SplashedWhite.pdf

Mutations in MITFand PAX3Cause ‘‘Splashed White’’

and Other White Spotting Phenotypes in Horses

Regula Hauswirth 1,2 . , Bianca Haase 3 . , Marlis Blatter 4 , Samantha A. Brooks 5 , Dominik Burger 4,6 ,

Cord Dr ¨ gem ¨ ller 1,2 , Vincent Gerber 7 , Diana Henke 8 , Jozef Janda 9 , Rony Jude 10 , K. Gary Magdesian 11 ,

Jacqueline M. Matthews 12 , Pierre-Andr ´ Poncet 4 , Vilhj ´ lmur Svansson 13 , Teruaki Tozaki 14 ,

Lorna Wilkinson-White 12 , M. Cecilia T. Penedo 15 , Stefan Rieder 4 , Tosso Leeb 1,2 *

1 Institute of Genetics, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 2 DermFocus, University of Bern, Bern, Switzerland, 3 Faculty of Veterinary Science,

University of Sydney, Sydney, Australia, 4 Swiss National Stud, ALP-Haras, Avenches, Switzerland, 5 Department of Animal Science, Cornell University, Ithaca, New York,

United States of America, 6 Swiss Institute of Equine Medicine, Vetsuisse Faculty, ALP-Haras and University of Bern, Avenches, Switzerland, 7 Swiss Institute of Equine

Medicine, Vetsuisse Faculty, University of Bern and ALP-Haras, Bern, Switzerland, 8 Division of Neurology, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 9 Division

of Experimental Clinical Research, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 10 Certagen GmbH, Rheinbach, Germany, 11 Department of Medicine and

Epidemiology, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America, 12 School of Molecular Bioscience, University of

Sydney, Sydney, Australia, 13 Institute for Experimental Pathology, University of Iceland, Reykjav´k, Iceland, 14 Department of Molecular Genetics, Laboratory of Racing

Chemistry, Utsunomiya, Japan, 15 Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America

Abstract

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.

Citation: Hauswirth R, Haase B, Blatter M, Brooks SA, Burger D, et al. (2012) Mutations in MITF and PAX3 Cause ‘‘Splashed White’’ and Other White Spotting

Phenotypes in Horses. PLoS Genet 8(4): e1002653. doi:10.1371/journal.pgen.1002653

Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America

Received November 4, 2011; Accepted February 28, 2012; Published April 12, 2012

Copyright: 2012 Hauswirth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: BH is a fellow of the Swiss Foundation for Grants in Biology and Medicine. This study was financed in part by a grant from Swiss National Science

Foundation (31003A_133034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: Tosso.Leeb@vetsuisse.unibe.ch

. These authors contributed equally to this work.

for the developing fetus [6]. If however too few of the migrating

melanoblasts survive, this will lead to partially or completely

unpigmented phenotypes [7]. Domestic animals with such

unpigmented phenotypes have been highly valued due to their

striking appearance and have often been actively selected in

animal breeding. Consequently, our modern domestic animals

provide a large repertoire of spontaneous mutants that allow the

dissection of the contributions of individual genes to migration,

proliferation, differentiation, and survival of melanocytes.

From a genetic point of view, white spotting is considered a

complex trait [8]. Phenotypes range from tiny white spots at the

extremities of the body to large unpigmented areas, in symmetrical

or asymmetrical patterns, up to completely unpigmented animals

Introduction

Coat color is a well-studied model trait for geneticists. Coat

color phenotypes are relatively easy to record, which facilitates

their analysis. In mammals melanocytes cover the entire body

surface and are responsible for the pigmentation of skin, hairs, and

eyes. Melanocytes are formed during fetal development from

melanoblasts, which originate in the neural crest and migrate

across the developing fetus in order to reach their final position on

the body [1]. This developmental program requires a delicate level

of regulation to ensure that the correct amount of cells reaches

their final destination [2–5]. An over-proliferation of cells that

have left their surrounding tissue might have fatal consequences

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Issue 4 | e1002653

MITF and PAX3 Mutations in Horse

by a large blaze of variable size and shape. Many splashed white

horses had an extremely large blaze extending over the eyes or

even covering parts of the cheeks; such a head pattern is termed

baldface. Splashed white horses also typically had high markings

that extend high up on their legs and occasionally little white belly

spots. Most of the splashed white horses had blue eyes or iris

heterochromia. Although many splashed white horses have a very

characteristic appearance, the phenotype is variable and in some

splashed white horses the unpigmented areas are so small that we

could not reliably distinguish these horses from horses with other

subtle

Author Summary

White spotting coat color phenotypes are the result of

aberrations in the development of melanocytes. The

analysis of domestic animals with heritable white spotting

phenotypes thus helps to better understand the compli-

cated genetic network controlling the proliferation,

migration, differentiation, and survival of pigment produc-

ing cells. We analyzed the so-called splashed white

phenotype in horses, which is characterized by a very

distinctive large blaze, extended white markings on the

legs, and blue eyes. Splashed white horses are also

frequently deaf. However, the phenotype is quite variable

and, in some horses with minimal expression, the splashed

white phenotype cannot be unambiguously discriminated

from the ‘‘common’’ white markings. We studied horses

from various breeds and found one mutation in the PAX3

gene and two mutations in the MITF gene that cause the

splashed white phenotype. A third mutation in the MITF

gene, which we found in a single Franches-Montagnes

horse, causes a new coat color phenotype, termed

macchiato. Similar mutations in humans cause either

Waardenburg or Tietz syndrome, which both are charac-

terized by pigmentation defects and a predisposition for

deafness. Our study reveals the molecular basis for a

significant proportion of white spotting phenotypes that

are intermediate between completely unpigmented hors-

es and common white markings.

depigmentation

phenotypes.

Some,

but

not

all

the

sampled splashed white horses were deaf.

In this Quarter Horse family the splashed white coat color

appeared to be inherited as an autosomal dominant trait. We

therefore genotyped 24 cases and 7 controls from this family on

the equine 50k SNP array and performed a linkage analysis

(Figure S1). Parametric analysis with a fully dominant model of

inheritance gave a maximum LOD score of 1.6 on ECA 6 with a

corresponding maximum a of 0.78. These results suggested locus

heterogeneity within the family. We also performed a case-control

genome-wide association study (GWAS) with the SNP genotypes

and found the strongest signal again on ECA 6 (p raw = 5.6610 27 ;

p genome = 0.003). The chromosomes with the next best associations

were 11 and 16, each having SNPs associated at p raw = 6.6610 25 ,

which is not genome-wide significant after permutation analysis

(p genome = 0.34).

The linked and associated region on ECA 6 contained PAX3 as

a strong functional candidate gene. We sequenced the nine exons

of the PAX3 gene in two cases and two controls and identified

seven polymorphisms including one missense mutation,

PAX3:c.209G.A (Table 1, Table S1). We then genotyped all

remaining animals of the Quarter Horse family and other

unrelated horses for this variant. We identified a total of 29

splashed white horses carrying the variant A-allele in heterozygous

state. All these horses traced back to a female Quarter Horse born

in 1987, whose genomic DNA from a hair-root sample tested

homozygous wild-type. Thus, the mutation most likely arose in the

germline of this animal. We did not detect the variant A-allele in

21 solid-colored Quarter Horses nor in 112 horses from 7 other

breeds. We also did not find any horse with the homozygous

variant A/A genotype (Table 2).

The PAX3:c.209G.A variant is predicted to result in a non-

conservative amino acid exchange p.C70Y in the so-called paired

domain, which together with the homeobox domain mediates the

DNA-binding of the transcription factor PAX3. The wild-type

cysteine at this position is conserved across all known PAX

paralogs in animals including Drosophila and Caenorhabditis elegans

(Figure 2). Based on the X-ray structure of the paired domain of

the

[9]. Different combinations of alleles at several loci can interact

and it is generally impossible to precisely predict the genotype of

an animal based only on a given white spotting phenotype.

Splashed white is a distinctive but nevertheless quite variable

white spotting pattern in horses, which is primarily characterized

by extensive depigmentation of the head. Splashed white horses

often have blue eyes and they are sometimes deaf [9]. This

phenotype has not yet been characterized at the molecular level.

Mutations in two genes have been identified to cause

pronounced depigmentation phenotypes in horses thus far. A

total of 19 different mutant alleles at or near the KIT gene were

reported to cause either completely white horses or horses with

pronounced depigmentation, such as the dominant white, tobiano,

and sabino-1 spotting patterns [10–14]. The frame overo spotting

pattern, which is phenotypically overlapping with the splashed

white phenotype, is caused by a missense mutation in the EDNRB

gene [15–17]. EDNRB mutations are also found in a small fraction

of human patients with Waardenburg syndrome (WS). WS is

characterized by pigmentation abnormalities, such as a typical

white forelock or white skin patches, strikingly blue or hetero-

chromatic irises, and varying degrees of sensorineural deafness and

skeletal dysmorphologies. Other forms of human Waardenburg

syndrome are caused by mutations in EDN3, MITF, PAX3, SNAI2,

and SOX10 [18]. Mouse mutants for these genes are available and

show largely similar although not completely identical phenotypes

[19].

In this study we report the identification of several independent

mutations in horses with striking depigmentation phenotypes that

show parallels to human Waardenburg syndrome.

human

PAX6

protein,

the

side-chain

this

cysteine

predicted to form a direct contact to the DNA backbone [20].

The initial GWAS indicated potential secondary signals on

ECA 11 and 16. We did not detect an obvious candidate gene on

ECA 11. However, the MITF gene on ECA 16 represents a strong

functional candidate for a white spotting phenotype. As the

PAX3:p.C70Y variant did not explain the phenotype in all

splashed white horses from the Quarter Horse family, we

sequenced all exons and the known promoter elements from the

MITF gene in two splashed white horses that did not have the

PAX3 C70Y allele and in two controls. We identified a total of 28

polymorphisms (Table S1). A variant in the proximal melanocyte-

specific MITF promoter replacing a thymine with 11 nucleotides

stood out as clear candidate for a non-coding regulatory mutation

(ECA16:g.20,117,302Tdelins11; Figure 3). This variant interrupts

a highly conserved binding site for PAX3 [21–23] and is located

Results

Splashed white in a Quarter Horse family

We obtained samples from a large Quarter Horse family

segregating for a striking white spotting pattern termed splashed

white (Figure 1). In our material this phenotype was characterized

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MITF and PAX3 Mutations in Horse

Figure 1. Phenotypes of splashed white horses from a Quarter Horse family. 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 PAX3 C70Y and MITF prom1 alleles with typical splashed white phenotypes. Horses carrying one copy of either PAX3 C70Y and/or

MITF prom1 show overlapping phenotypes. None of the horses in this figure carry the EDNRB I118K

(overo) allele.

doi:10.1371/journal.pgen.1002653.g001

close to the mutation causing white spotting in dogs [24]. The

variant allele was absent from 21 solid-colored Quarter Horses

and from 112 horses with minimal white markings from 7

additional breeds. We found 19 splashed white horses that carried

only the MITF prom1 allele and 20 splashed white horses that carried

both the MITF prom1 and PAX3 C70Y allele (Table 2).

We quantitatively analyzed the proportion of depigmented skin

in the face area of splashed white Quarter Horses in relation to

their underlying MITF and PAX3 genotypes as well as their base

coat color (Figure S2, Figure S3). This analysis indicated that one

copy of either the MITF prom1 or the PAX3 C70Y allele has a similar

effect on the face pigmentation. The presence of both the

MITF prom1 and the PAX3 C70Y alleles leads to a slightly more

pronounced depigmentation of the face on average than either

splashed white allele alone. This analysis also showed that the

average white face area is more extended in splashed white

chestnut horses compared to bay horses with the same splashed

white genotype (Figure 4; Table S2).

stallion also had a very large white blaze (Figure S4). The

MITF prom1 allele was also present in a Miniature Horse, a Shetland

Pony, and 11 Icelandic Horses with either splashed white or more

pronounced other white spotting phenotypes. Thus, the MITF prom1

allele is probably several hundred years old and arose before the

foundation of the modern horse breeds.

More pronounced other white spotting phenotypes:

Combinations of splashed white alleles

In our study we defined horses with $20% white face area and

#10% white area on the body as ‘‘splashed white horses’’. If a

horse had .10% white body area, it was considered to have a

more pronounced ‘‘other white spotting’’ phenotype. We noticed

that several horses with more pronounced ‘‘other white spotting

phenotypes’’ were offspring from two ‘‘splashed white’’ parents.

We identified a total of 24 horses from 5 breeds that were

homozygous for the MITF promoter variant. All these MITF prom1/prom1

horses showed very pronounced but still variable depigmentation

phenotypes. They had at least a white belly in addition to white

legs and the white head (Figure 5A–5C). One of these horses had

only small pigmented areas along the dorsal midline (Figure 5A).

One horse homozygous for the MITF prom1 allele and heterozygous

for the PAX3 C70Y

Breed distribution of the MITF prom1 allele

We sequenced the coding regions and proximal promoter

elements of the functional candidate genes KIT, MITF, and PAX3

in other horses with white spotting phenotypes in additional horses

from various breeds. We then noticed that the MITF promoter

polymorphism described above is much more widespread across

breeds than we had originally hypothesized. We found the

insertion allele in 58 Quarter Horses and American Paint Horses

with either the splashed white or more pronounced other white

spotting phenotypes (see below). We also found this allele in five

Trakehner horses with white spotting phenotypes. A detailed

pedigree analysis revealed that all these horses are descendants

from the Thoroughbred stallion Blair Athol born in 1861. This

allele was completely white and also deaf

(Figure 5D).

In our mutation analysis we identified one horse with a splashed

white phenotype including a white belly, which was wild-type for

both the MITF prom1 and the PAX3 C70Y alleles (Figure 5E). This

horse carried a small deletion in exon 5 of the MITF gene

(c.837_841del5). The variant leads to a frameshift and a severely

truncated MITF protein (p.C280Sfs*20), which might act in a

dominant negative fashion. A completely white offspring of this

horse was a compound heterozygote for the MITF prom1

and the

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MITF and PAX3 Mutations in Horse

Table 1. Causative mutations for white spotting phenotypes.

Allele designation

(genetic testing)

Variant a

Variant (genomic) b

Gene

Allele designation

Phenotype

MITF prom1

MITF

n.a.

ECA16:g.20,117,302Tdelins11

SW1

splashed white

MITF C280Sfs*20

MITF

c.837_841delGTGTC

ECA16:g.20,105,348_52del5

SW3

splashed white

MITF N310S

MITF

c.929A . G

ECA16:g.20,103,081T . C

macchiato

PAX3 C70Y

PAX3

c.209G . A

ECA6:g.11,429,753C . T

SW2

splashed white

a numbering refers to NM_001163874.1 (MITF) and XM_001494972.3 (PAX3).

b build EquCab 2.0.

doi:10.1371/journal.pgen.1002653.t001

MITF C280Sfs*20 alleles (Figure 4F). The MITF C280Sfs*20 allele was

absent from 21 solid Quarter Horses and 112 control horses from

7 additional breeds (Table 2).

In a few unrelated horses with large blazes we did not find any

candidate causative mutations in the KIT, MITF,orPAX3

candidate genes. These horses comprised 7 Quarter Horses, 1

Hanoverian, 2 Oldenburger, and 8 Thoroughbreds (Table 2).

suggested locus heterogeneity even within a family of closely

related Quarter Horses. Using a positional candidate gene

approach we subsequently identified three causative mutations

for splashed white phenotypes and one causative mutation for

another similar phenotype that we termed macchiato (Table 1).

Although we do not have functional proof for all of the

mutations, we obtained sufficient ancillary evidence to claim their

causality. For the PAX3 C70Y allele the arguments are that it (1)

occurs only in horses with the splashed white phenotype, (2) the

PAX3 locus was linked to this phenotype in a family, (3) mutations

within the PAX3 gene cause similar phenotypes in humans and

mice, and (4) the mutation affects a highly conserved cysteine

residue in the paired domain, that is predicted to participate in

DNA binding. We were able to determine that this variant arose in

1987 and to identify the specific founder animal.

For the three MITF mutations the support is even better than

for the PAX3 mutation: (1) Multiple mutations within the same

gene lead to comparable phenotypes, (2) the three variants occur

exclusively in horses with characteristic depigmentation pheno-

types, and (3) mutations within the MITF gene cause similar

phenotypes in humans, mice, and dogs [18,19,24]. MITF C280Sfs*20

is the result of a frameshift mutation, which is extremely likely to

affect the normal function of MITF as it truncates about half of the

protein. The MITF prom1 variant affects a region of the melanocyte-

specific promoter that has been shown to function as a PAX3

binding site in humans and mice [20–22]. For MITF N310S we

demonstrated that it represents a spontaneous de novo mutation in

the macchiato horse that was born out of solid-colored parents free

of this allele. We also provide functional evidence that the

MITF N310S protein has a reduced DNA binding capability. Taken

together these data strongly argue for the causality of the four

mutations in PAX3 and MITF that we report in this study.

Our findings will be of relevance to horse breeders. The

MITF prom1 allele arose at least several hundred years ago, it is

relatively common, and it occurs in several modern horse breeds.

Horses homozygous for this allele are viable and typically have a

more pronounced depigmentation phenotype than heterozygous

horses. The PAX3 C70Y allele is only 24 years old and occurs

exclusively in Quarter and Paint Horses. We did not find a horse

homozygous for this mutation, and based on data from mice it is

unlikely that a homozygous PAX3 C70Y/C70Y horse would be viable.

As PAX3 is required for several key steps in neural development,

homozygosity for this allele will most likely result in embryonic or

fetal lethality [26]. Therefore, the mating of two heterozygous

PAX3 +/C70Y horses is not recommended in order to avoid the

accidental production of an embryo homozygous for this allele.

The MITF C280Sfs*20 and MITF N310S alleles are extremely rare.

Data from mice again suggest that these alleles will most likely

result in severe clinical phenotypes such as e.g. microphthalmia in

Macchiato in a Franches-Montagnes Horse

In 2008 a colt with a striking white-spotting phenotype was born

out of two solid-colored bay Franches-Montagnes parents. The

coat color resembled a combination of white-spotting and coat

color dilution (Figure 6). The parentage of this colt was

experimentally verified using 13 microsatellite markers. Therefore,

we assumed the coat color of this colt to be the result of a

spontaneous de novo mutation and subsequently termed it

‘‘macchiato’’. In 2010, we performed a detailed clinical and

spermatological examination, which revealed that the two year old

macchiato stallion was deaf and had a low progressive sperm

motility.

We sequenced the coding regions of six functional candidate

genes in the macchiato stallion and his solid-colored parents. The

investigated candidate genes are involved in white spotting (KIT,

MITF) or coat color dilution (MLPH, PMEL, SLC36A1, and

SLC45A2). We identified a de novo missense mutation in exon 6 of

the MITF gene in the macchiato stallion (c.929A.G). We found

the mutant allele at approximately 50% intensity in DNA samples

from blood, hair roots and sperm, indicating that the macchiato

stallion is not a mosaic. The mutant allele was not present in DNA

from blood samples from either the mother or the father. The

mutation affects a highly conserved amino acid of the basic DNA

binding motif of the transcription factor MITF (p.N310S). We

analyzed the DNA binding properties of the mutant MITF in an

electrophoretic mobility shift assay and found that DNA binding

activity was reduced by about 80%, probably through changes to

the kinetics of binding (Figure 7, Table S3). A mutation at the

same residue in the human MITF protein leads to Tietz syndrome,

which is characterized by a more generalized depigmentation and

profound obligate hearing loss compared to the slightly milder

Waardenburg syndrome 2A, which is caused by many other

mutations in the human MITF gene [25]. The MITF:p.N310S

variant was absent from 96 solid-colored Franches-Montagnes

horses.

Discussion

As the splashed white phenotype is rather distinctive among the

many different variations of white spotting phenotypes in horses,

we started this investigation with the expectation of finding only a

single

causative

mutation. However,

the linkage/GWAS

data

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Issue 4 | e1002653

Table 2. Association of white spotting genotypes in different breeds.

Gene

Genotype

No mutation

found

EDNRB

I118K/ +

+ / +

MITF

+ / +

prom1/ +

prom1/prom1

prom1/ +

prom1/prom1

C280Sfs*20/ +

prom1/C280Sfs*20

+ / +

prom1/ +

prom1/prom1

PAX3

+ / +

C70Y/ +

Breed a

Phenotype

Splashed white

or other white

spotting

QH/AP b

4 c

7 c

Solid colored

n.d.

Results for the three newly described splashed white mutations and the overo mutation [15–17] are shown.

a QH, Quarter Horse; AP, American Paint Horse; HN, Hanoverian; IS, Icelandic Horse; MH, Miniature Horse; OL, Oldenburger; SP, Shetland Pony; TB, Thoroughbred; TR, Trakehner; AS, American Standardbred; FM, Franches-Montagnes;

HF, Haflinger; NO, Noriker; WB, European Warmblood.

b Gene flow exists between QH and AP. Several horses in our study had double registrations with both the American Quarter Horse Association (AQHA) and the American Paint Horse Association (APHA).

c The Icelandic Horses with the splashed white or other white phenotype were not experimentally tested for the absence of the EDNRB I118K , MITF C280fs*20 ,andPAX3 C70Y alleles. They were assumed to be homozygous wildtype at

these positions.

doi:10.1371/journal.pgen.1002653.t002

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