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Eur J Hum Genet. 2015 Apr;23(4):500-6. doi: 10.1038/ejhg.2014.149. Epub 2014 Jul 23.

Functional correction by antisense therapy of a splicing mutation in the GALT gene.

Author information

1
Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
2
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.
3
Department of Medicine, Santa Maria Hospital, Lisbon, Portugal.
4
Department of Pediatrics, Santa Maria Hospital, Lisbon, Portugal.
5
Metabolic Clinics, Pediatric Hospital, CHUC, Coimbra, Portugal.

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.

PMID:
25052314
PMCID:
PMC4666580
DOI:
10.1038/ejhg.2014.149
[Indexed for MEDLINE]
Free PMC Article

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