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

Discussiondepends on the requirement. Direct sequencing of human tumors can lead to anunderestimation of the methylation at CpG sites, since tumor samples often areheterogeneous and may contain normal cells which in most cases are unmethylated for thegene in question.Bisulfite sequencing is an important validation analysis that will reveal how representative theresults from each of the MSP assays are. In Paper Ib, we found hypermethylation of theMAL promoter in ~80% of colorectal carcinomas (Paper Ia and Ib) using MSP, whileanother study (Mori et al) reported only 6% for the same gene[132]. The primers used in ourMSP study were located very close to the transcription site while the other study usedprimers located a couple of hundred base pairs upstream. Bisulfite sequencing clearlyshowed that methylation is unequally distributed within the MAL promoter. A goodassociation between methylation status, as assessed by MSP, and the bisulfite sequences ofthe overlapping fragment for the MSP product located close to transcription start site(fragment A) was seen. In contrast, the region reported in the Mori study[132] was generallyunmethylated in the same cell line panel. Sequence data showed that only a minority of theCpG sites covered by the Mori antisense primer were methylated in the 19 colon cancer celllines analyzed, despite the fact that these were heavily methylated around the transcriptionstart site. The CpG sites analyzed by the Mori primer set were therefore not representativefor the methylation status of the promoter, leading to false negative results and anunderestimation of the methylation load of MAL. This finding illustrates the importance ofcombining MSP and bisulfite sequencing when analyzing new genes.Mutation analysesInnumerable methods for mutation detection are developed, and the methods can be dividedinto two crude categories, scanning methods and diagnostic methods. In general, adiagnostic method detects and identifies mutations in one analysis, while a scanning methoddetects sequence alterations without describing the mutation. Therefore, scanning methodsare often combined with a second step that identifies the mutation [18].47