being copied. When this happens, the change is called a mutation. Most mutations do not cause problems. Most DNA does not code for genes, so most changes are of little consequence (Chapter 6). Furthermore, the redundancy of the genetic code also prevents many mutations from having an effect on the protein. A mutation in the codon GAA changing it to GAG would still code for the amino acid, glutamic acid (see Table 4.1). These kinds of mutations are called silent mutations or neutral mutations or, more commonly synonymous mutations since they do not change the amino acid. When the DNA sequence change does substitute a different amino acid, this may or may not have an impact on the protein. These types of mutations are called non-synonymous mutations.
Another type of mutation is called a frameshift mutation, caused by an insertion or deletion of a base coding for an amino acid. As noted above, the genetic code specifies amino acids based on reading the DNA in frames of three bases. If a base is added or deleted, this causes a shift in the reading frame of the codon. As a result, the amino acids in the protein following the frameshift mutation are very likely to be changed and may include a stop codon, halting the translation process entirely. An example of a deletion causing a frameshift mutation is the variant responsible for severe combined immunodeficiency (SCID) in Arabian horses (see Chapter 16).
Effect of amino acid substitutions
The following table lists the chemical properties of the 20 amino acids that commonly appear in proteins. These are the amino acids that are specified by DNA/RNA using the genetic code as defined in Table 4.1. All amino acids have the same backbone, but they have side chains with different chemical properties as described in Table 4.2. These chemical properties are referred to as polar, non-polar, hydrophobic, acidic, basic, aromatic, or having other special properties. The chemical properties of the amino acid are key to performing the function of the protein.
Table 4.2. Chemical properties of amino acids based on their side chains.
| Chemical group | Amino acids |
| Hydrophobic (non-polar, uncharged) | Alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, valine |
| Polar (uncharged) | Serine, threonine, asparagine, glutamine |
| Aromatic | Tryptophan, phenylalanine, tyrosine |
| Basic (positively charged) | Lysine, arginine, histidine |
| Acidic (negatively charged) | Aspartic acid and glutamic acid |
| Special properties | Cysteine, proline and glycine |
If the amino acid has profoundly different chemical properties, this can change or even destroy the function of the protein. For example, the MC1R gene (also known as melanocortin 1 receptor), is responsible for pigment production in melanocytes (Chapter 7 on the Extension locus). There are two well-known variants of this gene, one associated with the production of red pigment and another with the production of black pigment. The differences are the result of a substitution of a T for a C in one of the codons (Marklund et al., 1996). The situation is illustrated in Fig. 4.2 taken from the paper of Marklund et al. (1996).
Fig. 4.2. This figure is taken from Marklund et al. (1996) and shows the alignment for two alleles in a region of the gene MC1R, the variant in codon 83 responsible for the difference between the E and e allele and the resulting amino acid change at position 83 from serine to phenylalanine (the amino acids are listed in the figure using three-letter codes for each amino acid).
This is called a non-synonymous mutation because it changes an amino acid. Furthermore, the change is chemically significant because phenylalanine is hydrophobic while serine is hydrophilic. Changing the amino acids in this position destroys the binding site of this receptor and, as a consequence, it cannot interact with melanocyte-stimulating hormone to create black pigment. The default pigment is red. When non-synonymous variants are found, one of the major questions is what impact this may have on gene function. Proof could come from doing gene editing and cell biology experiments. However, these experiments are costly, time-consuming, and not justifiable for every genetic variant that is discovered. Therefore, scientists turn to computer modeling to make predictions about the effects of a mutation.
Several of the more popular programs are SIFT (Kumar et al., 2009) and Polyphen-2 (Adzhubei et al., 2010) for simple substitutions and Provean for simple substitutions, deletions, and insertions (Choi et al., 2012). These programs assess the likelihood that changes in amino acids will alter protein function based on the physical and chemical properties of the amino acids. Scientists can enter the different series of amino acids associated with the two variants and the programs will return a prediction as to whether the variant will have no effect, a possible deleterious effect, or a probable deleterious effect.
Mutation versus variant
So far, we have used the terms “variant” and “mutation” interchangeably. This is common in genetics although the terms do have subtly different meanings. Variation denotes that different forms exist for a gene. A major theme for this book is to understand and appreciate the extent of genetic variation in the horse. The term “mutation” is a charged term, carrying the concept of normal DNA sequences versus those which are not normal. In the early 1900s scientists coined a term “wildtype” to denote what they considered to be the normal variant in a population. The alternative was mutation. The term wildtype is still occasionally used. The term “mutation” is frequently used when referring to disease-causing variants. In any case, the term mutation is appropriate at the moment when the mutational event is observed, for example, as a change in DNA sequence between parents and offspring.
While we now think of the chestnut allele as a variant, studies of ancient DNA plus studies of the function of the MC1R protein demonstrated that the ancestors of modern horses had black pigment and a mutation of the MC1R gene led to the creation of the chestnut variant. As noted in earlier chapters, early breeders liked some of these color variants and selected for them such that they have become characteristic of breeds. Chestnut is very common among Saddlebred horses. Likewise, gray coat color is the consequence of an ancient mutation but now very common among Arabian horses and Lippizan horses. We will follow the practice of calling the alleles for chestnut and gray as variants and not mutations although their origins were as mutations of what could be called the wildtype alleles.
Other roles of DNA: introns and exons, and regulatory DNA sequences
All
