EQUINE VETERINARY EDUCATION / AE / MAY 2018


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It is imperative to remember that SNPs are just part of the


road map of the genome. They are not in and of themselves necessarily mutations that have any effect on the organism, but they can. In molecular biology terms they are used to discover the location of a genomic region harbouring a disease related mutation. By way of analogy, a set of directions are like a SNP map. If I gave you directions to a clinic, they might be: “Head south on Holberry Lane, turn right at the Royal Horsehoes pub and it’s a mile further down on


the left hand side where the fir trees stand”. The pub and the fir trees have nothing to do with the clinic whatsoever but they are landmarks that enable you to be guided to the location: the clinic. SNPs that sit close together on a chromosome are likely to be inherited en bloc – a phenomenon termed genetic linkage. Since SNPs provide a roadmap of the genome, groups of statistically significant SNPs that are found at high frequency in an affected animal point to an area of the genome that warrants further investigation as an area where a relevant mutation may be located. This strategy has been especially useful when whole families are not known, too small in number, or breeding studies cannot be performed. The collection of SNPs that sit on a single chromatid of a chromosome pair that is associated is known as their haplotype. A chromatid is one copy of a duplicated chromosome. Selections of SNPs representing the whole genome have


been harnessed to design ‘SNP chips’ (SNP arrays, SNP beadchips). To date, the sequencing of equine genomes has identified over 10 million SNPs distributed throughout the 2700 million base pairs (Schaefer et al. 2014). Genetic studies in horses involves comparing groups of horses for these genetic variants. The first tool used to investigate SNPs was the Illumina 50K SNP chip. Approximately 55,000 SNPs were selected based on distribution among all chromosomes and the extent of variation that occurred among horses. Later a 70K SNP chip was developed, which included the SNPs from the original 50K SNP Chip plus additional markers to fill in gaps. More recently, a new tool was created by Affymetrix to assay 670,000 SNPs for horses. Depending on the study design and the cost of testing, scientists determine whether to use the 70K SNP chip or the 670K SNP chip. Once a disease phenotype is identified, DNA is harvested


from affected and unaffected animals. Related animals can be helpful and, where possible, avoiding breed variation can reduce ‘background noise’ by reducing breed related SNPs, which may produce false positive candidates. SNPs that colocalise in affected animals are not necessarily related to the disease process itself – they are simply landmarks of the particular genome region or regions that are different in animals with the disease phenotype. In these regions there may be genes or regulatory elements that are important in the disease process. SNPs provide a way to localise and refine the search for the genetic basis of the disease. Because the sample is interrogated across the whole genome, it is data-driven, thus limiting researcher bias. Studies using this approach are called genome wide association studies (GWAS; pronounced ‘gee-waahs’).


GWAS investigations in equine veterinary science


The SNP-GWAS approach has been used to localise regions of interest in the genome in such diseases as lavender foal syndrome (Brooks et al. 2010; Gabreski et al. 2012), recurrent


laryngeal neuropathy (Dupuis


et al.


2011), foal


immunodeficiency syndrome in the Fell and Dales pony (Fox- Clipsham et al. 2011a,b), osteochondrosis dissecans in Thoroughbreds (Corbin et al. 2012), guttural pouch tympany in Arabians and German Warmbloods (Metzger et al. 2012), recurrent uveitis in German Warmbloods (Kulbrock et al. 2013), and insect bite hypersensitivity (Schurink et al. 2012) and hydrocephalus in Friesians (Ducro et al. 2015) to name a few.


McCue et al. (2008a,b) employed the GWAS method to


study horses with polysaccharide storage myopathy (PSSM). Although their work was performed at a time when there were fewer SNP data available, they did have very well phenotyped horses with DNA samples taken from horses diagnosed with PSSM by muscle biopsy analysis, as well as detailed histories. They used a combination of microsatellite markers and the available SNPs to refine their search for the region of the genome that colocalised with affected horses as compared to control horses. Microsatellites are repeats of 2–5 nucleotides in the genome. They can be very long and vary between individuals to such an extent they are routinely used in human forensic examination and parentage suits. The variation in microsatellites originates when the DNA is replicated. The polymerase enzyme replicating the DNA is more likely to ‘slip’ in these repetitive regions. As it loses its place it starts over and can either repeat the section or skip forward by starting further downstream on the unwound DNA helix. As such, the number of repeats an individual can have is variable (even between monozygotic twins) but familial. Just like SNPs, these areas can act as landmarks for genome mapping. As a result of this work, PSSM horses were shown to have a missense, gain-of-function point mutation in the glycogen synthase gene, GYS1, (gene symbols tend to be written in italicised capital letters). A missense mutation is one where a different amino acid is incorporated into the protein coded for by that gene. Subsequent work has led to the subtyping of PSSM into type 1 and type 2. PSSM type 1 is associated with this mutation in GSY1 gene. Other DNA variations that can give rise to genetic


diseases include insertions and deletions. Collectively these can be termed ‘indels’. As discussed earlier, a triplet code is used to translate the mRNA into a polypeptide chain. An indel of one or two nucleotides gives rise to a shift in the reading frame (Fig 3). Such mutations are therefore called frame shift mutations. Lavender foal syndrome, a lethal inherited condition in Arabians, is an example of a deletion mutation leading to a frame shift mutation that causes a premature stop codon (Brooks et al. 2010). Using a combination of GWAS, pedigree analysis and gene sequencing, a group of highly significant SNPs were identified in affected animals. These SNPs were located in a 10.5 million


Normal reading frame – read in triplet code


Deletion or insertion of one (or 2) nucleotides shifts the reading resulting in a totally different code and amino acid sequence


Fig 3: An illustration of a nucleotide deletion and insertion mutations that cause reading frame shifts.


© 2016 EVJ Ltd


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