title: Functional characterization of the common c.-32-13T>G mutation of GAA gene: identification of potential therapeutic agents.
abstract: Glycogen storage disease type II is a lysosomal storage disorder due to mutations of the GAA gene, which causes lysosomal alpha-glucosidase deficiency. Clinically, glycogen storage disease type II has been classified in infantile and late-onset forms. Most late-onset patients share the leaky splicing mutation c.-32-13T>G. To date, the mechanism by which the c.-32-13T>G mutation affects the GAA mRNA splicing is not fully known. In this study, we demonstrate that the c.-32-13T>G mutation abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2 and that several splicing factors affect exon 2 inclusion, although the only factor capable of acting in the c.-32-13 T>G context is the SR protein family member, SRSF4 (SRp75). Most importantly, a preliminary screening using small molecules described to be able to affect splicing profiles, showed that resveratrol treatment resulted in a significant increase of normal spliced GAA mRNA, GAA protein content and activity in cells transfected with a mutant minigene and in fibroblasts from patients carrying the c-32-13T>G mutation. In conclusion, this work provides an in-depth functional characterization of the c.-32-13T>G mutation and, most importantly, an in vitro proof of principle for the use of small molecules to rescue normal splicing of c.-32-13T>G mutant alleles.
authors.affiliation: Regional Centre for Rare Diseases, University Hospital Santa Maria della Misericordia, Udine, Italy and International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Trieste, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Dardis
authors.forename: Irene
authors.initials: I
authors.lastname: Zanin
authors.forename: Stefania
authors.initials: S
authors.lastname: Zampieri
authors.forename: Cristiana
authors.initials: C
authors.lastname: Stuani
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pianta
authors.forename: Milena
authors.initials: M
authors.lastname: Romanello
authors.forename: Francisco E
authors.initials: FE
authors.lastname: Baralle
authors.forename: Bruno
authors.initials: B
authors.lastname: Bembi
authors.forename: Emanuele
authors.initials: E
authors.lastname: Buratti
mesh_terms: D002478:Cells, Cultured; D005091:Exons; D006009:Glycogen Storage Disease Type II / Q000235:genetics*; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D000072668:Splicing Factor U2AF; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C081934:SRSF4 protein, human; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D000068103:Serine-Arginine Splicing Factors; C509951:GAA protein, human; D000520:alpha-Glucosidases