# Nanopore Sequencing Reveals Novel Alternative Splice Variants of EZH2 in Pediatric Medulloblastoma

## TL;DR

Nanopore sequencing identified new EZH2 splice variants in medulloblastoma, with one variant linked to higher patient mortality.

## Contribution

First study to reveal the EZH2 alternative splicing profile in medulloblastoma using long-read sequencing.

## Key findings

- Seven EZH2 mRNA variants were identified in medulloblastoma, five of which are novel.
- High expression of the novel variant EZH2_RetI8 was associated with patient mortality.

## Abstract

Background: Medulloblastoma is the childhood tumor with the highest morbidity and mortality worldwide. This type of cancer is characterized by a high degree of heterogeneity that gives rise to different molecular groups with disparities in the clinical presentation and prognosis. Among the molecular differences, one of the most relevant factors is alternative splicing, as it is responsible for transcriptomic diversity. EZH2 is a gene processed by alternative splicing that functions as an epigenetic regulator. In cancer, certain EZH2 mRNA variants are associated with tumorigenesis; however, in medulloblastoma, the alternative splicing pattern of EZH2 has not been studied. Currently, the best tool for identifying alternative splicing variants is long-read sequencing. Methods: We amplified the most variable region of EZH2 alternative splicing and used nanopore sequencing to obtain the transcriptional profile of the gene in patients with medulloblastoma. We verified the variants identified with Sanger sequencing and digital RT–PCR. Finally, we studied the relationship between the expression levels and the clinical–biological characteristics of the patients. Results: We identified seven mRNA variants of EZH2 expressed in medulloblastoma patients, five of which had not been reported previously. In addition, high expression of the novel variant EZH2_RetI8 was associated with patient mortality (p < 0.05). Conclusions: This is the first evidence of the EZH2 mRNA variant profile in medulloblastoma, revealing seven alternative transcripts, one of which is associated with patient mortality. This is a clear example of the complexity of the transcriptome and how long-read sequencing can resolve alternative splicing patterns.