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Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.

Rivera-Barahona A, Sánchez-Alcudia R, Viecelli HM, Rüfenacht V, Pérez B, Ugarte M, Häberle J, Thöny B, Desviat LR - PLoS ONE (2015)

Bottom Line: The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

No MeSH data available.


Minigene analysis with specific primers for the natural or cryptic c.386+4 splice sites.A) Schematic drawing of the location of the primers, the murine and human cryptic splice sites and the sequence targeted by the AON. The star indicates the presence of the c.386G>A mutation. cVss: cryptic vector acceptor splice site. B) RT-PCR analysis using the denoted primer pairs obtained for murine wild-type (wt) or mutant (mut) minigenes cotransfected or not with a specific AON targeting the cryptic c.386+48 splice site. C) RT-PCR analysis using the denoted primer pairs obtained in Hep3B cells transfected with human wild-type or mutant minigenes. Sequencing analysis confirmed the identity of the bands and the use of the specific splice site in each case.
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pone.0122966.g003: Minigene analysis with specific primers for the natural or cryptic c.386+4 splice sites.A) Schematic drawing of the location of the primers, the murine and human cryptic splice sites and the sequence targeted by the AON. The star indicates the presence of the c.386G>A mutation. cVss: cryptic vector acceptor splice site. B) RT-PCR analysis using the denoted primer pairs obtained for murine wild-type (wt) or mutant (mut) minigenes cotransfected or not with a specific AON targeting the cryptic c.386+48 splice site. C) RT-PCR analysis using the denoted primer pairs obtained in Hep3B cells transfected with human wild-type or mutant minigenes. Sequencing analysis confirmed the identity of the bands and the use of the specific splice site in each case.

Mentions: In the spf/ash mouse and in human liver samples, some normally spliced transcripts (with the c.386G>A change) were detected (Fig 1), so we sought to confirm this in minigenes using a forward primer hybridizing to the junction of exon 4 and the vector exonic sequence (Fig 3A, NAT primer). In addition, the use of the c.386+4 cryptic splice site was also investigated using another specific primer that included the first four nucleotides of intron 4 (Fig 3A, CRYP primer). The normally spliced transcript could be amplified in all cases, although the amount was much lower with the mutant murine minigenes (Fig 3B). This was confirmed by semiquantitative PCR analysis (<30 cyles) (S2 Fig). The results confirm that some amount of normally spliced transcript is produced contributing to the mild phenotype observed in the mouse model and in patients with the mutation. Using the CRYP primer specific for the use of the cryptic c.386+4 splice site, we could also obtain amplified product in wild-type and mutant context, in both the human and murine minigenes, indicating this alternative splice site to be potentially active.


Functional characterization of the spf/ash splicing variation in OTC deficiency of mice and man.

Rivera-Barahona A, Sánchez-Alcudia R, Viecelli HM, Rüfenacht V, Pérez B, Ugarte M, Häberle J, Thöny B, Desviat LR - PLoS ONE (2015)

Minigene analysis with specific primers for the natural or cryptic c.386+4 splice sites.A) Schematic drawing of the location of the primers, the murine and human cryptic splice sites and the sequence targeted by the AON. The star indicates the presence of the c.386G>A mutation. cVss: cryptic vector acceptor splice site. B) RT-PCR analysis using the denoted primer pairs obtained for murine wild-type (wt) or mutant (mut) minigenes cotransfected or not with a specific AON targeting the cryptic c.386+48 splice site. C) RT-PCR analysis using the denoted primer pairs obtained in Hep3B cells transfected with human wild-type or mutant minigenes. Sequencing analysis confirmed the identity of the bands and the use of the specific splice site in each case.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390381&req=5

pone.0122966.g003: Minigene analysis with specific primers for the natural or cryptic c.386+4 splice sites.A) Schematic drawing of the location of the primers, the murine and human cryptic splice sites and the sequence targeted by the AON. The star indicates the presence of the c.386G>A mutation. cVss: cryptic vector acceptor splice site. B) RT-PCR analysis using the denoted primer pairs obtained for murine wild-type (wt) or mutant (mut) minigenes cotransfected or not with a specific AON targeting the cryptic c.386+48 splice site. C) RT-PCR analysis using the denoted primer pairs obtained in Hep3B cells transfected with human wild-type or mutant minigenes. Sequencing analysis confirmed the identity of the bands and the use of the specific splice site in each case.
Mentions: In the spf/ash mouse and in human liver samples, some normally spliced transcripts (with the c.386G>A change) were detected (Fig 1), so we sought to confirm this in minigenes using a forward primer hybridizing to the junction of exon 4 and the vector exonic sequence (Fig 3A, NAT primer). In addition, the use of the c.386+4 cryptic splice site was also investigated using another specific primer that included the first four nucleotides of intron 4 (Fig 3A, CRYP primer). The normally spliced transcript could be amplified in all cases, although the amount was much lower with the mutant murine minigenes (Fig 3B). This was confirmed by semiquantitative PCR analysis (<30 cyles) (S2 Fig). The results confirm that some amount of normally spliced transcript is produced contributing to the mild phenotype observed in the mouse model and in patients with the mutation. Using the CRYP primer specific for the use of the cryptic c.386+4 splice site, we could also obtain amplified product in wild-type and mutant context, in both the human and murine minigenes, indicating this alternative splice site to be potentially active.

Bottom Line: The equivalent nucleotide change and predicted amino acid change is found in OTC deficient patients.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

No MeSH data available.