DNA Methylation and Transcript Variant Analysis of CDKN2A Exon 2 Despite High Sequence Identity with CDKN2B Exon 2

The tumor suppressor p16^INK4a, encoded by CDKN2A, is frequently inactivated in cancer through genetic or epigenetic mechanisms. While promoter hypermethylation is the most common epigenetic cause, aberrant methylation of CDKN2A 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 CDKN2B. We developed a pyrosequencing assay to analyze CpGs in CDKN2A exon 2, which was previously found to be hypermethylated in breast cancer. Our novel primer set enabled co-amplification of the homologous regions in CDKN2A, including CpGs 1-24, and CDKN2B CpGs 1-23. By quantifying the proportion of CDKN2A, we could accurately determine methylation levels for CpGs in CDKN2A 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 CDKN2A^INK4a 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 CDKN2A expression. This novel method provides a valuable tool for future studies seeking a deeper understanding of CDKN2A regulation in cancer.