lavendowe zrebaki.pdf

Whole-Genome SNP Association in the Horse:

Identification of a Deletion in Myosin Va Responsible for

Lavender Foal Syndrome

Samantha A. Brooks 1 *, Nicole Gabreski 1 , Donald Miller 2 , Abra Brisbin 3 , Helen E. Brown 2¤ , Cassandra

Streeter 1 , Jason Mezey 3 , Deborah Cook 4 , Douglas F. Antczak 2

1 Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, United States of America, 2 Baker Institute for Animal

Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America, 3Department of Biological Statistics and Computational Biology,

College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, United States of America, 4M. H. Gluck Equine Research Center, Department of Veterinary

Science, University of Kentucky, Lexington, Kentucky, United States of America

Abstract

Lavender Foal Syndrome (LFS) is a lethal inherited disease of horses with a suspected autosomal recessive mode of

inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The

condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on

comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A).

Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses

were genotyped using a newly available single nucleotide polymorphism (SNP) chip containing 56,402 discriminatory

elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon

sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading

frame and introduces a premature stop codon. A PCR–based Restriction Fragment Length Polymorphism (PCR–RFLP) assay

was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for

this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency

of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first

successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in

avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals.

Citation: Brooks SA, Gabreski N, Miller D, Brisbin A, Brown HE, et al. (2010) Whole-Genome SNP Association in the Horse: Identification of a Deletion in Myosin Va

Responsible for Lavender Foal Syndrome. PLoS Genet 6(4): e1000909. doi:10.1371/journal.pgen.1000909

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

Received November 16, 2009; Accepted March 15, 2010; Published April 15, 2010

Copyright: 2010 Brooks 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: Funding was generously supplied by The Morris Animal Foundation as part of the Equine Consortium for Genetic Research (Grant ID # : D07EQ-500,

http://www.morrisanimalfoundation.org/) and the Arabian Horse Foundation (http://www.arabianhorsefoundation.org/home.html). The Harry M. Zweig Memorial

Fund for Equine Research (http://www.vet.cornell.edu/public/research/zweig/) and the Dorothy Russell Havemeyer Foundation (http://www.havemeyerfoundation.

org/) also provided some support. 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: Samantha.Brooks@cornell.edu

¤ Current address: Department of Biological Sciences, Warwick University, Coventry, United Kingdom

neonatal conditions including neonatal maladjustment syndrome

and encephalitis [2]. The inheritance of Lavender Foal Syndrome

is suspected to be recessive, although extensive pedigree analysis

has not, to date, been published. Outwardly healthy horses can

sire lethally affected foals; therefore a recessive mode of

inheritance for LFS is most likely.

Historically developed by the Bedouin tribesman on the

Arabian Peninsula, the Arabian horse is one of the oldest

recognized breeds of horse. Valued for its beauty and athleticism,

the Arabian has contributed to the development of many light

horse breeds, most notably the Thoroughbred, a breed used

extensively in horse racing across the world [4]. The majority of

documented cases of Lavender Foal Syndrome have been reported

in the Egyptian Arabian, a sub-group of the Arabian breed found

originally in Egypt but extensively exported and popular in the

United States. Egyptian Arabians have their own registry,

although they are also part of the main Arabian studbook. It is

estimated that

Introduction

Heritable disorders affect many domestic species, including the

horse. In the Arabian breed of horse a neurological disorder has

been reported that is lethal soon after birth [1]. Affected foals can

display an array of neurological signs including tetanic-like

seizures, opisthotonus, stiff or paddling leg movements and

nystagmus (Figure 1) [2]. Mild leucopenia is sometimes observed

[2,3]. These neurologic impairments prevent the foal from

standing and nursing normally and, if not lethal on their own,

are often cause for euthanasia. In addition to these abnormalities,

affected foals possess a characteristic diluted ‘‘lavender’’ coat color.

This resulting coat color, variously described as pale gray, pewter,

and light chestnut, as well as lavender, has coined the name

‘‘Lavender Foal Syndrome’’ (LFS) [2]. Also called ‘‘Coat Color

Dilution Lethal’’ [2], there is currently no treatment for LFS

available. Additionally, initial diagnosis can be difficult as the

clinical signs of LFS can easily be confused with a number of

there are 49,000 living registered Egyptian

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A MYO5A Deletion Leads to Lavender Foal Syndrome

element SNP chip for rapid whole genome scanning (Equine

SNP50, Illumina, San Diego, CA). SNP-based whole genome

association studies have proven exceptionally successful when

studying simple mendelian traits in domesticated species. Two

notable examples can be found in studies of coat traits in the dog

[10] and recessive diseases of cattle [11].

Previously described mutations in mice and humans provide

several comparative phenotypes similar to Lavender Foal

Syndrome. Two genes in particular, Ras-associated protein

RAB27a (RAB27A) and myosin Va (MYO5A), yield phenotypes

with striking parallels to LFS. These two proteins, along with

melanophilin (MLPH) are part of a transportation complex

responsible for the trafficking of melanosomes to the periphery

of the cell where they are transferred to the keratinocyte (reviewed

in [12]). The myosin Va transport complex is also utilized in the

dendrite of the neuron where it has been shown to move various

cargo, including mRNAs, glutamate receptors, and secretory

granules [13,14]. Disruption of these diverse functions could

explain the constellation of defects observed in RAB27A and

MYO5A mutants. In mice, 71 mutations in MYO5A and 106 in

RAB27A have been recorded in the MGD database [15]. In

humans, several unique recessive mutations in these two genes

have been shown to cause similar disorders. The severity of the

phenotype, known as Griscelli syndrome, varies with the gene and

location of the mutation [16]. Griscelli syndromes have been

divided in to three categories based on the gene responsible;

MYO5A in type 1, RAB27A in type 2, and MLPH in type 3 [17].

There are subtle differences in the phenotype of each of these

subtypes. For example, RAB27A mutations in both human and

mouse disrupt granule exocytosis in T lymphocytes. This leads to

immunodeficiency and leukocyte infiltration in to vital organs,

including the brain. Thus, although neurological defects are often

present in RAB27A mutants they are usually secondary to this

infiltration [17]. In contrast, MYO5A mutants exhibit a primary

neurologic dysfunction and have normal immune function. Based

on this distinction MYO5A was chosen as the primary candidate

gene for Lavender Foal Syndrome.

Author Summary

Genetic disorders affect many domesticated species,

including the horse. In this study we have focused on

Lavender Foal Syndrome, a seizure disorder that leads to

suffering and death in foals soon after birth. A recessively

inherited disorder, its occurrence is often unpredictable

and difficult for horse breeders to avoid without a

diagnostic test for carrier status. The recent completion

of the horse genome sequence has provided new tools for

mapping traits with unprecedented resolution and power.

We have applied one such tool, the Equine SNP50

genotyping chip, to a small sample set from horses

affected with Lavender Foal Syndrome. A single genetic

location associated with the disorder was rapidly identified

using this approach. Subsequent sequencing of functional

candidate genes in this location revealed a single base

deletion that likely causes Lavender Foal Syndrome. From

a practical standpoint, this discovery and the development

of a diagnostic test for the LFS allele provides a valuable

new tool for breeders seeking to avoid the disease in their

foal crop. However, this work also illustrates the utility of

whole-genome association studies in the horse.

Arabians worldwide (personal communication, Beth Minnich,

Pyramid Society). Identifying the genetic basis of this condition

and developing a diagnostic test for the LFS allele will enable

breeders to make more informed selection of mating pairs, thus

avoiding the production of affected foals and potentially lowering

the frequency of this allele in the population, without wholesale

culling of valuable stock.

Over the past 15 years the Horse Genome Project has produced

several generations of analytical and diagnostic resources (genetic

tools) that permit interrogation of polymorphisms across the entire

equine genome [5,6]. Previous mapping efforts using ,300

microsatellite markers yielded results for several heritable diseases

(for examples see [7,8]). However, this small number of markers

limited genetic studies in the horse to simple traits in closely related

families with fairly large numbers of samples. The recently

completed 6.8x whole genome sequence of the horse and the

associated identification of approximately 1.5 million Single

Nucleotide Polymorphisms (SNPs) located throughout the horse

genomic sequence [9] has enabled the construction of a 56,402

Results

Pedigree Analysis

Pedigree data from the six affected foals available at the time of

genotyping supported a recessive mode of inheritance. A single

common ancestor was identified six to eight generations from

these six affected foals (Figure S1). This common ancestor is

present on both sides of the pedigree in each foal. This stallion

may represent a founder among this group and this convergence

in the pedigree supports identity by descent for the LFS mutation.

Average inbreeding (F i ) was 0.0861 for affected foals, versus

0.0394 for parents of foals. The extended pedigree also allowed for

the calculation of the coancestry coefficient between each living

relative and the nearest affected foal in the pedigree. Based on this

calculation we predicted that the frequency of the LFS allele would

be 0.42 among the 30 relatives used for genotyping.

Association Mapping

Genotypic association tests using the six affected foals and their

30 healthy relatives revealed a single region on chromosome 1

(ECA1) with statistical significance above that of the rest of the

genome (Figure 2). These 14 highly significant SNPs encompassed

a region spanning 10.5 Mb (ECA1:129228091 to 139718117).

Although extensive inbreeding and relatedness between affected

individuals produced a high number of coincidentally significant

(p,0.05) SNPs across the genome, the high peak significance of

Figure 1. A foal with Lavender Foal Syndrome demonstrating

opisthotonus, one cardinal neurological sign of the disorder.

(Photo courtesy of Dr. Yael Giora).

doi:10.1371/journal.pgen.1000909.g001

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A MYO5A Deletion Leads to Lavender Foal Syndrome

Figure 2. P-values ( , 0.05) for SNP association with Lavender Foal Syndrome. Individual chromosomes are represented with various colors

in numerical order. The region with the most significant P-values (highlighted with a yellow bar) falls on the p arm of ECA1.

doi:10.1371/journal.pgen.1000909.g002

SNPs in the candidate region (p = 4.62e-6) was convincing

evidence for association. In total there were 14 SNPs at this locus

that were more significantly associated with the LFS trait than any

other region in the genome. The twelve LFS bearing chromo-

somes from the six affected horses represented only four unique

haplotypes for this 10 Mb candidate region. These four haplotypes

possessed one large block of 27 SNPs in common. This 1.6 Mb

region was homozygous in all six affected horses and heterozygous

in obligate carriers as well as many of the living relatives, as was

predicted by the coancestry in the pedigree. The linkage

disequilibrium (LD) structure and p-values in this likely location

for a recessive mutation are plotted in Figure 3. Only 10 Ensembl

Gene Predictions fell within this region, including MYO5A, but not

RAB27A (UCSC Genome Browser [9]).

Population Structure

Genome-wide observed homozygosity from the genotypes

obtained using the EquineSNP50 chip was on average 65.14%.

This was much higher than expected considering the homozygos-

ity of the inbred mare chosen for whole genome sequencing was

estimated at only 46% [9]. The ten founder Egyptian Arabian

individuals from this study, as well as an additional 10 unrelated

individuals from the Thoroughbred, Arabian (non-Egyptian) and

Saddlebred breeds were used to calculate average genome-wide

Figure 3. Linkage disequilibrium plot of the homozygous region associated with LFS. Plotted at the top are the corresponding p-values

for these 27 SNPs. The yellow shaded box highlights the SNP closest to MYO5A. Red diamonds represent D’ values equal to 1, lower values of D’ are

given in within the boxes in shades of pink to white, while non-significant associations are blue.

doi:10.1371/journal.pgen.1000909.g003

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A MYO5A Deletion Leads to Lavender Foal Syndrome

LD (Figure S2). This calculation revealed that the length of LD in

the Egyptian was similar to that of the Thoroughbred, a breed

with a long history of a closed studbook and relatively small

foundation population. LD in the Egyptian was also much longer

than that of the Arabian population as a whole, which was most

similar to the Saddlebred. The Saddlebred breed registry was

closed in 1917 and derived from fairly diverse types of horse

suitable for use as transportation under saddle and in harness.

Candidate Gene Sequencing

Individual PCR amplification and sequencing of the 39 exons of

MYO5A from a LFS affected foal revealed three SNPs and one

polymorphic microsatellite in intronic sequence, as well as a single

base deletion in exon 30 of MYO5A (Table 1). This deletion was

further confirmed by sequencing in a second foal and its

heterozygous parents (Figure 4). The deletion is termed ECA1

g.138235715del per Human Genome Variation Society (http://

www.hgvs.org/mutnomen/) nomenclature. This deletion changes

the reading frame, creating a premature stop codon in the

translation of exon 30, 12 amino acids following the mutation. A

multiple alignment of the predicted LFS exon 30 amino acid

sequence, as well as the wild type sequence from eight species,

shows that this region of the myosin Va protein is highly conserved

(Figure S3). The four intronic polymorphisms were not predicted

to change the function of myosin Va and were therefore not

investigated further.

Figure 4. Discovery of the Lavender Foal Syndrome–associated

single base deletion by sequencing. Pictured are aligned

chromatograms from horses with each of the three genotypes. The

deletion occurs at the base marked with the vertical line, causing a

mixed sequence from this point forward in the carrier.

doi:10.1371/journal.pgen.1000909.g004

Association and Frequency Estimates

We designed a PCR-RFLP assay using the Fau I restriction

enzyme to detect this deletion (Figure S4). Digestion of the PCR

product produces a positive control fragment of 289 bp in all

genotypes. Presence of the deletion abolishes a Fau I site, changing

the normal pattern of a 386 bp and a 90 bp fragment in to a single

476 bp product. All seven affected foals (the six originally

submitted for mapping plus one additional obtained after mapping

was completed) were homozygous for the deletion (Table 2). Eight

out of the 14 parents of these affected foals were available for

sampling and all carried the deletion. Among 23 relatives of

affected foals 16 were identified as carriers of the deletion. A

sample group of 114 Arabian horses was tested to provide a rough

estimate of the frequency of the MYO5A exon 30 deletion, and

therefore Lavender Foal Syndrome, in the breed as a whole

(Table 3). 10.3% of Egyptian Arabians (six out of 58 horses) and

1.8% of non-Egyptian Arabians (one out of 56 horses) were

identified as carriers.

for a genetic disorder in the horse. We have described a frameshift

mutation in the MYO5A gene that leads to Lavender Foal

Syndrome in the Egyptian Arabian breed of horse. This task was

made more challenging by the small number (six) of DNA samples

from available affected foals. We improved our chances of success

by using pedigree data to select control samples from the extended

family and by utilizing a genotype association rather than allelic

association statistic in combination with identification of regions of

homozygosity.

The extreme predicted impact on function resulting from the

single base deletion in MYO5A exon 30 makes it a very logical

cause of LFS. Indeed, an alignment of MYO5A exon 30 amino acid

sequences from 8 diverse species shows that the exon is completely

conserved in horses, humans, mice, dogs and cattle and contains

only a few changes in the possum, chicken, and zebrafish (Figure

S3). As LFS affected foals do not have an immunodeficiency

consistent with RAB27A mutations, and the genomic region

containing this gene was not

inherited as predicted by our

recessive model,

is doubtful

that

this gene plays a role in

Discussion

Lavender Foal Syndrome.

The newly discovered deletion in exon 30 of MYO5A leads to a

frame shift and premature termination of transcription. Loss of the

Here we describe the first successful use of the EquineSNP50

genotyping platform in identification of the mutation responsible

Table 1. Polymorphisms identified in the MYO5A sequence.

Table 2. Association of Lavender Foal Syndrome with ECA1

g.138235715del.

HGVS Nomenclature

Within Gene Location

Type

Genotypes a

+ / +

+ / 2

2 / 2

g.138148824A . G

Intron 3 + 100

SNP

g.138168098G . A

Intron 6 + 189

SNP

Affected (Homozygotes)

g.138230294C . T

Intron 27–156

SNP

Parents (Carriers)

g.138235715del a

Exon 30 + 148

Frameshift

Other Relatives (Unknown Genotype)

g.138253441GT(10_12)

Intron 35–79

(GT) satellite

Total

(a- LFS associated polymorphism).

doi:10.1371/journal.pgen.1000909.t001

(a- ‘‘ + ’’ stands for the wild type while ‘‘ 2 ’’ indicates the deletion).

doi:10.1371/journal.pgen.1000909.t002

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A MYO5A Deletion Leads to Lavender Foal Syndrome

negatively impact the breeding career of high-priced stallions and

lead to large economic losses. This estimate also assumes mating at

random. In the case of the Egyptian Arabian horse this is not a

realistic assumption given the commonplace use of inbreeding and

line-breeding in the industry. The allele frequency for LFS of 5.2%

is not unlike the frequencies of other heritable diseases in various

breeds of horse [21,22].

We identified a conserved block of 1.6 Mb in common in the

four LFS bearing haplotypes. This is somewhat smaller than would

be expected considering the average rate of decay of LD across just

the six to eight generations that separate these four haplotypes.

Indeed, upon further research of the pedigrees from carriers

identified during screening for the LFS allele in our sample of 107

Arabian horses, we identified carriers who did not possess this

candidate founder in their pedigree. Therefore it is likely the true

founder of this mutation occurred far earlier. The appearance of a

more recent common ancestor is not surprising given the prolific

use of popular sires and the prevalence of

Table 3. Frequency of the Lavender Foal Syndrome

associated deletion among a sample of Arabian horses.

Genotypes a

+ / +

+ / 2

2 / 2

LFS Allele Freq.

Egyptian Arabian

0.052

Other Arabian

0.0089

Total

107

0.031

(a- ‘‘ + ’’ stands for the wild type while ‘‘ 2 ’’ indicates the deletion.

doi:10.1371/journal.pgen.1000909.t003

379 amino acids at the C-terminus of the protein, which encode a

portion of the secretory vesicle-specific binding domains of the

globular tail, would likely impair binding of myosin Va to those

cargo organelles bearing the appropriate receptors [18]. Although

this truncation leaves intact the melanocyte specific alternative

exon, exon F, it has been previously shown that binding function is

nonetheless destroyed without the cooperative action of down-

stream motifs [19]. Additionally, the quantity of MYO5A protein

may be significantly reduced, as is often observed in experimen-

tally truncated constructs of this gene [19]. The resulting loss of

vesicle traffic could easily interfere with the normal function of

melanocytes and neurons. The neurologic deficits exhibited by

LFS affected foals are relatively more severe than the symptoms

reported in human cases of Griscelli Syndrome, which are most

often due to changes in a single amino acid rather than loss of a

significant portion of the transcript [16]. However, in the mouse a

broad spectrum of phenotypes are observed, owing to the variety

of causative mutations available for study.

There is some speculation that a mild, survivable epileptic

condition of young foals may represent a non-lethal phenotype of

LFS carriers [2] as the two conditions are often seen in the same

pedigrees. However, this association has not been scientifically

validated and samples from horses diagnosed with this condition

were not available for study at this time. Based on comparative

phenotypes in the mouse this is a plausible scenario. Several

MYO5A alleles in the mouse, most notably mutations of the

globular tail region like d-n and d-n2J, exhibit neurological and

behavioral defects in juvenile homozygotes [20]. These deficits

improve with age and are often survivable, as has been described

in the rumored condition of the horse. Discovery of the mutation

responsible for LFS will enable future studies to evaluate

association of this allele with juvenile neurological dysfunction.

Our results suggest the population frequency of carriers of this

deletion is 10.3% in the Egyptian Arabian. It is possible that this

may be an over estimation of carriers, as owners who suspect they

have LFS carrying horses may have been more motivated to

participate in the study. However it is equally as likely that this

figure is an underestimation as there is social stigma associated

with producing LFS foals, thus motivating breeders to hide the

carrier status of their breeding stock. Despite strict policies

regarding the confidential nature of identifying information in

research projects, this still influences some breeders to avoid

association with Lavender Foal Syndrome research out of fear of

being rumored to own carrier horses. Notably, three of the six

carriers identified were reported to be breeding stallions. Data

from the Egyptian Arabian horse registry indicates that approx-

imately 850 young horses are registered each year (personal

communication, Beth Minnich, Pyramid Society). Given our

estimate of the number of carriers in the population we expect that

around nine LFS foals would be born in the US each year. This is

a small number; however rumors of carrier status can very quickly

inbreeding in this

population.

Prevention of the economic and emotional losses associated with

lethal conditions in foals, included those affected with LFS is a

high priority among Arabian breeders. The market for Egyptian

Arabian horses particularly values certain popular bloodlines. This

leads to close breeding as owners seek to increase the percentage of

this ancestry in their foal crop. This breeding strategy thus

increases the need for vigilant prevention of recessive genetic

disorders. The test developed here will be a pivotal tool for

breeders seeking to breed within lines segregating for LFS, yet

minimize or eliminate the production of affected foals.

Widespread application of the EquineSNP50 chip in genetic

research is just beginning. In the case of Lavender Foal Syndrome,

the limited availability of samples had impeded the progress of

research using existing mapping tools for many years. Although

whole genome association using large numbers of SNP markers is

heralded as a tool for complex, polygenic traits, here we have

shown that it can be very successfully applied to a simple trait in a

small number of individuals. This work is the first use of the

EquineSNP50 genotyping chip to successfully identify a causative

mutation. While whole genome association is often the tool of

choice for mapping complex traits and QTLs, we have

demonstrated that it can also be a much anticipated solution for

simple traits that face additional challenges in phenotyping and/or

sample number. Testing for the LFS allele will be a valuable aid to

breeders seeking to avoid losing foals while still using many of the

popular lines that may carry Lavender Foal Syndrome. As the

Arabian horse was used to develop many of the modern light horse

breeds it is possible that the LFS allele is present in these breeds as

well. In future work we will test additional sub-types of Arabian, as

well as a variety of light horse breeds to better assess the

population frequency in these groups. It is possible that LFS

segregates in these groups at a low frequency without detection, as

it is easy to confuse with other neonatal disorders of the foal.

Materials and Methods

Ethics Statement

Procedures in living animals were limited to the collection of

blood by jugular venipuncture or hairs pulled from the mane or

tail. Both procedures were conducted according to standard

veterinary protocol and inflict minimal, if any pain. All samples

were voluntarily submitted by horse owners and/or attending

veterinarians to the Antczak or Brooks laboratories according to

protocols approved by the Cornell Institutional Animal Care and

Use Committee protocol #1986-0216.

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