CELLULAR AND GENETIC CHARACTERIZATION OF OCULAR MELANOSIS IN THE
CAIRN TERRIER DOG
By
Ethan Dawson-Baglien

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Genetics â Doctor of Philosophy
2017

PUBLIC ABSTRACT
CELLULAR AND GENETIC CHARACTERIZATION OF OCULAR MELANOSIS IN THE CAIRN TERRIER
DOG
By
Ethan Dawson-Baglien
Ocular melanosis (OM) is an inherited eye disease seen in the Cairn terrier dog breed.
The disease is very common in Cairn terriers â although the exact number of dogs affected by
the disease is not known, surveys of Cairn terrier breeders and owners frequently rank it near
the top of health concerns in the breed. The disease progresses through several stages, starting
off as a thickening and darkening of the iris. Eventually, dark brown-black pigment begins to
appear in patches in abnormal areas of the eye, such as the sclera (the whites of the eye).
These patches gradually grow and expand over time. Within the eye, pigmented material is
shed into the anterior chamber of the eye (the fluid-filled space between the iris and the front
of the eye). This pigmented material clogs up the eyeâs internal drainage pathways, and fluid
builds up within the anterior chamber, leading to an increase in pressure within the eye. This
increase in pressure can lead to painful glaucoma, and eventually blindness, in the dogs with
the most severe cases of OM. The underlying causes of OM are not currently known. Two
different methods were used to attempt to find out more about the disease â a cell culture
method and a gene sequencing method.
In the cell culture method, donated eyes from dogs with and without OM were used to
isolate and grow uveal melanocytes â the pigmented cells of the eye which grow and migrate in
OM. These cells were then tested using a variety of different cellular assays to determine how
the melanocytes from affected eyes differed from those in unaffected eyes. The only tests

where the melanocytes from the OM-affected dogs showed any difference from those in
unaffected dogs were in pigmentation â the melanocytes from OM-affected dogs had much
more pigment, and made new pigment more quickly, than those from unaffected dogs.
Gene sequencing methods were also used to try to find where in the genome the
mutation that causes OM was located. To determine the general location of the mutation, a
whole-genome SNP array was used to test 94 dogs at 170k markers from all around the 2.8
billion base pairs of the canine genome, to see if any of the markers was associated with the
disease. This identified a 7.5 million base-pair long region of chromosome 11 that was
significantly associated with the disease. Next, the entire genome of 10 dogs was sequenced, 5
OM-affected and 5 unaffected, to look for the exact mutation causing OM. Analysis of the
sequencing data failed to identify a likely causal variant, either within the identified region or in
known genes related to pigmentation disorders. Finally, RNA sequencing was performed on eye
tissues from 12 dogs; 7 OM-affected and 5 unaffected, to determine whether there were any
differences in gene expression between the two populations. Six genes were identified that
were expressed differently between the two populations that were in pathways known to be
associated with cancer metastasis.
Although a causal variation for OM has not yet been discovered, several promising new
clues have been identified that can be followed up on, including the general location of the
causal DNA mutation on chromosome 11, and a number of genes whose expression are altered
in OM-affected dogs. Following these leads may finally allow us to identify the underlying cause
of OM.

ABSTRACT
CELLULAR AND GENETIC CHARACTERIZATION OF OCULAR MELANOSIS IN THE CAIRN TERRIER
DOG
By
Ethan Dawson-Baglien
Ocular melanosis (OM) is an inherited eye disease characterized by a thickening of the
iris root due to expansion of a melanocyte population and an abnormal progressive deposition
of pigment in areas of the eye such as the sclera and episclera. The disease is inherited in an
autosomal dominant fashion, and occurs almost exclusively in Cairn terrier dogs, in whom the
disease is very common. OM is relatively recent in origin, leading us to suspect that it has a
single causative underlying mutation, which spread through the Cairn population via a founder
effect. In this work, two different approaches were used to attempt to characterize the OM
phenotype â a cellular and a genetic approach.
A method for isolating and culturing canine uveal melanocytes without contamination
from other cell types was initially developed. This was then used to isolate melanocytes from
OM-affected and control dogs which were then compared using various assays of physiological
behavior. These included a battery of standard cell behavior tests to evaluate doubling time,
migration rate, anchorage independence, ability to migrate through a membrane, extracellular
matrix preference, and melanin content and rate of production. In all aspects, melanocytes
from OM-affected dogs and those from unaffected dogs were identical in vitro, except for
melanin content and production rate â after initial culture, OM-affected melanocytes contained
much more melanin than unaffected cells, and produced more melanin as well. These changes

were eventually lost in later passages, but differences were statistically significant (p<0.05) in
early-passage cells.
Attempts to identify a causal mutation for OM based using a candidate gene approach
had not previously been successful, so a whole-genome SNP array was used to examine 94
Cairn terriers at ~170k evenly spaced markers throughout the genome. This identified a 7.5
megabase (Mb) region of chromosome 11 that was significantly associated with the disease.
Sanger sequencing of positional candidate genes selected from the region of interest did not
reveal any variants associated with the disease. Whole-genome sequencing was performed on
10 dogs; 5 affected and 5 unaffected, but no genes either within the region detected via the
SNP array or within a list of genes known to be associated with pigmentation disorders
contained any polymorphism that segregated with the disease. Finally, RNA sequencing was
performed on 12 samples, 7 affected and 5 affected, and the transcriptomes of OM-affected
and unaffected dogs were compared. Although no individual genes had a statistically significant
difference in expression level, pathway analysis of the genes with the lowest p-values revealed
6 genes with differential expression levels that were part of 1 of 2 pathways known to be
associated with cell migration in metastatic cancer.
Although further studies are needed, the identification of both a region associated with
OM and a putative pathway which may be involved in the migratory component of the disease
functions are major steps toward identifying the underlying cause of the disease.

Copyright by
ETHAN DAWSON-BAGLIEN
2017

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................................................viii
LIST OF FIGURES .............................................................................................................................ix
KEY TO ABBREVIATIONS .................................................................................................................xi
CHAPTER 1 INTRODUCTION ...........................................................................................................1
1.1 The dog as a model organism for inherited disease âŚ.âŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ..âŚâŚâŚâŚâŚâŚâŚ.2
1.1.1 The dog as a model organism .....................................................................................2
1.1.2 The canine genome âŚâŚ.âŚâŚâŚâŚ.âŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ..âŚ2
1.2 Anatomy of the canine eye and uvea âŚâŚ.âŚâŚâŚâŚâŚâŚâŚ.âŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ..âŚâŚâŚâŚ..4
1.2.1 General overview of the eye âŚâŚâŚâŚâŚ.âŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ...4
1.2.2 The fibrous tunic ........................................................................................................6
1.2.2.a The cornea ..................................................................................................6
1.2.2.b The sclera ...................................................................................................7
1.2.3 The uvea ....................................................................................................................8
1.2.3.a The choroid .................................................................................................8
1.2.3.b The ciliary body âŚ........................................................................................8
1.2.3.c The iris .........................................................................................................9
1.3 Melanocyte biology ................................................................................................................11
1.3.1 Melanocyte development ........................................................................................11
1.3.2 Melanin production .................................................................................................13
1.3.3 Uveal melanocytes ...................................................................................................16
1.3.4 Invasiveness in uveal melanocytes ...........................................................................16
1.4 Ocular melanosis ....................................................................................................................18
1.4.1 Physiological description .........................................................................................18
1.4.2 Identification of pigmented cells âŚ...........................................................................19
1.4.3 Breed prevalence and history ..................................................................................24
1.4.4 Inheritance pattern ..................................................................................................25
1.4.5 Comparisons to human disease âŚ............................................................................26
1.4.6 Comparisons to mouse models ................................................................................27
1.5 Target gene testing .................................................................................................................29
1.6 Conclusions ............................................................................................................................31
REFERENCES .................................................................................................................................32
CHAPTER 2 ISOLATION AND CULTIVATION OF CANINE UVEAL MELANOCYTES ...........................38
2.1 Abstract ..................................................................................................................................39
2.2 Introduction ...........................................................................................................................40
2.3 Materials and methods ...........................................................................................................42
2.3.1 Globe collection and harvesting of anterior uveal tissue ..........................................42
2.3.2 Isolation of uveal melanocytes ................................................................................42
v

2.3.3 Immunocytochemistry ............................................................................................45
2.4 Results ....................................................................................................................................48
2.4.1 Isolation and morphology ........................................................................................48
2.4.2 Cryopreservation .....................................................................................................49
2.4.3 Immunocytochemistry ............................................................................................49
2.5 Conclusions ............................................................................................................................52
2.6 Acknowledgements ................................................................................................................54
2.7 Funding Sources .....................................................................................................................54
2.8 Conflict of Interest ..................................................................................................................54
REFERENCES .................................................................................................................................55
CHAPTER 3 PHYSIOLOGICAL CHARACTERIZATION OF OCULAR MELANOSIS-AFFECTED CANINE
MELANOCYTES .............................................................................................................................58
3.1 Abstract ..................................................................................................................................59
3.2 Introduction ...........................................................................................................................61
3.3 Materials and methods ...........................................................................................................63
3.3.1 Animals used ............................................................................................................63
3.3.2 Immunohistochemistry ...........................................................................................65
3.3.3 Cell culture methods ................................................................................................67
3.3.4 Preparing ECM-treated plates .................................................................................68
3.3.5 Doubling time assay .................................................................................................68
3.3.6 Cellular melanin levels .............................................................................................68
3.3.7 Wound-healing rate .................................................................................................69
3.3.8 Cell adhesion assay ..................................................................................................70
3.3.9 Anchorage independence ........................................................................................71
3.3.10 Invasion assay ........................................................................................................72
3.4 Results ....................................................................................................................................73
3.4.1 Immunohistochemistry ...........................................................................................73
3.4.2 Cellular melanin levels .............................................................................................73
3.4.3 Doubling time ..........................................................................................................78
3.4.4 Anchorage independence ........................................................................................79
3.4.5 Invasion assay ..........................................................................................................80
3.4.6 Cell adhesion ...........................................................................................................81
3.4.7 Wound-healing rate .................................................................................................82
3.5 Discussion ...............................................................................................................................83
3.6 Acknowledgements ................................................................................................................91
REFERENCES .................................................................................................................................92
CHAPTER 4 OCULAR MELANOSIS LOCUS MAPPING AND CANDIDATE GENE SEQUENCING.âŚâŚ...95
4.1 Introduction ...........................................................................................................................96
4.2 Materials and methods ...........................................................................................................98
4.2.1 Sample collection .....................................................................................................98
4.2.2 Whole-genome canine SNP array ............................................................................98
4.2.3 Data analysis ..........................................................................................................102
vi

4.2.4 PCR-RFLP genotyping .............................................................................................102
4.2.5 Sanger sequencing of candidate genes ..................................................................103
4.3 Results ..................................................................................................................................104
4.3.1 Genome wide SNP array ........................................................................................104
4.3.2 Population SNP genotyping ...................................................................................107
4.3.3 Candidate gene selection .......................................................................................109
4.3.4 Sanger sequencing results .....................................................................................111
4.4 Discussion .............................................................................................................................112
REFERENCES ...............................................................................................................................115
CHAPTER 5 WHOLE-GENOME SEQUENCING OF OCULAR MELANOSIS-AFFECTED CAIRN
TERRIERSâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ..118
5.1 Introduction .........................................................................................................................119
5.2 Materials and methods .........................................................................................................121
5.2.1 Sample collection ...................................................................................................121
5.2.2 Whole-genome sequencing ...................................................................................121
5.2.3 Data analysis ..........................................................................................................122
5.2.4 Sanger sequencing of candidate genes ..................................................................125
5.3 Results ..................................................................................................................................126
5.3.1 PCR-based results ..................................................................................................126
5.3.2 PCR-free results .....................................................................................................131
5.4 Discussion .............................................................................................................................132
REFERENCES ...............................................................................................................................134
CHAPTER 6 TRANSCRIPTOME ANALYSIS OF OCULAR MELANOSIS-AFFECTED CAIRN TERRIER IRIS
TISSUE.âŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ..âŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚâŚ137
6.1 Introduction .........................................................................................................................138
6.2 Materials and methods .........................................................................................................140
6.2.1 RNA sample collection ...........................................................................................140
6.2.2 Extraction, purification, and evaluation of RNA from iridal tissue ..........................140
6.2.3 RNA sequencing .....................................................................................................142
6.2.4 Data evaluation ......................................................................................................144
6.2.5 Confirmatory qRT-PCR ...........................................................................................146
6.3 Results ..................................................................................................................................149
6.3.1 Expression level differences ...................................................................................149
6.3.2 qRT-PCR confirmation ............................................................................................150
6.3.3 Unannotated transcripts ........................................................................................151
6.3.4 RNA variant calling .................................................................................................151
6.4 Discussion .............................................................................................................................153
REFERENCES ...............................................................................................................................157
CHAPTER 7 DISCUSSION AND FUTURE DIRECTIONSâŚ................................................................162
REFERENCES ...............................................................................................................................169

vii

LIST OF TABLES

Table 1.1 â Primers used to amplify markers around candidate genes..........................................30
Table 2.1 â Antibody specifications for immunocytochemistry....................................................47
Table 2.2 â Characterization of cell lines using immunocytochemical labeling.............................50
Table 3.1 â Sources of globes used in this study...........................................................................64
Table 3.2 â Antibodies and positive control tissue........................................................................66
Table 3.3 â Percentage of the expansile round melanocyte population with immunoreactivity for
histiocytic and melanocytic markers within the anterior uvea or scleral plaques in Cairn terriers
with ocular melanosis ...................................................................................................................74
Table 3.4 â Average colonies per plate after 30 days incubation in soft agar.................................79
Table 3.5 â Average percent invasion of various cell lines as determined by Matrigel assayâŚâŚ.80
Table 4.1 â Dogs used in GWAS study..........................................................................................100
Table 4.2 â SNPs tested in trial genotyping..................................................................................108
Table 4.3 â Dogs tested in trial genotyping..................................................................................108
Table 4.4 â Population-wide SNP genotyping results..................................................................108
Table 4.5 â Primers for LINGO2 sequencing................................................................................110
Table 4.6 â Primers for ACO1 sequencing....................................................................................110
Table 4.7 â Primers for CDKN2A sequencing...............................................................................111
Table 4.8 â Dogs for candidate gene sequencing.........................................................................111
Table 5.1 â Dogs used for whole-genome sequencing.................................................................122
Table 5.2 â Dogs tested for variation in lncRNA32.......................................................................127
Table 5.3 â Dogs genotyped for variants in genes in the Color Genes database...........................128
Table 5.4 â Primers for genotyping variants in genes from the Color Genes database................128
Table 5.5 â Variants detected within genes from the Color Genes database via whole-genome
sequencing..................................................................................................................................129
Table 5.6 â Genotyping results for Color Genes variants.............................................................130
Table 6.1 â Information on dogs used for RNA sequencing..........................................................144
Table 6.2 â Primers used for qPCR...............................................................................................148

viii

LIST OF FIGURES

Figure 1.1 â Diagram of the three basic layers of the canine eye.....................................................5
Figure 1.2 â Layers of the canine cornea..........................................................................................6
Figure 1.3 â The canine limbus........................................................................................................7
Figure 1.4 â SEM image of the canine limbus and iridocorneal angle..............................................9
Figure 1.5 â Brief overview of mammalian melanocyte development...........................................13
Figure 1.6 â The melanogenesis pathway......................................................................................15
Figure 1.7 â Melanosome development by stage..........................................................................16
Figure 1.8 â Ocular melanosis in the Cairn terrier..........................................................................20
Figure 1.9 â Immunohistochemical expression of markers in sections of OM-affected and
unaffected globes .........................................................................................................................22
Figure 1.10 â Transmission electron micrograph of pigmented cells within the iris......................23
Figure 1.11 â A representative pedigree of a Cairn terrier lineage with ocular melanosis.............25
Figure 2.1 â Extracted canine uveal melanocytes develop dendritic processes after attachment to
culture plates................................................................................................................................48
Figure 2.2 - Extracted canine uveal melanocytes lose pigment over multiple passages................49
Figure 2.3 â Immunocytochemistry (ICC) of extracted first-passage canine uveal melanocytesâŚ.51
Figure 3.1 â A Cairn terrier with late-stage ocular melanosis.........................................................61
Figure 3.2 â Immunohistochemistry of ocular melanosis, anterior uvea or scleral plaque............75
Figure 3.3 â Immunohistochemistry of ocular melanosis, anterior uvea or scleral plaque............76
Figure 3.4 â Average melanin content of OM-uveal melanocytes.................................................77
Figure 3.5 â Average melanin produced per cell per day in OM-uveal melanocytes......................78
Figure 3.6 â Graph of doubling time (in hours) for melanocyte populations with different ECM
treatments....................................................................................................................................79
Figure 3.7 â Relative metabolic activity of melanocytes at 30-minute time point.........................81
Figure 3.8 â Rate of wound closure as a percentage of original scratch.........................................82
Figure 4.1 â Manhattan plots of p-values âŚâŚ..............................................................................106
Figure 5.1 â Whole-genome sequencing pipeline according to GATK best practices handbook..124
Figure 6.1 â RNA samples before and after CTAB-urea purification.............................................142
ix

Figure 6.2 â Graphical illustration of the variant calling pipeline for RNA data............................146
Figure 6.3 â Fold increase in gene expression as observed by qPCR............................................151
Figure 6.4 â Pigment patterns of various mice.............................................................................154

x

KEY TO ABBREVIATIONS

Mb â megabase pairs
bp â base pairs
OM â ocular melanosis
ICC â immunocytochemistry
IOP â Intraocular pressure
IHC â immunohistochemistry
SNP â single nucleotide polymorphism
WGS â whole-genome sequencing
qRT-PCR â quantitative real-time PCR

xi

CHAPTER 1
INTRODUCTION

1

1.1 The dog as a model organism for inherited disease
1.1.1 The dog as a model organism
Although a relative newcomer compared to the established traditional model organisms
of biology such as Drosophila melanogaster, Arabidopsis thaliana, and Mus musculus, the
domestic dog has many qualities which lend themselves well to disease research in the era of
genomic science. The dog combines a huge amount of phenotypic variation, in terms of size,
shape, and behavior, with a well-characterized and annotated genome and many excellent
tools designed to facilitate the analysis of large-scale genomic data. The wide variety of genetic
backgrounds present in the form of different dog breeds combined with certain breeding
practice leads to the occurrence of many spontaneous models of human disease. Since dogs
usually share an environment with their owners, and are more similar in size, behavior, and
physiology, orthologous diseases in the dog often more useful as models for human diseases
than those in traditional models like the mouse and rat.
1.1.2 The canine genome
In terms of raw size, the canine genome is roughly as large as the mouse genome (2.41
billion base pairs in dogs vs. 2.60 billion base pairs in mice), and slightly smaller than the human
genome (2.91 billion base pairs) (1-3). It is subdivided into 39 pairs of chromosomes, 38
autosomal pairs and one pair of sex chromosomes. The first high-quality draft of the canine
genome was completed in 2005 using whole-genome shotgun sequencing at a coverage of
approximately 7.5X to sequence the genome of a female boxer dog (1, 6).

2

In addition to having numerous and well-established tools that exist for working with
the canine genome, the dog genome also benefits from the unique population structure that
centuries of selective breeding have created. Through the process of selecting for certain
behavioral and physiological traits in certain breeds, dogs display an enormous amount of
variation between breeds, but very little variation within breeds (10). This can be expressed in
terms of intrabreed vs interbreed linkage disequilibrium (LD), the non-random association of
alleles at a given loci due to physical linkage. Intrabreed canine LD is on the order of
megabases, whereas the interbreed LD is on the order of kilobases, nearly 100 times shorter
than the LD seen within breeds. In fact, LD between breeds is similar to what is seen in some
well-characterized human populations (1). The large LD blocks seen within single breeds can be
very useful for applications where a disease inherited within a limited number of breeds is
being mapped â the large LD blocks seen in single breeds allow for marker-based studies like
genome-wide association studies to map genes with a high degree of power using only a very
small number of markers, around 10,000 for an average breed (1).
Although they may differ from humans in terms of LD, dogs are similar to them in terms
of the number of coding genes they possess. The dog genome was originally projected to
contain roughly 19,300 total genes, compared with around 22,320 in humans, 23,062 in mice,
and 24,147 in zebrafish, but recent studies suggest that up to 70% of the difference in gene
number between dogs and other established model organisms is the result of real canine genes
failing to be called by prediction algorithms, suggesting that the total number of true canine
genes is probably closer to 21,400 (1, 3, 11-13).

3

1.2 Anatomy of the canine eye and uvea
1.2.1 General overview of the eye
The canine eye is composed of three basic layers â the fibrous tunic, the uvea, and the
nervous coat (Figure 1.1) (5, 14-16). Fibrous tunic is a tough, mostly avascular outer layer which
helps the eye maintain its rigid shape. It is subdivided into the cornea and the sclera. The uvea
is the middle layer, and it is further subdivided into the choroid, the ciliary body, and the iris.
The uvea is heavily vascularized and pigmented, and helps to adjust intraocular light levels and
to provide nutrients to the other layers of the eye. The nervous coat is the innermost layer, and
it is composed of the retina and the optic nerve head. The retina contains the light-sensitive
cells that are responsible for converting light signals into electrical impulses which can then be
transmitted to the brain via the optic nerve (5, 14-16). For purposes of this work, we will
primarily be focusing on the various components of the uvea, as well as the fibrous layer to a
somewhat lesser extent.

4

I

L

PC

VC

AC
I

Figure 1.1 â Diagram of the three basic layers of the canine eye The fibrous tunic is shown
in yellow (Co, cornea) and white (S, sclera). The uvea is shown in black, consisting of the
choroid (Ch), the ciliary body (CB), and the iris (I). The nervous tunic is shown in grey, and is
composed of the retina (R) and the optic nerve (ON). Between the cornea and the iris lies
the anterior chamber (AC). The large cavity at the posterior of the eye is the vitreous
chamber, and the small space between the iris and the lens (L) is the posterior chamber
(PC). Modified from (5).

5

1.2.2 The fibrous tunic
1.2.2.a The cornea

Figure 1.2 â Layers of the canine cornea Anterior epithelium (AE), the stroma (S),
Descemetâs Membrane (DM), and the corneal endothelium (E). Figure reproduced from (5).
The cornea is the most anterior portion of the fibrous tunic, comprising roughly onefifth of the tunicâs total area. It is transparent, allowing the transmission of light through to the
lens and the retina, and is avascular. It receives the majority of its nutrients from the aqueous
humor. The cornea is made up of four layers: the anterior epithelium, the stroma, Descemetâs
membrane, and the corneal endothelium (Figure 1.2). The anterior epithelium is 25-40 Âľm thick
and consists of a single layer of basal columnar cells, 2-3 layers of polyhedral cells, and 2-3
layers of nonkeratizined squamous cells. The basal cell layer is attached to a basement
membrane which adheres the anterior epithelium to the underlying stroma. The stroma makes
up the bulk of the cornea, roughly 90% of the overall thickness, and it contains primarily of

6

transparent parallel sheets of collagen fibrils along with a small number of keratocytes which
form and maintain the lamellae. Descemetâs membrane is a homogenous acellular layer of
collagen fibrils produced by the basement layer of the corneal endothelium. Descemetâs
membrane is under tension, and helps to maintain the shape of the eye. The corneal
endothelium is the innermost layer of the cornea, and it consists of a single layer of
metabolically active cells which help to maintain Descemetâs membrane (5, 14, 17, 18).
1.2.2.b The sclera

Figure 1.3 â The canine limbus This image shows the junction between the highly regular,
parallel collagen fibers of the transparent cornea (C) and the irregular fibers of the opaque
sclera (S). Figure reproduced from (5).
The remaining four-fifths of the fibrous tunic compose the sclera, what is usually known
as the âwhiteâ of the eye. The fibrous tunic transitions from the transparent cornea to the
opaque sclera at a transition zone known as the limbus. At this transition zone, the layer that
had been the corneal epithelium expands and thickens and the stroma loses the regular parallel
arrangement of the collagen fibers that allowed it to transmit light, becoming opaque (Figure
1.3). The sclera is divided into four layers. The outermost layer is the episclera, a thin
collagenous membrane that connects to Tenonâs capsule (a thin sheath separating the eye from
the interior of the eyesocket), and the conjunctiva (the inner lining of the eyelids). Blood vessels
7

originating in the conjunctiva serve to vascularize the episclera. The remaining layers, the
stroma, the basal cell layer, and the endothelium, are continuous with and serve the same
function as their counterparts in the cornea, although unlike the cornea, the sclera contains
blood vessels (5, 14, 17-19).
1.2.3 The uvea
1.2.3.a The choroid
The choroid is the posterior portion of the uvea. It connects directly to the sclera, and is
composed of four layers. The outermost layer is the suprachoroidea, followed by the stroma
with large vessels, the stroma with medium-sized vessels and the tapetum (a layer of reflective
tissue which reflects light which has passed through the retina), and the choriocapillaris. Like
most of the uvea, the choroid is pigmented and heavily vascular (5, 14-16, 20).
1.2.3.b The ciliary body
The ciliary body, along with the iris, makes up the anterior uvea. It is located near the
limbus, and is roughly triangular in shape â one edge connects to the sclera, another to the lens
(via the lens zonular fibers), and the third to the iris (Figure 1.4). It helps to provide nutrients to
and remove waste from the avascular cornea by producing aqueous humor, an optically clear
fluid which contains diffuse proteins and nutrients for delivery to the cornea. Keeping the
proper volume of aqueous humor in the anterior chamber at all times is critical for proper
functioning of the eye â aqueous humor generates intraocular pressure (IOP). An impediment
to aqueous humor drainage leads to an increase in IOP which can eventually cause painful
glaucoma and blindness. Reduced aqueous humor production leads to a

8

A

B

Figure 1.4 â SEM image of the canine limbus and iridocorneal angle (A) is a SEM image of
the area near the limbus of a canine eye. Shown are the cornea (C), the stroma (S), the iris
(I), and the ciliary body (CM and CP). Notice the fine, porous meshwork near the junction of I
and CP. (B) is a SEM image of the canine iridocorneal angle. The iris is denotes with (I) near
the top of the frame, and the pectinate ligaments that bridge the gap between iris and
cornea are marked with a (PL). The arrowheads denote connections between the pectinate
ligaments and the finer trabecular meshwork behind them. Figure modified from (5).
drop in IOP, which can affect the eyeâs ability to maintain a rigid shape. The anteriormost
component of the ciliary body is the iridocorneal angle (ICA, Figure 1.4). At the ICA, pectinate
ligaments extend from the limbic junction down to the root of the iris (which extends anteriorly
from the ciliary body), attaching the iris to the fibrous tunic. Behind these ligaments is the
trabecular meshwork, a network of interwoven collagen cords through which aqueous humor is
filtered on its way out of the anterior chamber. Under normal circumstances, the aqueous
humor is produced from the ciliary body epithelium into the posterior chamber, flows through
the pupil into the anterior chamber, and exits through the iridocorneal angle/trabecular
meshwork (5, 15, 16, 20).
1.2.3.c The iris
The iris is a thin layer of pigmented, vascular tissue extending out from the ciliary body
that divides the anterior compartment of the eye into anterior and posterior chambers. It is
9

divided into four tissue layers â the anterior border layer, the stroma, the sphincter muscle, and
the posterior epithelium. The anterior border layer is a porous cell layer primarily composed of
fibroblasts and melanocytes. The stroma is a loosely arranged network of collagen fibers
containing fibroblasts. The stroma is organized into dense sheaths where it surrounds blood
vessels and nerves, and melanocytes tend to be found in close proximity to these features. The
iridal sphincter muscle is a thin band of unstriated muscle that changes the shape of the iris in
response to light levels, and the posterior epithelium, which is continuous with the choroid (5,
16, 20).

10

1.3 Melanocyte biology
Melanocytes are a type of cell specialized for the production of melanin, the pigment
which gives color to the skin, eyes, hair, and other tissues. They contain a specialized
membrane-bound organelle called a melanosome where melanin production can take place
sequestered from the remainder of the cell. This physical separation is required due to the fact
that some intermediates of melanin production are toxic to the cell. Although melanocytes
have been most extensively studied in the skin, physiologically distinct populations of
melanocytes also exist at a number of extracutaneous sites such as the ear, the meninges,
various mesenchymal tissues, and, most notably for this work, the eye (8, 21).
1.3.1 Melanocyte development
Melanocytes, regardless of the tissue in which they are found, are all originally derived
from the neural crest. Initially, neural crest cells differentiate into a mixed-fate progenitor cell
capable of developing into either a melanoblast or a glial cell progenitor (22). Both of these
lineages are specified by the transcription factors Paired Box 3 (PAX3) and SRY-Box 10 (SOX10).
The expression of two additional transcription factors, Forkhead-box D3 (FOXD3) and SRY-Box 2
(SOX2), determine whether this bipotent cell will further develop into a melanocyte or into a
glial cell. PAX3 and SOX10 are transcription factors that act synergistically to activate
microphthalmia-associated transcription factor (MITF), which is the master regulator of
melanocyte identity (8). FOXD3 is expressed in glial progenitor cells â it interacts with PAX3 and
prevents it from activating the MITF promoter. SOX2, on the other hand, regulates MITF
directly, by binding to its promoter and repressing its expression. Therefore, progenitor cells
develop into melanoblasts when FOXD3 and SOX2 are downregulated, although the

11

mechanisms for how these transcription factors are controlled are not well understood, and
appear to vary between organisms. Once MITF expression is induced, it appears to be selfreinforcing, as MITF expression causes the rapid downregulation of SOX2 (23). These cells also
begin to express dopachrome tautomerase (DCT, also commonly called TRP2), a melanocytespecific protein which is responsible for converting DOPAchrome to 5,6-dihydroxyindole-2carboxylic acid (DHICA) rather than the 5,6-dihydroxyindole (DHI) it would otherwise form
spontaneously (Figure 1.6) (24). The melanoblast cells follow two distinct migratory paths,
some ventrally, and some dorsolaterally (25). The gene for KIT receptor tyrosine kinase (KIT) is
expressed by the melanoblast throughout migration, and the presence of KIT and its ligand is
essentially for melanoblast survival during migration (26). Post-migration, although a large
number of melanoblasts differentiate into melanocytes, it has been shown that at least
mammalian melanocyte populations associated with hair follicles maintain reservoirs of
melanocyte stem cells, and that these follicular melanocyte stem cell reservoirs are capable of
replenishing depleted epidermal melanocyte populations (27). These stem cells are
dedifferentiated melanoblasts, who lose expression of MITF but maintain expression of DCT.
These stem cells can be activated to create differentiating, transit-amplifying daughter cells
which are then used to replenish depleted populations of differentiated melanocytes in the
epidermis or hair follicle (28).

12

Figure 1.5 â Brief overview of mammalian melanocyte development A neural crest cell
progenitor becomes specified as a SOX10-positive bipotent progenitor that can potentially
develop into either a melanoblast of a glial cell. Activation of MITF specifies the cell as a
melanoblast, which activated DCT. KIT is expressed during migration. Once migration is
complete, some portion of the cells differentiate into mature melanocytes and produce
melanin to transfer to keratinocytes. Another portion of the melanoblasts instead
dedifferentiates, becoming melanocyte stem cells. These stem cells replenish the
differentiated melanocyte population via MITF-positive, KIT-positive transit amplifying cells.
Modified from (8).
1.3.2 Melanin production
Melanocytes are capable of producing two different types of melanin, the black-brown
eumelanin (further subdivided into the darker-black DHI-melanin and the lighter-brown DHICAmelanin) and the red-yellow pheomelanin. The pathway for determining which type of melanin
is produced is illustrated in Figure 1.6 (7). As was previously mentioned, melanin is produced
within specialized membrane-bound organelles called melanosomes, which mature through
four stages of development during melanin synthesis (Figure 1.7). Initially, melanosomes are
small, spherical vesicles commonly thought to originate from the endoplasmic reticulum (stage
1), but this is still a matter of some debate (29). In stage II of melanosome development, the
previously spherical vesicle begins to elongate, and glycoprotein fibrils begin to form an

13

organized matrix. Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TRP1 and TRP2)
can be detected within the organelle. Melanogenesis begins in stage III, and the melanosome
takes on a light brown color as newly synthesized melanin begins to settle on the matrix fibrils.
By stage IV, the melanosome is densely pigmented and dark in color â it is completely filled
with melanin, and loses tyrosinase activity at this point (7, 30).

14

Figure 1.6 â The melanogenesis pathway Tyrosine is converted by tyrosinase (TYR) to LDOPA which is then converted to DOPAquinone. In the presence of cysteine, DOPAquinone
reacts and polymerizes to form the red-yellow pheomelanin. In the absence of cysteine,
DOPAquinone spontaneously cyclicalizes to DOPAchrome. If tyrosinase-related protein 2
(TRP2, or DCT) is present, the cell can convert DOPAchrome to DHICA, which is then
converted by tyrosinase-related protein 1 (TRP1) to the light brown DHICA-melanin, a
eumelanin. In the absence of TRP2, DOPAchrome oxidized to form a dark brown-black DHImelanin, the other eumelanin. Figure reproduced from (7).

15

Shape

Spherical

Elongated

Elliptical, ellipsoidal

Elliptical, ellipsoidal

Internal structure

â

Matrix fibrils are visible

Matrix fibrils are visible

Matrix fibrils are covered
by polymerized melanin

TYR

â

+

+

+

TYRP1

â

+

+

+

TYRP2

â

+

+

+

Melanin synthesis

â

â

Begins, settle on internal fibrils

Filled by melanin

Brown

Dark brown to black

Color

Figure 1.7 â Melanosome development by stage Figure reproduced from (7).
1.3.3 Uveal melanocytes
Uveal melanocytes differ in several significant ways from those seen in the epidermis or
in hair follicles. Epidermal and follicular melanocytes retain very little of their own cellular
melanin over the long term, transferring the melanin granules they produce to keratinocytes of
the skin or hair. These cells are highly dendritic and transfer melanosomes to large numbers of
associated keratinocytes â 30-40 keratinocytes for epidermal melanocytes, and 5-10 for
follicular ones (31). Uveal melanocytes, in contrast, have relatively few dendritic processes, and
retain most of their melanin throughout their life, producing more only under specific stimuli.
1.3.4 Invasiveness in uveal melanocytes
Aggressive metastatic uveal melanomas possess the ability to invade through the matrix
surrounding them, the basement membrane, and into the vasculature. Compared to normal

16

uveal melanocytes, melanoma cells that display this phenotype usually display elevated integrin
expression, and adhere more readily to endothelial cells and all types of extracellular matrix
(ECM) substrates (32). Due to the lack of lymphatics in the eye, many highly invasive uveal
melanomas are capable of vasculogenic mimicry â essentially creating their own pseudovasculature through a re-shaping of the surrounding ECM (33, 34). These channels are even
capable of undergoing anastomosis with existing blood vessels. Although invasive melanoma
cells are capable of harnessing existing ECM for these purposes, it should be noted that they
are often also capable of generating their own ECM components as necessary, expressing
fibronectin, laminin, collagen IV, and collagen VI as needed to provide the raw materials
necessary to create a pseudo-vasculature through which to migrate.

17

1.4 Ocular melanosis
Ocular melanosis (OM) is an inherited eye disease seen primarily in Cairn terrier dogs.
Briefly, the disease is characterized by the bilateral proliferation of abnormal pigmented cells
into the anterior uvea and into other areas of the eye such as the sclera. Over time, this
pigmented material can block the anterior chamber drainage angle, leading to a buildup of
pressure in the anterior chamber, as the aqueous humor is unable to properly drain. The
resulting rise in intraocular pressure is known as glaucoma. Glaucoma can cause pain and
blindness which may necessitate enucleation of the affected eyes to alleviate the pain. The
disease has a highly variable age of onset and progression, and appears to be autosomal
dominant in its mode of inheritance(4, 9, 35).
1.4.1 Physiological description
The clinical description of ocular melanosis includes four stages of development. The
changes associated with the transition through these stages are illustrated in Figure 1.8 (9). In
stage 1, a dark, donut-shaped thickening of the iris root is observed, but no other
morphological changes are apparent. Stage 2 is characterized by a continuing thickening of the
iris root, as well as the appearance of small pigmented plaques in the sclera and episclera. The
pigmented plaques initially appear spicule-shaped, but as the disease progressed more appear
and the existing ones enlarge and often become circular. Small deposits of pigment may also be
observed coating the ventral pectinate fibers at this stage of the disease. In stage 3, the iris
surface of some dogs takes on a lumpy, uneven appearance, while others continue to display
the circumferentially thickened donut-shaped iris observed in earlier stages. The previously

18

small scleral and episcleral plaques expand in size, becoming several millimeters in diameter. At
this stage, it is sometimes possible to see particles of free-floating pigment suspended in the
aqueous humor, and pigment deposition along the ventral drainage angle is much more
pronounced. Finally, stage 4 is usually characterized by the development of secondary
glaucoma as pigment completely obscures the drainage angle, leading to a buildup of pressure
as aqueous humor is prevented from draining out of the anterior chamber. At this point, most
changes observed are those typical of chronic glaucoma, such as loss of vision, lens subluxation,
and globe enlargement (4, 9, 36, 37). Additional pigment deposition into the sclera/episclera
patches is also observed at this stage of the disease. Ocular melanosis is always bilateral,
although there may be differences in the rate of pigment deposition and the development of
glaucoma between eyes.
1.4.2 Identification of pigmented cells
One of the most striking characteristics of OM is the appearance of large, densely
pigmented cells within the anterior segment and the sclera. Although the fact that the cells in
question were densely pigmented suggested a melanocytic origin, a clinically similar condition
observed in Boxers and Labradors had been shown to involve the accumulation of large
numbers of melanophages, phagocytic cells which have engulfed melanosomes, but which are
not themselves melanocyte-derived (36). Immunohistochemistry and transmission electron
microscopy were used to determine the identity of these cells in Cairn terrier uveal tissue (4).
Sectioned uveal tissue samples showed that the majority of the large, pigmented cells
characteristic of OM were immunoreactive for HMB45, MITF, and vimentin. HMB45 recognizes
gp100, an organelle-specific protein that localizes to immature melanosomes (38, 39). Normal
19

A

B

C

D

E

Figure 1.8 â Ocular melanosis in the Cairn terrier (A) shows a Cairn with stage 2 OM â the
thickening of the iris root can be seen ventrally, and the pigmented plaques on the sclera are
clearly visible. (B) is the view across the anterior chamber of the eye shown in (A). Notice the
thickening of the iris root. (C) shows a Cairn with stage 3 OM â the plaques on the sclera are
larger, and floating pigment particles can be seen in the pupil. The arrows highlight pigment
sedimentation on the corneal endothelium. (D) is the view across the anterior chamber of
the eye shown in (C). Notice the dense deposits of pigment particles that completely block
the drainage angle from view. (E) shows a stage 4 OM eye â there is heavy pigment
sedimentation on the corneal endothelium, and the pigment plaques take up a large portion
of the eyeâs surface area. Figure reproduced from (9).

20

adult uveal melanocytes are usually HMB45 negative, as resting adult melanocytes do not
usually produce melanin and therefore do not have any immature melanosomes to mark.
However, HMB45 does mark fetal melanocytes and melanoma cells that are actively producing
melanosomes, suggesting that these cells are melanocytic (Figure 1.9). This was further
confirmed by the fact that many of the large pigmented cells were also immunoreactive for
MITF, a melanocyte nuclear transcription factor critical for melanocyte development and
postnatal survival, and vimentin is a marker for mesenchymal cells expressed by melanocytes
(40). Taken together, this strongly suggests that the majority of the large abnormal pigmented
cells observed in the OM uvea are melanocytic in origin. Interestingly, some of the pigmented
cells also stained positively for CD18, an integrin known to be involved in mediating cell
adhesion.
Transmission electron micrographs of pigmented cells present in the iris also support a
melanocytic origin for the cells (Figure 1.10). The majority of the large, pigmented cells were
observed to contain melanosomes of varying sizes and stages of development in the cytoplasm
and rough endoplasmic reticulum, traits characteristic of melanocytes (4, 41, 42). A small
number of cells displayed multiple membrane-bound melanosomes, characteristic of
melanophages which have phagocytosed melanosomes, indicating the presence of at least
some melanophages in the uvea of affected dogs as well (4). In contrast, the uveal melanocytes
seen in eyes of unaffected Cairn terriers were less rounded than those seen in affected dogs,
and they contained only the mature melanosomes expected in a quiescent melanocyte (4).

21

Figure 1.9 â Immunohistochemical expression of markers in sections of OM-affected and
unaffected globes (a) shows MITF staining pigment-laden cells in the iris of an OM affected
dog â note the pink stained nuclei denoting positive cells, as MITF is a transcription factor.
(b) shows HMB45 staining in the ciliary body of an OM-affected eye. The ciliary body
epithelium is denoted by an arrowhead and is negative for HMB45, but the pigmented cells
seen below stain positively for HMB45 (pink). (c) shows the iris of an OM-affected eye
stained for HMB45. The arrowhead denotes the posterior iridal epithelium, which was
negative for HMB45. The iridal stroma, however, was infiltrated with many large pigmented
cells which stained positive for HMB45 (pink). (d) shows the anterior iris of an OM-affected
eye stained for CD18. Some staining in of the large, pigmented cells is observed (pink). Bar =
50 Âľm. Figure reproduced from (4).

22

Figure 1.10 â Transmission electron micrograph of pigmented cells within the iris (a) shows
the iris of a normal control dog, whereas b-e show the iris of an OM-affected Carin terrier.
Note the increased size of the pigmented cells in (b) and (c) relative to (a). Bar = 10 Âľm. (d)
shows a higher magnification image of the melanosomes of an affected dog â note the size
and shape differences indicating the presence of immature melanosomes. Bar = 2 Âľm. (e)
shows several membrane-bound melanosomes bound together as a single compound
melanosome (arrow), suggesting that the melanosomes in question may have been engulfed
by a melanophage. Bar = 1 Âľm.

23

1.4.3 Breed prevalence and history
The earliest reference to ocular melanosis in the literature was in a conference
proceedings from 1984, when Covitz et al described a condition they called pigmentary
glaucoma in Cairn terriers which was characterized by the proliferation of ocular pigment and
secondary glaucoma arising from a blockage of the anterior drainage angle by pigmented
material (43). Subsequent publications further describing the disease in Cairn terriers began to
use the term ocular melanosis to differentiate the disease from other known forms of
pigmentary glaucoma with different etiologies (35, 36, 44, 45). The fact that this disease had
not been reported in Cairn terriers prior to only a few decades ago suggests that the disease
may have a fairly recent origin, and has been propagated through the breed by means of a
founder effect (9).
Although the precise incidence of the disease is not known in Cairn terriers, its
prevalence within the breed is thought to be relatively high. A 2005 survey of the Cairn terrier
breeders and owners listed OM as the second most common health concern for the breed.
Fifty-eight percent of respondents agreed that OM was a major concern for the breed, and 14
percent of owners surveyed reported having owned at least one Cairn terrier with OM (46).
Additionally, the prevalence of glaucoma in Cairn terriers is estimated to be 1.82%, more than
double the species-wide prevalence of 0.89% (47). As no other predisposition to glaucoma is
known to exist in Cairn terriers, it is likely that the increase in glaucoma prevalence is primarily
due to glaucoma caused by late-stage OM (46).

24

Figure 1.11 â A representative pedigree of a Cairn terrier lineage with ocular melanosis
Male dogs are represented as squares, females as circles. Filled symbols represent OMaffected dogs, and open symbols represent unaffected dogs age 8+. Symbols with a slash are
of unknown phenotype. The dog indicated with a * is the unaffected offspring of two affected
parents, suggesting that the disease is autosomal dominant rather than recessive. Figure
reproduced from (9).
1.4.4 Inheritance pattern
Based on pedigree analysis, ocular melanosis appears to be an autosomal dominant
disease possibly with incomplete penetrance (4, 9). A representative pedigree is shown in
Figure 1.11 (9). The disease does not skip any generations, as would be expected from a
recessive condition, and the disease occurs in roughly equivalent proportions of male and
female dogs, suggesting that it is not sex-linked. No instances have been observed of
unaffected parents producing affected offspring, although there have been a small number of
instances of two affected parents producing an offspring which appears to be unaffected, which
suggests that the disease is likely autosomal dominant. However, as previously noted, this is
complicated by the fact that OM has a widely variable age of onset. The average lifespan of a
Cairn terrier is estimated at 12-15 years, and there have been multiple instances where a Cairn

25

terrier which had previously been diagnosed as OM-free will first begin to show signs of the
disease at age 12 or later (9).
1.4.5 Comparisons to human disease
Ocular melanosis has similarities to certain forms of pigment dispersion syndrome (PDS),
an eye disease seen in humans. PDS, like OM, is characterized by a release of pigment into the
anterior chamber which eventually blocks drainage pathways and leads to secondary glaucoma
(48). The disease appears to be genetically heterogeneous, however, as different loci have been
mapped with the disease in different populations (49, 50). The disease appears to have
different etiologies in different populations â the disease has been most extensively studied in
white populations, where it is thought that pigment is released due to mechanical shearing
caused by the lens rubbing against the iris due to a defect in iris which is not seen in Cairn
terriers (51, 52). However, a distinct form of PDS is seen in black patients which lacks the iris
defect thought to cause the disease in white populations (53, 54). According to an analysis of
PDS inheritance in black populations, this form of PDS has an autosomal dominant mode of
inheritance, although it may not be completely penetrant (55). The exact prevalence of disease
in black populations is not known, it is thought that the disease is under-diagnosed, as
difficulties in detecting the increased pigmentation against the darker iris background seen in
black populations make the disease very hard to detect (55, 56). Although there have not to
date been any studies done to determine whether any of the loci mapped in white populations
associate with the form of the disease seen in black populations, the differences in disease
phenotype suggest that it is unlikely that they share a causative mutation (53).

26

Currently, the available treatments for PDS primarily treat the symptoms of the
secondary glaucoma caused by the disease rather than the disease itself. The most common
treatment is the non-specific reduction of IOP using drugs designed to suppress new aqueous
production, although there has been some suggestion that these drugs worsen pigment
dispersion (53, 57). When this approach fails, selective laser trabeculoplasty has been
successful for reducing IOP in patients suffering from PDS, although the exact mechanism, by
which it does so is not well understood (58). Surgical intervention to remove pigment debris
from the trabecular meshwork is also possible, although it has not been successful at
controlling PDS long-term (59).
1.4.6 Comparisons to mouse models
The DBA/2J mouse has an interesting phenotype which appears at least superficially
similar to OM. These mice develop severe glaucoma as a result of increased intraocular
pressure caused by pigmented cell debris occluding the ocular drainage angles, but the rate of
disease progression is variable and can develop at different speeds between eyes of the same
affected mouse, similar to the variable progression we see in OM (60, 61). This phenotype is the
result of two separate mutations in genes related to the normal functioning of the
melanosomes, Gpnmb and Tyrp1 (60). On their own, both of these mutations produce a milder
phenotype â mice with mutations in Gpnmb alone caused a milder form of pigmentary
glaucoma, similar to PDS in humans. Mutations in Tyrp1 alone produced atrophy of the iris
stroma. The GPNMB and TYRP1 proteins share many structural similarities, and are thought to
be part of a common gene family â specifically, a family of glycoproteins responsible for
maintaining the structure of the developing melanosome. Epistasis experiments showed that
27

the presence of these mutations in mice which produced low levels of pigmentation did not
experience the glaucoma phenotype, or shed cell debris into the anterior chamber. This
suggests that the phenotype seen in the DBA/2J mice is dependent on the production of
pigment. It is hypothesized that the severe glaucoma associated with the disease arises from
melanocyte cell death in the iris of affected mice caused by structurally weakened
melanosomes releasing cytotoxic melanogenesis intermediates.

28

1.5 Target gene testing
Based on the phenotype that ocular melanosis presents in affected Cairn terriers, 11
genes either known to have a role in melanocyte and melanosome development or known to
be associated with other ocular pigmentation disorders were chosen as potential candidate
genes to be screened for possible association with ocular melanosis, including the genes
implicated in the DBA/2J mouse phenotype (60, 62). A complete list of candidate genes and the
markers used to genotype them is shown in Table 1.1. All candidate genes were assayed by
performing an association-based exclusion analysis. In an association-based exclusion analysis,
variable genomic markers (in this case, SNPs and microsatellites) located near the gene of
interest are assayed in a large number of dogs to determine whether a particular variant for any
of these markers segregates completely with the disease. The failure of nearby markers to
segregate completely with the disease phenotype excludes the gene near the marker as a
possible candidate gene. This test is based on the following underlying assumptions:
-

All affected dogs share the same underlying causative mutation for ocular melanosis
that is identical by descent due to a founder event

-

There has been no recombination between the marker and the causative mutation
since the mutation originally appeared

-

There have been no new mutations in either the marker or the gene

No single shared allele was detected at any of the 22 marker sites selected around the 11
candidate genes, showing that it is highly unlikely that a variant in any of them is causative for
OM (62).

29

Table 1.1 â Primers used to amplify markers around candidate genes Reproduced from (62).

30

1.6 Conclusions
Ocular melanosis is a major health concern within the Cairn terrier population.
Identification of the causal mutation would be greatly beneficial to owners and breeders, as it
would allow for the creation of a screening test to be created for the disease which would
facilitate the eradication of the disease from the breed by means of selective breeding.
Discovering the pathway underlying OM may also provide insight into the as-yetunmapped form of PDS commonly seen in black populations which shares many phenotypic
similarities to the disease as it manifests in the Cairn terrier. The fact that this disease seems to
effect only melanocytes of the eye, but does not alter behavior of melanocytes at all other body
locations suggests that this knowledge could also help to explain why uveal melanocytes
behave so differently from all other melanocytes despite having a nearly identical
embryological origin.
In the following chapters, the results of efforts to identify the underlying mechanisms
behind OM are described. These efforts include the cultivation of uveal melanocytes from both
OM-affected dogs and unaffected controls, various physiological assays performed to
characterize the in vitro behavior of those cells, and sequencing-based efforts to map the
causative mutation and determine what changes OM causes to the transcriptome of affected
dogs.

31

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32

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Miller PE. Chapter 11 - Uvea. Slatter's Fundamentals of Veterinary Ophthalmology
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Dubielzig RR, Ketring K, McLellan GJ, Albert DM. Chapter 9 - The uvea. Veterinary Ocular
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cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial
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melanocytes. Experimental cell research. 2010;316(8):1397-407.
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melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns.
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skin pigmentation. Pigment Cell Melanoma Res. 2011;24(3):401-10.
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role of the niche in melanocyte stem-cell fate determination. Nature. 2002;416(6883):854-60.
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domestic dog is governed by variants in three genes. Science. 2009;326.
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America CTCo. Cairn Terrier Club of America 2005 Breeder-Owner Survey. 2005.

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Balu G. Selective Laser Trabeculoplasty.

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candidate genes for ocular melanosis in Cairn terriers. J Negat Results Biomed. 2013;12:6.

37

CHAPTER 2
ISOLATION AND CULTIVATION OF CANINE UVEAL MELANOCYTES

Dawson-Baglien EM, Winkler PA, Bruewer AR, Petersen-Jones SM, Bartoe JT. Isolation and
cultivation of canine uveal melanocytes. Veterinary Ophthalmology. 2015;18(4):285-290

38

2.1 Abstract
Objectives: To establish a method for isolation and culture of canine uveal melanocytes.
Animals studied: Uveal explants from five mixed-breed dogs.
Procedures: Donor globes were dissected, and the anterior uvea removed. The uveal explants
were placed in trypsin solution for enzymatic digestion. Extracted cells were cultured in
modified F12 media. Immunocytochemistry was performed to confirm the identity of the
extracted cells.
Results: Melanocytes were successfully isolated from uveal explants. Contaminating cell types
were not observed. Repeated passaging of the melanocytes resulted in a gradual decrease in
intracellular pigment. Melanocyte cell lines could be cryopreserved, thawed, and cultures
successfully reestablished.
Conclusions: This extraction technique allows for generation of large populations of canine
uveal melanocytes in a relatively short period of time. This technique could be a useful tool for
future studies investigating both normal cellular characteristics and alterations found in
melanocytes from dogs with ocular melanocytic disorders.

39

2.2 Introduction
The uveal tract or vascular tunic of the mammalian eye is comprised of the adjoining iris,
ciliary body, and choroid (1). Melanocytes within the uveal tract synthesize and store photonabsorbing melanin pigments. Melanin plays a critical role in protection of all other
nonpigmented cell types within the eye through a combination of physical and biochemical
mechanisms (2). Physical protection is mediated by the âphoto-screening effectâ in which
melanin directly absorbs incident ultraviolet radiation (UV), whereas biochemical protection is
provided by the antioxidant/free radical scavenging properties of melanin resulting in
deactivation of reactive oxygen species generated from UV-exposure or other biochemical
processes.
Dysregulation of normal uveal melanocyte physiology is thought to contribute to a
number of breed-related ocular disorders in dogs. For example, potentially heritable iris
melanoma has been reported in Labrador retrievers (3, 4). Uveodermatologic syndrome, which
was first reported in the Akita, appears similar to Vogt-Koyanagi-Harada syndrome in humans
resulting from autoimmune destruction of melanocytes (5). Recently, a heritable pigmentary
uveitis/glaucoma condition has been reported in Golden retrievers (6). While the specific
etiology of the disease remains elusive, thickening of the iridal stroma, posterior synechiae, and
pigment deposition on the anterior lens capsule have been described in affected dogs (6, 7).
Ocular melanosis (OM) in the Cairn terrier appears to segregate as an autosomal dominant trait
and is characterized by proliferation and enlargement of melanocytes within the uveal tract (8,
9). Ultimately these atypical melanocytes can infiltrate into the trabecular meshwork of the
ciliary cleft resulting in secondary glaucoma.
40

Characterization of these diseases has primarily been limited to clinical and histological
descriptions of the abnormal phenotypes. More recently potential candidate genes for Cairn
terrier ocular melanosis have been screened (10), and genome-wide association studies are
planned or underway for Golden retriever pigmentary uveitis (W.M. Townsend, personal
communication) and for Cairn terrier ocular melanosis in our laboratory. We believe in vitro
uveal melanocyte cultures will provide a critical resource for investigation of the intracellular
pathways which become dysregulated in dogs with in ocular pigmentary diseases. A greater
understanding of intracellular pathway dysregulation may highlight potential candidate genes
to screen for causative mutations or suggest interventional treatment targets. The aim of the
current study was to develop a technique for extraction and culture of canine uveal
melanocytes.

41

2.3 Materials and methods
2.3.1 Globe collection and harvesting of anterior uveal tissue
Eight globes were collected aseptically from five normal, adult, mixed-breed dogs
following euthanasia for reasons unrelated to the current study. Upon collection, the globes
were placed in chilled, sterile phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO,
USA) for transport back to the laboratory.
Any residual extraocular tissue was trimmed, globes submersed in 10% povodone iodine
solution (Novaplus, Irving, TX, USA) for 5 min and rinsed in sterile physiological saline.
Observing sterile dissection techniques, a full-thickness sclera stab incision was made using a
no. 11 Bard-Parker blade (Fisher Scientific, Pittsburgh, PA, USA) 3 mm posterior to the limbus
and advanced circumferentially around the globe with tenotomy scissors. The anterior
segment, including the lens, was separated from the posterior segment, including the vitreous
humor. Using tissue dressing forceps and gentle traction, the residual uveal structures were
carefully separated as a single piece from the corneoscleral shell.
2.3.2 Isolation of uveal melanocytes
A custom isolation protocol was developed through modification of methods reported
by Hu et al (11). in the 1993 manuscript describing isolation and purification of human uveal
melanocytes. Anterior uveal explants were placed in 5 mL of a 0.0625% trypsin solution and
stored overnight at 4Â°C. This solution was made by diluting 2.5% trypsin solution (SAFC Global,
St. Louis, MO, USA) in Dulbeccoâs modified eagle medium with D-glucose and glutamine
(DMEM; Gibco, Grand Island, NY, USA) to a concentration of 0.0625%. Although the protocol

42

used by Hu et al. used trypsin at a concentration of 0.25%, preliminary experiments in our
laboratory showed the lower concentration (0.0625%) to be effective. Uveal explants were
incubated for 1 h in a 37Â°C water bath then transferred to 5 mL of fresh 0.0625% trypsin and
returned to the water bath for another hour. The used trypsin media was centrifuged at 2000 g
for 5 min to collect any suspended cells released from the uvea. Cell pellets were resuspended
in 2.5 mL of culture media and plated into 25-cm2 culture flasks (Corning, USA). Culture media
consisted of F12 media with L-glutamine (Gibco, USA) 10% fetal bovine serum (FBS, Gibco,
USA), 100 units/mL of penicillin (Gibco, USA), 100 Âľg/mL streptomycin (Gibco, USA), 20 ng/mL
basic fibroblast growth factor (Sigma-Aldrich, USA), and 10 ng/mL cholera toxin (SigmaAldrich,
USA). Cholera toxin is a known inhibitor of fibroblast growth in cell culture conditions (12). This
process was repeated for four sequential trypsin treatments, after which the remaining uveal
explant was cut into small pieces and placed into a 100x20 mm cell culture dish (Corning, NY,
USA) with 5 mL of culture media. All cultures were incubated at 37 Â°C in 5% CO2 with media
changed every 2â3 days, as determined by change in media color indicator. During the first 2
weeks, spent media was centrifuged at 2000 g for 5 min to collect nonadherent cells which
were resuspended and returned to the original flask; subsequently spent media was directly
aspirated using a Pasteur pipette (Fisher Scientific) attached to culture hood vacuum apparatus.
Cells were observed every other day using an Olympus IX71 inverted microscope and passaged
at 80% confluence.
When passaging cells, spent media was aspirated, and the cells were washed with 2.5
mL of PBS. This PBS was then aspirated, and 2.5 mL of 0.25% trypsin EDTA solution was added
(Gibco, USA). The cells were incubated at 37 Â°C for 5 min with intermittent agitation to

43

separate them from the flask. Following incubation, the trypsin was neutralized by addition of
2.5 mL of culture media to the flask. Repeated aspiration with a 5-mL pipette (Fisher
Scientific) was used to further dislodge any cells that remained adherent. The resultant
cellular suspension was transferred to a 15-mL centrifuge tube and spun at 2000 g for 5 min.
The media was aspirated, and the cell pellet resuspended in culture media. Cells were
replated at densities of 1x103â1x104 cells/cm2 as counted by hemocytometer. Any remaining
cells were cryopreserved for later use. Cells grown in 5-mL culture dish from the macerated
uveal explants were collected in an identical manner and transferred to a 25-cm2 culture
flasks.
When preparing cells for cryopreservation, pelleted cells were resuspended at a
concentration of 1â1.5x106 cells/ mL in freezing media consisting of F12 media with Lglutamine supplemented with 9% DMSO (J.T. Baker, Center Valley, PA, USA) and 15% FBS.
This cell suspension was then aliquoted into 2-mL cryotubes (Corning, USA), placed in a 80 Â°C
freezer for 24 h, and ultimately transferred to a liquid nitrogen immersion dewar for long
term storage.
When thawing cryopreserved cells, cryotubes were thawed in a 37Â°C waterbath.
Immediately after thawing, 1 mL of FBS was added to the suspension. The cell suspension was
centrifuged at 2000 g for 5 min to pellet the cells, and the freezing media was aspirated. The
cell pellet was then resuspended in 2.5 mL of culture media and transferred to a 25 cm 2
culture flask.

44

2.3.3 Immunocytochemistry
Immunocytochemistry (ICC) was performed using antibodies recognizing S100, Melan-A,
cytokeratin, and vimentin to confirm the identity and purity of the extracted cell cultures. For
ICC, freshly harvested cells were grown to 80% confluence; at the first passage, a subset of cells
was plated and cultured for 48 h in Nunc 8 chamber slides (Sigma-Aldrich, USA) resulting in cell
density of approximately 80% confluence. ICC was also performed under identical conditions on
several later-passage cell populations (table 2.2). Chambered cells were fixed in a 1:1 mixture of
100% methanol and 99.8% acetone for 10 min at 20 Â°C. Blocking was carried out in a 5% bovine
serum albumin (BSA, Sigma-Aldrich, USA) solution in PBS (Sigma-Aldrich, USA) with 0.025%
Triton X-100 (Sigma-Aldrich, USA) for 30 min at room temperature. Details and dilutions for
both primary and secondary antibodies are shown in table 2.1. All primary antibody incubations
were carried out at 37 Â°C for 1 h. All secondary antibody incubations were carried out at room
temperature for 1 h in a light-proof container. As a control to delineate any nonspecific binding
of the secondary antibody, ICC was performed identically on a set of slides in which the primary
antibody was omitted. All cells types were counterstained with 4â,6-diamidino-2-phenylindole
(DAPI; Sigma-Aldrich, USA). S100 is a calcium-binding protein and used as a cell marker for
neural crest-derived cells. Anti-S100 antibodies have been shown in previous studies to label
ocular melanocytes, but not fibroblasts or epithelial cells (11). Anti-MelanA recognizes a
melanocyte surface protein and labels melanocytes with greater specificity than S100 (13).
Vimentin is expressed by mesenchymal and neuroectodermal-derived cells in normal tissues,
and antibodies directed against it have been shown to label both fibroblasts and melanocytes
(11, 14). Cytokeratins are a class of intermediate filaments commonly found in epithelial cells,

45

but absent in melanocytes and fibroblasts (11). A broadspectrum cytokeratin antibody which
labels positively for many different types of cytokeratin was used to ensure that any epithelial
cell contamination was detected (11). Fibroblast cells from canine testes and MAC-T bovine
mammary epithelial cells were used as positive controls for vimentin and cytokeratin,
respectively (11, 14, 15). Fibroblast and MAC-T epithelial cells were cultured in DMEM media
with D-glucose and glutamine supplemented with 10% FBS, 100 units/mL of penicillin, and 100
Âľg/mL streptomycin.

46

Table 2.1 - Antibody specifications for immunocytochemistry
Antibody
Name

Wide
spectrum
AntiCytokeratin
Antivimentin
Anti-S100

MelanA
(A103)

Primary
Antibody
Manufacturer

Type

Expected Targets

Working
Secondary
Concentration Antibody
(Primary
Antibody)
1/100
goat antirabbit

Abcam, ref.
ab9377

Rabbit
Polyclonal

Cytoskeleton

Millipore, ref.
AB5733
Santa Cruz
Biotechnology,
ref. sc-71994
Abcam, ref.
ab79672

Chicken
Polyclonal
Mouse
Monoclonal

Cytoskeleton

1/5000

Diffuse
cytoplasmic
signal
Endomembrane
system
components,
melanosomes

1/100

Mouse
Monoclonal

1/100

47

goat antichicken
rabbit
antimouse
goat antimouse

Secondary
Antibody
Manufacturer
Life
Technologies,
ref. A11070
Abcam, ref.
ab6875
Life
Technologies,
ref. 11062
Life
Technologies,
ref. 11001

Working
Concentration
(Secondary
Antibody)
1/500

1/2500
1/500

1/500

2.4 Results
2.4.1 Isolation and morphology

Figure 2.1 - Extracted canine uveal melanocytes develop dendritic processes after
attachment to culture plates Cells were photographed 7 days after extraction. Size bar
equals 100 Âľm.

Viable cells were isolated from both the sequential trypsin treatment suspensions and
the macerated uveal explant cultures. Initially, the extracted cells were free-floating and
contained dark brown pigment. The vast majority of these cells had adhered to the bottom
surface of the culture flask within 7 days of isolation, although viable free-floating cells were
observed as late as 3 weeks post isolation. After adhesion, cells developed a variable number of
dendritic processes (figure 2.1), similar to what had previously been reported for human uveal
melanocytes (11). An average of approximately 2x106 cells were obtained from each uveal

48

(b)

(a)

(c)

Figure 2.2 - Extracted canine uveal melanocytes lose pigment over multiple passages
Passage 1 (a), passage 3 (b), and passage 5 (c). Size bar equals 100 Âľm.

explant prior to initial passaging. In all subsequent passages (with a typical time of 1â2 weeks
between passages), the cells re-adhered within 1â2 days, quickly regaining the dendritic
morphology. The observed pigment was gradually lost from cultured cells over 3â5 passages
(figure 2.2). This is similar to previous reports for human uveal melanocytes (11, 16). The
longest-lived cell line to date has been successfully passaged 14 times. These cells do not yet
show visible characteristics of senescence, which we anticipate would include an enlarged,
plate-like appearance, and a loss of dendritic processes (11).
2.4.2 Cryoperservation
All four cell lines were successfully revived following cryopreservation regardless of
passage number at the time of freezing. The cell line which has been cryopreserved the most
often has undergone four successful freeze/thaw cycles.
2.4.3 Immunocytochemistry
Immunocytochemistry results are displayed in figure 2.3 and summarized in table 2.2. Cells
extracted from canine uveal tracts labeled positively for: MelanA, S100, and vimentin

49

antibodies, but were not labeled by the cytokeratin antibody. These findings are consistent with
previous descriptions of melanocyte cultures (11, 14). Control cell lines labeled as expected;
canine testes fibroblasts labeled positive for vimentin and negative for MelanA, S100, and
cytokeratin; MAC-T epithelial cells positive for cytokeratin, and negative for MelanA, vimentin,
and S100.
Table 2.2 - Characterization of cell lines using immunocytochemical labeling
Cell Line

Cell Type

Number of
Passages
N/A
3
6

MelanA
(A103)
+

S100 Cytokeratin Vimentin

MAC-T
Epithelial control
+
Fibroblast Fibroblast control
MLN-M Extracted uveal
+
melanocytes
MLN-J
Extracted uveal
3
+
+
melanocytes
MLN-R
Extracted uveal
5
+
+
melanocytes
MLN-U Extracted uveal
5
+
+
melanocytes
(+) denotes a detectable signal above that seen in negative control lines. (-) denotes no
detectable signal above that seen in negative control lines.

50

+
+
+
+
+

Figure 2.3 - Immunocytochemistry (ICC) of extracted first-passage canine uveal
melanocytes Extracted first-passage melanocytes (âMelanocytesâ column) label positively
for: vimentin (âVimentinâ row; Texas Red, red color), S100 (âS100â row; Alexa Fluor 594, red
color), and MelanA (âMelanAâ row; Alexa Fluor 488, green color); however, are negative for
cytokeratin (âCytokeratinâ row; Alexa Fluor, green color). MAC-T epithelial cells (âEpithelial
Cellsâ column) label positively for cytokeratin and canine testes fibroblasts (âFibroblastsâ
column) label positively for vimentin. The âControlâ column shows first passage canine uveal
melanocytes incubated without primary antibodies to demonstrate any secondary antibody
nonspecific binding. All ICC samples were counterstained with DAPI (blue color). Unlabelled
cells were imaged using a bright field microscope to demonstrate typical appearance in
vitro. Size bar equals 100 Âľm.

51

2.5 Conclusions
The isolation and culture techniques described here successfully generated viable uveal
melanocytes from canine globes. In developing a technique to isolate human uveal
melanocytes, Hu et al. observed widespread contamination of their cultures by other cell types
prevalent in the uvea, most notably fibroblasts and epithelial cells. This necessitated treatment
of the cultures with the selective cytotoxic agent geneticin (11). Although they did observe
instances of uveal melanocytes outcompeting contaminant cells (ultimately leading to the
disappearance of the contaminants), this occurred in only a minority of total cultures. Unlike
the situation reported for human uveal melanocyte isolation, we did not encounter-mixed cell
populations and it was not necessary to use geneticin. Our melanocyte cultures did not contain
cytokeratin-positive cells, showing they were free of epithelial cell contamination. The
melanocytes labeled positively for both S100 and MelanA, antibodies which do not label
fibroblasts. This provided further evidence that our cultures were free of the contaminants
observed by Hu et al. There were two principal differences between our culture procedures and
those used by Hu et al. First, the concentration of trypsin used to treat the uveal explants
differed. Hu et al. used a 0.25% trypsin solution, whereas we used only a 0.0625% trypsin
solution. It is possible that at lower trypsin concentrations, the total number of cells released by
the uveal explants is biased toward melanocytes, causing them to make up a much higher
percentage of the total cells released than other cell types. Second, our study was performed
with canine uveal tracts rather than human uveal tracts. It is possible cells of the canine uvea
have different nutritional requirements than their human counterparts, and canine
melanocytes will tend to outgrow other cell types in the specific culture media used in this

52

study. Additional study is required to definitively identify why we observed a significant
reduction in contaminating cell types with our extraction methods.
In conclusion, we have established a successful method for isolation and culture of
canine uveal melanocytes. We believe the techniques described here will be useful for
investigation of various ocular disorders arising from aberrant melanocyte growth and
replication. A future aim for our laboratory is utilization of the described techniques to
generate melanocyte cell lines from OM-affected uveal tracts. We have established a donor
registry of Cairn terriers affected by ocular melanosis. Following death by natural causes,
owners have consented to allow us to collect OM-affected globes. Comparison of the in vitro
growth characteristics of melanocyte cultures from normal and OM-affected uveal tracts may
highlight critical differences. Identification of such differences may suggest potential candidate
genes for the mutation causing ocular melanosis or suggest potential pharmacological targets
for therapeutic intervention.

53

2.6 Acknowledgements
We wish to thank Drs. Lorraine Sordillo, Vilma Yuzbasiyan-Gurkan, and Steven Arnoczky
for providing cell lines used as ICC controls. This study was supported by grants from the
Foundation of the Cairn Terrier Club of America, the Myers-Dunlap Endowment for Canine
Health, the Battle Creek Kennel Club though the Michigan State University College of Veterinary
Medicine Endowed Research Funds, and Michigan State University College of Veterinary
Medicine Faculty Development Funds.

2.7 Funding Sources
The funding sources had no role in the design of this study, the collection, analysis, or
interpretation of data, or the writing and submission of the manuscript.

2.8 Conflict of Interest
The authors declare no competing interests for this study.

54

REFERENCES

55

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Prince JH. Comparative anatomy of the eye. Springfield, Ill.: Thomas; 1956.

2.
Hu DN, Simon JD, Sarna T. Role of ocular melanin in ophthalmic physiology and
pathology. Photochemistry and photobiology. 2008;84(3):639-44.
3.
Cook C, Lannon A. Inherited iris melanoma in the Labrador Retriever dogs. Transactions
of the American College of Veterinary Ophthalmologists. 1997;28:106.
4.
Donaldson D, Sansom J, Scase T, Adams V, Mellersh C. Canine limbal melanoma: 30
cases (1992-2004). Part 1. Signalment, clinical and histological features and pedigree analysis.
Vet Ophthalmol. 2006;9(2):115-9.
5.
Akakura S, Takahashi T, Onishi T. Vogt-Koyanagi-Harada syndrome (uveitis diffusa acuta)
in the dog. Journal of the Veterinary Medicine. 1977.
6.
Sapienza JS, Simo FJ, Prades-Sapienza A. Golden Retriever uveitis: 75 cases (1994-1999).
Vet Ophthalmol. 2000;3(4):241-6.
7.
Esson D, Armour M, Mundy P, Schobert CS, Dubielzig RR. The histopathological and
immunohistochemical characteristics of pigmentary and cystic glaucoma in the Golden
Retriever. Vet Ophthalmol. 2009;12(6):361-8.
8.
Petersen-Jones SM, Forcier J, Mentzer AL. Ocular melanosis in the Cairn Terrier: clinical
description and investigation of mode of inheritance. Vet Ophthalmol. 2007;10 Suppl 1:63-9.
9.
Petersen-Jones SM, Mentzer AL, Dubielzig RR, Render JA, Steficek BA, Kiupel M. Ocular
melanosis in the Cairn Terrier: histopathological description of the condition, and
immunohistological and ultrastructural characterization of the characteristic pigment-laden
cells. Vet Ophthalmol. 2008;11(4):260-8.
10.
Winkler PA, Bartoe JT, Quinones CR, Venta PJ, Petersen-Jones SM. Exclusion of eleven
candidate genes for ocular melanosis in Cairn terriers. J Negat Results Biomed. 2013;12:6.
11.
Hu DN, McCormick SA, Ritch R, Pelton-Henrion K. Studies of human uveal melanocytes
in vitro: isolation, purification and cultivation of human uveal melanocytes. Investigative
ophthalmology & visual science. 1993;34(7):2210-9.
12.
Eisinger M, Marko O. Selective proliferation of normal human melanocytes in vitro in
the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci U S A. 1982;79(6):2018-22.
13.
Clarkson K, Sturdgess I, Molyneux A. The usefulness of tyrosinase in the
immunohistochemical assessment of melanocytic lesions: a comparison of the novel T311
antibody (anti-tyrosinase) with S-100, HMB45, and A103 (anti-melan-A). Journal of Clinical
Pathology. 2001;54(3):196-200.

56

14.
Koenig A, Wojcieszyn J, Weeks BR, Modiano JF. Expression of S100a, vimentin, NSE, and
melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of
canine melanoma. Vet Pathol. 2001;38(4):427-35.
15.
Huynh HT, Robitaille G, Turner JD. Establishment of bovine mammary epithelial cells
(MAC-T): an in vitro model for bovine lactation. Experimental cell research. 1991;197(2):191-9.
16.
Hu DN, McCormick SA, Orlow SJ, Rosemblat S, Lin AY, Wo K. Melanogenesis by human
uveal melanocytes in vitro. Investigative ophthalmology & visual science. 1995;36(5):931-8.

57

CHAPTER 3
PHYSIOLOGICAL CHARACTERIZATION OF OCULAR MELANOSIS-AFFECTED CANINE
MELANOCYTES

58

3.1 Abstract
Objective: Cairn terriers with ocular melanosis (OM) accumulate large, heavily
pigmented melanocytes in the anterior uvea. Darkly pigmented plaques develop within the
sclera, leading us to hypothesize that OM uveal melanocytes may have an abnormal migratory
capacity.
Animals studied: Globes from OM-affected Cairn terriers and unaffected control eyes
enucleated for reasons unrelated to this study were used for immunohistochemistry and to
culture melanocytes for in vitro cell behavior assays.
Procedures: The scleral plaques of six dogs were immunolabeled for HMB45, MelanA,
PNL2, CD18, CD204, and Iba-1 and compared with the pigment cells accumulated within the
irides. Cultured uveal melanocytes from OM-affected and control dogs were compared using
conventional assays measuring cell proliferation, invasion capability, and melanin production.
Results: Melanocytes isolated from OM eyes had significantly elevated levels of per-cell
melanin content and production compared to controls. The majority of pigmented cells in the
scleral plaques were HMB45 positive indicating a melanocytic origin. Many were also CD18
positive. No differences were observed between cultured melanocytes from OM-affected and
control uvea for standard in vitro proliferation or invasion assays.
Conclusion: Pigmented cells which accumulate in the sclera of OM-affected Cairn
terriers are predominantly melanocytes, however in vitro assays of uveal melanocytes did not
reveal differences in migratory behavior between OM and control cells. Migratory behavior of

59

OM-melanocytes may be environment dependent. We suggest that RNA sequencing and
differential expression analysis would be a useful next step in understanding this disease.

60

3.2 Introduction
Ocular melanosis (OM) in Cairn Terriers is a hereditary condition characterized by a
bilateral accumulation of large, heavily pigmented, discrete round cells predominantly within
the anterior uvea. These cells cause circumferential expansion and peripheral thickening of the
iris root and are associated with the progressive development of pigmented scleral/episcleral
plaques (figure 3.1) (1). With progression there is deposition of similar large, round, pigmented
cells in other sites of the affected eye, such as the surface of the optic nerve head, the choroidal
stroma, where it may obscure the tapetum, and even the meninges around the optic nerve.
There is also release of pigment into the aqueous, which leads to deposition within the
drainage angle and along the ventral corneal endothelial surface. Eventually the ventral
opening into the ciliary cleft can be completely obscured and aqueous drainage impeded to
such an extent as to cause secondary glaucoma. Our previous studies have shown that the
majority of the large, pigmented cells throughout the uvea in affected dogs are melanocytes,

Figure 3.1 - A Cairn terrier with late-stage ocular melanosis There are extensive scleral
pigmented plaques sclera adjacent to the limbus (a). The iris stroma is expanded and
obscured by dense populations of large, round, densely pigmented cells; few smaller,
spindloid to stellate uveal melanocytes representing normal resident populations are at the
left of the image (b). Similar large round, densely pigmented cells form plaques between
bundles of scleral collagen (c). Dog No. 4. Hematoxylin and eosin, post bleached, Bar =
40Âľm.

61

although a variable, but usually small number of melanophages are often also present (2). The
abnormal expanded population of melanocytes in OM-affected dogs differ in morphology from
normal uveal melanocytes, appearing round instead of spindle-shaped (although some spindleshaped melanocytes can still be observed in the anterior uvea of OM-affected eyes), and
packed with melanosomes of varying size. It is thought that the darkly pigmented cells that
accumulate in the sclera/episclera migrate there from the uvea; however, there are no
reported immunohistochemical studies to investigate whether these cells in scleral plaques are
melanocytes or melanophages. The apparent invasive or migratory behavior of the cells
extending into sclera and other sites in OM-affected eyes has been likened to a neoplastic
behavior, but it is unclear if the large round pigment laden cells that typify ocular melanosis are
overtly neoplastic. In contrast to classic anterior uveal melanocytoma in dogs which typically
presents as a single expansile lesion, the large round melanocytes of ocular melanosis occur
diffusely and circumferentially throughout the iris root.
We hypothesize that the pigmented cells in the scleral plaques are also melanocytes,
and that the abnormal anterior uveal melanocytes of OM-affected dogs will have increased
migratory capacity and melanogenesis in vitro. In this study, we used immunohistochemistry
(IHC) to characterize the cells within the scleral/episcleral plaques in OM-affected eyes and
compared them with those expanding the uvea. We also isolated and cultured melanocytes
from OM-affected eyes and compared their behavior under a battery of assays testing
migratory and neoplastic characteristics, with uveal melanocytes from normal control eyes.

62

3.3 Materials and methods
3.3.1 Animals used
Forty-eight globes from 30 dogs; 27 from 18 OM-affected dogs and 21 from 12 clinically
normal dogs were used in this study. The OM-affected globes were donated by the dogâs
owners after being enucleated to manage painful OM-related glaucoma or when an OMaffected dog was euthanized for other health related reasons. OM was diagnosed by a
veterinary ophthalmologist. Control eyes were from either elderly unaffected Cairn Terriers or
crossbred dogs with dark irides which were euthanized for reasons unrelated to this study. The
unaffected Cairn Terriers had been examined and found to be OM unaffected by a veterinary
ophthalmologist. Forty-two globes were used for uveal culture (21 OM-affected and 21
controls) and 6 OM-affected globes were used for immunohistochemical examination. Table 3.1
provides details of the dogs used in this study.

63

Table 3.1 - Sources of globes used in this study
Number

Dog

Age and Sex

Breed

OM Status

1
2
3
4

CA76
CA166
D1
CA599

15,M
12,F
Unknown
16,M

Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier

Affected
Affected
Affected
Affected

# Globes
Donated
2
2
1
2

5
6

CA569
CA538

8,F
11,F

Cairn Terrier
Cairn Terrier

Affected
Affected

2
2

Cell Culture
Cell Culture
Cell Culture
Cell Culture
and IHC
Cell Culture
Cell Culture

7
8
9
10
11
12
13
14
15
16

CA319
CA524
CA518
CA239
CA67
CA537
CA609
CA393
CA576
D2

13,F
9,F
10,F
16,F
15,M
8,F
16,M
15,F
16,F
1,F

Affected
Affected
Affected
Affected
Affected
Affected
Affected
Unaffected
Unaffected
Unaffected

2
1
1
2
2
1
2
2
2
2

Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture

17

D3

1,M

Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Curly Coat
Retriever Mix
Curly Coat
Retriever Mix
Mixed Breed
Mixed Breed
Mixed Breed
Unknown
Unknown
Mixed Breed
Mixed Breed
Mixed Breed
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier
Cairn Terrier

Unaffected

2

Cell Culture

Unaffected
Unaffected
Unaffected
Unaffected
Unaffected
Unaffected
Unaffected
Unaffected
Affected
Affected
Affected
Affected
Affected

1
1
1
2
2
2
2
2
1
1
1
1
1

Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
Cell Culture
IHC
IHC
IHC
IHC
IHC

18
19
20
21
22
23
24
25
26
27
28
29
30

D4
Unknown,M
D5
Unknown,M
D6
Unknown,F
D7
Unknown
D8
Unknown
D9
Unknown
D10
Unknown
D11
Unknown
CA530
10,M
CA608
12,M
CA535
12,F
CA607
12,F
CA642
10,F

64

Usage

3.3.2 Immunohistochemistry
Six formalin fixed globes from OM-affected dogs (# 4, 26-30) were processed for IHC.
They were sagittally bisected and one or both halves embedded in paraffin. 5Âľm sections were
pre-bleached and routinely stained with hematoxylin and eosin (H&E), or processed for
immunohistochemistry (IHC) and labeled for HMB45, MelanA, PNL2, CD18, CD204, or Iba-1 and
post-bleached and counterstained with Gillâs III hematoxylin. Details of IHC methods and
antibodies are provided in table 3.2. Bleaching was by incubation of slides with 0.5% Potassium
Permanganate for 20 minutes and 5% Oxalic Acid for 45 seconds. H&E and IHC sections were
evaluated by two pathologists (DGS, EN). For each IHC marker, the percentage of the
pigmented round cells in the anterior uveal stroma and within the scleral/episcleral plaques
positive for antibody labelling was recorded.

65

Table 3.2 - Antibodies and positive control tissue

Antibody

Type/Host

Supplier

Dilution

Retrieval
method

Chromogen

Positive
tissue

Kit

Melan A

PNL-2

HMB45

Mouse
monoclonal

Mouse
monoclonal

Mouse
monoclonal

Low pH PT1

ChromoMap
V-red
Melanoma
(Alkaline
Phosphatase)

Santa Cruz 1:500

Low pH PT1

ChromoMap
V-red with
amplification Melanoma
(Alkaline
Phosphatase)

Dako

1:10

ChromoMap
V-red with
3
CC1 , 32 min amplification Melanoma
(Alkaline
Phosphatase)

1:20

ChromoMap
V-red
Histiocytic
(Alkaline
sarcoma
Phosphatase)

Dako

1:20

CD18

Mouse
monoclonal

Peter
Moore

CD204

Mouse
monoclonal

Tans Genic
Inc./
1:1000
Cosmo Bio

IBA-1

Wako
Rabbit polyclonal Chemical
USA, Inc.

1:1000

1 Heat

Low pH PT1

ChromoMap
V-red
Histiocytic
CC13, 64 min
(Alkaline
sarcoma
Phosphatase)

Low pH PT2

FLEX (AEC)

Histiocytic
sarcoma

retrieval at low pH on the Dako PT link (Discovery Ultra, Ventana, Tucson, AZ, USA)
retrieval at low pH on the Dako PT link (Agilent Technologies, Santa Clara, CA, USA)
3 Heat retrieval using CC1 (Cell Conditioning 1, Discovery Ultra, Ventana, Tucson, AZ, USA)
2 Heat

66

3.3.3 Cell culture methods
Melanocytes were isolated and cultured as previously described (3). Briefly, the anterior
uvea was excised from donor eyes, placed in 5 ml of 0.0625% trypsin (SAFC Global, St. Louis,
MO, USA) in Dulbeccoâs modified eagle medium with D-glucose and glutamine (DMEM, Gibco,
Grand Island, NY, USA) and incubated at 37Â°C for one hour and the incubating media collected.
This process was repeated 3 additional times resulting in 4 total aliquots of collected media.
The media was centrifuged at 2000g for 5 minutes to collect the released cells. The cell pellets
were resuspended in 2.5 ml of melanocyte media (F12 with L-glutamine [Gibco, USA], 10% fetal
bovine serum [Gibco, USA], 100 units/ml of penicillin [Gibco, USA], 100 Âľg/ml streptomycin
[Gibco, USA], 20 ng/ml basic fibroblast growth factor [Sigma-Aldrich, St. Louis, MO, USA], and
10 ng/ml cholera toxin [Sigma-Aldrich, USA]) and plated in 25 cm2 culture flasks (Corning,
Corning, NY, USA). The resulting cultures were incubated at 37Â°C in 5% CO2. Media was replaced
every 2-3 days, using the media pH associated color-change indicator as a guide.
Cells were passaged upon reaching approximately 80% confluence. They were washed
with PBS, and incubated with 2.5ml of 0.25% Trypsin-EDTA (Gibco, USA) at 37Â°C for 5 minutes.
Trypsin was neutralized with 2.5 mL of culture media, the cells pelleted (2000 g for 5 min at
room temperature), resuspended in 5-10 mL of culture media and quantified with a
hemocytometer They were then either used for assays described below, cryopreserved for later
use, or re-plated for further growth.

67

3.3.4 Preparing ECM-treated plates
Plates were treated with extracellular matrix components (ECM) as described by
Wagner et al 1997 for use in doubling time, scratch and cell adhesion assays (4). Five types of
extracellular matrices were used: fibronectin (Sigma-Aldrich, USA), laminin (Sigma-Aldrich,
USA), collagen I (Sigma-Aldrich, USA), collagen III (Millipore, Billerica, MA, USA), and collagen IV
(Sigma-Aldrich, USA). All were reconstituted according to the manufacturerâs instructions. For
collagens I, III, and IV, 10 Âľg/cm2 was added to each well of a culture plate, then air dried
overnight in a laminar flow hood. Fibronectin and laminin coatings were applied at 2 Âľg/cm2
and incubated at 37Â°C for one hour, after which any remaining solution was removed.
3.3.5 Doubling time assay
Five OM-affected (# 1-3, 5, 7) and 5 control uveal cell lines (# 20-24) were used. One 24well plate per ECM component was used for each cell line, plus one untreated control. Seeded
plates (1x104 cells in 250 Âľl media) were incubated at 37Â°C with 5% CO2. Every 24 hours for the
next 8 days, adherent cells in three wells were trypsinized and collected for counting. The total
number of cells in culture each day was charted, and data points from the exponential growth
phase of the curve used to calculate a doubling time using a least squares fitting method (5).
3.3.6 Cellular melanin levels
As cells were passaged, 3 ml of cell suspension was taken for cellular melanin
concentration testing performed in 3 one ml technical replicates. Cells were pelleted and
dissolved in 1N NaOH. The absorbance of each sample at 475 nm was measured using a

68

Beckman DU 650 spectrometer. The average absorbance of 3 readings from 3 technical
replicates was calculated and compared to a standard curve generated by measuring the
absorbance of known amounts of synthetic melanin (Sigma-Aldrich, USA) to determine the total
melanin present (6). Total melanin was divided by the previously counted number of cells/ml to
determine a melanin/cell ratio. Canine testes fibroblasts were used as an unpigmented cell
control, and their average absorbance value was subtracted from each sample to account for
the background absorbance of non-melanin cell components. Melanin production per cell was
calculated using the following equation:

đđđś =

đđśđĄđ â đđśđ
1.3đˇ(đ â 1)

where MPC is melanin produced per cell per day (in ng/cell/day), MCt is the melanin content (in
ng/cell) at time t (in days), MCo is the melanin content per cell (ng/cell) at time 0, P is the
population increase (in cells) during time t, and D is the average doubling time of the cell type
(affected or control, in days) (7). Twelve OM-affected (# 1-3, 5-13) and 10 control uveal
melanocyte cell lines (# 14-15, 18-25) were used.
3.3.7 Wound-healing rate
Seven OM-affected (# 3-4, 6-7, 10, 12-13) and 4 control uveal melanocyte cell lines (#
17, 21, 24-25) were used. Cells were plated into 24-well culture plates at a concentration of
~1x105 cells/well. Three technical replicates were performed for each cell line for each ECM
component and an untreated control plate. Cells were incubated at 37Â°C with 5% CO2 until
confluent, with media being changed every 2-3 days as needed. A 10 Âľl pipette tip was then

69

used to create a scratch (wound) in the cell monolayer, and a razor blade was used to make a
cut perpendicular to the scratch so that the same area could be reliably located for observation.
An inverted microscope (Nikon Eclipse TS100) was used to capture images of the scratched area
over the course of the next 72 hours (8). The program TScratch was used for automated
analysis and comparison of the acquired images (9).
3.3.8 Cell adhesion assay
Cell adhesion rate was measured using a modified tetrazolium colorimetric assay (10,
11). Four OM-affected (# 1-3, 8) and 4 control uveal melanocyte cell lines (# 15, 22, 24-25) were
used. 7.5x103 cells in melanocyte media per well were plated into ECM coated 96-well plates or
an untreated plastic control. Plates were incubated at 37Â°C with 5% CO2 to allow cellular
attachment, and every 10 minutes for 60 minutes post-plating, the media was aspirated from 3
wells of each cell type and replaced with serum-free media to remove any unattached cells. At
the end of 60 minutes, 50 Âľl of a 1mg/ml solution of MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), Sigma-Aldrich, USA) was added to each well. The plates were
then incubated for 4 hours at 37Â°C with 5% CO2 to allow attached cells to reduce MTT to a blue
formazan crystal. Remaining media was aspirated, each well gently washed twice with BSS to
remove any unreduced MTT, and then 10% SDS-0.01 N HCl solution used to dissolve the MTT
formazan. Sample absorbance at 550nm and 655nm was measured using a BioRad model 3550
plate reader. The 655nm reading was subtracted from the 550nm reading to account for
background absorbance. Results were reported as a percent metabolic activity relative to the
control. Additional cells from each cell line were plated at the same concentration in ECM

70

treated plates and allowed to attach for 2 hours to determine a maximum attachment. The
previously measured metabolic activity data was compared to that of the maximum attachment
samples to determine a % maximum attachment for each time point.
3.3.9 Anchorage independence
Seven OM-affected (# 1-2, 5-6, 8-9, 12) and 6 control uveal cell lines (# 15, 21-25) were
used. Each well of a 6-well plate was coated with 2.5 ml of a base layer containing 0.5% agarose
dissolved in melanocyte growth media as described above with 1% or 10% FBS for melanocyte
lines. A negative control of canine testes fibroblasts and a positive control of MDA-MB-231
human breast adenocarcinoma cells were cultured in DMEM media with 100 units/ml of
penicillin, 100 Âľg/ml streptomycin and 1% or 10% FBS. The base layer was allowed to cool until
solid. A second layer containing 2.5x104 cells in 2.5 ml of growth media with 0.35% agarose was
added to the top of the base layer, and allowed to cool until solid. The plate was incubated
overnight at 37ÂşC with 5% CO2. Then 2.5 mL of growth media was added. Media was replaced
every 3 days for 30 days. Plates were then imaged using an inverted microscope and a
STEMgrid-6 plate-counting grid (STEMCELL technologies, Cambridge, MA, USA). Colonies
defined as cluster of > 20 cells were counted in ten 2mm2 grid squares for each sample, and the
averages of these counts were multiplied by the number of grid squares per plate to give a
colonies-per-plate count.

71

3.3.10 Invasion assay
Five OM-affected (# 1-2, 7-9) and 5 control uveal cell lines (# 20, 22-25) were used.
Plates for Matrigel in vitro cell invasion assay with 8 Âľm pore diameter size supports (Corning,
USA) were prepared as per the manufacturerâs instructions. The assay was performed in
triplicate with canine testes fibroblasts as a negative control, and MDA-MB-231 cells a positive
control. The chemoattractant used was 10% FBS in F12 media, and 2.5x104 cells were plated in
each well, as recommended in the manufacturerâs protocol. Cell invasion chambers were
incubated at 37ÂşC with 5% CO2. After 24 hours, any remaining cells were removed from the
upper chamber, the membranes were stained with Diff-Quik (Sigma-Aldrich, USA) and cells that
had invaded were quantified by counting four fields near the center of each replicate.

72

3.4 Results
3.4.1 Immunohistochemistry
The scleral plaques within all 6 OM-affected globes examined consisted of large, round,
densely pigmented cells similar in appearance to those expanding the anterior uvea. IHC (Table
3) showed the majority of these cells were immunoreactive for HMB45, but not for PNL2 or
Melan A (figure 3.2). In contrast, rare round, pigmented cells in the anterior uveal stroma of 4
(# 4, 27, 29, 30) of the 6 dogs were PNL2 and MelanA immunoreactive. Between 30 and 90% of
pigmented round cells both in the anterior uvea and 80 to 90% of those within the scleral
plaques were immunoreactive for CD18 (figure 3.3). Rare round pigmented cells in the anterior
uvea, but not in the scleral plaques, of the eye of one dog (# 29) were immunoreactive for Iba-1
and/or CD204. This eye, however, also had a more significant inflammatory response than
other cases with perivascular infiltrates of lymphocytes, plasma cells, and histiocytes occurring
throughout the anterior uvea. None of the large pigmented round cells in the uvea or scleral
plaques of the other 5 dogs (# 4, 27-28, 3026-29, 31) were immunoreactive for Iba-1 or CD204.
3.4.2 Cellular melanin levels
Cellular melanin levels were significantly higher in early-passage melanocytes from OMaffected dogs relative to unaffected control melanocytes, both in terms of total melanin
content and per-cell melanin production (figures 3.4 and 3.5). For the first three passages
following isolation, OM-uveal melanocytes had an average per-cell melanin content that was
more than twice the amount found in unaffected cells, including a 3.9-fold average increase in
the first passage following isolation (0.37Âą0.19ng/cell vs. 0.094Âą0.064ng/cell, p=0.00185). The

73

Table 3.3 - Percentage of the expansile round melanocyte population with immunoreactivity for histiocytic and melanocytic
markers within the anterior uvea or scleral plaques in Cairn terriers with ocular melanosis
Dog
No.
4
26
27
28
29
30

CD18
anterior scleral
uvea
plaque
30
80
30
80
60
90
90
90
90
90
90
90

CD204
anterior scleral
uvea
plaque
0
0
0
0
0
0
0
0
<10
0
0
<10

IBA-1
anterior scleral
uvea
plaque
0
0
0
0
0
0
0
0
<10
0
0
0

74

HMB45
anterior scleral
uvea
plaque
60
70
60
95
60
60
60
90
30
70
60
70

PNL2
anterior scleral
uvea
plaque
<10
0
0
0
20
0
0
0
<10
0
0
0

Melan A
anterior scleral
uvea
plaque
10
0
0
0
10
0
0
0
10
0
10
0

Figure 3.2 â Immunohistochemistry of ocular melanosis, anterior uvea or scleral plaque
Ocular melanosis, anterior uvea (a, c, e) or scleral plaque (b, d, f), dog. Large round
pigmented cells show diffuse cytoplasmic red labeling for HMB45 in the anterior uvea (a)
and in scleral plaques (b). Dog No. 26. IHC post bleached with hematoxylin counterstain.
Only spindle to stellate shaped cells label for PNL2 (c) or Melan A (e) in the anterior uvea.
Round cells are not immunoreactive for PNL2 (d) or Melan A (f) within the scleral plaques.
Dog No. 4. IHC post bleached with hematoxylin counterstain. Bar = 40Âľm.
75

Figure 3.3 â Immunohistochemistry of ocular melanosis, anterior uvea or scleral plaque
Ocular melanosis, anterior uvea (a, c, e) or scleral plaque (b, d, f), dog. The large round
pigmented cells widely show strong perimembranous to cytoplasmic labeling for CD18 in both
the anterior uvea (a) and scleral plaque (b). Dog No. 4. IHC post bleached with hematoxylin
counterstain. These cells are not immunoreactive for CD204 in either the anterior uvea (c) or
scleral plaque (d). Only small round cells consistent with inflammatory cells label for Iba-1 in
the anterior uvea (arrow) and in the scleral plaque (f). Dog No. 29. Bar = 40Âľm.

76

Figure 3.4 â Average melanin content of OM-uveal melanocytes Average melanin content
(ng/cell) of OM-uveal melanocytes compared with unaffected melanocytes at different
passages in culture. Significant differences exist between the two populations for the first
three passages post-isolation (*p<0.05), after which melanin concentration in both
populations equalizes. For each timepoint, n is between 5 and 9.
difference remained significant in passages 2 (2.3-fold increase, 0.094Âą0.025ng/cell vs.
0.041Âą0.022ng/cell, p=0.0052) and 3 (2.9-fold increase, 0.04Âą0.0066ng/cell vs.
0.014Âą0.011ng/cell, p=0.0017) (figure 3.4), but ceased to be significant in passage 4 and
beyond. The average per-day melanin production of OM-uveal melanocytes between passage 1
and 2 was 2.8-fold higher than the unaffected control average (0.046Âą0.014ng/cell/day vs.
0.016Âą0.0084ng/cell/day, p=0.003). This increase remained significant between passages 2 and
3 (2.5-fold increase, 0.019Âą0.0032ng/cell/day vs. 0.0076Âą0.0067ng/cell/day, p=0.0021), after
which there was no significant difference between the two populations (figure 3.5).

77

Figure 3.5 â Average melanin produced per cell per day in OM-uveal melanocytes Average
melanin produced per cell per day (ng/cell/day) in OM-uveal melanocytes compared with
unaffected melanocytes at different passages in culture. Significant differences exist
between the two populations until after the third passage (*p<0.05), after which the
populations equalize. Note that individual doubling time data was not available for all cell
populations, so the average doubling time for affected or unaffected melanocytes was used
where appropriate. For each time point, n is between 4 and 7.

3.4.3 Doubling time
No significant difference was detected between the doubling times of OM-uveal
melanocytes (44Âą5 hrs) and those of the unaffected control melanocytes (47Âą3 hrs) (p=0.3851,
figure 3.6). Similarly, no difference was detected between the average doubling time of OMuveal melanocytes and unaffected controls in any of the 5 different ECM treatment groups
(figure 3.6). Additionally, none of the ECM components tested caused a significant change in
the doubling time of the uveal melanocytes when compared to the untreated control plates.

78

3.4.4 Anchorage independence
Both OM-uveal melanocytes and unaffected controls formed colonies at very low rates,
and these rates were not significantly different from that of the negative control (canine testes
fibroblasts) or from one another in either the 1% FBS or the 10% FBS soft agar treatments (table
3.4).
Table 3.4 â Average colonies per plate after 30 days incubation in soft agar
Cell Type

Colonies/plate (1% FBS)

Colonies/plate (10% FBS)

MDA-MB-231 (+ control)
Fibroblasts (- control)
Affected Avg. n=7
Unaffected Avg. n=6
p-value (A vs. U)

285Âą34
15Âą13
19Âą18
20Âą19
0.9523

503Âą52
38Âą13
49Âą29
43Âą30
0.7246

Figure 3.6 â Graph of doubling time (in hours) for melanocyte populations with different
ECM treatments For all samples, n=5.

79

3.4.5 Invasion assay
Although both OM-uveal melanocytes and unaffected controls had migration
percentages that were higher than the negative control, neither sample was very invasive
compared to the positive control (table 3.5). Furthermore, there was no significant difference
between the average migration percentage of OM-uveal melanocytes and melanocytes from
unaffected dogs.
Table 3.5: Average percent invasion of various cell lines as determined by Matrigel assay
Cell Type

Percent Invasion

MDA-MB-231
Fibroblasts
Affected Avg. n=5
Unaffected Avg. n=5
p-value (A vs. U)

86Âą4
5Âą2
12Âą3
13Âą4
0.6870

80

Figure 3.7 - Relative metabolic activity of melanocytes at 30-minutes time point Metabolic
activity of unaffected melanocytes on the untreated plastic control was defined as 1. The
difference between fibronectin the plastic control is significant at p=0.0001 as calculated by
two-tailed t-test. There were no significant differences between uveal melanocytes from
OM affected eye and those from control eyes. For all samples, n=4.

3.4.6 Cell adhesion
Fibronectin was the preferred binding substrate for attachment for both sets of uveal
melanocytes (figure 3.7) as evidenced by cells reaching their maximum degree of attachment
soonest, with cells reaching 80-100% of their maximum attachment percentage within 30
minutes of plating, compared to 40-50 minutes in trials with other ECM substrates (data not
shown). No other ECM component had a rate of attachment that was significantly different
from untreated plastic. There were no differences in attachment rate between OM-uveal
melanocytes and unaffected controls for any of the 5 ECM substrates tested.

81

3.4.7 Wound-healing rate
No significant differences were detected in the rate of wound closure for OM-uveal
melanocytes and unaffected controls on any of the different ECM substrates tested (figure 3.8).
Although the OM-uveal melanocyte lines consistently had a higher average closure rate than
the control lines, the difference was not statistically significant, and both types of cell line
displayed high levels of individual variation between the different cell lines tested.

Figure 3.8 - Rate of wound closure as a percentage of the original scratch Percentage of
wound remaining open after 12 or 24 hours post-scratch. No significant difference was
detected for any cell type or treatment. For affected melanocytes, n=7. For unaffected
melanocytes, n=4.

82

3.5 Discussion
Melanocytes originate from neural crest cells which migrate during development with
those in the eye reaching the iris, ciliary body and choroid by following branches of the
trigeminal nerve (12). Despite a common origin, melanocytes at different body sites exhibit
different behavior and different responses to stimuli (13, 14). Uveal melanocytes are typically
considered to be relatively quiescent, although ocular inflammation and in certain species,
drugs such as prostaglandin analogues can result in an increase in iridal pigmentation. Cairn
terriers with ocular melanosis develop a characteristic circumferential expansion of the iridal
root primarily due to an expanded population of large melanosome-laden melanocytes (2). The
reason for the altered behavior of the iridal melanocytes in OM-affected dogs is not known. The
abnormal melanocytes in OM-affected eyes appear to be able to migrate to other sites in the
eye, albeit at a very slow rate. This abnormal, slow expansion of the population of iridal
melanocytes with a potential local migratory phenotype was the impetus to isolate and study
the behavior of these cells in a number of different in vitro assays for growth and migration.
One of the prominent additional sites of pigment accumulation in OM-affected Cairn terriers is
the sclera and episclera. These lesions start as small, flat, bone-spicule shaped black pigment
accumulations that over a period of years expand into larger, slightly raised plaques. We
wanted to investigate whether the cells that had apparently migrated to this site, perhaps via
the aqueous drainage pathways, were melanocytes or melanophages, a cell type also found in
varying numbers within the irides of affected dogs. IHC for a number of antibodies
demonstrated that the predominant cells in the scleral/episcleral pigmented plaques were

83

large, round, densely pigment melanocytes appearing morphologically very similar to those
within the irides.
IHC demonstrated that a high proportion of the large, round, densely pigmented cells
expanding the anterior uvea and forming the pigmented scleral plaques were positive for
HMB45. This monoclonal antibody is highly specific for a glycosylated form of Pmel17 (gp100)
which is present in immature melanosomes; for example, melanocytes in embryonic tissues,
many melanomas and some proliferative melanocytic lesions (15). Previous EM studies
identified all stages of melanosomes including many large melanosomes in these cells (2).
Immunoreactivity for HMB45 conclusively shows that the abnormal pigmented cells in OMaffected dogs are of melanocytic origin. The iris stroma of affected dogs also has a background
population of spindle-shaped melanocytes that morphologically resemble the melanocytes of
the normal dog iris. These cells were also identified by IHC as melanocytes; typically
immunoreactive for PNL2 and Melan A, but not HMB45. This suggests that the diffusely
present, large, round pigmented cells are present in addition to the typical spindle-shaped
melanocytes that can be found within normal iris stroma. A high proportion of the large round
melanocyte cell population of ocular melanosis eyes were also positive for CD18, integrin beta2, which plays a role in cell adhesion. No CD18 expression was detected in the spindle-shaped
melanocytes within the iris stroma. Although commonly used as a leukocyte marker in dogs,
CD18 can rarely be expressed in proliferative melanocytic lesions, including melanomas and we
have previously reported such expression in the abnormal melanocytes of Cairn Terriers with
OM (2). CD18 is one part of Leukocyte Function-Associated antigen-1 (LCA1) that is important
for transendothelial migration, a feature of malignant melanomas. It is suggested that tissue

84

inflammatory cells provide LCA1 to melanoma cells because most melanoma cell lines do not
express the LCA1 antigens (including CD18) (16). Expression of CD18 on the abnormal OM
melanocytes may explain the apparent migratory behavior of the cells, although no differences
in adhesion to various ECM components were detected between OM melanocytes and
unaffected controls in vitro (figure 3.7). The lack of immunoreactivity for histiocyte markers
CD204 and Iba-1 further supports that the majority of the round cells of OM are melanocytes
rather than melanophages, although there were small numbers of CD18, CD204, and Iba-1
positive melanophages scattered throughout the uvea of some cases. This is consistent with our
previous observations in the uvea of OM-affected dogs in which varying numbers of
melanophages were also observed, admixed with the large round melanocytes (2). Clinically,
OM-affected dogs have periodic episodes of anterior uveitis typically accompanied by the
shedding of pigment into the aqueous, so the presence of a population of melanophages in
affected tissue is expected.
Melanin content and rate of melanin production were greatly elevated in OM-uveal
melanocytes relative to unaffected controls for the first several passages in vitro. During the
period of statistically significant elevation, OM-uveal melanocytes had an average melanin
content ranging from 0.37Âą0.19 to 0.04Âą0.0066 ng/cell, compared to a range of 0.094Âą0.064 to
0.014Âą0.01 ng/cell in unaffected controls. In comparison, growth-phase human uveal
melanocytes from different iris colors had average per-cell melanin content ranging from
0.1020 ng/cell from melanocytes cultured from eyes with a dark iris (brown or dark green) to
0.0140 ng/cell from melanocytes cultured from eyes with a light iris (blue or light green) (6).
The unaffected control average seems to correspond roughly to the normal range observed in

85

humans, with the melanocytes from OM-affected dogs showing levels of pigmentation ranging
from well above those seen in the normal human eye down to the upper half of the normal
human range. The melanin content in both unaffected control and OM-uveal melanocytes
decreased rapidly in early passages before eventually stabilizing, a pattern also observed in
human uveal melanocyte cultures. OM-uveal melanocytes also displayed elevated levels of
melanin production, producing melanin at an average rate ranging from 0.046Âą0.014 to
0.019Âą0.0032 ng/cell/day during the significantly elevated period, compared with a range of
0.016Âą0.0084 to 0.0076Âą0.0067 ng/cell/day in unaffected control cells. The unaffected control
canine melanocytes were found to be intermediate between growth-phase pigment at
production levels of dark and moderately dark human uveal melanocyte melanin production
rates (0.0180 and 0.0071 ng/cell/day, respectively). The OM-uveal melanocytes produced
melanin at a rate higher than melanocytes from dark human irides during this period.
As the precise pathogenesis of OM and nature of the round cells comprising the lesion
are presently not very well understood, a number of established in vitro assays were employed
to evaluate basic behavioral aspects of these cells. As uveal tissue in OM-affected dogs displays
increased cellularity, the doubling time of OM-uveal melanocytes was measured to determine if
their replication rate was intrinsically different from that of unaffected controls. No significant
difference in doubling rate was detected. The doubling rate of both OM and control uveal
canine melanocytes (44Âą5 and 47Âą3 hrs respectively) were slightly faster than the range of
doubling times reported for human uveal melanocytes (48-72hrs) (6). Doubling time assays
were repeated in the presence of common ocular ECM components, as different ECM
components are known to have differential effects on cell morphology and behavior in vitro,

86

but this had no significant impact on the doubling time of OM-uveal melanocytes or unaffected
controls. OM-uveal melanocytes appear to migrate into the sclera, so a matrigel invasion assay
was used to evaluate their ability to invade through a membrane, and a wound-healing assay
was used to analyze their migration rate. Although no human data is available for comparison,
neither test showed any significant difference between OM-uveal melanocytes and unaffected
controls. Matrigel assay results for both cell types showed very little invasive potential relative
to that of a known invasive cell line used as a positive control. Wound-healing assays showed
no significant differences in migration rate between OM-uveal melanocytes and unaffected
controls on untreated plastic or any of the different ECM substrates tested. Wound-healing
assay measurements averaged from combined samples had large standard deviations,
suggesting that most differences in migration rate were primarily attributable to individual
variation rather than OM-affected status. Soft agar assays were performed to test the
anchorage-independent growth potential of OM-uveal melanocytes. Average colony formation
rates from both OM-affected and unaffected cell lines were very low, and not significantly
different from the negative control, indicating that OM-uveal melanocytes are not capable of
anchorage-independent growth. Previous studies have shown that transformed human uveal
melanocytes have a higher attachment rate on a wider variety of extracellular matrix (ECM)
components than normal uveal melanocytes, so we evaluated OM-uveal melanocytes on their
attachment rates to various ECM components found in the canine eye. In humans, normal uveal
melanocytes displayed a preference for fibronectin, adhering at a rate 1.5-fold greater than
they adhere to untreated plastic (4). They attached significantly less well to collagens I, III, and
IV, binding at roughly 80% of their attachment rate to untreated plastic. In contrast, human

87

uveal melanoma cells bound collagens I, III, and IV at the same rate they bound fibronectin, a
1.5-fold increase over untreated plastic. Both OM-uveal melanocytes and unaffected controls
displayed a strong preference for fibronectin, adhering at a 2.5-fold greater rate over untreated
plastic. No other ECM component caused a binding rate that was significantly different from
untreated plastic. Although it was surprising that canine uveal melanocytes appear to bind
collagens I, III, and IV at a rate higher than that reported for human uveal melanocytes, the
difference was consistent across both OM-uveal melanocytes and unaffected controls.
Although IHC shows the pigmented scleral plaques to be composed primarily of
melanocytes, which most likely have migrated there from the expanded population in the
anterior uvea, presumably along aqueous drainage pathways, all of our assays related to cell
migratory behavior showed that there were no significant differences between uveal
melanocytes from OM-affected dogs and normal controls. Our melanogenesis assays, however,
showed a clear difference in melanin content and production between OM-affected and
unaffected dog-derived melanocyte cultures was present when examining early-passage cells,
but that that difference quickly disappeared in culture, diminishing nearly 50% between
passage 1 and 2 and disappearing by the fourth passage at the latest. Although we endeavored
to use as early a cell passage as possible for all assays presented in this paper, the difficulties of
culturing a sufficient number of melanocytes for each assay was such that most of the cells
used in the assays presented here were between passage number 2 and 4 when being tested.
Ocular melanosis is a very slowly progressing disease, often developing over the course of many
years, so any differences in cell migration behaviors between the melanocytes of affected and
unaffected eyes may be quite small in magnitude. If the difference in cell behavior being

88

measured experiences the same 50% reduction in effect size after a single passage in culture as
melanogenesis does, this would make any difference between the two cell populations
extremely difficult to detect. While OM-uveal melanocytes clearly appear to have a migratory
phenotype in vivo based on the IHC data, we likely cannot replicate the phenotype in vitro due
to changes in phenotype caused by differences between the intraocular environment and the
environment in cell culture. In an attempt to investigate some possible environmental factors,
we performed some of our assays in the presence of different uveal ECM components, but did
not see any changes in cell behavior as a result of this.
Testing our hypothesis that local environmental effects may be responsible for the
apparent altered behavior of OM-uveal melanocytes could require creating a uveal co-culture
or similar set of conditions that mimic the uveal environment closely enough to cause the OMuveal melanocytes to behave as they do in vivo. As we have no way of determining which
factors are more likely to be driving the behavioral difference seen in the OM-uveal
melanocytes to design a specific assay, a more plausible next step in investigating the
underlying causes of the disease would be to perform RNAseq analysis on uveal tissue from
both affected and unaffected Cairn Terriers and look for differential expression of individual
genes or pathways that relate to either melanogenesis or cell proliferation, and direct further
cell studies once a putative pathway has been identified.
In conclusion, we demonstrated that the large pigmented cells present in the pigmented
scleral plaques of OM-affected dogs are primarily melanocytes, and that the characteristic
uveal melanocytes of OM-affected eyes produce greatly elevated levels of pigment in culture

89

initially, before equalizing with the level produced by control uveal melanocytes after several
passages. Despite their apparently invasive phenotype in vivo, we did not find evidence of
invasive behaviors in vitro. Identification of the causal mutation for ocular melanosis and
altered gene expression in affected eyes may allow us to understand the mechanism underlying
this phenotype.

90

3.6 Acknowledgements
We would like to thank Dr. Vilma Yuzbasiyan-Gurkan for cells that were used as controls
in this paper. This research was supported by grants from the Foundation of the Cairn Terrier
Club of America, the Myers-Dunlap Endowment for Canine Health, and Michigan State
University College of Veterinary Medicine Endowed Research funds. We would also like to
thank Stephanie Eldredge and Hillary Domenech-Barreto for their contributions to some of the
cell culture assays.

91

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92

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Aoki H, Yamada Y, Hara A, Kunisada T. Two distinct types of mouse melanocyte:
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Li L, Hu DN, Zhao H, McCormick SA, Nordlund JJ, Boissy RE. Uveal melanocytes do not
respond to or express receptors for alpha-melanocyte-stimulating hormone. Investigative
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Kapur RP, Bigler SA, Skelly M, Gown AM. Anti-melanoma monoclonal antibody HMB45
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Ghislin S, Obino D, Middendorp S, Boggetto N, Alcaide-Loridan C, Deshayes F. LFA-1 and
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94

CHAPTER 4
OCULAR MELANOSIS LOCUS MAPPING AND CANDIDATE GENE SEQUENCING

95

4.1 Introduction
Ocular melanosis (OM) is an inherited eye disease of Cairn terrier dogs that is
characterized by a bilateral proliferation of uveal melanocytes (1, 2). These melanocytes
frequently migrate to abnormal areas of the eye, such as the sclera. Additionally, many dogs
with late-stage OM develop glaucoma as a result of the eyeâs anterior drainage angles
becoming blocked by pigmented material and cell debris, preventing the normal draining of the
aqueous humor (1).
Ocular melanosis only began to reported in Cairn terrier populations relatively recently
(the first cases were reported in 1984) but nonetheless is present in a significant proportion of
the overall population (3, 4). This suggests that the disease is probably the result of a single
mutation that arose once in a popular sire dog that spread through the breed by means of a
founder effect (1). Initial candidate gene-based attempts to map the causative locus for OM
examined 11 genes known to have a role in melanocyte and melanosome development or
known to be associated with other pigmentation-related diseases, including a pair of genes
(Gpnmb and Tyrp1) which have known mouse variants that, together, produce a mouse
phenotype which strongly resembles OM as seen in Cairn terriers (5, 6). Examination of SNP
and microsatellite markers near these genes showed that no single common allele was present
in all OM-affected Cairn terriers for any of these 11 genes. As pedigree analysis has shown OM
to be autosomal dominant, the lack of a common shared allele at these 11 loci provides
evidence to exclude the loci as harboring a variant causative for OM (1, 5).

96

With failure of the initial candidate gene approach, we decided to take a whole-genome
approach to identifying regions of the genome potentially linked to OM. Here, we report the
results of a two-stage genome-wide association study performed on 94 total Cairn terriers (62
affected, 32 unaffected) using the Illumina CanineHD Beadchip system to genotype each dog at
over 170,000 evenly spaced SNP markers across the canine genome. This approach identified a
7.5 megabase (Mb) region of chromosome 11 significantly associated with OM.

97

4.2 Materials and methods
4.2.1 Sample collection
All DNA used in this study was extracted from blood samples of Cairn terriers donated
with the consent of the dogâs owner. DNA was extracted using a modified version of Qiagenâs
PureGene commercial DNA extraction kit (Qiagen, Germantown, MD). Briefly, DNA was mixed
twice with a red blood cell lysis buffer (0.32 M sucrose, 5 mM MgCl2, 1% Triton-X 100, 10 mmM
Tris-HCl, pH 8.0, filter sterilized) to remove red blood cell contamination. The remaining cells
were pelleted and dissolved in PureGene cell lysis solution (Qiagen) to lyse white blood cells.
PureGene protein precipitation solution (Qiagen) was added to remove protein. The DNAcontaining supernatant was mixed with isopropanol to precipitate DNA and washed with 70%
ethanol before ultimately being resuspended in DNA hydration buffer (Qiagen).
4.2.2 Whole-genome canine SNP array
For our whole-genome SNP array, we genotyped a total of 96 Cairn terriers (94 unique
dogs, 2 dogs which were genotyped twice to assess reproducibility) using the Illumina
CanineHD Genotyping BeadChip. We utilized two different vendors to perform this genotyping
â GeneSeek (Lincoln, Nebraska) performed genotyping on an initial 48 samples using a model of
the Illumina CanineHD BeadChip which used 173,662 single-nucleotide polymorphism markers
(SNPs). The second 48 samples were genotyped by the University of Minnesota Genomics Core
Facility (Minneapolis, Minnesota) using a newer model of the BeadChip with 220,853 markers,
including all of the original 173,662 markers on the previous BeadChip. Of the 94 dogs
genotyped, 62 were diagnosed as OM-affected, and 31 were unaffected control cairn terriers.

98

One dog possessed an atypical phenotype which superficially resembled OM but was missing
several key indicators. All dogs used for genotyping are shown in Table 4.1. Since OM has a
highly variable age of onset, the oldest unaffected Cairn terriers that had been examined by a
veterinary ophthalmologist and shown not to have any clinical indications of OM and were
therefore most likely to be true unaffecteds, were used as controls. The ages of the unaffected
dogs used for controls ranged from 7 to 16 years, with a mean age of 11.9 years (Table 4.1). All
62 affected dogs that were used in this study had been diagnosed by a veterinary
ophthalmologist as being OM-affected. When selecting OM-affected samples for genotyping,
preference was given to more severely affected samples, as it was thought that the high
variability seen in the OM phenotype might be due to zygosity differences in affected Cairn
terriers.

99

Table 4.1 - Dogs used in GWAS study
Dog

OM Status

CA2
CA6
CA12
CA13
CA16
CA23
CA28
CA36
CA43
CA44
CA45
CA52
CA54
CA57
CA66
CA68
CA76
CA81
CA90
CA124
CA138
CA142
CA148
CA150
CA158
CA167
CA175
CA178

A
A
A
U
A
A
A
A
A
A
U
U
U
U
A
A
A
U
A
U
A
U
A
A
A
A
A
U

Age at
Sex Glaucoma
Last Exam
Status
12
F
Yes
9
F
Yes
12
F
No
15
F
No
11
F
Yes
13
M
No
8
M
Yes
12
F
No
9
F
Yes
5
M
Yes
11
M
No
13
F
No
9
F
No
12
M
No
14
F
No
11
F
Yes
11
M
No
9
F
No
12
M
Yes
13
M
No
13
F
No
9
F
No
11
F
Yes
10
M
Yes
15
F
Yes
14
M
No
9
F
Yes
9
F
No

CA188
CA190
CA193
CA194
CA195
CA196
CA197
CA198
CA204
CA208
CA216
CA217
CA229
CA239
CA240
CA245
CA251
CA314
CA318
CA366
CA367
CA369
CA370
CA374
CA390
CA393
CA394
CA405
CA423
CA424

100

A
A
U
A
A
U
U
U
U
A
A
A
U
A
A
U
U
A
U
U
U
U
U
U
U
U
U
A
A
A

13
10
10
14
12
9
15
12
12
10
10
11
16
11
14
15
13
12
11
7
10
12
12
12
14
13
13
12
8
14

F
F
F
M
F
F
M
M
F
F
M
M
M
F
M
F
F
F
M
F
M
M
F
F
M
F
M
F
M
F

Yes
Yes
No
No
No
No
No
No
No
No
Yes
Yes
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes

Table 4.1 (contâd)
Dog

OM Status

CA483
CA486
CA488
CA489
CA490
CA491
CA494
CA509
CA511
CA518
CA524
CA540
CA541
CA558
CA582
CA593
CA595
CA599
CA602
CA605
CA606
CA611
CA614
CA615
CA616
CA617
CA618
CA619

A
A
A
A
A
A
A
A
A
A
A
U
A
U
A
U
A
A
U
A
A
U
A
A
A
A
A
A

Age at
Sex Glaucoma
Last Exam
Status
Unknown
F
Unknown
13
F
No
9
F
No
12
F
Yes
12
M
Yes
8
M
No
10
M
Yes
12
F
Yes
8
F
Yes
10
F
Yes
9
F
Yes
11
F
No
10
F
Yes
16
M
No
10
F
Yes
14
F
No
16
F
No
12
M
No
16
F
No
10
M
No
12
F
Yes
7
M
No
14
F
Yes
14
M
Yes
11
F
Yes
12
F
Yes
10
F
Yes
18
M Unknown

CA620
CA622
CA624
CA625
CA626
CA628
CA630
CA631

101

A
A
A
A
A
A
A
Atypical

10
12
8
13
16
11
9
6

M
M
F
F
F
M
M
M

Yes
Yes
Yes
Yes
No
Yes
Yes
Yes

4.2.3 Data analysis
Whole-genome SNP data was initially analyzed using the PLINK statistical software
package (7). Quality cutoffs used for this data analysis removed SNPs with a minor allele
frequency (MAF) of less than 5% or which were missing calls in greater than 10% of all samples
genotyped. Dogs which genotyped at less than 90% of all possible SNPs were excluded from
analysis as low-quality samples. Analysis was carried out using PLINKâs standard case/control
association test as well as PLINKâs model-based association test, with correction for multiple
testing carried out using the max(T) permutation procedure with 10,000 permutations. In order
to control for population stratification, the program GEMMA was used to estimate a centered
relatedness matrix from the genotypes for each phenotype, and the association test was
performed again using the same screening criteria for SNPs with a univariate linear mixed
model with the relatedness matric, phenotype, genotype, and sex all taken into account as
factors, and the Wald frequentist test being used to test for significance (8-11).
4.2.4 PCR-RFLP genotyping
The UCSC genome browser (https://genome.ucsc.edu/) canfam3 reference sequence
was used to retrieve sequence information near known SNPs. The webtool NEBcutter was used
to determine whether the SNP being investigated led to the loss or gain of a restriction enzyme
cut site (http://www.labtools.us/nebcutter-v2-0/). Primers flanking the area to be amplified
were designed using the Primer3 online primer design tool (http://bioinfo.ut.ee/primer30.4.0/).

102

4.2.5 Sanger sequencing of candidate genes
The UCSC genome browser (https://genome.ucsc.edu/) canfam3 reference genome was
used to retrieve the exon sequence data for genes of interest. Primers were designed flanking
each exon using the Primer3 online primer design tool (http://bioinfo.ut.ee/primer3-0.4.0/).
Sanger sequencing of fragments was carried out by the MSU Research Technology Support
Facility (RTSF) using both the forward and reverse primers, and the resulting sequence data
analyzed using the program Sequencher (Gene Codes Corporation, Ann Arbor, Michigan). A
total of three affected and three unaffected dogs were sequenced for each gene.

103

4.3 Results
4.3.1 Genome wide SNP array
The total number of markers shared between the two SNP arrays that survived the
pruning conditions described above was 118,937. The filters removed 4,657 SNPs due to low
genotyping rate (> 10% of samples not genotyped) and 50,065 SNPs due to low minor allele
frequency (MAF < 5%). Of the 94 dogs genotyped, 88 dogs passed the genotyping rate
threshold (61 affected dogs, and 27 unaffected dogs). The total genotyping rate for the 88
remaining dogs was >99.4%. Dogs number CA13, CA36, CA142, CA370, CA374, and CA394 were
filtered out for genotyping rates of less than 90%. Initial naĂŻve analysis using PLINK had an
elevated genomic inflation factor (1.69) which was corrected for using GEMMA. Following this
correction, the genomic inflation factor was reduced to 1.10, which is below the level at which
further adjustment for population stratification is generally required, especially as small
increases in genomic inflation factor can be caused by true associations, and this effect is more
pronounced in animals like dogs with large linkage disequilibrium blocks (12-14).
According to the Bonferroni correction for multiple testing, the genome-wide
significance threshold for a test involving 118,937 markers is p < 4.2x10-7. Twenty-four SNPs
from the combined BeadChip data had p-values below this threshold, ranging from 1.32x10-7 to
4.17x10-7. These SNPs formed a continuous block on chromosome 11, delineating a ~7.5
megabase (Mb) region that was associated with the OM phenotype at statistically significant
levels, chr11:43,919,606-51,409,494 in the canfam3 build. No SNPs from other areas of the
genome reached statistical significance (Figure 4.1), although many more SNPs from

104

throughout the 7.5 Mb region approached genome-wide significance. No significant differences
were observed when comparing the glaucoma-only population of dogs with either the nonglaucoma affected dogs or the unaffected dogs.
Examination of the transcripts annotated on both the dog and human genomes, the
7.5Mb region contains a total of 82 putative transcribed elements, some of which have multiple
documented splice variants. About a third of these are Ensemble protein predictions with no
supporting documentation or predicted function. There are 12 genes in the region that have
been validated in dog as part of the RefSeq gene database, and an additional 32 predicted
canine genes that have been validated as RefSeq genes in other species.

105

A

B

Figure 4.1 â Manhattan plots of p-values A. Manhattan plot of the âlog of the p-values
plotted against the genomic location for each of the 118,937 SNP markers genotyped in 88
dogs from the Illumina CanineHD BeadChip. The red line represents statistical significance (log(4.2x10-7)). B. Manhattan plot of chromosome 11 only. The red line represents statistical
significance.

106

4.3.2 Population SNP genotyping
To determine how common this haplotype was within the entire population of OMaffected dogs, it was decided to genotype one SNP located within this cluster of significant SNPs
in all dogs in our database. To select this SNP, flanking primers (Table 4.2) were designed
around five candidate SNPs located near the SNP with the lowest p-value as determined by
PLINK, and 10 OM-affected and 10 unaffected Cairn terriers that had not been part of the SNP
array (Table 4.3) were genotyped via Sanger sequencing. These SNPs were perfectly concordant
in all 20 dogs, the OM affected dogs all being heterozygous for the OM-associated SNP, and the
unaffected dogs all being homozygous for the SNP not associated with OM. The remaining Cairn
terrier samples that we have registered in our database were genotypes at the SNP with
genomic position chr11:46,303,228 (Table 4.4), where the variant associated with the affected
phenotype introduces a HpyCH4IV cut site. The majority of the affected dogs we have in our
database (72/83, 86.7%) have at least one copy of the allele thought to be linked to the
dominant causative mutation. A lower percentage of unaffected dogs (131/184, 71.2%) were
homozygous for the allele thought to correspond to recessive normal phenotype. Much of the
discordance in the unaffected dog population may be due to the high variability of OMâs age of
onset â we have many younger Cairn terriers in our database that currently show no signs of
OM and are therefore provisionally listed as unaffected, but who may end up developing the
disease later in life. A chi-square test comparing the allele frequencies of these two populations
produced a p-value < 0.0001, confirming the linkage we had observed with the genome-wide
SNP array.

107

Table 4.2 â SNPs tested in trial genotyping
SNP Location
chr11:46303175
chr11:46303228
chr11:46413975
chr11:46345226
chr11:46424658

Ref.
Allele
G
C
A
G
C

Var.
Allele
A
T
C
A
T

F Primer

R Primer

tgggacctaagtcacagtgg
atcgcatctcagcttccatt
tgggacctaagtcacagtgg
atcgcatctcagcttccatt
gaagaggtgcctttctctgc
ggcctctggagtagctgttg
ttgttgcagatttgatttgga
ttctgccaacctcagattctc
aagcagactagagatgaggctgt gtgtgccttagcaattgtgg

Fragment
Size
248
248
221
296
224

Table 4.3 â Dogs tested in trial genotyping
OM Affected
CA570
CA548
CA539
CA540
CA551
CA552
CA556
CA571
CA402
CA527

OM Unaffected
CA526
CA519
CA514
CA243
CA239
CA220
CA511
CA250
CA485
CA421

Table 4.4 â Population-wide SNP genotyping results (A) represents the allele believed to be
linked to the dominant OM causal gene, (a) represents the recessive wild type unaffected
allele Affected Status
Genotype
Number of Dogs
Unaffected

Affected

AA
Aa
aa
AA
Aa
aa

108

10
43
131
17
55
11

4.3.3 Candidate gene selection
Examining the 7.5 Mb region, several genes within and near the region were considered
to be potential positional candidate genes. The SNPs with the highest p-values were located
within the introns of LINGO2 (leucine-rich repeat and Ig Domain Containing 2), a developmental
gene thought to be restricted to neuronal tissue with several known variants that are
associated with Parkinsonâs disease and essential tremor (15, 16). Although LINGO2 did not
seem like a good candidate gene based on the phenotypes of the diseases it is known to be
associated with, we selected it for sequencing because the most highly significant SNPs were
located within it. Also within the region is ACO1 (aconitase 1), a gene which codes for a TCAcycle enzyme that also plays a role in the regulation of cellular iron levels. Inherited mutations
in ACO1 have been linked to an increased risk of cutaneous melanoma in humans, although the
mechanism by which ACO1 mutations can contribute to melanoma development is largely
speculative at this time (17). Finally, CDKN2A was also selected for sequencing. Although
upstream of the significantly associated region by about 1.5 Mb (chr11:44225749-41264280),
CDKN2A represented by far the best candidate gene near the region identified by the genomewide SNP array. CDKN2A codes for two different proteins, p16INK4a and p14arf, inhibitors of
cyclin-dependent kinases CDK4 and CDK6. By blocking the activity of these proteins, the
products of CDKN2A block the cellâs transition into S phase. It is a tumor suppressor gene,
mutations in which have been linked to high-penetrance familial melanoma, as well as many
other cancers (18, 19). Given the migratory behavior of OM melanocytes, and the general
proximity to the region identified by our genome-wide SNP array, CDKN2A appeared to be a
very good candidate, and so its coding region was sequenced as well.

109

Table 4.5 â Primers for LINGO2 sequencing
Name
Exons 1-3
Exon 4
Exon 5
Exon 6
Exon 7 pt. 1
Exon 7 pt. 2
Exon 7 pt. 3
Exons 8-13
Exons 14-16

F Primer

R Primer

tactcggcatccctcctaga
aaggaaaagctcggactcgt
ttcccaagagtttttgtttca
gctggacctaggacatggtt
tcttctcttcctgccttctcc

tcctctcttcctctggctga
ccggggagatcatcctaact
cattctccaaaggccaacat
tgatcagatgccttcctcaa

atatgcccgtgtatgccttt
gctagtggatgaagggcaga

ttgagatgcttcagatgtaggc
ggtgtcacccaggaaatcac
cttccatggaccctgcttt

tgagacagctggcacagtaaa
ggcggagatatgcagaatga

catatctccgcccatagctc
gcattccaattcttgatctcc

Amplicon Size
453
197
175
250
696
697
691
599
390

Table 4.6 â Primers for ACO1 sequencing
Name
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
Exon 11
Exon 12
Exon 13
Exon 14
Exon 15
Exon 16
Exon 17
Exon 18
Exon 19
Exon 20
Exon 21 and 22
Exon 23 and 24

F Primer

R Primer

aggttcctgaggtccagctt
tgctttctcctttttccctaga
gtggaggcaaaagctgaaaa
agcaagcctgcagacaaagt
gtgttctttccattctcaatcct
agcatccttgtgtcatgctg
tggagtctcaagtttttctcttgtt
tcatcacgaaatcctaatgtctgt
gtccgttcctgagctcctta
tctcagccagtgacttttgtt
ttgtcggtgtttacattgcac
attccccacattgctgttct
ccttctctgggtgacctgat
ggctggatgtgatcatgaaa
gaatggaatgtgattagatttgct
actgacccgctgtgttcttt
tttgctccggtcactaacct
ccttgcttttctcgtcctca
gtcctgtcttggctcctcag
cctccctgattgtcctctgtt
gtgtgctgcccacgtcac
cacggtaggcgaacacttg

gactcttgattgctgtgagaaca
gacaagggaggcagtgaaga
gaaaacccaagtatccgtcct
atctcctcggcacctctctt
acagaataggatcctggaacaa
gtgcacttcctccctgtcac
actttgcacagggcattctg
gaggaaatgcacgctctctc
ttcctcatcagagggtgaaaa
gagggattcacatgcactca
aagctgacacatgctacaagg
ggctcctctgcaattacctg
tagctgcacaaatgcaacct
acgcaggaaggcagagtg
tcaaagcaagggcaaagagt
gccttagtccttctccgtca
accacccagaggctgataaa
tcaccactcgagggaaagc
ttccactcatttctcggtga
gctggctagtggcgcttc
tgtcagtcagccccactgta
tccaaaaccacctccaaagt

110

Amplicon Size
231
236
246
249
238
230
248
179
249
351
249
209
220
225
249
199
228
242
290
212
338
250

Table 4.7 â Primers for CDKN2A sequencing
Name
Exon 1
Exon 2

F Primer
gcagagcggctccgagat
ctgtccctgtcctgaccact

R Primer
ggaggcctttcttacctgct
ggaagctctccgagttccaa

Amplicon Size
405
381

Table 4.8 â Dogs for candidate gene sequencing
Name
CA318
CA366
CA367
CA489
CA490
CA494

OM Status
Unaffected
Unaffected
Unaffected
Affected
Affected
Affected

Sex
M
F
M
F
M
M

Age
11
13
15
15
14
11

4.3.4 Sanger sequencing results
The three candidate genes selected on the basis of the genome-wide SNP array were all
sequenced in three affected and three unaffected dogs. No coding region variation was
detected in any of the exons that segregated with the phenotype or was present in all
sequenced affected dogs.

111

4.4 Discussion
A genome-wide association assay identified a 7.5 Mb portion of chromosome 11 that is
significantly associated with the ocular melanosis phenotype. Although there was genomic
inflation suggestive of population stratification present in the initial sample of 88 dogs that
remained after removing dogs with low quality scores, the region remained significantly
associated with OM even after correcting for this. A single SNP located in the most statistically
significant region was genotyped in all dogs present in our database, and was present in one or
two copies in 86.7% (72/83) of all dogs affected with OM. Although that is a very high
percentage, the fact that 11 of 83 affected dogs did not have the marker indicates that even the
most significant SNPs from our mapping study are not completely linked with the disease. We
have been unable to find a marker which is completely linked with the affected phenotype
(data not shown). This suggests four possibilities: 1. that some of the samples we have listed as
OM affected have been mislabeled and are not true affected dogs, 2. that some of them have
been misdiagnosed, 3. that environmental effects are producing a phenocopy in some dogs,
and 4. that there is genetic heterogeneity. Mislabeling may happen due to human error,
although it is unlikely that nearly 15% of our affected dog samples have been misfiled.
Misdiagnosis is also a possibility, as OM can be very difficult to detect in earlier stages, although
the OM phenotype becomes quite dramatic as the disease progresses. Typically, misdiagnoses
will be unaffected dogs which are incorrectly called as affected. Many of the discordant
affected dogs had advanced OM, making a misdiagnosis in those cases unlikely. It is also
possible that environmental factors could create a phenocopy of OM â although rare, a disease
with similar clinical signs to OM has been observed in other breeds. Our lab has received

112

samples from a spitz, a Jack Russel terrier, a Staffordshire terrier, two boxers and two Labrador
retrievers that display similar clinical signs to OM. These breeds are quite diverse, and none
have a documented history of a heritable OM-like condition, suggesting the cause may be
environmental rather than genetic. Finally, genetic heterogeneity of the disease in Cairn terriers
seems more likely in light of a dog (CA 631) that was recently examined at MSU after previously
having been diagnosed as OM affected elsewhere (this dog was included on a whole-genome
SNP array). Upon examination by Dr. Petersen-Jones, it became apparent that while the dog
presented some of the clinical signs commonly seen in OM, such as glaucoma and
pigmentation, the dog did not have a thickened iris root which has been considered a
diagnostic sign. This could perhaps be a phenocopy as suggested earlier, but it could also
indicate that there is genetic heterogeneity as is seen in many other hereditary conditions in
dogs. This would greatly complicate genetic analysis of the disease. However, even with those
potentially confounding factors, we were able to map a region of the genome which is
associated with the disease.
Despite finding a genomic region significantly associated with OM, we have so far been
unable to identify the causative mutation via a candidate gene approach. Several genes within
the mapped locus could plausibly contribute to the phenotype seen in OM based on their
annotated function, but those that had no coding region variation that was shared between all
affected dogs. Many genes within the region have very little annotation information, and none
of the remaining well-annotated genes have a function that suggests they would be good
candidates. Our next step at this stage was to perform next-generation sequencing of the
genome of several affected and unaffected Cairn terriers, as this would allow us to examine all

113

remaining exonic variation as well as to look for variation in intronic and regulatory sequences
within the region, and also to interrogate regions of the genome other than our mapped region.

114

REFERENCES

115

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Petersen-Jones SM, Forcier J, Mentzer AL. Ocular melanosis in the Cairn Terrier: clinical
description and investigation of mode of inheritance. Vet Ophthalmol. 2007;10 Suppl 1:63-9.
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Petersen-Jones SM, Mentzer AL, Dubielzig RR, Render JA, Steficek BA, Kiupel M. Ocular
melanosis in the Cairn Terrier: histopathological description of the condition, and
immunohistological and ultrastructural characterization of the characteristic pigment-laden
cells. Vet Ophthalmol. 2008;11(4):260-8.
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Covitz D, Barthold S, Diters R, Riis R. Pigmentary glaucoma in the cairn terrier.
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America CTCo. Cairn Terrier Club of America 2005 Breeder-Owner Survey. 2005.

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Winkler PA, Bartoe JT, Quinones CR, Venta PJ, Petersen-Jones SM. Exclusion of eleven
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Anderson MG, Smith RS, Hawes NL, Zabaleta A, Chang B, Wiggs JL, et al. Mutations in
genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice. Nat Genet.
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Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool
set for whole-genome association and population-based linkage analyses. Am J Hum Genet.
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Zhou X, Stephens M. Genome-wide Efficient Mixed Model Analysis for Association
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Devlin B, Roeder K. Genomic Control for Association Studies. Biometrics.
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Eu-ahsunthornwattana J, Miller EN, Fakiola M, Wellcome Trust Case Control C, Jeronimo
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Clayton DG, Walker NM, Smyth DJ, Pask R, Cooper JD, Maier LM, et al. Population
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14.
Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB, Kamal M, et al. Genome
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VilariĂąo-GĂźell C, Wider C, Ross OA, Jasinska-Myga B, Kachergus J, Cobb SA, et al. LINGO1
and LINGO2 variants are associated with essential tremor and Parkinson disease.
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Wu Y-W, Prakash K, Rong T-Y, Li H-H, Xiao Q, Tan LC, et al. Lingo2 variants associated
with essential tremor and Parkinsonâs disease. Human genetics. 2011;129(6):611-5.
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Yang XR, Liang X, Pfeiffer RM, Wheeler W, Maeder D, Burdette L, et al. Associations of
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Hayward NK. Genetics of melanoma predisposition. Oncogene. 0000;22(20):3053-62.

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Borg Ak, Sandberg T, Nilsson K, Johannsson O, Klinker M, MaĚsbaĚck A, et al. High
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117

CHAPTER 5
WHOLE-GENOME SEQUENCING OF OCULAR MELANOSIS-AFFECTED CAIRN TERRIERS

118

5.1 Introduction
Ocular melanosis (OM) is an inherited eye disease commonly seen in the Cairn terrier
dog breed. This disease is characterized by a bilateral proliferation of densely pigmented
melanocytes, both within the uvea and in other areas of the eye such as the sclera and episclera
(1, 2). According to pedigree analysis, the disease shows an autosomal dominant mode of
inheritance, and the fact that the disease appeared relatively recently in a single breed of dog
(the disease was first reported in 1984) suggests that the disease is caused by a single mutation
which has been propagated throughout the breed by means of a founder effect (3).
Previous attempts to identify a causative gene for OM based on a candidate gene
approach were unsuccessful (4). Our previous studies using whole-genome SNP array
genotyping on a set of 94 Cairn terriers (see Chapter 4) allowed us to identify a 7.5 megabase
(Mb) region on chromosome 11 that was significantly associated with the disease, however,
there were no genes within our region whose function suggested them as obvious candidates.
Sanger sequencing was performed on several genes within the region near the most strongly
associated SNPs, as well as several genes that had previously been shown to have some
connection to pigmentation or cell migratory disorders, but none of these genes showed any
coding-region variation that segregated with the OM phenotype.
Without a specific target to examine, we decided to perform whole-genome sequencing
(WGS) to look for the causative variant in OM, focusing most intently on genes within our
previously identified region and genes known to be involved in pigmentary disorders. We
initially sequenced 6 dogs using traditional PCR-based WGS, and added another 4 dogs

119

sequenced via PCR-free WGS, as PCR-free methods have been shown to have more even
coverage of the genome and to provide better coverage of GC-rich regions that can be poorly
represented using PCR-based methods (5).

120

5.2 Materials and methods
5.2.1 Sample collection
To ensure the highest quality DNA for sequencing and to ensure that no sample mix-ups
had occurred, the owners of all dogs sequenced in this study were asked to submit a fresh
blood sample from their dog, which was extracted immediately prior to sequencing using the
DNA extraction protocol outlined in Chapter 4.
5.2.2 Whole-genome sequencing
Whole-genome sequencing was carried out on 10 total dogs (5 affected and 5
unaffected, see Table 5.1 for a complete list of dogs used). Sequencing was carried out using
two different protocols, one using traditional PCR-based methods for library creation, and the
other using PCR-free methods. Six samples (3 affected and 3 unaffected) were sent to DNA
LandMarks for library preparation and sequencing (DNA LandMarks, Saint-Jean-sur-Richelieu,
Quebec, Canada). Library preparation was carried out using the Illumina TruSeq kit (Illumina,
San Diego, CA), and the samples were run on 4 lanes of an Illumina HiSeq 2500 sequencer
(Illumina) in 100bp paired-end mode. Each affected sample was run as an individual lane,
resulting in an average coverage of ~12X. The three unaffected samples were pooled and
sequenced across a single lane, resulting in an average per-sample coverage of ~4X. The
sequencing of the remaining 4 dogs (2 affected and 2 unaffected) was carried out at the
University of Minnesota Genomics Core Facility (University of Minnesota, Minneapolis,
Minnesota). Library preparation was carried out using the Illumina TruSeq PCR-Free library
preparation kit (Illumina), and sequencing was performed on an Illumina HiSeq 2500 sequencer

121

(Illumina) using v4 chemistry in 125bp paired-end mode. One sample (CA229) was sequenced
on a half-lane of a high-output flow cell, resulting in an average coverage of ~26X. The
remaining three samples were pooled and run on a single lane of a high-output flow cell,
resulting in an average coverage of ~17X.
Table 5.1 â Dogs used for whole-genome sequencing
Dog
CA189
CA204
CA229
CA488
CA491
CA511
CA593
CA602
CA628
CA630

OM Status
Unaffected
Unaffected
Unaffected
Affected
Affected
Affected
Unaffected
Unaffected
Affected
Affected

Age and Sex
16,M
12,F
16,M
9,F
8,M
8,F
14,F
16,F
11,M
9,M

Library Prep Method
TruSeq PCR-Free
TruSeq
TruSeq PCR-Free
TruSeq
TruSeq
TruSeq
TruSeq
TruSeq
TruSeq PCR-Free
TruSeq PCR-Free

5.2.3 Data analysis
NGS data was analyzed using two pipelines. The first pipeline was used on the 6 dogs
which used PCR-based library preparation methods. The first step in sequencing involved using
Sickle (https://github.com/najoshi/sickle) at default settings in paired-end mode to trim low
quality score reads, reads containing multiple Ns, and very short reads. Read alignment to the
genome was performed using SHRiMP (http://compbio.cs.toronto.edu/shrimp/) (6). SAMtools
(http://www.htslib.org/) was used to convert the SAM alignment files these programs produced
and convert them to BAM files (7). Variant calls were performed using the Freebayes toolset (8,
9) with a minimum coverage filter of 2. Annotation of called variants was done using ANNOVAR

122

and SnpEff (10, 11). SOAPsv (http://soap.genomics.org.cn/SOAPsv.html) was used to examine
the genome for structural variation according to the program documentation (12). Finally,
mapped reads from the 7.5 Mb region of interest Âą 1.5 Mb were extracted and examined
manually in the integrative genome viewer (IGV) to look for small indels that algorithm-based
variant callers sometimes have difficulty detecting (13).
Later, all data (both the original PCR-based data and the new PCR-free data) was also
analyzed according to the GATK Best Practices handbook as of June 2016 (figure 5.1). Raw
FASTQ files were trimmed using Trimmomatic
(http://www.usadellab.org/cms/?page=trimmomatic) to remove Illumina sequence adapters,
remove reads shorter than 25 bp, to remove runs of leading or trailing bases with quality scores
below 2, and to scan reads with a 4 bp window and trim where the average quality score drops
below 3 (14). These reads were then mapped to the reference using the BWA-MEM BurrowsWheeler aligner (http://bio-bwa.sourceforge.net/) (15). The PicardTools package
(https://broadinstitute.github.io/picard/) was used to add read group information and mark
optical duplicates. Base quality score recalibration was done using the BQSR tool in the
genome analysis toolkit (GATK) (8). Calling of variants was done using GATKâs Haplotypecaller in
GVCF mode. At this point, the per-sample GVCFs were combined for joint genotyping using the

123

Figure 5.1 â Whole-genome sequencing pipeline according to GATK best practices
handbook Reproduced from: (https://software.broadinstitute.org/gatk/bestpractices/bp_3step.php?case=GermShortWGS) on June 2016
GenotypeGVCFs tools and variants were filtered according to the following criteria: SNPs were
dropped if quality by depth (QD) was less than 2.0, Fisher Strand (FS) Bias Score greater than
60.0, Mapping Quality Rank Sum Test (MQRankSum) score was less than -12.5, Root Mean
Square mapping quality (MQ) was less than 40.0, of alternate allele Rank Sum Test
(ReadPosRankSum) was less than -8.0, and if the stand odds ratio (SOR) was greater than 3.0.
Indels were dropped if QD was less than 2.0, if FS was greater than 200, ReadPosRankSum was
less than -20.0, or SOR was greater than 10.0. Called variants were annotated using ANNOVAR
and SnpEff (10, 11).
Once variants were called, two primary screening methods was used to examine them.
Variants within 1.5 Mb of the previously mapped 7.5 Mb region of interest were examined for
variants that would cause a coding region or splicing change. The list of whole-genome variants
124

was also compared to a list of genes from the Color Genes database, an online resource
containing location information on 171 mapped genes that have been shown to be associated
with pigmentation changes in mice, humans, and zebrafish. An additional database of
annotated canine non-coding RNAs was provided to us by the Broad Institute was also used in
these screenings.
5.2.4 Sanger sequencing of candidate genes
Sanger sequencing of regions of interest was carried out according to the protocol
previously described in Chapter 4.

125

5.3 Results
5.3.1 PCR-based results
In total, there were 22,931 unique variants identified within the 7.5 Mb region of
interest Âą 1.5 Mb across all 6 dogs genotyped. The vast majority of these, 17,197, were
intergenic variants. Of the total variants present, 1,943 (1,826 intergenic variants) were present
in all affected dogs. Once variants were screened to remove any variants which were also
present in at least one unaffected dog sample, the total number of variants was reduced to
1,155 â 1,154 intergenic variants, and a single variant present in a long non-coding RNA, lncRNA
32, a gene which had been identified as a putative tumor suppressor gene based on data from
mouse cell line expression data (16, 17). This variant (chr11:45,414,338-45,414351) was called
as containing a single base-pair insertion within an intron, converting a run of 13 adenine
residues to a run of 14 adenine residues. Sanger sequencing of an additional 20 dogs (13
affected, 7 unaffected) showed that dogs were often heterozygous for this region, having runs
of 11-15 adenine residues within the indicated intron (F Primer: TTTTTCGGTTGCCTTCATTC, R
Primer: CCTCGGTCCCCTCTAATCTT, 192 bp amplicon). No particular sequence length segregated
with the disease phenotype, and the region was not predicted to have any impact on splicing.

126

Table 5.2 â Dogs tested for variation in lncRNA32
Dog Number
CA483
CA486
CA489
CA509
CA519
CA524
CA525
CA576
CA577
CA578
CA593
CA595
CA596
CA602
CA604
CA614
CA615
CA616
CA617
CA619

OM Status
A
A
A
A
A
A
A
U
U
U
U
A
U
U
U
A
A
A
A
A

Age
Unknown
13
12
12
10
10
12
15
11
16
14
15
13
13
13
13
11
11
12
18

Sex
F
F
F
F
F
F
M
F
M
F
F
F
M
F
F
F
M
F
F
M

Fifteen coding-region variants were detected in 10 different genes (Apc, Frem2, Pcbd1,
Fgfr2, Ntrk1, Dock7, Mlph, Rxra, Atp7a, and Ercc2) from the Color Genes database which were
present in at least one copy in each affected dog. None of these genes were within our mapped
region on chromosome 11. Of these, 12 variants were present in at least one unaffected dog as
well, and 6 were present in multiple unaffected dogs. PCR primers were designed around these
regions to determine whether or not they were associated with the disease phenotype. Each
fragment was sequenced in 15 total dogs, 8 affected and 7 unaffected. None of the variants
called via whole genome sequencing segregated with the affected phenotype.

127

Table 5.3 â Dogs genotyped for variants in genes in the Color Genes database
Dog Number
CA195
CA207
CA308
CA393
CA477
CA489
CA491
CA509
CA519
CA541
CA593
CA596
CA613
CA617
CA622

OM Status
A
U
U
U
U
A
A
A
A
A
U
U
U
A
A

Age
12
11
15
13
13
12
8
12
10
10
13
13
13
12
13

Sex
F
M
M
F
M
F
M
F
F
F
M
M
M
F
M

Table 5.4 â Primers for genotyping variants in genes from the Color Genes database
Primer Name
F Primer
R Primer
Amplicon Size
Apc
ATCTCAAAGGGTGGGAAAGG
TCTTGGCGAGCAGATGTAAA
170
Frem2
TTGAGGGACAGATGGGAAAG
GTTACCCTGCTGGAGCTCTG
218
Pcbd1
CGAGGGTAAGGGTGTTGAAG GCGTCTGTGTGTCTGAGTGTG
219
Fgfr2
ATGGAGCAAGCGACGAAGT
GCGAGTTGCAGCAAAGTTAG
240
Ntrk1
ACGCTCTCACCAGAAACCAC
ATGTGCCAAGACCCACTCTC
191
Dock7
TCCAAGGCAAAAATGTAGTTCC TGGAGCAAACTCCTATTCCA
250
Mlph
CTGAACGCGGATCTCCTTG
AGGTCTGACCCTTCCTCCTC
164
Rxra
CTTCCTGCCTCCCTTTTCTT
AACCCAGCCAGCTTCTTTCT
153
Atp7a pt.1
AAGGACGAGTAAAGGCCACA
ATCTCACACACCGGGAGAAG
167
Atp7a pt. 2
GGTATCACCTTCCTGCCTCA
AGACTGCCAAATGCAGCAC
238
Ercc2
ACGTGACCAGTTCCAGATCC AGGAAGAGGGCAAGGAAGAG
226

128

Table 5.5 â Variants detected within genes from the Color Genes database via whole-genome sequencing
Variant Name

Location

Apc â a
Apc â b
Frem2
Pcbd1
Fgfr2 â a
Fgfr2 â b
Ntrk1 â a
Ntrk1 â b
Dock7
Mlph â a
Mlph â b
Rxra

3:266419
3:266427
25:2129042
4:21723757
28:31410838
28:31410986
7:41148019
7:41148023
5:47482462
25:48160377
25:48160434
9:50557377

Atp7a â a
Atp7a â b
Ercc2

Ref Seq

Variant

TCAA
CCAAA
ATT
AT
TGG
TG
C
T
A
C
A
G
TG
TCG
CTT
CT
A
G
CCTACGGTT
GCTGCAGTGTCTC
AGCTT
AGGAAG
CGTGTGTGTGTGTGTG
CGTGTGTGTGTGTGTG
TGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTG
X:60203445
A
T
X:60260558
TACA
TACACA
1:110189305
GAA
GAAA

129

In
Unaffecteds?
Yes (2)
Yes (1)
Yes (3)
Yes (1)
Yes (1)
No
Yes (1)
Yes (1)
No
No
Yes (2)
Yes (2)
Yes (1)
Yes (3)
Yes (3)

Variant Type
Frame shift
Frame shift
Frame shift
Start gain
Start gain
Start gain
Frame shift
Frame shift
Splicing
Frame shift
Frame shift
miRNA variant within
intron
Start Gain
Frame shift
Frame shift

Table 5.6 â Genotyping results for Color Genes variants
Dog#
CA195
CA207
CA308
CA393
CA477
CA489
CA491
CA509
CA519
CA541
CA593
CA596
CA613
CA617
CA622

OM
Status
A
U
U
U
U
A
A
A
A
A
U
U
U
A
A

Apc
-a
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V

Apc
-b
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V

Frem
2
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V

Pcbd
1
V/V
V/V
V/V
R/R
V/V
R/V
V/V
R/R
V/V
R/V
V/V
V/V
-/V/V
V/V

Fgfr2
-a
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R

Fgfr2
-b
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V

Ntrk1
-a
R/R
R/R
V/V
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R

Ntrk1
-b
R/R
R/R
V/V
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R
R/R

Dock
7
V/V
V/V
R/V
V/V
V/V
V/V
R/V
R/V
V/V
V/V
V/V
R/V
-/V/V
V/V

Mlph
-a
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V

Mlph
-b
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V

Rxr
a
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*

Atp7a
-a
R/R
V/V
-/V/V
R/V
V/V
V/V
V/V
R/R
V/V
V/V
V/V
V/V
R/V
R/V

Atp7a
-b
R/R
V/V
-/V/V
R/V
V/V
V/V
V/V
R/R
V/V
V/V
V/V
V/V
R/V
R/V

Ercc
2
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
V/V
-/V/V
V/V

* more than one variant allele was present for these dogs â they possessed a variable number of GT repeats, ranging from 13-18,
with no particular repeat length associated with the disease phenotype
- denotes a dog that was not genotyped
âRâ denotes the genotype listed as âRef Seqâ in Table 5.5
âVâ denotes the genotype listed as âVariantâ in Table 5.5

130

5.3.2 PCR-free results
A comparison of the variants called in affected dogs that had been sequenced by PCRfree methods to variants called in affected dogs sequenced by traditional PCR-based
sequencing showed very little difference within the region of interest. Across the 7.5 Mb region,
20,629 total variants were called in at least one affected dog. Of those, only 59 variants were
present in PCR-free sequenced dogs in regions where the traditionally sequenced dogs had
lacked sufficient coverage to make a call. None of these variants were exonic. In contrast, there
were 24,081 total variants called in at least one unaffected dog. Of those, 634 were unique to
the dogs sequenced via PCR-free methods, although this is more likely attributable to the
greater depth of coverage at which the two PCR-free unaffected samples were sequenced
rather than a result of the differing sequencing techniques. The greater number of unique
variants present in the PCR-free unaffected dogs should help reduce the number of variants
shared by all OM-affected dogs which are absent in unaffected dogs from the 1,154 that were
seen in the PCR-based data, but analysis of the PCR-free data has not yet been completed.

131

5.4 Discussion
Despite the presence of a strong statistical association between a 7.5 Mb pair region of
chromosome 11 identified via whole-genome SNP array, no coding region variation was
observed in any of the annotated genes in that region which segregated with our phenotype.
Further, no variants which matched our expected inheritance pattern were detected in intronic
regions likely to affect splicing or in known regulatory RNAs. The remaining variation in the
region which followed our expected inheritance pattern is all intergenic. The total number of
intergenic variants (1,154) are too numerous to assay individually. Additionally, no variants
were found in any of 171 genes known to cause pigmentation disorders which segregated with
the phenotype when tested in additional dogs.
Taken together, this suggests that the causal mutation for OM likely to be regulatory â
either a cis-acting regulatory factor which is altering the expression of a gene in or near our 7.5
Mb region of interest, or a variant in a trans-acting regulatory element which is not annotated
within our region. Because interrogating each of the remaining 1,154 intergenic variants is
impractical, we decided to pursue a whole-transcriptome approach to determine which genes
were being differentially regulated, anticipating that a single pathway would be implicated
which would reduce the number of potential regulatory sites we would have to examine, while
providing some clues to OMâs mode of action.
The lack of any detected coding region change in our whole-genome sequencing data
does not completely preclude the possibility that our variant is a coding change, however.
Although quite good for a semi-model organism, the canine genome is not as well annotated as

132

the extensively studied mouse and human genomes, for example. De novo assembly of our
sequence data should be performed, to help to uncover any errors that may exist in the canine
reference annotation. Additionally, although SOAP was used to look for structural variants with
the genome, the short-read nature of Illumina sequencing is ill-suited for finding large-scale
structural changes (18, 19). Although our RNA sequencing may be able to uncover changes in
gene regulation resulting from a structural rearrangement, long-read sequencing is the most
robust method for detecting them (20).

133

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134

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136

CHAPTER 6
TRANSCRIPTOME ANALYSIS OF OCULAR MELANOSIS-AFFECTED CAIRN TERRIER IRIS TISSUE

137

6.1 Introduction
Ocular melanosis (OM) is an eye disease commonly seen in Cairn terrier dogs that is
characterized by a bilateral proliferation of uveal melanocytes, their migration into abnormal
areas of the eye such as the sclera and episclera, and the release of pigmented material into the
anterior chamber of the eye (1, 2). Over time, this pigmented material builds up in the drainage
pathways of the anterior chamber and impedes drainage of the aqueous humor. This leads to
painful secondary glaucoma and often blindness. The pedigree analysis indicates that the
disease has an autosomal dominant mode of inheritance, although it may not be completely
penetrant. It has a variable rate of progression, and the age at first onset can vary widely from
2-12 years of age.
Attempts to map a causal gene using a candidate gene approach examining genes
known to be involved in pigment-related disorders was unable to find any common alleles
shared by all affected dogs, and whole-genome sequencing of OM-Affected dogs was unable to
identify any variants that seem likely to be causal for the disease (see Chapter 5) (4). A 7.5
megabase region of chromosome 11 has been identified as significantly associated with the
disease (see Chapter 4), which confirms the notion that the disease has a genetic basis, and is
not a purely environmental effect which has not been identified.
Taken together, the fact that a) there is a region of chromosome 11 that is strongly
associated with the OM and b) there seem to be no coding or splicing changes within the region
that can account for the OM phenotype suggest that the variant being mapped is regulatory in
nature. Unfortunately, the annotation of regulatory elements present within the canine

138

genome is quite poor, and the sheer number of intergenic variants detected via NGS that fit the
expected inheritance pattern for OM makes screening them impractical. Thus, we decided to
perform RNA sequencing and transcriptome analysis on a number of OM affected and
unaffected Cairn terriers to determine what differences in expression level are associated with
OM. In the event that the DNA variant mapped to chromosome 11 is cis-acting, we would
expect to see changes in expression levels of genes within the 7.5 Mb mapped region Âą 1.5 Mb,
as that is the maximum distance at which cis-acting regulatory elements have been shown to be
able to act (5). In the event that it is a trans-acting regulator, differences in expression levels
should be observed in gene pathways under regulation. The alignments within the 7.5 Mb
region identified were also assessed by eye to determine if any unannotated transcripts are
present in the region.

139

6.2 Materials and methods
6.2.1 RNA sample collection
All RNA samples used in this study were extracted from uveal tissue donated with the
consent of the owner from Cairn terrier dogs being euthanized for health reasons unrelated to
OM or from eyes being enucleated for therapeutic reasons from late-stage OM dogs that had
developed glaucoma. Prior to euthanasia/enucleation, veterinarians performing the operation
were sent an aliquot of RNAlater (Fisher Scientific, Pittsburgh, PA), a preservation solution used
to rapidly stabilize RNA in tissue samples (6). Operating veterinarians were instructed to
immediately remove the anterior segment from the eye and fully submerge it in the provided
RNAlater solution to allow it to penetrate into the iris, and ship the tissue back overnight in an
insulated (but not refrigerated) box, as per the RNAlater usage instructions. Upon receipt of
these tissue samples, the anterior chamber was removed from RNAlater. The iris was then
excised from the remainder of the anterior chamber and stored at -80Â°C as per the RNAlater
usage instructions.
6.2.2 Extraction, purification, and evaluation of RNA from iridal tissue
RNA was extracted from the collected iris tissue using a Qiagen miRNeasy mini-prep kit
(Qiagen, Germantown, MD) according to the manufacturerâs protocol, including the optional
DNase digestion steps. RNA extracted in this way showed very high levels of melanin
contamination in the final product, such that the sample was visibly dark brown in color (figure
6.1). Melanin complicates most downstream applications for which RNA is used. The fact that is
absorbs large amounts of light complicates attempts to quantify the RNA using a

140

spectrophotometer, and melanin has been shown to bind to a wide variety of DNA polymerases
and reverse transcriptases and inhibit their activity (7, 8).
The RNA samples were purified of melanin using a modified CTAB-Urea purification protocol (3,
9). Cetyl-trimethylammonium bromide (CTAB) is a cationic detergent. The cationic micelles that
CTAB forms selectively complex with the anionic nucleic acids, leaving the uncharged melanin
remaining in the supernatant. The presence of urea greatly increases the specificity of the
reaction, and although the method by which it does so is not completely understood, it is
thought to help sequester hydrophobic molecules that may otherwise disrupt the CTAB
micelles (3). In brief, the purification was performed as follows: RNA purified via the RNeasy kit
was diluted with water to a final volume of 200 Âľl. To the RNA, 65 Âľl of 5M NaCl was added,
followed by 800 Âľl of CTAB-urea solution (1% CTAB [Sigma-Aldrich, St. Louis, MO], 4 M urea, 50
mM Tris-HCl, 1 mM EDTA, pH 7.0) and mixed via pipetting. Samples were left overnight at 4Â°C,
and then centrifuged at 15,300g at 4Â°C for 15 minutes. The pigment-containing supernatant
was removed, and the precipitated RNA was resuspended in 200 Âľl of 7 M guanidine
hydrochloride (Sigma). The guanidine hydrochloride replaces the RNA via ionic exchange, and
allowed the RNA to be precipitated by adding 400 Âľl of 100% ethanol (10, 11). The resulting
mixture was incubated on ice for 1 hour, and pelleted via centrifugation at 15,300g at 4Â°C for 15
minutes. The pellet was then washed with 400 Âľl of 70% ethanol, and centrifuged for 10 min at
20,800g at 23Â°C. The remaining ethanol was removed, and the RNA pellet resuspended in 100
Âľl of water. This greatly reduced the level of melanin present in the samples (figure 6.1), but it
also appeared to have an adverse effect on overall RNA quality. Following purification, RNA was
quantified using a Qubit fluorimeter (Fisher) and the RNA integrity number (RIN) was

141

ascertained using a model 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RIN values
varied between samples (8.3-5.7), but most were lower than those reported for human ocular
tissue that had been preserved in RNAlater under similar conditions (12). Additionally, some
samples retained a small amount of pigment following the initial CTAB-urea purification which
were subjected to a second round of CTAB-urea purification. There samples uniformly produced
RNA samples with unusably low RIN values (4.8-1.1).

Figure 6.1 â RNA samples before and after CTAB-urea purification Six RNA samples before
(left-side tubes) and after (right-side tubes) CTAB-Urea purification. Figure reproduced from
(3).

6.2.3 RNA sequencing
RNA samples from 12 total dogs (7 affected and 5 unaffected, see Table 6.1 for a
complete list of dogs used) were sent to Michigan State Universityâs Research Technology
Support Facility for RNA sequencing. The dogs were subdivided into two groups â a pilot group
of 4 dogs to determine whether the RNA purified via CTAB-urea precipitation was of sufficient
quality for sequencing, and a second group of 8 dogs to provide the power necessary for the
study. The initial four dogs had higher-quality RNA as determined by RIN value. Library

142

preparation was performed using the Illumina TruSeq stranded mRNA library kit (Illumina). The
samples were barcoded and pooled, and sequenced using an Illumina HiSeq 2500 Rapid Run
flow cell v2 (Illumina, San Diego, CA) in 100bp paired-end mode. The average coverage after QC
was 42.5 million reads per sample with an average yield of 8.52 Gbp of sequence data per
sample. The second set of dogs used slightly lower-quality RNA, as determined by RIN value.
Traditional library creation kits like the TruSeq kit used for the pilot samples isolate
polyadenylated mRNA via the use of oligo-dT beads, and this can results in a strong 3â bias in
degraded samples. To compensate for this, their library was created using the NuGEN Ovation
RNA-Seq system (NuGEN, San Carols, CA). This library creation kit uses pseudo-random primers
for first-strand cDNA synthesis, allowing for even coverage of the sample while avoiding rRNA.
Samples were barcoded and pooled and sequenced on a single lane of an Illumina HiSeq 2500
High Output v4 (Illumina) in 125bp paired-end mode. The average coverage after QC was 37.5
million reads per sample with an average yield of 9.38 Gbp of sequence data per sample.
According to most sources, this level of coverage should be sufficient for allowing technically
precise detection of differential expression in most genes (13, 14).

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Table 6.1 â Information on dogs used for RNA sequencing
Dog Number OM Status Age in years and Sex RIN Library Preparation Coverage in read-pairs
CA614
A
14,F
7.6
Ovation
30,779,315
CA197
U
15,M
7.6
TruSeq
31,299,299
CA455
U
13,F
7.5
TruSeq
58,973,062
CA619
A
18,M
7.0
TruSeq
32,173,667
CA615
A
14,M
6.6
Ovation
35,331,298
CA621
A
10,M
7.0
TruSeq
47,856,741
CA229
U
16,M
8.3
Ovation
39,821,306
CA624
A
8,F
6.0
Ovation
31,795,705
CA456
U
16,M
6.7
Ovation
44,895,038
CA314
A
12,F
5.9
Ovation
43,249,458
CA558
U
16,M
5.9
Ovation
38,414,521
CA626
A
16,F
5.7
Ovation
35,897,872

6.2.4 Data Evaluation
RNA sequence data was used for two primary applications â examining differences in
expression level, and using the data as pseudo-exome data to look for variants. All data was
trimmed prior to use using Trimmomatic to remove Illumina adapter sequence, remove very
short reads (less than 25bp), to trim leading or trailing bases with a quality score of below 2,
and to scan reads with a 4bp sliding window and cut where the average quality score drops
below 3 (15).
Expression analysis was carried out using the spliced-read mapper TopHat
(http://ccb.jhu.edu/software/tophat/index.shtml) and the statistical analysis software R
(https://www.r-project.org/) (16, 17). TopHat was run with the mate inner distance setting
changed to reflect the insert size of each sample and with other settings set to default values.
The mapped read files generated by TopHat were loaded into R and analyzed using several
elements of the Bioconductor toolset (https://www.bioconductor.org/about/). The

144

GenomicFeatures, GenomicRanges, GenomicAlignments, Rsamtools and org.Cf.eg.db packages
were used to generate per-gene read-count tables for each sample based on ensemble gene
annotation (18).Tables for the affected and unaffected dogs were then compared across
disease status using two different software packages - DESeq (to perform a base mean
comparison between two conditions) and edgeR (to perform a genewise exact test for
differences) (19-21). Genes with p-values below 0.05 (DESeq) or 0.01 (edgeR) were analyzed
using PANTHER (http://pantherdb.org) to determine if any known gene families or pathways
were overrepresented in the resulting data (22).
For direct data visualization, a subset of the mapped read files was created which
contained the 7.5 Mb mapped region Âą 1.5 Mb on either side. The IGV software package
(http://software.broadinstitute.org/software/igv/) was used to visualize the data (23). All
mapped reads from the 7.5 Mb region Âą 1.5 Mb on either side, were extracted and loaded into
IGV along with the ensemble canine gene annotation track (ver. CanFam3.1.87). Potential
unannotated transcripts were called by eye in regions where more than 5 dogs had multiple
reads with a mapping quality greater than zero map to the same region where no gene was
annotated in the ensemble track. Sequence data from these regions was then viewed in UCSC
genome browser, and converted to human (build hg38) or mouse (build mm10) to look for
annotation in other organisms if necessary.
Variant calling was carried out according to the GATK Best Practices handbook as of
1/21/2017 (https://software.broadinstitute.org/gatk/documentation/article.php?id=3891).
Briefly, the RNAseq reads were mapped to the reference genome (CanFam3.1) using the STAR
2-pass method, which runs an initial alignment to detect splice junctions that it then uses to
145

guide the final alignment (24, 25). Picard (http://broadinstitute.github.io/picard/) was used to
add read groups, mark duplicates, and index the mapped reads. The genome analysis toolkit
package (GATK) was used to clip out regions where exon sequence inappropriately overhangs
into intronic regions, realign around indels, and perform base quality score recalibration (26).
GATKâs HaplotypeCaller tool was used to variants. The resulting raw variants were filtered to
remove any variants with a Fisher Strand value of less than 30 or a quality by depth score of less
than 20, and variant annotation was performed using ANNOVAR and SnpEff (27, 28).

Figure 6.2 â Graphical illustration of the variant calling pipeline for RNA data Figure
reproduced from
(https://software.broadinstitute.org/gatk/documentation/article.php?id=3891)
6.2.5 Confirmatory qRT-PCR
To confirm differences observed in RNAseq data, quantitative real-time PCR (qRT-PCR)
was carried out using. For qRT-PCR, first-stand cDNA synthesis was done using the Transcriptor
first-strand cDNA synthesis kit (Roche, Indianapolis, IN) according to the manufacturerâs

146

instructions. The qRT-PCR itself was performed using a pre-made SYBRGreen mastermix
(SsoFast EvaGreen Supermix with low ROX, Biorad, Hercules CA) according to the
manufacturerâs protocol and run on an Applied Biosystems 7500 Fast Real-Time PCR (Fisher).

147

Table 6.2 â Primers used for qPCR
Gene Target
COL3A1
COL6A3
COL14A1
CXCL10
PREX1
PIK3C2G Pair 1
PIK3C2G Pair 2
PIK3C2G Pair 3
GAPDH

Exon Junction
Spanned
49-50
36-37
44-45
1-2
35-36
27-28
18-19
6-7
None

Forward Primer

Reverse Primer

GGTGGACAGATTCTGGTGCT CCTTCATTTGACCCCATCAG
GGTGAACGTCAAGGAGGTGT AACCAATCGCACTGTTTCCT
CCAGCCAATCCTCATCAGTT ACTCTCCCCTGAAGGTCCTG
GCAGAGGAACCTCCAGTCAC ACGATGGACTTGCAGGAATC
GGCCCTGAAGGTTGTCTTCT GCCCTGAGTTTGCGGTAATA
TGCAACCAGGTCAATTCAAA AGTGCCACCAATGAGGAAAC
CGTAAAGTGGCAGTTCGACA AGCTGCCAACAGCTTCTGAT
TCAGAAAAATGGGGAAGTGC TTGCTGGGTTTTCAATAGGC
ACAGTCCATGCCATCACTGCC GCCTGCTTCACCACCTTCTTG

148

Product Size
250
186
245
195
225
200
234
178
266

6.3 Results
6.3.1 Expression level differences
Expression levels were compared across 21,709 total annotated canine genes, giving a
Bonferroni-corrected p-value of 2.3x10-6. No genes reached this threshold using either analysis
method. The lowest p-value for DESeq was 9.4x10-3, and the lowest p-value calculated by edgeR
was 2.0x10-4. None of the 200 smallest p-value results from either analysis were from
transcripts located within the 7.5 Mb region of interest mapped via GWAS (see Chapter 4). The
transcripts with the lowest calculated p-values from each program were analyzed with
PANTHER to determine if any known gene pathways or gene families were overrepresented. A
cutoff p-value of 0.1 was used for DESeq samples (53 transcripts) and 0.01 for edgeR (93
transcripts). In both samples, two pathways had multiple genes map to them â integrin
signaling pathways, and chemokine and cytokine-mediated inflammatory pathways. Taking
both analyses into account, 4 genes classified by PANTHER as being related to chemokine or
cytokine-mediated inflammation, and 4 genes related to integrin signaling were present in the
lowest p-value transcripts, with 2 genes present in both groups. C-X-C Motif chemokine 10
(CXCL10), collagen type XIV alpha 1 chain (COL14A1), phosphatidylinositol 4-phosphate 3-kinase
C2 domain containing subunit gamma (PIK3C2G), and phosphatidylinositol 3,4,5-triphosphatedependent RAC exchanger 1 protein (PREX1) were classified by PANTHER as being involved
chemokine and cytokine-mediated inflammatory pathways. COL14A1 and PIK3C2G were also
classified genes involved in integrin signaling pathways, along with collagen type III alpha I chain
(COL3A1) and collagen type VI alpha 3 chain (COL6A3). All genes listed here had higher levels of
expression in OM-affected dogs than in unaffected dogs. Observed average fold increases for
149

each gene were 4.78 (CXCL10), 4.04 (COL14A1), 3.19 (PREX1), 3.15 (COL3A1), and 3.38
(COL6A3). No PIK3C2G expression was observed in unaffected samples, so no fold change can
be calculated. PIK3C2G expression was observed in 5 of 7 affected dogs sequenced, with a
mean of 44.7 copies per dog observed among all OM affected dogs.
6.3.2 qRT-PCR confirmation
To confirm the changes in expression for each sample obtained from the RNAseq data,
we used quantitative real-time PCR (qRT-PCR). Some of the samples used for RNAseq were
mildly degraded (table 6.1) and replication of the results via qRT-PCR would help to confirm
that the observed expression differences were not the result of library creation bias. We
performed qRT-PCR on the 12 samples that we initially sent for RNA sequencing. For 5 of the 6
genes (COL3A1, COL14A1, CXCL10, PREX1, and COL6A3) an expression difference was observed
that was significant at P < 0.05. For PIK3C2G, no fold change could be calculated. PIK3C2G
expression was detected at very low levels in our RNAseq data (it was detected in 5 of 7 OMaffected samples, and in none of our unaffected samples). When performing qRT-PCR, we used
3 different primer pairs to attempt to amplify PIK3C2G, but got no amplification above the
negative control background in all samples except for CA314 and CA626, both of which were
OM-affected.

150

Fold Change In Gene Expression

12
10
8
6
4
2
0
COL3A1

COL14A1

CXCL10

Affected

PREX1

COL6A3

Unaffected

Figure 6.3 â Fold increase in gene expression as observed by qPCR Fold increase in
expression level for each gene as observed by qPCR. The average unaffected expression level
was defined as 1.0. PIK3C2G had no detectable expression in unaffected samples from which
to compute a fold change.
6.3.3 Unannotated transcripts
Twenty-one regions of the genome fit our criteria for further investigation (multiple
mapped reads with positive mapping quality scores from 5 or more dogs at the same location
with no annotated transcript in ensemble or refgene). Upon further examination, each of these
21 regions mapped to within a known canine long interspersed nuclear element (LINE). No
regions were observed that suggest an unannotated transcript.
6.3.4 RNA variant calling
Examining variants called in genes with the 7.5 Mb region identified by whole-genome
SNP array, only 3 total variants were called which were present in all affected samples. There
variants were also present in all unaffected samples. The genome-wide list of variants was
compared to a list of genes known to be associated with pigmentation changes in human, mice,

151

or zebrafish (http://www.espcr.org/micemut/). A total of 17 variations were observed in all
affected samples, 14 of which were present in all unaffected samples as well, and 3 of which
were present in 4 of 5 unaffected samples.

152

6.4 Discussion
All 6 of the genes identified by PANTHER as part of a common pathway have been
shown to have some effect on cell migration or invasiveness in various cancer models,
suggesting that, while they may not explain the excessive pigment production observed in OM,
they may provide important clues into the nature of the migratory behavior the affected
melanocytes display when they migrate to the sclera.
Of the 6 genes identified, PIK3C2G seems to be the furthest upstream in this pathway â
it is a member of the phosphoinositide 3-kinase (PI3K) family of proteins, which are known to
play a key regulatory role in a wide variety of cellular processes, including cell survival,
proliferation, migration, and oncogenic transformation (29). Changes in the activity of PI3K
proteins have been linked to many different forms of cancer (29-31). Although this family of
proteins is in general very well characterized, PIK3C2G falls into the category of class II PI3Ks,
which are not as extensively characterized as class I PI3Ks. Class II PI3Ks are known to be
activated by cytokine receptors, integrins, and receptor tyrosine kinases, but the exact cellular
function of class II PI3Ks remains unclear.
PREX1 (often seen as P-REX1) is a guanine nucleotide exchange factor for the Rac
subfamily of Rho GTPases. PREX1 activity is activated by phosphatidylinositol-3,4,5-triposphate,
the product of PI3K proteins, suggesting that the increase PREX1 expression that we see in
affected OM samples may be the result of downstream result of higher PIK3C2G levels (32, 33).
Elevated PREX1 expression has been observed in a number of different human cancer types,
including breast, prostate, and ovarian cancers, but the most interesting facet in relation to OM
is that PREX1 is upregulated and drives migration and invasion in many human and mouse

153

Figure 6.4 â Pigmentation patterns of various mice Note that PREX1-/- mice display a lack of
belly and feet pigmentation regardless of background. Modified from (34).
melanomas, and that it has an important developmental role in normal melanoblast migration
(34, 35). PREX1-/- mice on a C57BL6 background displayed a completely penetrant
depigmentation of belly, tail and feet â the furthest points for melanoblast migration from the
neural crest during embryogenesis(figure 6.4), suggesting that the loss of PREX1 resulted in
impaired
melanoblast migration (rather than a deficiency in proliferative or melanogenic capacity) (34).
Further mouse studies confirmed this assessment, and also showed that PREX1 deficiency
significantly impairs melanoma metastasis in Tyr::NrasQ61K/âŚ;INK4a-/- mice, a genetically modified
mouse model for malignant melanoma. However, metastatic cell populations driven by PREX1
overexpression tend to be primarily anchorage independent (32). Cultured OM melanocytes did
154

not display anchorage independent growth characteristics in vitro when tested via soft agar
assay (see Chapter 3).
CXCL10 is a chemokine which has been found to be overexpressed in many different
types of metastatic cancer (36). This overexpression has typically been mediated by PI3K
signaling pathways, suggesting that CXCL10 overexpression is a downstream effect of the
observed increased PIK3C2G activity, but PREX1 has also recently been shown to have a
regulatory effect on CXCL10 (33). CXCL10 functions via activation the CXCR3 receptor. There
exist two different types of CXCR3 receptor, and which kind CXCL10 binds to determine its
downstream effects. CXCR3-A is the predominant isoform of the receptor, and upon binding
CXCL10 it induces cell proliferation and chemotaxis. The rarer CXCR3-B isoform has the
opposite effect, inhibiting cell proliferation and migration.
The expression differences in the three collagen subunits are more difficult to directly
integrate into the pathway, as none of their expression is directly linked to the three proteins
noted above. However, extracellular matrix rearrangement and microenvironment alteration
are known to be important in all forms of cancers and cellular migratory diseases (37). The
specific collagens shown to be upregulated in OM in this study have been shown to be
associated with various cancer types. Mutations in COL3A1 are known to be associated with
melanoma, COL3A1 overexpression has been shown to be linked to drug resistance in ovarian
cancers, and multiple carcinoma lines have been shown to secrete COL3A1 into the ECM at high
levels (38-40). However, tumors implanted into COL3A1+/- mice metastasize more aggressively
than those implanted into wild-type mice, suggesting that normal levels of COL3A1 provide

155

some protective effect (41). Likewise, COL14A1 has been shown to act as a tumor suppressor in
renal carcinomas (42). COL6A3 has been shown to often be secreted in uveal melanomas (43).
Although qRT-PCR confirms that these genes are expressed at increased levels in OM
affected dogs, important follow-up studies still remain to be done. Most of the OM-affected
eyes used for RNA collection were enucleated from OM affected dogs in order to treat painful
secondary glaucoma. As a result of this, most of the affected eyes used in this study were
currently either inflamed or being treated for inflammation at the time of their submission.
Although a literature search does not show that expression changes in any of these six genes
have been directly linked to glaucoma, some of them, such as CXCL10, are directly involved in
inflammation pathways (36, 44). If possible, immunohistochemistry of an OM affected eye that
has not yet begun to experience changes related to glaucoma could show how the location and
prevalence of these proteins changes between OM-affected and normal tissues, and would be
useful in establishing the presence of these expression changes in early-stage OM.
Taken together with the mapping data from the genome-wide SNP array (see Chapter
4), this data supports the hypothesis that the causal mutation for OM is trans-acting and
regulatory in nature. None of the six genes with elevated expression in OM affected dogs are on
chromosome 11, and none of the regions that these six genes are located in are mapped as
significant by the genome-wide SNP array. No genes known to regulate any of the six genes
with elevated expression are within 1.5 Mb of the 7.5 Mb region, ruling out a cis-acting
regulatory element or an undetected coding region change in a gene which regulates this
pathway. Analysis of the mapped RNA reads by eye did not reveal any unannotated transcripts
within this region.

156

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CHAPTER 7
DISCUSSION AND FUTURE DIRECTIONS

162

Although we have yet to identify a causal mutation, we have made great strides in
understanding the genetic and physiological changes that underlie ocular melanosis (OM) as a
result of the work done in this study. To summarize, we have â
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Developed methods to isolate and culture uveal melanocytes from canine eyes without
contamination from other cell types

-

Extensively studied the in vitro phenotypic differences between melanocytes cultured
from OM affected eyes and those from unaffected controls

-

Mapped a statistically significant region of chromosome 11 which is associated with the
disease

-

Performed whole-genome sequencing to interrogate possible causative mutations in
OM affected dogs

-

Eliminated the possibility that the primary cause of the disease is a coding-region
variation in a known canine gene

-

Used transcriptome sequencing to identify a putative change in pathway regulation
present in OM affected dogs which suggests a possible method by which the migratory
portion of the OM phenotype functions

From these results, three avenues for further investigation readily suggest themselves â
As was discussed in Chapter 3 of this work, there were no detectible in vitro changes in
cell behavior in OM affected melanocytes related to cell migration and proliferation, only
pigment production. We theorized that this loss of the migratory phenotype might be due to a
lack of necessary signaling from other cell types usually present in the uvea. We suggest that

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cellular co-culture experiments may be useful to attempt to re-create the in vivo phenotype in
vitro. There have been numerous recorded instances of melanocyte and melanoma-derived cell
lines losing certain physiological characteristics in culture which are restored in co-culture â
three lines of metastatic melanoma cells which normally disseminate throughout the body via
extravasation were shown to regain lost expression of LFA-1 (an integrin involved in
extravasation) when cultured in the presence of human umbilical vein endothelial cells (1).
Cultured human melanocytes have been shown to undergo melanogenesis when exposed to
UVB radiation only when cultured with keratinocytes (2). Especially given that the causal
change in OM appears to be a regulatory one (see Chapter 6), performing co-culture
experiments to attempt to re-create in vivo conditions would be a useful next step. Although
there are many different cell types present in the uveal with which co-culture could be
attempted, uvea melanoma / endothelial cell interactions have been previously characterized,
and suggest that uveal melanoma growth and metastasis is reliant on an interplay between
uveal melanoma cells and cytokines released by endothelial cells (3). PIK3C2G, one of the
transcripts shown to be upregulated in OM uveal tissue, is known to be activated by cytokine
receptor activity, so endothelial cell co-culture represents a logical starting point for these
studies (4, 5). Although some of the tests outlined in Chapter 3 would be difficult to perform
under co-culture conditions, two elements critical to the OM phenotype could be tested fairly
easily â the ability to migrate through a membrane and elevated melanin production. There are
already well-developed protocols for examining transmigration of melanoma cells in co-culture
with endothelial cells using a simple transwell assay, and for measuring differences in melanin
synthesis rates for co-cultured cells (2, 3). Furthermore, if these co-culture experiments prove

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successful in recreating the in vivo phenotype, analysis of culture media to obtain a cytokine
profile and use of anti-cytokine antibodies to prevent cytokine signaling could help to provide
additional clues to the exact pathways which must be active to produce the OM phenotype,
and provide insight into how to best treat affected dogs.
Another possible avenue of investigation involves determining whether the six proteins
corresponding to the six genes shown by RNAseq to have elevated expression levels, PREX1,
PIK3C2G, COL31A, COL6A3, COL14A1, and CXCL10 are actually present in elevated levels in the
uvea of OM affected dogs. As many of these genes are known to be associated with
inflammation, it would be best if these studies could be done on early-to-mid stage OM
affected eyes rather than eyes which were enucleated as the result of glaucoma. While it is rare
for us to receive such eyes via donation, an appeal could be made to the breed club that any
owner with an older affected dog which is clear for glaucoma should keep our lab in mind in the
even that their dog needs to be euthanized for therapeutic reasons unrelated to OM. Ideally,
part of each sample can be used to extract total protein to quantitatively analyze protein levels
via Western blot, and the remainder can be used for immunohistochemistry to determine
whether or not these proteins co-localize with the large, pigmented cells characteristic of OM.
Given the difficulties in obtaining sample tissue for RNA sequencing, one possible
alternative is to extract RNA from early-passage cultured melanocytes. Although OM affected
melanocytes do lose their pigmented phenotype in vitro, they retain it for the first few passages
immediately following isolation (see Chapter 3). Although these cells are likely undergoing
expression changes in culture, they are also not in an actively inflammatory environment, as
they would be in a glaucomatous eye, which may help to decrease background noise when
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comparing differences between cell populations. Additionally, comparing expression changes
between affected cells in culture and affected iris tissue may highlight which changes in
expression are causing the loss of the pigmented phenotype in cultured cells.
We can also attempt to leverage our existing whole-genome sequencing data in new
ways. To date, our study has focused primarily on genes near the 7.5 MB region mapped by the
whole genome SNP array, and genes with a known connection to pigmentary disorders. There
has been no extensive screening done of genes known to be involved in cytokine or integrin
signaling pathways. The results of our transcriptome analysis indicate that signaling in these
two pathways may be driving the changes in OM melanocyte migratory behavior that we
observe in vivo. Both of these pathways are fairly well understood, which give us a good pool of
target genes to examine for possible causative variants.
The 7.5 MB mapped region on chromosome 11 continues to present questions,
however. No genes within that region showed significant expression differences as determined
by either method of transcription level analysis, and whole-genome sequencing has not
revealed any variants in that region that would seem to explain OM. Though the region is
statistically significant in its association with OM, no annotated element within it is a strong
candidate to be linked with the OM phenotype. To date, we have also been unable to find a
completely linked marker within the region that segregates perfectly with the OM phenotype,
finding at best ~85% concordance between a marker and the affected phenotype. Although we
have observed a recent clinical case of a Cairn terrier with a phenotype that appears similar to
OM but lacks some of the distinctive features, the vast majority of dogs diagnosed with OM by
other physicians have been confirmed as true cases when examined by Dr. Petersen-Jones. This
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makes it unlikely that misdiagnosis is occurring at a high enough rate to account for the
discrepancy. At present, this seems like an impasse. We propose to use de novo assembly of our
existing RNA sequencing data to try to detect the presence of any further unannotated
transcripts within the region which may explain this difference.
Although we found no structural rearrangements when examining the sequencing data
near the mapped region of interest, the short-sequence reads employed in Illumina-based
workflows for efficient whole-genome sequencing create technical problems that make
structural variation very difficult to detect systematically with existing software packages (6). In
order to more fully investigate the possibility of a structural rearrangement within the mapped
region, we suggest sequencing several OM-affected and unaffected Cairn terriers using a
sequencing method which produces long average read lengths, such as Pacific Bioscienceâs
SMRT-sequencing platform (Pacific Biosciences, Menlo Park, CA), which has an average read
length of 10,000 bp (7, 8). This method would also allow us to span the gaps in the canine
genome via de novo sequencing.
Finally, although we would obviously prefer to identify the underlying cause of OM, for
owners and breeders, simply finding a perfectly linked marker would be sufficient to begin the
process of selectively breeding away from the disease. Although the ~170k beadchip used in
this study was dense enough to allow us to map a 7.5 MB region, increasing our marker density
may allow us to narrow down the region even further, and allows us to test a large number of
markers across a large number of dogs for concordance with the disease simultaneously. A new
canine SNP array using ~1.2 million SNP markers is currently in development by Affymetrix
(Santa Clara, CA). Although the commercial release of the chip will have less markers than that,
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it should still have enough to allow us to look for mapped regions at a much higher resolution
than had previously been possibly with the ~170k beadchip.
In addition to these other proposed studies, it may also be beneficial for us to try
incorporating samples from another, closely related breed into some of the other analyses we
have already performed. The West Highland white terrier dog breed is very closely related to
the Cairn terrier, but does not develop ocular melanosis. Sequencing of several West Highland
white terriers (Westies) would allow us to generate a set of background variants which could be
subtracted from our Carin terrier variants to help us remove most of the variation unique to
these particular dog breeds. Additionally, melanocytes from unaffected Westie iris tissue may
provide better controls for cell culture studies, as they are very closely related to Cairn terriers,
but there are no doubts as to their OM status, unlike the older Cairn terriers we have previously
used. It should also be easier to procure these samples, as samples could be taken from dogs of
any age.

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