Novel genetic and epigenetic alterations in ... - Ous-research.no

DiscussionSince MSP only gives information on the methylation status of the gene in question based onthe CpG-sites covered by the primers, it is important that these specific sites arerepresentative for the gene promoter. In cases where you are looking for promotermethylation that affects the gene transcription, CpG sites in the close proximity to thetranscription start point will usually be most suitable. Hypermethylation of such promoterregions have in general shown a strong association with loss of or reduced gene expression.In contrast, hypermethylation within the body of the gene may be associated with an activetranscriptional state[142]. In the case of MLH1 only a few bases are correlated withtranscriptional repression, while the other CpG sites play a marginal role[143]. Even thougha similar result was seen for the MAL gene (see bislufite sequencing page 46), both casesrepresent exceptions to the general link between promoter methylation and transcription. Atthe same time they underline the importance of good study design and careful technicalvalidation.Scoring of MSP resultsThe MSP products were separated with agarose gel electrophoresis, stained with ethidiumbromide and visualized with UV. Samples with equal or stronger band intensity than thepositive control in the methylation specific reaction were denoted strongly methylated (++),while samples with less intense bands than the positive control were categorized as weaklymethylated. Samples with very weak band intensity and those with no visible PCR product inthe methylation-specific reaction were regarded as unmethylated. We considered carcinomaswith strong band intensities (++) as methylation positive for the gene promoter in question,while the benign lesions and normal mucosa are scored as positive also when weaklymethylated (+ and ++). The rationale for this is based on the clonal expansion theory (page12). In a carcinoma, tumorigenic alterations have already accumulated. Hence, only genesmethylated in the majority of tumor cells (++) would be functionally/biologically interesting.In contrast, benign precursors may need additional alterations and/or time in order todevelop a malignant potential. Therefore, methylation changes present only in a smallfraction of the adenoma cells may provide these cells with a future growth advantage leadingto a selection and clonal expansion. By scoring the normal samples and early lesions with a45