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Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

Plog S, Klymiuk N, Binder S, Van Hook MJ, Thoreson WB, Gruber AD, Mundhenk L - PLoS ONE (2015)

Bottom Line: Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals.Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins.Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

ABSTRACT
The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

No MeSH data available.


Related in: MedlinePlus

A deletion mutation in the CLCA4b gene leads to transcripts with a premature termination codon.A 10 base pair (bp) deletion was found at the splice acceptor site of exon (ex) 8. The mutated gene (mut) coded for different alternatively spliced mRNA species (cd smut 1 to 3) with an insertion (grey box), loss of exon 8 (grey cross) or the insertion (grey box) and loss of exon 8 (grey cross). All alternatively spliced transcripts had a premature termination codon (PTC) with shortened predicted ORFs.
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pone.0140050.g002: A deletion mutation in the CLCA4b gene leads to transcripts with a premature termination codon.A 10 base pair (bp) deletion was found at the splice acceptor site of exon (ex) 8. The mutated gene (mut) coded for different alternatively spliced mRNA species (cd smut 1 to 3) with an insertion (grey box), loss of exon 8 (grey cross) or the insertion (grey box) and loss of exon 8 (grey cross). All alternatively spliced transcripts had a premature termination codon (PTC) with shortened predicted ORFs.

Mentions: In contrast to these findings, however, there was occasional evidence of a 10 bp deletion in CLCA4b, involving the 5´-end of exon 8 as well as the upstream intronic region (Fig 2), resulting in the loss of the splice site acceptor site of intron 7. As a consequence, variations of alternative splicing of the CLCA4b transcripts were observed in pigs bearing this naturally occurring mutation (Fig 2). While transcripts from wild-type CLCA4b showed the predicted nucleotide sequence with a coding sequence (cds) of 2,766 bp, alternative splicing to an AG-site 38 bp upstream of exon 8 resulted in an insertion of 28 bp into the transcript that causes a frame shift and generates a premature termination codon (PTC) within exon 8 (Fig 2, cds mut 1). Alternatively, splicing out exon 8 (178bp) resulted in a frame shift and a PTC within exon 9 (Fig 2, cds mut 2). Occasionally, outsplicing of exon 8 concurrently occurred with an alternative splicing to exon 4, introducing a sequence of 105 bp to the cds (Fig 2, cds mut 3), and again causing a deleterious modification of the transcript. However, such transcript variant was only found in a single individual and we did not further examine whether this finding was specific for the mutated variant or a common effect of CLCA4b.


Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

Plog S, Klymiuk N, Binder S, Van Hook MJ, Thoreson WB, Gruber AD, Mundhenk L - PLoS ONE (2015)

A deletion mutation in the CLCA4b gene leads to transcripts with a premature termination codon.A 10 base pair (bp) deletion was found at the splice acceptor site of exon (ex) 8. The mutated gene (mut) coded for different alternatively spliced mRNA species (cd smut 1 to 3) with an insertion (grey box), loss of exon 8 (grey cross) or the insertion (grey box) and loss of exon 8 (grey cross). All alternatively spliced transcripts had a premature termination codon (PTC) with shortened predicted ORFs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608703&req=5

pone.0140050.g002: A deletion mutation in the CLCA4b gene leads to transcripts with a premature termination codon.A 10 base pair (bp) deletion was found at the splice acceptor site of exon (ex) 8. The mutated gene (mut) coded for different alternatively spliced mRNA species (cd smut 1 to 3) with an insertion (grey box), loss of exon 8 (grey cross) or the insertion (grey box) and loss of exon 8 (grey cross). All alternatively spliced transcripts had a premature termination codon (PTC) with shortened predicted ORFs.
Mentions: In contrast to these findings, however, there was occasional evidence of a 10 bp deletion in CLCA4b, involving the 5´-end of exon 8 as well as the upstream intronic region (Fig 2), resulting in the loss of the splice site acceptor site of intron 7. As a consequence, variations of alternative splicing of the CLCA4b transcripts were observed in pigs bearing this naturally occurring mutation (Fig 2). While transcripts from wild-type CLCA4b showed the predicted nucleotide sequence with a coding sequence (cds) of 2,766 bp, alternative splicing to an AG-site 38 bp upstream of exon 8 resulted in an insertion of 28 bp into the transcript that causes a frame shift and generates a premature termination codon (PTC) within exon 8 (Fig 2, cds mut 1). Alternatively, splicing out exon 8 (178bp) resulted in a frame shift and a PTC within exon 9 (Fig 2, cds mut 2). Occasionally, outsplicing of exon 8 concurrently occurred with an alternative splicing to exon 4, introducing a sequence of 105 bp to the cds (Fig 2, cds mut 3), and again causing a deleterious modification of the transcript. However, such transcript variant was only found in a single individual and we did not further examine whether this finding was specific for the mutated variant or a common effect of CLCA4b.

Bottom Line: Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals.Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins.Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

ABSTRACT
The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

No MeSH data available.


Related in: MedlinePlus