title: Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants.
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.
authors: Faculty of Medicine Department of Genetics and Institute for Biomedical Research, Rouen University Hospital, Inserm U614, IFRMP, University of Rouen, Northwest Canceropole, Rouen, France.
authors: Pascaline
authors: P
authors: Gaildrat
authors: Audrey
authors: A
authors: Killian
authors: Alexandra
authors: A
authors: Martins
authors: Isabelle
authors: I
authors: Tournier
authors: Thierry
authors: T
authors: Frébourg
authors: Mario
authors: M
authors: Tosi
mesh_terms: D017398:Alternative Splicing / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D017930:Genes, Reporter; D030342:Genetic Diseases, Inborn / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D005821:Genetic Techniques; D014644:Genetic Variation / Q000502:physiology*; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D012333:RNA, Messenger