Here we describe the first successful use of the EquineSNP50 genotyping platform in identification of the mutation responsible for a genetic disorder in the horse. We have described a frameshift mutation in the MYO5A gene that leads to Lavender Foal Syndrome in the Egyptian Arabian breed of horse. This task was made more challenging by the small number (six) of DNA samples from available affected foals. We improved our chances of success by using pedigree data to select control samples from the extended family and by utilizing a genotype association rather than allelic association statistic in combination with identification of regions of homozygosity.
The extreme predicted impact on function resulting from the single base deletion in MYO5A
exon 30 makes it a very logical cause of LFS. Indeed, an alignment of MYO5A
exon 30 amino acid sequences from 8 diverse species shows that the exon is completely conserved in horses, humans, mice, dogs and cattle and contains only a few changes in the possum, chicken, and zebrafish (Figure S3
). As LFS affected foals do not have an immunodeficiency consistent with RAB27A
mutations, and the genomic region containing this gene was not inherited as predicted by our recessive model, it is doubtful that this gene plays a role in Lavender Foal Syndrome.
The newly discovered deletion in exon 30 of MYO5A
leads to a frame shift and premature termination of transcription. Loss of the 379 amino acids at the C-terminus of the protein, which encode a portion of the secretory vesicle-specific binding domains of the globular tail, would likely impair binding of myosin Va to those cargo organelles bearing the appropriate receptors 
. Although this truncation leaves intact the melanocyte specific alternative exon, exon F, it has been previously shown that binding function is nonetheless destroyed without the cooperative action of downstream motifs 
. Additionally, the quantity of MYO5A
protein may be significantly reduced, as is often observed in experimentally truncated constructs of this gene 
. The resulting loss of vesicle traffic could easily interfere with the normal function of melanocytes and neurons. The neurologic deficits exhibited by LFS affected foals are relatively more severe than the symptoms reported in human cases of Griscelli Syndrome, which are most often due to changes in a single amino acid rather than loss of a significant portion of the transcript 
. However, in the mouse a broad spectrum of phenotypes are observed, owing to the variety of causative mutations available for study.
There is some speculation that a mild, survivable epileptic condition of young foals may represent a non-lethal phenotype of LFS carriers 
as the two conditions are often seen in the same pedigrees. However, this association has not been scientifically validated and samples from horses diagnosed with this condition were not available for study at this time. Based on comparative phenotypes in the mouse this is a plausible scenario. Several MYO5A
alleles in the mouse, most notably mutations of the globular tail region like d-n
, exhibit neurological and behavioral defects in juvenile homozygotes 
. These deficits improve with age and are often survivable, as has been described in the rumored condition of the horse. Discovery of the mutation responsible for LFS will enable future studies to evaluate association of this allele with juvenile neurological dysfunction.
Our results suggest the population frequency of carriers of this deletion is 10.3% in the Egyptian Arabian. It is possible that this may be an over estimation of carriers, as owners who suspect they have LFS carrying horses may have been more motivated to participate in the study. However it is equally as likely that this figure is an underestimation as there is social stigma associated with producing LFS foals, thus motivating breeders to hide the carrier status of their breeding stock. Despite strict policies regarding the confidential nature of identifying information in research projects, this still influences some breeders to avoid association with Lavender Foal Syndrome research out of fear of being rumored to own carrier horses. Notably, three of the six carriers identified were reported to be breeding stallions. Data from the Egyptian Arabian horse registry indicates that approximately 850 young horses are registered each year (personal communication, Beth Minnich, Pyramid Society). Given our estimate of the number of carriers in the population we expect that around nine LFS foals would be born in the US each year. This is a small number; however rumors of carrier status can very quickly negatively impact the breeding career of high-priced stallions and lead to large economic losses. This estimate also assumes mating at random. In the case of the Egyptian Arabian horse this is not a realistic assumption given the commonplace use of inbreeding and line-breeding in the industry. The allele frequency for LFS of 5.2% is not unlike the frequencies of other heritable diseases in various breeds of horse 
We identified a conserved block of 1.6 Mb in common in the four LFS bearing haplotypes. This is somewhat smaller than would be expected considering the average rate of decay of LD across just the six to eight generations that separate these four haplotypes. Indeed, upon further research of the pedigrees from carriers identified during screening for the LFS allele in our sample of 107 Arabian horses, we identified carriers who did not possess this candidate founder in their pedigree. Therefore it is likely the true founder of this mutation occurred far earlier. The appearance of a more recent common ancestor is not surprising given the prolific use of popular sires and the prevalence of inbreeding in this population.
Prevention of the economic and emotional losses associated with lethal conditions in foals, included those affected with LFS is a high priority among Arabian breeders. The market for Egyptian Arabian horses particularly values certain popular bloodlines. This leads to close breeding as owners seek to increase the percentage of this ancestry in their foal crop. This breeding strategy thus increases the need for vigilant prevention of recessive genetic disorders. The test developed here will be a pivotal tool for breeders seeking to breed within lines segregating for LFS, yet minimize or eliminate the production of affected foals.
Widespread application of the EquineSNP50 chip in genetic research is just beginning. In the case of Lavender Foal Syndrome, the limited availability of samples had impeded the progress of research using existing mapping tools for many years. Although whole genome association using large numbers of SNP markers is heralded as a tool for complex, polygenic traits, here we have shown that it can be very successfully applied to a simple trait in a small number of individuals. This work is the first use of the EquineSNP50 genotyping chip to successfully identify a causative mutation. While whole genome association is often the tool of choice for mapping complex traits and QTLs, we have demonstrated that it can also be a much anticipated solution for simple traits that face additional challenges in phenotyping and/or sample number. Testing for the LFS allele will be a valuable aid to breeders seeking to avoid losing foals while still using many of the popular lines that may carry Lavender Foal Syndrome. As the Arabian horse was used to develop many of the modern light horse breeds it is possible that the LFS allele is present in these breeds as well. In future work we will test additional sub-types of Arabian, as well as a variety of light horse breeds to better assess the population frequency in these groups. It is possible that LFS segregates in these groups at a low frequency without detection, as it is easy to confuse with other neonatal disorders of the foal.