Long-Read Transcriptome Sequencing and Functional Validation Reveals Novel and Oncogenic Gene Fusions in Fusion...

Area of Science:

• Genomics
• Oncology
• Neuroscience

Background:

• Gliomas are heterogeneous central nervous system tumors driven by gene fusions (GFs).
• Current GF detection methods, like targeted short-read sequencing panels, have limitations in identifying novel or full-length fusion transcripts.
• This necessitates advanced sequencing approaches for comprehensive GF discovery in gliomas.

Purpose of the Study:

• To identify novel and full-length gene fusions (GFs) in previously fusion-negative gliomas.
• To analyze isoform structures and alternative splicing patterns associated with detected GFs.
• To functionally validate the oncogenic potential of candidate GFs using an in vivo model.

Main Methods:

• Whole-transcriptome RNA sequencing using Oxford Nanopore Technologies (ONT) long-read sequencing on 49 glioma samples.
• Transcriptome-wide fusion discovery and isoform-level transcript analysis.
• Functional evaluation of candidate GFs in Drosophila melanogaster, assessing ventral nerve cord (VNC) morphology.

Main Results:

• Identified additional known and potentially novel oncogenic GFs beyond the constraints of targeted panels.
• Resolved full-length fusion transcripts and associated isoforms, revealing alternative splicing near GF breakpoints.
• Eight of 15 tested candidate GFs demonstrated significant VNC abnormalities in Drosophila, indicating oncogenic potential.

Conclusions:

• Untargeted long-read sequencing enables comprehensive GF discovery in gliomas, surpassing limitations of short-read panels.
• Isoform-level analysis and in vivo functional validation are crucial for identifying and prioritizing novel, clinically relevant GFs.
• This approach enhances understanding of glioma oncogenesis and provides potential biomarkers for diagnosis and therapy.