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Original research
Comprehensive RNA and protein functional assessments contribute to the clinical interpretation of MSH2 variants causing in-frame splicing alterations


Background Spliceogenic variants in disease-causing genes are often presumed pathogenic since most induce frameshifts resulting in loss of function. However, it was recently shown in cancer predisposition genes that some may trigger in-frame anomalies that preserve function. Here, we addressed this question by using MSH2, a DNA mismatch repair gene implicated in Lynch syndrome, as a model system.

Methods Eighteen MSH2 variants, mostly localised within canonical splice sites, were analysed by using minigene splicing assays. The impact of the resulting protein alterations was assessed in a methylation tolerance-based assay. Clinicopathological characteristics of variant carriers were collected.

Results Three in-frame RNA biotypes were identified based on variant-induced spliceogenic outcomes: exon skipping (E3, E4, E5 and E12), segmental exonic deletions (E7 and E15) and intronic retentions (I3, I6, I12 and I13). The 10 corresponding protein isoforms exhibit either large deletions (49–93 amino acids (aa)), small deletions (12 or 16 aa) or insertions (3–10 aa) within different functional domains. We showed that all these modifications abrogate MSH2 function, in agreement with the clinicopathological features of variant carriers.

Conclusion Altogether, these data demonstrate that MSH2 function is intolerant to in-frame indels caused by the spliceogenic variants analysed in this study, supporting their pathogenic nature. This work stresses the importance of combining complementary RNA and protein approaches to ensure accurate clinical interpretation of in-frame spliceogenic variants.

  • Genetic Predisposition to Disease
  • Genetic Variation
  • Loss of Function Mutation
  • Sequence Analysis, RNA
  • Sequence Analysis, Protein

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.

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