Evidence in disease and non-disease contexts that nonsense mutations cause altered splicing viamotif disruption.

Liam Abrahams,Bethan Young,Grzegorz Kudla,Laurence D Hurst,Rosina Savisaar,Christine Mordstein

doi:10.1093/nar/gkab750

Abstract

Transcripts containing premature termination codons (PTCs) can be subject to nonsense-associated alternative splicing (NAS). Two models have been evoked to explain this, scanning and splice motif disruption. The latter postulates that exonic cis motifs, such as exonic splice enhancers (ESEs), are disrupted by nonsense mutations. We employ genome-wide transcriptomic and k-mer enrichment methods to scrutinize this model. First, we show that ESEs are prone to disruptive nonsense mutations owing to their purine richness and paucity of TGA, TAA and TAG. The motif model correctly predicts that NAS rates should be low (we estimate 5–30%) and approximately in line with estimates for the rate at which random point mutations disrupt splicing (8–20%). Further, we find that, as expected, NAS-associated PTCs are predictable from nucleotide-based machine learning approaches to predict splice disruption and, at least for pathogenic variants, are enriched in ESEs. Finally, we find that both in and out of frame mutations to TAA, TGA or TAG are associated with exon skipping. While a higher relative frequency of such skip-inducing mutations in-frame than out of frame lends some credence to the scanning model, these results reinforce the importance of considering splice motif modulation to understand the etiology of PTC-associated disease.

Highlights

Understanding the molecular mechanisms that underpin genetic diseases is core to genetic-based medicine
TCAAGA→TGAAGA reflects an exonic splice enhancers (ESEs) transitioning to another ESE via a nonsense mutation, both hexamers featuring in the INT3 set
A significant number, 339/541 (62.66%), have positive Z scores (P = 0.004, one-tailed exact binomial test; note here a positive Z score indicates increases in RPMskip over the simulants). This result is robust to missense mutations being matched by their distance to the exon boundary (381/557, P < 2.2 × 10–16, one-tailed exact binomial test). These results suggest that the association between premature termination codons (PTCs) and exon skipping cannot be explained solely by nonsensemediated decay (NMD), as NMD should not affect the absolute count of reads that support skipping

Summary

Introduction

Understanding the molecular mechanisms that underpin genetic diseases is core to genetic-based medicine (e.g. see [1,2,3]). A PTC may result in the synthesis of a truncated protein with potentially problematic loss of function or gain of toxicity [5,6,7]. Nonsensemediated decay (NMD) [8,9] targets some PTC-containing transcripts for degradation, potentially avoiding any toxic effects of truncated proteins in heterozygotes, but largely abolishing expression in PTC homozygotes. Just as synonymous and nonsynonymous mutations can cause disease by altering splicing [14,15], so too PTCcontaining exons can cause exon skipping (reviewed in 15). With the PTC bearing exon removed, the PTC in question is not subject to NMD (N.B. as PTCs are usually defined at the DNA level under an assumption of canonical splicing, we retain the language of PTCs even if they induce exon skipping)

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Results

Conclusion