Seven novel KIT mutations in horses with white coat colour phenotypes

Introduction

Pigment cells in developing vertebrates are derived from a transient and pluripotent population of cells located in the neural crest. In vertebrates, the melanocyte precursor cells migrate along the dorso-lateral pathway to the distal sites of the body, proliferate and subsequently differentiate into pigment-producing melanocytes (Erickson 1993). Different signalling pathways influencing the development, migration and proliferation of melanocytes have been identified. Key factors include the endothelin B receptor (EDNRB) and its ligand endothelin-3 (EDN3) (Baynash et al. 1994; Hosoda et al. 1994; Yamada et al. 2006), and the transcription factors PAX3 (Potterf et al. 2000), SOX10 (Pingault et al. 1998) and MITF (Hodgkinson et al. 1993). In addition to these factors, the KIT receptor, a type III receptor protein tyrosine kinase also referred to as steel factor, plays an essential role in melanocyte development (Chabot et al. 1988; Geissler et al. 1988). KIT signalling is crucial for the development of haematopoietic, gonadal and pigment stem cells and acts as an essential survival factor for migrating and proliferating melanoblasts (Blume-Jensen et al. 1991; Steel et al. 1992). The influence on various cell lines explains the frequently observed pleiotropic effects of KIT mutations.

In humans, mice, pigs and horses, various KIT gene mutations have been determined. Besides pigmentation disorders, KIT mutations often cause pleiotropic effects. In mice, KIT mutations lead to modification of coat colour, anaemia and male sterility (Geissler et al. 1981, 1988; Reith et al. 1990; Guerif et al. 2002). In humans, KIT mutations have been identified as the cause of piebaldism, an autosomal dominant disorder of pigmentation (Giebel & Spritz 1991). Gain-of-function KIT mutations have been implicated in gastrointestinal stromal tumours, acute myelogenous leukaemia and systemic mastocytosis (Hirota et al. 1998; Isozaki & Hirota 2006). The white coat colour of hundreds of millions of commercially produced pigs and the belted coat colour phenotype of the Hampshire pig breed are also caused by mutations at the KIT locus (Marklund et al. 1998; Giuffra et al. 1999).

KIT gene mutations in the horse show a broad range of phenotypic expression. Sabino-1, tobiano, roan and dominant white are depigmentation phenotypes caused by KIT gene mutations. The sabino-1 spotting pattern is caused by an intronic mutation causing partial skipping of exon 17 (Brooks & Bailey 2005). Horses heterozygous for the sabino-1 mutation are characterized by intense white patches on the legs and face usually combined with white body spots and roan areas. Horses homozygous for the sabino-1 mutation are almost completely white. The tobiano spotting pattern is caused by a large chromosomal inversion disrupting a regulatory element of the KIT gene (Brooks et al. 2007; Haase et al. 2008). Horses homozygous or heterozygous for the tobiano mutation are phenotypically indistinguishable. Tobiano horses generally have white lower legs, and the white body patches cross the dorsal midline. The roan phenotype in horses is characterized by intermingled white and pigmented hair on the body. Roan is caused by an as yet unknown KIT mutation (Marklund et al. 1999).

The dominant white coat colour is phenotypically very similar to the coat colour in homozygous sabino horses. The skin and hair of dominant white horses are completely or almost completely unpigmented (Pulos & Hutt 1969). Dominant white is inherited as a monogenic autosomal dominant trait. It is hypothesized to be embryonic lethal in the homozygous state. To date, no homozygous horse could be identified. We previously reported four different mutations in the coding sequence of the KIT gene in white horses of different horse breeds (Haase et al. 2007). Currently, there is little known about possible pleiotropic effects of KIT mutations in horses. Haematological parameters in horses carrying the p.Tyr717X mutation were no different from those in horses with wildtype KIT alleles (Haase et al. 2009). In the present study, we report the genetics and coat colour phenotypes of new segregating white coat colour phenotypes in independent horse families.

Materials and methods

Results

We recently identified four equine KIT mutations in dominant white horses (Haase et al. 2007) and propose to designate these as W1–W4. We now analysed the KIT gene in nine new horse families from five different breeds segregating for a white or partially white coat colour phenotype. We sequenced all 21 KIT exons and their flanking sequences in 192 horses. Among these, 35 horses had a white or partially white coat colour phenotype (Fig. 1). We tested these 35 horses for the absence of known KIT mutations including sabino-1 and tobiano. Comparative sequencing revealed a total of 30 new polymorphisms in the equine KIT gene (Table 1). Among these, we identified seven new mutations affecting the KIT coding sequence in the white horses of seven different families and designated these as W5–W11 (Table 1, Fig. S2).

Table 1. KIT gene polymorphisms.

Discussion

This study indicates that white phenotypes occur in many different horse breeds because of independent mutations in the KIT gene. Although we do not have functional data on the different KIT alleles, the knowledge of KIT mutations in other species and the data on the previously published equine KIT mutations (Brooks & Bailey 2005; Brooks et al. 2007; Haase et al. 2007) strongly suggest that the seven newly discovered mutations affecting the KIT coding sequence are indeed causative for the observed white or partially white phenotypes. These seven candidate causative mutations were absent from 112 solid-coloured Franches-Montagnes Horses. One can argue that these mutations might represent breed-specific polymorphisms, which would not be expected to occur in the Franches-Montagnes breed. However, of the other 37 presumably neutral polymorphisms in the KIT gene, 22 (59%) also segregate in the Franches-Montagnes breed. Therefore, the absence of all seven candidate causative mutations from solid-coloured Franches-Montagnes Horses provides additional suggestive support for the causality of these mutations.

Two of the seven candidate causative mutations are frameshift mutations (c.2193delG; c.1126_1129delGAAC) and three represent splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). It seems highly likely that such mutations affect the KIT protein function. The remaining two candidate causative mutations are missense mutations (c.856G>A corresponding to p.Gly286Arg and c.1789G>A corresponding to p.Gly597Arg). For the two missense mutations, it is more difficult to predict whether they indeed affect the KIT function. The p.Gly286Arg mutation affects the extracellular ligand-binding domain and the p.Gly597Arg mutation the first intracellular tyrosine kinase domain. The wild-type glycine residues at both positions are conserved in the human and murine KIT proteins. Both mutations cause an exchange of glycine to arginine, which are extremely dissimilar amino acids. Therefore, it is conceivable that these mutations also affect the KIT function.

As the genotype–phenotype correlations of the different KIT alleles in horses are complex, it is not possible to predict the exact genotype of a white or partially white horse from its phenotype. We therefore propose to use the allele designations W1–W11 for the characterized KIT mutations to distinguish these horses. We observed a remarkable phenotypic variability for some of the equine W alleles. The alleles W1, W5 and W10 occur in completely white horses but also in horses with substantial residual pigmentation (Haase et al. 2007; Fig. 1). All horses carrying the W2, W3, W4, W6, W7, W9 and W11 alleles were completely white. However, only very few horses with these alleles were available. Therefore, it cannot be excluded that these alleles may also result in partially white horses. The reason for the variability in the coat colour phenotype of horses with the W1, W5 and W10 alleles is not known. The lack of melanocytes in these horses might be because of haploinsufficiency of the KIT gene, and subtle variations in the amount of residual KIT protein might already cause the observed phenotypic variability. On the other hand, it is also possible that one or more trans-acting modifier genes determine the amount of residual pigmentation in these horses.

The pedigree records allow a precise dating of the individual mutation events for this dominant trait. Whenever a white foal is born out of solid-coloured parents, the most likely explanation is a KIT mutation in the germline of one of its parents or alternatively a mutation in the early developing embryo itself, which might lead to mosaic foals. Many of the described W alleles arose during the last 10 years; therefore, our study included several founder animals where mosaicism cannot be excluded. One example for such a scenario is the W8 allele observed in a single ‘mottled’ Icelandic horse, which represents the founder animal for this mutation (Fig. 1g). This horse might be a mosaic, and it remains to be determined whether it will consistently produce offspring with the mottled phenotype.

The equine KIT gene shows a remarkable allelic diversity. Obviously, the striking coat colour phenotypes of the horses and the dominant inheritance increase the chances that spontaneous mutations in this gene are recognized in the first place. Therefore, the observed allelic diversity does not necessarily implicate a particularly high mutation rate. To date, 11 different W mutations, the sabino-1 mutation and the tobiano mutation are characterized as functional mutations of the equine KIT locus (Brooks & Bailey 2005; Brooks et al. 2007; Haase et al. 2007). In addition, the as yet uncharacterized roan mutation is also linked to the KIT locus (Marklund et al. 1999). Finally, in the two families where we did not find any coding mutations, further non-coding regulatory mutations at the KIT locus or mutations in other coat colour genes might be responsible for the observed white phenotypes. Thus, there are at least 14 functionally relevant KIT alleles in horses with white or white spotted coat colour. According to our knowledge, this is the largest observed allelic spectrum for any gene in domestic animals, although other examples of more limited allelic heterogeneity are also well known in domestic animals (Schmutz et al. 2002; Drögemüller et al. 2007).

In conclusion, we confirmed the significant role of KIT mutations in white horses and identified seven new candidate causative mutations. This will allow an improved classification of white horses based on their genotypes. The identified KIT mutations alone do not explain the variable phenotypic expression in some horse families.