Ocular Anomalies.pdf

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Mamm Genome (2011) 22:353–360
DOI 10.1007/s00335-011-9325-7
Targeted analysis of four breeds narrows equine Multiple
Congenital Ocular Anomalies locus to 208 kilobases
Lisa S. Andersson • Katarina Lyberg • Gus Cothran •
David T. Ramsey • Rytis Juras • Sofia Mikko •
Bj ¨rn Ekesten • Susan Ewart • Gabriella Lindgren
Received: 29 November 2010 / Accepted: 8 March 2011 / Published online: 5 April 2011
The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract The syndrome Multiple Congenital Ocular
Anomalies (MCOA) is the collective name ascribed to
heritable congenital eye defects in horses. Individuals
homozygous for the disease allele (MCOA phenotype)
have a wide range of eye anomalies, while heterozygous
horses (Cyst phenotype) predominantly have cysts that
originate
peripheral retina. MCOA syndrome is highly prevalent in
the Rocky Mountain Horse but the disease is not limited to
this breed. Affected horses most often have a Silver coat
color; however, a pleiotropic link between these pheno-
types is yet to be proven. Locating and possibly isolating
these traits would provide invaluable knowledge to scien-
tists and breeders. This would favor maintenance of a
desirable coat color while addressing the health concerns of
the affected breeds, and would also provide insight into the
genetic basis of the disease. Identical-by-descent mapping
was used to narrow the previous 4.6-Mb region to a 264-kb
interval for the MCOA locus. One haplotype common to
four breeds showed complete association to the disease
(Cyst phenotype, n = 246; MCOA phenotype, n = 83).
Candidate genes from the interval, SMARCC2 and IKZF4,
were screened for polymorphisms and genotyped, and
segregation analysis allowed the MCOA syndrome region
to be shortened to 208 kb. This interval also harbors
PMEL17, the gene causative for Silver coat color. How-
ever, by shortening the MCOA locus by a factor of 20, 176
other genes have been unlinked from the disease and only
15 genes remain.
from
the
temporal
ciliary
body,
iris,
and/or
Electronic supplementary material The online version of this
article (doi: 10.1007/s00335-011-9325-7 ) contains supplementary
material, which is available to authorized users.
L. S. Andersson K. Lyberg S. Mikko G. Lindgren ( & )
Department of Animal Breeding and Genetics, Swedish
University of Agricultural Sciences, Undervisningsplan 4A,
P.O. Box 7023, 750 07 Uppsala, Sweden
e-mail: Gabriella.Lindgren@slu.se
G. Cothran R. Juras
Department of Veterinary Integrative Biosciences, College
of Veterinary Medicine and Biomedical Sciences, Texas A&M
University, College Station, TX 77843-4458, USA
D. T. Ramsey
The Animal Ophthalmology Center, PLLC, 1300W.
Grand River Avenue, Williamston, MI 48895, USA
B. Ekesten
Department of Clinical Sciences, Swedish University
of Agricultural Sciences, P.O. Box 7054, 750 07 Uppsala,
Sweden
Introduction
Multiple Congenital Ocular Anomalies (MCOA) syndrome
is a congenital nonprogressive syndrome described in
horses. The most frequent feature of this disease is fluid-
filled cysts of variable sizes (2–20 mm) in the posterior iris
and ciliary body epithelium within the eye. Two distinct
ocular phenotypes exist: (1) cysts that originate from the
posterior iris, temporal ciliary body, and/or peripheral ret-
ina (Cyst phenotype), and (2) cysts in combination with
additional
S. Ewart
Department of Large Animal Clinical Sciences, College of
Veterinary Medicine, Michigan State University, East Lansing,
MI 48824, USA
Present Address:
K. Lyberg
Unit of Clinical Immunology and Allergy, Karolinska University
Hospital, L2:04, 171 76 Stockholm, Sweden
ocular
defects
including
iridocorneal
angle
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354
L. S. Andersson et al.: Equine MCOA locus
abnormalities, cornea globosa, iris hypoplasia, congeni-
tal cataracts, lens subluxation, focal areas of retinal
detachment, microphthalmia, and macropalpebral fissures
(MCOA phenotype). Horses with MCOA have abnormal
pupillary light reflexes and pupils do not dilate after
administration of mydriatic drugs (Ramsey et al. 1999a , b ).
Individual MCOA-affected horses may or may not have the
complete set of congenital defects described above. Both
the distinct subdivision of phenotypes and the transmission
of the disease within our pedigrees are consistent with a
mutant allele displaying incomplete dominance. The Cyst
horses are heterozygous and have an intermediate pheno-
type compared to the horses with multiple anomalies that
carry two copies of the disease allele (Ewart et al. 2000 ;
Andersson et al. 2008 ).
Conditions have been favorable for the mutation causing
MCOA to be enriched in the Rocky Mountain Horse breed
(Ewart et al. 2000 ). This breed originated from a very lim-
ited number of founder horses, which were used extensively
to develop the breed. In fact, many Rocky Mountain horses
can be traced back to a single foundation stallion. The horses
within the breed have been selected for a distinctive four-
beat gait and the Silver coat color has been highly favored.
The fact that an intensive selection process can lead to
amplification of undesirable traits has been demonstrated in
several other horse breeds [e.g., hyperkalemic periodic
paralysis (Rudolph et al. 1992 ), hereditary equine regional
dermal asthenia (Tryon et al. 2007 ), severe combined
immunodeficiency (Shin et al. 1997 ), and Overo Lethal
White Syndrome (Santschi et al. 1998 )]. In the Rocky
Mountain Horse breed, selection of horses with the highly
desirable Silver coat color has simultaneously increased
MCOA syndrome as these traits are linked on horse chro-
mosome 6 (Andersson et al. 2008 ). The Silver coat color in
horses is characterized by dilution of black pigment in the
hair and revealed to be associated with a missense mutation
in pre-melanosomal protein 17 or PMEL17 (Brunberg et al.
2006 ). Additional horse breeds that have been diagnosed
with MCOA include the Kentucky Mountain Saddle Horse,
Mountain Pleasure Horse (both closely related to the Rocky
Mountain Horse), Belgian Draft, Morgan Horse, Shetland
Pony, American Miniature Horse (Ramsey et al. 1999a ;
Grahn et al. 2008 ; Komaromy and Rowlan 2009 ), and the
Icelandic Horse (B. Ekesten, unpublished).
Cysts are found in most affected horses and are usually
bilateral. Horses with cysts usually have normal functional
vision irrespective of cyst size since cysts are either
translucent or lightly pigmented. A small number of
juvenile horses that have cornea globosa as a component of
multiple ocular defects have a considerable refractive error
and subsequently poor vision. This refractive error is cor-
rected
The primary objective of this research was to identify
the gene and mutation that regulate the MCOA syndrome
in horses. The results would have practical implications
such as enable genetic testing for precise diagnosis of
carrier horses and horses with the multiple eye defects.
We previously mapped the locus for equine MCOA to a
4.9-Mb interval on horse chromosome 6 by linkage map-
ping (Andersson et al. 2008 ). In the present study we have
significantly limited the interval for the MCOA locus and
selected two additional candidate genes in the interval,
IKZF4 and SMARCC2, for mutation screening. IKZF4 was
selected as a candidate gene because it is expressed in
ocular tissue, is a member of the IKAROS family of
transcription factors, and interacts with the gene micro-
phthalmia-associated transcription factor, MITF (Hu et al.
2007 ). MITF influences both pigmentation and ocular
development (Hodgkinson et al. 1993 ). Mutations in MITF
can cause Waardenburg syndrome type 2A (Nobukuni
et al. 1996 ), an auditory-pigmentary disorder that affects
ocular development. In addition, a mutation in the PAX3
gene causes Waardenburg syndrome type 1, and it has been
suggested that it occurs through a failure to regulate MITF
(Watanabe et al. 1998 ). The other candidate gene,
SMARCC2, was selected because it encodes a 170-kDa
protein that is one of 12 proteins in the SWI/SNF complex
(Dechassa et al. 2008 ). The ATPase component Brm of this
complex regulates differentiation of early retinal stem cells
(Das et al. 2007 ). In humans, the SWI/SNF chromatin
remodeling complex is involved in the regulation of the
CRYAB (ab-crystallin) gene that is highly expressed in the
vertebrate lens where cataracts are commonly associated
with crystalline protein deficiencies (Liu et al. 2001 ;
Duncan and Zhao 2007 ). The present study defines a
208-kb genomic interval for the MCOA locus by identical-
by-descent (IBD) mapping of multiple horse breeds.
Materials and methods
Horse material
Four hundred sixty-five horses were genotyped in this study
(Table 1 ), including 362 Rocky Mountain horses, 57
American Miniature horses, 22 Kentucky Mountain Saddle
horses, and 24 Icelandic horses. All Rocky Mountain horses
evaluated in this study were derived from four half-sibling
families and have been described previously (Ewart et al.
2000 ). The distributions for phenotypes of Rocky Mountain
horses were 72 with MCOA, 222 with Cyst phenotype, and
68 unaffected horses. Distribution of phenotypes for the
remaining breeds
by
emmetropization
as
the
juvenile
horse
eye
was
as
follows:
American
Miniature
Horse:
3
MCOA,
14
Cyst,
40
unaffected;
Kentucky
achieves adult size (Ramsey et al. 2000 ).
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L. S. Andersson et al.: Equine MCOA locus
355
Mountain Saddle Horse: 4 MCOA, 4 Cyst, 14 unaffected;
and Icelandic Horse: 4 MCOA, 14 Cyst, 6 unaffected.
Profiler ver. 2.4 (Amersham Bioscience, GE Healthcare
Bio-Science Corp.). Parentage testing was performed
according to standard procedures (17 microsatellite mark-
ers, Equine Genotypes TM Panel 1.1, Finnzymes Oy, Espoo,
Finland)
Phenotype assessment
at
the
Animal
Genetics
Laboratory,
Swedish
Eye examinations were performed as described previously
(Ramsey et al. 1999a ). Briefly, direct and indirect pupillary
light reflexes were first assessed. Following pharmacologic
mydriasis with 1% tropicamide administered topically, a
complete ophthalmic examination consisting of slit lamp
biomicroscopy, indirect ophthalmoscopy, and applanation
tonometry was performed. The study was approved by the
Michigan State University Institutional Animal Use and
Care Ethics Committee for all breeds except the Icelandic
Horse; for that breed, the study was approved by the Ethics
Committee for Animal Experiments in Uppsala, Sweden.
University of Agricultural Sciences, Uppsala.
SNP genotyping
Six single nucleotide polymorphism (SNP) markers were
selected in regions lacking microsatellite markers. Custom
TaqMan SNP Genotyping assays [Applied Biosystems
(ABI), Foster City, CA] were used for genotyping five of
the SNPs. Probe and primer designs were obtained from the
cadt/ ) using the custom genotyping assays order option.
The ABI PRISM 7900 HT sequence detection system for
384-well format (ABI) was used for the analysis. The six
selected SNP markers were genotyped in 123–465 horses
(Table 2 ). One additional SNP was investigated in 33
horses by traditional Sanger sequencing (see the next
subsection). The PMEL17 SNP in exon 11 was interrogated
using pyrosequencing as described previously (Andersson
et al. 2008 ).
Microsatellite genotyping
Fourteen microsatellite markers spanning approximately
12 Mb on ECA6q were used for genotyping (Table 2 ,
Supplementary File 1). Seven of these markers were
identified previously (TKY570, TKY412, TKY284, UPP5,
UPP6, UPP7, and TKY952), while seven were novel and
developed from the horse genome sequence (EquCab2)
(UCSC Genome Browser, http://genome.ucsc.edu/ ) by
displaying tracks from simple tandem repeats recognized
by Tandem Repeats Finder (Benson 1999 ). Initially,
genotyping was done on a limited number of horses for a
wide interval. Subsequently, an increased number of
horses were genotyped for a smaller region that was
approximately 2–3 Mb wide. After identifying recombinant
horses, further marker genotyping focused on these sam-
ples. Table 2 summarizes the number of horses that were
genotyped for each marker. Primers for novel microsatel-
lites were designed using Primer3 (Rozen and Skaletsky
2000 ). PCR reactions were performed as described previ-
ously (Andersson et al. 2008 ). Amplified fragments were
multiplexed when possible and separated using a Mega-
BACE TM 1000 instrument (GE Healthcare Bio-Science
Corp., Piscataway, NJ) according to the manufacturer’s
recommendations. Results were analyzed using Genetic
Sanger sequencing of SMARCC2 and IKZF4
Two candidate genes, SMARCC2 and IKZF4, within the
identified genomic interval were sequenced using the
Sanger method. Three horses with MCOA and two unaf-
fected controls were used to sequence SMARCC2, while
two MCOA horses and one unaffected control were used
for sequencing IKZF4. The generated DNA sequences
were also compared with the horse reference genome
(Wade et al. 2009 ). Since none of the genes are well
annotated in the horse genome, exon number, size, and
position were deduced by displaying the track ‘‘None-
Horse mRNA’’ from GenBank. Sequencing primers were
designed to amplify 500–700-bp fragments using Primer3
(Rozen and Skaletsky 2000 ). Primer sequences are listed in
Supplementary File 1. All exons, as well as some introns,
the untranslated regions (UTRs), and certain evolutionary
Table 1
Number of horses investigated in the present study
Breed
MCOA phenotype
Cyst phenotype
Unaffected
Unaffected (Silver)
Total
Rocky Mountain Horse
72
222
59
9
362
Kentucky Saddle Horse
4
4
13
1
22
American Miniature Horse
3
14
37
3
57
Icelandic Horse
4
14
3
3
24
Total
83
254
112
16
465
The unaffected horses that are Silver represent the nonpenetrance horses in this study
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356
L. S. Andersson et al.: Equine MCOA locus
Table 2
Genotyping results for 21 markers among horses grouped according to phenotypic status
Position
Marker
Total
MCOA
Cyst
Unaffected
n
n
f (MM)
f (Mm)
f (mm)
n
f (MM)
f (Mm)
f (mm)
n
f (MM)
f (Mm)
f (mm)
66793555
TKY570
82
26
0.31
0.62
0.08
35
0.11
0.57
0.31
21
0.00
0.43
0.57
70589359
TKY412
178
41
0.29
0.44
0.27
105
0.03
0.43
0.54
26
0.00
0.04
0.96
72902566
MS18
433
74
0.27
0.39
0.34
231
0.02
0.46
0.52
120
0.01
0.19
0.80
73607795
MS1
434
75
0.85
0.13
0.01
231
0.22
0.71
0.08
120
0.03
0.27
0.71
73640494
N1
33
18
0.78
0.22
0.00
3
0.00
1.00
0.00
12
0.83
0.17
0.00
73658168
MS14
428
79
0.97
0.03
0.00
230
0.45
0.55
0.00
114
0.26
0.40
0.33
73665305
Pmel17-ex11
462
83
0.98
0.02
0.00
245
0.00
1.00
0.00
125
0.00
0.13
0.87
73722621
MS3
453
83
0.98
0.02
0.00
238
0.08
0.92
0.00
124
0.02
0.25
0.73
73726092
8345
135
34
0.97
0.03
0.00
30
0.43
0.57
0.00
52
0.37
0.48
0.15
73768488
TKY284
365
64
0.97
0.03
0.00
189
0.06
0.94
0.00
105
0.00
0.15
0.85
73788011
8377
129
32
0.97
0.03
0.00
40
0.70
0.30
0.00
50
0.64
0.32
0.04
73835084
MS13
447
77
0.97
0.03
0.00
234
0.03
0.97
0.00
124
0.01
0.15
0.85
73904952
8453
123
31
0.65
0.29
0.06
39
0.03
0.56
0.41
50
0.10
0.56
0.34
73968182
8475
130
33
0.67
0.27
0.06
45
0.02
0.60
0.38
49
0.00
0.24
0.76
74029118
MS21
392
71
0.82
0.14
0.04
198
0.37
0.57
0.06
115
0.19
0.51
0.30
74063248
4963
123
32
0.66
0.28
0.06
34
0.03
0.56
0.41
50
0.10
0.56
0.34
74667009
UPP6
422
71
0.86
0.14
0.00
228
0.02
0.91
0.07
115
0.00
0.10
0.90
75475234
UPP7
340
69
0.93
0.07
0.00
209
0.24
0.75
0.01
54
0.15
0.43
0.43
76228564
UPP8
184
46
0.85
0.15
0.00
101
0.06
0.89
0.05
37
0.00
0.16
0.84
78856446
MS4
91
32
0.66
0.31
0.03
36
0.50
0.44
0.06
23
0.61
0.35
0.04
79472875
TKY952
83
26
0.58
0.42
0.00
36
0.11
0.61
0.28
21
0.05
0.29
0.67
The 264-kb associated haplotype is marked in bold. The disease allele is depicted as M and an alternative allele as m. Sixteen nonpenetrance
horses are included as unaffected individuals
conserved elements were sequenced (Supplementary File
1). Sequencing reactions were carried out as described
previously (Brunberg et al. 2006 ) and results were analyzed
using CodonCode software (CodonCode Aligner ver.
1.6.3, CodonCode Corporation, Dedham, MA). Consensus
sequences were deposited in GenBank (accession Nos.
HQ331540
data of chromosomes that carry the mutation is depicted in
Table 3 . Forty-eight percent of the individual genotypes
were inferred based on surrounding marker information.
This was performed only when the haplotype could be
assessed with certainty. In total, a combination of 457
affected and unaffected horses of four breeds were used to
define the interval (see schematic figure in Supplementary
File 2). This corresponds to 409 analyzed chromosomes
that carry the MCOA mutation (including horses with both
the MCOA and the Cyst phenotype). Of 83 MCOA horses
included in this study, 81 were homozygous for the dis-
ease-associated haplotype; the remaining two horses were
diagnosed clinically with MCOA but carried only one copy
of the disease haplotype. Of the 254 horses with the Cyst
phenotype, 245 were heterozygous for the disease-associ-
ated haplotype and one horse had two copies of the disease
haplotype. The remaining eight horses had individually
unique haplotypes (see Supplementary File 2). These
horses were parentage-tested to determine whether DNA
and/or blood sample error had occurred. However, par-
entage results were accurate. Our genetic data also revealed
16 horses in which the Cyst genotype showed nonpente-
trance. These horses represent only 6% of all genetically
defined heterozygous horses.
and
HQ331541
for
SMARCC2
and
IKZF4,
respectively).
Results
Haplotype analysis
Genotyping of 14 microsatellite and 7 SNP markers firmly
identified a 264-kb genomic interval for the MCOA locus
(Tables 2 , 3 haplotype A, B, C1, and D1). SNP markers N1
and 8453 represented the 5 0 and 3 0 borders of the interval,
respectively. Four of the microsatellite markers (MS14,
MS3, TKY284, and MS13) and three SNPs (PMEL17ex11,
8345, and 8377) reside within the interval. Table 2 displays
the results from the genotyping of each marker. With the
exception of markers PMEL17 and TKY284, disease-asso-
ciated alleles are also common in unaffected horses. Phased
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L. S. Andersson et al.: Equine MCOA locus
357
Table 3
Phased data of 13 markers
Haplotype
No.
of Chr
MS1
N1
MS14
PMEL17-
ex11
MS3
8345
TKY284
8377
MS13
SMARCC-
int24
SMARCC-
int19
8453
8475
Breed
A
355
269
T
247
T
233
C
177/175
C
222
G
G
A
T
RH, KY
B
12
263
G
247
T
233
C
177
C
222
G
G
G
A
RH, KY
C.1
10
263
G
247
T
233
C
177
C
222
G
G
A
A
MINI
D.1
21
263
G
247
T
231
C
177
C
222
G
G
GAIS
D.2
1
263
G
247
T
231
C
177
C
222
G
T
GAIS
C.2
10
263
G
247
T
233
C
177
C
222
A
T
G
A
MINI
Position a
0 33 50 58 115 118 161 180 227 240 242 297 360
Six haplotypes were identified in the four analyzed breeds. The number of chromosomes (Chr) and the respective breed are listed for each
haplotype. The 264-kb associated haplotype is marked in italics, while the 208-kb haplotype that includes the horses recombinant at the
SMARCC2 locus is marked in bold
a The chromosome position of marker MS1 is 73607795 and is here set as the reference point zero (0). The distance to the following markers are
given in kilobases
Sanger sequencing of IKZF4 and SMARCC2
The disease-associated allele for this SNP is also guanine
but the C2 haplotype holds an adenine at this position.
These horses thus shortened the genetic interval by 57 kb,
providing a minimum shared haplotype of 208 kb.
In total, 13.6 kb of IKZF4 (87%) were evaluated for
polymorphisms, including all exons and conserved
sequences within introns and 3.6 kb upstream of the gene
(Supplementary File 1). Comparison among MCOA horses
and unaffected control horses revealed three intronic
polymorphic sites (Supplementary File 3). Two of these
were SNPs (introns 3 and 4) and not completely associated
with the disease. The third polymorphism was a homozy-
gous adenine insertion (intron 5) in both MCOA horses that
was not present in either of the unaffected controls or the
reference genome.
Resequencing of the SMARCC2 gene covered 12.6 kb
(76%) and included all 29 exons (Supplementary File 1)
and intronic conserved elements. Ten SNPs were detected
and two of them were positioned within coding regions
(Supplementary File 3). Only two SNPs, in intron 19 and
24, respectively, matched the correct mode of inheritance
in the horses selected for resequencing. Since SMARCC2 is
positioned close to the 3 0 downstream border of the 264-kb
IBD interval, the intron 19 SNP was genotyped in recom-
binant horses. These are the horses with the shortest IBD
region compared with the majority of horses (Table 3 ,
haplotypes B-D). The disease-associated allele for this SNP
is G, but genotyping revealed nine genetically well-defined
Cyst horses as being TT (Table 3 , haplotypes C2 and D2).
Two additional American Miniature horses displaying the
Cyst phenotype can be phased as either C1 or C2 but have
been labeled as C2 due to a close relatedness to the other
horses carrying the C2 haplotype. Furthermore, a subset of
horses, including the newly identified recombinants, was
also genotyped for the SNP in intron 24. SMARCC2 is
positioned on the minus strand and hence intron 24 is
upstream of intron 19 according to the depicted interval.
Discussion
Identical-by-descent mapping was used to firmly define a
208-kb genomic interval on horse chromosome 6 for the
MCOA locus. This has shortened the previous 4.9-Mb
MCOA genetic interval by a factor of 20, which has
unlinked 176 other genes from the disease. In addition, a
large number of horses were utilized to verify that the
mutation causing MCOA syndrome does have a clear
additive effect and that the two distinct phenotypic cate-
gories, Cyst and MCOA, are caused by one and two copies
of the mutant allele, respectively (p = 2.2 9 10 -16 ).
Results of the study reported here are based on analysis of
21 genetic markers in 465 horses from four different
breeds. The identified interval is a gene-dense area of the
genome and includes 15 genes (see Supplementary File 4).
Based on information on gene function from studies in
other species, two candidate genes within the interval,
SMARCC2 and IKZF4, were evaluated for DNA poly-
morphisms using Sanger sequencing. Two intronic SNPs
that segregated with the disease phenotype were detected in
SMARCC2. After analysis of these SNPs in recombinant
horses, the interval for the MCOA syndrome was shortened
by 57 kb (from 265 to 208 kb). Sequencing also revealed
one disease-associated insertion in intron 5 of IKZF4.
However, since this polymorphism resides within a non-
coding region, it is less likely than the PMEL17 mutation to
have a phenotypic consequence. Nevertheless, this variant
will be tested in a larger number of horses.
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