Use of Splicing Reporter Minigene Assay to Evaluate the Effect on Splicing of Unclassified Genetic Variants

  • Pascaline Gaildrat
  • Audrey Killian
  • Alexandra Martins
  • Isabelle Tournier
  • Thierry Frébourg
  • Mario Tosi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 653)

Abstract

The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for “unclassified variant”) found in genetic screenings represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patient genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing. This method represents a complementary approach to reverse transcription-PCR analyses of patient RNA, for the identification of pathogenic splicing mutations.

Key words

Cancer molecular diagnosis Minigene construct Splicing mutations Unclassified genetic variants 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Pascaline Gaildrat
    • 1
  • Audrey Killian
    • 1
  • Alexandra Martins
    • 1
  • Isabelle Tournier
    • 1
  • Thierry Frébourg
    • 1
  • Mario Tosi
    • 1
  1. 1.Faculty of Medicine Department of Genetics and Institute for Biomedical Research, Rouen University Hospital, Inserm U614, IFRMPUniversity of Rouen, Northwest CanceropoleRouenFrance

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