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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
  1. Laëtitia Meulemans1,
  2. Stéphanie Baert Desurmont2,
  3. Marie-Christine Waill3,
  4. Gaia Castelain1,
  5. Audrey Killian1,
  6. Julie Hauchard1,
  7. Thierry Frebourg2,
  8. Florence Coulet3,4,
  9. Alexandra Martins1,
  10. Martine Muleris3,4,
  11. Pascaline Gaildrat1
  1. 1 Normandie Univ, UNIROUEN, Inserm U1245, Normandy Centre for Genomic and Personalized Medicine, F-76000 Rouen, France
  2. 2 Normandie Univ, UNIROUEN, Inserm U1245, Normandy Centre for Genomic and Personalized Medicine, and CHU Rouen, Department of Genetics, F-76000 Rouen, France
  3. 3 Department of Genetics, AP-HP.Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France
  4. 4 Inserm UMR-S 938, Centre de Recherche Saint-Antoine, CRSA, Paris, France
  1. Correspondence to Dr Pascaline Gaildrat, Normandie Univ, UNIROUEN, Inserm U1245, 76183 Rouen, France; pascaline.gaildrat{at}


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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Data availability statement

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

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  • Deceased Thierry Frebourg deceased

  • Contributors Conceptualisation: LM, FC, AM, MM and PG; data curation: LM, SBD, GC, AK, JH, MM and PG; formal analysis: LM, SBD, MM and PG; funding acquisition: TF, AM and PG; investigation: LM, SBD, M-CW, GC, AK, JH, MM and PG; methodology: LM, M-CW, FC, AM, MM and PG; project administration: LM, FC, AM, MM and PG; resources: all authors; supervision: FC, AM, MM and PG; validation: LM, SBD, M-CW, FC, AM, MM and PG; visualisation: LM, SBD, FC, AM, MM and PG; writing of the original draft: LM, SBD, AM, MM and PG; writing (review and editing): LM, SBD, FC, AM, MM and PG. PG acts as a guarantor for this manuscript.

  • Funding This work was financially supported by the Fédération Hospitalo-Universitaire (FHU) Normandy Centre for Genomic and Personalised Medicine (NGP), the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (Gefluc, #R18064EE), a translational research grant from the French National Cancer Institute and the Direction Generale de l’Offre des Soins (INCa/DGOS, AAP/CFB/CI, FASDEC), as well as the European Union and Région Normandie. Europe gets involved in Normandie with the European Regional Development Fund. L. Meulemans was funded by an FHU-NGP PhD fellowship and benefitted from two short-term mobility fellowships, one from Cancéropôle Nord-Ouest and the other from EDnBISE.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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