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A new dominant white allele was suspected when two Thoroughbred horses with minimal white marking on the coat produced a colt with a large amount of coat depigmentation. Because of its association with similar patterns in other horses, the KIT gene was selected as a candidate gene, and all 21 exons were sequenced in the colt. A novel 5-bp deletion was discovered in exon 3 and was confirmed with allele-specific PCR. The mutation introduced a pre-mature stop codon, resulting in truncation of the protein. The deletion was not present in either parent and is suspected to be responsible for the extensive white coat colour in the colt. Additionally, a previously described missense mutation was detected in exon 14 of both the colt and sire but is not believe to be causative. Parentage testing was conducted as required by The Jockey Club for Thoroughbred registration, and the foal qualified for the stated parentage. This novel deletion in exon 3 is the 12th discovered dominant white allele in the horse.
The KIT gene in mammals encodes a tyrosine kinase receptor whose signalling is involved in the development of erythrocytes, melanocytes, germ cells, mast cells and interstitial cells of Cajal (ICC) (Kitamura & Hirotab 2004). Homozygous loss of function mutations in mice have been shown to cause areas of depigmentation, impairment of hemopoiesis, sterility, gastrointestinal tumours and early lethality (Bult et al. 2008). Gain of function mutations identified in humans and mice have been shown to cause tumours in mast cells, germ cells and ICCs (Kitamura & Hirotab 2004).
Mutations in KIT leading to unpigmented skin and hair have been identified in humans, mice, pigs and horses (Haase et al. 2009a). Many mutations in KIT can produce the dominant white phenotype that was first reported by Pulos & Hutt (1969). So far, 14 polymorphisms involving the KIT gene have been reported for horses, with phenotypes ranging from small areas of depigmentation to white over the entire body (Haase et al. 2009a). Two of these mutations, Sabino-1 and Tobiano, produce viable homozygotes and are not classified as dominant white (W) alleles (Brooks & Bailey 2005; Brooks et al. 2007). There are no defects other than depigmentation documented for horses with a single copy of a W allele, although an embryonic lethal condition is expected for homozygotes and compound heterozygotes with some, if not all, of these alleles. KIT expression also affects development of mast cells, and a study of haematological parameters in the Franches Montagnes breed found no statistically significant effects of the W1 KIT allele compared with wild-type individuals (Haase et al. 2009b).
A novel mutation in the KIT gene was suspected when the mating of two Thoroughbreds produced a colt with a large amount of white patterning. Approximately half of the proband’s body was depigmented, with four high white stockings that extended over his midline, irregular markings on his body and a white blaze that covered his muzzle (Fig. 1). Both parents had small white markings; a white stripe on the face and three white pasterns in the case of the sire, and only one white fetlock in the case of the dam. As of March 2010, the sire had produced over 400 registered foals with no recorded dominant white offspring. The dam had produced three solid colour foals prior to this colt.
Figure 1. One-month-old dominant white Thoroughbred colt and dam. Both parents have minimal white markings and had previously only produced solid coloured foals.
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Hair samples were obtained from the 1-month-old Thoroughbred colt and his dam for genetic analysis of the KIT gene as a candidate for his unusual colour. DNA was obtained from these two samples using a hair lysis protocol previously described by Brooks et al. (2007). DNA from the sire was provided from a previously banked sample. The proband was born healthy and appeared to develop normally up to 5 weeks of age, when the samples were obtained.
Eighteen primer pairs were created with Primer 3 based on the gapped alignment of KIT mRNA (NM_001163866) to equCab2 genomic sequence and were used to amplify the 21 KIT exons by PCR (Rozen & Skaletsky 2000). Detailed primer information is given in Table S1. Sequencing direction was selected based on the distance of the exon to the primers. PCRs were prepared by scaling the manufacturer’s protocol to 20 μl reactions with 2 μl template, with the exception of using only 1.0 U of FastStart Taq polymerase (Roche Diagnostics Corp.). Reactions were carried out on an eppendorf Mastercycler gradient using the recommended procedure from Roche. The PCR products were sent to Cornell University’s Life Sciences Core Laboratories Center for Sanger sequencing in one direction. The trace files were analysed and aligned to sequencing data from an unrelated solid colour horse as well as the equCab2 genome assembly with phredPhrap (Green Group, University of Washington). The electropherograms were visually inspected for polymorphisms using consed (Green Group). The complementary strand of the exon 3 amplicon was sequenced and added to the exon 3 data to confirm the presence of a polymorphism (accession number HQ256561).
Two allele-specific primers were designed to detect a 5-bp deletion in the 408-bp region containing exon 3 (Table S1). The PCR was scaled down to 10 μl and included 1 μl of each 5 μm primer. The wild-type allele produced a 273-bp fragment, whereas the mutant allele produced a 147-bp fragment. The region was amplified for each sample and was visualized on a 3% agarose gel containing 1× SYBR Safe (Invitrogen).
KIT exon screening by sequencing detected a deletion in exon 3 (c.559_563delTCTGC), which would result in early termination of translation (p.Ser187ArgfsX10). The allele-specific primers produced the deletion fragment in the colt and only the wild-type fragment in the dam, sire and an unrelated control (Fig. 2). One copy of a previously described polymorphism in exon 14 (c.2045A>G, p.His682Arg) was the only other polymorphism detected in the colt (Haase et al. 2009a). The exon 14 polymorphism has been found in multiple breeds and is not associated with a dominant white phenotype. The sire was heterozygous at this locus, whereas the dam was homozygous for the wild type.
Figure 2. Three per cent agarose gel visualized with SYBR Safe showing the allele-specific PCR products. The 408-bp control and 272-bp wild-type fragment are seen in the control horse (lane 1), the colt (lane 2), the dam (lane 3) and the sire (lane 4), although the 147-bp deletion fragment is only present in lane 2.
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The deletion detected in exon 3 has not previously been documented in horses. However, there are four previously identified mutations leading to truncation of the KIT protein which have been linked to dominant white phenotypes, two of which are the result of nucleotide deletions (Haase et al. 2007). The W3 allele is the most similar to the mutation detected in the colt; it is a nonsense mutation in exon 4 that results in receptors lacking the intercellular portion, transmembrane domain, ligand binding domain and part of the extracellular domain (Haase et al. 2007). The W3 allele was only found in the heterozygous state and had no phenotype other than depigmentation reported. Similarly, the proband’s mutation is heterozygous, and thus he is still able to produce some full-length KIT transcripts, which accounts for the pigmented areas and his apparent health. The depigmentation is likely the result of the deletion in exon 3 leading to severely truncated KIT receptors.
Unfortunately, around 5 weeks of age the colt was found dead in his stall. The body was sent to the University of Kentucky Livestock Disease Diagnostic Center. Necropsy found mild focal suppurative bacterial omphalitis, severe chronic gastric ulceration and mild multifocal hepatitis. The death was attributed to cardiovascular shock and ventricular arrhythmia of undetermined aetiology.
Gastric ulcers have been documented for some human and mouse KIT mutants (Kitamura & Hirotab 2004). No similar cardiovascular defects were documented for murine allele variants (Bult et al. 2008). It is possible that the exon 14 polymorphism only has a mild effect on phenotype that is not usually detected; there were no detrimental effects reported in the original study documenting this allele (Haase et al. 2009b). However, if the mutation was present in the non-truncated copy of KIT, thus making the colt a compound heterozygote, the receptor’s function may have been affected enough to cause detrimental effects that could have contributed to the foal’s death. Unfortunately, as only limited tissue samples were available from the colt, it cannot be concluded with certainty that effects of the exon 3 deletion in KIT contributed to his death.
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Table S1 Primer pairs used to amplify the 21 exons of the equine KIT gene.
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