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An insertion in the coding region of the myostatin (MSTN) gene affects carcass conformation and fatness in the Norwegian Spaelsau (Ovis aries).

ABSTRACT: BACKGROUND: A phenotype of increased muscle mass (IMM) and reduced fat, comparable to reported effects of deleterious mutations in the myostatin gene (MSTN), has been observed in the Norwegian Spaelsau breed. However, the genotyping of five AI rams producing descendants with this phenotype, failed to reveal any of the known functional MSTN mutations. FINDINGS: In the present study, the coding region of the MSTN gene was sequenced in a Spaelsau ram lamb with this particular phenotype. A one base-pair insertion mutation (c.120insA) producing a premature stop codon in amino acid position 49 was identified. The consequence of this mutation is that the bioactive carboxy-terminal end of the protein is not translated, and a completely non-functional myostatin protein is produced. Among the 98 available AI rams of this breed, all five individuals having descendants with this particular phenotype were found to be heterozygous for the c.120insA mutation. The probability that these five selected AI rams should be heterozygous carriers of the c.120insA mutation purely by chance was calculated to be 3.1 x 10-7. In total, 7 AI rams were found to be heterozygous carriers of c.120insA. The estimated breeding values (EBVs) for EUROP carcass conformation and fat class for these 7 individuals also points towards a strong phenotypic effect of this mutation. CONCLUSION: Based upon the completely deleterious effect this novel c.120insA mutation has on myostatin protein function, and the documented phenotypic effect of comparable MSTN mutations in Norwegian White Sheep and other species, we conclude that this mutation is the functional explanation underlying the IMM phenotype in Norwegian Spaelsau. The allele distribution among the 98 genotyped AI rams support this conclusion, and also suggests that c.120insA is the most common reason for this phenotype in the Norwegian Spaelsau breed.

SUBMITTER: Boman IA

PROVIDER: S-EPMC2709116 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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An insertion in the coding region of the myostatin (MSTN) gene affects carcass conformation and fatness in the Norwegian Spaelsau (Ovis aries).

Boman Inger A IA Våge Dag I DI

BMC research notes 20090608

<h4>Background</h4>A phenotype of increased muscle mass (IMM) and reduced fat, comparable to reported effects of deleterious mutations in the myostatin gene (MSTN), has been observed in the Norwegian Spaelsau breed. However, the genotyping of five AI rams producing descendants with this phenotype, failed to reveal any of the known functional MSTN mutations.<h4>Findings</h4>In the present study, the coding region of the MSTN gene was sequenced in a Spaelsau ram lamb with this particular phenotype ...[more]

PMID: 19505313

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Project description:BackgroundMyostatin, encoded by the MSTN gene comprising 3 exons, is a potent negative regulator of skeletal muscle growth. Although a variety of myostatin inhibitors have been invented for increasing muscle mass in muscle wasting diseases, no effective inhibitor is currently available for clinical use. Myostatin isoforms in several animals have been reported to inhibit myostatin, but an isoform has never been identified for the human MSTN gene, a conserved gene among animals. Here, a splice variant of the human MSTN gene was explored.MethodsTranscripts and proteins were analysed by reverse transcription-PCR amplification and western blotting, respectively. Proteins were expressed from expression plasmid. Myostatin signalling was assayed by the SMAD-responsive luciferase activity. Cell proliferation was assayed by the Cell Counting Kit-8 (CCK-8) assay and cell counting. Cell cycle was analysed by the FastFUCCI system.ResultsReverse transcription-PCR amplification of the full-length MSTN transcript in CRL-2061 rhabdomyosarcoma cells revealed two bands consisting of a thick expected-size product and a thin additional small-size product. Sequencing of the small-size product showed a 963-bp deletion in the 5' end of exon 3, creating exon 3s, which contained unusual splice acceptor TG dinucleotides. The novel variant was identified in other human cell lines, although it was not identified in skeletal muscle. The 251-amino acid isoform encoded by the novel variant (myostatin-b) was identified in CRL-2061 rhabdomyosarcoma cells. Transfection of a myostatin-b expression plasmid into CRL-2061 and myoblast cells inhibited endogenous myostatin signalling (44%, P < 0.001 and 63%, P < 0.001, respectively). Furthermore, myostatin-b inhibited myostatin signalling induced by recombinant myostatin (68.8%, P < 0.001). In remarkable contrast, myostatin-b did not inhibit the myostatin signalling induced by recombinant growth differentiation factor 11 (9.2%, P = 0.70), transforming growth factor β (+3.1%, P = 0.83) or activin A (+1.1%, P = 0.96). These results indicate the myostatin-specific inhibitory effect of myostatin-b. Notably, the expression of myostatin-b in myoblasts significantly enhanced cell proliferation higher than the mock-transfected cells by the CCK-8 and direct cell counting assays (60%, P < 0.05 and 39%, P < 0.05, respectively). Myostatin-b increased the percentage of S-phase cells significantly higher than that of the mock-transfected cells (53% vs. 80%, P < 0.05).ConclusionsWe cloned a novel human MSTN variant produced by unorthodox splicing. The variant encoded a novel myostatin isoform, myostatin-b, that inhibited myostatin signalling by myostatin-specific manner and enhanced myoblast proliferation by shifting cell cycle. Myostatin-b, which has myostatin-specific inhibitory activity, could be developed as a natural myostatin inhibitor.

Dataset Information

An insertion in the coding region of the myostatin (MSTN) gene affects carcass conformation and fatness in the Norwegian Spaelsau (Ovis aries).

Publications

An insertion in the coding region of the myostatin (MSTN) gene affects carcass conformation and fatness in the Norwegian Spaelsau (Ovis aries).

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