The Impact of the Mutation Causing Overo Lethal White Syndrome on White Patterning in Horses

The Impact of the Mutation Causing Overo Lethal

White Syndrome on White Patterning in Horses

Paul D. Vrotsos, BS, CVT; Elizabeth M. Santschi, DVM, Diplomate ACVS; and

James R. Mickelson, PhD

The mutation responsible for Overo Lethal White Syndrome usually results in frame overo white

patterning in carriers. In 10 –20% of carriers, this phenotype is unapparent due to suppression by

other color genes or because of fusion with additional white patterning genes. To be certain of

genotype in individuals, allele-speciﬁc PCR analysis must be performed. Breeders can use the

genotype information when planning matings to eliminate the possibility of producing a lethal white

foal. Authors’ addresses: Peterson and Smith Equine Hospital, 4747 SW 60

Ave, Ocala, FL,

34474 (Vrotsos); Department of Surgical Sciences, University of Wisconsin, School of Veterinary

Medicine, 2015 Linden Drive West, Madison, WI, 53706 (Santschi); and Department of Veterinary

AAEP.

1. Introduction

White patterning (large white patches on the body

and limbs) is a desirable trait in American Paint

Horses, Pintos, Appaloosas, and Miniature Horses.

It is an undesirable trait in the American Quarter

Horse. American Paint horses have irregular,

asymmetric white coat color patches on the body

that are distinct from the small, symmetrical white

spotting characteristic of the Appaloosa. The two

main types of white patterning recognized by the

American Paint Horse Association (APHA) are overo

and tobiano.

Overo horses are characterized by

white coloration on the abdomen that can extend to,

but does not cross, the dorsal midline between the

withers and tail. The heads of overo horses usually

have extensive white markings. In contrast, the

white body markings of tobiano horses cross the

dorsal midline, and the white on the head is most

often limited to stars, snips, and blazes. Horses

can be registered as toveros (or tob-overos) when

they have characteristics of both tobiano and

overo. Additionally, the APHA registers horses of

Paint, Quarter Horse, and Thoroughbred breeding

without white patterning and Paint horses that are

all white as breeding stock.

Many coat color genes have been identiﬁed in lab

animals that inﬂuence embryonic neural crest cells

that are the precursors of melanocytes.

2,3

In mice,

several genes control the location or amount of white

in the coat,

2,3

and additional genetic loci modify the

amount of white.

In Paint horses, different genes

are thought to control overo and tobiano white

patterning.

However, overo is a broad category of

white patterning, with several subtypes including

frame, calico, splashed white, and sabino.

Occasionally, all-white foals are produced from

Paint horse matings.

6,a

A small number of these

horses have normal gastrointestinal function and

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are registered as breeding stock white, but most are

affected by myenteric aganglionosis and fatal func-

tional intestinal obstruction.

This condition is

called overo lethal white syndrome (OLWS). Con-

ditions similar to OLWS occur in mice, rats, and

humans (Hirschprung’s disease).

8 –10

All rodent

conditions and some forms of Hirschprung’s in hu-

mans result from mutations in the endothelin recep-

tor B (EDNRB) or one of its ligands, endothelin

8 –10

The endothelin-signaling pathway is essen-

tial for the development and migration of neural

crest cells that ultimately form melanocytes and

enteric neurons.

2,11

It is now known that substitu-

tion of lysine for isoleucine at residue 118 of EDNRB

is responsible for OLWS in Paint horses,

and this

mutation has been associated with the parental

frame overo phenotype.

Most solid-colored horses

are homozygous for the Ile118 allele of EDNRB (wild

type), all parents of OLWS foals are heterozygous,

and all OLWS foals are homozygous for the Lys118

allele.

12–14

It has been suggested that all overo horses carry a

copy of the Lys118 allele.

If true, 25% of overo

breedings would result in the birth of an OLWS

affected foal. Paint horse breeders report that the

incidence of OLWS from overo breedings is much

lower, and that some overo stallions never produce

foals affected with OLWS. In a small breeding trial

conducted in 1978, 6 of 76 (7.9%) overo breedings

resulted in the birth of a foal affected with OLWS.

One explanation for the lower than 25% incidence of

foals with OLWS is that not all overo horses carry

the Lys118 allele, suggesting that overo white pat-

terning is the result of more than one gene. We

report here the EDNRB genotype and white pattern-

ing phenotype of 1000 horses to determine the inci-

dence of the Ile118Lys EDNRB and describe its

association with types of white patterning.

2. Materials and Methods

Horses

Genomic DNA was obtained from horses admitted to

the University of Minnesota Veterinary Teaching

hospital for treatment, from client samples submit-

ted for detection of the Lys118 allele, and from

breeders. The vast majority (⬎90%) of samples

were obtained from registered American Paint

horses. A few samples were obtained from Pintos

(white-patterned horses other than American Paint

Horse, Quarter Horse, or Thoroughbred), Thorough-

breds, Mustangs, and American Miniature Horses.

Paint Horse breeding farms tested included those

that had a high incidence of births of foals with

OLWS and those that had no OLWS. The solid-

colored horses tested were of Quarter Horse and

Thoroughbred breeding.

Genotyping

Genomic DNA was obtained from blood

or hair root

bulbs (3 roots in 20

␮

l of 200 mM NaOH for 15

minutes at 70°C, followed by neutralization with 20

␮

l of 200 mM HCl and 20

␮

l of 100 mM Tris, pH

8.5). The EDNRB alleles were detected by use of

allele-speciﬁc PCR, as described,

allowing each

horse to be genotyped as homozygous for Lys118 or

Ile118, or as heterozygous.

Phenotyping

1,5,15

Horses were phenotyped by 2 authors (PDV, EMS)

who observed the live horse or pictures taken of both

sides of the horse including facial markings.

Horses without white patterning that did not de-

scend from white-patterned lines were designated as

solid. Horses of white-patterned bloodlines were

ﬁrst classiﬁed phenotypically by use of the American

Paint Horse color classiﬁcations of tobiano, overo,

tovero, or breeding stock. Overo horses were fur-

ther classiﬁed into 6 subtypes, and breeding stock

horses were classiﬁed as solid breeding stock or

white breeding stock.

Tobiano

White markings on the head are similar to solid-

colored horses (Fig. 1). Typically, all 4 limbs are

white. Body markings are distinct and regular, of-

ten with round or oval patterns. Flanks are dark

and the tail is often of two colors. White patches

are often vertically oriented, cross the dorsal mid-

line, and can appear draped over shoulders.

Overo

The head is often extensively marked with white

(Fig. 2). Typically, at least 1 limb is dark. White

markings vary from distinct crisp white patches on

the body to large irregular or roan areas. White

coloration does not cross the back between the with-

ers and tail. The tail is one color.

Tovero

These horses have tobiano and overo characteristics.

The overo characteristic is most commonly apparent

Fig. 1. A tobiano.

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on the head, but can also be expressed by irregular

body markings.

Breeding Stock

These are solid-colored horses of Paint lineage, in-

cluding American Miniature Horses. Horses were

subtyped as breeding stock white if they were adult

all-white horses of Paint breeding.

Subtypes of Overo

Horses that ﬁt the general description of overo have

great variation in color pattern that allows fur-

ther division into 6 distinct phenotypic subtypes:

frame, calico, splashed white, sabino, medicine hat,

and bald-faced.

Frame

A frame is an overo horse with body spots on the

lateral aspect of the body (neck, shoulder, abdomen,

ﬂank, and hip) (Fig. 2). Body spots are distinct,

with sharp borders, and do not connect with limb

markings. At least 1 limb (often more) is dark.

White limb markings do not usually extend up the

carpus or tarsus. The head is usually extensively

marked with white, which often extends laterally to

the eyes. White spots that are primarily on the

ventral midline or only involve the axilla are not

typical of frame horses, especially when associated

with extensively marked white legs.

Calico

A calico is an overo horse with white markings that

are scattered and have irregular borders (Fig.

3). One or more limbs are white, and the white

limb markings have narrow extensions up the limb.

White markings on the limbs often connect to white

markings on the body. Head markings are irregu-

lar and may be wide, but do not usually involve the

eyes. Calico horses have the widest variation in

the amount of white patterning. White-patterned

horses without white patterning on the body but

with irregular white limb markings extending above

the tarsus and carpus were considered minimally

marked calico overos. Horses with irregular limb

markings above the tarsus and carpus and irregular

white body patches on ⬍75% of their body were

considered calico overos, and horses with irregular

limb markings above the tarsus and carpus and

irregular white markings on ⬎75% of their body

were considered loud calico overos.

Splashed White

A splashed white horse is an overo horse with ex-

tensive white markings on the head and large, crisp

white patches on the ventral aspect of the neck and

thorax that often connect with extensive white

patches on the forelimbs (Fig. 4). The hind limbs

also have white markings that extend dorsally above

the tarsus, and the ventral aspect of the abdomen is

Fig. 3. A loud calico overo.

Fig. 4. A splashed white overo.

Fig. 2. A frame overo.

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white. A splashed white horse is often described as

a horse dipped in white.

Sabino

A sabino is an overo horse with one or more white

limbs and white markings on the face (Fig. 5). An

extensive area of roan coloring (mixing of white

hairs within colored patches) is the major character-

istic of sabinos. Colored areas are also irregularly

shaped and ﬂecked with white that blends with

small white patches.

Bald-Face

These are overo horses with white face markings

that extend ventral to a line that connects the point

of the mouth and the poll. The body coloring is

solid, and white limb markings distal to the tarsus

and carpus may be present.

Medicine Hat

This is a rare color pattern in which the coat is

almost entirely white (Fig. 6). Pigmented areas are

found primarily on the ears and poll, but may also

appear on the thorax, ﬂank, dorsal midline, and tail

head. Medicine hat horses can arise from overo or

tovero bloodlines; when of overo bloodlines, medicine

hat horses may have pigment that is quite faint on

the dorsal midline.

Overo Blends

Overo blends are those overo horses that have evi-

dence of two or more overo white patterns (Fig.

7). There are two recognizable subdivisions of

overo blends. The most common is the frame blend,

which has frame characteristics such as lateral body

spots, solidly colored limbs, and wide blazes, in ad-

dition to patterning of another overo subtype, which

is usually calico. Less common are the other (non-

frame) overo blends, which have two other types of

overo patterning.

OLWS

Foals affected with OLWS are almost entirely white

(Fig. 8). In most foals with OLWS, pigmentation is

restricted to the retina. However, small pigmented

areas may be present, usually in the mane or tail.

Nonpaint

These are registered Thoroughbreds and Quarter

Horses that do not have excessive white on their

faces or white limb markings proximal to the carpus

or tarsus.

Statistics

Data were summarized by use of two-way tables.

Horses were categorized by pattern versus 3 geno-

types (homozygous Ile118, homozygous Lys118, and

heterozygous). Chi-square analysis was used to

test for association between pattern and genotype.

Subtables were analyzed by use of chi-squared anal-

Fig. 5. A sabino overo.

Fig. 7. A frame blend overo.

Fig. 6. A medicine hat overo.

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ysis or the Fisher exact test. Signiﬁcance was es-

tablished as p ⬍ 0.05.

3. Results

All foals with OLWS were homozygous for the

Ile118Lys EDNRB mutation, and adults that were

homozygous were not found. White patterning was

strongly associated with EDNRB genotype (p ⬍

0.001). Color patterns with highest incidence

(⬎94%) of heterozygotes (carriers) were frame overo,

highly white calico overo, and frame blend overo.

White-patterned bloodlines with lowest incidence of

carriers (⬍21%) were tobiano, sabino, minimally

white calico overo, splashed white overo, nonframe

blend overo, and breeding stock solid. The muta-

tion was not detected in solid-colored horses from

breeds without white patterning (Table 1). Looked

at from another perspective, the white patterns ap-

peared to segregate into three groups based on their

incidence of carriers (Fig. 9). In the group where

the gene was very common, it appears to be essential

for the production of that pattern. In those color

patterns with a middle incidence of carriers, the

gene was non-essential and did not interfere with

the production of that pattern, and in color patterns

where the incidence was low, the gene is not essen-

tial and may be deleterious to the production of that

phenotype.

4. Discussion

Of all frame and frame-blend overos tested in the

study reported here, 96% possessed the Lys118 al-

lele, strongly supporting the conclusion that in the

heterozygous state, the Ile118Lys EDNRB mutation

commonly causes the frame phenotype. However,

calico overos with a large amount of white coloring

had 100% incidence of the Lys118 allele, indicating

that these largely white calico horses are actually

frame blends. The frame patterning is unapparent

because it magniﬁes the calico white pattern. Ad-

ditionally, some heterozygote horses for the Lys118

allele do not express the frame overo pattern.

Causes for non-frame phenotype heterozygotes in-

clude fusion with other white patterns such as tobi-

anos (10% heterozygotes), splashed white overos

(12%), calico overos (55%), and sabino overos

(20%). Another cause is variable expression of the

frame phenotype (bald-faced and breeding stock solid),

possibly because of the inﬂuence of other genes.

Breeding stock horses of American Paint Horse lin-

eage that carry the Lys118 allele always have some

white markings, but 2 heterozygote American Min-

iature horses had no white markings, suggesting

that there is a modiﬁer gene present in the Ameri-

can Miniature horse population that strongly sup-

presses white patterning.

In addition to the nonframe heterozygotes, there

are a few horses of apparent frame phenotype (5% of

frame overos) that do not carry the Lys118 al-

lele. The most common marking on these horses is

a single distinct white patch on the ventral aspect of

the abdomen that extends laterally up the frame.

Variable expression of other white patterning genes

is most likely the cause for these patterns. For

example, splashed white horses have white body

patches with sharp borders, and minimal expression

of this pattern could look like frame patterning.

There appears to be 4 distinct overo patterns:

frame, calico, splashed white, and sabino, and these

subtypes of overo are under the control of different,

independently assorting genes.

Our data support

that hypothesis. The Lys118 allele causes the

frame pattern, and splashed white, calico, and

Table 1. Distribution of Genotypes for Endothelin Receptor B in 945

White-Patterned Horses and 55 Solid-Colored Horses

Color Pattern

Number of

Horses N/N N/L (%) L/L

Overos

Frame 188 10 178 (95) 0

Calico Loud 37 0 37 (100) 0

Calico 38 17 21 (55) 0

Minimal 67 61 6 (9) 0

Splashed White 26 23 3 (12) 0

Sabino 15 12 3 (20) 0

Bald faced 17 11 6 (35) 0

Medicine Hat 13 4 9 (69) 0

Overo Blends

Frame blend 158 5 153 (97) 0

Non-frame blend 14 14 0 (0) 0

OLWS foals 28 0 0 (0) 28

Tobiano 109 98 11 (10) 0

Tovero 84 35 49 (58) 0

Breeding stock

Solids 146 120 26 (18) 0

All-white 5 1 4 (80) 0

Solid-colored horses 55 55 0 (0) 0

N/N ⫽ wild type genotype; N/L ⫽ heterozygote; L/L ⫽ homozy-

gous for OLWS mutation.

Fig. 8. A frame overo mare and her overo lethal white foal.

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sabino horses have a signiﬁcantly different inci-

dence of the Lys118 allele, indicating a distinct ge-

netic basis. Additionally, these 4 overo patterns

commonly appear blended, as would be expected

from independently segregating overo genes. When

combined with other overo white patterns, the

Lys118 allele appears to add more white on the head

and frame of the body. This is best seen in calico

overos in which the percentage of heterozygotes in-

creases from 9% in minimally white calicos, to 55%

in moderately white individuals, to 100% in the

whitest calicos.

Tobiano is known to have a genetic basis distinct

from overo, and there is low incidence (10%) of the

Lys118 allele in these horses. However, if breeders

choose a tobiano mate for a horse that is heterozy-

gous for Lys118 to avoid OLWS, they will not always

achieve their goal. Our experience is that tobiano

horses are only heterozygous for the Lys118 allele

when their pedigree contains overo horses. The

18% incidence of heterozygotes in breeding stock

solid horses again suggests that the frame pheno-

type either has variable expression or may be sup-

pressed by other genes. Nonframe blends are

horses that have two overo patterns other than

frame, most commonly sabino and calico. Their ex-

istence and lack of heterozygotes among them sup-

ports the hypothesis that overo horses are

genetically heterogeneous.

Additional support for genetic heterogeneity of

overo patterning can also be inferred from the ⬍25%

incidence of OLWS foals from overo breeding.

the study reported here, 73% percent of all overo and

overo blend horses were heterozygous for the Lys118

allele. If our samples were representative of overos

in general, the predicted incidence of births of foals

affected by OLWS in overo ⫻ overo breeding would

be 13.3% (i.e., 0.73 ⫻ 0.73 ⫻ 25%). This ﬁgure

agrees with anecdotal reports from breeders of Paint

horses, but is slightly higher than the ﬁgure of 7.9%

detected in a small breeding trial.

Our ﬁnding

that 27% of overo horses did not possess the Lys118

allele explains why some overo breedings never pro-

duce foals affected by OLWS.

A few white-patterned horses of breeds other than

American Paint horses (Thoroughbreds, American

Miniature Horses, and half Arabians) were het-

erozygous for the Lys118 allele. This may be delib-

erate, because breeders outcross to American Paint

Horses to add white patterning. When heterozy-

gotes are used for outcrossing, this gene will be

introduced into the breed, along with the possibility

of producing foals affected by OLWS. However, the

appearance of the Lys118 allele in breeds unrelated

Fig. 9. EDNRB genotype associations among horses tested. White patterns on the x-axis have a similar incidence of heterozygotes

as white patterns on the y-axis where they share a color bar.

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to American Paint Horses is more difﬁcult to ex-

plain. Undocumented outcrosses to white-pat-

terned horses are a possibility. De novo mutations

have also been suggested to be responsible for the

birth of white-patterned horses from solid par-

ents,

13,16

but this is yet unproven. We believe that

these horses may also result from white patterning

genes in solid breeds that are minimally expressed

because of variable expression or suppression by

other genes. If this is true, the Lys118 allele en-

tered these breeds many years ago.

Of greater importance, at least to some breeders,

is the occurrence of white patterning in Quarter

Horses. Foals with excessive white markings born

to registered Quarter Horse parents are known as

crop-outs, and are ineligible for registration by

the American Quarter Horse Association. It is

known

or suggested by breeding results

that at

least some crop-out Quarter Horses are heterozy-

gous for the Lys118 allele. This study tested 25

Quarter Horses that were either crop-outs or par-

ents of a white-patterned horse, and did not ﬁnd any

heterozygotes, lending further support to the con-

cept that other genes control overo coloration. As

genetic testing becomes available for more overo

genes, testing of crop-outs and their parents will

help determine whether the crop-out white pattern-

ing is the result of de novo mutations or whether

white patterning genes exist in solid-colored Quar-

ter Horses.

5. Conclusion

The Ile118Lys EDNRB mutation causes OLWS

when a foal inherits a copy from each parent. Car-

riers of the mutation most commonly exhibit a frame

overo phenotype, but the frame pattern can be sup-

pressed or combined with other white patterns,

making accurate estimation of EDNRB genotype

by visual inspection difﬁcult. Determination of

EDNRB genotype by use of a DNA test is the only

way to determine with certainty whether white-

patterned horses can produce a foal affected with

OLWS. If carrier-to-carrier matings are prevented,

breeders can eliminate the production of lethal

white foals.

Supported by the Minnesota Agricultural Experi-

ment Station.

The authors thank Judith L. Morris, L. A. Feich-

tinger, A. Purdy, and Drs. J. Hurtgen, J. S. Juzwiak,

M. Ketchum, C. Landa, N. McEachern, and S. J.

Valberg for technical assistance, and Drs. Murray K.

Clayton and Ryland B. Edwards for statistical

analysis.

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