title: Functional correction by antisense therapy of a splicing mutation in the GALT gene.
abstract: In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia.
authors: Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Ana I
authors: AI
authors: Coelho
authors: Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Sílvia
authors: S
authors: Lourenço
authors: Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Matilde
authors: M
authors: Trabuco
authors: 1] Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal [2] Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Maria João
authors: MJ
authors: Silva
authors: Department of Medicine, Santa Maria Hospital, Lisbon, Portugal.
authors: Anabela
authors: A
authors: Oliveira
authors: Department of Pediatrics, Santa Maria Hospital, Lisbon, Portugal.
authors: Ana
authors: A
authors: Gaspar
authors: Metabolic Clinics, Pediatric Hospital, CHUC, Coimbra, Portugal.
authors: Luísa
authors: L
authors: Diogo
authors: 1] Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal [2] Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Isabel
authors: I
authors: Tavares de Almeida
authors: 1] Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal [2] Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: João B
authors: JB
authors: Vicente
authors: 1] Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal [2] Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
authors: Isabel
authors: I
authors: Rivera
mesh_terms: D000818:Animals; D019556:COS Cells; D016022:Case-Control Studies; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D053938:DNA Fragmentation; D016373:DNA, Antisense / Q000494:pharmacology*; D005693:Galactosemias / Q000235:genetics / Q000628:therapy*; D005820:Genetic Testing; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D012322:RNA Precursors / Q000235:genetics; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics*
chemical_list: D016373:DNA, Antisense; D009841:Oligonucleotides; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase