# Long-Read MDM4 Sequencing Reveals Aberrant Isoform Landscape in Metastatic Melanomas

## TL;DR

This study uses long-read sequencing to identify and quantify different MDM4 isoforms in melanoma tumors, revealing new insights into their expression patterns.

## Contribution

The study introduces a novel MDM4 isoform and provides a detailed characterization of MDM4 transcript diversity in melanoma using long-read sequencing.

## Key findings

- High expression levels of MDM4-S, MDM4-FL, MDM4-A, and MDM4-209 were observed in melanoma tumor samples.
- A novel MDM4 isoform lacking exons 6 and 9, named MDM4-A/S, was identified.
- RT-PCR and RT-qPCR confirmed the presence and expression levels of novel splice junctions in melanoma specimens.

## Abstract

MDM4 is upregulated in the majority of melanoma cases and has been described as a “key therapeutic target in cutaneous melanoma”. Numerous isoforms of MDM4 exist, with few studies examining their specific expression in human tissues. The changes in splicing of MDM4 during human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Compounding this, studies relying on short reads lose “connectivity” data, so full transcripts are frequently only inferred from the presence of splice junction reads. To address this problem, long-read nanopore sequencing was utilized to read the entire length of transcripts. Here, MDM4 transcripts, both alternative and canonical, are characterized in a pilot cohort of human melanoma specimens. RT-PCR was first used to identify the presence of novel splice junctions in these specimens. RT-qPCR then quantified the expression of major MDM4 isoforms observed during sequencing. The current study both identifies and quantifies MDM4 isoforms present in melanoma tumor samples. In the current study, we observed high expression levels of MDM4-S, MDM4-FL, MDM4-A, and the previously undescribed Ensembl transcript MDM4-209. A novel transcript lacking both exons 6 and 9 is observed and named MDM4-A/S for its resemblance to both MDM4-A and MDM4-S isoforms.