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

## TL;DR

Researchers developed a new method to study DNA methylation in CDKN2A exon 2, which is challenging due to its similarity with CDKN2B, and found that methylation in this region affects gene regulation in cancer.

## Contribution

A novel pyrosequencing assay was developed to distinguish and analyze methylation in CDKN2A exon 2 despite its high sequence similarity with CDKN2B.

## Key findings

- The new method enabled accurate quantification of CDKN2A exon 2 methylation in cancer cell lines.
- Both promoter and exon 2 methylation contribute to the regulation of CDKN2A expression in breast cancer.
- Epigenetic and genetic alterations, including exon 2 skipping, were observed in breast cancer cell lines.

## Abstract

The tumor suppressor p16INK4a, 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 CDKN2AINK4a 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.