Cream (C) is inherited as an incompletely dominant gene. In the heterozygous condition cream dilutes phaeomelanin from red to yellow, which results in palominos, buckskins, and smoky seal browns. Homozygotes are lighter in color (cremello and perlino). In addition, in the homozygous condition eumelanin is also diluted, which results in smoky cream (dilute black) horses (Sponenberg, 2009). The cream locus was mapped to ECA21q and a single base substitution in exon 2(G457A) of the solute carrier 45 family A2 (SLC45A2, also known as membrane-associated transporter protein or MATP) is thought to be responsible for the cream dilution phenotypes (Locket et al., 2001; Mariat, Taourit, & Guerin, 2003). The precise function of SLC45A2 is unknown, but in mice, mutations in this gene disrupt the processing and trafficking of tryosinase (a key enzyme in pigment production) to the melanosome (Costin et al., 2003).

Pearl color dilution in breeds of Iberian origin, or “Barlink factor” as it is known in Paints and Quarter Horses, is also caused by a mutation in SLC45A2 (Penedo et al., 2011). However unlike cream, the coat (both eumelanin and phaeomelanin) is diluted only in the homozygous condition, thus pearl is described as a recessive mutation (Sponenberg, 2009). However, in the heterozygous condition, while the hair remains unchanged, the skin is lighter and small pale spots may be present. The causative mutation for pearl was discovered only recently (Penedo et al., 2011), and a DNA test is available.

Champagne (CH) dilutes phaeomelanin and eumelanin in both the heterozygous and homozygous condition and is therefore caused by a dominant allele. Often homozygotes and heterozygous are phenotypically indistinguishable from each other and from that of cream and pearl dilutes. In addition to the diluted coat color, champagne horses often have amber-colored eyes and pumpkin-colored skin, which can sometimes help delineate these animals from other dilute phenotypes. CH was mapped to ECA14 and a SNP in exon 2 (resulting in an amino acid substitution T63R) of another solute carrier, SLC36A1 (Solute Carrier 36 family A1), is thought to be responsible (Cook et al., 2008). While the precise function of SLC36A1 remains to be determined, the authors postulated that this gene may help in regulating pH and thus in the maturation of the melanosome.

The silver dilution (Z), also known as silver dapple, dilutes eumelanin only (phaeomelanin is unaffected) to a “silver” or “chocolate” color, usually leaving the main and tail much lighter than the body hairs. In addition, horses with silver dilution frequently have “dapples” or darker pigment outlining lighter areas. Silver is inherited as a dominant allele and homozygotes may be more dilute (Brunberg et al., 2006). Silver was mapped to ECA6 and the positional and functional candidate gene PMEL17 (pre-melanosomal protein 17) or SILV (silver homolog) and was subsequently sequenced by two separate groups (Brunberg et al., 2006; Reissmann, Bierwolf, & Brockmann, 2007). A missense mutation in exon 11 (DQ665301:g.1457C>T) resulting in an amino acid substitution (Arg618Cys) is believed to cause this dilution (Brunberg et al., 2006). PMEL17 is thought to be involved in the biogenesis of the pre-melanosome (Yasumoto et al., 2004; Hoashi et al., 2005). Equine multiple congenital ocular anomalies (MCOA) have been associated with the silver phenotype. MCOA is characterized by a diverse set of ocular phenotypes, with large cysts being the predominant phenotype (Ewart et al., 2000; Grahn et al., 2008). Incomplete penetrance of this disorder has made studying the molecular mechanism difficult, and it remains unknown whether PMEL17 and/or the mutation Arg617Cys also causes MCOA (Andersson et al., 2008; Grahn et al., 2008).

Dun (D) is a dominantly inherited dilution of all base coat colors (Adelsteinsson, 1978). Similarly to the CH, both eumelanin and phaeomelanin pigments are lightened and heterozygotes are indistinguishable from homozygotes. In contrast to any of the other dilute phenotypes, Dun horses also have dorsal stripes and other primitive markings. While the exact genetic mechanism remains unknown, the genetic locus for D was mapped to ECA8 (Bricker, P. C. M., Millon, & Murray, 2003). A genetic test based on the zygosity of linked markers is commercially available and is useful for identifying homozygotes.

Coat Color Dilution Lethal (CCDL), also known as Lavender Foal syndrome (LFS), is a recessive condition that reduces pigmentation in homozygous individuals, creating a softened hue described as pale gray, pewter, and light chestnut, as well as lavender (Bowling, 1996). Unfortunately, it is also responsible for a neurological disorder that is lethal soon after birth (Bowling, 1996). Affected foals have various neurological signs including tetanic-like seizures, opisthotonus, stiff or paddling leg movements, and nystagmus (Fanelli, 2005). Mild leucopenia is also occasionally present (Fanelli, 2005; Page et al., 2006). These neurological impairments prevent the foal from standing and nursing normally and, if not lethal on their own, are often the causes for euthanasia. LFS is most frequently reported in the Egyptian subgroup of the Arabian horse (Bowling, 1996).

Mapping and mutation detection for LFS was accomplished in 2010 as the first published successful use of the Equine SNP50 SNP set in the horse (Brooks et al., 2010). The discovered deletion in exon 30 of myosin VA (MYO5A) leads to a frame shift and premature termination of transcription (ECA1 g.138235715del). Loss of the C-terminus of the protein, which encodes a portion of the secretory vesicle-specific binding domain of the globular tail, would likely impair binding of the myosin 5a motor to specific cargo organelles bearing the appropriate receptors (Pashkova et al., 2005). The resulting loss of vesicle traffic is likely to interfere with the function of dendritic cells like melanocytes and neurons, thus creating the associated phenotypes.

White Spotting and Depigmentation Patterns (Frame, Tobiano, Sabino, Dominant White, Leopard Complex, Gray, Roan, and White face and leg markings)

The genetics of white spotting in horses was among the first traits to be studied. White spotting can occur on any base color and can occur in combination with dilution and other white spotting patterns. White spotting often results from the absence of melanocytes due to defects in melanocyte differentiation and/or proliferation during embryonic development. It can also occur by the loss of pigment and/or depletion of pigment cells throughout life (depigmentation). Several white spotting and/or depigmentation genes have similar phenotypes and often horses are erroneously classified; thus DNA testing is a useful tool, and new tests will undoubtedly aid in correct classification. Most white spotting patterns vary in the amount of white present on the horse and much of this variation is thought to be caused by as yet uncharacterized modifier genes. Similarly to other species, several white spotting patterns are associated with pleiotropic effects (reviewed in Bellone, 2010; Rieder, 2009).

Frame overo patterning, inherited by a dominant allele (O), defines white spotting that typically occurs in the middle of sides of the flank, neck, and ventrally but not dorsally, so that pigmentation “frames” the horse. Homozygosity results in a lethal condition known as lethal white foal syndrome (LWFS or OLWS). This syndrome is characterized by foals that are born with a nearly complete white coat and are affected with intestinal aganglionosis. This lack of enervation causes intestinal obstruction and death soon after birth (Hultgren, 1982; McCabe et al., 1990). In Quarter Horses the frame pattern has also been associated with deafness (Magdesian, 2009). Using a candidate gene approach, three research groups independently identified the dinucleotide missense mutation (NM_001081837.1:c.353_354delinsAG) in the first exon of endothelin receptor type B (EDNRB) (Metallinos, Bowling, & Rine, 1998; Santschi et al., 1998; Yang et al., 1998). The resulting protein substitution (Ile118Lys) is thought to disrupt functioning of melanocytes and enteric ganglia cells, but the exact mechanism is not completely understood.

The Tobiano pattern (TO), a dominant trait, is characterized by depigmented patches of skin and the associated hair, usually crossing the dorsal midline and covering the legs (Figure 9.1a). The phenotype can vary from minimal body white with some leg markings to depigmentation of all but perhaps 10% of the body surface. Homozygous TO/TO horses often have dime- to quarter-sized spots of pigment interspersed among the larger white patches (frequently termed “ink spots” or “paw prints”). However, these markings are not a reliable indicator of zygosity (Sponenberg, 2009). Tobiano is present in many diverse breeds of the horse. Linkage of TO to the blood protein locus Albumin (ALB) was first reported in a pony family (Trommershausen-Smith, 1978). Further work extended this association to a conserved haplotype on ECA3: Albumin (ALB)-B and Vitamin D binding factor (GC)-S (Bowling, 1987). To explain the unusually strong linkage disequilibrium between the TO, ALB and GC loci Bowling et al. proposed that an inversion on ECA3 could be preventing recombination in this region (Bowling, 1987).

Figure 9.1 The Tobiano pattern (a) and Sabino pattern (b) are both attributed to the KIT locus in the horse.

Early work by Raudsepp et al. (1999) did not detect such an inversion. However, in 2007, subsequent FISH studies with additional markers discovered a large pericentric inversion on ECA3q in TO horses (Brooks et al., 2007). This inversion spans more than 47 Mb beginning approximately 100 kb from the distal end of the KIT gene, a gene involved in white spotting phenotypes in several species, and does not appear to disrupt any coding sequence. However, similar to inversions in the mouse producing the Rump-white and Sash spotting patterns, disruption of sequences near KIT are thought to cause aberrant special or temporal expression of the receptor and thus result in improper migration of melanocytes (Stephenson et al., 1994; Nagle et al., 1995). The inversion is completely associated with TO in several American and European breeds of the horse (Haase et al., 2008).

Another white spotting pattern, termed Sabino, is characterized by irregularly bordered white patches (hair and skin) that begin at the extremities and face (Figure 9.1a). These white patches often extend to include the belly and midsection, either as distinct areas of white hair or as a diffuse scattering of white hairs resembling roan (Kunst et al., 1992). While phenotypically similar, there appears to be several different genetic mechanisms for Sabino, and the genetics of one type of Sabino patterning, Sabino 1 (SB1), is incompletely dominant. Heterozygotes have the typical Sabino pattern while homozygotes are white or nearly white with some pigmentation persisting around the topline. In the United States, SB1 is found among many light horse breeds, notably in the Tennessee Walking Horse and Missouri Foxtrotter (Sponenberg, 2009). Genomic DNA sequencing of the KIT gene revealed a base substitution in intron 16 (KI16+1037A) responsible for SB1 (Brooks & Bailey, 2005). When the KI16+1037A SNP is present, KIT is transcribed lacking exon 17, making this trait a notable example of polymorphic exon-skipping. While exon-skipping is not complete, as some normal gene products are produced, it has been proposed that this SNP reduces the overall strength of the 3′ splicing consensus sequence and that loss of exon 17 results in impaired protein function (Brooks & Bailey, 2005). Interestingly, unlike KIT mutant mice strains, SB1 horses appear to have no health defects (Geissler, McFarland, & Russell, 1981).

It has been observed that the Sabino-type pattern has heterogeneous genetic origins as SB1 does not explain all Sabino phenotypes (Brooks & Bailey, 2005). Presumably, variation at other genetic sites within KIT or another gene is responsible for other Sabino-type patterns. For example, Clydesdale and Shire draft horses are well known for their Sabino phenotype, but the SB1 mutation was not found among a sample group of those breeds (Brooks & Bailey, 2005).

“White” horses uniquely possess a predominantly white coat and dark eyes. Dominant White (W), so named for its mode of inheritance and the orthologous murine locus, was first identified as a homozygous lethal among a horse herd in Nebraska (Pulos & Hutt, 1969). Mau et al. (2004) studied dominant white in Swiss Franches-Montagnes Horses and identified linkage with the KIT gene. In sharp contrast to the other known coat color loci in the horse, many novel mutations were subsequently identified in one gene (KIT) and attributed to the same phenotype (Haase et al., 2007; Haase et al., 2009; Holl, Brooks, & Bailey, 2010). The twelve total W mutations (discovered in several breeds) encompass phenotypes ranging from a splotched pattern possessing much of the underlying base color to a completely white coat. In each case the mutation occurred fairly recently; either a founder was identified based on breed records or completely de novo, with neither confirmed parent carrying the variant allele. The W alleles are therefore breed or, in some cases, family specific. For this reason, although DNA testing is available, it is not broadly applicable. To date no horse has been identified as homozygous for any W allele (or compound heterozygous for two W alleles), suggesting that the hypothesis of homozygous lethality put forward by Pulos and Hutt (1969) is true. No hematopoietic abnormalities have been identified in the one W allele that has been scrutinized for pleiotropic effects (Haase et al., 2010).

Leopard complex spotting (LP; also referred to as appaloosa spotting) defines a group of white spotting patterns that occur in several breeds of horses including Appaloosa, Noriker, Knabstrupper, American Miniature Horse, British Spotted Pony, and Pony of the Americas (Bellone et al. 2010; Haase et al., 2010). Notably, LP is among the few ancient coat color patterns that have been detected in ∼25,000-year-old ancient DNA samples originating from wild, pre-domestic horses of Western and Eastern Europe (Pruvost et al., 2011). Leopard spotting is inherited by a single incompletely dominant gene. Homozygotes tend to have few spots of pigment (termed leopard spots) in their white patterned areas (Figure 9.2a), while the opposite is true of heterozygotes (Figure 9.2b). In either zygotic condition, the amount of white on the coat can range from very minimal (white flecks on rump, Figure 9.2c) to a coat that is almost entirely white (few spot, Figure 9.2a), and this variation is thought to be determined by several modifier genes. One of such modifiers, which is associated with the “Leopard”-specific pattern (denoted as PATN1, Figure 9.2b), was recently mapped to ECA3 (Archer et al., 2007). In addition to the patterning in the coat, LP is thought to be responsible for the associated traits of mottled skin, striped hooves, white sclera, and LP-specific progressive roaning (also termed varnish roan). This roaning is separate and distinct from classical roaning (described below), thus making LP both a white spotting and depigmentation pattern.

Figure 9.2 Leopard complex spotting: (a) LP/LP homozygote (b) LP/lp heterozygote (c) minimally marked LP horse whose genotype is difficult to distinguish by phenotype alone.

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