Methylation is an enzyme-mediated chemical modification that adds methyl groups at selected sites on proteins, RNA and DNA. DNA methylation primarily affects cytosine bases when they are part of the dinucleotide pG. It is a modification of the genome as opposed to being part of the genome so it is known as 'epigenetics'. The function of DNA methylation remains incompletely understood. Over the years, a role for DNA methylation has been proposed in multiple systems, including control of gene expression, control of chromosomal integrity and of recombinational events. Most recently, it has been proposed that DNA methylation evolved primarily as a defence mechanism against invading parasitic DNA sequences in the genome. Approximately 70-80% of the cytosine guanosine sites that are candidates for methylation are methylated in the DNA. The exceptions are so called CpG islands, these are CpG-rich areas very frequently found in the proximal promoter regions of almost half the genes in the genome. Most of the CpG islands associated with such promoters are protected from methylation in normal cells and the absence of methylation indicates normal transcriptional activity.
In the case of both solid tumours and leukaemia, hypermethylation has now been shown to occur in the promoter CpG islands of a growing list of genes. The de novo methylation of CpG islands occurs early during the process of carcinogenesis and is associated with an unfavourable state for transcription or expression of the associated genes. Recent research has shown that methylation is at least as common as mutation as a mechanism of gene silencing in currently identified tumour-related genes. Genes frequently silenced in association with hypermethylation are for example the retinoblastoma gene, p16, E-cadherin and the BRCA1 gene
From a clinical perspective, DNA methylation changes in cancer represent an attractive therapeutic target, as epigenetic alterations are more readily reversible than genetic events. 5-Azacytidine is a powerful inhibitor of DNA methyltransferases. This agent reverses hypermethylation at the start site of genes and reactivates genes involved in tumour suppression. However, the great strength of DNA methylation in the clinic promises to be in the area of molecular diagnostics and early detection. Cancer-specific DNA methylation patterns can be detected in tumour-derived free DNA in the blood stream and in epithelial tumour cells shed into the lumen, offering a promising approach to the early detection of cancer.
Various methods have been developed to analyse DNA methylation. Amongst these methods are restriction enzyme- and sodium bisulphite based approaches which directly detect methylation at the level of a single gene or the whole genome. Restriction-enzyme based methods are based on the inability of methylation sensitive restriction enzymes to cleave methylated cytosines in their recognition site. The identification of the methylation status relies on Southern hybridization techniques or PCR and is based on the length of the digested DNA fragment. The inability to digest methylated sequences results in longer fragments, indicating a methylated CpG dinucleotide. Although restriction-enzyme based methods are simple, rapid and highly sensitive, the technique is limited to specific restriction sites and requires a substantial amount of high quality DNA. In addition, incomplete digestion can lead to false positive results. Treatment of single-stranded DNA with sodium bisulphite results in sequence differences due to deamination of unmethylated cytosines to uracil under conditions whereby methylated cytosines remain unchanged. The difference in methylation status marked by bisulphite reactivity can accurately be determined and quantified by PCR-based technology. Bisulfite sequencing techniques provide qualitative data on the methylation status of 5-methylcytosines in the amplicon between the sequence primers. Although this approach provides detailed information on the methylation status of all CpG-sites, the method is laborious and time-consuming. Methylation-specific PCR (MSP) is based on the use of two distinct methylation specific primer sets for the sequence of interest. The unmethylated (U) primer will only amplify sodium bisulphite converted DNA in unmethylated condition, while the methylated (M) primer is specific for sodium bisulfite converted methylated DNA. MSP provides a sensitive (detection of 1 methylated allele in a background of 1000 unmethylated alleles), quick and cost-effective test to analyze the methylation status of CpG dinucleotides in a CpG-islands making the technique suitable for high-throughput analysis of clinical samples.
Since the publication of the original MSP protocol, several variants have been developed. To increase the sensitivity of MSP for detection of methylated DNA in a background of unmethylated DNA, a nested, two-stage MSP was developed. The first round PCR is performed using primers which amplify sodium bisulphite-modified DNA, although these primers do not discriminate between methylated and unmethylated alleles. The obtained PCR product is subjected to second round PCR reactions using U and M specific MSP primers. Nested MSP allows a more sensitive (1 methylated allele in >50,000 unmethylated alleles) detection of methylation in clinical samples harbouring small amounts of poor quality DNA. Another advantage of this technique is that the first round primers of different genes can be multiplexed allowing simultaneous analysis of multiple gene promoters.
In our laboratory the DNA methylation profile of inflammatory breast tumours is investigated. To this purpose, a multiplex nested MSP approach is used. Genomic DNA is specifically extracted from inflammatory breast tumour cells using laser capture microdissection of paraffin embedded tissues (SLµCut system, MMI) and treated with sodium bisulfite (ZymoResearch). The methylation status of the promoter regions of genes known to be involved in breast pathogenesis such as APC, E-cadherin, THBSP-1, DAPK, GSTP1, RASSF1A, BRCA1, ESR1, caveolin-1 and -2 are being investigated using a multiplex nested MSP method. DNA isolated from normal peripheral lymphocytes from healthy individuals serves as a negative methylation control. In vitro methylated DNA serves as the positive methylation control. MSP products are analyzed on agarose gel electrophoresis, and are determined to have methylation if a visible band is observed in the methylation reaction.
