# Synonymous and non-synonymous variants at splice junctions can disrupt splicing and are frequently linked to disease associated loss of function genes

## TL;DR

This study shows that genetic variants near RNA splice junctions can disrupt splicing and are often found in genes linked to disease through loss of function.

## Contribution

The study identifies that synonymous and missense variants near splice junctions are frequently associated with disease-related loss-of-function genes.

## Key findings

- Variants at splice donor and acceptor sites are enriched in genes intolerant to protein truncation.
- G > A and G > T mutations at splice junctions are common and often missense.
- Splice junction variants are rarely observed in population data, suggesting disease relevance.

## Abstract

RNA splicing facilitated by the spliceosome complex, relies on specific motifs at exon-intron junctions of pre-mRNAs to generate mature mRNAs. Mutations in splice junctions can disrupt splicing, potentially leading to premature protein truncation. Nucleotides within the exonic component of the junction are also essential for splicing. Evaluation of silent and missense variants in the exonic splice junction on RNA splicing is essential to investigate the significance of such variants in disease pathogenesis.

We analyzed cancer-associated silent and missense variants reported in the COSMIC database that are located within three nucleotides of splice donor and acceptor sites. We examined the prevalence of these variants in genes for which loss of function is a known mechanism of disease development. We also studied the performance of splicing impact prediction tools and evaluated their clinical relevance as well as their alignment with experimentally validated splicing outcomes.

Nucleotide composition analysis revealed a high preference for the nucleotide G at the donor 1 (d1) and acceptor (a1) positions, 87% and 69%, respectively. We observed a high prevalence of G > A and G > T variants at d1 and a1 positions. Interestingly, 66% to 86% of the identified variants at these positions are missense mutations, with G > T variants being specific for this type of mutation. Evolutionary conservation analysis indicates high nucleotide conservation for these positions at donor and acceptor sites, highlighting their importance at the nucleotide level. The frequently occurring variants are associated with tumor suppressor genes, and 58 of the top 100 genes have LOEUF scores below 1, indicating low tolerance to protein truncation. In contrast, such genes are rarely observed among population variants.

Our data driven computational study emphasizes the importance of evaluating silent and missense variants at splice junctions to understand their impact on RNA splicing. These variants may have a neutral effect on protein function. However, evaluating their effect at the RNA level is essential to understanding the significance of these variants in disease pathogenesis. This is particularly important for genes in which loss of function is the mechanism of disease development.

The online version contains supplementary material available at 10.1186/s12864-025-12466-0.