title: Genomic definition of a pure intronic dystrophin deletion responsible for an XLDC splicing mutation: in vitro mimicking and antisense modulation of the splicing abnormality.
abstract: We characterised a dystrophin gene rearrangement in a previously described family with X-linked dilated cardiomyopathy and we demonstrated that it represents an 11 kb deletion occurring within intron 11. This unique deletion joined two physiologically distant intronic regions and brought adjacent two cryptic splice sites, generating a 159 bp sequence recognised as a novel alternative exon and spliced into the dystrophin transcript. Comparative analysis of the intronic region involved in the breakpoint revealed the presence of a LINE1 element (L1P_MA2), containing a 5' unconventional region (L1M1_5). This region provides the 5' cryptic splice site utilised by the novel exon, includes part of the region spliced into the dystrophin transcript and contains two short GA rich regions compatible with splicing motifs. We performed an in vitro splicing assay by using a minigene containing the patient minimal genomic rearrangement and we reproduced the inclusion of the novel alternative exon seen in the patient tissues. Antisense splicing modulation targeting the 3' cryptic splice site succeeded in restoring the canonical splicing. This represents a novel intronic mutational mechanism affecting the dystrophin gene and generating a splicing pathology. The definition of this mechanism might open perspectives in unravelling splicing regulatory motifs and their involvement in human genetic diseases.
authors.affiliation: Sezione di Genetica Medica, Dipartimento di Medicina Sperimentale e Diagnostica, Università di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Gualandi
authors.forename: Paola
authors.initials: P
authors.lastname: Rimessi
authors.forename: Barbara
authors.initials: B
authors.lastname: Cardazzo
authors.forename: Luisa
authors.initials: L
authors.lastname: Toffolatti
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Dunckley
authors.forename: Elisa
authors.initials: E
authors.lastname: Calzolari
authors.forename: Tomaso
authors.initials: T
authors.lastname: Patarnello
authors.forename: Francesco
authors.initials: F
authors.lastname: Muntoni
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ferlini
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002311:Cardiomyopathy, Dilated / Q000235:genetics*; D016189:Dystrophin / Q000235:genetics*; D019143:Evolution, Molecular; D015321:Gene Rearrangement; D005796:Genes / Q000235:genetics; D040181:Genetic Diseases, X-Linked; D006801:Humans; D007438:Introns / Q000235:genetics*; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D010802:Phylogeny; D011323:Primates / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D016189:Dystrophin; D016376:Oligonucleotides, Antisense