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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

Effect of AON targeting the c.386G>A cryptic splice site on the splicing profile of murine minigenes.Wild-type (wt) and mutant (mut) murine minigenes transfected in Hep3B cells were cotransfected with 20 μM of AON and RT-PCR analysis performed after 24 h. SCR, scrambled oligonucleotide. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis. Grey boxes represent vector sequences (V) and white boxes indicate exon 4. Numbers indicate the position of the cryptic splice sites used. The box denoted as cV indicates a stretch of vector DNA that is retained in the mRNA due to the use of a vector cryptic splice site. The star indicates the presence of the c.386G>A mutation. The arrow points to the band corresponding to the use of the cryptic c.386+48 site in the mutant minigenes.
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pone.0122966.g004: Effect of AON targeting the c.386G>A cryptic splice site on the splicing profile of murine minigenes.Wild-type (wt) and mutant (mut) murine minigenes transfected in Hep3B cells were cotransfected with 20 μM of AON and RT-PCR analysis performed after 24 h. SCR, scrambled oligonucleotide. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis. Grey boxes represent vector sequences (V) and white boxes indicate exon 4. Numbers indicate the position of the cryptic splice sites used. The box denoted as cV indicates a stretch of vector DNA that is retained in the mRNA due to the use of a vector cryptic splice site. The star indicates the presence of the c.386G>A mutation. The arrow points to the band corresponding to the use of the cryptic c.386+48 site in the mutant minigenes.

Mentions: As can be seen in Fig 4, in the presence of AON the band corresponding to the insertion of 48 intronic nucleotides disappeared indicating that the AON efficiently and specifically blocks the use of the cryptic splice site. No effect was visible using a scrambled oligo. Concomitantly, an increase in the band corresponding to intron 4 retention was detected. We did not observe an increase in the band which corresponds by size to the use of the natural splice site or the cryptic c.386+4 site. To corroborate this, we used specific primers NAT and CRYP (Fig 3). As can be seen in Fig 3B, AON treatment did not result in any detectable increase of these specific amplified products.


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)

Effect of AON targeting the c.386G>A cryptic splice site on the splicing profile of murine minigenes.Wild-type (wt) and mutant (mut) murine minigenes transfected in Hep3B cells were cotransfected with 20 μM of AON and RT-PCR analysis performed after 24 h. SCR, scrambled oligonucleotide. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis. Grey boxes represent vector sequences (V) and white boxes indicate exon 4. Numbers indicate the position of the cryptic splice sites used. The box denoted as cV indicates a stretch of vector DNA that is retained in the mRNA due to the use of a vector cryptic splice site. The star indicates the presence of the c.386G>A mutation. The arrow points to the band corresponding to the use of the cryptic c.386+48 site in the mutant minigenes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122966.g004: Effect of AON targeting the c.386G>A cryptic splice site on the splicing profile of murine minigenes.Wild-type (wt) and mutant (mut) murine minigenes transfected in Hep3B cells were cotransfected with 20 μM of AON and RT-PCR analysis performed after 24 h. SCR, scrambled oligonucleotide. The schematic drawings on both sides show the identity of the bands which were characterized by sequence analysis. Grey boxes represent vector sequences (V) and white boxes indicate exon 4. Numbers indicate the position of the cryptic splice sites used. The box denoted as cV indicates a stretch of vector DNA that is retained in the mRNA due to the use of a vector cryptic splice site. The star indicates the presence of the c.386G>A mutation. The arrow points to the band corresponding to the use of the cryptic c.386+48 site in the mutant minigenes.
Mentions: As can be seen in Fig 4, in the presence of AON the band corresponding to the insertion of 48 intronic nucleotides disappeared indicating that the AON efficiently and specifically blocks the use of the cryptic splice site. No effect was visible using a scrambled oligo. Concomitantly, an increase in the band corresponding to intron 4 retention was detected. We did not observe an increase in the band which corresponds by size to the use of the natural splice site or the cryptic c.386+4 site. To corroborate this, we used specific primers NAT and CRYP (Fig 3). As can be seen in Fig 3B, AON treatment did not result in any detectable increase of these specific amplified products.

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