At the GAC, equipment for the EpiTYPER analysis includes pipette robotics, a nanodispenser, a mass spectrometer and the EpiTYPER software. The samples are processed in 384-well Microplates. An amount of 500 - 1000ng of genomic DNA per sample are needed for about 50 – 90 reactions.