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Updated: Sep 5, 2025

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Identification of Spliceogenic Variants beyond Canonical GT-AG Splice Sites in Hereditary Cancer Genes

Vita Šetrajčič Dragoš1,2, Ksenija Strojnik3, Gašper Klančar1

  • 1Department of Molecular Diagnostics, Institute of Oncology Ljubljana, SI-1000 Ljubljana, Slovenia.

International Journal of Molecular Sciences
|July 9, 2022
PubMed

Insights

Variants of uncertain significance (VUS) in hereditary cancer genes can now be clarified. RNA sequencing successfully reclassified 50% of VUS, improving genetic testing and patient management.

Area of Science:

  • Genetics
  • Molecular Biology
  • Bioinformatics

Background:

  • Pathogenic variants in cancer susceptibility genes often disrupt RNA splicing.
  • Variants outside canonical splice sites are frequently classified as variants of uncertain significance (VUS) without RNA studies.
  • A high VUS burden complicates genetic testing interpretation and clinical management.

Purpose of the Study:

  • To reduce the VUS burden in genetic testing for hereditary cancer syndromes.
  • To functionally characterize VUS predicted to affect RNA splicing.
  • To reclassify VUS in key cancer susceptibility genes using mRNA analysis.

Main Methods:

  • Bioinformatic prediction of splicing defects for novel VUS in 732 patients.
  • Selection of 12 VUS for mRNA analysis based on predicted splicing impact.
  • RNA sequencing (RNAseq) to analyze splicing patterns of selected variants in APC, ATM, FH, LZTR1, MSH6, PALB2, RAD51C, and TP53 genes.

Main Results:

  • Functional characterization of 12 VUS located beyond the canonical splice site.
  • Successful reclassification of 50% of the investigated VUS.
  • 25% of VUS were downgraded to likely benign, and 25% were upgraded to likely pathogenic.

Conclusions:

  • mRNA analysis is effective in reclassifying VUS, reducing diagnostic uncertainty.
  • Reclassification of VUS significantly improves clinical management for patients and families.
  • This approach aids in identifying pathogenic variants crucial for hereditary cancer risk assessment.
Keywords:
RNAseqVUSVUS reclassificationaberrant splicingdeep intronic variantshereditary cancer syndromein silico prediction toolspseudoexonreclassificationsplice variantspliceogenicspliceogenic variant

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