title: Functional characterization of putative novel splicing mutations in the cardiomyopathy-causing genes.
abstract: Molecular diagnosis of cardiomyopathies remains difficult not only because of the large number of causative genes and the high rate of private mutations but also due to the large number of unclassified variants (UVs) found in patients' DNA. This study reports the functional splicing impact of nine novel genomic variations previously identified in unrelated patients with cardiomyopathies. To identify splice variants among these UVs, a combination of in silico and in vitro hybrid minigene tools was used as transcript is not available. Using this two-step approach, these UVs were reclassified as splicing mutations (MYBPC3-c.655-25A>G, MYBPC3-c.1790G>A (p.Arg597Gln), MYBPC3-c.2414-36G>T) or as mutations with a majority of abnormally spliced transcripts (MYBPC3-c.1182C>A, TNNT2-c.460G>A (p.Glu154Lys), and TNNT2-c.822-3C>A) or as variations with a weak splicing effect (TNNT2-c.1000-38C>A). For the two remaining variations in intron 11 of the TNNT2 gene in the vicinity of the acceptor splice site (c.571-7G>A, c.571-29G>A), a minigene assay was inconclusive as exon 12 is neither recognized as an exon by HeLa nor by H9c2 cells. Our study highlights the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional splicing impact of identified genetic variants if the RNA sample from the patient is not easily available.
authors: 1 Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est-Hospices Civils de Lyon , Bron, France .|2 EA4612, Université Claude Bernard Lyon 1 , Lyon, France .
authors: Gilles
authors: G
authors: Millat
authors: 2 EA4612, Université Claude Bernard Lyon 1 , Lyon, France .
authors: Estèle
authors: E
authors: Lafont
authors: 1 Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est-Hospices Civils de Lyon , Bron, France .
authors: Séverine
authors: S
authors: Nony
authors: 3 Centre de Biotechnologie Cellulaire, CBPE-Hospices Civils de Lyon , Bron, France .
authors: Isabelle
authors: I
authors: Rouvet
authors: 1 Laboratoire de Cardiogénétique Moléculaire, Centre de Biologie et Pathologie Est-Hospices Civils de Lyon , Bron, France .
authors: Dominique
authors: D
authors: Bozon
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002311:Cardiomyopathy, Dilated / Q000235:genetics*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D022821:RNA Splice Sites; D051381:Rats; D017422:Sequence Analysis, DNA; D020107:Troponin T / Q000235:genetics; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D022821:RNA Splice Sites; D020107:Troponin T; D000099163:Myosin Binding Protein C; C545568:TNNT2 protein, human