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.
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Acknowledgments
This work has been generously supported by the Foundation de France (including a fellowship to Pascaline Gaildrat) and is presently supported by the French North West Canceropole.
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Gaildrat, P., Killian, A., Martins, A., Tournier, I., Frébourg, T., Tosi, M. (2010). Use of Splicing Reporter Minigene Assay to Evaluate the Effect on Splicing of Unclassified Genetic Variants. In: Webb, M. (eds) Cancer Susceptibility. Methods in Molecular Biology, vol 653. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-759-4_15
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DOI: https://doi.org/10.1007/978-1-60761-759-4_15
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Publisher Name: Humana Press, Totowa, NJ
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