A research team at Ghent University has developed a new way to improve genetic diagnoses using RNA sequencing. Their method focuses on a small blood sample and a simple lab treatment that makes it easier to detect problems with how genes are spliced, especially in people with neurodevelopmental disorders like epilepsy and intellectual disability.

While traditional DNA sequencing looks at the genetic code itself, RNA sequencing reveals how genes are actually used to produce proteins. Some mutations affect how genes are spliced, leading to faulty or incomplete proteins. These changes can be hard to spot with DNA sequencing alone. By culturing peripheral blood mononuclear cells (PBMCs) for a short time and treating them with a drug called cycloheximide, the team was able to preserve faulty RNA transcripts long enough to detect them.

Overview of the proposed diagnostic workup for RNA-sequencing

A Wet lab workflow. B–D Decision trees for variant classification/evaluation for respectively FRASER (B 1/ pre-set parameters and 2/gene-specific evaluation), OUTRIDER (C), and mono allelic expression (D).

The researchers tested their approach in 46 individuals with suspected genetic conditions and 15 of their parents. They found that in 9 patients with known or suspected splicing variants, RNA sequencing revealed clear defects in 6 of them. This allowed them to reclassify 7 genetic variants, giving patients and their families more accurate information. Additional testing confirmed most of these results, although it missed a couple of more complex RNA changes. Importantly, the team created a practical flowchart that labs can use to include this RNA-seq protocol in everyday diagnostics.

This new method is easy to use, doesn’t require a tissue biopsy, and gives clinicians more information when evaluating rare diseases. It shows how RNA sequencing is becoming a powerful tool for making sense of tricky genetic variants.