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The callipyge mutation causes postnatal muscle hypertrophy in heterozygous lambs that inherit a paternal callipyge allele (+/CLPG). Our hypothesis was that the up-regulation of one or both of the affected paternally expressed genes (DLK1 or PEG11) initiates changes in biochemical and physiological pathways in skeletal muscle to induce hypertrophy. The goal of this study was to identify changes in gene expression during the onset of muscle hypertrophy to identify the pathways that are involved in the expression of the callipyge phenotype. Gene expression was analysed in longissimus dorsi total RNA from lambs at 10, 20, and 30 days of age using the Affymetrix Bovine Expression Array. An average of 40.6% of probe sets on the array was detected in sheep muscle. Data were normalized and analysed using a two-way anova for genotype and age effects with a false discovery rate of 0.10. From the anova, 13 genes were significant for the effect of genotype and 13 were significant for effect of age (P < 0.10). No significant age-by-genotype interactions were detected (P > 0.10). Of the 13 genes indicating an effect of genotype, quantitative PCR assays were developed for all of them and tested on a larger group of animals from 10 to 200 days of age. Nine genes had significantly elevated transcript levels in callipyge lambs. These genes included phosphofructokinase, a putative methyltransferase protein, a cAMP phosphodiesterase, and the transcription factor DNTTIP1.

Background: The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele (paternal heterozygotes) have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression., Results: There was a significant effect of genotype (p < 0.0001) on the transcript abundance of DLK1, PEG11, and MEG8 in the muscles of lambs with the callipyge allele. DLK1 and PEG11 transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The PEG11 locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as PEG11AS, in skeletal muscle. PEG11AS transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the PEG11 start codon and spanning the entire open reading frame. Analysis of PEG11 expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals. PEG11 transcripts were 14-fold more abundant than PEG11AS transcripts in the gluteus medius of paternal heterozygous animals. PEG11AS transcripts were expressed at higher levels than PEG11 transcripts in the gluteus medius of animals of the other three genotypes., Conclusions: The effect of the callipyge mutation has been to alter the expression of DLK1, GTL2, PEG11 and MEG8 in the hypertrophied skeletal muscles. Transcript abundance of DLK1 and PEG11 was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of PEG11 and PEG11AS transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in PEG11 gene regulation and polar overdominance in callipyge sheep.

The inheritance pattern of the skeletal muscle hypertrophy phenotype caused by the callipyge gene has been characterized as polar overdominance. We hypothesized that this trait may be caused by a gain or loss of gene expression because of the reversible nature of the phenotype in paternal vs. maternal inheritance. Suppression subtraction cDNA probes were made from skeletal muscle mRNA of normal (NN) and callipyge (C(Pat)N(Mat)) animals and hybridized to Southern blots containing bacterial artificial chromosomes (BACs) that comprise a physical contig of the callipyge region. The CN-NN probes hybridized to two ovine and seven bovine BACs. Sequence analysis of fragments within those BACs indicated short regions of similarity to mouse gene trap locus (gtl2). Northern blots analysis of RNA from hypertrophy-responsive muscles show a population of GTL2 mRNA centred around 2.4 kb that were abundantly expressed in 14-day prenatal NN and C(Pat)N(Mat) lambs but were down-regulated in day 14 and day 56 postnatal NN lambs. The expression of GTL2 remained elevated in 14- and 56-day-old C(Pat)N(Mat) lambs as well as in 56-day-old N(Pat)C(Mat) and CC lambs. Expression of GTL2 in the supraspinatus, which does not undergo hypertrophy, was very low for all genotypes and ages. Isolation of cDNA sequences show extensive alternative splicing and a lack of codon bias suggesting that GTL2 does not encode a protein. The mutation of the callipyge allele has altered postnatal expression of GTL2 in muscles that undergo hypertrophy and will help identify mechanisms involved in growth, genomic imprinting and polar overdominance.