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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.


Related in: MedlinePlus

RT-PCR analysis in liver samples from spf/ash mouse and from an OTCD patient carrying the analogous c.386G>A mutation.The figure shows the result of amplifying the full length cDNA transcript in mice and a fragment encompassing exons 1 to 5 for the human samples. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis (see S1 Fig). The faint bands in the mouse samples could not be sequenced. The star indicates the presence of the c.386G>A mutation. Numbers indicate the position of the cryptic splice sites used.
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pone.0122966.g001: RT-PCR analysis in liver samples from spf/ash mouse and from an OTCD patient carrying the analogous c.386G>A mutation.The figure shows the result of amplifying the full length cDNA transcript in mice and a fragment encompassing exons 1 to 5 for the human samples. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis (see S1 Fig). The faint bands in the mouse samples could not be sequenced. The star indicates the presence of the c.386G>A mutation. Numbers indicate the position of the cryptic splice sites used.

Mentions: The effect of the c.386G>A mutation on OTC exon 4 splicing was analysed in liver samples from spf/ash mice and from a male patient with the equivalent variation. RT-PCR analysis and subsequent DNA sequencing confirmed a splicing defect in both samples (Fig 1 and S1 Fig). For the mutant mouse, the analysis confirmed previously published results [10] as we detected transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic splice site as well as normally spliced transcripts, always containing the c.386G>A (p.R129H) variant. In contrast, in the OTC patient we observed exon 4 skipping or variant (c.386G>A; p.R129H) exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC enzyme activities in liver were 5.6% of normal control (7,2 versus 127,3 μmol/mg/hr) in mouse and 3.7% (0.4 versus 10,9 μmol/mg/hr) in human tissues.


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)

RT-PCR analysis in liver samples from spf/ash mouse and from an OTCD patient carrying the analogous c.386G>A mutation.The figure shows the result of amplifying the full length cDNA transcript in mice and a fragment encompassing exons 1 to 5 for the human samples. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis (see S1 Fig). The faint bands in the mouse samples could not be sequenced. The star indicates the presence of the c.386G>A mutation. Numbers indicate the position of the cryptic splice sites used.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122966.g001: RT-PCR analysis in liver samples from spf/ash mouse and from an OTCD patient carrying the analogous c.386G>A mutation.The figure shows the result of amplifying the full length cDNA transcript in mice and a fragment encompassing exons 1 to 5 for the human samples. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis (see S1 Fig). The faint bands in the mouse samples could not be sequenced. The star indicates the presence of the c.386G>A mutation. Numbers indicate the position of the cryptic splice sites used.
Mentions: The effect of the c.386G>A mutation on OTC exon 4 splicing was analysed in liver samples from spf/ash mice and from a male patient with the equivalent variation. RT-PCR analysis and subsequent DNA sequencing confirmed a splicing defect in both samples (Fig 1 and S1 Fig). For the mutant mouse, the analysis confirmed previously published results [10] as we detected transcripts corresponding to partial intron 4 retention by the use of the c.386+48 cryptic splice site as well as normally spliced transcripts, always containing the c.386G>A (p.R129H) variant. In contrast, in the OTC patient we observed exon 4 skipping or variant (c.386G>A; p.R129H) exon 4 retention by using the natural or a cryptic splice site at nucleotide position c.386+4. The corresponding OTC enzyme activities in liver were 5.6% of normal control (7,2 versus 127,3 μmol/mg/hr) in mouse and 3.7% (0.4 versus 10,9 μmol/mg/hr) in human tissues.

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.


Related in: MedlinePlus