Allele-specific expression / NMD escape test

[bloo-berries]

What it tests

Whether the mutant c.138del transcript escapes nonsense-mediated mRNA decay (NMD). If the truncated mRNA is stable, the 54-amino-acid peptide is produced and could interfere with holoenzyme assembly (Model 3) or serve as substrate for translational reinitiation (Model 2).

Method

• Allele-specific RNA-seq from peripheral blood (PBMCs)
• Quantify mutant:wild-type transcript ratio at the c.138del locus
• Apply NMDetective predictions (Lindeboom et al., 2019) as orthogonal validation
• The c.138del PTC is in exon 3 of 49 exons — classic NMD-triggering position (>50nt upstream of last exon-exon junction), but NMD efficiency varies by tissue and transcript

Expected outcomes by model

• Mutant mRNA degraded (ratio << 0.5): NMD active → Models 2 and 3 weakened (no mRNA template for reinitiation, no truncated peptide produced)
• Mutant mRNA stable (ratio ≈ 0.5): NMD escape → Models 2 and 3 remain viable; truncated peptide and/or reinitiation products may be produced
• Tissue-variable NMD: May explain tissue-specific phenotype differences

Priority justification

Tier-1: Achievable from a single blood draw. Determines whether the mutant transcript is available for reinitiation or peptide production. Directly gates the feasibility of Models 2 and 3.

Key references

• Lindeboom RGH, Supek F, Lehner B (2016) The rules and impact of NMD in human cancers. Nature Genetics 48:1112–1118.
• Lindeboom RGH et al. (2019) The impact of NMD on genetic disease, gene editing and cancer immunotherapy. Nature Genetics 51:1645–1651.