Mutations in MITF and PAX3 Cause “Splashed White” and Other White Spotting Phenotypes in Horses

During fetal development neural-crest-derived melanoblasts migrate across the entire body surface and differentiate into melanocytes, the pigment-producing cells. Alterations in this precisely regulated process can lead to white spotting patterns. White spotting patterns in horses are a complex trait with a large phenotypic variance ranging from minimal white markings up to completely white horses. The “splashed white” pattern is primarily characterized by an extremely large blaze, often accompanied by extended white markings at the distal limbs and blue eyes. Some, but not all, splashed white horses are deaf. We analyzed a Quarter Horse family segregating for the splashed white coat color. Genome-wide linkage analysis in 31 horses gave a positive LOD score of 1.6 in a region on chromosome 6 containing the PAX3 gene. However, the linkage data were not in agreement with a monogenic inheritance of a single fully penetrant mutation. We sequenced the PAX3 gene and identified a missense mutation in some, but not all, splashed white Quarter Horses. Genome-wide association analysis indicated a potential second signal near MITF. We therefore sequenced the MITF gene and found a 10 bp insertion in the melanocyte-specific promoter. The MITF promoter variant was present in some splashed white Quarter Horses from the studied family, but also in splashed white horses from other horse breeds. Finally, we identified two additional non-synonymous mutations in the MITF gene in unrelated horses with white spotting phenotypes. Thus, several independent mutations in MITF and PAX3 together with known variants in the EDNRB and KIT genes explain a large proportion of horses with the more extreme white spotting phenotypes

Novel variants in the KIT and PAX3 genes in horses with white-spotted coat colour phenotypes.

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part-bred Arabians with sabino-like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white-increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18-W20 to provide a simple and unambiguous nomenclature for future genetic testing applications

Functional validation of the MITF:p.N310S mutation.

<p>(A) The basic DNA binding domain in the vicinity of the mutation is 100% identical in vertebrates from mammals to fish. (B) Electrophoretic mobility shift assay (EMSA). Increasing concentrations (given in µM) of recombinant wild-type and mutant MITF protein from <i>E. coli</i> were incubated with a radioactively labeled double-stranded oligonucleotide and run on a non-denaturing polyacrylamide gel. The mutant MITF protein shows a weaker retention band than the wild-type MITF, which indicates a partially defective DNA binding activity (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002653#pgen.1002653.s008" target="_blank">Table S3</a>).</p

Causative mutations for white spotting phenotypes.

a<p>numbering refers to NM_001163874.1 (<i>MITF</i>) and XM_001494972.3 (<i>PAX3</i>).</p>b<p>build EquCab 2.0.</p

Phenotypes of splashed white horses from a Quarter Horse family.

<p>Note that the expression of the phenotype is quite variable. Splashed white may be caused by different mutations, even within closely related horses. (A) Splashed white bay horse. The unpigmented areas are relatively small, but the horse has blue eyes. (B) Typical expression of the splashed white phenotype in a chestnut horse with blue eyes. (C) A splashed white chestnut horse with normal eye color and a relatively small blaze. (D) Splashed white coat color and brown eyes in a chestnut horse. (E, F) Two horses carrying both the <i>PAX3^C70Y</i> and <i>MITF^prom1</i> alleles with typical splashed white phenotypes. Horses carrying one copy of either <i>PAX3^C70Y</i> and/or <i>MITF^prom1</i> show overlapping phenotypes. None of the horses in this figure carry the <i>EDNRB^I118K</i> (overo) allele.</p

Phenotypes of horses with different combinations of splashed white alleles.

<p>(A) American Paint Horse with a very pronounced depigmentation phenotype. In addition to being homozygous for the <i>MITF^prom1</i> allele, it also carries a private allele at the <i>KIT</i> gene (p.H40Q), which may enhance the depigmentation phenotype. The functional significance of the p.H40Q variant is unclear at the moment. (B) This Quarter Horse is also homozygous for the <i>MITF^prom1</i> allele, but has substantially more residual pigmentation than the horse shown in panel A. (C) A Trakehner horse homozygous for the <i>MITF^prom1</i> allele. (D) A completely white horse with multiple splashed white alleles. (E) Quarter Horse with the rare <i>MITF^C280Sfs*20</i> allele. This horse has a very pronounced splashed white phenotype with blue eyes and a largely unpigmented head and belly. (F) A compound heterozygote for two different <i>MITF</i> mutant alleles is completely white. None of the horses in this figure carry the <i>EDNRB^I118K</i> (overo) allele.</p

White face area in splashed white Quarter Horses with specific genotypes.

<p>Raw phenotypes for individual horses are indicated as black circles. The average values for each genotype class are indicated by red lines. While there is considerable variation in the phenotypic expression, the average proportion of white face area is higher in chestnut horses versus bay horses and it is also higher in horses carrying both the <i>MITF^prom1</i> and the <i>PAX3^C70Y</i> allele than in horses carrying only one of the two splashed white alleles (p<0.05, two-sided Welch t-test). The white face areas of all horses and the p-values between all different genotype classes are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002653#pgen.1002653.s007" target="_blank">Table S2</a>.</p