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8/01 cJCIRC/DateDuopGS—sz

HEREDITARY DEAFNESS AND MAST CELL TUMORS 1N DOGS: SEQUENCE
ANALYSIS OF CANDIDATE GENES FROM THE MELANOCYTE
DEVELOPMENT PATHWAY
By

Daniel Zemke

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology and Molecular Genetics

2003

ABSTRACT
HEREDITARY DEAFNESS AND MAST CELL TUMORS IN DOGS: SEQUENCE
ANALYSIS OF CANDIDATE GENES FROM THE MELANOCYTE
DEVELOPMENT PATHWAY
By

Daniel Zemke

The dog is the result of centuries of breeding by humans. Various genetic
diseases are prevalent in various breeds of dog as compared to mixed bred dogs. One
example is a hereditary sensorineural deafness that is often accompanied by abnormal
pigmentation, seen primarily in breeds with merle or white Spotting coat patterns.
Examination of deaf dogs has revealed an absence of melanocytes in the cochleas of
affected ears. Among other functions, melanocytes produce the pigment melanin, which
determines coat color. Similar conditions of hearing loss and pigmentation in other
species are linked to mutations in genes from the melanocyte development pathway.
Mutations in these genes also cause other diseases besides hearing loss and pigmentation.
The purpose of this study was to examine selected members of this set of genes at the
molecular level in the dog and evaluate their potential role in hereditary deafness and
selected other conditions. Candidate genes were selected based on similarity of the
phenotype seen in other species to that in the dog. The genes for endothelin receptor B
(EDNRB), microphthalmia—associated transcription factor (MIT F), KIT and its ligand
(KIT LG) were examined by sequencing the entire coding region in 1 normal and 7 deaf
dogs. No causative mutations were found during the course of the sequencing, however

other features such as alternative splicing events, single nucleotide polymorphisms

(SNPs), a microsatellite, and a pseudogene were identiﬁed. SNPs in KIT and EDNRB
were not found to be associated with deafness in the Jack Russell Terrier. It appears
unlikely that any of the candidate genes are responsible for deafness in dogs. In addition
to deafness, the KIT gene was examined for its involvement in mast cell tumors.
Examination of 88 tumors revealed duplications and deletions that appeared to be
associated with tumors of higher grade. Duplications similar to those in this study result
in constitutive activation of KIT. Other activating mutations of KIT have been shown to
result in tumor formation. Further study is needed to determine the exact nature of its
involvement, including the effect of drugs targeting such activation on the biological

behavior of these tumors.

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................... vi

LIST OF FIGURES ........................................................................................................ vii
CHAPTER 1

LITERATURE REVIEW ............................................................................................... 2

Hereditary deafness ............................................................................................ 3

EDNRB .............................................................................................................. 14

MITF .................................................................................................................. 18

KlT/KITLG ........................................................................................................ 21

References .......................................................................................................... 25
CHAPTER 2

CANDIDATE GENE SEQUENCIN G AND EVALUATION FOR DEAFNESS ........ 34

Introduction ........................................................................................................ 34

Materials and Methods ....................................................................................... 34

Results ................................................................................................................ 35

EDNRB .................................................................................................. 35

MITF ...................................................................................................... 48

KIT ......................................................................................................... 66

KITLG .................................................................................................... 69

Analysis of candidate genes’ roles in hereditary deafness .................... 79

Discussion. ......................................................................................................... 84

References .......................................................................................................... 89
CHAPTER 3

A SINGLE NUCLEOTIDE POLYMORPHISM AND A (GA)n
MICROSATELLITE IN INTRON 6 OF THE CANINE ENDOTHELIN

RECEPTOR B (EDNRB) GENE ................................................................................... 91
Source/description .............................................................................................. 91
Primer Sequences ............................................................................................... 91
PCR conditions ................................................................................................... 92
Polymorphism .................................................................................................... 92
Chromosomal Location ...................................................................................... 93
Mendelian Inheritance ........................................................................................ 93
References .......................................................................................................... 94

CHAPTER 4

PREFACE TO EVALUATION OF KIT MUTATIONS TN MAST CELL

TUMORS ....................................................................................................................... 96

CHAPTER 5
CHARACTERIZATION OF AN UNDIFFERENTIATED MALIGNANCY AS A
MAST CELL TUMOR USING MUTATION ANALYSIS IN THE PROTO-

ONCOGENE c-KIT ........................................................................................................ 99
Abstract .............................................................................................................. 99
Introduction, results, and discussion .................................................................. 100
Sources and Manufacturers ................................................................................ 109
References .......................................................................................................... 109

CHAPTER 6

MUTATIONS IN THE IUXTAMEMBRANE DOMAIN OF c-KIT ARE

ASSOCIATED WITH HIGHER GRADE MAST CELL TUMORS IN DOGS ........... 113
Abstract .............................................................................................................. 113
Introduction ........................................................................................................ 114
Materials and Methods ....................................................................................... 116
Results ................................................................................................................ 121
Discussion .......................................................................................................... 125
References .......................................................................................................... 129

CHAPTER 7

SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS ................................. 133

CHAPTER 8

MATERIALS AND METHODS ................................................................................... 140

APPENDIX
APPENDD( A
DCT AND EDNRB MAP TO DOGMAP LINKAGE GROUP L07 ................. 149

Source/description .................................................................................. 149
Primer Sequences ................................................................................... 149
Chromosomal Location .......................................................................... 149
Comment. ............................................................................................... 150
References .............................................................................................. 1 50
REFERENCES ............................................................................................................... 153

LIST OF TABLES

PRIMERS DESIGNED WITHIN EDNRB EXONS ..................................................... 36
PCR FRAGMENTS USED TO OBTAIN THE EDNRB CODING REGION ............. 36
PRIMERS DESIGNED WITHIN EDNRB INTRON 6 ................................................ 47
PRIMERS DESIGNED WITHIN MIT F EXONS ......................................................... 49
PCR FRAGMENTS USED TO OBTAIN THE MITF—M CODING REGION ............ 49
PRIMERS DESIGNED WITHIN MIT F INTRONS ..................................................... 55
PCR F RAGMENTS USED TO OBTAIN THE MIT F-M EXONS ............................... 64
PRIMERS DESIGNED FOR THE KIT GENE ............................................................. 68
PCR FRAGMENTS USED TO OBTAIN THE KIT CODING REGION ..................... 69
PRIMERS DESIGNED FOR THE KIT LG GENE ........................................................ 79
PCR FRAGMENTS USED TO OBTAIN THE KIT LG CODING REGION ............... 79
GENOTYPE RESULTS FOR A SNP IN INTRON 6 OF EDNRB .............................. 84
GENOTYPE RESULTS FOR A SNP IN INTRON 10 OF KIT .................................... 84

GRADE DISTRIBUTION OF MAST CELL TUMORS IN VARIOUS DOG
BREEDS ......................................................................................................................... 122

GRADE DISTRIBUTION OF DUPLICATIONS AND DELETIONS IN c-KIT
AMONG CANINE MAST CELL TUMORS ................................................................ 124

DISTRIBUTION OF DUPLICATIONS AND DELETIONS IN c-KIT AMONG
DIFFERENT DOG BREEDS ........................................................................................ 125

LIST OF FIGURES

BAER TEST RESULTS FROM A CATHOULA LEOPARD DOG WITH

UNILATERAL DEAFNESS ......................................................................................... 6
AUDITORY STRUCTURES OF THE MIDDLE AND INNER EAR ......................... 8
GENE PATHWAY OF MELANOCYTE DEVELOPMENT ....................................... 14
STRUCTURES OF THE mRNAs OF THE CANDIDATE GENES ............................ 15
STRUCTURE OF THE EDNRB PROTEIN ................................................................. 16
STRUCTURE OF THE MITF PROTEIN ..................................................................... 20
STRUCTURE OF THE KIT PROTEIN ........................................................................ 22
STRUCTURE OF THE KITLG PROTEIN ................................................................... 23
ALIGNMENT OF THE cDNA SEQUENCES OF DOG AND HUMAN EDNRB

WITH MOUSE Ednrb ................................................................................................... 39
ALTERNATIVE SEQUENCES IN CANINE EDNRB ................................................ 43
SEQUENCES FROM CANINE EDNRB INTRONS ................................................... 45

ALIGNMENT OF DOG AND HUMAN MIT F -M CODING REGION
SEQUENCES WITH MOUSE MiIf-m AND THE CANINE MIT F PSEUDOGENE

(PSG) .............................................................................................................................. 51
SEQUENCES FROM CANINE MITF INTRONS ....................................................... 57
ALIGNMENT OF H1 AND Blb SEQUENCES FROM DoG AND HUMAN MITF
WITH MOUSE Mitf. ...................................................................................................... 62
ALIGNMENT OF THE cDNA SEQUENCES OF M AND HUMAN KIT WITH
MOUSE Kit .................................................................................................................... 71
SEQUENCES OF CANINE KIT INTRONS 10 AND 11 ............................................. 78

ALIGNMENT OF THE cDNA SEQUENCES OF DOG AND HUMAN KIT LG
WITH MOUSE Kit] ....................................................................................................... 81

vii

TWO PERCENT AGAROSE GEL SHOWING SNP (LANES 2-7) AND
MICROSATELLITE (LANES 8-13) RESULTS FOR A FAMILY OF SIX DOGS... 93

MACROSCOPIC LESIONS OBSERVED IN A 6.5-YEAR-OLD FEMALE
BOXER .......................................................................................................................... 101

HEPATIC NODULE FROM A 6.5-YEAR-OLD FEMALE BOXER. ......................... 102

SEPARATION OF AMPLIFIED BANDS FROM THE JUXTAMEMBRANE
DOMAIN OF c-KIT IN A FEMALE BOXER .............................................................. 106

GENOMIC SEQUENCE OF THE JUXTAMEMBRANE DOMAIN OF CANINE c-
KIT AND FLANKING REGIONS (AMINO ACIDS 518-606) ................................... 120

AGAROSE GEL SHOWING PRODUCTS OBTAINED BY AMPLIFICATION OF
THE JUXTAMEMBRANE REGION OF c-KIT FROM DIFFERENT SOURCES ..... 122

viii

CHAPTER 1

LITERATURE REVIEW

Humans have been responsible for the genetic manipulation of a number of plant
and animal species. Individuals that exhibit desirable phenotypic traits are preferentially
used for breeding, and breeding from individuals with undesirable traits is avoided. The
domestic dog is the result of thousands of years of such selective breeding. Although all

dogs share a single common ancestor, the American Kennel Club recognizes 145

different breeds, and it has been suggested that over 1,000 breeds exist worldwidez’53
Many of these breeds were speciﬁcally bred to perform certain tasks, and the number of
breeds reﬂects the varied roles that dogs play in human societies. To ensure that
particularly advantageous traits become permanently established, breeders generally
resort to some measure of inbreeding. Dogs with champion status are often bred to
multiple partners, including their own offspring and siblings. Formal breed clubs
generally discourage mating between dogs of different breeds. The effect of inbrwding
is to increase the frequency of certain alleles at a trait locus and increase the number of
homozygotes for a particular allele. Unfortunately, this means that the frequency of
normally rare recessive traits, some of which may be disadvantageous, can be quite high
within a particular breed.

In many dog breeds, there is often an increased risk of inheriting one or more
genetic diseases, due to the decreased gene pool that results from inbreeding. For

example, the Labrador Retriever is prone to progressive retinal atrophy and narcolepsy,

and the German Shepherd is prone to spinal muscle atrophy and hemophilia. 13 Inherited
diseases in dogs are known for nearly every organ system in the body. These diseases
may be fatal, disabling, or merely uncomfortable to an affected dog. The cost of

treatment in some cases can be so high that the dog may be euthanized. Unfortunately,

the genetics of these diseases are often not well understood, and those recognized as
recessive traits are hard to remove from the population because breeders do not have a
means to identify carriers. Some breeders ignore or deny the fact that there are problems
within their breed. As a result, many continue the same breeding practices that created
the problem in the ﬁrst place. Molecular biology, however, can be used to speciﬁcally
identify the genetic basis of a trait of interest. This would provide the ability to screen for
carriers and lower the frequency of undesirable traits by removing carriers from the
breeding population. If a mutation leading to the production of an abnormal protein is
shown to cause a particular disease, this may yield the opportunity to develop methods of
prevention or treatment. For example, a mutant protein that is constitutively active could
be treated with drugs that inhibit it. If a disease if found to have to the same molecular
basis in both hmnans and dogs, development of a treatment for dogs may also result in a
treatment for humans. The study of a gene at the molecular level can also increase the
understanding of its role in complex mammalian systems and how it interacts with other

genes.

Hereditary deafness
The main focus of this study is hereditary hearing loss, a genetic disease prevalent

in a number of dog breeds. There are three main types of hearing loss, categorized by the

location of the physiological defect that causes it.24 Conductive hearing loss stems ﬁ'om
defects within the outer and middle ear, including blockage of the ear canal and defects
that prevent the conduction of sound from the eardrum to the inner ear. Central hearing

loss is due to defects in the brain and auditory nerves that prevent the proper reception or

processing of auditory stimuli. Sensorineural hearing loss is the result of defects within
the inner ear that prevent the detection of sound. Sensorineural hearing loss is ﬁrrther
classiﬁed into two subcategories based on which tissues within the inner ear are affected.
Neuroepithelial deafness is the result of defects within the sensory hair cells and their
supporting tissues alone. Cochleosaccular deafness is the result of degradation of
multiple tissues within the cochlea and saccules, oﬁen including the hair cells as well.

Other criteria used to evaluate hearing loss are its heritability, age of onset,

progressiveness, and whether or not it is syndromic.24 Hereditary hearing loss can be
passed ﬁ'om parent to offspring and is caused by mutations within the DNA of the
individual. Acquired hearing loss is caused by outside factors and cannot be inherited.
The normal hearing loss that occurs at old age is considered to be acquired hearing loss.
Other non-hereditary causes of hearing loss include physical trauma and exposure to
various chemicals. Congenital hearing loss is deafness that is apparent at birth or soon
after, whereas other forms of hearing loss do not appear until later in life. In progressive
hearing loss, the severity of the loss increases with time. Non-progressive hearing loss
does not change in severity. Finally hearing loss can be syndromic on non-syndromic. In
syndromic hearing loss, other systems of the body are affected in addition to the auditory
system. Non-syndromic hearing loss refers to cases where deaﬁress is the only symptom.

Acquired deafness cannot be passed from one individual to another, and therefore
does not present any threat to the population as a whole. Because hereditary deafness can
be propagated through a lineage, it is important to discover the underlying cause and

screen for it within breeding stock. In dogs, congenital deafness has been reported in at

least 69 breeds.74 However, inheritance has not been proven in some of these breeds,

therefore the actual number with hereditary deafness is unknown. Hereditary deafness in
different breeds may also have different etiologies; therefore a broad study of hereditary
deafness in all dogs is not feasible. The focus of this study was on hereditary deafness in

breeds that show inheritance of the merle (for example Collies and Australian Cattle

Dogs) and spotting (for example Boxers and Dalmatians) coat color loci.73’80 These
breeds share a common pattern of deafness that is hereditary, cochleosaccular, congenital,
non-progressive, and syndromic. Hearing loss, when present, can occur in either one ear
(unilateral) or both ears (bilateral).

The ﬁequency of deafness is different in each breed and may even vary between
different populations within a breed. Hereditary deafness has been best described in the

Dalmatian. In one estimate, 8% of Dalmatians have bilateral deafness and 22% have

unilateral deafness.74 The mode of inheritance in Dalmatians is unclear because

different studies have produced conﬂicting results. Some studies have suggested that

deafness is due to a single autosomal recessive gene with incomplete penetrance.32’56

Another study suggests that multiple loci are involved and that unilateral and bilateral
deafness may have different genetic causes.23 Some studies have found that deafness is

signiﬁcantly more prevalent in females than in males.38 The mode of inheritance in

other breeds has not been extensively reported, however one study of the Australian

Cattle Dog suggests that 2 recessive genes may be involved.73

The canine ear continues to develop after birth, and the ear canal does not Open

until about 2 weeks of age.73 Testing is usually not performed until at least 6 weeks of

age, when mature hearing patterns have been established and the processes leading to the

form of deafness under study are complete. The preferred way to assess hearing loss is
the brainstem auditory evoked response (BAER) test, also known as the brainstem
auditory evoked potential (BAEP) or auditory brainstem response (ABR).73 An auditory
stimulus is sent to the car by ear-pieces in the form of a series of clicks. Electrodes
positioned on the head receive the electrical signal produced in response to this stimulus,
and the signals from multiple stimulatory clicks are recorded and processed by a
computer. Sample results ﬂour a BAER test are shown in Figure 1. In a normal auditory
response, 4 or 5 distinct peaks are produced corresponding to passage of the signal

through different parts of the nervous system. The ﬁrst peak is produced in the cochlea

3| .ZIIIV ‘1 Al/CZ

32 .2uv _ Al/CZ

33 .ZIuV A271:
34 .2Iuv 42111

Figure 1. BAER test results ﬁom a Catahoula Leopard Dog with unilateral

 

deafness. Two recordings were made from each ear. The upper two lines are from the
right ear and show a normal auditory response. The lower lines show the lack of a

response for the deaf left ear. The position of peaks I and V are shown on the graph.

and the adjacent end of the cochlear nerve, and the remaining peaks are produced in
different parts of the brain. If the ﬁrst peak is present but any of the following peaks are
absent, central hearing loss is indicated. Absence of the ﬁrst peak indicates either
sensorineural or conductive hearing loss. A physical examination of the car can be used
to ensure that the stimulus was properly transmitted to the cochlea and rule out
conductive hearing loss. Alternatively, a stimulus can be transmitted through bone
directly to the cochlea.

A diagram of the inner ear is shown in Figure 2. The cochlea consists of three
ﬂuid ﬁlled chambers. The scala vestibuli and scala tympani contain a ﬂuid known as
perilymph, and the cochlear duct contains a ﬂuid known as endolymph. In a normal ear,
sound vibrations entering the ear impact upon the tympanic membrane and are

transmitted to the scala vestibuli by the action of the bones of the middle ear on the oval

vvindow.73 The resulting pressure waves in the perilymph of the scala vestibuli travel the
length of the cochlea, return via the scala tympani, and exit at the round window. This
movement of pressure waves causes a shearing force on the sensory hair cells within the
organ of Corti of the cochlear duct. The stereocilia of these hair cells, which are
embedded in the tectorial membrane, are deﬂected. This open ion channels within the
hair cells that allows potassium ions from the endolymph to enter. The inﬂux of
potassium ions depolarizes the hair cells, causing them to release neurotransmitters that
stimulate neighboring nerve endings. The electrical impulses generated travel along the
cochlear nerve to the brain and are interpreted as sound. High frequency sounds
maximally stimulate the basal turns of the cochlea and low frequency sounds stimulate

the apical turns. This allows the brain to distinguish different frequencies of sound.

 

 

Figure 2. Auditory structures of the middle and inner car. (A) Overall view of
the middle and inner ear structures. (B) Cross section view of the cochlea. Diagrams are

based on structures published by Evans, 1993, but have been modiﬁed.

The pathology in deaf dogs has been found to be similar in the Dalmatian, Collie,

Border Collie, Australian Shepherd, and Great Dane.33940’48 These represent breeds
with inheritance at both the spotting and merle loci. The earliest detectable defect in

affected ears of deaf dogs is a degeneration of the stria vascularis in the cochlea of the

inner ear (Figure 2).41 This degeneration can be seen as early as one day after birth, and

is therefore considered to be a congenital defect. The stria vascularis is a layer of highly

vascularized tissue that secretes potassium ions into the endolymph.73 As previously
mentioned, it is the inﬂux of potassium ions into the hair cells that depolarizes them and

leads to nerve stimulation. In affected ears, the stria vascularis is reduced in thickness

with little or no blood supply.5’48 Atrophy of the stria vascularis is followed by collapse
of the vestibular membrane, hair cell degeneration, and collapse of the saccule. In later
stages, cell loss has also been noted in the spiral ganglion that is a part of the auditory

nerve. Further examination reveals that pigmentation in the stria vascularis due to

melanocytes is diminished.48

Melanocytes are found within the stria vascularis as part of a layer of intermediate
cells that lies between the marginal epithelial cells that line the inner surface of the
cochlea and the basal cells that supply them with nutrients. In mice, it has been found

that marginal cells normally form extensive interdigitations with both the intermediate

and basal cells.70 When melanocytes in the inner ear are absent, it has been found that
the extent of interdigitations and number of blood vessels supplying the marginal cells
are reduced. The endocochlear potential in these cases is often zero. The endocochlear
potential is an ionic gradient that is a result of the secretion of potassium ions by the stria
vascularis into the endolymph. This gradient is necessary for the inﬂux of those ions into
the hair cells. It is unclear whether melanocytes directly secrete potassium ions or are
merely supporting the cells that do.

Another function of melanocytes is the production of the pigment melanin, which
is partially responsible for determining the color of the skin, hair, and eyes. Deafness in

Dalmatians has been shown to be positively associated with blue eye color and negatively

associated with the presence of colored patches.23 In many breeds, deaf dogs generally

have a larger proportion of white in the coat than normal.72 In some cases the dog may
be completely white. White coat color can be produced by the merle and spotting loci,

both of which are associated with deafness. White coat color can also be produced by the

albino locus, however this locus is not associated with deafness. The merle phenotype is

dominantly inherited, and produces a dilution of color in the heterozygous state.80 In
homozygotes, extreme dilution produces a coat that is nearly completely white. Many
breeds that have inherited merle also inherited alleles at the spotting locus; therefore
white spots in these breeds may be due to either locus. There is some evidence of

reversion of the merle phenotype, and it has been suggested that merle may be due to a
transposable element.66 The tweed and harlequin phenotypes are modiﬁcations of the

merle phenotype, and harlequin in particular produces white spots.67968 The Irish

spotting, piebald, and extreme piebald phenotypes result from the inheritance of recessive

alleles at the spotting locus.80 Since the inheritance of deafness is generally considered
to be recessive, the spotting locus would therefore appear to be a better candidate than
merle.

In one study, melanocytes were undetectable in hair bulbs taken from white areas

of a Dalmatian and a nonmerle Shetland Sheepdog, both of which have inherited alleles

at the spotting locus.63 Melanocytes were found in hair bulbs from white areas ofa
Shetland Sheepdog with the harlequin modiﬁcation of merle, but they were immature.

Homozygous merle dogs were not examined. In contrast, the white hair produced in

albinism is the result of mature melanocytes that have no tyrosinase activity.34 The

10

absence of melanocytes in the white areas of dog breeds with Spotting alleles, and a
similar absence of melanocytes in the inner ears of deaf dogs ﬁom these breeds, suggests
that a common cause might be responsible for both. It is possible that the immature
melanocytes seen in the harlequin merle are less capable of surviving in the inner car. It
is also possible that immature melanocytes are exclusive to the harlequin modiﬁer, and
that homozygous merles are white due to an absence of melanocytes. The merle locus
can’t be ruled out as being associated with deafness, therefore, without further studies.

In the Dalmatian, it has been suggested that white coat color, blue eyes, and

deaﬁress can all be explained by the action of the spotting locus.16 The effect of this
locus might be a decreased viability of melanocyte precursors. If precursors in the skin
or iris die, white hair color or blue eyes result. If precursors in the ear die, deaﬁress
results. In places where the precursors manage to survive, normal coloration or hearing
results. The randomness in the survivability of these precursors might explain the
difﬁculty in determining the mode of inheritance of deaﬁress. It does not explain,

however, why deaf parents are more likely to produce deaf offspring than normal

parents.23 The situation might be the same for the merle locus. This discrepancy might
be explained by the inheritance of either modiﬁer genes or mutations within the spotting
or merle genes themselves that affect precursor survival and increase the risk of deaﬁress.
The genes for spotting, merle, or their modiﬁers are not known in the dog. Therefore,
similar phenotypes of abnormal pigmentation and deafness in other species must be used
as models to determine possible candidates for study in the dog.

The dominant spotting (W) phenotype in mice is a coat color variant

characterized by reduced or absent pi grnentation. Affected mice have a stria vascularis

ll

that is thinner than normal, which is accompanied by a lack of intermediate cells

including melanocytes.21’64 Collapse of the cochlear duct and saccule follow, along
with degeneration of the hair cells and nerve ﬁbers. The microphthalmia (mi) locus in

the mouse has many different phenotypic alleles, many of which exhibit dilute or white

coat color and smaller than normal or absent eyes.71 Some of these alleles, such as
microphthalmia-white (Mi-wh), also have inner ear defects that lead to cochleosaccular
deafness. This is interesting with respect to dog breeds that show inheritance of the merle

trait. In the Great Dane and Collie, dogs with a predominantly white coat have been seen

that exhibit both microphthalmia and deafness.33
In the human, Waardenburg syndrome is one potential model for deafness in

dogs. Common symptoms include cochleosaccular deafness, patchy white skin, a white

forelock of hair, and blue eyes or heterochromia irides.7 Heterochrorrria irides refers to a
condition in which one eye is normally pigmented and the other eye is blue, or in which
the iris of a single eye contains both normal and blue colored segments. There are 4
types of Waardenburg syndrome currently described. Waardenburg syndrome type I
(W 81) exhibits the common symptoms plus dystopia canthorum, a facial phenotype in
which the eyes are spaced farther apart than normal. Waardenburg syndrome type 11
(W S2) does not exhibit dystopia canthorum. Waardenburg syndrome type III (W S3) is
similar to type I, but with deformities of the limbs. Waardenburg syndrome type IV
(WS4), also known as Shah-Waardenburg syndrome, consists of the common symptoms
plus aganglionic megacolon. W81, W82, and WS3 are dominantly inherited, whereas
WS4 is recessively inherited. Since aganglionic megacolon and physical deformities

have not been described in deaf dogs, WS2 appears to be the best model for deafness in

12

dogs. Because of its variegated nature, it has been suggested that the merle locus is a

better model of Waardenburg syndrome than spotting.63 Merle would also be a better
model for WSI, WS2, and W83 because of a similar mode of inheritance. In addition to

Waardenburg syndrome, piebaldism in humans also results in a white forelock of hair. In

some cases, human piebaldism has been known to exhibit hearing 1oss.59 Therefore, this
condition should also be considered as a model for what is seen in dogs.

Many of the conditions mentioned above have been linked to mutations within
genes that are a part of the melanocyte development and melanin synthesis pathway.
This pathway is shown in Figure 3. The genes PAX3, endothelin receptor B (EDNRB)
and its ligand endothelin 3 (EDN3), microphthalmia-associated transcription factor
(MIT F), KIT and its ligand (KIT LG), and SOX10 are all important for the survival,
proliferation, migration, and differentiation of melanocyte precursors. Multiple names
and symbols are used for these genes between and even within species, depending on the
author. In this dissertation, the official human and mouse symbols will be used, except

Where different in the previously published manuscripts. Waardenburg syndrome types I

and III are caused by PAX3 mutations.54 Waardenburg syndrome type II is caused by
mutations in human MIT F , and the mouse microphthalmia phenotype is the result of
defects in the Mitf gene. Waardenburg syndrome type IV is the result of mutations in the

EDNRB, EDN3, and SOXI 0 genes. Human piebaldism is caused by KIT mutations, and

the mouse W phenotype is caused by defects in Kit.76 Due to the number of genes
involved, not all could be examined. The EDNRB, MIT F , KIT, and KIT LG genes were
chosen as the best candidates for the investigation of deafness in the dog. Diagrams of

their gene structures are shown in Figure 4.

l3

Mdnh synthesis

/T\

 

TRPl l Tyrant-no I I DCT I
I \
1 \
’ \
’ \

x
I
I T ‘

 

 

 

 

 

 

 

 

Figure 3. Gene pathway of melanocyte development. The major genes involved
are shown in boxes with solid arrows indicating known interactions and dashed arrows
indicating proposed interactions. The development pathway of melanocytes is shown at

the bottom.

EDNRB
Endothelin was ﬁrst discovered as a vasoactive peptide of 21 amino acids secreted

by endothelial cells that is similar in structure to some neurotoxins that act on membrane

ion channels.81 Three different endothelins are currently known: endothelin 1 (ETl or
EDNl), endothelin 2 (ET2 or EDN2), and endothelin 3 (ET3 or EDN3). Two receptors

named endothelin receptor A (EDNRA or ETA) and endothelin receptor B (EDNRB or

14

(I...

w

MITF-A

MITF-C

MITF-B

DIRT-D

MITF-M

exons. The coding region of each gene is shown in white, untranslated regions in black.

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Figure 4. Structures of the mRNAs of the candidate genes. Boxes represent

21

Exon designations are shown below each box, and the sizes of the coding sequences in

base pairs are shown above. Sizes of untranslated regions are given where known. Sizes

are for the human genes, except where noted otherwise. MIT F isoforrns are shown with

the optional 18 bp in exon 6 present.

15

ETB) have been identiﬁed that bind to endothelins. A third receptor, endothelin receptor
C, has been identiﬁed in Xenopus.43 EDNRA binds EDNI and EDN2 strongly, but has a

much weaker afﬁnity for EDN3.52 EDNRB, on the other hand, binds all three

endothelins with equal afﬁnity. Subtypes of EDNRB have been identiﬁed in some

Species that differ in their sensitivity to various competing agents. ”’46

The EDNRB protein belongs to the G-protein-coupled receptor family and
consists of 7 helical transmembrane domains (Figure 5).52 It is expressed in many

tissues, including the brain, kidney, thyroid, liver, uterus, and endothelial cells.61 The

 

 

C

Figure 5. Structure of the EDNRB protein. The transmembrane helices are

designated H1 — H7.

16

EDNRB gene has been mapped to Chromosome 13q22 in the human, and Ednrb is on
Chromosome 14 in the mouse”,59 In the human, the EDNRB gene has been reported to

contain 7 exons.6 In the rat, however, alternative splicing of a non-coding exon has been

seen in the 5’ untranslated region and has led to a gene structure consisting of 8

exons.”,1 8 The ﬁrst two exons in the rat structure correspond to exon 1 in the human.
For the purpose of this study, the structure in the rat consisting of 8 exons was used. The
coding region in the human is 1329 bp in length and codes for a protein of 442 amino
acids.61

Activation of EDNRB by EDN3 in melanocyte precursors increases their rate of

cell proliferation, and temporarily prevents them from differentiating.44 EDNRB

inhibitors have been shown to slow the rate of tumor growth in malignant melanoma
cases.45 EDNRB activation is also responsible for the migration of certain precursor

cells from the neural crest during early embryonic development.42 In particular, the
precursors of enteric ganglion cells and melanocytes fail to migrate to distal portions of
the body in the absence of proper signaling. A lack of ganglion cells within the distal
portions of the gastrointestinal tract leads to a condition known as aganglionic
megacolon. In this condition, the large intestine is unable to contract due to a lack of
innervation, resulting in intestinal blockage that is often fatal at an early age.

Aganglionic megacolon has been found to be caused by mutations within EDNRB or

within the receptor tyrosine kinase RE T.3 ’29 In humans, aganglionic megacolon is

known as Hirschsprung’s Disease.

17

The failure of melanocyte precursors to properly migrate results in abnormal
pigmentation, due to a lack of melanocytes within the affected area. In many cases, both
innervation of the colon and pigmentation are affected. The piebald-lethal phenotype in

the mouse and the spotting lethal phenotype in the rat are both due to deletions in the
Ednrb gene. 1 7’39 In the horse, EDNRB mutations have been associated with lethal white

foal syndrome (LWFS).82 These foals are the result of matings between horses with the
overo coat color phenotype. They have a completely white coat and usually die a few

days after birth due to aganglionic megacolon. Mutations in either EDN3 or EDNRB

have been found in many Shah-Waardenburg patients.8’37

MITF
The microphthalmia-associated transcription factor (MIT F) gene has been mapped
human Chromosome 3pl4.1-p12.3, and Mitf is found on Chromosome 6 in the

mouse.36’75 The entire gene produces two major transcripts of about 5.5 kb and 5.7

kb.36 A total of 8 MIT F isoforrns are currently known and are produced by alternative
splicing of a single MIT F gene. The coding regions of these isoforms contain 9 exons
and range ﬁ'om 1.3 to 1.6 kb in size (Figure 4). All of the isoforrns share exons 2 through
9 in common and differ only in their exon 1 sequences. The exon 1 in each isoform
consists of a unique 5’ end and, except for MITF-M, the common sequence segment Blb.

An alternative splicing site also exists at the 5’ end of exon 6, and results in the optional

removal of the initial 18 base pairs of that exon.36 The effect of this splicing on MITF

function is not known.

18

The different isoforms of MIT F all have different expression patterns, which are

most likely mediated by their different 5’ ends. MIT F -M is expressed exclusively within
melanocytes.76 The MIT F -M promoter contains binding sites for the PAX3 and SOX10
transcription factors, which are also involved in the regulation of pigmentation. 10 MITF-
A is widely expressed but is enriched in the retinal pigment epithelium.1 MH‘ F —H is also
widely expressed but is enriched in the heart. MIT F -C is expressed in a number of
tissues, but not melanocytes.27 MIT F -D is primarily expressed in the retinal pigment
epithelium. It is also expressed in macrophages and osteoclasts, but not in melanocytes

or natural killer cells.77 MIT F-E and MIT F -MC are expressed in mast cells.58’78 The

ﬁnal isoform, MIT F -B, has been found in retinal pigment epithelium, melanoma, and

cervical cell cancer cell lines by reverse transcription PCR (RT-PCR) only.79
The MITF protein consists of a basic-helix-loop-helix-leucine zipper (bHLH-Zip)

structure (Figure 6) and is closely related to TFE3 and TFEB, transcription factors that

bind the E box in the irnmunoglobulin heavy chain enhancer.36 MITF is involved in the

differentiation of many cell types, including neural crest-derived melanocytes, mast cells,

osteoclasts, and optic cup-derived pigment epithelium.7l MITF activates the tyrosinase
and tyrosinase related protein 1 (TRPI) genes of the melanin synthesis pathway (See

Figure 3) by binding to an 11 base consensus sequence AGTCATGTGCT, known as the

M box, that is present in their promoters.84 The M box contains within it the consensus
sequence CANNT G, known as the E-box, which is recognized by all bHLH-Zip proteins.

The DOPAchrome tautomerase (DCT or TRPZ) gene, which is also involved in melanin

synthesis, contains an M box in its promoter as well. 1 5 It is not conclusive, however,

19

 

 

 

 

Figure 6. Structure of the MITF protein. Two MITF molecules are Shown,

depicting a functional homodimer.

whether or not MITF activates DCT. MITF also increases the expression of KIT, which

in turn activates the microtubule-associated protein (MAP) and RSK-l kinases.35’76

These kinases phosphorylate MITF at Ser73 and Ser409, respectively. The
phosphorylated MITF recruits the cofactor p300/CBP and is ubiquinated, resulting in a
short burst of activity followed by degradation.62

A total of 21 Mirf mutant alleles are currently known in the mouse.55 Phenotypic
effects may include small or absent eyes, loss of pigmentation or abnormal pigmentation
in various parts of the body, deafness, mast cell deﬁciency, and loss of secondary bone

resorption. In the human, MITF mutations have been linked to Tietz albinism-deafness

20

syndrome and Waardenburg syndrome type 11, both of which are characterized by

deafness and abnormal pigmentation of the hair, skin, or eyes.76 In the dog, the MIT F
gene is a candidate for both hereditary deaﬁiess associated with abnormal pigmentation

and microphthalmia.

KIT/KITLG

The proto-oncogene KIT (sometimes referred to as c-KIT) is homologous to the v-

kit gene found in the Hardy-Zuckerman 4 feline sarcoma virus.9 KIT has been mapped to

Chromosome 4q1 1-q12 in the human, and Kit is located on Chromosome 5 in the
mousezo’83 The gene consists of 20 exons and has a coding region of 2931 bp in the

human, encoding 976 amino acids.83 The KIT gene is expressed primarily in the brain,
heart, skeletal muscle, kidney, and lungs. The gene product is a membrane bound protein
that is a member of the type III receptor tyrosine kinase family, which also includes the

receptors for the colony stimulating factor (CSF- 1) and platelet-derived growth factor
(PDGF).60 The extracellular portion of the protein consists of 5 immunoglobulin-like

folds (Figure 7).14 The 3 folds closest to the amino terminus form the ligand-binding
domain, and the fourth fold is believed to be necessary for dimerization. The intracellular
portion of the protein consists of a juxtamembrane domain and a tyrosine kinase domain.
The ATP binding and phosphotransferase regions of the tyrosine kinase domain are
separated by a kinase insert sequence.

The kit ligand (KITLG, Kitl) is alternatively known as the mast cell growth factor

(MGF), Steel factor (SF or SLF), and stem cell factor (SCF). The human KIT LG gene is

21

 

 

 

Figure 7. Structure of the KIT protein. The major structural domains are labeled.
Immunoglobulin-like folds are indicated as crescent shapes, the transmembrane helix by

a cylinder, and the kinase domain by cones.

located on Chromosome 12q22-12q24, and the mouse Kill gene is located on

Chromosome 10.4919 The KIT LG gene in humans consists of 8 exons comprising a total
coding region of 822 base pairs and encoding 273 amino acids.51 Exon 6 is alternatively
spliced and contains a proteolytic cleavage site aﬁer Ala165 in the mouse. 14 When exon

6 is present, cleavage at this site removes the transmembrane domain and produces the
soluble form of the protein. When exon 6 is absent, the cleavage Site is also absent and
the membrane-bound form of KITL is produced. The soluble form of KITL in the mouse

can be produced by cleavage at either the homologous site in exon 6 or at an alternative

22

cleavage site in exon 7. Endothelial cells and ﬁbroblasts constitutively express the
KITLG protein. 14 KITLG is also expressed in the brain, bone marrow hematopoietic
stem cells and stromal cells, skin keratinocytes, gut epithelial cells, the thymus, and the
testis. The ratio of soluble to membrane-bound KITLG varies, but the soluble form is the

predominant form in most tissues. The structure of the KITLG protein consists of 2 B-

pleated sheets and a bundle of 4 tit-helices (Figure 8). 14 A total of 4 cysteine residues are
present within the KITLG protein and participate in 2 intramolecular disulﬁde bonds.
The cytoplasmic domain, parts of the ﬁrst, third, and fourth helices, and both disulﬁde
bonds have been determined to be essential for normal function, particularly amino acids

1-141. The active form of KITLG is a homodimer that is heavily glycosylated.

 

£9

9

o it

 

C

Figure 8. Structure of the KITLG protein. Shown is a single monomer of
KITLG; the functional form is a homodimer. The cylinders labeled H1 - H4 depict

helices. Arrows depict beta pleated sheets.

23

Normal signaling by the KIT receptor is the result of KITLG binding. Upon
binding of KITLG, KIT dimerizes, which induces its autophosphorylation activity. 14
Activation of KIT results in the initiation of multiple signal cascades. The

phosphorylated KIT phosphorylates and activates JAKs, which are non-transmembrane

tyrosine kinases.11 The JAKs further activate the signal transducers and activators of
transcription (STAT) proteins STATla, STATSA, and STATSB, causing them to
dimerize, translocate to the nucleus, and bind to speciﬁc promoter response elements.
Activated KIT also results in the activation of MITF as described above. Signalling by
KIT/KITLG is involved in the development and maturation of a number of cell types,
including hematopoietic stem cells, mast cells, germ cells, and melanocytes.

Mutations that decrease the activity of KIT may result in anemia, a reduced

number of mast cells, decreased fertility, or a lack of pigmentation.14 Lack of
pigmentation is usually displayed as a patch of white hair or skin, and is usually the result
of a complete lack of melanocytes within the affected area. Human piebaldism, the white
spotting phenotype in mice, the white belt and dominant white phenotypes in pigs, and
roan coat color in horses are all associated with mutant “12830313950 Mutations

that increase KIT activity are associated with mast cell leukemia and mastocytosis with

associated hematologic disorders.26’57 In the human mast cell leukemia cell line, these

mutations were found to result in constitutive phosphorylation of KIT in the absence of
KITLG.26 Similar results have also been obtained for the mutation found in a canine

mastocytoma cell line.“ The association between KIT and mast cell tumors will be

discussed in later chapters. KIT LG mutations have been linked to the roan coat color

24

phenotype in certain breeds of cattle.65 Kit] mutations are also the basis for the Steel
phenotype in the mouse, which involves abnormal pigmentation.25 Other mutations may

cause anemia, a lack of mast cells, and decreased fertility.14

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30

67.

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Sponenberg DP. Inheritance of the harlequin color in Great Dane dogs. J Hered
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Sponenberg DP, Larnoreux ML. Inheritance of tweed, a modiﬁcation of merle, in
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31

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32

CHAPTER 2

CANDIDATE GENE SEQUENCIN G AND EVALUATION FOR DEAFNESS

33

Introduction

The purpose of this study was to determine whether mutations in the candidate
genes EDNRB, MIT F , KIT, and KIT LG are responsible for hereditary deafness in dogs.
In order to later identify sequence mutations, it was ﬁrst necessary to obtain the normal
sequences of these genes ﬁom a dog without deafness. The coding regions of the genes
were chosen for study because mutations within them could potentially affect the
structure or function of the proteins that they encode. The polymerase chain reaction
(PCR) was used to amplify fragments of the coding regions that were then isolated and
sequenced. Once the coding region of a gene had been sequenced in a normal dog, it was
also sequenced in a number of deaf dogs and examined for sequence differences. In
addition to the coding regions, the promoter regions and intronic sequences such as splice
sites were also potential sites of mutations causing deaﬁiess. PCR was used where
possible to obtain introns and untranslated regions where possible and sequence them.
Any sequence differences seen were reexamined to determine whether they represented
experimental error, neutral polymorphisms, or mutations. Polymorphisms were used as
markers to facilitate mapping of the genes and to determine if there was an association

between a particular candidate gene and deafness.

Materials and Methods

Materials and methods for this portion of the study are presented in Chapter 8.

34

Results
EDNRB

Prior sequence for the canine EDNRB gene was not available; therefore the
human and murine sequences were used as a basis for the isolation of the canine
homolog. Human EDNRB and murine Ednrb sequences were retrieved ﬁom GenBank
(Accession numbers L06623 and NM_007904) and aligned using a software program. It
was believed that portions of the gene that were conserved between the human and the
mouse were likely to also be conserved in the dog. Regions of high sequence identity
between the human and mouse were selected for the placement of primers for the
polymerase chain reaction (PCR). Primers were designed to be approximately 20 bp in
length with a GC content of about 50%. Where possible, pairing between a certain
primer and itself or its opposite primer was limited to no more than 4 consecutive base
matches or 8 total. Where sequence differences between the human and mouse were
unavoidable, the human sequence was used for the primer.

Initially, 2 sets of primers were designed within individual exons so that they
could be ampliﬁed from genomic DNA. These primers were ﬁrst tested on human DNA
to ensure that they functioned properly, then on canine DNA. The PCR products
obtained were sequenced to determine that they did represent the correct ﬁagments from
the EDNRB gene. Additional primers were then designed to allow the ampliﬁcation of
the entire coding region of the canine EDNRB gene ﬁom cDNA in overlapping
ﬁagments. The primers designed for this gene are given in Table 1. The fragments used
for ampliﬁcation of the coding region are given in Table 2. Total RNA was extracted

from a frozen sample of spleen taken from a Labrador Retriever with normal hearing and

35

Table 1. Primers designed within EDNRB exons. Location is given relative to the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

start of the canine coding region.

Name Sequence 5’ end Direction
ETB X4OF CCAAGTTTCCCACTGGCGCG -194 Forward
ETB 11F TGACCCAAGTGTCCTTGCTT -90 Forward
ETB 11R GCTGCTACCTGCTCCAGAA -1 Reverse
ETB 134F TAATGACGCCACCCACTAAGA 134 Forward
ETB 204E TGCGGAGGTGCCTAAAGGAG 204 Forward
ETB 298R AAGTCTCCTTGATCTCGATGGAT 298 Reverse
ETB 321F GTCCTGTCTAGTGTTCGTGCTGG 321 Forward
ETB 365R AGTGTGGAGTTCCCGATGAT 365 Reverse
ETB 398BR CGCATGCACTTGTTCTTGTAGA 398 Reverse
ETB 12F GGAGACCTGCTGCACATCA 436 Forward
ETB 12R CTTCTGTATGAAAGGCACCA 546 Reverse
ETB 13F GCCTCCGTGGGAATCACTGTGT 547 Forward
ETB 601 F CGAGCTGTTGCTTCTTGGAG 601 Forward
ETB 661R CTACTGCTGTCCATTTTGGAACCC 661 Reverse
ETB 14F ATTGACTACAAAGGACGTTAC 733 Forward
ETB 799R GCATGAAAGCTGTCTTCTGAA 799 Reverse
ETB 14R GCAGAAATAGAAACTAAATAGC 849 Reverse
ETB ISF GTTGAGAAAGAAGAGTGGCA 900 Forward
ETB 952E AGACGGGAAGTGGCCAAAAC 952 Forward
ETB 15R AAGCCAGCACAGGGCAAAGA 1011 Reverse
ETB l 127BR GAGGCCATATTGATGCCGAT l 127 Reverse
ETB 17F ATAGCTCTGTATTTGGTGAG 1 150 Forward
ETB 1233F ACAGTCCTTAGAGGAAAAGCA 1233 Forward
ETB 1303R ACCGGAAGTTGTCATATCCGTGAT 1303 Reverse
ETB I7R GTTTTAATGACTTCGGTCCA 1386 Reverse

 

 

 

 

Table 2. PCR fragments used to obtain the EDNRB coding region

 

 

 

 

 

 

 

 

 

Forward Primer Reverse Primer Temperature (°C) angent size (hp)
ETB X40F ETB 298R 62 493
ETB 204F ETB 365R 56 162
ETB 321F ETB 661R 66 341
ETB 601F ETB 1303R 58 703
ETB 17F ETB 17R 52 237

 

 

36

 

used for reverse transcription into cDNA. Fragments of the coding region were ampliﬁed
from the cDNA by PCR, puriﬁed by extraction from agarose gels, and sequenced.

The EDNRB sequence determined from the normal dog is given in Figure 1.
Most of the EDNRB coding region sequence has been submitted to GenBank (Accession
number AF 034530). The coding region is 1329 bp in length and is 94.1% and 83.5%
identical to the human and mouse, respectively, at the nucleotide level. At the amino acid
level, it is 97.2% identical to the human and 88.2% identical to the mouse. A discrepancy
was noted in the sequence obtained from two of the overlapping fragments. The PCR
fragments ETB X40F — ETB 298R and ETB 204F - ETB 365R both share bases 224-275
within the coding region. The ETB X40F — ETB 298R ﬁagrnent showed a single
sequence in these bases that closely matches the human sequence. The ETB 204F — ETB
365R fragment also showed a single sequence, but this sequence differed from that of the
other fragment. A total of 14 single base differences were seen in the region of overlap
between the two fragments, as shown in Figure 2. A total of 8 dogs from different breeds
were sequenced in this region and the same results were obtained in all cases.
Interpretation of this ambiguity is saved for the discussion.

Sequence from the 3’ end of the coding region initially could not be achieved.
Rapid ampliﬁcation of cDNA ends (RACE) was used unsuccessfully in an attempt to
obtain this end. The 3’ end was eventually obtained by the design of a new set of primers
based on human and mouse homology. During the RACE procedure, however, several
ampliﬁed ﬁagrnents were isolated and sequenced. The sequence of one of these
fragments matched the EDNRB cDNA sequence up until the 3’ end of exon 6. The ﬁrst 8

bases of the ﬁagrnent sequence beyond this point matched the ﬁrst 8 bases of intron 6 in

37

Figure 1. Alignment of the cDNA sequences of dog and human EDNRB with
mouse Ednrb. Vertical lines indicate exon breaks and the ends of the coding region.
Base positions relative to the start of the canine coding region are given to the left.

Nucleotides identical between species are shaded.

38

men:L

D09
Bu-
Mbu

Hun
Nbu

Bun
Mbu

Doe
Bun
“bu

Bun
Mbu

Hun
Mbu

Hun
Mbu

D09
nun
Mbu

Dog
Hun
Mbu

-174

-124

-76

~28

22

72

119

169

219

   
    

   
 

 

j aaacttgagttacttttgagcgtggatactggcga_gaggctgcgggc

Uaadcf aﬁffactfttgagcg égatacfggégaégéggctgcggqci
.achtagﬁgEEaﬂg ~~~~~~~~~ gcgggggtagaggcaaccg

‘eeac

 

“tattagcgtftgcagcqacttggcthggcagctgaCC5~~Eagtgtcd
tattagcgtttgcagcgacttggctcgggcagctgaccgcaaagtgt~

ctagtltnrtmtmaigmmagmtm~~tthaattstc.

1 II 2
gtcttccttcctctgcttgtctctaggctctgaaS~Etgcgga Eggc
gtcttccttcctctgcttgtctctaggctctgaaa~ctgcgg~~ ggct

RQEREEQaCEQQCEaItIgHSQLQCEﬂaEﬂ~“Eééﬂaﬁaﬂﬁaiig

| START
ccggacgctE~Etggagcaggtagcagcatgcagccgcctccaagtcti

 

 

 

 

ccggacgctq ctggagcaggtagcagcatgcagccgcctccaagtct
_EgagggtEEcaiaﬁgggggalctItlaEatgcgatEg_gcgEaagcl

 

gcggacgcgccctggttgcgctggttcttgcctgcggcctgtcgcgga
gcggacgcgccctggttgcgctggttcttgcctgcggcctgtcgcgga
c c tEggEgﬁcgctgclgEEgncctgtﬁggtlcﬂtlgggg

 
  

 

tggggagaggagagaggcttcccgcctgacagggccactg ~~~ gctt
tggggagaggagagaggcttcccgcocgacagggccact§ ~~~ gctt

agggggggggalaggggga tcc_aEctgclcaahccaggﬁ tgtﬁa

 

gggacgggagagataatgacgccacccactaagacctEgtggcccaa

gcaaaccgcagagataatgacgccacccactaagaccttatggcccaag
QE
.cgggegtaaagéggtaatgacgccacccactaagaccgccEggaEggga

 

gttccaacgccagtctggcgcggtcgttggcacctgcggaggtgcctaI
gttccaacgccagtctggggcggtcgtEggcacctgcggaggtgccta

ttccaa tEcagtctgath_gttgcﬁccﬁcacctgcgga gg tgaic

P. 1.121.111.11le

ggagacaggacggcaggEEEEééagﬁgCgCaCEEEEEEEECEcccccg_
ggagqgaggggggcaggagggccgccacgggccgEgtcccctcccccgj

a ggggggghggthgggtctcgccga'atbg~~~ﬁtgcctcgth

 
  

39

Figure l (cont’d).

Doe
Hun
Mbu

D09
Hu-
Mbm

Doe
Hun
Mbu

269

319

369

419

469

519

569

619

669

 

 

gEagg3tECatcgagatcaaggagactttca3gE3tEtcaacacggﬁg
ccaaggacgcatcgagatcaaggagactttcaaatacatcaacacggtg

E9 aacasaatertaagetcegcaeaeqttttaaeta catcaaqagga;

 

 
   

tgtcctgccttgtgttcgtgctggggatcatcgggaactccacacttc'

tgtccttEEaEtgttcgtgctgggcatcatcgaaéactccacaEEéEg
EQ£QGEQQQECQESLWWQ§QP a CQEQQEEQQQnathcaQQEth

 

 

agaattatctacaagaacaagtgcatgcgaaacggtcccaatatcttg

EagaatcSEtEacaagaacaagtgcatgcgaaacgﬁcEBtEatatcttgf
aaaamtcatstesssaesuwcaa t cat C Castingssssatatctt

 

EaﬁccagcctggctctgggagacctgctgcacatcatcattgacatcCEa
xcgccagqttggctctgggagacctgctgcacatcgtcattgacatccdt

£29.95.th®& GagﬁgqatcgEanacagaggé
2 ll 3

EEEgcfgtctacaagctgcﬂtgatﬁaggactggcchEEggaatEgaga
§tcaagg§ctacaagctgctggcagaggactggccatttggagctgaga
EEtagcacEtacaagtEg_Ecﬁcagaggactggccattt a ct a a

 

 

 

EWEEaSEEEEEEEEEEEm“Eacagaaggc“EmE§E§§§53E33"Emf§t

tgtaagctggtgcctttcatacagaaagcctccgtgggaatcactgtg
htgtaagctggtgcgcgtcatacagaagggtEgtﬁtgggaatcaga t

3 1 1 4
. m1t-Mtsia'tacbdtetaabraacbacaga't‘é‘t'acraé‘ac‘f't'gEC-‘fé’é‘i9
t

   
   

gagtctatgtgctctgagtattgacagatatcgagctgttgcttcttg

- a to tEgEggtctaagtatt aca atatc a ct tt cttct

 

 
  
 
  

gtagaattaaaggaattggggttccaaaatggacagcagtagaaattg;

WEEbaéEfaﬁﬁﬁﬁagffgﬁﬁﬁffbﬁgﬁaafdﬁggaEEEEféﬁgﬁnggQ
t gaattaaaggaatpggggttccaaaagggaca ca ta aaatt

 

 

 

 

Ettaattfngtgqtcfgcﬁfgqff tggctjtccctgggﬁcagﬁgﬁgt:
gttgatttgggtggtctctgtggttctggctgtccctgaagccataggt‘
ﬁttgggtn ggg, tctct Lg ttct Ct tgggc aa ccata..tj

 

40

Figure 1 (cont’d).

Bun
Mbu

Dog
Hu-
Mbu

Bun
Mbu

an.
Mbu

Bun
Mbu

Hun
Mbu

Dog

Hun
Mbu

Dog

Bun
Mbu

009

Hum
Mbu

719

769

819

869

919

969

1019

1069

1119

 

tgatataattacgggggactacaaaggaagmggtctgggggtctgctt
t atatgattacgtcggactacaaaggagagccgggaaﬂggtctgﬁa

4 ll 5
ﬁttcatcataccEagaaaacagccttcatgcagttttacaagacag t§3
tttcatccagficagaagacagqtttcatgcagttttacaagacagcaaa
EwgagtccgtgtcagaaaacaggcttcatgcagttttacaagacaQQCQg

Efﬁ3€3¥§3€aaccwﬂtﬁaﬁfacaaaggScEttacctgcgaafctgctt3

 

 

 

 

gattggtggctgttcagtttctatttctgcttgccggtggccatcact

Egattggtggcfafﬂtﬁgtttctatttctgcttgccattggccatcact
1QéttggtggctgttcaqtttCtQCEtQtQCttQCGQCIaEQQBtQagti

 

,cfttttttataccctgatgacctgtgaaatgttgagaaagaagagtggg
:atttttttataCactaatgacctgtgaaatgttgagaaagaaaagtggﬁ

‘agﬁcixgnq$accaxgataapqnncaaaatactcaagnaqaagaacnat

5 ll 6
tgcagattgctttaaatgScEacttaaagcagagacgggaagﬁtﬁcca3
tgcagattgctttaaatgatcacctaaagcagagacgggaagtggccaa

EEQQQQQLLQQLt5gaﬁLQQLQQGQLQQQQCQQQQQQQaEQQQLQQEgai

 

 

 

gacagtcttttgcctggtccttgtctttgcccﬁgtgctggcttcccctEq
ﬁaccgtcttttgcctggtccttgtctttgccctctgctggcttccccttd

awmgggxscggchaggccﬁcusggttaQtE._m ttsscttccccttd

 

 

 

Etctcagcaggatettgaagtha-EﬁaittatgatCagaatgatcccagﬁ
@cctcagcaggattctgaagctcactctttataatcagaaggatcccaaﬁ

acctcaggcggmgcgtgaagctcagcptgga,ggcg_g§gcagtcgacac

6 II 7
EZatgtgaacttttgagctttttgttggthtgg tEEtgﬁcﬁacﬁmEa

 

gatgtgaacttttgagctttctgttggtattggactatattggtatca
gEg_g§gE;chgachEEttgttggEtEt gga ctgcgttggtatcag

t5? jacfmcEfgaatﬁccEgca?%aafcatgfangEE§€33Efgg‘ma
tatggcttcactgaattcctgcattaacccaattgctctgtatttggtgd

E__ggc ttgttEg_§cpcctg Ecaatccaqgcgctctgtatttgg_gé

4|

Figure l (cont’d).

D09
Hun
Mbu

D09
Hun
Mbu

D09
Bun
me

Dog
Hun
Mbu

7 ll 8
1169 gcaaaagattcaaaaactgctttaagtcatgcttatgctgctggtgccag
Ecaaaagattcaaaaactgctttaagtcatgcttatgctgctggtgcca

 

Qaaaagattcaaaaastgctttaagtcatqtangaggxgctqgngcqga

 

 

 

1219 ﬁcatttgaagaaaaacagtcctEaﬁéggaaaagcagtaafgcttaaagtq
tcatttgaagaaaaacagtccttggaggaaaagcagtdgtgcttaaagt

aEggggggg21_1§991g£§£§tggeggggaéggégggctggchgégggﬁ

 

1269 Eaaagctaatgatcacggatatgacaacttccgttccagtaataaataci
aaagctaatgatcacggatatgacaacttccgttccagtaataaataca

FaaaascaacaataaggaaLALaagaag;ngqgtagaacaamaﬁaxaca

STOP |
1319 EctcatctEahaagaaqgaataﬂcEacEtEatEcEEEEEEEEEEtEgc
ctcatcttgaaagaagaactattcactgtatttcattttctttatat

DQLQQS. 911$ QﬂégicﬂégéﬁclcﬁwﬁgﬁCIaQEC’é 9130895 ﬁcicﬁﬁﬁg

 

42

A ac ggﬁg'ﬁaE—g—atctEEéEEEEEESEEaEEtEéEbEEEEEEgEEECSEgg
B 99 aégaggcggdggcgggccacgcacgcggagggcpgcgQQC;gggaqaa

Figure 2. Alternative sequences in canine EDNRB. The region shown spans
bases 224-275 in the canine coding region. Sequence A was obtained from fragment
ETB X40F — ETB 298R and sequence B was obtained from fragment ETB 204F — ETB

365R Bases that are identical between the sequences of the two fragments are shaded.

the rat; therefore it was thought that this fragment may contain the canine intron 6. The
presence of an intron may be explained by genomic DNA contamination of the cDNA
preparation or by an unprocessed RNA from which the introns had not been removed.
Primers ﬂanking the break separating exons 6 and 7 were used to amplify the intervening
intron from genomic DNA. The entire intron was sequenced and found to contain only a
single base difference from a sequence in GenBank (Accession number AF 026088)
identiﬁed as canine EDNRB intron 4. Comparison of the ﬂanking exonic sequences
determined that this represented intron 6 in the numbering scheme used in this study.

The sequence obtained is given in Figure 3 and is available from GenBank (Accession
number AF134188). Within this intron was found a microsatellite and a single nucleotide
polymorphism (SNP). Primers used for investigation of these polymorphisms are given
in Table 3. The characterization of these polymorphisms is described in the following

chapter. The microsatellite was used to screen a panel of dogs ﬁ'om the DogMap group,

and the results were used to locate the EDNRB gene to canine Chromosome 22.7 The

manuscript of the resulting publication is provided in the Appendix. Another canine

43

Figure 3. Sequences from canine EDNRB introns. Base numbering is given
where introns have been completely sequenced. The remaining introns could not be
completely spanned and are shown by partial sequences obtained ﬁ'om the ends, with dots
representing the unsequenced central portions. The unsequenced regions are of unknown

size due to the unknown size of these introns in the dog. R = G or A.

IEgne3.

51
101

InﬂonZ

gtaaagggctcctattgacgggagggtgcctagttaggaggagggaggtg
ggagagcctttgggggatcttcttactcaagaagatcatcgcctcctaaa

atcagtgaacattggacataaattc .........................
ccactaagtaatgcagttcagagggcactgtgtgaatatctccctaacac
acctcatgtcttctcaatgcag

lnﬂon3

gtaagagcataaatttaagccaggtatatcctgaacactatactagtgat
tgctatgttacatagaaaataaaccatgtaattcagtaaaagcaggctct
gcagcttgacagataattctattttgttcttcag

Inhon4

gtgaattttaccattttctttcctttctgttcttgccttataaatattta
gctactt ...........................................
ttactttgcatttagtatatagatttttttctacagggaaatgttaatct
tataatactctcttttccatag

InhonS

gtaagagaacagaagtatgtgctgactcatgattacagtgatgattatga
ataaaaaa ..........................................
aaaaatgaatttgtagttattttagttctaattcatattaactattcaat
ttaaaggtcagtgttctggatttttacaaataccactgactttttgtaaa
caatattaagtgttctatttgggaagagagaacgttgtgattatctgaac
catctatttaatttctgactatggttttatttcag

45

Figure 3 (cont’d).

51
101
151
201
251
301
351
401
451
501
551

Inuon6

gtaagaaaggtaaaagagagttttgtaggtaactgccattcagaagtttt
tttcattgatccctttcataggcagagagagagtatcattttgctagtct
ttagggagcaaggagtcaagattctcattttttatcttcacccctattgt
agagaaaggaataaaggcgacttatgagaacactgggaatggagagccag
agctgtcaaaatgttgagtgggtaccacaattggaaatatcagctccaat
tctcctttgcacctcacaaagtcttttctcagccagccccagcacttcca
gatgagattttattgtaaaagagagaattgggcatgggcagaagggcatg
atgacagagagagagagagagagagagacagagacagagagagagacaga
gagggggaaagagagagagaggtcatcattagcaaagaccagtgataaag
tcaaaaattatgratatgggtaatttttttcttcacaaaaactcaaaagt
ttgcgagaatacatacttaaagcaatgtattgttcacagacatattttgg
gtggttttttgcag

Inhon7

gtaggagtatttcaaaataaaaactctttttggcctagcatcaaatataa
cctttccaaactattcatatttctatttaaagacatttcgtaaattgttt
tatagttt ..........................................

atcacatgtgaactgatgtgaatgtgtaacaagttattttgctttgtaca
9

46

Table 3. Primers designed within EDNRB intron 6. Location is given relative to the

 

 

 

 

 

 

start of the intron.
Name Sequence 5’ end Direction
ETB 16F AGGTAACTGCCATTC 27 Forward
ETB RPTF GAGAATTGGGCATGGGCAGA 323 Forward
ETB I6GAF GACCAGTGATAAAGTCAAAAATCAT 437 Forward
ETB RPTRZ TGACTTTATCACTGGTCTTTG 453 Reverse

 

 

 

 

 

mapping project has also located EDNRB to syntenic group 7, which was later identiﬁed

as part of Chromosome 22.25

A 740 bp genomic DNA fragment spanning bases 952-1127 in the coding region
and intron 6 was used to probe a canine genomic bacterial artiﬁcial chromosome (BAC)
library. Clones producing positive signals were cultured, and a single colony from each
clone was boiled to make a crude preparation of DNA. One of the clones was determined
by PCR to contain the EDNRB gene and pure DNA preparations were made of this clone.
Primers were designed facing toward exon boundaries to allow sequencing of bordering
intronic sequences (Table 1). The BAC clone DNA was directly sequenced using these
primers, and the resulting sequences are given in Figure 3. Intron 6 had already been
sequenced by other means as mentioned above and intron 3 was small enough to be
completely sequenced from the BAC. Partial sequences were obtained at each end of
introns 2, 4, 5, and 7, however no sequence could be obtained from intron 1. It is

possible that the BAC clone does not contain this portion of the gene.

47

MIT F

The MIT F gene had not been previously sequenced in the dog; therefore, human-
mouse homology was used for the design of primers to allow ampliﬁcation of the canine
sequence by PCR. Human MIT F -M and murine Mrﬂ-m sequences for the melanocyte-
speciﬁc forms were retrieved ﬁ'om GenBank (Accession numbers Z29678 and Z23066)
and aligned using a software program. Primer design was performed as previously
described. Primer pairs within individual exons were ﬁrst tested from genomic DNA to
ensure that the MIT F gene could be correctly ampliﬁed in the dog, and then additional
primers were designed to allow the ampliﬁcation of the coding region of the gene from
cDNA using overlapping ﬁagments. The primers designed for MIT F within exons are
given in Table 4. The fragments used to amplify the coding region of MT F are given in
Table 5. The same normal Labrador Retriever used for EDNRB was also used as a source
of cDNA for ampliﬁcation of MIT F . The ampliﬁcation fragments were puriﬁed by gel
extraction and sequenced.

The sequence obtained from the normal dog is given in Figure 4. The coding
region is 1260 bp in length. At the nucleotide level, it is 93.8% identical to the human
and 87.6% identical to the mouse. At the amino acid level, it is 97.6% identical to the
human and 93.0% identical to the mouse. Two different size bands were obtained for the
cDNA fragment Ml 206F - MI 955R. These bands were found to represent the different
MITF forms resulting ﬁ'om the previously mentioned alternative splice site in exon 6.

The 5’ end of the MIT F -M gene could not be obtained from cDNA due to the
presence of a second sequence of unknown origin that strongly ampliﬁed at the same

size. Therefore, the sequence of the 5’ end was obtained from genomic DNA. The M1

48

Table 4. Primers designed within MT F exons. Locations are given relative to

the start of the canine coding region.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Name Sequence 5’ end Direction
N11 11 MP GTCTACCGTCTCTCACTGGA -43 Forward
MI 51F AAACCCCACCAAGTACCACA 51 Forward
MI I 1R CTGCCTTTGGGCTTGCTGTA 90 Reverse
MI 206F GCGCACCCAACAGCCCCATG 206 Forward
Ml 261R CTCTTTTTCACAGTTGGAGTT 261 Reverse
MI 12R GGCATTCACTTTCCGCCCTG 307 Reverse
MI 13F CAGGGCGGAAAGTGAATGC 288 Forward
MI 13R ATCCCAGGATTTCTTCATTATA 409 Reverse
MI 14F ATAATGAAGAAATCCTGGGA 389 Forward
NH 431F TGGCAAATACGTTACCTGTCTCT 43] Forward
MI 14R CCTTGGTTGCCATAAAGAT 485 Reverse
MI 522F TCCAGCCAACCTTCCCAACA 522 Forward
MI 16F TCTGAAGCGAGAGCATTGGC 580 Forward
MI 613R TTTGCCTCTCTTTAGCCAATG 612 Reverse
NH 16R GGATCATTTGACTTGGGAATC 710 Reverse
NH 17F ATTCCCAAGTCAAATGATCC 691 Forward
MI 17R GATATAGTCCACAGATGCTT 762 Reverse
M1 18F GAAGAAATTGGAGCACGCCA 813 Forward
MI 887F GACTTTCCCTTATTCCATCCAC 887 Forward
MI 18R CCCGTGGATGGAATAAGTGA 911 Reverse
MI 955R GTTCCTGCTTGATGATCCGATTC 955 Reverse
N11 1232R CTCATACTGCTCCTCCGGCT 1232 Reverse
M1 X1466R ATCAAGAAAACCCCTTCAGGTA 1345 Reverse
Table 5. PCR fragments used to obtain the MIT F -M coding region
Forward Primer Reverse Primer Temperature (°C) Fragment Size (hp)
MI 11 MF MI 11R 58 134
Ml 51F Ml 261R 54 211
M1 206F MI 955R 64 750
Ml 887F MI X1466R 58 459

 

 

 

 

 

49

 

 

Figure 4. Alignment of dog and human MIT F-M coding region sequences with
mouse Mitf-m and the canine MIT F pseudogene (Psg). Vertical lines indicate exon
breaks and the ends of the coding region. Base positions relative to the start of the canine
coding region are given to the leﬁ. Nucleotides identical between species are shaded.
The pseudogene sequence given represents the two alleles that contain mutations. The
third pseudogene allele is not shown, as the sequence is identical to the normal canine

sequence. Y=CorT,M=AorC,W=AorT.

50

Iﬁgne4.

Dog
Hun
“bu

Peg

Bun
Mbu
Psg

D09
Bun
Mbu
P39

P39

Hun
Mbu
Peg

l START
taaaaaaaaaaaaaaagtaataafaafaaaaaaab“aaaaemeaatCai
,tggtgcggcctaaaacattgttatgctggaaatgctagaatataatcaé
Egggggtggggg aa ctggctatgctggaaatgctagaatgcigggg’

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

-23

     

1 ll 2
28 atcaggtgcagacccacctcgaaaaccccaccaagtaccacatacagc
atcaggtgcagacccacctcgaaaaccccaccaagtaccacatacagc
chaggngcagacccaccaguaaaacccQaccaagtacgagatacagc
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 11;.a..<.=.a.g.c

 

 

gcccachgcagcaggtaaagcagtacctttctaccactttagcaaat
gotcagaggcaccaggtaaagcagtacctttctaccactttagcaaat

Egcccaaaggcagcaggtaaagcagtacctttctaccactttagcaaat

 

128 mmfgccaaccaadfCCEMEEEEEQCENE faggggggagggfgﬁgﬁa

racatgccaaccaagtcctgagctQgccatgtccaaaccagcctggcga

acatgccagccaagtcctgagctcaccatgtccaaaccagcctggcga
vacatgccaaccaagtcctg_gcttgccatgtccaaaccagcct c

78 gcccaaaggcagcaggtaaagcagtacctttctaccactttagcaaati
c

 

 

a-_1._..:i

‘atgtcatqccaccagtqcegqggagcagcgcacccaacagccccatgg”
atgtcatgccacdggtgccggggagcagcgcacccaacagccccatgg
atgccatgccaccagtgccggggagcagcgcacccaacagccdthtgg

‘aEQEEQEQ$%%%EQE£92£9999§9£§9§9YEQQSQQQQQﬁQﬁEﬁEQQf

2 ll 3
228 atgctcacacttaactccaactgtgaaaaagagggattttataagttt
atgcﬁtacgcttaactccaactgtgaaaaagagggattttataagttt
atgctcagtbttaactccaactgtgaaaaagaggcattttataagttt

cttagggccaactgtgaaaaagagggattttataa ttt

178

 
  
 

 

 
 
    

278

   
      

agagcaaiEEgﬁﬁﬁEﬁﬁggaﬁfﬁggfﬁcccaaEEEE§33333§35€F

Iagagcaaaacagggcagagagcgagtgcccaggcatgaacadabattc,
ggagcagagcagggcagagagtgagtgcccagqtatgaacacgcactQt
a .Qaaaaca .c aaa t aatgcccaaccaggaacacgcattca

 

51

Figure 4 (cont’d).

Hun
Nbu
Peg

Bun
“bu
Peg

Peg

Bun
Mbu
Peg

nun
Mbu
Peg

328

378

428

478

528

578

627

  

 

 
 
  
  

 

3|l4
Taaacategﬁaearqeagafééataatataartaafaacateaftagccﬁ
gagcgtdctdthtgcagatggatgatgtaancgatgacatcattagccg

 

pgagcgtcgtgcatgcagatggatgatgtaattgatgacatcaucagcc
.aaasaaagmga “gamma; qataaa aaca ataaaa.

 

 
   
    

gaatcaagttataatgaagaaatcctgggattgatggatcctgctttga
Egaatcaagttataatgaggaaatcttgggcttgatggatcctgctttg4
ggaatcaagttataatgaagaaautttgggcttgatggatccggdcttga
Eggatcaa-ttataatoaaoaaatcct-ooatt-atooatccto ’

4 || 5
aatggcaaatacgttacctgtctctggaaatctgattgatctttatgg§
aatggcaaatacgtﬁgcctgtctnggaaacttgattgatctttatgda
aatggcaaatacgttacocgtctctggaaacttgabcgaccﬂctacazj
aatggpaaatacg:tacctgtctctggaaatctgattg__ctttat

 

 
 
  
  
 
  
 

a~aaa~aqaaaaqac€ccaa-aagaeﬁaaaaaaaaaaaa‘aeaaaa‘aaaaa.
accaaggtptgcqccqaccaggcctcaccatcagcaactcctgtccaga
’accagggcctgCGaedgtcaggccmtaccatcagcaactcctgtccaga

‘ aaaamasmgamc aamqttmmaaaammaa g.

5 ll 6
uaaccttcccaacataaaaagggagctcacagcgtgtatttttcccaca
Iaaccttcccaacataaaaagggagctcacagcgtgtatttttcccaca
aaccttcccaacataaaaagggagctcacagcgtgtattttccccaca

7aaccgggggggggpaaaaagggagctcacagcgtgtatttttccc

 

 

atctgaagcgagagcattggctaaagagaggcaaaaaaa~ §gacaatc
'gtctgaagcaagagcactggdcaaagagaggcagaaaaa~ ggacaatc

'gtctgaagcaagqgcattggctaaagagaggcagaaaa ~ggacaatc
atct aa a a tagattggctaaagagaggcaaaaaaga .
, 6 II 7 ,

I‘a‘aa‘trcjat'fa‘aaagaaaa‘aaaa‘cjartfaa aaraaaeaaeaa‘aaﬁaaa
aacctgattgaacgaagaagaagatttaacataaatgaccgcattaaa

aacttgattgaacgaagaagaagatttaacataaacgaccgcattaag
laaaaLaaLLaaaaaaasassaaaatQaaagaLaaagaagaggaasaaa

 

 

52

Figure 4 (cont’d).

Peg

Peg

D09
Hun

Peg

Hun
“bu
Peg

Hun
“bu
Peg

677
727
777
827
877

927

977

7 II 3
EactaggtactttgaEEc6333§€3§335gaEccagacaE§3§€Egééﬁi

 

actaggtactttgattcccaagtcaaatgatccagacatgcgctggaa1
gctaggtacnctgatccccaagtcaaatgatccagacatgcggggaaa
actaggtactttgattcccaaggagaatgatccagacatgggggggggﬁ

 

 

 

'agggaaeﬁatéftaaaageattfgtggactatatéégaaagttqcaac.
agggaaccatgttaaaagcatccgtggactatatccgaaagttgcaac
agggaaccattctcaaggcctctgtggactacatccggaagttgcaac

mtaaatt aaamsatammaaaaaamaaaamagaaa

 

 

gaacagcaacgtgcaaaagaacttgaaaatEgacagaagaaattggag
gaacagcaacchcaaaagaacttgaaaaccgacagaagaaactggag
ggaacagcaacgagctaaggaccttgaaaaccgacagaagaagctggag
Egaacagcaacgtgcaaaagaacttgaaaaccgacagaaggt‘tt

 
 
  

8 || 9
cgccaaccggcatttgttgctcagaatacaggaacttgaaatgcang\
cgccaaccggcatgtgttgctcagaatacaggaacttgaaatgcaggc
tgogaaccggcacctgctgctcagagtacaggagctggagatgcaggc
7c ccaa gggcafﬁtgttgctcagaatacaggaacttgaaatgcaggcﬁ

  

’é'ﬁﬁé’fﬁ‘ﬁt‘g‘fj‘a‘éf‘ff8368851:86838665368ﬁ’é‘c‘i’f‘ﬁ‘tﬁ'ﬁ‘t‘ét“ "83633:
gagctcatggactttcccttattccatccacgggtctctgctctccag”
agagdgcatggactttcccttaﬁcccatccaccggtctctgctCgbctg

EmmggamacaagamagaL95;asasagaagaggﬁggtaaag.

 

éttggtgaatcggatcatcaagcaggaacccactcttgagaactgcaacq
gttggtgaatcggatcatcaagcaagaacccgttcttgagaactgcagcé
?cpggtgaatcggatcatcaagcaagaaccagttcttgagaactgcagcé

éwiggtgaatcggatcatcaagcaggaagggégggtt a aa wﬁggggg

 

 

 

 
   
 

a§EEEEEéEtcagcatcatgcagacCtSECttgtacgacgacgcttgéﬁ
'agacctccttcagcatcatgcagacctaaCctgtacaacaactctcgad
ggaactpgtacagcaccaggcagacctgacatgtacgacaactctggaﬁ
agacctccttcagcatcatggagacctagcttgtacgacgggggggggﬁ

53

Figure 4 (cont’d).

009

Hum
“bu
Peg

D09
Bun
“bu
Peg

Dog

Hun
“bu
Peg

Dog
Hum
“bu

Peg

Dog
nun
“bu
Peg

Dog

Hun
Ian:
Peg

1027

1077

1126

1176

1226

1276

 

 

Etéacagatggcagcatcaccttcaacaacaaccttggagccggqaccgg
Etcacggatggcaccatcaccttcaacaacaacctcggaaotgggactg“
ctcacggchgtaccatcacctstuccaacaacctcggcaccatgccgg”

gtegggggkﬁgg_gcatpaccttcaacaacaaccttggagccggg %q

 

 

 

agtagccaagcctatagcgtccccacgaaaaE~ Eggatccaaactgga‘
gccaaccaagcctatagtgtccccaqaaaaat~ gggatccaaagtgga
' agcagcacggcctacagdatccccaggaagat~ gggctccaacttgga

agtagccaagcctatagpgtccccgyEaaaagtEggatccaaactgga

ﬁaEﬁEé‘fgatggatgﬁéﬁbteff?8%8686??§§E”f3368§388%8cg

acatcctgatggacgac_accctttctcccgtcggtgtcactgatccacg
gacatcctgatggacgatgccctctcapetgttggagtcadcgacccacu
Eacatcctgatgg__gacactctttctcccgttggtgtaactgacccacg

 

 

 

 

chttcateaqtgtcceetgqaqetteeaaaaeaagea§EEgaaggaqc;
pctttcctcagtgtcchcEgagcttccaaaacaagcagccggaggagc
gcﬁgtcatcagtgtqgccaggagcttcahaaacaagcagccggaggagc
Egaaaqaaeaamﬁamt Wmmm ’

STOP l
catgagcatggaagaaaccgatcaggcttgttagcagggcctccctgeﬁ
tatgagcagggaagagacggagcacabttgttagcgaatectccctgda
tatgagcgcagaagaaacggagcatgcgtgttagcgagcetgtdttgcﬁ
ccctcccpgcg

 

 

 

   
  
 

EEQESEEEEEEEEEEEEEEEEEEEEEcttgattcgtaggtttcataéEZ
tg attcgcacaaactgcttcctttcttggttcgtagatttaataacg
tg cEcEgcadggBGchﬂcCcuctcttctﬁcaggagactutgtaaté

Egggggtttcaaaaactgcttcctttcttgattcgtaggtttcataatﬁ

54

Figure 4 (cont’d).

 

Dog 1027 Etﬁacaqatgqcaqcatcaccttcaacaacaaccttggannggaccge

 

Hun tcacggatggcaccatcaccttcaacaacaacctcggaaatgggaqtgA
Bk“: tcacggacggtaccatcacctﬁtlctaacaacctchcacCatgcng'
P89 §tqggagakngggg9§t0@ccttcaacaaqgépctt92é922292_mcg;

 

 

Dog 1077 vggtagccaagcctatagcgtccccacgaaaaE~§ggatccaaactgga

  
  

Hun, gccaaccaagcctatagtgtccccaqaaaaat gggatccaaaptgga
“bu ‘agcagcccggcctacagcatccccaggaagat~ gggctccaacttgga
Peg a ta ccaagcctatagcgtcgpggyhaaaggtEggatccaaactg a

 

Dog 1126 "acafcufgmfﬁégfégbadtcf?EEtcccﬁffﬁafﬁfaEEEEacccacﬁ
Hun acatcctgatggacgacaccctttctcccgtcggtgtcactgatpcac

“bu acatcctgatggacgatgccctctaacctgttggagtcadcgacccacé
Peg Eacatcctgatggatgacactctttctcccgttggtgtaactggcccacd

 

 

 

Dog 1176 EctttcatCaqtqtcccctggagcttccaaa‘Ea'géagccgaaggagcv

 

 

Bum pctLtcctcagtgtcccchgagcttccaaaacaagcagccggaggagc
“bu gtbgtcatcagtgtqgccaggagcttcahaaacaagcagccggaggagc2
P89 cqpgggaggagggnykggtgaaag:ngaagacaaqgaaggqgaggaag
STOP |
Dog 1226 catgagcatggaagaaaccgatcapgcttgttagcagggcctccctgcﬂ
Hun ‘tatgagcangaagagacggagcacabttgttagcgdattctccctgda
“bu ‘tatgagcgcagaagaaacggagcatgdgtgttagcgagcdtgtdttgc
Peg catgagcafggaagaaaccgatcatgcttgttagcaggccéggpc 9%

chain?!

Dog 1276 Efgcgc'ffcaaaaacfgcttccEEtcttgattcgtaggtttcataatE
Hun tgdattcgcacaaactgcttcctttcttggttcgtagatttaataacg
“bu tgécuctgcacggacchUCcctctcttcttcaqgagactttgtaata

Peg Etgcgctttcaaaaactgcttcctttcttgattcgtaggtttcataati

 

  
  
  

54

exon of MIT F is the closest to the rest of the gene within the genomic DNA, therefore it
was possible to amplify a fragment containing the M1 exon, the intron following it, and
part of exon 2. The entire sequence of the intron was obtained by designing additional
primers within the intron itself (Table 6). The sequence obtained is given in Figure 5.
RACE was used in an attempt to obtain the 5’ alternatively spliced exons from the other

MIT F isoforms. Kidney was used as a source of cDNA because the Miijf-a, Migf-h, and

Table 6. Primers designed within MIT F introns. Position numbers of the 5’ end of
the primer are given. Positive numbers are relative to the 5’ end of the intron and

negative numbers are relative to the 3’ end.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Name Sequence Intron 5’ end Direction
MI ElR CTGCACTTACTGGAAAGAG l 99 Reverse
MI IlBF AAGGAGGAAAAATACCCTGG 1 187 Forward
MI EZF AGCCGACCGAACTCACAAA 1 811 Forward
MI 11 BR GTCTAACTCTCAGGATTTGG 1 697 Reverse
NH EZR CACAGTTGAGTGGGGGAATA 2 7O Reverse
MI IZBF ACCGGGTTATTGGGTTGTGT 2 160 Forward
MI IZBR GATGACAAATACGGACAGAG 2 -l78 Reverse
MI E3F CATCAGCCTCGTGTGAACAT 2 -80 Forward
MI E3R TCATTACAAAGAGTTACATCC 3 45 Reverse
N11 E4F TTGGGTGGCTTTGCACAGTT 3 -63 Forward
M1 E4R GAGAGAACACTGGAAATATC 4 60 Reverse
MI MUTF TTGCTCAGTAGTTCATTTCTG 4 -40 Forward
M1 ISBF GACAAGTGAGGTTATCAAAG 5 51 Forward
MI MUTR CTTTGATAACCTCACTTGTC 5 70 Reverse
MI 15 3’ F TAGTGTGCGTCATTGTGTGC 5 -63 Forward
MI ISBR CTATAAAACATCTCATTTTC 5 -61 Reverse
MI E6F TGTGCGTCATTGTGTGCCTT 5 -60 Forward
M1 E6R TCAATTTCCTCCCAAGAGAT 6 90 Reverse
MI E7F TAACAAGATACACTAAATGCG 6 -69 Forward
MI E7R TCGCAACAATATGAATAAGCA 7 71 Reverse
M1 E8F GAGTGCTCTGGATAATGAAT 7 -73 Forward
M1 ESR AGCGACATATTGGAAACCCT 8 4O Reverse
MI E9F AATCCTCTGTAAACCACCTCT 8 -54 Forward

 

 

 

 

 

55

 

Figure 5. Sequences from canine MIT F introns. Base numbering is given where
introns have been completely sequenced. The remaining introns could not be completely
spanned and are shown by partial sequences obtained from the ends, with dots
representing the unsequenced central portions. The unsequenced regions are of unknown
size due to the unknown size of these introns in the dog. The string of thymine residues
at the 3’ end of intron 8 causes polymerase slippage, therefore the exact length of this

stretch is unknown.

56

FﬁpueS.

51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851

Innonl

gtgagctttattcttattcatatttagtgtctgaaatatatgcaatacat
tgagtaattcaccttttcatgttattgtactctttccagtaagtgcaggt
ctactactttgatcgtgtttactgtttgataccatgagtatcactgattt
aaaggatttttaattctgtattaatgtttataagaaaggaggaaaaatac
cctggcggcttaatagtcctgcttttaaaaacacatacatgtaagtgtga
ggcatctgaaagaaactgcttcatgtagaattgctttttatgttgagttc
aagatcttaattaaaatgttgaataatcatgtccaaatgaaagtgggagg
aattaatctacaactagttgatttaatcatcagagtgtttctgtttattt
tctacaaccgttttacagctggctcttaaggaggtttgttgttgttgttt
ttaaacagaaagctgttctgtttgccaaagaaagtaaaataaattgttat
gctcctttttttagattgttgttctgatctcagtaaatcctttcagtcgt
gttggggaaaattttgccctccagatggttgcaaattttaaattattaga
catgaacaaaagggcacaagttttcaaagagatgtgcagctaattagcag
ggaaataaaacagggcaaagtattaccccaaatcctgagagttagacttt
aaacttcagtaatatcaaaatccattagcacagtgcctggtacataacag
gggcttaataatttattctgttggtggactggccagtctcatgtttgtgc
ctgagaaaagagccgaccgaaactcacaaataacggcgctgtcttctctt
ccctccgtggctatgttcag

InﬂonZ

gtaattcatgtctcctcccctctcctgtcttcttacactaaatgaatgtc
tgtcggatattattcccccactcaactgtggactctgcggggccacacac
gccggtctgtgtcccagattctgctatgtggcctccaccctaggcctctt
gacaccatcaccgggttattgggttgtgtggctcaggatggg ........
tcccctgtgcccaaaccaacctgtcttatttttctctgtccgtatttgtc
atctatcaagctcatttgacggaagttctttctgcattattttatttctc
ggtcagattctactttgtgaaagctttcttagtccatcttgttgctgcac
catcagcctcgtgtgaacatgtcattgaaaagtcatttgcaaatccaagt
catgggctgattttgcttgtgtttttgcag

57

Figure 5 (cont’d).

Innon3

gtactgaatgactcggcagtgcaaggatgtaactctttgtaatgagaatc
ta ................................................
tctctctttgggtggctttgcacagtttttgcttacatttatctctcttc
cattgccctttttcctacag

Inhnn4

gtattgatgactttttttttttttttaaagaaaatcttgagatatttcca
gtgttctctccctttccctgaact ..........................
agataaagtctggaatagatctgggtgctagctgaataacctaggaattg
ctcagtagttcatttctgttattgcttctctctctag

Intron 5

gtaaatactggcttgtgtgcctcttcctggggattttctgtttattttct
gacaagtgaggttatcaaagttgtgacctctagactatt ...........
gatatggttccatatatatctagaaaatgagatgttttatagtgtgcgtc
attgtgtgccttaaacagttcccgtttctaattacttcattcacgtgcac

ag

Inhon6

gtaagttggttttatgttcatgatgttgatattggagtgaatgttccccc
ttgtatcaaaattgtttaaaatctcttgggaggaaattgatatacat...
aggtaatgcacacatggctttaacaagatacactaaatgcgtatgtggtg
ctgttactaatagtcccttcctatgctcttttcttgaag

58

Figure 5 (cont’d)

Inﬂon7

gtgagtacaattccatgttaatctgcatcgtatattttttggtaccttaa
tgcttattcatattgttgcgaaaaatgcacagttatagaaactagagtca
agaagcaccccctctccctttgattccatttctggtatacttct ......
catagttcaacttgtcattgttaccttgtctttagaaattgagtgctctg
gataatgaattttcattgtgcctcaaatcccgaaaaaggttgttttcttc
ctctttgttacag

Inuons

gtatggggcatgtgttgtgtagggtttccaatatgtcgctgacatggagg
tgggaggagaggggatataataagccatgagggacttgatttacatgatt
ctatatagtag .......................................
ttggtgttatacctcctgtgagtttgaacaaatatgtaataacatgtatg
cgtcattatagtatatcatatatagtactttgccctaaaatcctctgtaa
accacctcttgaaacgtgattttttttttacttttattttag

59

Mitf-c isoforms are all expressed in kidney in the mouse.3 A fragment containing the
5’end of the MITF-H isoform was isolated and sequenced to obtain the sequences of the
H1 and Blb exons (Figure 6). At the nucleotide level, the H1 exon is 98.2% identical to
the hmnan and 91.1% identical to the mouse. The Blb exon is 96.4% identical to the
human and 96.0% identical to the mouse.

During the course of sequencing the MIT F coding region, variations were often
seen that seemed to indicate the presence of point mutations and frameshift mutations.
When RNA isolated from tissue was treated with DNase prior to reverse transcription,
these variations were not seen. This indicated that alternative sequences were being
ampliﬁed ﬁom genomic DNA. In order to further examine these seqeunces, cDNA
derived from DNased RNA and genomic DNA treated with RNase were used with PCR
primers designed to amplify the coding region of the MIT F gene and sequenced in 4
dogs. The sequence obtained from cDNA closely matched the human and mouse
sequences and was considered to be the normal canine sequence. The fragments
ampliﬁed from genomic DNA were the same size as those from cDNA, and sequencing
conﬁrmed that no introns were present within these ﬁagments. Since the normal
genomic MH’F gene was known to contain introns, it was hypothesized that these
alternative products represented a processed pseudogene.

Two of the dogs were found to be homozygous for a sequence that was identical
to the normal sequence obtained from cDNA. Since the genomic DNA had been treated
with RNase prior to PCR and the introns were absent, this indicates that this sequence is
indeed from the pseudogene and not from RNA contamination or the normal genomic

copy of MIT F . In the other two dogs, mutations were seen in the fragments ampliﬁed

60

Figure 6. Alignment of H1 and Blb sequences from dog and human MIT F with
mouse Migﬁ Base positions relative to the start of the canine coding region in H1 are

given to the left. Nucleotides identical between species are shaded.

61

lﬁgme6.

Hﬂ

Dog

Bun
lkwl

Hun
Mbu

Dog
Hun
Mbu

Blb

Dog
Hun
Mbu

Dog
Bun
Mbu

D09
Bun
Mbu

Dog
Bun
Mbu

Dog
Hun
Mbu

-79

-29

20

 

ﬂu] \Jnm

gtgacacagccagtgccagaactaactttgactttcactcttcgccaag
tgacaggggqegtgcgageacteactttsacttrcastcttcgccaa

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

 gcttgcagaacaccttaaaggaaaaa§~~patggaggcgcttagagttq
ctt ca aacaccttaaaggaaaaaa~~gatggaggcgcttagagttq

~~~~~~~~~~~~~~~ LLaaaggaaagaaaaaaLQQEEQQQSLLaﬂﬂtiC9

 

 

 

‘gatgttcatgccctgctcctttgaaaqcttgtatCE
gatgttcatgccatgctcctttgaaagcttgtatcﬁ
t CﬁgégOscarsthctLnggggggggﬁgscE

 
  

 

 

 

EagttCcdag§§§catcctggggcctccaagcctccgataagct65€EE§
ragttccgccgagcatcctggggcctccaagcctccgataagctcctccq

.Qggggt2%QSQSEQEQQnggggggggégggngQl1...-11 n.

 

 

tatgacatcacgcatcttgctacgccagcaactcatgcgtgagcagat

E:atgacatcacgcatcttgctacgccagcaactcatgcgtgagcia’ﬁ’f”w
1'QLQQQQFQQQSQQECtLQQE@999cegcaagacatgggagaggear

 

Eaggagcaggagcgcagggagcagcagcagaagctgcaggcggcccath
Eaggagcaggagcgcagggagcagcagcagaagctgcaggcggcccath
EaggagpaggagqgcagggagcagcagcagaagctgqaggcgggggagtQ

 

 

EatgcaacagagagtngtEtQaacEégaacEcachétaaacgtcaac
atgcaacagagagtgcccgtgagtcagacaccagccataaacgtcagt
gargcaacagagagtgcccgtgagtcagacaccagccataaacgtcagt

 

 

gcccaccacccttccctctgccacgcaggtgccgatggaagtccttaa

ﬁééoEéécaédéftEéEfétdecaacﬁhdgtEEEQaEQQaEQEéCEcga“
gcccaccacccttccctctgccacgcaggtgccgatggaagtccttaa

 

 

62

from genomic DNA. The locations of the mutations are given in Figure 4. These
mutations were not seen in the same dogs in fragments ampliﬁed from cDNA. The
mutations consisted of single base insertions and point mutations. The insertions resulted
in frameshifts and one of the point mutations created a stop codon, both of which would
be predicted to create a nonfunctional protein. In both of these dogs, the insertions and
some of the point mutations were homozygous, but the remaining point mutations were
heterozygous. This appears to indicate that two mutant pseudogene forms exist. The
homozygous mutations are present on both forms, but the remaining mutations are
present on only one of the two forms. Therefore, a total of 3 pseudogene forms were
found, two mutants and one apparently normal except for the absence of introns. It is not
known whether these represent a single pseudogene with multiple alleles or multiple
pseudogenes in different locations.

A 1295 bp MI 51F — MI X1466R cDNA ﬁ'agment was used to probe the canine
genomic BAC library. A total of 14 clones were found by PCR to contain the MIT F
gene. Ampliﬁcation from these clones using primers that would produce different size
fragments from the real gene and the pseudogene indicated that all of the clones
contained the real gene. This was also conﬁrmed by sequencing the ﬁ'agments obtained
ﬁ'om the clones. One of the clones was used for the preparation of high quality DNA,
and primers were designed facing exonic boundaries to allow the sequencing of bordering
intronic sequences (Table 4). The BAC DNA was directly sequenced using these primers
and partial sequence was obtained ﬁ'om each end of introns 2-8. The sequences of these
introns are given in Figure 6. PCR primers were then designed within the introns facing

outward to allow the ampliﬁcation of individual MIT F exons and their bordering splice

63

sites from genomic DNA (Table 6). The primers used to amplify MIT F exons are given
in Table 7. Exons M1 and 2-9 were ampliﬁed from the normal Labrador Retriever and
sequenced. No differences were seen compared to the sequence obtained from cDNA,
conﬁrming that the sequence was correct. As the primers were based within introns, no
pseudogene sequence was seen.

In order to further characterize the MT F gene, an attempt was made to determine
the location of the gene in the canine genome. During the course of sequencing MIT F in
the dog, no polymorphisms were found that would allow it to be mapped in the same
manner as EDNRB. It was thought that if polymorphisms could be identiﬁed in a closely
linked gene, mapping of this gene would identify a location that likely contains MIT F as
well. In the current human map, MIT F is located on Chromosome 3 at a position of
approximately 68.5 Mb. The two genes T MF 1 (67.6 Mb) and R030] (76.6 Mb) ﬂank
MT F in the human genome. Multispecies primers already existed in the lab for these
genes and a canine-hamster radiation hybrid panel was available. The conservation of

MTF in the clones of this panel was tested by PCR ampliﬁcation of a genomic ﬁ‘agment

Table 7. PCR fragments used to obtain the MITF-M exons.

 

 

 

 

 

 

 

 

 

 

 

Forward Primer Reverse Primer Temperature (°C) Fragment Size (bp)
M111 MF MI ElR 56 175
Ml E2F MI E2R 56 368
Ml E3F MI E3R 52 209
M] E4F MI E4R 52 219
Ml MUTF MI MUTR 52 228
Ml E6F MI E6R 52 225
Ml E7F MI E7R 52 216
M1 E8F MI E8R 52 261
Ml E9F MI X1466R 56 541

 

 

 

 

 

containing exon 7 and portions of its ﬂanking introns. This fragment ampliﬁes only from
canine DNA and not hamster DNA; therefore the conservation of the canine gene could
be determined.

The TMFI and ROBOI primers were designed to span introns within those genes.
Since intron sequences usually are not conserved between species, they were expected to
produce ﬁagments of different sizes from dog and hamster. This was conﬁrmed ﬁ'om
dog and hamster genomic DNA, however the hamster Tmﬂ ﬁ'agment was preferentially
ampliﬁed from the panel clones and canine T MF 1 could not be seen. A canine TMFI
fragment from genomic DNA was sequenced and new primers were designed within the
introns so that the new ﬁagment could only be ampliﬁed from canine DNA. The
conservation of the canine TMFI and ROBOI genes were then tested in the panel. A
similar pattern of conservation was seen for MIT F and T MFI , whereas the pattern for
ROBOI was dramatically different. The conservation of the three genes in the panel
clones was input into the RHMAP software. Signiﬁcant linkage was found between
MT F and TMFI (LOD score = 13.09), but not between MIT F and ROBOI (LOD score
= 0.06). It appears that ROBOI might be located on a different canine chromosome than
the other two genes. MITF and TMFI are both being examined for polymorphisms that
could be used for mapping. The conservation of the MITF pseudogene is also being
tested to determine if it is closely linked to MIT F or located elsewhere in the genome, but

so far has not been successfully ampliﬁed from the radiation hybrid panel.

65

Two canine KIT sequences were already available in GenBank (Accession
numbers AF 044249 and AF 099030). The sequence AF 044249 was used for the design of
PCR primers. The primers were designed for use in both the ampliﬁcation of the c-KIT
coding region from cDNA and the potential ampliﬁcation and sequencing of the introns.
The primers designed for the KIT gene are given in Table 8, and the ﬁagments used for
the ampliﬁcation of the coding region are given in Table 9. Coding region fragments
were ampliﬁed, puriﬁed and sequenced from the same normal dog as the other genes.
The sequence obtained from the dog is given in Figure 7 and is available in GenBank
(Accession number AF 448148). The coding region of the KIT gene is 2940 bp in length.
At the nucleotide level, it is 88.3% identical to the human and 81.9% identical to the
mouse. At the amino acid level, it is 88.5% identical to the human and 82.0% identical to
the mouse. The sequence contained numerous differences from both of the previously
published canine sequences. The same sequence was obtained in all dogs tested,
indicating that the differences in the published sequences are likely to be sequencing
errors and not polymorphisms.

During the sequencing of the coding region, 6 neutral SNPs were found within the
gene. None of these polymorphisms correspond to the differences seen with the other
two published canine sequences. In addition, introns 10 and 11 were small enough to be
ampliﬁed and sequenced from genomic DNA. Intron 10 contained 1 SNP, and intron 11
contained 4 SNPs. The locations of the SNPs in the coding region are shown in Figure 7,
and the sequences and SNP locations for the introns are given in Figure 8. The sequences

of introns 10 and 11 are also available in GenBank (Accession numbers AF 448146 and

66

Table 8. Primers designed for the KIT gene. The location is given relative to the

start of the canine coding region.

67

Table 8. Primers designed for the KIT gene.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Name Sequence 5’ end Direction
KIT 11F TCAGAGTCTATCGCAGCCAC -24 Forward
KIT IlR TGCTGGATGGATGGATGGGA 132 Reverse
KIT 12F TGCACCAACAGAGATGGCTT 295 Forward
KIT 12R TTGCCTTCTTTCCCATACAA 395 Reverse
KIT 13F GACGGTGCTGTCCAAGAAAT 585 Forward
KIT 13R TCAGGAGAGAGCTTGTTTTG 676 Reverse
KIT 14F GATGTGTCTAGTTTCGTGGA 715 Forward
KIT 14R AATTGAAGTCAC CAT GATGC 814 Reverse
KIT I5F GGATCAGCAAATGTCACAAC 898 Forward
KIT 15R ACTCATCATGGGGAAGATAT 969 Reverse
KIT 16F AGAT TATCCCAAGTCTGACA 1089 Forward
KIT 16R TGTAAGTGCCTCCTTCGTTC 1180 Reverse
KIT 17F GTGTCCAATTCCGATGTCAA 1188 Forward
KIT 1241F CAAAAC CAGAAATCCTGACT 1241 Forward
KIT 17R TGCAACCACACACTGGAGCA 1302 Reverse
KIT 18F TGGTTGCAGGATTCCCAGAG 1295 Forward
KIT 1355R CTCTGCTCAGCTCCTGGACA 1355 Reverse
KIT 18R ACAGAGAC GAGTTTTGCATC 1408 Reverse
KIT 19F ACAACAATGTAGGCAGGAGT 1490 Forward
KIT MutF CAAATCCATCCCCACACCCTGTTCAC 1552 Forward
KIT 19R ACAAAGCCAATCAGCAAAGG 1598 Reverse
KIT 11 1 F GAGGAGATCAATGGAAACAA 1690 Forward
KIT 1731 CT CAAATCCATCCCCACACCCTGAT CAC 1731 Forward
KIT 1806R CACTTTCCCGAAGGCACCAGCACCCA 1818 Reverse
KIT 112F CTGATTAAGTCGGATGCGGC 1840 Forward
KIT 112R TAGGGCTTCTCGTTCGGTTA 1920 Reverse
KIT Il3F TGTGAATCTTCTTGGAGCGT 1968 Forward
KIT 1] 3R AGAT CACCATAGCAACAATAT 2042 Reverse
KIT I14F AGGAAGAT CACGGAGAAGTG 2090 Forward
KIT 114R AACGTAAGAAACGCCGGGTT 2202 Reverse
KIT 115F GCGTTTCTTACGTTGTGCCA 2189 Forward
KIT I15R GCCAACTCATCATCTTCCAT 2297 Reverse
KIT I 16F AGAGGACTTGCTGAGCTTTT 2307 Forward
KIT 116R TTCGACCATGAGTAAGGAGG 2422 Reverse
KIT 11 7F TCTAGCCAGAGACAT CAAGA 2445 Forward
KIT 117R CAGTTGAAAATGCTCTCAGG 2540 Reverse
KIT 118F GAAAGTGATGTCTGGTCCTA 2554 Forward
KIT 118R TTTGAATCGACTGGCATCCC 2642 Reverse
KIT 119F GGGATGCCAGTCGATTCAAA 2623 Forward
KIT 119R AACGTCGGCCTTTTCAGGG 2759 Reverse
KIT I20F CAGATCGTGCAGCTAATTGA 2764 Forward
KIT 120R AACCAAAAGAACAGGGATCG 2995 Reverse

 

68

 

Table 9. PCR ﬁagments used to obtain the KIT coding region.

 

 

 

 

 

 

 

 

 

 

 

Forward Primer Reverse Primer Temperature (°C) Fragment Size (hp)
KIT IlF KIT 12R 52 419
KIT 12F KIT 13R 54 382
KIT 13F KIT 15R 52 385
KIT 15F KIT 17R 54 405
KIT 17F KIT 19R 54 409
KIT 19F KIT 112R 54 419
KIT 112F KIT 115R 54 457
KIT I15F KIT 117R 54 352
KIT Il7F KITIl9R 54 315
KIT 119F KIT I20R 54 373

 

 

 

 

 

 

AF 448147). The allele frequencies of the intronic SNPs and one of the SNPs in the
coding region were determined as part of a study on mast cell tumors, described in
Chapter 6. A 644 bp genomic DNA fragment containing bases 1552-1818 of the canine
KIT coding region and introns 10 and 11 was used to screen the canine genomic BAC
library. A total of 17 clones were found that potentially contain the KIT gene and are
currently under investigation. This same 644 bp region was found to contain duplications
or deletions in some canine mast cell tumors. The analysis of these tumors will be

presented in later chapters.

KIT LG

The human KIT LG and murine Kitl sequences were retrieved from GenBank
(Accession numbers NM_000899 and NM_013598) and aligned using a software
program. Regions of homology between the human and murine sequences were chosen
for the creation of PCR primers as previously described. Primers were designed for use

in both the ampliﬁcation of the coding region from cDNA and the potential

69

Figure 7. Alignment of the cDNA sequences of dog and human KIT with mouse
Kit. Vertical lines indicate exon breaks and the ends of the coding region. Base positions
relative to the start of the canine coding region are given to the left. Nucleotides identical
between species are shaded. SNPs in canine c-KIT are shown as white capital letters on a

black background. R = G or A, Y = C or T.

70

lﬁgne7.

Bun
Hon

D09
Hm-
Mbu

au-
Mbu

Bun
Mon

D09
an.
Mbu

009
Run
Mbu

Dog
Bun
Mbu

D09
Bun
Mon

Bun
“bu

47

97

147

197

243

293

343

393

| swan:

ygcga gagaggcgc'cgcggcgccfbg aEEEEE EEEEEEECCEQCECG
wgcgatgagaggcgctcgcggcgcctgggattttctctgcgttctgctEQ
s cct-eeaaciggtctgcgtcctﬂtigg

   

   

   

1 ll 2 __1__1_LH_11__
ggf tgwﬁgatgctcggggﬁccagacag §é¥foﬁﬁéaﬁbéaﬁCQ§fgag
LactgcQtl- ~~~~~~ cgtccagacaggctcttctcaaccatctgggag
Ecgtgcgcﬁ ~~~~~~ EtiggcagacachaﬁggqtcgggcatCLgCa,.

 

-n*—-

7caggggaaccgtcf5tcCcatccatccatccagcaaaafcagagttaaﬁ
caggggaaccgtctccaccatccatccatccaggaaaatcagacttaag

ggcgtctcdgbcatcaatgcagwﬁ_gg§cggggeggggtea

   

 

 

    
 
 

gtchchgggcgacg_gattaggctgntatgcactgatccgggcttt

gt"a3t$€c§§|ﬁacq396ttaggctgtchgéa8c§3cEEa§§aEEE§
SEE96aICtE_CQ§QaCCEBCEQCEEQaigtﬂﬁatLﬂﬂLQdclaitﬁE

 

 

   
 

Ca“gfggaettttgag§ca ctg ~~~~gEc53c§gaatg3g33cﬁc\c”
caaatggactttEgagatht ~~~~5tgaaacgaatgagaataagCJ

.qagaLQaasttacaagd~ anatttcaaﬁﬂﬂﬂﬂiﬂgttgﬁggagaggaE

 

 

 

cﬁacﬁaatggatcaaaﬁagEaggcagaggBtggEcacacgggcaatEacQ
gEatgaatggatcacggaaaaggcagaagcééccaacaccggcaaatac}

ammmcamamcmaetﬁgawcamﬂ

 

 

 

   
  

gtgcaccaacag3g5tggcth5§cadgECcatttatgtgtttgﬂcigQ
gtgcaccaacaaacacggcttaagcaattccatttatgtgtttgttagQ

gceéséﬂcaa.gsscaCicgagLLgtatt_gcat ttt ttarﬁ

     

II 3
atcctg a55§cttttcchgttgaccttcccttgtatgggaaagaagg
atcctgccaagcttttccthttgaccgcttcttgtatgggaaagaag

atcctgccagagttttgmggﬁttgggcttccctgggtgggc aa aa

 

 

   
   

 

 

 

*aacgacacgctggtccgctgtgctctcacagacccagaagtgaccaat

ﬂtﬁatﬁcgctggtccgctlctctgaégﬁacccagaagtgaccaatQ
geg_ggggctggtccgctdct_pctgacagaccc§chﬁ_gtw caat

71

Figure 7 (cont’d).

Dog
Bun
“bu

Hun
“bu

Hun
“bu

Dog
Bun
“bu

nun
“bu

Bun
“bu

Bun
“bu

Bun
“bu

Hu-
“bu

443

493

543

593

643

693

743

790

840

 

 

 

 

 

attCCanccccaaggcgggcatcatgatcaaaagtgtgaaacgcgcct
EEEgsaaﬁcqpcaagacngggatcaqcaxsagaaacctgaénaaasc9L

W95 Eggtﬁc‘éaictqgé‘atéa'cqétngﬁa‘acgtqaagc"QCdagE3

 

 

 

  

tEatcggctct5cthcactchctgcggaccagaEgggcagEa‘ .
Ecatcggctctgtctgcattgttctgtggaccaggagggcaagtcagtga

CacssqctsEgugicsghﬁangcaaﬁtCigﬂgtnicndtlcaﬁgﬁctgf

3 ll 4
gt ca3g5aattcaccctgaaagtgagggcagccatc3g5gcthaEEa
gtc gbaaaaattcatcctgaaagtgaggccagccttcaaagctgtgcca

attstaaca.mtt9a9¢crcaeaaaﬂcnadaaasgaEgaagnssaﬁcsc

ttgtgtaﬁﬁafccaaaacaagcﬁctctcCQgEaggaaggggaagacF
ttgtgtctgtgtccaaagcaagctatcttcttagggaaggggaagaat

ttgtgtct g gc'tggaacaagtclcgtcgggi agaﬁ_gggg§caEgt

 

 

 

 

 

   
 

tEtEtgatgtgdtttﬁtaaaagatgtgtctagtEtEgtagﬁctagEEgX
acagtgacgtgcadaataaaagatgtgtctagttctgtgtactcaacg

t§§gﬁ_ggtgtgcagcataaaagatgtgtctgcagccgtgahgmgc‘t]

4 II 5

gataaaggaaaacaicEagcagacta3tgc ~~~E33acﬁcﬁaagfa aEE
gaaaagagaaaacag~~~tcag ctaaaci

~~~ aggagaaatataa
ciaaadath_gcctE;ggctcagcicatggcc tﬁaadcic

Echgg33333tEgEgaCQEC33EEttgaacgtcaggaaéag EE gEtEé
Igctggcatcacggtgacttcaattatgaacgtcaggcaacgttgacta
ct acta

c cigghg_gacttcaattatgaacgcggggjga_gc

 

 

 

  
   

agttcagCgagagttaatgattctggagtgttcatgtgttatgccaat

«agdfcagcaagagff aEgaEEcEggagEgEEca g g ccC3ﬁ 33
,Ct

gﬁcaagagtggIcgattctggagtgttcatgtgttatggcaat

 

72

Figure 7 (cont’d).

“bu

009
Hu-
“bu

Bun
“bu

Dog
in.
“bu

890

940

990

1040

1090

1140

1190

7

1240

1290

5 II 6
35%€5€3§3€E§3€3§3€856363583385Ef§33§3f5§€3§5€3§'
tacttttggatcagcaaatgtcacaacaaccttggaagtagtagataa
tactttgggatcagcaaatgtcacaacaaccttga§§g§§g£§g§a “

   
   
 
 

95%€%£%%3353t8tf5686§fg§t§ag§EEEéﬁﬁatafff§€333tEC
gattcattaatatcttcccggtgagaaacactacagtatttgtaaacg

,attca'cggcatpgctggtgtgjéhaacactacqgtatttggggc

  

 

 

  
  

ggég gﬁétgﬁgﬁéﬂcfgéffgffgaatafgﬁggéétgtEéﬁééﬁéag:
ggagaaaatgtagatttgattgttgaatatgaagcatthccaaacqt
. canagetttggtgggggééggcnggggctacccaaaaaéc

 

 

gcaccagcagtggatctatatgaacagaaCCttcaétgataaatgggaE
acaccagcagtggatctatatgaacagaacctgggctgataaatggga

agaaaaagaanaaaﬁataLangaagaagasgﬂcggbﬁaicagagﬂaalg

6 ll 7 _
attatcccaagtctgacﬁatgaaagtaatatcagatatgtgagtgaacﬁ

1-

 

 

attatggcaagtctgdghaggaaagtaatatcagataegtaagggaac‘
ﬁmﬁgtmawtﬁcamﬁdcmaCCB@9..

 

 

qtctaacgagattaaaaggcaccgaaggaggcacttacacatnccta

E;atctaaccagattaaaagagIaEgaaggaggcacttacacatttaa
c
' gCEﬂgEQSQQQLLQQQQQQQQQaﬁéégsagggéeﬁkitEQCL§EQ§9

 

 

gtccaattdcﬁéﬁgtcEéEtEtﬁagﬁtggcatttaatgtttatgtgaag
gtccaattctgacgtcaatgctgccatagcatttaatgtttatgtgaat

tccacEctgatgcgggggcttccgtgaqgggcagcgtttgcgggggﬁ

caaaaccagaaatcctgacﬂcltﬁaaEQtEtcaclﬁatggcatgctccf
'caaaaccagaaatcctgaqttacgacaggctcgEgaatggcatgctcci

caaaacca aaatcct a gEacgacaggctCQQagatggcatgctccﬁ

  

 

 

 
      

 

 

iEgEgEgatpﬁcaggattcccagagccégEagEagattggtattﬂcEEF"
atgtgtggéagqgggattcccagagcccacaatagattggtatttttgt:

EEQEQEQQEQQa9HQQEEEEQghESESEQEEEEEQQEEQSEQEELELSEa

73

Figure 7 (eont’d).

Bu-
“bu

Bu-
“bu

§

§

§E§ NE? E

ﬁé’

1340

1390

1440

1490

1540

1590

1640

1690

1740

 

8 II 9
gqagétgégcagagatgttbtatCE~3aEtgg§CC3aEQGatqudas
aggaactgagcagagatgctctgcttctg actpccagtggatgtgcae
.ggggsaneggaaangs_§aﬁcag_g2§gﬁctCagsgggggacnsa;av

 

 

 
  
 
 

 

caﬁtpaagggatctgggccaccgtttggaaagctagtggttcagagttV

EEgEaaaactEgEEchgtcaccgﬂctggaaaactagtggttcagagtta
gichgaat9taLsLaLaiaggsatLLaaaaassign$aaLLsaaaaLLj

 

 

 

Eaﬂcﬁatiatagtgccttcaagcacaatggcaﬂaﬁﬂcﬁagtgtagﬁgctﬁ
tatagattctagtgcattcaagcacaatggcacggttgaatgtaaggctg

EggagackacaacltE$990gReagaaCageaggaﬁgﬁssisxaaaggc<

 

EcaaaaEthaEgcagﬁagttctgccttttttaactttgcatttaaaggg
g

caacgatgtgggcaagacttctgcctattttaactttgcatttaaag
c aac at t caa a tEgcaEggﬁcEﬁtlaactttgc atttaaqg~~

"WIND-"'1

 

9 || 10
EacagEaaag3aEaaatccatccccacaccctgftcaéaﬁctttgctggg

 

gagggggagcaaatccatccccacaccctgttcadtcctttgctga
~~~~~~~~~~ Egcaaatccgggcccaca gtEtgttcaggEgchg_§c

 

 

ggctttgtgatcgcagctgdaﬁtgatgtgcattaﬁcgtgatgattcﬁt\
gtttcgtaatcgtagctggcatgatgtgcattattgtgatgattctg_

gEcgcagctggggcgatggngﬁchttgtgatggﬁgEgc‘

10 ll 11
E333333g53ﬁctacagaaacccatgtatgaagtacagtggaaggttgtg
ictacaaatatttacagaaacccatgtatgaagtacagtggaaggttgtr
ictacaaatattggEggaaacccatgtatgaagtacga aa tt tc

 

   

aggagataaatggaaacaattatgtttacatagacccaacacaacttc:

aggagacEaEggaa333aEEaEgEEE33aa 33333aca63g5310“
a ataaatggaaacaattatgtttacatagaccggggg aacttc

11 ll 12

  
 

‘ .a E3E3hafgggagf3f3333gaaac3§bcfgaac _
tatgatcacaaatgggagtttcccagaaacaggctgagttttgggaaa‘

Latgétcacaaaggggggtttcccagaaacaggctgggttttggaggg‘

 

 

74

Figure 7 (cont’d).

D09
Hun
“bu

1790

1840

1890

1940

1990

2040

2090

2140

2190

 

 

 
 
 
 

gt:tgggtQCtggthCttnggadaEEgEtthaEccactgcatatggl
EcctgggtgctggaQCtttcgggaaggttgttgaggcaactgcttatggw

VaEEEgga ct t ccttc cggga aggEcé

 

 

12 ll

cfﬁattaagtcggatgcggccatgactgttgcbéEtEEba gc caaac
taattaagtcagatgcggccatgactgtcgdtgtaaagatgctcaagc

t attaagpcggEEgEt coat a aﬁttgccgEgE_gatgctcaaac

 

 

13

Fag?gCEEEEEEEEECEEEcgagaachCfEEEEECEQEgEEcéEEEEEt
gagtgcccatttgacagaacgggaagccctcatgtctgaactcaaagtc
Eagtgcccatttaacag_§a§_g§ghccctaatgtEggaacEgEEg tc

 

  
  

IgadEEEECECEEEmEEEEtEEgaaE EE EgaéicftEEE'gagEgqu
gagttaccﬁtbgtaatcacatgaatattgtgaatctacttggagcctg

a cEaccEghchatcacatga atattg_ggchEgEEEggcgEa

13 ll 14 7%
ggéﬁffg gagéﬁEécaEEtEggE ‘fEEEtEaaE Efﬁffgdf Eggtg:
W cqattggagggcccaccctggtcattacagaatattgttgctatggtg'
‘ ﬁnghgggggcccaccctggtcattacag_atatt ti 9 ‘

 
 
   

 
  
   

 

 

; c‘ftttaa‘ﬁ t? fft’EC5'539%533’5‘53f‘f‘é‘ﬁ‘fff'E'E'E’E’cj'ﬁ't c‘aa ag
.Cttttgaattttttgagaagaaaacgtgattcatttatttgttcaaag'

IcgtLtgéattLtgxggggggggagEngchggtttatLﬂt £9333.

 

 

   
 

9§33Qatcéc gﬁqaaﬂtﬁgcactttataagaaccttctgcattaaaaq
.ggaagatcat cagaagatgcactttataagadttttctgcattcaaae

a ._ ._ ~_g§dgi939mcm9399uam93999 L993393€L9330

14 || 15
agtCttcctgcagtgacagtactaatgaatacatggacatgaaaCECf¥
agtcttcctgghgcgatagtactaatgagtacatggacatgaaacctg
Cm9gcm~~~imﬁtlammdtmmmgm9

  

 

 

 

   

   

 

agtttcttatgttgtcccaaccaaggccgacaaaaggagatctgtgaga

kgtttcttacgttgtgccaaccaaggcagacaaaaggagatctgcgagig
599999999499999999999999a999993999999999cg§amg

75

Figure 7 (cont’d).

Bun
Mbu

nu-
“bu

Dog
Hun
Mbu

Hu-
“bu

Bu-
Mbu

Bu-
Mbu

Bun
Mbu

Dog
Bun
Mbu

Dog
Bun
Mbu

15 II 16
2240 Eaaéctcatacatagaaaagﬁatgtgactcctgccatcatggaagatgat

 

 

aggctcatacatagaaagagatgtgactcccgccatcatggaggatgag
[agaEgggmacaLaaaaagaaicn;aagtgg§9994$0409§a49909§9

 

 

agttggccctagadttagqagacttgctgagcttttcttaccaggtggi
ClﬂﬂﬁﬁsﬁgﬂsﬁgigﬁdtﬁitELSQLQEQQ£SC£QC£§$$$EQEQS

16 II 17
2 3 4 O Eaaggdtﬁtggﬂagtcct‘i‘gﬁcctdgﬁ'attgtattcacagagacttga

2290 Eagttggctctagatﬁtagﬁggacttgctgagcttttcttaccaggtgig

 

 

 

aaaggQCathQtttcctcgcctccaagaattgtattcacagagacttg
éaéggcgthgégi;9g£93992g222922£gggégggagégagit t

 

agccagaaatatcctccttactcatggtcggatcacaaagatttgtgaﬁ

2390 tﬁatﬁgaaatatcctéEttactcatggtcdaﬁtcacaaagatttgtgaﬂ
ea cca ggatagggggggcaggggcggggggatcacaaagatttgcggﬁ

 

 
  

ttggtctagccagagacatcaagaatgattctaattatgtggttaaagi
~cggggtagccagagacatcgggaatgattggaattgc t tcaaa

17 II 18

2490 aacchcagE?§EEEgE§3agtggatggchEEgagagc3?fE%%%éb§
iaacgctcgactacctgtgaagtggatggcacctgaaagcattttcaacg
a‘tggazgacggEQCEtgaagtggatggcacgagagagcatggtcgggﬁ

2440 E;tggtctagccagagacatcaagaatgattctéattatgtggtcaaag

 

2540 g afﬁaéngffm353§E§§E§E€E§EEEEfﬁfﬁﬁﬁifffE€€Eg€3“
tgtatacacgtttgaaagtgacgtctggtcctatgggatttttctttga
Ecgggtacacatttgaaagtgatgtctggtcctatgggatttgcgﬁcg
18 ll 19
2590 'éaétéfﬁdictttagqaagéaﬁaccCEECCéﬁggéatgcaaatégafﬁa
Eigctgttctctttaggaagcagcccctatcctggaatgccggtcgattﬁ
a ctcttcth;taggaagcagcccctaccgagggatgccggtcgﬁcgg

 

 

 

t agttctacaagatgatcaaggaaggcttccggatgctcagccctgaa

2640 a aqttctacaaqatgatéaaﬁgaagagftccggatﬁbtéaéccctﬁégi
C égtEgtacaaQQEQatcaaggaaggctgccggatgg;cagccqgaag'

76

Figure 7 (cont’d).

Dog
Hun
Mbu

Dog
Bun
Mbu

909

Run
Mbu

Dog
Hun
Mbu

Dog
Hun
Mbu

Dog
Bun
Mbu

19 ll 20

2690 tgcaccfgctgaagigtatgacatcatgaagaagtgctgggatgctgaﬁ
cgcacctgctgaaatgtatgacataatgaagacttgctgggatgcagaQ

gEggggcgaaatgtatg_ggEcatgaagacttgctggggcgctggc

 

 

2740 EccCtgaaaaggcggQEgEtcaagcagaﬁcﬁfgﬁagctaattgagaagcQ
ccctaaaaagaccaacattcaagcaaattgttcagctaattgagaagcQ

 

 

E_gt&gaaaaggccaacattcaagca§g£_g§ceaaﬁstEggggge_ggé
20 ll 21

 

 

 

atttcagagagcaccaatcatatttactccaacttagcaaactgcagcq

2790 EatttcagatﬁgcaccaatcatatttStE“”§EEc€c§Eg§actgcagca
aﬁcﬁqgﬂécﬁgcaccaQQQQCEtttactccaactgggcaaagtggaapq

 

 

2840 EcaacccagaacgEcccgtggQ~~~Egaccattccgtgcggatcaattcc
Ecaaccgacagaagcccgtggq~~~agaccattctgtgcggatcaattQt

RCQQEQQQQﬂgéﬂQGQPQLﬂgﬁggthgﬂQQﬁtQQﬂglﬂaﬂqgtgaﬁcﬁég

 

 

tcggcagcaccgcttcctcctcccagcctctgcttgtgcacgacgath

2887 Etgggcagcagcgagfcttccacccagcctctgcﬁgﬁﬁabacgaagatgg
raggcagngggadctcttqt§99séggqcsgsctangsaggaaggnac

STOP

2937 gE_3a§ca§g§gg5gtaccggﬁggtatEEéaaaEEaﬁma

Etga~gcagaatcagtgtE~tgggtgapccctccaggaa
Etg§~acaga§aEccaag ~ccaacaggEtt§gEtlctIt

77

Intron l0

1 gtaatcattcatttgttctctaccctaagtgctataatgatcgaaatgtt
51 attcattaaaagatgatctltctctcttttctccccccaccag

Intron l 1

1 gtcagtatgaaa'aggggctttccatgtaacctttttgtgtacgtgtaac
51 aatgactttagggaaccccattagcttcctttgttctgttccaactgaga
101 caataagtattttctgtgaagtttcatca'ttttgatagattcc'cataa
151 agcaccttatagagaaatgtccttagctggatttgtccttaattccttaa
201 caattccttgattgttgactttgaaattacccagatgctcctttggtcct
251 a'caccaccccttactcttttcttcctttctgcag

Figure 8. Sequences of canine KIT introns 10 and l 1. SNPs are shown as white

text on a black background. R = G or A, Y = C or T, K = G or T.

ampliﬁcation and sequencing of the introns. The primers designed for the KIT LG gene
are given in Table 10, and the fragments used to amplify the coding region are given in
Table 11. Fragments of the coding region were ampliﬁed, puriﬁed, and sequenced from
the same normal dog as the other genes. The sequence obtained from the canine KIT LG
coding region is given in Figure 9. The coding region of the canine KIT LG gene is 825
bp in length. The coding region sequence is identical to a canine KIT LG sequence that
was later found in GenBank (Accession number $53329). It is 91.0% and 87.1%
identical to the human and mouse, respectively, at the nucleotide level. At the amino acid

level, it is 85.4% identical to the human and 80.7% identical to the mouse.

78

Table 10. Primers designed for the KIT LG gene.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Name Sequence 5’ end Direction
MGF IIF AGCTAAACGGAGTCGCCACA -57 Forward
MGF IlR CGAGAGGATTAAATAGGAGC 67 Reverse
MGF 12F GGAATCGTGTGACTAATAAT 89 Forward
MGF 12R GGACATTTATGAGGGTTATC 175 Reverse
MGF 13F GATAACCCTCAAATATGTCC 156 Forward
MGF 13R TCCAGAAGATCAGTCAAGCT 257 Reverse
MGF 14F ATTCCATCAGATACAAACTTG 293 Forward
MGF 15R CTGCAACAGGGGGTAACATA 571 Reverse
MGF 16F TATGTTACCCCCTGTTGCAG 552 Forward
MGF 16R GCTCCAAAAGCAAAGCCAAT 704 Reverse
MGF 17F ATTGGCTTTGCTTTTGGAGC 684 Forward
MGF 17R AAACATGAACTGTTACCAGC 882 Reverse
Table 11. PCR fragments used to obtain the KIT LG coding region.
Forward Primer Reverse Primer Temperature (”C) Fragment Size (hp) 1
MGF 11F MGF 13R 54 314
MGF 13F MGF 16R 52 546
MGF 16F MGF 17R 52 331

 

 

 

 

 

 

Analysis of candidate genes ' roles in hereditary deaﬁress

Total RNA was extracted from tissue samples ﬁom one deaf dog in each of 7

different breeds: Dalmatian (spleen), Australian Cattle Dog (spleen), Catahoula Leopard

Dog (ovary), Boxer (testis), Great Dane (testis), Springer Spaniel (uterus), and Jack

Russell Terrier (ovary). The RNA was treated with DNase and reverse transcribed into

cDNA. The coding regions of EDNRB, MITF-M, KIT, and KITLG were completely

sequenced in all 7 dogs. The individual exons of the MIT F -M gene were ampliﬁed and

sequenced ﬁom genomic DNA in an Australian Shepherd with both deafness and

79

Figure 9. Alignment of the cDNA sequences of dog and human KIT LG with
mouse Kit]. Vertical lines indicate exon breaks and the ends of the coding region. Base
positions relative to the start of the canine coding region are given to the left.

Nucleotides identical between species are shaded.

80

Iﬁgme9.

D09
Hun
Mbu

Hun
Mbu

Hun
“bu

Doc
Bu-
Mbu

Hu-
Mbu

Bu-
Mbu

I START
26588688€gggc gga acagc§“%388£f£'“EF‘Eﬁﬁzagaaacac
Jcact9E;tgtgctggatcgcagcgctgcctttccttatgaagaagacac
'c at cct , ct atc ca c ct cctttccttatgaagaagacag

-37

   
   

 

1 ll 2
14 asaffa‘wfféfcaéffg“mftfatoff“é§8taaf88t§f%€3§%ccﬁ
aacttggattctcacttgcatttatcttcagctgctcctatttaatccQ
aacttggattatcacpggcatttatcttcgaptgctcctatttaatccﬁ

 

 

 

6 4 Ega'ﬁ‘caé‘é‘a’éfa arggﬁqaestﬁgagggaaaE‘ﬁ-a ﬁﬁt'ﬁ'ﬁﬁgﬁf‘é‘fg? ,

7tcgtcaaaacggaagggatctgoaggaatcgtgtgactaataatgtaa
.‘titcaaaaﬂCBagiaBétctgcgggaatgcggggactgataatgtaa-

2 II 3
114 gE_EgttaeaaaattggtquaaatcttccaaaagactafaaﬁétagE a
gacgtcaotaaattggtggcaaatcttccaaaagactacatgataacca

gagaﬁsagaaéactm, aacaaatcnggggaat QLaLaLgataaec

3 || 4 ,
164 .caaatatgtccccgggatggatgttttgcctagtcattgttggataagi
_caaatatgtccccgggatggatgttttgcdaagtcattgttggataag

caacmmgnmmtmammmcmcla

   

 

 

 

214 tEatggEglaﬁEggEtgtcagtcagcttgactgatcttctggadaath
agatggtagtacaattgtcaqacagcttgactgatcttctggacaagtQ

tmammat.sacmg.m§na ergtxgmmsg

 

 

264 tcaaatatttctgaaggactgagtaattattatﬁtcatagacaaacttP
tcaaatatttctgaaggcttgagtaattattccatcatagacaaactt

Gammatatggct aa ctt a taatLactggggcggaw

4
314 gaaaatagtggatgdtbttgtggagtgchagaaagltEctcaﬁth
gaatatagtcgatgaccttgtggggtgcgEcaaagaaaactcatctaa

gﬁaaaatagtggatgaccchEgttaggggggﬁgagaaaaggggcﬁg a

I 5
364 E_Egtaaaaaaag5acatEagagcccagaaatEaggcﬁtfmtéctcct:3

 

 

 

gatctaaaaaaatcattcaagagcccagaacccaggptctttactcctg

E_§a§aaaggﬁ_§gtEﬁgaagagggcagagalt33atcEtttactcct

81

 

Figure 9 (cont’d).

Dog
Hun
Mbu

Bun
Mbu

Dog
Bun
Mbu

Dog
Hun
Mbu

Dog
Hun
Mbu

Dog
Bun
Mbu

Dog
Hun
Mbu

D09
Bum
Mbu

009
Hum
Mbu

414

464

514

564

614

664

7

714

764

814

 

 

gaattctttagaatttttaataqatccanﬁatgcctttaagqacthg
gaattctttagaatttttaatagatccattgatgccttcaaggacttQ~

aaaxtetttaatatttgcmanaganscatggagggggEggggs§9§§g~

 

 

 

 

 

aggcﬁgtggcatcQaaaEgEagtgiantgtggtttcttcaanttaagQ
~~gQagtggcatctgaaactagthtEgtgtggtttcttcaacattaagQ

~~§tggtggeatctgchcnagtgacggtgtchc;cutcaacattqggé

5 ll 6
EctgﬂtﬁaagattccagagtcagtgtcacaaaaccatttatgttaccccQ
Ectgagaaagattccagagtcagtgtcacaaaaccatttatgttaccccq

EﬁcmlﬁﬁﬂﬂmﬁﬁﬁﬁsméﬁﬁWQSFCQ

6 || 7
gttgcagccagctcccttaggaatgacagcagtagcagtaataggaagi

 

 

‘gttgcagccagctcccttaggaatgacagcagtagcagtaataggaag
,g tgggmggcagggccct§_ggg3;ggcagcagtagc agtaatagga 9a

 

 

EtEaaattEEattggagactccaaltfacaatgggcagccatggcatte
EcaaaaaQccccctggagactccagcctacactgggcagccatggcattw

ECgEmagggEgcctgaﬁgactggggcctacaatggagagccat catlte

 

Ecagcattcttttctcttgtaattjagtttgcttttggagccttatact
Egagcattgttttctcttataattggctttgcttttggagccttatact

ghgtcEgaEEEEgEEEgEaattggcttEggttt tggagccttatact

ll 3
aagaagaaacaaEcaialdEcEcaagaaEngtgaaaaEatacagatt3

   
 

aagaagagacagccaagtcttacaagggcagttgaaaatatacaaatt
aa aa aaaca tEaagtcttacaaqggcagttqaaaatatacaggEEﬁ

:yﬁﬁgﬁﬁggafaifg aaafaagEaQgEfgcaagagaaagagaEgﬁég QEQ
tgaagaggataatgagataagtatgttgcaagagaaagagagagagttQ
t aa a ataat a ataagtatgttggaacagaaagagagaggagﬁ

 

STOP 1
aégaggtqtaQ~Ff§EggEfEcEatEéaéééfaffééﬁfQtﬁﬁﬁﬁcfta
-aagQabtgtaQattgtgggjgggtatcaacactgttactttcgtacatt~
éagaggtgta§~tgg£99ac~922292§s§tt9ttagcﬁthcegggt'

82

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microphthalmia. Individual exons were also ampliﬁed from the 7 dogs above. For each
exon, the ampliﬁed fragments from all 7 dogs were pooled together and the pooled DNA
was used as a template for sequencing. An additional 7 deaf Jack Russell Terriers were
similarly pooled and sequenced, and 21 other deaf dogs were partially sequenced for
MIT F in pools of 7 as well. No mutations relative to the normal sequences were found
for any of the candidate genes in the dogs tested. For those dogs in which individual
MIT F exons were sequenced, no mutations were found in the bordering splice sequences
either.

Since polymorphisms had been found for the EDNRB and KIT genes, these could
be used as markers that would allow the investigation of whether either of these genes
was associated with deafness. DNA from cheek swabs was available for 59 Jack Russell
Terriers, 51 of which belonged to a single pedigree. Among these dogs, 7 were
conﬁrmed to be deaf and 41 were conﬁrmed to have normal hearing by BAER testing.
The remaining 11 dogs had not been tested, but had not been reported with hearing
problems and were considered to be normal. The regions containing the SNPs in intron 6
of EDNRB and intron 10 of KIT were ampliﬁed by PCR for each dog. Restriction digests
that would differentially cut each allele determined the genotypes of the two SNPs in
each dog. The results of these experiments are shown in Table 12 and Table 13. For the
SNP in intron 6 of EDNRB, 3 of the deaf dogs were homozygous for a G allele, 1 was
homozygous for an A allele, and 3 were heterozygous. The region containing the SNP in
intron 10 of KIT could only be ampliﬁed from 56 dogs. The samples that did not amplify
represented 1 normal and 2 deaf dogs. Of the remaining deaf dogs, 2 were homozygous

for an A allele, 1 was homozygous for a G allele, and 2 were heterozygous.

83

Mk

 

A chi-

Dis

[hes
S€qU

0f (ht

Table 12. Genotype results for a SNP in intron 6 of EDNRB.

 

GG GA AA Total
Deaf 3 3 1 7
Normal 1 8 22 12 52
Total 21 25 13 59

 

 

 

 

 

 

 

 

 

 

Table 13. Genotype results for a SNP in intron 10 of KIT.

 

AA GA GG Total
Deaf 2 2 1 5
Normal 23 18 10 51
Total 25 2O 1 1 56

 

 

 

 

 

 

 

 

 

 

A chi-squared analysis could not be performed on the data because expected values
among the deaf dogs were less than 5; therefore Fisher’s exact test was used to examine
the genotype distributions of both SNPs in deaf and normal dogs. The probability that
the distribution for the SNP in EDNRB was produced by random chance was 1, and the
probability for the SNP in KIT was 0.823. Therefore, no signiﬁcant distribution
difference was seen, and there does not appear to be an association between genotype at

these SNPs and deafness.

Discussion

The coding regions of 4 genes were completely sequenced in the dog. Two of
these genes had not been previously sequenced in the dog, and mistakes in the published
sequences for a third gene were corrected. Partial intronic sequences were obtained for 3

of the 4 genes, and some introns were completely sequenced. Polymorphisms were

frag, 1

identiﬁed

mutations

 

addition. n
the genes.
1‘.
dogs fror
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genesi
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identiﬁed in two of the genes, and a processed pseudogene was identiﬁed in the third. No
mutations were found linking any of these genes to hereditary deafness in dogs. In
addition, no association was seen between deafness and polymorphic markers in two of
the genes.

The absence of mutations within the coding regions of the candidate genes in deaf
dogs from 7 breeds indicates that these genes are not likely the cause of hereditary
deafness in these breeds. Mutations within the promoter regions or splice sites of these
genes, however, may cause altered expression that could lead to deafness. For the MIT F
gene, the splice sites were examined and found to contain no mutations. Most of the
splice sites of EDNRB are available for screening due to the bordering intronic sequences
obtained from a BAC clone. Potential clones have also been identiﬁed for KIT, which if
conﬁrmed could be sequenced to get the splice sites for this gene as well. If mutations
cannot be found within the splice sites of the candidate genes, this would strengthen the
case for excluding these genes as candidates for deafness in the breeds studied. Although
the sequences obtained for the genes in this study extend into the 5’ untranslated regions,
the promoter sequences of these genes have not yet been obtained. Further study of these
genes should include sequencing of the promoters if possible. In the MIT F gene, each
isoform is thought to be under the control of its own speciﬁc promoter. It is not known
whether any of the other genes may also have multiple promoters. Altered expression of
these genes could also be tested for by Northern blots.

No association was seen in the Jack Russell Terrier breed between deafness and
polymorphic markers in the EDNRB and KIT genes. For this experiment, it is assmned

that a mutation causing deafness in these genes would be close enough to the SNPs that

85

__._-—~— _._ _ _

recombin;
disequiiit
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inherita
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have

recombination between them has not occurred. This is a reasonable assumption since the
disequilibrium of loci 50 kb apart, a distance similar to the genomic sizes of the genes in

this study, does not decay appreciably for thousands of years, longer than most modern

dog breeds have been established.8 It is also assumed that a mutation causing deafness
will have occurred only once and that deafness in this breed is due to a founder effect by
inheritance from a single common ancestor; therefore no allelic heterogeneity exists.
This means that such a mutation should be associated with only one allele of a SNP and
not the other. Finally, it is assumed that only one gene locus is involved, there are no
phenocopies, and gene conversion has not occurred. Under these assumptions, the
ﬁnding that dogs homozygous for either allele of these SNPs can be deaf suggests that
neither of these genes contain a mutation causing deafness. It is possible, however, that
the mutations causing the SNPs in these genes happened after the deafness mutation was
already established in the population, meaning that deafness could be associated with
both alleles. Therefore, these genes can’t be conclusively excluded as candidates for

deafness. Recently, however, the EDNRB and KIT genes have been excluded as the basis

of white spotting in the Border Collie.6 Since deafness is believed to be associated with
white spotting and Jack Russell Terriers have inherited the piebald allele at the spotting
locus, this is further evidence that these genes are not involved in deafness.

The possibility does exist that some of the genes in this study have multiple
copies in the genome and that these alternate copies contain mutations. Multiple copies
have not been identiﬁed in other species for MIT F , KIT, or KITLG, although the
possibility still exists for multiple copies in the dog. For EDNRB, however, subtypes

have been identiﬁed clinically by differential binding afﬁnity for ET3 competitors in

86

some species, including dogs.1 In the quail, these subtypes have been sequenced, but

were found to share only 74% homology at the amino acid level and so are likely

produced by different genes.4 In this study, two different sequences were found within
one portion of the canine EDNRB gene. These consisted of 14 base changes in a 52 bp
region of overlap between two PCR fragments and would be predicted to cause 6 amino
acid changes. This may potentially represent EDNRB subtypes present in the dog.
Alternately, there may be an EDNRB pseudogene in the canine genome. One of the
fragments can be ampliﬁed from genomic DNA, whereas the other cannot. The use of
RNA that had been treated with DNase, however, means that preferential ampliﬁcation of
a genomic pseudogene could not have occurred with this fragment. Further study of this
region is necessary to clarify what is actually occurring, which may include Southern
blots to determine if more than one EDNRB gene exists.

A potential pseudogene has been identiﬁed for MIT F , however. The apparent
presence of three different sequences for this pseudogene cannot be explained at present
and requires further study. The extent of the pseudogene is also not currently known.
The sequence acquired to date covers much of the coding region of the real MIT F gene.
If this sequence extends further upstream, it is possible that this might actually be a copy
of the MITF gene that can be expressed. The presence of nonsense and frameshift
mutations would result in a nonfunctional protein that might have a dominant negative
effect. Even if this is the case, however, an association with deafness is unlikely. One of
the dogs in which the proposed pseudogene sequence appeared normal was deaf. As with
EDNRB, Southern blots should be used to determine whether multiple copies or

pseudogenes of MIT F exist in the canine genome. Besides the potential pseudogene, it is

87

possible that one of the known MT F isoforms is involved in deaﬁiess, particularly those
that are expressed in melanocytes. In this study, the speciﬁc exon 1 sequences could only
be obtained for 2 MIT F isoforms. Attempts should be made to obtain the 5’ ends of the
other isoforms so that they may be screened for mutations.

As the investigation of the candidate genes in this study was primarily performed
by PCR and sequencing, it is not known whether altered expression of one of these genes
might play a role in deafness. Defects in a regulatory gene might lead to changes in the
level or timing of expression of one of the candidate genes. Although these genes
continue to be expressed even after birth, they are speciﬁcally required for melanocyte
development in even the earliest embryonic stages. If possible, fetal tissues should be
examined to determine if there is altered expression at this time. It is also possible that
altered expression may only occur in certain tissues. The dogs used in this study were
privately owned and euthanization was not an option in most cases. Therefore, in many
cases the tissues used were reproductive organs that were removed when the dogs were
sterilized. Attempts should be made to obtain tissues from more relevant locations,
particularly the inner ear, to investigate gene expression in those areas.

Although ﬁirther studies are required in order to provide conclusive evidence, it
seems most likely at this time that none of the genes studied are responsible for
hereditary deafness in dogs, at least in the breeds studied. Additional candidate genes
remain based on phenotypic similarity to other species, however, namely EDN3, PAX3,
SOXI 0, and RE T. In the event that these genes are also excluded as candidates, other
methods will have to be employed to determine the true cause. During the course of this

study, substantial pedigrees were acquired for the Jack Russell Terrier and Catahoula

Leopard Dog breeds. Although not ideal in their current state, with further cooperation
from breeders and owners they could be completed to the point where they would be
useful for linkage studies. If deaﬁiess could be linked to a particular region of the canine
genome, positional cloning could be used to identify new candidate genes based on

genetic information.

References

1. Brooks DP, DePalma PD, Pullen M, Gellai M, Nambi P. Identiﬁcation and
ﬁmction of putative ETB receptor subtypes in the dog kidney. J Cardiovasc

Pharmac0126 Suppl 3:S322-S325, 1995.

2. Canine Radiation Mapping Project, Universite de Rennesl, France. World Wide
Web URL: http://www-recomgen.univ-rennesl .fr/Dogs/maquette.html. Retrieved
January 6, 2003.

3. Fuse N, Yasurnoto K, Takeda K, Amae S, Yoshizawa M, Udono T, Takahashi K,
Tamai M, Tomita Y, Tachibana M, Shibahara S. Molecular cloning of cDNA
encoding a novel microphthalmia-associated transcription factor isoform with a
distinct amino-terminus. J Biochem (Tokyo) 12621043-1051, 1999.

4. Lecoin L, Sakurai T, Ngo MT, Abe Y, Yanagisawa M, Le Douarin NM. Cloning
and characterization of a novel endothelin receptor subtype in the avian class.
Proc Natl Acad Sci U S A 95:3024-3029, 1998.

5. Mellersh CS, Hitte C, Richman M, Vignaux F, Priat C, Jouquand S, Werner P,
André C, DeRose S, Patterson DF, Ostrander EA, Galibert F. An integrated
linkage-radiation hybrid map of the canine genome. Mamm Genome 11:120-130,
2000.

6. Metallinos D, Rine J. Exclusion of EDNRB and KIT as the basis for white
spotting in Border Collies. Genome Biol lzRESEARCHOOO4, 2001.

7. Schmutz SM, Moker J S, Yuzbasiyan-Gurkan V, Zemke D, Sampson J, Lingaas F,
Dunner S, Dolf G. DCT and EDNRB map to DogMap linkage group L07. Anim
Genet 32:321, 2001.

8. Suarez BK, Hampe CL. Linkage and association. Am J Hum Genet 54:554-559,
1994.

89

CHAPTER 3

Zemke D, Yuzbasiyan-GurkanV. A single nucleotide polymorphism and a (GA)n
microsatellite in intron 6 of the canine endothelin receptor B (EDNRB) gene. Anim
Genet 30:390, 1999.

90

Source/description: Consensus PCR primers to the published human and mouse
sequences were designed to amplify the intron between exons 6 and 7 of the canine

endothelin receptor B (EDNRB) gene. The numbering of the exons is the same as for the

published human and murine sequences1 ’2. A total of 700 bp of canine sequence was
obtained from the resulting PCR product, including the entire intron and both 5' and 3'

exonic sequences. The intron sequence (GenBank accession no. AF 1 34188) matched

that of an intron previously sequenced by others3 and reported to lie between exons 4 and
5 (Genbank accession no. AF 026088). The two sequences were nearly identical,
including a (GA)n microsatellite, except for the presence of an extra G at base 235 in

AF 026088. Comparison of the surrounding exonic sequences reported by Liu et al.

(1998)3 with the nearly complete canine EDNRB cDNA sequence obtained by this
laboratory (Genbank accession no.AFO34530) conﬁrmed that this intron indeed lies
between exons 6 and 7. During sequencing, a single nucleotide polymorphism (SNP)
was found, resulting in a G—A transition as 40 nucleotides downstream of the polymorphic

microsatellite (GenBank accession no. AF134188).

Primer Sequences:

Forward primer, F: 5’-AGACGGGAAGTGGCCAAAAC-3’

Reverse primer, R: 5’-GAGGCCATATTGATGCCGAT-3’

SNP detection primer, S: 5’-GACCAGTGATAAAGTCAAAAATCAT-3’

Microsatellite forward primer, MF: 5'-GAGAAT‘TGGGCATGGGCAGA-3'

Microsatellite reverse primer, MR: 5'-TGAC'I'I' I ATCACTGGTC'I'I 'I'G-3'

91

PCR conditions: All PCR reactions were carried out in 25 uL reactions containing 0.6 U

Taq polymerase, 0.8 p.M of each primer, 1.5 mM MgC12, and 100 M dNTPs. Reactions

were denatured at 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, the chosen
annealing temperature for 2 min, and 72°C for 3 min, followed by a ﬁnal extension at
72°C for 8 min. The initial PCR was performed using primers F and R and 3 pL of
genomic DNA at a 66°C annealing temperature. The resulting band was sampled by
stabbing into the gel with a pipette tip and transferring it to 15 pL of water. Three
microliters of this sample was used as template in the second PCR with primers S and R

for SNP detection and primers MF and MR for microsatellite sizing, both at a 60°C

annealing temperature. The expected size of the initial product was 740 bp, the SNP
detection product was 170 bp, and the microsatellite product z 131 bp. The SNP
detection primer was designed to introduce a partial BspHI restriction site next to the
single nucleotide polymorphism, which is completed when the polymorphic base is A
and not completed if the polymorphic base is G. Following BspHI digestion, the G allele

is represented as a 170-bp band and the A allele as two bands of z 146 and 24 bp.

Polymorphism: Genomic DNA from 36 dogs, representing 11 mixed bred animals and 25
different pure breeds was tested for both the GA and the SNP polymorphisms. Four
alleles were observed with the GA microsatellite and two with the SNP. The observed
heterozygosity (HET) was 0.42 for the microsatellite and 0.36 for the SNP. The third
largest (GA)n microsatellite repeat allele was found to be associated in all cases with an

A at the SNP site. All other repeat alleles were associated with a G at this site.

92

Chromosomal Location: Unknown.

Mendelian inheritance: Testing of a family of dogs (Fig. 1) segregating for both the SNP
and the microsatellite supported the inheritance of both of these variations in a Mendelian

fashion.

 

in!

1'2345678910111213

Fig. 1. Two percent agarose gel showing SNP (lanes 2-7) and microsatellite
(lanes 8-13) results for a family of six dogs. The sire (lanes 2 and 8) was homozygous
for the A SNP allele and the third largest microsatellite allele. The dam (lanes 3 and 9)
was heterozygous for both SNP alleles and the second and third largest microsatellite
alleles. The offspring are shown in lanes 4-7 and 10-13. Lane 1 contains a 100-bp DNA

ladder.

Acknowledgments: This work was supported in part by grants from the American Kennel
Club Canine Health Foundation, the Dalmatian Club of America and the Jaqua

Foundation.

93

References
l Elshourbagy N.A. et al. (1 993) J Biol Chem 268, 3873-3879.
2 Hosoda K. et al. (1994) Cell 79, 1267-1276.

3 Liu P-C. et al. (1998) Anim Genet 29, 236.

Correspondence: V. Yuzbasiyan-Gurkan (e-mail: yuzbasiyan@cvm.msu.edu)

94

CHAPTER 4

PREFACE TO EVALUATION OF KIT MUTATIONS IN MAST CELL TUMORS

95

Many dog breeds appear to exhibit an increased risk for the development of
cancer when compared to other breeds. It is possible that the cause of the increased risk

in these breeds has a genetic component. The Boxer in particular has a high incidence of
many types of cancer, especially mast cell tumors.2 Besides its role in the development

of melanocytes, KIT also controls the development of mast cells.1 Human mast cell

leukemia has been linked to mutations in KIT, and mouse mastocytoma cell lines have

been shown to have Kit mutations},6 It was hypothesized that mutations in KIT might
be responsible for mast cell tumors in dogs as well.

Since the KIT gene had already been sequenced in the dog as part of the
investigation into hereditary deafness, most of the materials required for a study of this
gene in mast cell tumors were already available. It was therefore decided to pursue this
study as an adjunct to the main project on deaﬁless. While in the preliminary stages of
the investigation, our suspicions were conﬁrmed by the publication of two articles

reporting the discovery of mutations in the juxtamembrane domain of KIT in mast cell

tumors.4’5 At this point, a female Boxer had been acquired that had previously been

treated for a mast cell tumor and was later euthanized after the development of numerous

additional tumors. The following two chapters constitute reprints of published

manuscripts resulting from studies of this dog and additional mast cell tumor cases.7i8

References
l. Broudy VC. Stem cell factor and hematopoiesis. Blood 90: 1 345-1 364, 1997.
2. Cohen D, Reif J S, Brodey RS, Keiser H. Epidemiological analysis of the most

prevalent sites and types of canine neoplasia observed in a veterinary hospital.
Cancer Res 34:2859-2868, 1974.

96

. F uritsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, Sugahara H,
Butterﬁeld JH, Ashman LK, Kanayama Y, Matsuzawa Y, Kitamura Y, Kanakura
Y. Identiﬁcation of mutations in the coding sequence of the proto-oncogene c—kit

in a human mast cell leukemia cell line causing ligand-independent activation of
c-la't product. J Clin Invest 92:1736-1744, 1993.

. London CA, Galli SJ, Yuuki T, Hu ZQ, Helfand SC, Geissler EN. Spontaneous
canine mast cell tumors express tandem duplications in the proto-oncogene c-kit.
Exp Hematol 27:689-697, 1999.

. Ma Y, Longley Bl, Wang X, Blount JL, Langley K, Caughey GH. Clustering of
activating mutations in c-KITs juxtamembrane coding region in canine mast cell
neoplasms. J Invest Dermatol 112:165-170, 1999.

. Tsuj irnura T, Furitsu T, Morimoto M, Isozaki K, Nomura S, Matsuzawa Y,
Kitamura Y, Kanakura Y. Ligand-independent activation of c-kit receptor
tyrosine kinase in a murine mastocytoma cell line P—815 generated by a point
mutation. Blood 83:2619-2626, 1994.

. Zemke D, Yamini B, Yuzbasiyan—Gurkan V. Characterization of an
undifferentiated malignancy as a mast cell tumor using mutation analysis in the
proto-oncogene c-KIT. J Vet Diagn Invest 13:341-345, 2001 .

. Zemke D, Yamini B, Yuzbasiyan-Gurkan V. Mutations in the juxtamembrane
domain of c-KIT are associated with higher grade mast cell tumors in dogs. Vet
Pathol 39:529-535, 2002.

97

 

CHAPTER 5

Zemke D, Yamini B, Yuzbasiyan-Gurkan V. Characterization of an undifferentiated
malignancy as a mast cell tumor using mutation analysis in the proto-oncogene c-KIT.
J Vet Diagn Invest 13:341-345, 2001.

98

Abstract. A 6.5-year-old female Boxer was euthanized and presented for
necropsy following rapid clinical decline concomitant with the development of
numerous tumor masses. The largest of these masses was in the same location as a
mast cell tumor that had been previously removed from this dog. Gross examination
revealed the presence of nodules ﬁom 5-200 mm in diameter throughout the body,
including the lymph nodes. Histologic analysis showed an inﬂux of round cells with
no granules, leading to the provisional diagnosis of systemic lymphosarcoma.
Immunohistochemical staining for B- and T-lymphocyte antigens was negative.
Molecular tests were used to identify a tandem duplication in the c-KIT proto-
oncogene from both the earlier mast cell tumor and the current nodules, implicating a
common origin. Addition of molecular testing to conventional necropsy evaluations

allowed a deﬁnitive diagnosis of mast cell tumors.

Mast cell tumors, or mastocytomas, are one of the most common types of skin
cancer seen in dogs, accounting for an estimated 7-21% of all cases.3’10’1 7 Although

mast cell tumors are rare and generally nonthreatening in humans,17 they are often more
aggressive and constitute a signiﬁcant health risk in dogs. Canine mast cell tumors

develop swiftly, are quick to metastasize, and frequently recur after having been
removed. 1 7 Moreover, certain breeds such as the Boxer appear to have some

predisposition toward their development.3 To investigate the possibility that the Boxer
breed has an inherited increased susceptibility to certain types of cancer, particularly mast
cell tumors, cases of mast cell tumors in Boxers were solicited and examined.

A 6.5- year-old female Boxer had a growth removed ﬁom the left shoulder in
April 1998. This growth was diagnosed as a grade H mast cell tumor by routine
histopathologic analysis. In early 1999, numerous additional nodules developed rapidly
over the entire body in a matter of a few weeks, according to the owner and the
veterinarian, and the dog was euthanized. The body was frozen and placed in storage
until it could be transferred to Michigan State University, after which it was thawed for
study. Postmortem examination revealed multiple round to oval nodules ﬁom 10-30 mm
in diameter on the skin of the medial and caudal thighs, perineal region, humerus, femur,
and thoracic and abdominal walls. Subcutaneous nodules 10-50 mm in diameter were
found in the inguinal region (Fig. 1A). Multiple nodules 10-20 mm in diameter were
present in the lateral and caudal musculature of the hip. A very large mass about 200 mm
in diameter was found in the left subscapular region, approximately the same site from
which the original mast cell tumor had been removed; this mass extended deep into the

thoracic musculature. Nodules 5-30 mm in diameter were also found in the heart (Fig.

l00

1B), liver (Fig. 1C), kidney (Fig. 1D), and lungs. The mesenteric and mediastinal lymph
nodes were enlarged. Samples of nodules ﬁom the skin, liver, a lymph node and samples
from the large subscapular mass were taken for both microscopic examination and

molecular studies.

 

Figure l. Macroscopic lesions observed in a 6.5-year-old female Boxer. Note

white, ﬁrm nodules of various sizes in inguinal region (A), heart (B), liver (C) and

kidney (n).

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Figure 2. Hepatic nodule from a 6.5-year-old female Boxer. Round cells have no

 

intracytoplasmic granules. Giemsa stain. A. 100 X. B. 200 X.

Histologic examination of the nodules revealed a diffuse inﬁltration of medium-
sized neoplastic round cells with large hyperchromatic nuclei. The normal architecture of
the lymph node was obliterated by inﬁltration of neoplastic cells. The mitotic index was
moderate, with an average of 5 mitoses/high-power ﬁeld. No eosinophils were observed
during the microscopic examination. Special stains (Giemsa, toluidine blue) were used,
and no mast cell granules were identiﬁed (Fig. 2). The absence of granules in sections
stained with hematoxylin and eosin and with special stains and the extent and pattern of
nodules found during the gross examination resulted in a preliminary diagnosis of

malignant round cell tumor, highly suggestive of systemic lymphosarcoma. For

102

conﬁrmation, immunohistochemical immunophenotyping for B and T lymphocytes was
performed. Formalin-ﬁxed, parafﬁn-embedded sections of neoplastic tissues were tested.
Monoclonal antibodies to CD79a for B lymphocytes and polyclonal antibodies to CD3
for T cell lymphocytes were used, with negative results. Molecular testing was therefore
utilized in an attempt to clarify the diagnosis.

Tissue samples from the same nodules used for microscopic examination and a
sample of spleen that appeared to contain only normal tissue were chosen to search for
potential mutations in the juxtamembrane domain of c—KIT . The c-KIT gene encodes for

a receptor tyrosine kinase that is required for the proper development, survival, and

maturation of hematopoietic stem cells, melanocytes, and mast cells.2’12’1 8 The
receptor consists of an extracellular domain of 5 immunoglobulin—like folds and an

intracellular kinase domain separated by transmembrane and juxtamembrane

domains.1 1’19 Point mutations, deletions, and duplications have been identiﬁed in the

juxtamembrane domain of c-KIT in certain canine mast cell tumors and mast cell tumor

cell lines.6’8 Some of these changes have been shown to result in constitutive activation

of c-KIT in the absence of binding of its nomal ligand, alternatively known as Steel
factor, mast cell growth factor, or stem cell factor.2 Activating mutations have also been
identiﬁed in both human4 and rodentld”16 mast cell tumor cell lines, and in situ in cases

of human mastocytosis associated with other disorders.7’9 Similar mutations cause

factor- independent cell growth and aggressive behavior characteristic of tumor formation

in murine cell lines.5

103

Molecular characterization of normal and tumor tissue from this dog was carried

out as follows. A guanidinium-based solution (T rizola) was used for the isolation of
genomic DNA and total RNA from fresh tissue, following the manufacturer’s protocol.

Contaminating genomic DNA was removed from the RNA preparation by treatment with

RNase-free DNase as previously described”. The sample was precipitated in ethanol
and resuspended in deionized water to the same volume originally obtained from the
extraction procedure. The RNA was then reverse transcribed using random hexamers as

primers. A reaction mixture was prepared containing 10 mM DTT, 2.4 pg of random
hexamers, 0.32 mM dNTPs, 1X ﬁrst-strand buffera, 8 uL of RNA, and 800 U of

Moloney murine leukemia virus reverse transcriptase3 in 100 uL total volume. All
reagents except the ﬁrst-strand buffer and enzyme were combined and incubated at 70 C
for 5 minutes to denature the RNA secondary structure and then placed on ice for 5
minutes. The buffer and enzyme were then added, and the entire mixture was incubated
for 1 hour at 37 C, followed by inactivation at 90 C for 5 minutes.

Primers were designed for polymerase chain reaction (PCR) ampliﬁcation of the

juxtamembrane domain of c-KIT based upon the published canine sequence (GenBank

no. AF 099030).6 Ampliﬁcations were performed with the forward primer 5’-
ACAAATCCATCCCCACACCCTGTTCAC-3’ and the reverse primer 5’-

CACTTI‘CCCGAAGGCACCAGCACCCA-3’. PCR reactions were in a total volume of

25 uL consisting of IX PCR buffer,a 1.5 mM MgClz, 0.2 mM dNTPs, 20 pmol of each

primer, 0.625 U of Taq polymerase,a and either 3 uL of cDNA template or 5 uL of

genomic DNA template. Cycling conditions included an initial denaturation of 4 minutes

104

at 95 C followed by 35 cycles of 95 C for 1 minute, 66 C for 2 minutes, and 72 C for 3

minutes and a ﬁnal polymerization step of 8 minutes at 72 C. The expected size of the

ampliﬁed fragment was 267 bp from cDNA and 645 bp ﬁ'om genomic DNA.
Ampliﬁed products were separated on 2% agarose gels in Tris-acetate-

ethylenediaminetetraacetic acid (EDTA) buffer and puriﬁed using a commercial gel
extraction kit.b Extracted bands were eluted in 30 pL of 10 mM Tris (pH 8), and 5 uL
was used for manual sequencing using a commercial dideoxy kit with 33P-radiolabelled

terminators,° using the manufacturer’s protocol. For the cDNA fragments, the same
primers used for PCR ampliﬁcation were used for the sequencing. For the genomic DNA
templates, an additional internal primer, 5’-GAGGAGATCAATGGAAACAA—3’ was

used to sequence the entire fragment.
For archived tumors, a slight modiﬁcation of a microwave-based lysis method1

was used to extract DNA. A sample of approximately 1-3 mm3 was removed from each
parafﬁn block and placed into 400 pL of a solution of 50 mM Tris (pH 8.5), lmM
EDTA, and 0.5% Tween. The samples were heated to 95 C for 10 minutes and then
microwaved twice for 30 seconds each, with vortexing between each heating step. The
samples were allowed to cool, and 75 pg of proteinase K was added to each. After
incubation overnight at 42 C, the enzyme was inactivated by heating to 95 C for 10
minutes. The samples were centrifuged for 10 minutes at 12,000 rpm in a
microcentrifuge, and the supernatant was transferred to a clean tube. The amount of
DNA obtained was variable; therefore, multiple dilutions of each sample were tested until

PCR was successful. Typical samples ampliﬁed correctly at a 1:25 or 1:50 dilution of the

105

original DNA. This diluted DNA was used for PCR and sequencing as described for
DNA from fresh tissue.

Ampliﬁcation of both cDNA and genomic DNA templates from normal tissue
produced a single band of the expected size, whereas tumor tissue produced two bigger
bands in addition to the expected band, approximately 30 and 50 bp bigger than expected
(Fig. 3). In both cDNA and genomic DNA templates, sequencing of the band ﬁom

normal tissue conﬁrmed that it contained the normal canine sequence. Sequencing of the

 

Figure 3. Separation of ampliﬁed bands from the juxtamembrane domain of c-

KIT in a female Boxer. Lane 1 contains a 100-bp DNA ladder, with the 600-bp marker
(‘) to the right. Sample lanes represent PCR products from normal spleen (lanes 2 and 7)
and 4 tumor nodules from various locations (lanes 3-6, 8-11). Ampliﬁcations from
cDNA are shown in lanes 2-6, and those from genomic DNA are in lanes 7-11. M =
molecular weight marker; N = normal tissue, T = tumor, with subscripts C and G

indicating cDNA and genomic DNA templates, respectively.

3 bands obtained from tumor tissue determined that they represented 1) the normal
sequence, 2) a sequence with a 45-bp tandem duplication, and 3) a mixture of the 2
sequences. The duplication was similar to those previously seen in canine mast cell

tumors and in approximately the same location, consisting of bases 1721-1765, as per the

canine c-KIT sequence.6 This suggested that the tumor in the presented case was a mast
cell tumor rather than a lymphosarcoma, as initially diagnosed.

To test the possibility that such mutations might also occur in lymphosarcomas,
parafﬁn-embedded samples of mast cell tumors and lymphosarcomas, including the
original mast cell tumor from the Boxer, were selected from the archives in the Animal
Health and Diagnostic Laboratory at Michigan State University. RNA was not
obtainable ﬁom the tissue blocks; therefore, only genomic DNA was studied for these
cases. A total of 31 lymphosarcomas and 15 mast cell tumors were successfully
ampliﬁed. None of the lymphosarcomas contained duplications in this region of c-KIT;
however, 2 of the mast cell tumors, including the original mast cell tumor ﬁom the
Boxer, had tandem duplications. The duplication from the original Boxer tumor was
identical to that ﬁ'om fresh tissue in the tumor nodules. It is not known whether or not
this particular duplication causes constitutive activation of c-KIT; however, it appears that
duplications in this region can be used as markers to positively identify mast cell tumors
in some cases, regardless of their implication as the cause of the tumor.

The results suggest that the tumor and nodules in this Boxer represent a highly
undifferentiated and aggressive mast cell tumor that resembles systemic lymphosarcoma.

Highly undifferentiated grade HI mast cell tumors have a signiﬁcantly decreased number

of granules” however, the granules in the present tumors were completely absent. To

107

rule out the possibility that the absence of granules was due to the ﬁ'eezing and thawing
process and was not the original state of the tumor, a sample from a newly diagnosed
mast cell tumor was subjected to similar conditions of freezing and thawing. No change
in granule content was observed in this sample tumor even after 2 months of storage in
the ﬁ'eezer, indicating that the tumor in this Boxer most likely never contained granules.
Because the presence of granules is often key in the identiﬁcation of mast cell tumors, it
is reasonable to assume that the tumor could have been mistyped as a lymphosarcoma.
However, the lymphocyte antigen staining results brought this diagnosis into question.
Molecular tests appear to have produced a deﬁnitive determination of the actual tumor
type seen in this dog. Because the duplication was only seen in tumor DNA and not in
normal constitutive DNA, it must have arisen de novo in the tumor and was not an
inherited trait. Because it is highly unlikely that 2 independent events would produce
identical mutations in 2 different cell types, the current tumors must have been derived
from the original tumor and therefore are mast cell tumors.

This case illustrates a potentially beneﬁcial role for the use of molecular tests to
aid in diagnosis when the results of conventional tests are in question. The exact test
used in this study represents a rare situation that could not occur in all cases because it
entailed the identiﬁcation of a mutation and the availability of a sample ﬁom a primary
tumor that had been deﬁnitively identiﬁed. However, results from a larger scale study
currently underway appear to indicate that duplications such as that seen in this dog may
be common in the more highly aggressive mast cell tumors, suggesting that molecular
tests might be useful in these particular cases if the diagnosis is in doubt, even without a

previous tumor for comparison. Furthermore, molecular testing may be useful as an

108

adjunct prognostic indicator and may be important in determining therapeutic options.
However, such applications will require further studies. With the increasing number of
genetic alterations known or suspected to cause neoplasms, the identiﬁcation of many
different tumor types could be facilitated in the near future by molecular testing.

Acknowledgements. We thank the AKC Canine Health Foundation and the Jaqua
Foundation for their ﬁnancial support, Dr. José A. Ramos-Vara (University of Missouri)
for performing the lymphocyte antigen tests, and Ms. Marianne Balzer and Dr. Chris

Jones for referring this case to our laboratory.

Sources and manufacturers
a. Gibco BRL, Gaithersburg, MD.
b. QIAEX II, Qiagen, Valencia, CA.
c. Thermo Sequenase Radiolabelled Terminator Cycle Sequencing Kit, USB,
Cleveland, OH.

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109

5. Kitayama H, Kanakura Y, F uritsu T, et al.: 1995, Constitutively activating
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6. London CA, Galli SJ, Yuuki T, et al.: 1999, Spontaneous canine mast cell tumors
express tandem duplications in the prom-oncogene c-kit. Exp Hematol 27: 689-
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7. Longley D], Tyrrell L, Lu S-Z, et al.: 1996, Somatic c-KIT activating mutation in
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18. Valent P, Spanblbchl E, Sperr WR, et al.: 1992, Induction of human mast cells
from bone marrow and peripheral blood mononuclear cells by recombinant
human stem cell factor / kit-ligand in long-term culture. Blood 80: 2237-2245.

19. Yarden Y, Kuang W-J, Yang-Feng T, et al.: 1987, Human prom-oncogene c-kit:

a new cell surface receptor tyrosine kinase for an unidentiﬁed ligand. EMBO J 6:
3341-3351.

111

CHAPTER 6

Zemke D, Yamini B, Yuzbasiyan-Gurkan V. Mutations in the Juxtamembrane Domain
of c-KIT are Associated with Higher Grade Mast Cell Tumors in Dogs. Vet Pathol
39:529-535, 2002.

112

Abstract. Mast cell tumors are among the most commonly seen tumors of the
skin in dogs and are more highly aggressive than mast cell tumors of other species. Some
breeds display a markedly higher incidence of mast cell tumor development than others
and appear to have some genetic predisposition. Recently, mutations have been found in
canine mast cell tumor tissues and cell lines within the juxtamembrane domain of the
proto-oncogene c-KIT. In previous studies utilizing a small number of cases, no
association between the presence of a mutation and the breed of dog or grade of the
tumor could be identiﬁed. An expanded study with a larger sample set was performed in
order to explore this possibility. The juxtamembrane domain of c-KIT was ampliﬁed
using the polymerase chain reaction from genomic DNA preparations of 88 parafﬁn-
embedded mast cell tumors from selected breeds. Mutations, consisting of duplications
and deletions, were found in 12 of the tumors. A signiﬁcant association was found
between the presence of a mutation and a higher grade of tumor but not between breed

and grade or between breed and the presence of a mutation.

Key words: c-kit receptor; dogs; DNA; mastocytoma; mutation; polymerase chain

reaction; sequence analysis; single nucleotide polymorphism.

113

Mast cell tumors are one of the most frequently seen skin neoplasms in dogs,

accounting for up to 21% of cases.5’12’18 Most of these tumors are benign, developing

slowly and persisting for years without increasing in size or metastasizing. However, a
large number are highly aggressive and present a signiﬁcant threat to canine health.18 In

contrast, mast cell tumors in humans are rare and usually benign.l8 Some dog breeds

appear to be at relatively little risk, whereas others such as the Boxer have been reported

to have a high incidence of mast cell tumor development.5 This difference among breeds
indicates a possible genetic inﬂuence on both susceptibility to tumors and tumor

aggressiveness.

Mast cell tumors are usually graded on a histologic scale.ll Grade I tumors are
highly differentiated, with regular nuclei, rare or no mitotic ﬁgures, and a large number
of metachromatic granules. They are generally considered to be benign. Grade 111
tumors are highly undifferentiated, with large misshapen nuclei, many mitotic ﬁgures,
and few cytoplasmic granules. This is the most aggressive of the three grades. Grade II
tumors are intermediate between the other two types. Mast cell tumors are often also
identiﬁed by clinical stage based on the extent of their growth. Incompletely excised
tumors are designated stage 0, single tumors without and with lymph node involvement

are designated stage I and stage II, respectively, multiple tumors comprise stage III, and
recurrent or metastatic tumors make up stage IV. 1 8 Both histologic grading and clinical

staging are good predictive factors for the ﬁnal outcome. 1 8

The genes involved in the development of mast cell tumors are currently under

study. Analyses of human7 and rodent1 5'17 mast cell tumor cell lines have revealed a

114

number of mutations in the proto-oncogene c-KIT. The c-KIT gene encodes for a cell
surface receptor, which upon binding of its cognate ligand induces a signal transduction

cascade responsible for the development, maturation, and survival of many cell lineages,

including hematopoietic stem cells, melanocytes, and mast cells.3’14’19 The c-KIT

receptor consists of an extracellular domain of ﬁve immunoglobulin-like loops and an

intracellular tyrosine kinase domain. 1 3’20 Mutations in the kinase domain or the
neighboring juxtamembrane domain have been shown to cause constitutive activation of
c-KIT in the absence of ligand binding. Constitutive activation of c-KIT in certain murine

cell lines leads to the uncontrolled cell growth and aggressive behavior typical of tumor

development.8 The mutations identiﬁed to date in humans and rodents are conﬁned to
the kinase and juxtamembrane domains of c-KIT and consist of point mutations and small
deletions. Many of these mutations cause constitutive activation of c-KIT in vitro and
some have been identiﬁed in situ.

Mutations in canine c-KIT have only recently been identiﬁed, and all have been

found exclusively in the juxtamembrane domain. Ma et al.10 discovered point mutations

and small deletions in three of seven tumors and a duplication in two of three cell lines.

London et al.9 found duplications in 5 of 11 tumors; however, they did not see any of the
other types of mutations. Although the duplications encompassed approximately the
same area of c-KIT, no two were identical. Both groups found mutations in
approximately 50% of the animals studied. In a recent study, 2 of 15 mast cell tumors
tested contained juxtamembrane domain duplications, and in one of these dogs, a

recurrence of a tumor with the exact same duplication allowed molecular conﬁrmation of

the tumor type when the diagnosis based on pathology was questionable.21 Because

115

duplications in the juxtamembrane domain of c-KIT as seen in canine mast cell tumors
have not been identiﬁed in any other species to date, these duplications may be related in
some way to the increased aggressiveness of these tumors in dogs. In the previous
studies, the limited number of animals involved made it impossible to accurately
determine the percentage of canine mast cell tumors with detectable mutations or to
identify any correlation between the type of mutation seen and the breed of dog or the
aggressiveness of the tumor. The present study of c-KIT duplications was conducted on a

much larger scale to determine the signiﬁcance of these mutations.

Materials and Methods

Sample selection

Mast cell tumor cases were selected from tissue samples submitted to the
Michigan State University Animal Health and Diagnostic Laboratory (AHDL) during the
period of 1998-1999. Cases were selected from breeds with the highest number of cases
so that differences between breeds could be examined. The Boxer and Boston Terrier
were chosen based on their suspected predisposition for mast cell tumors, whereas the
Labrador Retriever, Golden Retriever, and mixed-breed dogs were not suspected to be
predisposed and were chosen for comparison. The 15 dogs from the preliminary study
were included; this group consisted of a Pit Bull in addition to the bmds listed above.
For each dog, a single block of forrnalin-ﬁxed, parafﬁn-embedded tumor tissue was

retrieved from the AHDL archives.

116

Paraffin block DNA isolation

Sections of each block were cut and stained with hematoxylin and eosin. Stained
sections were examined under a microscope by a certiﬁed veterinary pathologist (B.
Yamini) to determine the borders of the tumor, which were marked on the block. A small
piece of tissue approximately 2 mm in diameter was excised ﬁ'om each block within the

boundaries of the tumor. DNA was isolated by a modiﬁed version of a previously

described method.1 The tissue was placed into 400 uL of digestion buffer (50 mM Tris,
pH 8.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Tween). The parafﬁn in
the samples was melted by heating at 95 C for 10 minutes and heating for 30 seconds
twice in a microwave at full power, with thorough vortexing after each heating step. The
samples were allowed to cool, and 5 uL of 15 mg/mL proteinase K was added to each.
The samples were then incubated at 42 C overnight or until the piece of tissue was
completely digested. The proteinase K was inactivated by heating at 95 C for 10
minutes, and the samples were centrifuged at 12,000 rpm in a microcentrifuge for 10

minutes. An aliquot of 200 uL was then transferred to a clean tube, avoiding the transfer

of parafﬁn.

Amplification of the juxtamembrane region of c-KI T

For each sample, the undiluted DNA preparation and dilutions of 1:10, 1:25, and
l :50 were used as templates for polymerase chain reaction (PCR) ampliﬁcation of the
juxtamembrane domain of c-KIT. PCRs with a total volume of 25 uL were set up using 5

uL of template, 20 pmol of each primer, 0.625 U Taq polymerase (Gibco BRL), and ﬁnal

117

concentrations of 100 uM dNTPs, 1.5 mM MgC12, 50 mM Tris-Cl (pH 8.3), and 10 mM

KCl. Ampliﬁcation was performed using primers JuxtF and JuxtR (Fig. 1). PCR
conditions consisted of an initial denaturation step of 4 minutes at 94 C, followed by 35
cycles of 1 minute at 94 C, 2 minutes at 66 C, and 3 minutes at 72 C, and a ﬁnal

extension step of 8 minutes at 72 C.

Puriﬁcation of ampliﬁcation products

The PCR ampliﬁcation products were separated on 2% agarose gels in Tris-
acetate-EDTA buffer. Individual bands were stabbed with a pipette tip and transferred to
15 1.1L of water, 5 pL of which was used as a template in an additional PCR with the same
conditions as the initial ampliﬁcation. The secondary ampliﬁcation products were
separated on 2% agarose gels, and the resulting bands were excised and puriﬁed using the
QIAEX II kit (Qiagen) following the manufacturer’s protocol, with each sample

resuspended in a ﬁnal volume of 30 uL of 10 mM Tris, pH 8.

Sequencing of puriﬁed bands

Manual sequencing was performed using the Thermo Sequenase radiolabelled
terminator cycle sequencing kit (U SB). Samples were sequenced from both the JuxtF
and JuxtR primers used for PCR ampliﬁcation, plus an internal primer JuxtM (Fig. l).
Sequencing followed the manufacturer’s standard protocol for the dGTP termination mix
except for an annealing temperature of 50 C, using 5 uL of template DNA and 0.5 pmol
of primer per sample. The reaction products were separated on 6% denaturing

polyacrylamide gels and visualized by exposure to x-ray ﬁlm.

118

Fig. l. Genomic sequence of the juxtamembrane domain of canine c-KIT and
ﬂanking regions (amino acids 518-606). The amino acid sequence of the exons is given
beneath the DNA sequence. Single nucleotide polymorphisms are shown in boxed letters
(R = A or G; Y = C or T; K = G or T). Primers used for ampliﬁcation and sequencing are
shown in bold type, with arrows indicating the direction of the primer. Exon sequences
are shown in uppercase letters; intron sequences are in lowercase letters. Deletions are

denoted by open bars; duplications are denoted by solid bars.

119

\‘Cﬂ

Cllfli

12am

1'

ICCS

JuxtF

 

Exon 10 >
CAAAICCAICCCCACACCCIGTTCACACCTTTGCTGATTGGCTTTGTGATCGCAGCTGGAATGATGTGCATTATCGTGATGATT
Q I H P H T L F T P L L I G F V I A A G M M C I I V M I

520 525 530 535 540 545

Intron 10
CTTACCTACAAGTATCTACAGgtaatcattcatttgttctctaccctaagtgctataatgatcgaaatgttattcattaaaagatgatc
L T Y K Y L Q
550

Em" 11 [:1 Delz
SNP! l J Del 1m”

dﬁtctctcttttctccccCcaccagAAACCCATGTATGAAGTACAGTGGAAGGTTGTTGAGGAGAICAAIGGAAACAATTATGTTTACA
K P M Y E V Q W K V V E E I N G N N Y V Y
555 560 565 570

 

 

 

_Dupl Intron 11 gm
TAGACCCAACACAGCTTCCTTAEGATCACAAATGGGAGTTTCCCAGAAACAGGCTGAGCTTTthcagtatgaaaﬂaggggctttccat

I D P T Q L P Y D H K W E F P R N R L S F
575 580 585 590

gtaacctttttgtgtacgtgtaacaatgactttagggaaccccattagcttcctttgttctgttccaactgagacaataagtattttct

SNP3 SN" .
gtgaagtttcatcaEktttgatagattcdgcataaagcaccttatagagaaatgtccttagctggatttgtccttaattccttaacaat

SNPS Exon 12
tccttgattgttgactttgaaattacccagatgctcctttggtcctaEkaccaccccttactcttttcttcctttctgcagGGAAAACT
G K T
595

 

JuxtR
f
TTGGGTGCTGGTGCCTTCGGGAAAGTG
L G A G A F G K V
600 605

120

Automated sequencing was performed on an ABI Prism 377 DNA Sequencer
using Big Dye terminators. Samples were sequenced from JuxtF and, when readable
length produced was insufﬁcient, JuxtM. Cycle sequencing was performed using 10 uL
of template DNA and 3.2 pmol of primer per sample, following the manufacturer’s
protocol. Unincorporated terminators were removed by isopropanol precipitation
following the manufacturer’s protocol, except for the addition of 1 uL of 20 mg/mL
glycogen as a carrier. Samples were resuspended in 4 uL of loading dye, 2 uL of which

was loaded onto the gel.

Results

The juxtamembrane domain of c-KIT was successfully ampliﬁed from a total of
88 canine mast cell tumor cases. The number of dogs of each breed and the grade
distribution of tumors within those breeds is shown in Table 1. PCR ampliﬁcation
revealed that 8 of the 88 cases contained two larger bands in addition to the expected
band when separated on agarose gels, indicating possible duplications. An additional case
also contained two extra bands, one below and one above the expected band, indicating a
possible deletion. All bands from these cases were puriﬁed and sequenced manually.
For each sample, the three bands represented the normal sequence, either a duplication or
deletion, and a mixture of the two sequences. Figure 2 shows an example of a
duplication and a deletion as seen on an agarose gel. The remaining samples were
sequenced either manually or with an automated sequencer to screen for mutations too
small to be seen as separate bands. Three additional deletions were found during the

course of this sequencing.

121

Table 1. Grade distribution of mast cell tumors in various dog breeds.

 

 

Breed No. Tumors Gra_de I Grade II Grade III
Boston Terrier 8 2 6 0
Boxer 29 7 20 2
Golden Retriever 13 l 10 2
Labrador Retriever 18 6 1 l 1
Pit Bull 1 1 0 0
Mixed Breed 19 7 11 1

 

 

Fig. 2. Agarose gel showing products obtained by ampliﬁcation of the
juxtamembrane region of c-KIT from different sources. Lane M contains a 100 bp DNA
ladder. The 600-bp band is marked with an asterisk. For lane 1, the lower band contains
normal sequence and the middle band contains a 45-bp duplication (Dup2). The upper
band is a heteroduplex of normal and mutant sequence. Lane 2 is an example of
ampliﬁcation from normal tissue. In lane 3, the lower band contains a 30-bp deletion
(Dell), the middle band is normal, and the upper band is a heteroduplex of normal and

mutant sequence.

122

Figure 1 shows a portion of the juxtamembrane domain sequence (GenBank
accession Nos. AF 448146, AF448147, and AF 448148) with the positions of the
duplications indicated. The duplications were all in frame, ranged in size from 44 to 69
base pairs (bp), and were located near the 3’ end of exon 11, with ﬁve of them (Dup3,
Dup4, Dup6, Dup7, Dup8) extending into the neighboring intron 11. In two of the dogs,
the duplicated sequences (Dup7 and Dup8) were identical. Three of the duplications
cause tandem repeats within the protein sequence: Dupl , residues Asp575 - Arg589;
Dup2, residues Pr0576 - Asn590; Dup5, residues Pr0576 - Arg591. Dup4 resulted in the
insertion of a Gly residue aﬁer Phe594, followed by a direct repeat of residues Pro576 -

Phe594. The numbering of the amino acid residues is based on the normal canine c-KIT

cDNA sequence (GenBank accession No. AF448148). The remaining four duplications
involve duplication of part or all of the consensus splice sequence at the 5’ end of intron
ll, creating the potential for alternative splicing events. For all four duplications, no
change to the protein is made if the upstream site is used. If the downstream site is used,

then Dup3 causes an insertion of Gly-Gln-Tyr plus a repeat of residues Asp575 - Phe594,
Dup6 causes an insertion of Gly plus a repeat of residues Gln578 - Phe594, and
Dup7/Dup8 cause an insertion of Gly-Gln plus a repeat of residues Tyr5 81 - Phe594,
with the insertion occurring aﬁer Phe594 in all four cases. RNA studies are required to

determine the splicing pattern in these tumors.
Figure 1 also shows the location of the identiﬁed deletions. Dell encompasses 30
bp, including the last few bases of intron 10 and part of the 5’ end of exon 11. Because

the deletion removes part of the 3’ splice site sequence for intron 10, the exact effect of

123

this deletion on the protein is not known. Ifthe ﬁrst AG following the deletion is used
for splicing, the result would be a deletion of Lys 55 3 - Lys561; however, other potential
splice acceptors are present in the area. A study of the RNA ﬁom such cases is necessary
to determine which of these sites, if any, is used. Del2 removes 6 bp and causes the
substitution of a Phe residue for Trp560 - Va1562 in the protein sequence. Del3 and Del4

remove 7 bp but are followed by the insertion of a single G, so no frameshift is created.

They cause the substitution of an Arg residue for Gln559 - Lyssa.

The distributions of the different mutation types by breed and tumor grade are
shown in Tables 2 and 3. None of the mutations identiﬁed were from grade I tumors. All
of the deletions were found within grade H tumors, and equal numbers of duplications
were found in grade II and grade III tumors. The mutation frequencies for the three

grades were signiﬁcantly different from each other as determined by a minimum chi-
square analysis (x2 = 21.68, 95% conﬁdence level = 9.49). With respect to breed, two of
the four deletions were found in Labrador Retrievers (1‘ able 3). Half of the duplications

were identiﬁed in Boxers, and the remaining half consisted of single tumors in each of

the other breeds. No signiﬁcant association was seen between presence of a mutation and

the breed of dog (x2 = 6.06, 95% conﬁdence level = 18.31).

Table 2. Grade distribution of duplications and deletions in c-KIT

among canine mast cell tumors.

 

Grade No. Tumors Duplications Deletions

 

I 24 0 0
II 58 4 4
III 6 4 0

 

124

Table 3. Distribution of duplications and deletions in c-KIT among different dog

 

 

 

breeds.
Breed No. Tumors Duplications Deletions

Boston Terrier 8 1* 1*
Boxer 29 41' 0
Golden Retriever 13 II 11
Labrador Retriever 18 1§ 2§
Pit Bull 1 0 0
Mixed Breed 19 Ill 0

“ Dup5 and Dell

1' Dup2, Dup3, Dup7, Dup8

I Dup6 and Del4

§ Dup4, De12, Del3

ll Dupl

During the course of sequencing work on c-KIT for this and other projects, a
number of single nucleotide polymorphisms (SNPs) were found throughout the gene, six
of which are located in the region shown in Figure 1. Five of these SNPs are located in
introns and have no effect on the protein. The allele frequencies of the SNPs among 61
of the dogs in this study were evaluated and determined to be 35.2% A + 64.8% G for
SNP], 49.2%T + 50.8% C for SNP2, 48.4% C + 51.6% T for SNP3, 48.4% G + 51.6% T
for SNP4, and 47.5% C + 52.5% T for SNPS. The sixth SNP is located within exon 11,
but is silent, causing no amino acid change. The allele frequency of this SNP was 24.6%

T + 75.4% C.

Discussion
Duplications and deletions of sufﬁcient size can readily be distinguished by

agarose gel electrophoresis (Fig. 2). The smaller 6 or 7-bp deletions were only

125

identiﬁable by direct sequencing. All of the duplications and the largest of the deletions
identiﬁed in this study produced a pattern of three bands similar to the examples in
Figure 2. The lower two bands represent the normal and mutant sequences, indicating
that the tumors were heterozygous for their respective mutations. The uppermost band
represents heteroduplexes containing one strand each of normal and mutant DNA, as
determined by sequencing. In heteroduplexes, unpaired bases in one of the strands form

a bulge in the DNA, which retards the migration of the heteroduplexes during

electrophoresis and causes them to appear as if they were larger fragments.2

The results of this study suggest that there is a relationship between tumor grade
and the presence of c-KITjuxtamembrane domain mutations, particularly duplications.
No mutations were found in any of the grade 1 tumors tested, and <10% of the grade II
tumors had mutations (Table 2). Among grade III tumors, however, four of six contained
mutations in the juxtamembrane domain. Equal numbers of duplications were seen in
grade II and grade III tumors, in spite of the fact that nearly 10 times more grade II
tumors than grade III tumors were tested. J uxtamembrane duplications appear to be
associated with only the most aggressive tumors.

With respect to breed, there does not appear to be an association between the

presence of mutations and the breed of dog in which they were identiﬁed (Table 3), and

no signiﬁcant association was observed between breed and tumor grade (x2 = 7.97, 95%
conﬁdence level = 18.31). This ﬁnding is in contrast to those of previous studies in
which a higher percentage of mast cell tumors in Boxers were of a low grade as
compared with other breeds. A compilation of records for all cases submitted to AHDL

during 1999 revealed that the tumor grade distribution in Boxers was nearly identical to

126

that seen in mixed-breed dogs. Of 69 mast cell tumors in Boxers, 29.0% were grade I,
62.3% grade II, and 8.7% grade H1. The distribution for 99 mixed-breed dogs was 29.3%
grade I, 62.6% grade II, and 8.1% grade III.

In both of the previous studies, the canine mastocytoma cell line C2 containing a

c-KITjuxtamembrane domain duplication exhibited constitutive phosphorylation of c-

KIT .9’10 Because the duplications identiﬁed in this study all overlap with the duplication
found in that cell line, the present duplications probably also would cause activation of c-

KIT. Mouse cell lines with c-KIT-activating mutations can induce growth behavior

typical of tumor formation.8 Uncontrolled growth seen in tumors should be expected
when a receptor regulating cell growth is constitutively active. This ﬁnding implicates
the duplications found in this study as potential causes of the aggressiveness of the
tumors in which they were identiﬁed. The role of the deletions requires further
investigation.

In the present study, approximately 13.6% of canine mast cell tmnors tested

contained mutations within the coding region for the juxtamembrane domain of c-KIT.
This frequency is much lower than those reported by Ma et al.,10 who found mutations in

three of seven tumors and two of three cell lines, and London et al.,9 who found
duplications in 5 of 11 tumors. Our study was based upon a larger sample set, which is

less susceptible to ascertainment bias, and tumors of all grades were included. London et

al.9 studied only grade 11 tumors, some of which may have been at the higher end of that
range (borderline grade 111). If that were the case, our ﬁnding of mutations in four of six

grade III tumors would agree more closely with the results of London et al. The grades

of the tumors studied by Ma et al. were not reported. 10

127

A signiﬁcant portion of canine mast cell tumors currently have no identiﬁed
genetic defect in the juxtamembrane domain of c-KIT. In addition to the possibility that

other genes are involved, there is also the chance that some of these tumors may contain
other mutations in c-KIT. In this study and that of London et al.,9 only the

juxtamembrane domain was screened for mutations; however, Ma et al.10 also tested the

phosphotransferase domain. The phosphotransferase domain of c-KIT has been reported

to harbor mutations in some human7 and rodent”'17 mast cell tumors. In addition to the
duplications and deletions, our identiﬁcation of numerous SNPs in c-KIT, both in the
juxtamembrane domain and in other regions of the c-KIT gene, suggests that c-KIT as a
whole may be a hotspot for mutations in dogs, with the potential for mutations in mast
cell tumors outside of the juxtamembrane domain.

Our ﬁnding of c-KIT mutations in grade II and grade 111 but not grade I tumors
indicates that c-KIT may be a good marker for tumor status and suggests a role for c-KIT
in at least part of the tumorigenic process, providing a potential target for intervention.

Tyrosine kinase inhibitors are currently in development for treatment of a number of

cancer types. Very recently, one of these tyrosine kinase inhibitors, STI-5716 (Gleevec,
Novartis Pharmaceuticals), has been approved by the Food and Drug Administration for
the treatment of chronic myologenous leukemia (CML) in humans. In CML, a
translocation causes a hybrid tyrosine kinase made up of parts of the genes bar and ab] to
be constitutively active.6 This drug may also be a good inhibitor of c-KIT .4 c-KIT
inhibitors such as these and others could be used on tumors both with and without

mutations to evaluate their effect on tumor grth and their potential as therapeutic

agents. We are planning to launch such studies at the molecular and cellular level,

128

followed by clinical studies. If reduced tumor growth were observed, c-KIT activation,

such as that caused by these mutations, would be considered important for the

development of mast cell tumors. These ﬁndings would pave the way for an additional

medical treatment for the management of these tumors.

Acknowledgements

We thank Dr. Joseph Hauptrnan for assistance with the statistical analyses. This work

was funded in part by the Jaqua Foundation, the Animal Health and Diagnostic

Laboratory, and the Elizabeth Eddy Fund of Michigan State University.

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activation of c-kz't receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3.
Int Arch Allergy Immunol 106:377-385, 1995

17. Tsuj irnura T, Morimoto M, Hashimoto K, Moriyama Y, Kitayama H, Matsuzawa
Y, Kitamura Y, Kanakura Y: Constitutive activation of c-kit in FMA3 murine
mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane
domain. Blood 87:273-283, 1996

18. Vail DM: Mast cell tumors. In: Small Animal Clinical Oncology, eds. Withrow
SJ, MacEwen EG, 2nd ed., pp. 192-210. W.B. Saunders, Philadelphia, 1996

19. Valent P, Spanbldchl E, Sperr WR, Sillaber C, Zsebo KM, Agis H, Strobl H,
Geissler K, Bettelheim P, Lechner K: Induction of human mast cells from bone
marrow and peripheral blood mononuclear cells by recombinant human stem cell

factor / kit-ligand in long-term culture. Blood 80:2237-2245, 1992

20. Yarden Y, Kuang W, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, Chen E,
Schlessinger J, F rancke U, Ullrich A: Human prom-oncogene c-kit: a new cell
surface receptor tyrosine kinase for an unidentiﬁed ligand. EMBO J 6:334]-
3351, 1987

21 . Zemke D, Yamini B, Yuzbasiyan-Gurkan V: Characterization of an
Undifferentiated Malignancy as a Mast Cell Tumor Using Mutation Analysis in
the Proto-oncogene c-KIT. J Vet Diagn Invest 13:341-345, 2001

Request reprints from Dr. Yuzbasiyan-Gurkan, D-208 Veterinary Medical Center,

Michigan State University, East Lansing, MI 48824-1314, (USA).

E-mail: yuzbasiyan@cvm.msu.edu

131

CHAPTER 7

SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS

132

The genes examined in this study have multiple roles and affect different cell
types, meaning that defects in these genes could have effects on many systems of the
body. These genes share a common role in the development and maintenance of
melanocytes, cells that control pigmentation in many parts of the body and that were
hypothesized to also be involved in some aspect of normal hearing. The examination of
these genes in dogs with hereditary hearing loss found no mutations within the coding
regions. In the MIT F gene, splice sites were also evaluated and no mutations were found.
Future studies will involve examination of the splice sites in the EDNRB and KIT genes,
for which intronic sequences have been obtained. The absence of mutations within the
genes studied reduces the likelihood that any of them are involved in hereditary deafness
in the breeds studied, although important regions of these genes outside of the coding
region have yet to be studied. The involvement of EDNRB and KIT in deafness in the
Jack Russell Terrier is particularly unlikely given the lack of an association between
deafness and markers within these genes. This breed shows inheritance of the piebald
allele at the canine spotting locus. In addition to the spotting locus, the merle locus is
also possibly linked with deafness in dogs. The Catahoula Leopard Dog, which shows
inheritance of the merle locus, is currently being tested with these markers to determine
whether they are associated with deaﬁiess in that breed.

Although no potential cause for deafness was found, the study of these genes
identiﬁed several features that may be useful for future studies. The EDNRB and MIT F
genes had not been sequenced in the dog prior to this study, and probable errors in the
published sequences of KIT were identiﬁed. Furthermore, polymorphisms were found

within the EDNRB and KIT genes. These polymorphisms can be used as markers both

133

for linkage studies and for mapping of the genes. Using a polymorphism found in

EDNRB, this gene was mapped to canine Chromosome 22.9 Mapping efforts are
currently underway for KIT and MIT F . The study of these genes also identiﬁed other
interesting sequence features, namely a potential pseudogene for MITF and a sequence
variation in EDNRB that is currently unexplained. Further examination of these features
is important for the full characterization of these genes.

Besides melanocytes, the KIT gene is also important for the development of mast
cells. Investigation of this gene has determined that mutations within it are found in
some mast cell tumors in the dog. Most of these mutations involved duplications of a
particular region of the gene and are predicted to cause constitutive activation of the
receptor. This study provided evidence for association of these mutations with high
grade tumors. Any role that KIT possible plays in deafness is probably unrelated to mast
cell tumors. Deafness, and the lack of pigmentation that is associated with it, are most

likely the result of a loss of function in a particular gene. Mast cell tumors, on the other

hand, appear to involve the constitutive activation of KIT.5’6 Another group has

recently published a study of mast cell tumors in a number of dogs and also found that

duplications were more frequent in high grade tumors.2 However, this difference was
not determined to be signiﬁcant. Further studies are necessary to determine the role of
such mutations in mast cell tumors. In fact, a prospective study is being planned
incorporating mutation studies in KIT, studies of proliferation markers and other
histological attributes, and the biological behavior of mast cell tumors in dogs.

The identiﬁcation of duplication events at the same site in independent cases is of

interest. While it is likely that these events are selected for, the exact nature of how

134

duplications within this particular region of KIT are generated is currently unknown. An

oncogene that was identiﬁed in a feline sarcoma virus shows considerable homology to a
portion of the KIT gene.1 The duplications found in KIT are in approximately the same

region where this viral homology exists.6 It is possible that sequences mediating
recombination exist in this area and are responsible for the creation of duplications. This
does not explain, however, why the majority of the duplications have different endpoints
from each other. It also does not explain the deletions found in this study that lie
upstream of this region. The effect of these mutations in KIT and their role in mast cell

tumor development is also unclear. Studies have shown that one canine mast cell tumor

cell line with a KIT duplication exhibits constitutive phosphorylation of the receptor.6’8
This is likely to be true for other duplications in the same region as well. The

juxtamembrane domain has been proposed to contain an a-helix that inhibits receptor

phosphorylation.7 Duplications may disrupt this structure and lead to activation. The

effect of deletions in this region is not known at present. Activating mutations of KIT in

other species have been shown to result in tumor formation.5 These mutations differ in
type and location from those seen in the dog, however. The phosphotransferase domain

of KIT, one location where these mutations are seen in other species, was found not to

contain mutations in canine tumors.8 Future evaluation of such mutations in
experimental systems will need to be carried out to further deﬁne the role of such
mutations in mast cell tumor development and behavior.

In the mouse, mutations in the Mitf gene are responsible for the microphthalmia

phenotype.4 Many of the known Mitf alleles result in abnormal pigmentation.

135

Depending on the particular allele, the phenotype may also include microphthalmia,

deafness, or other abnormalities.11 Microphthalmia is prevalent in a number of dog

breeds, including those that have inherited the merle trait, and can be associated with

white coat color and deafness.3 Based on the similarity to the mouse phenotype, it is
possible that microphthalmia in the dog is also due to mutations in the MIT F gene. This
gene is currently being screened in dogs from a number of affected breeds to determine if

this is the case. The microphthalmia black-eyed white allele in mice is the result of the
insertion of a LINE element within one of the introns of Migﬂlz It has been suggested

that the merle trait in dogs may be due to a transposable element.10 It is possible that
this may also be responsible for deafness and microphthalmia in these dogs. As well as
the coding region and the splice sites, efforts should be made to obtain full sequences for
the introns, so that they can be examined as well.

Besides the genes involved in this study, focus should also be placed on other
candidate genes for deaﬁress. The PAX3 gene was originally included in this study,
however difﬁculties prevented sufﬁcient progress ﬁom being obtained. The EDN3 gene
was also attempted without success. Work should be continued on these genes, and other
genes such as SOX 10 should be investigated as well. Genes that modify the activity of
the current candidiates should be studied. Attempts should also be made to build a
pedigree of dogs with deafness that could be used for linkage studies. With linkage
results, positional cloning could be employed and the number of candidate genes to be
studied should be reduced. It would also allow the identiﬁcation of candidates that so far
have not presented themselves. With the current state of knowledge of the canine

genome and the number of genetic markers being produced, this is far more feasible now

136

than it was when this study was initiated. Finally, the examination of the candidate genes
to this point has been primarily at the sequence level. Studies of expression and large-
scale genomic studies such as Northern and Southern blots should be performed to

determine whether other levels are involved in deafness.

References

l. Besmer P, Murphy JE, George PC, Qiu F, Bergold PJ, Lederman L, Snyder HW
Jr, Brodeur D, Zuckerman EE, Hardy WD. A new acute transforming feline

retrovirus and relationship of its oncogene v-kit with the protein kinase gene
family. Nature 320:415, 1986.

2. Downing S, Chien MB, Kass PH, Moore PE, London CA. Prevalence and
importance of internal tandem duplications in exons 11 and 12 of c-kit in mast cell
tumors of dogs. Am J Vet Res 63:1718-1723, 2002.

3. Gwin RM, Wyman M, Lim DJ, Ketring K Jr, Werling K. Multiple ocular defects
associated with partial albinism and deafness in the dog. J Am Anim Hosp Assoc
17:401-408, 1981.

4. Hodgkinson CA, Moore KJ, Nakayama A, Steingrimsson E, Copeland NG,
Jenkins NA, Amheiter H. Mutations at the mouse microphthalmia locus are

associated with defects in a gene encoding a novel basic-helix-loop—helix-zipper
protein. Cell 74:395-404, 1993.

5. Kitayama H, Kanakura Y, Furitsu T, Tsujimura T, Oritani K, Ikeda H, Sugahara
H, Mitsui H, Kanayama Y, Kitamura Y, Matsuzawa Y. Constitutively activating
mutations of c-kit receptor tyrosine kinase confer factor-independent growth and
tumorigenicity of factor-dependent hematopoietic cell lines. Blood 85:790-798,
1995.

6. London CA, Galli SJ, Yuuki T, Hu ZQ, Helfand SC, Geissler EN. Spontaneous

canine mast cell tumors express tandem duplications in the proto-oncogene c-kit.
Exp Hematol 27:689-697, 1999.

137

7. Ma Y, Cunningham ME, Wang X, Ghosh I, Regan L, Longley BJ. Inhibition of
spontaneous receptor phosphorylation by residues in a putative a-helix in the KIT
intracellular juxtamembrane region. J Biol Chem 274:13399-13402, 1999.

8. Ma Y, Longley B], Wang X, Blount JL, Langley K, Caughey GH. Clustering of
activating mutations in c-KITs juxtamembrane coding region in canine mast cell
neoplasms. J Invest Dermatol 112:165-170, 1999.

9. Schmutz SM, Moker J S, Yuzbasiyan-Gurkan V, Zemke D, Sampson J, Lingaas F,
Dunner S, Dolf G. DCT and EDNRB map to DogMap linkage group L07. Anim
Genet 32:321, 2001.

10. Sponenberg DP. Germinal reversion of the merle allele in Australian shepherd
dogs. J Hered 75:78, 1984.

ll. Steingrimsson E, Moore KJ, Larnoreux ML, Ferré-D'Amaré AR, Burley SK,
Zimring DCS, Skow LC, Hodgkinson CA, Amheiter H, Copeland NG, Jenkins
NA. Molecular basis of mouse microphthalmia (mi) mutations helps explain their
developmental and phenotypic consequences. Nat Genet 8:256-263, 1994.

12. Yajima I, Sato S, Kimura T, Yasurnoto K, Shibahara S, Goding CR, Yarnamoto
H. An Ll element intronic insertion in the black-eyed white (Mithmi-bwD gene:
the loss of a single Mitf isoform responsible for the pigrnentary defect and inner
ear deaﬁiess. Hum Mol Genet 8:1431-1441, 1999.

138

CHAPTER 8

MATERIALS AND METHODS

139

Genomic DNA Isolation From Tissue

Genomic DNA was obtained ﬁ'om frozen tissues using either a guanidine
isothiocyanate solution (Trizol, Gibco BRL) or a guanidine-detergent solution (DNAzol,
Gibco BRL).

For isolation with Trizol, approximately 200-400 mg of tissue was homogenized
in 3 mL of Trizol with a glass homogenizer and allowed to sit for 5 minutes at room
temperature. The homogenized tissue was split into 4 microcentrifuge tubes and 150 uL
of chloroform was added to each. The tubes were shaken by hand for 15 seconds,
allowed to sit for 3 minutes at room temperature, and then centrifuged at 4 °C for 15
minutes at 12,000 rpm. The upper phase was removed and the lower two phases were
mixed with 225 pL of 95% ethanol by inversion. The samples were allowed to sit for 3
minutes at room temperature, then centrifuged at 4 °C for 5 minutes at 2000 rpm. The
supernatant was removed and 750 uL of 0.1 M sodium citrate in 10% ethanol was added.
The samples were allowed to sit for 30 minutes at room temperature with periodic mixing
by inversion, then centrifuged at 4 °C for 5 minutes at 2000 rpm. The supernatant was
removed and a second wash with the sodium citrate solution was performed. After
removal of the supernatant, 1.5 mL of 75% ethanol was added to each pellet, allowed to
sit for 20 minutes at room temperature with periodic mixing by inversion, then
centrifuged at 4 °C for 5 minutes at 2000 rpm. The supernatant was removed, the pellets
were air-dried for 10 minutes, and then resuspended in 300 uL of 8 mM NaOH. The 4
tubes were then combined for a total volume of 1.2 mL of DNA from each tissue sample.

. For isolation with DNAzol, approximately 50—100 mg of tissue was

homogenized in 1 mL of DNAzol with a glass homogenizer in as few strokes as possible.

140

The homogenized tissue was transferred to a microcentrifuge tube and spun at 4 °C for 10
minutes at 10,000 rpm. The supernatant was transferred to a clean tube and mixed with
500 uL of 95% ethanol by inversion. The sample was allowed to sit for three minutes at
room temperature, then a pipette tip was used to spool up the DNA and transfer it to a
clean tube. The DNA was washed twice by adding 1 mL of 75% ethanol, mixing by
inversion, allowing the DNA to settle to the bottom of the tube by gravity, then removing
the supernatant. The pellet was air-dried for a few seconds and resuspended in 1 mL of 8

mM NaOH.

Genomic DNA Isolation From Cheek Swabs

A sterile cytology brush was placed between the lip and gum and used to brush
the inside surface of the cheek for approximately 15 seconds. The brush was then stored
in the original packaging until the DNA was isolated. The tip of each swab was cut off
and placed into a microcentrifuge tube containing 600 uL of 50 mM NaOH. The tube
was mixed by vortexing, heated at 95 °C for 5 minutes, and mixed again. The tube was

spun brieﬂy, followed by the addition of 60 uL of 1 M Tris, pH 8.

Total RNA Isolation

Total RNA was isolated ﬁom ﬁozen tissues using a solution of guanidine
isothiocyanate and phenol (Trizol, Gibco BRL). Approximately 200-400 mg of tissue
was homogenized in 3 mL of Trizol with a glass homogenizer and allowed to sit for 5

minutes at room temperature. The homogenized tissue was split into 4 microcentrifuge

tubes and 150 uL of chloroform was added to each. The tubes were shaken by hand for

141

15 seconds, allowed to sit for 3 minutes at room temperature, and then centrifuged at 4
°C for 15 minutes at 12,000 rpm. The upper phase was transferred to a clean tube and
375 uL of isopropanol was added. The samples were mixed by inversion, allowed to sit
for 10 minutes at room temperature, then centrifuged at 4 °C for 10 minutes at 12,000
rpm. The supernatant was removed and 750 uL of 75% ethanol was added to each pellet.
The samples were mixed by vortexing and centifuged at 4 °C for 5 minutes at 7500 rpm.
The supernatant was removed, the pellets were air-dried for 10 minutes, and were
resuspended in 75 uL of water each. The 4 tubes were then combined for a total volume

of 300 uL total RNA ﬁom each tissue sample.

DNase Treatment of Total RNA Samples

A 400 uL reaction mixture was set up containing 300 uL RNA, 50 mM Tris pH
7.5, 10 mM MgC12, 50 ug/mL bovine serum albumin, and 40 U RNase-free DNase I
(Boehringer Mannheim). The mixture was incubated for 30 minutes at 37 °C, then

ethanol precipitated and resuspended in 300 pL deionized water.

Reverse Transcription

A 76 uL reaction mixture was prepared containing 10 mM DTT, 2.4 ug random
hexamers, 0.32 mM dNTPs, and 8 pL of RNA. This mixture was incubated at 70 °C for
5 minutes to denature the RNA secondary structure, then placed on ice for 5 minutes.
After incubation on ice, 20 uL ﬁrst strand buffer and 800 U MMLV reverse transcriptase
(Gibco BRL) were added for a total volume of 100 uL, and the entire mixture was

incubated for 1 hour at 37 °C, followed by inactivation at 90 °C for 5 minutes.

142

Polymerase Chain Reaction

Standard PCR reactions had a total volume of 25 uL and contained 20 pmol of
each primer, 0.625 U Taq polymerase (Gibco BRL/Invitrogen), and ﬁnal concentrations
of 100 uM dNTPs, 1.5 mM MgC12, 50 mM Tris-Cl (pH 8.3), and 10 mM KCl. Typically,
3 uL of template were used for ampliﬁcation ﬁ'om cDNA and 5 uL of template were used
for ampliﬁcation from genomic DNA. Standard PCR conditions consisted of an initial
denaturation step of 4 minutes at 94 °C, followed by 35 cycles of 1 minute at 94 °C, 2
minutes at the appropriate annealing temperature, and 3 minutes at 72 °C, with a ﬁnal

extension step of 8 minutes at 72 °C.

Gel Purification of Ampliﬁed Bands

Ampliﬁcation products were separated on agarose gels, and the resulting bands
were excised. DNA was isolated from the excised gel slices using a system based on
silica beads and chaotropic salts (QIAEX II, Qiagen). Each gel slice was placed into a
microcentrifuge tube, to which 500 uL of buffer QXl and 5 uL of beads were added.
The samples were incubated for 10 minutes at 55 °C, with mixing by vortexing every 2
minutes. The samples were then spun brieﬂy in a microcentrifuge at maximum speed.
The supernatant was removed, the pellet was resuspended in 500 uL of buffer QXl by
vortexing, and the sample was spun brieﬂy again. This step was repeated two more times
using buffer QX2. The supernatant was removed, the pellet was air-dried for 10 minutes,
then resuspended in 30 uL of 10 mM Tris, pH 8 by vortexing. The sample was allowed
to sit at room temperature for 5 minutes then spun brieﬂy in a microcentrifuge at

maximum speed. The supernatant containing DNA was then transferred to a clean tube.

143

Sequencing of Puriﬁed DNA

Manual sequencing was performed based on a modiﬁed version of the Sanger

sequencing method using dideoxy terminators labeled with 33P (Thermo Sequenase kit,
USB). A 20 uL reaction mixture was prepared containing 5-15 uL of template DNA, 2
uL of reaction buffer, 0.5 pmol of primer, and 2 uL of Thermo Sequenase enzyme. A 4.5
uL aliquot of this mixture was transferred to each of 4 separate tubes containing 2 uL of
dNTP solution and 0.5 uL (approximately 225 nCi) of labeled ddNTP. The samples were
cycled for 30 cycles of 95 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 1
minute. In the case of template sequences containing sequence compressions, a dNTP
solution containing dITP instead of dGTP was used and the samples were cycled at 95 °C
for 30 seconds, 50 °C for 30 seconds, and 60 °C for 10 minutes. The reaction products
were mixed with 4 uL of stop solution, denatured at 70 °C for 2-10 minutes, and 4 uL of
each was electrophoresed on a 6% polyacrylamide/7 M urea gel at a constant 60 W. The
gel was transferred onto Whatman 3MM paper, dried under vacuum at 80 °C, and
visualized by exposure to x-ray ﬁhn.

Automated sequencing was performed by the author on an ABI Prism 377 DNA
Sequencer using Big Dye terminators. A 20 uL sequencing reaction was prepared
containing 8 uL of the Big Dye reaction mix, 5-10 uL of template DNA, and 3.2 pmol of
primer. Samples were cycled for 25 cycles of 96 °C for 30 seconds, 50 °C for 15
seconds, and 60 °C for 4 minutes. Unincorporated terminators were removed by
precipitation with 75% isopropanol plus 1 uL of 20 mg/mL glycogen as a carrier. A

solution of 25 mM EDTA pH 8 + 50 mg/mL blue dextran was mixed with 5 volumes of

144

 

deionized formamide to create a loading buffer. Each sample was resuspended in 8 pL of

loading buffer, denatured for 2 minutes at 95 °C, and a 1.5 pL aliquot was loaded onto

the gel.

Preparation of Radioactive Probes by the Incorporation of 32P-labeled dCTP
A 28 pL mixture containing 9.6 pg of random DNA hexamers and 200 ng of

probe DNA was boiled for 3 minutes then placed on ice. To this sample, 5 pL of a

 

mixture containing 0.5 mM each of dGTP, dATP, and dTTP was added. Next, 5 pL of ;

Klenow buffer, 8 U of Klenow ﬁagrnent, and 50 pCi 32P-labeled dCTP were added and
the reaction brought up to a total volume of 50 pL. The reaction was incubated for 4

hours at 37 °C, then stopped by the addition of 2 pL 0.5 M EDTA and 200 pL TE.

DNA Isolation From BAC Clones

A bacterial stock containing each clone was used to inoculate 2 mL of LB broth
containing 20 pg/mL chloramphenicol, which was incubated at 37 °C overnight with
shaking at 250 rpm. The overnight cultures were spun at 1000 G for 10 minutes at room
temperature, and the cell pellet was resuspended in 300 pL of 15 mM Tris pH 8, 10 mM
EDTA, 100 pg/mL RNase. To each sample, 300 pL of 0.2 N NaOH, 1% SDS was
added. Each tube was mixed by inversion and placed at room temperature for 5 minutes.
This was followed by the addition of 300 pL of 3 M KOAc pH 5.5 to each tube, mixing
by inversion, and incubation on ice for 5 minutes. The samples were spun at 12,000 rpm
for 10 minutes in a microcentrifuge at 4 °C. The supernatant was transferred to a tube

containing 0.8 mL of ice-cold isopropanol. The samples were mixed by inversion,

145

incubated on ice for 5 minutes, then spun for 15 minutes at 4 °C. The supernatant was
removed and the pellets were washed with 500 pL of 70% ethanol, then spun at for 5
minutes at 4 °C, followed by removal of the supernatant. The pellets were air dried and

resuspended in 40 pL TE.

Genomic DNA Isolation From Paraffin-Embedded Mast Cell Tumors

Sections of the embedded tumor were stained with haematoxylin and eosin, and
viewed by a veterinary pathologist to determine the borders of the tumor. DNA was
isolated from the tumors by a microwave-based method similar to that initially developed
by Banerjee, et al. A modiﬁed protocol was developed that is summarized as follows. A
small piece of tissue approximately 2 mm in diameter was excised ﬁom each block
within the boundaries of the tumor. The tissue was placed into 400 pL of digestion
buffer (50 mM Tris pH 8.5, 1 mM EDTA, 0.5% Tween). The parafﬁn in the samples was
melted by heating them to 95 °C for 10 minutes, then heating for 30 seconds twice in a
microwave at ﬁrll power, mixing thoroughly by vortexing after each heating step. The
samples were allowed to cool, and 5 pL of 15 mg/mL proteinase K was added to each.
The samples were then incubated at 42 °C overnight or until the piece of tissue was
completely digested. The proteinase K was inactivated by heating at 95 °C for 10
minutes, and the samples were centrifuged at 12,000 rpm in a microcentrifuge for 10
minutes. An aliquot of 200 pL was then transferred to a clean tube, avoiding the transfer

of paraffm as best as possible.

146

APPENDIX

147

APPENDIX A

Schmutz SM, Moker J S, Yuzbasiyan-Gurkan V, Zemke D, Sampson J, Lingaas F,
Dunner S, Dolf G. DCT and EDNRB map to Dong linkage group L07. Anim Genet
32:321, 2001.

 

148

Source/description: Dopachrome tautomerase (dopachrome-isomerase, tyrosine-related
protein 2) is the gene referred to as slaty in the mouse1 which is thought to dilute

eumelanin from black to greyz.

Primer sequences: Published dog primer sequences for a microsatellite in an intron of If

DCT3 were used. EDNRB4, CXX.2795, FH21096, and LE10057, were also genotyped

and mapped to the L07 linkage groups.

 

Chromosomal location:

All the DogMap families8 were genotyped for this purpose by various members. CRI-
MAP was used to analyse linkage data. DCT was found to be linked to all four markers
previously mapped to L07 with a LOD > 3. The sex averaged map generated by CRI-

MAP was CXX.279 - LEIOOS - DCT - F112109 - EDNRB. DCT maps to human
chromosome 13q31-q329, to mouse chromosome 141, to cattle chromosome 1210, and to
pig 111 1. EDNRB, another coat colour gene, has also been mapped to cattle chromosome

12q22 by in situ hybridization12 and to human 13q22-q3113.

Recently painting of dog chromosomes with human chromosome-speciﬁc paints

and painting of human chromosomes with dog-specifc chromosome paints”,1 5 suggests
human l3q21-qter is homologous to the entirety of dog chromosome 22. At the ISAG

2000 meeting, the DogMap workshop endorsed the chromosome numbering system

proposed by the International Committee for the Standardization of the dog Karyotype”.

149

Therefore, the linkage mapping of DCT reported here suggests that L07 from the

Dong studies is assigned to dog chromosome 22.

Comment: An autosomal recessive disease, Black Hair Follicular Dysplasia which alters
black pigmented hair to grey in addition to other abnormalities, did not segregate

concordantly with DCT in a family of Large Munsterlanders affected with this
condition"). EDNRB alleles did not segregate with pups that were plated (recessive) vs.

ticked (dominant) following the terminology of Little16 for r locus phenotypes.

Acknowledgements: The Natural Science and Engineering Research Council provided

funding in Canada and the AKC Canine Health Foundation provided funding in the USA.

References

1 Jackson U. et al. (1992) EMBO 11, 527-35.

2 Budd P. & Jackson 1.]. (1995) Genomics 29, 35-43.

3 Liu P.-C. et al. (1998) Anim Genet 29, 327.

4 Zemke D. & Yuzbasiyan-Gurkan V. (1999) Anim Genet 30, 390.
5 Ostrander E. et al. (1993) Genomics 16, 207-13.

6 Francisco L.V. et al. (1996) Mamm Genome 7, 359-62.

7 Lingaas F. et al. (1997) Mamm Genome 8, 218-21.

8 Lingaas F. et al. (2001) J Anim Breed Genet 118, 3-19.

9 Sturm RA. et al. (1994) Genomics 21, 293-6.

10 Schmutz SM. et al. (1998) Can Vet J39, 644-6.

150

 

11 Chowdhary B.P. et al. (1993) Chromosome Res 1, 175-9.

12 Schlipfer J. et al. (1997) Mamm Genome 8, 380-1.

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Correspondence: S. Schmutz (e-mail: schmutz@sask.usask.ca)

151

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