title: Functional analysis of three splicing mutations identified in the PMM2 gene: toward a new therapy for congenital disorder of glycosylation type Ia.
abstract: The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N-glycosylation. The most prevalent form of CDG-type Ia-is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256-1G>C and c.640-9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640-15479C>T). Cell-based splicing assays strongly suggest that all these are disease-causing splicing mutations. The c.256-1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640-9T>G mutation was found responsible for the activation of a cryptic intronic splice-site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine-rich (SR) proteins. Finally, the deep intronic change c.640-15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation-specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen.
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biología Molecular-SO UAM-CSIC, Universidad Autónoma de Madrid,Campus de Cantoblanco, Madrid, Spain.
authors.forename: Ana I
authors.initials: AI
authors.lastname: Vega
authors.forename: Celia
authors.initials: C
authors.lastname: Pérez-Cerdá
authors.forename: Lourdes R
authors.initials: LR
authors.lastname: Desviat
authors.forename: Gert
authors.initials: G
authors.lastname: Matthijs
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ugarte
authors.forename: Belén
authors.initials: B
authors.lastname: Pérez
mesh_terms: D001483:Base Sequence; D002239:Carbohydrate Metabolism, Inborn Errors / Q000201:enzymology* / Q000235:genetics / Q000628:therapy*; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D017875:Phosphotransferases (Phosphomutases) / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D017875:Phosphotransferases (Phosphomutases); C519582:phosphomannomutase 2, human