szampanski.pdf

Missense Mutation in Exon 2 of SLC36A1 Responsible for

Champagne Dilution in Horses

Deborah Cook 1 *, Samantha Brooks 2 , Rebecca Bellone 3 , Ernest Bailey 1

1MH Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, United States of America, 2 Department of Animal

Science, Cornell University, Ithaca, New York, United States of America, 3 Department of Biology, University of Tampa, Tampa, Florida, United States of America

Abstract

Champagne coat color in horses is controlled by a single, autosomal-dominant gene (CH). The phenotype produced by this

gene is valued by many horse breeders, but can be difficult to distinguish from the effect produced by the Cream coat color

dilution gene (CR). Three sires and their families segregating for CH were tested by genome scanning with microsatellite

markers. The CH gene was mapped within a 6 cM region on horse chromosome 14 (LOD = 11.74 for h = 0.00). Four candidate

genes were identified within the region, namely SPARC [Secreted protein, acidic, cysteine-rich (osteonectin)], SLC36A1 (Solute

Carrier 36 family A1), SLC36A2 (Solute Carrier 36 family A2), and SLC36A3 (Solute Carrier 36 family A3). SLC36A3 was not

expressed in skin tissue and therefore not considered further. The other three genes were sequenced in homozygotes for

CH and homozygotes for the absence of the dilution allele (ch). SLC36A1 had a nucleotide substitution in exon 2 for horses

with the champagne phenotype, which resulted in a transition from a threonine amino acid to an arginine amino acid

(T63R). The association of the single nucleotide polymorphism (SNP) with the champagne dilution phenotype was

complete, as determined by the presence of the nucleotide variant among all 85 horses with the champagne dilution

phenotype and its absence among all 97 horses without the champagne phenotype. This is the first description of a

phenotype associated with the SLC36A1 gene.

Citation: Cook D, Brooks S, Bellone R, Bailey E (2008) Missense Mutation in Exon 2 of SLC36A1 Responsible for Champagne Dilution in Horses. PLoS Genet 4(9):

e1000195. doi:10.1371/journal.pgen.1000195

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

Received February 11, 2008; Accepted August 8, 2008; Published September 19, 2008

Copyright: 2008 Cook 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: This work was funded by Morris Animal Foundation.

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

* E-mail: deborah.cook@uky.edu

Introduction

CR in that; 1) CH dilutes both pheomelanin and eumelanin in its

heterozygous form and 2) heterozygotes and homozygotes for CH

are phenotypically difficult to distinguish. The homozygote may

differ by having less mottling or a slightly lighter hair color than the

heterozygote. Figure 1 displays images of horses with the three base

coat colors chestnut, bay and black and the effect of CH upon each.

Figure 2 shows that champagne foals are born with blue eyes, which

change color to amber, green, or light brown and pink ‘‘pumpkin

skin which acquires a darker mottled complexion around the eyes,

muzzle, and genitalia as the animal matures [6]. Foals with one copy

of CR also have pink skin at birth but their skin is slightly darker and

becomes black/near black with age. The champagne phenotype is

found among horses of several breeds, including Tennessee Walking

Horses and Quarter Horses. Here we describe family studies that

led to mapping the gene and subsequent investigations leading to

the identification of a genetic variant that appears to be responsible

for the champagne dilution phenotype.

Many horse breeders value animals with variation in coat color.

Several genes are known which diminish the intensity of the

coloration and are phenotypically described as ‘‘dilutions’’. Two of

these are a result of the Cream (CR) locus and Silver (Z) locus. The

molecular basis for Cream is the result of a single base change in

exon 2 of the SLC45A2 (Solute Carrier 45 family A2, aka MATP for

membrane associated transport protein) on ECA21 [1,3]. This change

results in the replacement of a polar acidic aspartate with a polar

neutral asparagine in a putative transmembrane region of the

protein coded for by this gene [3,2]. CR has an incompletely

dominant mode of expression. Heterozygosity for CR dilutes only

pheomelanin (red pigment) whereas homozygosity for CR results in

extreme dilution of both pheomelanin and eumelanin (black

pigment) [4].

The Silver dilution is the result of a missense mutation of PMEL17

(Premelanosomal Protein) on ECA6. The base change causes

replacement of a cytosolic polar neutral arginine with non-polar

neutral cysteine in PMEL17 [2]. In contrast to CR,theZ locusisfully

dominant and affects only eumelanin causing little to no visible

change in the amount of pheomelanin regardless of zygosity. The

change in eumelanin is most apparent in the mane and tail where the

black base color is diluted to white and gray [5].

The coat color produced by the CH locus is similar to that of the

CR locus in that both can cause dilution phenotypes affecting

pheomelanin and eumelanin. However, the effect of CH differs from

Results

Linkage Analyses

Table 1 summarizes the evidence for linkage of the CH gene to a

region of ECA14. The linkage phase for each family was apparent

based on the number of informative offspring in each family.

Recombination rates (h) were based on the combined recombina-

tion rate from all families. Four microsatellites showed significant

linkage to the CH locus: VHL209 (LOD= 6.03 for h = 0.14),

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SLC36A1 Variant Responsible for Champagne Horses

base 76 of exon 2 (c.188C.G) (Figure 5). These SLC36A1 alleles

were designated c.188[C/G], where c.188 designates the base pair

location of the SNP from the first base of SLC36A1 cDNA, exon 1.

Sequencing traces for the partial coding sequence of SLC36A1

exon 2 with part of the flanking intronic regions for one non-

champagne horse and one champagne horse were deposited in

GenBank with the following accession numbers respectively:

EU432176 and EU432177. This single base change at c.188 was

predicted to cause a transition from a threonine to arginine at

amino acid 63 of the protein (T63R).

Author Summary

The purpose of this study was to uncover the molecular

basis for the champagne hair color dilution phenotype in

horses. Here, we report a DNA base substitution in the

second exon of the horse gene SLC36A1 that changes an

amino acid in the transmembrane domain of the protein

from threonine to arginine. The phenotypic effect of this

base change is a diminution of hair and skin color intensity

for both red and black pigment in horses, and the resulting

dilution has become known as champagne. This is the first

genetic variant reported for SLC36A1 and the first evidence

for its effect on eye, skin, and hair pigmentation. So far, no

other phenotypic effects have been attributed to this

gene. This discovery of the base substitution provides a

molecular test for horse breeders to test their animals for

the Champagne gene (CH).

Protein Alignment

Figure 6 shows the alignment of the protein sequence for exons

1 and 2 of SLC36A1 for seven mammalian species with sequence

information from Genbank (horse, cattle, chimpanzee, human,

dog, rat and mouse). Alignment was performed using AllignX

function of Vector NTI Advance 10 (Invitrogen Corp, Carlsbad,

California). The alignment demonstrates that this region is highly

conserved among all species. At position 63, the amino acid

sequence is completely conserved among these species, with the

exception of horses possessing the champagne phenotype. This

replacement of threonine with arginine occurs in a putative

transmembrane domain of the protein [9].

TKY329 (LOD= 3.64 for h = 0.10), UM010 (LOD= 5.41 for

h = 0.04) and COOK007 (LOD= 11.74 for h = 0.00).

Figure 3 identifies the haplotypes for offspring of a single sire

showing recombination between the genetic markers and the CH

locus. Pedigrees of the three sire families and haplotype

information are provided in Figure S1 and Table S1 respectively.

The CH locus maps to an interval between UM010 and TKY329

with microsatellite. No recombinants were detected among 39

informative offspring between the CH and COOK007 locus.

Population Data

The distribution of c.188G allele among different horse breeds

and among horses with and without the champagne phenotype

was investigated. Table 2 is a compilation of the population data

collected via the genotyping assay. All dilute horses (85) which did

not have the CR gene, tested positive for the c.188G allele with

genotypes c.188C/G or c.188G/G. No horses in the non-dilute

control group (97) possessed the c.188G allele. The horses used for

the population study were selected for coat color and not by

random selection; therefore measures of Hardy-Weinberg equi-

librium are not applicable and were not calculated.

Candidate Genes

Candidate genes were selected on the basis of proximity to the

marker COOK007 and as genes previously characterized in other

species as influential in the production or migration of pigment

cells.

SPARC was located closest at ,90 kb downstream from

COOK007 and is coded for on the plus strand of DNA. It has

been implicated in migration of retinal pigment epithelial cells in

mice [7].

SLC36A family members are solute carriers and other solute

carrier families have been found to play a role in coat color.

SLC36A1 is located ,250 kb downstream from COOK007. It is the

first and most proximal to COOK007 of three genes in this family

and is coded for on the minus strand of DNA.

SLC36A2 and SLC36A3 are coded for on the plus strand of DNA

and are approximately 350 k and 380 k downstream from

COOK007 respectively. A2 and A3 have been found to be

expressed in a limited range of tissues in humans and mice [8].

Discussion

Family studies clearly showed linkage of the gene for the

champagne dilution phenotype within a 6 cM region on ECA14

[10] (Table 1). Based on the Equine Genome Assembly V2 as

viewed in ENSEMBL genome browser (http://www.ensembl.org/

Equus_caballus/index.html) this region spans approximately

2.86 Mbp [11]. Within that region, four candidate genes were

investigated; one based on known effects on melanocytes (eg.

SPARC) and three for their similarity to other genes previously

shown to influence pigmentation (eg, SLC36A1, A2, and A3). While

SNPs were found within the exons of SPARC, none were

associated with CH. Of the other 3 candidate genes, only SLC36A1

and SLC36A2 were found to be expressed in skin cells. Therefore,

the exons of those two genes were sequenced. A missense mutation

in the second exon of SLC36A1 showed complete association with

the champagne phenotype across several breeds. While SNPs were

found for SLC36A2, none showed associations at the population

level for the champagne dilution phenotype.

This observation is the first demonstration for a role of SLC36A1

in pigmentation. Orthologous genes in other species are known to

affect pigmentation. For example, the gene responsible for the

cream dilution phenotypes in horses, SLC45A2 (MATP), belongs to

a similar solute carrier family. In humans, variants in SLC45A2

have been associated with skin color variation [12] and a similar

missense mutation (p.Ala111Thr) in SLC24A5 (a member of

potassium-dependent sodium-calcium exchanger family) is impli-

cated in dilute skin colors caused from decreased melanin content

RT-PCR

RT-PCR (reverse transcription-polymerase chain reaction) was

used to determine if SLC36A1, SLC36A2 or SLC36A3 were expressed

in skin. SLC36A1 and SLC36A2 were expressed in skin and their

genomic exons were sequenced. SLC36A3 was not detected in skin

and therefore not investigated for detection of SNPs. Results for RT-

PCR of these three genes are shown in Figure 4.

Sequencing

All 9 exons of SPARC were sequenced. Three SNPs were found

in exons but none showed associations with the champagne

phenotype and are shown in Table S2.

SLC36A2 was sequenced with discovery of 9 SNPs in exons.

None of the SNPs showed associations with CH. These SNPs and

all other variations detected are described in Table S2.

SLC36A1 was sequenced. Only one SNP was found, a missense

mutation involving a single nucleotide change from a C to a G at

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SLC36A1 Variant Responsible for Champagne Horses

Figure 1. Effect of Champagnegene action on base coat colors of horses (chestnut, bay, and black). A) Chestnut – horse only produces

red pigment. B) Chestnut diluted by Champagne =Gold Champagne. C) Bay – black pigment is limited to the points (e.g. mane, tail, and legs)

allowing red pigment produced on the body to show. D) Bay diluted by Champagne = Amber Champagne. E) Black – red and black pigment

produced, red masked by black. F) Black diluted by Champagne = Classic Champagne.

doi:10.1371/journal.pgen.1000195.g001

among people of European ancestry [13]. The same gene,

SLC24A5 is responsible for the Golden (gol) dilution as mentioned

in the review of mouse pigment research by Hoekstra (2006) [14]

It is proposed, here, that the missense mutation in exon 2 of

SLC36A1 is the molecular basis for champagne dilution pheno-

type. While this study provides evidence that this is the mutation

responsible for the champagne phenotype, the proof is of a

statistical nature and a non-coding causative mutation can not be

ruled out at this point. SLC36A1, previously referred to by the

name PAT1 (proton/amino acid transporter 1) in human and

mouse [15], is a proton coupled small amino acid transporter

located and most active in the brush border membranes of

intestinal epithelial cells. This protein has also been characterized

in rats under the name LYAAT1 (lysosomal amino acid transporter 1).

LYAAT1 is localized in the membrane of lysozomes in association

with LAMP1 (lysosomal associated protein 1) and in the cell

membrane of post-synaptic junctions. In lysozomes it allows

outward transport of protons and amino acids from the lysozome

to the cytosol [16]. During purification and separation of early-

stage melanosomes LAMP1 is found in high concentrations in the

fraction containing stage II melanosomes [17],. Perhaps SLC36A1

plays a role in transitions from lysozome-like precursor to

melanosome. Since organellular pH affects tyrosine processing

and sorting [18], an amino acid substitution in this protein may

affect pH of the early stage melanosome and the ability to process

tyrosine properly. There must be an increase in pH, before the

tyrosinase can be activated. The cytosolic pH gradient must also

be maintained for proper sorting and delivery of the other proteins

required for melanosome development

[19]. Thus,

the pH

gradient of the cell may be altered by this mutation.

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September 2008 | Volume 4 |

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SLC36A1 Variant Responsible for Champagne Horses

Figure 2. Champagne Eye and Skin traits. A, B and C) Eye and skin color of foals. D and E) Eye color and skin mottling of adult horse.

doi:10.1371/journal.pgen.1000195.g002

Table 1. Linkage Analysis between the Champagne Dilution and Microsatellite Markers; UM010, COOK007, TKY329 and VHL209.

alleles

Sire contribution

Statistics

Sire Family

(CH)

microsatellite

a/b

a +

a 2

b +

b 2

LOD score

( + / 2 )

UM010

124/108

5.41

S =

5.41

( + / 2 )

COOK007

332/334

4.21

( + / 2 )

COOK007

332/334

2.41

( + / 2 )

COOK007

332/324

5.12

S =

11.74

( + / 2 )

TKY329

117/139

1.92

( + / 2 )

TKY329

111/137

1.34

( + / 2 )

TKY329

117/139

3.64

S =

6.9

0.1

( + / 2 )

VHL209

95/93

1.49

( + / 2 )

VHL209

91/93

0.46

( + / 2 )

VHL209

95/93

4.08

S =

6.03

0.14

N= the number of informative meiosis.

H = recombination frequency between that microsatelite and the champagne gene for all families combined.

S = LOD score for which 1/10 S = the odds the association between the phenotype and the marker is due to chance.

doi:10.1371/journal.pgen.1000195.t001

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SLC36A1 Variant Responsible for Champagne Horses

Figure 3. Example of Recombinant Haplotypes. Linear relationship from top to bottom between the microsatellites, phenotype, and genotype

of recombinant offspring for study sire # 3. Phenotype is noted in top row with offspring’s ID # .

doi:10.1371/journal.pgen.1000195.g003

This variant, discovered in association with a coat dilution in the

horse, is the first reported for the SLC36A1 gene. The phenotype

resulting from this mutation, a reduction of pigmentation in the

eyes, skin and hair, illustrates previously unknown functions of the

protein product of SLC36A1. Furthermore, now that a molecular

test for champagne dilution is established, the genotyping assay

can be used in concert with available tests for cream dilution and

silver dilution to clarify the genetic basis of a horse’s dilution

phenotype. This will give breeders a new tool to use in developing

their breeding programs whether they desire to breed for these

dilutions or to select against them.

Hair and blood samples from horses with the champagne

dilution phenotype were submitted by owners along with pedigree

information and photographs showing the champagne color and

characteristics of each horse. Samples were collected from the

following breeds (85 total): American Miniature Horse (9),

American Cream Draft cross (1), American Quarter Horse (27),

American Paint Horse (13, in addition to the family), American

Saddlebred (2), Appaloosa (1), ASB/Friesian cross (1), Arabian

crossed with APHA or AQHA horses (3), Missouri Foxtrotter(4),

Mule (2), Pony (1), Spanish Mustang 1), Spotted Saddle Horse (1),

Tennessee Walking Horse (20, in addition to the families).

Materials and Methods

Color Determination

To be characterized as possessing the champagne phenotype,

horses exhibited a diminished intensity of color (dilution) in black

or brown hair pigment and met at least two of the three following

criteria: 1) mottled skin around eyes, muzzle and/or genitalia, 2)

amber, green, or light brown eyes, or 3) blue eyes and pink skin at

birth [6]. This was accomplished by viewing photo evidence of

these traits or by personal inspection. Due to potential confusion

between phenotypes of cream dilution and champagne dilution, all

DNA samples from horses with the dilute phenotype were tested

for the CR allele and data from those testing positive were not

included in the population data.

Horses

Three half-sibling families, designated 1, 2 and 3, were used for

mapping studies. Family 1 consisted of a Tennessee Walking Horse

(TWH) stallion, known heterozygous at the Champagne locus (CH/ch),

and his 17 offspring out of non-dilute mares (ch/ch). Family 2

consisted of an American Paint Horse stallion (CH/ch) and his 10

offspring out of non-dilute (ch/ch) mares. Family 3 consisted of a

TWH stallion (CH/ch), 23 offspring and their 12 non-dilute dams

(ch/ch) and 1 dilute (buckskin) dam (ch/ch, CR/cr).

To investigate the distribution of the gene among dilute and

non-dilute horses of different horse breeds, 97 non-champagne

horses were chosen from stocks previously collected and archived

at the MH Gluck Equine Research Center. These horses were

from the following breeds: TWH (20), Thoroughbreds (TB, 35),

American Paint Horses (APHA, 32), Pintos (5), American

Saddlebreds (ASB, 2), one American Quarter Horse (AQHA),

one pony, and one American Miniature (AMH) Horse.

DNA Extraction

DNA from blood samples was extracted using Puregene whole

blood extraction kit (Gentra Systems Inc., Minneapolis, MN)

according to its published protocol. Hair samples submitted by

owners were processed using 5–7 hair bulbs according to the

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