DNA Methylation and Transcript Variant Analysis of Exon 2 Despite High Sequence Identity with Exon 2.

The tumor suppressor p16, encoded by , is frequently inactivated in cancer through genetic or epigenetic mechanisms. While promoter hypermethylation is the most common epigenetic cause, aberrant methylation of exon 2 has also been associated with various tumor types. However, analyzing DNA methylation of exon 2 is challenging due to its high sequence similarity with . We developed a pyrosequencing assay to analyze CpGs in exon 2, which was previously found to be hypermethylated in breast cancer. Our novel primer set enabled co-amplification of the homologous regions in including CpGs 1-24, and CpGs 1-23. By quantifying the proportion of , we could accurately determine methylation levels for CpGs in exon 2. This method was applied to patient-derived glioma cells and commercial breast cancer cell lines. To reveal the role of exon 2 methylation in gene regulation, we additionally examined promoter methylation and expression at both mRNA and protein levels in breast cancer cell lines. We observed a range of (epi)genetic alterations, including homozygous deletions, transcript-specific expression, and exon 2 skipping. Our findings indicate that both promoter and exon 2 methylation contribute to regulation of expression. This novel method provides a valuable tool for future studies seeking a deeper understanding of regulation in cancer.