title: Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.
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.
authors: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Instituto de Investigación Biomédica Hospital La Paz (IdiPAZ), Madrid, Spain.
authors: Ana
authors: A
authors: Rivera-Barahona
authors: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Instituto de Investigación Biomédica Hospital La Paz (IdiPAZ), Madrid, Spain.
authors: Rocío
authors: R
authors: Sánchez-Alcudia
authors: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital, Zürich, Switzerland.
authors: Hiu Man
authors: HM
authors: Viecelli
authors: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital, Zürich, Switzerland.
authors: Veronique
authors: V
authors: Rüfenacht
authors: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Instituto de Investigación Biomédica Hospital La Paz (IdiPAZ), Madrid, Spain; Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Madrid, Spain.
authors: Belén
authors: B
authors: Pérez
authors: Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Instituto de Investigación Biomédica Hospital La Paz (IdiPAZ), Madrid, Spain; Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Madrid, Spain.
authors: Magdalena
authors: M
authors: Ugarte
authors: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital, Zürich, Switzerland.
authors: Johannes
authors: J
authors: Häberle
authors: Division of Metabolism and Children's Research Centre (CRC), University Children's Hospital, Zürich, Switzerland.
authors: Beat
authors: B
authors: Thöny
authors: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Instituto de Investigación Biomédica Hospital La Paz (IdiPAZ), Madrid, Spain; Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Madrid, Spain.
authors: Lourdes Ruiz
authors: LR
authors: Desviat
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D005091:Exons; D006801:Humans; D007438:Introns; D008099:Liver / Q000201:enzymology; D051379:Mice; D009154:Mutation; D009954:Ornithine Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D020163:Ornithine Carbamoyltransferase Deficiency Disease / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics*
chemical_list: D022821:RNA Splice Sites; D009954:Ornithine Carbamoyltransferase