Allelic heterogeneity at the equine KIT locus in dominant white (W) horses

Abstract

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from approximately 50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the approximately 82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations.

Figure 1. Dominant White Phenotype in Horses

(A) Franches-Montagnes mare with little residual pigmentation. (B, C) Dominant white Franches-Montagnes stallion showing partial depigmentation as a colt and almost complete depigmentation at 4 years of age. (D) Dominant white Thoroughbred stallion. (E) Dominant white Arabian stallion. (F) Camarillo White Horse. (G) Immunohistochemistry using a polyclonal KIT antibody on a skin biopsy from a solid-colored horse. Blue staining indicates KIT expression throughout the epidermis. Melanin produced by melanocytes is visible as brown granules. (H) Immunohistochemistry on a skin biopsy of a white horse. Note the weak blue staining and the complete absence of melanocytes and melanin. The bars correspond to 50 μm.

Figure 2. Mutations in the Equine KIT Gene

Direct sequencing of genomic PCR products from solid-colored and dominant white horses revealed four nonsynonymous polymorphisms that were exclusively found in white horses. (A) Exon 15, c.2151C>G mutation in a white Franches-Montagnes Horse. (B) Exon 4, c.706A>T mutation in a white Arabian. (C) Exon 12, c.1805C>T mutation in a Camarillo White Horse. (D) Exon 13, c.1960G>A mutation in a White Thoroughbred. (E) Electropherograms of exon 15 sequences from a dominant white Franches-Montagnes Horse derived from genomic DNA and cDNA. The presence of the polymorphism is clearly visible on the genomic DNA and on the transcript level. The reduced signal intensity of the mutant G allele in the cDNA sequence indicates a reduction of the mutant transcripts compared to the wild-type transcripts of about 50%.

Figure 3. Western Blot Analysis of KIT Protein Expression in Skin Samples

A strong band of the expected size of glycosylated wild-type KIT protein was detected in the protein sample from the solid-colored horse. Protein extract from the skin of a dominant white horse yielded two very weak bands. The sizes of the two bands corresponded to wild-type and truncated KIT protein, respectively. As the skin sample from the dominant white horse did not contain melanocytes, the weak bands presumably were due to other KIT-expressing cells that may have been present in the skin biopsy.