|Freking, Bradley - Brad|
|Smith, Timothy - Tim|
Submitted to: Genome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2004
Publication Date: 4/2/2005
Citation: Murphy, S.K., Freking, B.A., Smith, T.P., Nolan, C.M., Wylie, A.A., Evans, H.K., Jirtle, R.L. 2005. Abnormal postnatal maintenance of elevated prenatal DLK1 levels in callipyge sheep. Mammalian Genome. 16:171-183. Interpretive Summary: The impact of a mutation in sheep, named callipyge, with large effects on lean and fat development as well as eating quality of meat was evaluated for altered gene expression levels. Expression of muscle hypertrophy is inherited in a unique parent-of-origin manner referred to as polar overdominance. Specifically, animals exhibiting characteristic muscle hypertrophy must inherit the previously identified mutated allele from the sire, and not from the dam, making callipyge a unique phenomenon. We have provided evidence that the callipyge phenotype correlates with altered temporal expression of the nearby DLK1 gene in muscle. Specifically, the timing of appearance of the phenotype coincides with the time that DLK1 expression is abnormally elevated in the affected sheep yet is down regulated in all of the nonaffected genotypes, including the homozygous mutation carriers that are phenotypically normal. Furthermore, DLK1 elevation is not seen in muscle that does not hypertrophy or in liver; therefore this does not represent a global deregulation of this gene. This, together with the role of DLK1 in cellular differentiation, strongly suggests that DLK1 is an effector molecule of the muscle hypertrophy phenotype and that the nearby callipyge mutation is somehow negating the normal ability to down regulate expression of DLK1 postnatally. Further investigation will be required to determine how the callipyge mutation mechanistically influences the expression of the imprinted genes in the callipyge region. We identified the specific causative mutation by sequencing key inbred animals identical-by-descent for a 210-Kb region known to contain the gene. A single base change was revealed as the only variable position in the sequenced region within these two rams that were progeny tested as heterozygous carriers. Additional genotypic information concluded that this variable base completely described the inheritance of the muscle hypertrophy phenotype and was specific to descendants from the animal where the mutation was first observed. Identification of this mutation has led to the subsequent new discovery of a previously unknown expressed gene product. The discovery of this mutation and a new gene product encompassing the mutation will focus new investigations on both genetic and regulatory aspects of this important genomic region.
Technical Abstract: The underlying mechanism of the muscular hypertrophy phenotype in sheep (Ovis aries) known as callipyge is not presently understood. This phenotype, characterized by increased glycolytic type II muscle proportion and cell size accompanied by decreased adiposity, is not visibly detected until approximately three to eight weeks after birth. The muscular hypertrophy results from a single nucleotide change located at the telomeric end of ovine chromosome 18, in the region between the two imprinted genes Maternal Expressed Gene 3 (MEG3) and Delta , Drosophila , Homolog-like 1 (DLK1). Muscle hypertrophy is evident only when this mutation is paternally inherited and present in a heterozygous individual. We have examined the pre- and postnatal expression of MEG3 and DLK1 in sheep of all four possible allele combinations for the mutation, in affected and nonaffected muscles as well as in liver. We determined that the callipyge phenotype correlates with abnormally high DLK1 expression during the postnatal period in the affected sheep, and that this elevation is specific to the affected fast twitch muscles. These results are the first to show anomalous gene expression that coincides with both the temporal and spatial distribution of the callipyge phenotype, and suggests that the effect of the callipyge mutation is to interfere with the normal postnatal down regulation of DLK1 expression.