Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man
Entity
UAM. Departamento de Biología MolecularPublisher
Public Library of ScienceDate
2015-04-08Citation
10.1371/journal.pone.0122966
PLoS ONE 10.4 (2015): e122966
ISSN
1932-6203 (print)DOI
10.1371/journal.pone.0122966Funded by
This work was supported by Grant SAF2010-17272 from Ministerio de Economia y Competitividad (to LRD), Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa, University fellowship (to AR), and a Postdoctoral fellowship from Centro de Diagnóstico de Enfermedades Moleculares (to RS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptEditor's Version
http://dx.doi.org/10.1371/journal.pone.0122966Subjects
Animal model; Enzyme activity; Exon; Missense mutation; Ornithine transcarbamylase deficiency; Biología y Biomedicina / BiologíaRights
© 2015 Rivera-Barahona et al.Abstract
The spf/ash mouse model of ornithine transcarbamylase (OTC) deficiency, a severe urea cycle disorder, is caused by a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a cryptic splice site 48 bp into the adjacent intron. The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients. Here we have used liver tissue and minigene assays to dissect the transcriptional profile resulting from the "spf/ash" mutation in mice and man. For the mutant mouse, we confirmed liver transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic site and to normally spliced transcripts, with exon 4 always containing the c.386G>A (p.R129H) variant. In contrast, the OTC patient exhibited exon 4 skipping or c.386G>A (p.R129H)-variant exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC tissue enzyme activities were between 3-6% of normal control in mouse and human liver. The use of the cryptic splice sites was reproduced in minigenes carrying murine or human mutant sequences. Some normally spliced transcripts could be detected in minigenes in both cases. Antisense oligonucleotides designed to block the murine cryptic +48 site were used in minigenes in an attempt to redirect splicing to the natural site. The results highlight the relevance of in depth investigations of the molecular mechanisms of splicing mutations and potential therapeutic approaches. Notably, they emphasize the fact that findings in animal models may not be applicable for human patients due to the different genomic context of the mutations
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Google Scholar:Rivera-Barahona, Ana
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Sánchez-Alcudia, Rocío
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Viecelli, Hiu Man
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Rüfenacht, Veronique
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Pérez, Belén
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Ugarte, Magdalena
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Häberle, Johannes
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Thöny, Beat
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Ruiz Desviat, Lourdes
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