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base long region on ECA1. After taking into account familial associations, the region was narrowed down to 1.6 million bases. Ten predicted genes mapped to this region, including MYO5. Mutations in MYO5 in man have been expressed as neurological dysfunction. By sequencing the area of interest a nucleotide deletion mutation was found. This deletion caused a shift in the triplet code reading frame leading to a premature stop codon, rendering the RNA message ineffective. A recent study into the autosomal recessive trait of


hydrocephalus in Friesian horses has identified the genetic basis for the disease (Ducro et al. 2015). This disease has been observed to cause stillbirth and dystocia. By performing a GWAS an area of interest was identified. Genome sequencing discovered a point mutation in the B3GALNT2 gene. The change of a C to a T in one exon results in the codon becoming a stop codon rather than coding for glycine. A mutation that generates a premature stop codon is called a missense mutation.


Molecular technologies


Polymerase chain reaction (PCR) is a versatile technique, for which Mullis and Smith won a Nobel Prize in 1993, and is very familiar to all equine clinicians as a diagnostic test and this example further demonstrates its flexibility (Bartlett and Stirling 2003). PCR relies on specific sequences that are unique to the gene of interest in the case of a genetic disease, or the organism of interest in the case of an infectious agent. The test’s specificity lies in the probability that, for example, an 18 nucleotide long sequence specific primer has an infinitesimally small chance of finding the same sequence unrelated to the region of interest by chance – indeed the chance is 1 in 7 9 1010. This is the key to the sensitivity of PCR as a technique, but it is worth noting that because DNA is such a persistent and robust molecule it can still be detected by PCR even when the organism is no longer viable.


Sequencing


So far much of our discussion has centred on DNA based investigations. However, it is worth mentioning other tools featured in the literature that relate to gene expression. DNA microarrays were popular screening tools to compare RNA isolated from two samples. These arrays computationally generate a heat map indicating equal, increased or decreased comparative levels of expression in the genes profiled on the microarray. This technology has rapidly lost ground in the wake more affordable and accessible sequencing platforms. Frederick Sanger won not one, but two Nobel prizes. The


first was for amino acid sequencing the protein insulin, and the second was for developing a method to sequence DNA (Walker 2014). Indeed, the methods that Sanger invented for DNA sequencing, popularly called ‘Sanger sequencing’, made the Human Genome Project possible. The Human Genome Project was completed using Sanger sequencing; however, in recent years new technologies have been developed to sequence DNA more rapidly and at a lower cost. These technologies are referred to as next generation sequencing (Finno and Bannasch 2014). In order to sequence RNA, it must be reliably converted into a more stable molecule so that it does not change or


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decay during transportation and sequencing. To do this a complimentary DNA (cDNA) molecule is made. This technique was made possible by another Nobel winning discovery in 1970: the discovery of a retrovirus enzyme referred to as reverse transcriptase. This amazing enzyme makes a complimentary strand of DNA from an RNA template. Importantly, it does so in a one-to-one ratio thus preserving the relative amounts of the starting RNA molecules – a critical feature for gene expression studies. In this way, RNA sequencing (RNA-seq) can be performed. The ability to sequence all of the RNA transcripts (the


‘transcriptome’) of a cell or tissue type has exponentially expanded the ability to delve into exactly how the genome is used by different cell types, under different conditions, and in different diseases. All of the transcript variants can be examined and the blinkers are removed from the researcher as the resulting data drive the investigation rather than the investigator focusing on a handful of known genes of interest. Huge datasets are generated, which can be challenging for researchers trained in biological disciplines. These datasets, especially in the horse where the genomics tools are not as advanced as they are in man or mice, require an integrated approach. Collaboration with bioinformatics groups to help computationally organise and annotate these huge data sets is vital. RNA-seq is such a powerful tool that it is revolutionising genomic molecular biology and starting to dominate gene expression studies. Recently, RNA-seq was employed in combination with other tools such as DNA sequencing to elucidate the genetic basis of Congenital Stationary Night Blindness and Leopard Complex Spotting found in Appaloosas (Bellone et al. 2013). A large sequence of repetitive DNA (long terminal repeat) was found to be interfering with proper mRNA transcription of the gene TRPM1, which is involved with night vision capability and melanocytes, although neither mechanism is fully understood.


Epigenetics


The more precise and discovery-driven ‘-omics’ approach, such as transcriptomics, offers more insight into the perplexing question of how the genome is selectively used in different cell types, at different developmental stages, and during disease processes. No discussion of genomics would be complete without mention of epigenetics in this vein. Epigenetics is the study of changes to the genome that do not involve a change in the sequence of the DNA itself. However, they can be heritable changes. The genome is only partially used at any given time. Sequences, such as protein coding sequences, that are active and those that are inactive are in that state as a result of how tightly the DNA is wrapped around, and attracted to the histone proteins (hair curlers!). It is also related to the reversible addition of small chemical groups to the DNA itself – such as adding methyl groups. It is fascinating to note that this is a significant part of the chemical basis of memory, and these modifications are heritable. Other epigenetic controls do not involve the DNA


template per se, but affect matters downstream of it. One significant area of control is at the level of mRNA transcript stability. Regulatory elements that are often in the noncoding region of the DNA are transcribed and put under the umbrella term of noncoding RNA. One important group of noncoding RNAs that are getting a lot of press currently is


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