The whippet breed was developed in the late 1800s specifically for the sport of racing [3
]. Despite its comparatively small stature it is a very fast dog capable of running up to 35 miles per hour [3
]. We have discovered a 2-bp deletion in the whippet MSTN
gene that in the homozygote state results in a double-muscling phenotype commonly referred to as the “bully” whippet. This deletion causes a premature truncation of the protein at amino acid 313, removing the latter 17% of the protein.
has been mapped to canine Chromosome 37 (CFA37) and consists of three exons spanning 5083 bp (http://genome.ucsc.edu
). It is highly conserved across species [9
] and in the human genome is located on Chromosome two. The gene is a member of the transforming growth factor β family and encodes the myostatin protein. Studies of Mstn
knockout mice demonstrate that the gene is a negative regulator of skeletal muscle mass [9
]. This is the result of a cascading pathway triggered by MSTN
signaling that prevents myoblast cell progression from the G1
to S phase of the cell cycle. MSTN
therefore controls the total number of muscle fibers by regulating overall myoblast proliferation [14
]. In the absence of functional protein, greater numbers of muscle fibers are made [14
Double muscling has been described in several breeds of cattle [5
] and muscular hypertrophy (an increase in muscle-fiber size) has been described in sheep [7
]. Muscular hypertrophy has also been described in the domestic cat [18
]; however, a deficiency in dystrophin is the cause in this species rather than a mutation in MSTN
. To date, six different mutations in the bovine MSTN
gene have been reported to cause double muscling [5
]. While all these mutations result in a loss of MSTN
function, a subset of them and the one we describe here in the whippet likely change the three-dimensional shape of the protein by disrupting the “cysteine knot,” a structure important in the folding of all transforming growth factor β family member proteins [5
]. The mutation in the whippet also removes nearly 20% of the protein.
We sequenced genomic DNA not only from whippets but also from multiple dogs from each of 14 additional breeds in order to determine the haplotype background on which this mutation arose (). In each dog, 15 PCR amplicons that spanned the MSTN gene and amplicons spanning known dog SNPs within 50 kb of the MSTN mutation were sequenced. Using the resulting data we observed two haplotypes, termed two and seven, that occurred in a large number of breeds and that were identical except at position 3,676,629, which is located outside the gene, 15,801 bp downstream from the 2-bp deletion. The mh mutation occurs only on haplotype six, which is identical to haplotype seven except for the deletion itself. Not surprisingly, the golden jackal sequence has only the wild-type allele at the position of the mutation, indicating the mh allele represents the derived state. We conclude therefore that haplotype six likely derives from haplotype seven (). Haplotype seven is the most common and widely dispersed haplotype spanning the gene and was found in 12 out of 15 breeds sequenced. Interestingly, we did not observe haplotype seven in the Afghan hound, Basenji, or boxer.
Our data do not exclude the possibility that the mutation occurs in breeds other than the whippet. However, we screened for the 2-bp deletion in several mastiff type breeds (rottweiler, bulldog, Presa Canario, miniature bull terrier, American Staffordshire terrier, Staffordshire bull terrier, and bullmastiff) and did not find it. These data argue that the changes in musculature exhibited by the whippet are unique and caused by the effects on MSTN associated with the deletion described in this study.
An excess of the mh/+ genotype was observed among the fastest racers, as defined by the highest racing grade achieved during a dog's career. This demonstrates that the heterozygote state carries a performance-enhancing polymorphism that provides a competitive edge. The optimal study of racing performance would use the racing points acquired by each whippet during their career as a quantitative measure of performance. However a dog's total career points are a function of the number of races run throughout their career and, as such, whippets of different ages are not easily compared. To compensate, the total number of points accrued over a lifetime of racing could be averaged over the number of races entered. However, as dogs age their performance declines. Some owners stop racing their dogs after their performance declines while others continue to race their dogs for months or even years longer. Using the average number of points accrued during a specific year of the dog's life, for instance age two or three, presents similar problems. Dogs reach their racing prime at different ages and the number of points will always reflect the number of races entered. While an average is satisfactory if many races are run in a given year, the average will be inaccurate if few races are run.
While cattle breeders have long selected for individuals that are homozygous for mutations in MSTN because of their increased musculature, which is optimal for beef production, this is the first example of breeders unknowingly selecting for individuals with a single polymorphism that increases athletic performance. Of interest, the trait appears to confer an undesirable appearance upon dogs competing in conformation. Only two mh/+ dogs were found among the dogs reported to compete in conformation events, and those dogs were reported to show poorly. This is consistent with the association seen between a dog's genotype and their relative muscle mass as defined by either a ratio of mass (kg) to height at the withers (cm) (p = 7.43 × 10−6; Kruskal-Wallis test) (A) or the direct measure of an individual's neck girth (p = 3.47 × 10−5; Kruskal-Wallis test) (B) or chest girth (p = 0.001462; Kruskal-Wallis test) (C). We acknowledge that there are more accurate methods to measure muscle mass. However, many of these methods are either invasive, such as a muscle biopsy, or would need to be conducted post-mortem, neither of which was an option. These measurements were not designed to specifically eliminate contributions from body fat. However, obesity is rare in the whippet; indeed, the breed is characterized by an overall low body fat content. Thus, these measurements are the best achievable metrics of the phenotype.
Greyhounds and whippets share a common ancestral gene pool and as a result the breeds are difficult to separate in genetic clustering analyses [11
]. This, together with the fact that both were bred to excel at racing, suggested that the mutation might also be found in racing greyhounds. However, none of the greyhounds tested carried the mutation. There are three possible explanations for this result. First, an insufficient number of samples have been tested if the mutant allele is relatively rare in the greyhound population. Second, the mutation may only be present in a subset of greyhound lines, none of which were among those tested. Finally, the mutation may not be carried in the greyhound population at all, indicating that it is a relatively new mutation in the purebred dog population. This may be because the mutation offers no advantage to greyhound racers. Indeed, it may even be disadvantageous. Studies of muscle composition in Mstn
knockout mice demonstrate a higher proportion of both fast type II and glycolytic fibers, versus slow type I and oxidative fibers when compared to wild-type mice [19
]. While this change in muscle composition may offer an advantage to whippets, which typically race a short sprint of 200–300 m, it may be disadvantageous to greyhounds, whose races extend to 900 m and where endurance is more important. In addition, Belgian Blue cattle that are homozygous for a MSTN
mutation display a decrease in the size of several organs, including the lungs [20
]. If heterozygous dogs have even a slightly reduced lung capacity, it is possible that a MSTN
mutation would actually be disadvantageous for racing longer distances as greyhounds do. Finally, it remains to be determined whether additional health problems are associated with being a carrier of this mutation.
We examined the microsatellite data set for evidence of population substructure and found that there is not random gene flow across the racing classes. All groups display positive FST values with the greatest found between the grade A racers and all others. This is not unexpected. The very presence of the “bully” phenotype is evidence that breeders choose to mate dogs with increased musculature to one another. Reducing the mating population of a breed to a small proportion of the whole population has consequences, particularly for genetic mapping of complex traits. This is evidenced by our analysis of the same marker set for association with racing grade. While we find low p-values at many of the alleles, only one of 73 had a p-value smaller than the 2-bp deletion, confirming that the association between racing grade and the MSTN mutation is not simply a spurious result of population structure. Overall, these results suggest that the population structure within breeds is likely to have an important confounding effect on association mapping in the domestic dog.
Our findings have implications for competitive and professional sports. Here, we show that a disruption in the function of the MSTN
gene increases an individual's overall athletic performance in a robust and measurable way. To date, the muscular hypertrophy phenotype has been described in a single human child [8
]. This child possessed two copies of a G-to-A transition in the noncoding region of the human MSTN
gene. This mutation results in the mis-splicing of precursor mRNA, which most likely truncates the myostatin protein. The child's mother, a former professional athlete, was heterozygous for this mutation and also appeared muscular, although not to the same degree as her child. Perhaps additional mutations in MSTN
have yet to be discovered in other species that competitively race, such as the horse or humans. As discussed by others [21
], human athletes could undergo so-called gene doping via disruption of MSTN
. The potential to increase an athlete's performance by disrupting MSTN
either by natural or perhaps artificial means could change the face of competitive human and canine athletics. Given the poorly understood consequences for overall health and well-being, caution should be exercised when acting upon these results.