Molecular genetic analysis of canine congenital

Aus dem Institut fuumlr Tierzucht und Vererbungsforschung der

Tieraumlrztlichen Hochschule Hannover

Molecular genetic analysis of canine congenital sensorineural deafness

in Dalmatian dogs

INAUGURAL-DISSERTATION

zur Erlangung des Grades einer

DOKTORIN DER VETERINAumlRMEDIZIN

(Dr med vet)

durch die Tieraumlrztliche Hochschule Hannover

Vorgelegt von

Katharina Mieskes aus Goumlttingen

Hannover 2006

Scientific supervisor Univ-Prof Dr Dr O Distl

Examiner Univ-Prof Dr Dr O Distl

Co-examiner Univ-Prof Dr H Y Naim

Oral examination 18 Mai 2006

This work was supported by a grant from the Gesellschaft zur Foumlrderung

Kynologischer Forschung (GKF) eV Bonn Germany

To my family

Parts of this work have been submitted for publication in the following journals

1 Gene

2 Journal of Heredity

3 Animal Genetics

Contents

1 Introduction 1

2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans 5

Abstract 7

The structure of the ear 7

Deafness in man 8

Deafness in dogs 9

The canine genome project 11

3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs 23

Abstract 25

Introduction 25

Material and methods 26

Results and discussion 28

4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs 41

Abstract 43

Introduction 43

5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 63

Abstract 65

Introduction 65

Materials and Methods 67

Results and Discussion 69

6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81

Abstract 83

Introduction 83

Results 86

Discussion 87

7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97

Abstract 99

Introduction 99

8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107

Abstract 109

Introduction 109

9 General Discussion 117

The candidate gene approach 119

Linkage and association analysis 120

CFA1 122

CFA31 123

CFA10 123

10 Summary 125

11 Erweiterte Zusammenfassung 129

12 References 145

13 Appendix I 14 List of publications XIII

Abbreviations

List of abbreviations

A adenine

Acc No accession number

ACTG1 actin gamma 1

AEP akustisch evozierte Potentiale (acoustically evoked potentials)

APS ammonium persulphate

AT annealing temperature

BAC bacterial artificial chromosome

BAER brain stem auditory evoked response

BLAST Basic Local Alignment Search Tool

bp base pair

C cytosine

CCSD canine congenital sensorineural deafness

CDH23 cadherin related 23

cDNA copy desoxyribonucleic acid

CFA chromosome of Canis familiaris

CLDN14 claudin-14

cM centiMorgan

COCH coagulation factor C homolog cochlin

COL11A2 collagen type XI alpha 2

CRYM crystallin mu

CSD cochleosaccular degeneration

CX connexin

DFN x-linked deafness locus

DFNA autosomal dominant deafness locus

DFNA5 deafness autosomal dominant 5

DFNB autosomal recessive deafness locus

DIAPH1 diaphanous homolog 1 (Drosophila)

DMSO dimethyl sulfoxide

Abbreviations

DNA deoxyribonucleic acid

dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)

EDN3 endothelin 3

EDNRB endothelin receptor type B

EDTA ethylenediamine tetraaceticacid

EMBL European Molecular Biology Laboratory

ESPN espin

EST expressed sequence tag

EYA4 eyes absent homolog 4 (Drosophila)

F forward

FISH fluorescence in situ hybridisation

G guanine

GJA1 gap junction protein alpha 1 43kD (connexin 43)

GJB2 gap junction protein beta 2 26k (connexin 26)

GJB3 gap junction protein beta 3 31kDa (connexin 31)

GJB6 gap junction protein beta 6 (connexin 30)

GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the

Advancement of Cynological Research)

HET observed heterozygocity

HE expected heterozygosity value

HSA chromosome of Homo sapiens

IBD identical by descent

IRD infrared dye

KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4

Kb kilobase

LD linkage disequilibrium

LINE long interspersed nuclear element

LOD logarithm of the odds

M molar

Mb megabase

Merlin multipoint engine for rapid likelihood inference

MITF microphthalmia-associated transcription factor

Abbreviations

MS microsatellite

MTRNR1 mitochondrially encoded 12S RNA

MTTS1 mitochondrially encoded tRNA serine 1 (UCN)

MYH9 myosin heavy polypeptide 9 non-muscle

MYH14 myosin heavy polypeptide 14

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

NCBI National Center for Biotechnology Information

NMMHC-A nonmuscle myosin heavy chain-A

ODDD oculodentodigital dysplasia

OMIM Online Mendelian Inheritance in Man

OTOA Otoancorin

OTOF otoferlin

P error probability

PAX3 paired box gene 3 (Waardenburg syndrome 1)

PCDH15 Protocadherin-15

PCR polymerase chain reaction

PIC polymorphism information content

POU3F4 POU domain class 3 transcription factor 4

PRES solute carrier family 26 member 5 (prestin)

QTL quantitative trait locus

R reverse

RACE rapid amplification of cDNA ends

RH radiation-hybrid

RLM RNA ligase-mediated

RNA ribonucleic acid

RT-PCR reverse transcription polymerase chain reaction

SAS Statistical Analysis System

Abbreviations

SH1 Src homology 1

SINE short interspersed nuclear element

SLC26A4 solute carrier family 26 member 4 (pendrin)

SLC26A5 solute carrier family 26 member 5 (prestin)

SNP single nucleotide polymorphism

SOX10 SRY (sex determining region Y)-box 10

STRC stereocilin

STS sequence-tagged site

T thymine

TBE tris-borate-ethylenediamine tetraacetic acid

TECTA tectorin alpha

TEMED NNNrsquoNrsquo-tetramethylenediamine

TFCP2L3 transcription factor CP2-like 3

TJ tight juncions

TMC1 transmembrane channel-like gene 1

TMIE transmembran inner ear gene

TMPRSS3 transmembrane protease serine 3

U unit

USH1C Usher syndrome 1C

UTR untranslated region

WFS1 Wolfram syndrome 1 (wolframin)

wgs whole genome shotgun

WHRN whirlin

WS Waardenburg syndrome

Chapter 1

Introduction

Introduction 3

Introduction

Canine congenital sensorineural deafness (CCSD) has often been reported in the

literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog

showing the highest incidence The inheritance and segregation of a major gene in

CCSD has been demonstrated in different Dalmatian dog populations But although

several studies have demonstrated the mode of inheritance in Dalmatian dogs no

universally accepted mode of inheritance for the other dog breeds affected by CCSD

has yet been identified

Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and

sires is forbidden by paragraph 11b of the German animal welfare laws and thus the

hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight

weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs

are very difficult to raise and often become aggressive and snappish from fear most

puppies suffering from bilateral hearing loss are euthanized However it has been

shown in recent years that auditory testing does not seem to be an effective way of

clearly reducing the high incidence of deafness in this breed Thus prevention of

CCSD cannot be achieved alone by exclusion of affected animals from breeding

Consequently a molecular genetic approach toward unravelling the responsible

genes in carriers is urgently needed

Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate

a high level of clinical and molecular similarity Therefore the mutated genes in

human hereditary deafness seemed to be appropriate candidates for canine

congenital sensorineural deafness

The objective of the present study is to localize the gene that is involved in the

development of CCSD in Dalmatian dogs In order to achieve this goal successively

32 canidate genes were evaluated by means of linkage analyses using microsatellite

markers and single nucleotide polymorphisms (SNPs) This candidate gene

approach using gene-associated markers for linkage studies in families segregating

for deafness turned out to be little effective Therefore the canine chromosomes

(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers

Additionally single nucleotide polymorphisms (SNPs) were developed for fine

mapping the identified CCSD regions

Introduction 4

Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary

hearing impairment in humans Parallels and differences in canine and human

deafness are shown including the clinical signs inheritance patterns and

histopathology We located the humane deafness genes in the canine genome and

discussed the advantages of comparative genomics and different molecular genetic

approaches

In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate

genes were used for a non-parametric linkage analysis with congenital sensorineural

deafness (CCSD) in Dalmatian dog families segregating for deafness

In Chapter 4 newly developed SNP markers associated with in total eight candidate

genes were evaluated for CCSD in Dalmatian dogs

In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide

9 non-muscle (MYH9) gene on dog chromosome 10q232 is described

Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families

segregating for congenital sensorineural deafness using microsatellite markers on

canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping

regions linked with the CCSD phenotype using newly developed SNPs

Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8

Chapter 10 is a concise English summary of this thesis while Chapter 11 is an

expanded detailed German summary which takes into consideration the overall

research context

Chapter 2

A comparative overview of the molecular genetics of non-syndromic deafness

in dogs and humans

Non-syndromic deafness in dogs and humans 7

A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans

Abstract

In man as in different dog breeds deafness is an often diagnosed disorder with the

Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly

heritable and the presence of a recessive major gene affecting the disorder was

shown in several Dalmatian dog populations

This Chapter provides an overview of the identified 39 mutated genes causing

human non-syndromic hereditary hearing impairment as well as of the five genes

responsible for Waardenburg syndrome in humans We point out their cytogenetic

and genomic localisations in man and dog and compare the genomic and mRNA

sequences of these genes between man and dog Moreover an overview is given on

deafness genes-associated markers identified in Dalmatian dogs and on candidate

genes characterized in dogs

The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea

whereas the vestibule and the semicircular canals are concerned with vestibular

function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane

and the basilar membrane divide the cochlea longitudinally into three scalae the

scala vestibule the scala media and the scala tympani The process of transduction

occurs in the structures within scala media sitting on the basilar membrane and

comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala

media is more or less triangular formed by Reissneracutes membrane basilar

membrane and a structure called the stria vascularis The fluid that fills scala

tympani and scala vestibule is called perilymph the fluid that fills scala media is

called endolymph The organ of Corti rests on the basilar membrane within scala

media The cochlea contains an array of highly specialized cells arranged in a highly

specialized manner Two types of cells in the organ of Corti are support cells and

hair cells The hair cells are the receptor cells that trancsduce sound

When a sound wave brings physical displacement of the membranes separating the

perilymph from the endolymph they cause the organ of Corti to move and the hair

cells on it are scraped along the bottom of the tectorial membrane The tectorial

membrane is firmly anchored to the bone Relative movement of the organ of Corti

and its hair cells with respect to the tectorial membrane is the source of the

deformation of the hair cells microvilli The hair cells are so constructed that any

deformation of their microvilli will cause a change in the overall membrane potential

of the cell This signal is detected by the fibers from the cells in the spiral ganglion

These fibers are neural elements and they carry their own depolarization wave into

the auditory region of the brain

Deafness in man There are various ways to categorise deafness The two main types of deafness are

classified based on which portions of the auditory system are affected conductive

hearing loss occurs when when sound is not conducted efficiently through the outer

andor middle part of the ear Much more common is the sensorineural hearing loss

Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)

or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases

of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)

Hearing loss can be present at birth (congenital) or become evident later in life

(acquired) Congenital deafness similarly may or may not be genetic In fact more

than half of congenital hearing loss is inherited Alternatively congenital deafness

may be due to a condition or infection to which the mother was exposed during

pregnancy Furthermore congenital hereditary deafness may occur as part of a

multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular

system (non-syndromic) As non-syndromic hereditary hearing impairment is almost

exclusively caused by cochlear defects affected patients suffer from sensorineural

hearing loss In Table 1 and 2 the genes underlying human hereditary non-

syndromic deafness as a result of cochlear defects in consequence of primary

defects in hair cells non-sensory cells and the tectorial membrane or unknown cell

type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of

congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this

non-syndromic form of deafness shows an autosomal recessive inheritance followed

by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases

are X-linked A maternally inherited form may also occur (Van Camp and Smith

Non-syndromic forms of hereditary deafness are classified by their mode of

inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X

chromosome-linked autosomal dominant and autosomal recessive modes of

transmission respectively

Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al

2001) Up to 1 of the human genes are estimated to be necessary for hearing

(Friedmann and Griffith 2003) Today approximately 120 genes for human

hereditary deafness have been identified approximately 80 for syndromic and 39 for

non-syndromic hereditary deafness which is suspected to be one-third of the total

(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic

hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes

cause autosomal recessive and 15 genes cause autosomal dominant forms six

genes are involved in both recessive and dominant forms one gene causes X-linked

and two a maternally inherited form (The Hereditary Hearing Loss Hompage

httpwebhostuaacbehhh)

Furthermore several hundred forms of syndromes with hearing loss have been

documented in humans (Van Camp and Smith 2003) One is the human

Waardenburg syndrome (WS) which manifests itself with sensorineural deafness

and pigmentation defects in the iris hair and skin The WS is classified into four

types depending on the presence or absence of additional symptoms which are

caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10

respectively These genes are shown in Table 3 They are known to be expressed in

the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes

(MITF) and are inter alia involved in migration differentiation or survival of

melanocytes respectively (Bondurand et al 2000)

Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal

species other than humans ranging from mice to dogs guinea pigs and mink

Canine congenital deafness has often been reported in the literature and occurs in

more than 54 different breeds of dogs according to Strain (1996 and 2004) The

breeds with the highest incidence include Dalmatian dogs Bull Terrier English

Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-

Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine

congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit

unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997

Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance

and segregation of a major gene in canine congenital sensorineural deafness

(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et

al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies

have demonstrated the mode of inheritance in Dalmatian dogs no universally

accepted mode of inheritance for the other dog breeds affected by CCSD has yet

been identified

Congenital sensorineural hearing impairment can be recognised in dogs at four to

eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs

have shown that the degeneration of the inner ear structures begins as early as one

day after birth and is histologically clearly evident by four weeks of age (Johnsson et

al 1973) In 70 of the cases with human hereditary deafness the histological

pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)

commonly known as Scheibe dysplasia with preservation of the pars superior of the

membranous labyrinth and an unremarkable bony labyrinth As in man also in many

affected dog breeds the histological pattern of congenital sensorineural deafness is

known as cochleo-saccular degeneration

weeks old using the brainstem auditory evoked response (BAER) test that detects

electrical activity in the cochlea and auditory pathways in the brain Although the

BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it

does not seem to be an effective way of clearly reducing the incidence of deafness in

affected breeds particularly in a recessive mode of inheritance so that hearing dogs

can still be genetic carriers Furthermore deaf dogs are very difficult to raise and

often become aggressive and snappish from fear consequently most puppies

suffering from bilateral hearing loss are euthanized Thus prevention of CCSD

cannot be achieved alone by exclusion of affected animals from breeding and

consequently a molecular genetic approach toward unravelling the responsible

Over the past decade it has become increasingly clear how far structural and

functional homologies at the gene level extend across even distantly related species

Much is known about deafness-causing gene mutations in humans and mice

including the fact that the clinical and histopathological findings are often very similar

to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of

human non-syndromic deafness the genes that are responsible for non-syndromic

congenital hereditary deafness in humans (Table 1) seemed to be appropriate

candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The

genes that are mutated in the human WS (Table 2) were selected as candidates

because the WS phenotype where the deafness is associated with pigmentation

defects seems to be similar to the phenotype of most affected dog breeds (Strain

and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have

demonstrated that patched Dalmatians are less likely to be deaf than unpatched

animals and blue-eyed Dalmatians are more likely to be affected from hearing

impairment than brown-eyed animals

In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers

Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak

et al 2002a 2002b 2003) already mapped 24 potential candidate genes for

sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation

hybrid panel to 16 different canine chromosomes

The canine genome project In December 2005 an international research team led by scientists at the Broad

Institute of MIT and Harvard achieved the completion of a high-quality genome

sequence of the domestic dog together with a catalog of 25 million specific genetic

differences across several dog breeds (Lindblad-Toh et al 2005) The authors found

that humans share more of their ancestral DNA with dogs than with mice confirming

the utility of dog genetics for understanding human disease Furthermore the

physiology disease presentation and clinical response of dogs often mimic human

diseases closely As indicated above hearing impairment seemed to be no

exception

In the last years most projects have exploited canine traits for which either direct

candidate genes could be proposed and evaluated or for which large informative

pedigrees were available to enable linkage mapping to identify candidate regions A

major component of such research efforts comprised the cloning sequencing and

mapping of individual canine homologs of genes either proposed as candidate

genes or expected to be located in candidate regions This was necessary to

identify new informative polymorphisms (eg single nucleotide polymorphisms

(SNPs) microsatellites) for high resolution mapping of candidate regions and to

examine each exon and exonintron boundary for positional candidates Availability

of the second version of the dog genome assembly (build 21) of the NCBI database

shortcut this effort and increase the investigators efficency

The current RH map with 3200 markers provides a good estimate of the order and

physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon

et al 2003) and was recently complemented by the construction of a 4249-marker

integrated canine genome RH map that consists of 900 genes 1589 microsatellites

and 1760 BAC end markers (Breen et al 2004) all included and available in the

NCBI database The second version 1 of the NCBIs genome annotation consists of

large contigs covering all canine chromosomes given with their located markers and

genes The great majority of genes are derived by automated computational analysis

using the gene prediction method GNOMON

With this help either additional candidate genes for canine CSD can be found directly

by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene

is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus

the canine whole genome shotgun (wgs) sequence resource can be used to obtain

the sequence of the canine genomic contigs containing the human homologous

gene The localisation of all 39 known human non-syndromic hereditary deafness

genes in the canine genome with the corresponding accession numbers of the contig

and if available the accession number of the genomic sequence and mRNA of the

canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and

human or mouse mRNA is shown in Table 5 The average identity of canine and

human mRNA is with 088 percent higher than the average identity of canine and

mouse mRNA with 084 percent Canine sequences that correspond to the human

candidate gene can now be used to find microsatellite or SNP markers associated to

the respective canine gene These markers can be used for linkage and haplotype

studies in dog families segregating for deafness

Table 7 shows the microsatellite and SNP markers developed for in total 32

candidate genes for CCSD

The candidate genes for which a set of in total 43 microsatellite marker were

designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH

COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF

MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10

TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24

candidate genes were used for linkage and haplotype studies in Dalmatian dog

families segregating for deafness (Chapter 3) These 24 genes are known to be

involved either in human non-syndromic deafness or in the human Waardenburg

syndrome For another eight candidate genes including TMC1 TMIE USH1C

MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed

(Chapter 4) and subsequently used for linkage and association analyses in

Dalmatian dog families segregating for deafness These genes are also involved in

human non-syndromic deafness

Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in

humans

Inheritance Gene Gene product Type of molecule Locus namea

ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26

COCH Cochlin extracellular matrix component DFNA9

COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13

CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28

Autosomal dominant

WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14

CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16

TMIE TMIE transmembrane domain- containing protein DFNB6

TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18

Autosomal recessive

WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11

TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21

Autosomal dominant and autosomal recessive

TMC1 TMC1 transmembrane channel-like protein

DFNB7 DFNB11DFNA36

X-linked POU3F4 POU3F4 transcription factor DFN3

MTRNR1 Mitochondrial 12S rRNA not defined

nomenclature Mitochondrial

MTTS1 Mitochondrial 12S rRNA not defined

nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA

Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary

Primary defect

Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C

WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN

KCNQ4 TMC1 OTOF POU4F3

Non-sensory cells

GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14

COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4

Tectorial membrane

COL11A2 TECTA

Unknown

DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1

Table 3 Genes involved in the human Waardenburg syndrome

Inheritance Gene Gene product Type of molecule Type

EDN3 endothelin 3 vasoconstricted peptide WS type IV4

EDNRBendothelin

receptor type B receptor protein WS type IV4

microphthalmia-

associated

transcription

factor

transcription factor WS type II2

PAX3 paired box 3 DNA-binding protein WS type I1and III 2

SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease

(autosomal recessive inheritance)

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of

the genomic sequence and mRNA and if available the percent identity of canine and

human or mouse mRNA Canine candidate gene

Acc No canine genomic sequence

Acc No canine mRNA (predicted)

Canine mRNA (bp)

Identity () of canine and human mRNA

Identity () of canine and mouse mRNA

ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170

COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890

DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown

KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940

MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610

MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780

PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740

STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510

TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910

TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716

EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861

SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs

Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)

of canine candidate genes for canine congenital sensorineural deafness in

Dalmatian dogs

Canine candidate gene

Number of gene-associated

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

MYO7A 3 0 OTOF 1 0 PAX3 1 0

POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3

TMPRSS3 2 0 USH1C 0 2 WHRN 0 3

Chapter 3

Linkage analysis of gene-associated microsatellite markers with

congenital sensorineural deafness in Dalmatian dogs

Linkage analysis of gene-associated microsatellites 25

Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs

Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an

existing set of 43 microsatellite markers associated with in total 24 candidate genes

for canine congenital sensorineural deafness (CCSD) were used for linkage and

haplotype analyses in a large Dalmatian dog population with frequent occurrence of

CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As

linkage was found for different candidate genes in different families the results of

these test statistics indicate that the inheritance of non-syndromic deafness in

Dalmatian dogs is heterogenic in origin

Introduction

Canine congenital sensorineural deafness (CCSD) has been reported to occur in

more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds

the most commonly observed histological pattern of degenerative inner ear changes

is known as the cochleo-saccular or Scheibe type of end organ degeneration

The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-

30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and

Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The

inheritance and segregation of a major gene in CCSD has been demonstrated in

different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be

pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a

2003b Mair 1976 Strain et al 1992 Strain 1996)

No gene mutation has yet been identified that is responsible for CCSD in Dalmatian

dogs or in one of the various other dog breeds that suffer from inherited hearing

impairment Since mutations in various genes have already been found to be the

cause of sensorineural hearing impairment in humans or mice 24 of these genes

Linkage analysis of gene-associated microsatellites

were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)

Details of the 24 candidate genes are given in Table 1

Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et

al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural

deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel

Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA

markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2

DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6

MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and

TMPRSS3 (Table 2)

Among the 24 candidate genes seven genes cause autosomal dominant non-

syndromic forms of deafness seven cause autosomal recessive forms and five

genes cause both recessive and dominant forms of non-syndromic deafness in

different human families segregating for either forms

The functions of these 19 deafness-causing genes are diverse and include gap

junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)

and ion channel activators (TMPRSS3) Included are also unconventional myosins

(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as

extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton

regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with

unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also

include five genes which are mutated in the human Waardenburg syndrome (WS)

The WS is classified into four types depending on the presence or absence of

additional symptoms which are caused by mutations in the five genes EDN3

EDNRB MITF PAX3 and SOX10 respectively The objective of the current study

was to use this set of markers developed by Rak (2003) for a non-parametric linkage

analysis with CCSD in a German and French Dalmatian dog population

Material and methods Pedigree material

For the linkage analysis we used DNA from altogether 215 animals belonging to a

total of 24 Dalmatian dog families The families included 22 full-sib families and one

large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a

large paternal half-sib family of French Dalmatian dogs All families were segregating

for CCSD The genotyped families included all affected dogs (unilaterally and

bilaterally deaf) their parents if available and one to four unaffected animals At least

two of the full sibs of each family were unilaterally deaf

In total these 24 families included 402 individuals with an average family size of 168

ranging from 5 to 116 animals and covering two to four generations The hearing

status of 344 dogs was examined by veterinarians using the BAER (brain stem

auditory evoked response) test and the other animals included in the pedigree being

not BAER tested were used to construct relationships among CSD affected dogs

The prevalence of CSD in this pedigree was 285

Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for

linkage analysis This set included 36 markers developed by Rak (2003) and 7

markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml

For most of the 24 candidate genes two markers were available for two of the

candidates three markers were available but for seven candidate genes the set

contains only one marker The marker set is composed of 33 perfect repeats two

imperfect six compound-perfect and two compound-imperfect repeats

The majority (674) of the 43 markers in the set was represented by dinucleotide

repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23

pentanucleotide repeats In addition one marker (23) was a compound di-

tetranucleotide and another one (23) was a compound tetra-pentanucleotide

repeat The average number of alleles was 35 with a minimum of 2 and a maximum

of 8 different alleles per marker

PCR reactions for microsatellites were carried out in 12 microl reaction mixtures

containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg

Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq

Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward

primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out

in MJ Research thermocyclers with the following program 4 min at 94 degC followed

by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a

final extension of 45 sec at 72degC PCR-products were diluted with formamide loading

buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on

automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using

denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe

Germany)

To localize the 24 candidate genes and their associated microsatellites exactly the

canine candidate gene sequences were derived from sequences deposited in the

current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank

by BLAST (Basic Local Alignment Search Tool) search

(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence

(Table 3)

Linkage analysis

Multipoint linkage and haplotype analyses were performed using the MERLIN

software version 0102 (multipoint engine for rapid likelihood inference Center for

Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test

statistics Z-mean and Lod score were used to test for the proportion of alleles shared

by affected individuals identical by descent (IBD) for the considered marker loci

Linkage analyses were performed regarding the marker set consisting of 43 gene-

associated microsatellite markers Linkage analysis was at first carried out for all 24

families conjoined After this the families were scanned separately

The data of the genotypes was additionally analyzed using SASGenetics (Statistical

Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify

the number of alleles of each marker the allele frequency the observed (HET) and

expected (HE) heterozygosity and the polymorphism information content (PIC)

(Table 4 and 5)

Results and discussion

Test statistics for all families conjoined are given in Table 6 Significant CCSD loci

were located on different chromosomes The loci were located on canine

chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even

higher test statistics for subgroups of families (Table 7) Scanning only families with

Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9

on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different

Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes

in their flanking regions are involved in the development of the disease in the

respective familes The results of this test statistics indicate that the inheritance of

non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as

it is in humans Genetic heterogeneity means that different mutations cause the same

phenotype or disease the different mutations can either be found at the same locus

(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)

The extreme heterogeneity of human deafness often hampered genetic studies

because many different genetic forms of hearing loss give rise to similar clinical

phenotypes and conversely mutations in the same gene can result in a variety of

clinical phenotypes In man genes that transport ions across membranes to maintain

appropriate solute concentration and pH as well as regulatory genes mainly

transcription factors and genes that play a part in structural integrity are essential for

the hearing process

However this study was a first step in identifying genes responsible for CCSD in

Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will

be further analyzed with a combined approach using microsatellites and single

nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different

candidate genes in different families subsequently only the families indicating

linkage will be chosen for further molecular analyses of the respective gene

To confirm the result of this study the density of the intragenic markers has to be

increased The current RH map with 3200 markers provides a good estimation of the

order and physical spacing (ie in base pairs) of markers along canine

chromosomes (Guyon et al 2003) and was recently complemented by the

construction of a 4249-marker integrated canine genome RH map which consists of

900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all

included and available in the NCBI database (httpwebncbinlmnihgov)

Thus microsatellites derived from the NCBI database could be used to confirm the

linkage Alternatively BLAST searches versus the canine whole genome shotgun

(wgs) sequence resource were perfomed to localize the genes exactly and to obtain

the sequence of the canine genomic contigs containing the human homologous gene

The results of the BLAST searches of the 24 candidate genes against the Boxer

genome assembly 21 are shown in Table 3 The genomic sequence of the

respective candidate gene can now be used to search for intragenic SNPs as these

polymorphisms are the most abundant and useful markers for fine mapping

Development of SNPs requires sequencing of DNA for the respective genomic

regions of the parents with the aim to identify heterozygous base pair exchanges

After a heterozygous base pair is found the whole family can be genotyped for this

informative SNP marker These polymorphisms can than be used for linkage

analyses as well as association studies

Fine mapping using SNP markers for all genes indicating linkage with CCSD

identified by this study should enable us to detect mutations responsible for CCSD in

parts of the Dalmatian dog population

Table 1 Details of the 24 selected human candidate genes

Symbol Gene name Locus name1 Most important reference

CDH23 cadherin related 23 DFNB12 Bork et al 2001

CLDN14 claudin 14 DFNB29 Wilcox et al 2001

COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998

COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999

DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998

DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997

EDN3 endothelin 3 WS type IV Edery et al 1996

EDNRB endothelin receptor type B WS type IV Attie et al 1995

EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001

2 Liu et al 2001

GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997

GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999

Del Castillo et al 2002

MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994

MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000

MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003

MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997

MYO15A myosin XVA DFNB3 Wang et al 1998

OTOF otoferlin DFNB9 Yasunaga et al 1999

PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji

et al 1992

POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998

SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998

SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998

TECTA tectorin alpha DFNB21 DFNA8DFNA12

Mustapha et al 1999 Verhoeven et al 1998

TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001

1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine

congenital sensorineural deafness were localized using BLAST searches against the

Boxer genome assembly 21 The accession numbers (AccNo) of the whole

genome shotgun (WGS) contigs containing the genes as well as their associated

microsatellites are given

Microsatellite Acc No of WGS contig CFA Location of

Gene (Mb) Location of

MS (bp) Location of

MS in relation to gene

CDH23_MS1 NW_876311 4 2541025780 25510 intragenic

CLDN14_MS1 NW_876295 31 3379533796 33790 proximal

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS1 NW_876327 8 1321513232 13225 intragenic

COCH_MS2 NW_876327 8 1321513232 13290 distal

COL11A2_MS1 NW_876254 12 56315659 5608 proximal

DFNA5_MS1 NW_876258 14 4116941237 41135 proximal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic

DIAPH1_MS2 NW_876292 2 3933039430 39552 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal

EYA4_MS1 NW_876269 1 2928029550 29531 intragenic

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic

GJB2_MS2 NW_8762781 25 2093620942 20863 proximal

GJB2+6_MS1 FH2324 25 2093620942 17543 proximal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MITF_MS2 NW_876271 20 2485324884 24844 proximal

MITF_MS3 REN100J13 20 2485324884 25668 distal

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

MYO15A_MS1 NW_876313 5 4436944419 44330 proximal

MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic

MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal

MYO7A_MS3 AHTH298 21 2454324609 24594 distal

OTOF_MS1 NW_876263 17 2350223595 23463 proximal

PAX3_MS1 NW_876304 37 3134831445 31426 intragenic

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

SOX10_MS2 NW_876251 10 2975129762 29740 proximal

TECTA_MS1 NW_876312 5 1588515954 15910 intragenic

TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal

TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal

Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and

polymorphism information content (PIC) for the developed marker-set

Feature Mean SD Min Max

No of alleles 75 31 3 14

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

Table 5 Number of alleles per microsatellite locus and their PIC () values of the

developed marker-set

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped

families conjoined Zmeans and LOD scores are given with their respective error

probabilities for the gene-associated markers of the candidate genes CLDN14

COL11A2 GJA1 MITF MYH9 and SOX10

Marker Location on canine chromosome (CFA)

Zmean pZmean LOD score pLOD

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Table 7 Significant test statistics for linkage analyses carried out each family

separately Zmeans and LOD scores are given with their respective error probabilities

for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1

Gene-associated

marker

Number of families with significant linkage to

Number of corresponding

family members

Zmean pZmean LOD-score pLOD

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one

German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family

Chapter 4

Evaluation of eight candidate genes for canine congenital sensorineural deafness

in Dalmatian dogs

Evaluation of eight candidate genes for CCSD 43

Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract

In this study we have been focusing on genomic loci that encode various enzymes

and transporters involved in the hearing process in humans We developed intragenic

markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C

and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have

been shown to be responsible for human hereditary deafness and to employ these

newly developed markers for non-parametric linkage analyses with canine congenital

sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian

dogs which represent the parents of four families for single nucleotide polymorphisms

(SNPs) in the eight candidate genes by means of direct sequencing combined with a

polymerase chain reaction method for amplifying genomic DNA We characterized 20

SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we

additionally genotyped one microsatellite marker each The families used for

subsequent genotyping of the markers included 39 members from four full-sib

families with frequent occurrence of CCSD We concluded that mutations in ESPN

MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in

CCSD of the Dalmatian dog population investigated here

Introduction Over the past ten years significant progress has been made in the identification of

genes causing different forms of human deafness Currently 39 of the genes

responsible for non-syndromic hearing impairment have been identified in different

human populations (The Hereditary Hearing Loss Hompage

httpwebhostuaacbehhh Van Camp and Smith 2003)

Since non-syndromic hereditary hearing impairment is almost exclusively caused by

cochlear defects affected patients suffer from sensorineural hearing loss

Evaluation of eight candidate genes for CCSD

The most common histopathologic finding in cases of profound congenital deafness

in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in

approximately 70 of cases in man and also in dog breeds the histological pattern is

known as cochleosaccular degeneration

a high level of clinical and molecular similarity Therefore genes responsible for

human hereditary deafness seem to be appropriate candidate genes for CCSD

especially in Dalmatian dogs

In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two

microsatellite markers in altogether eight selected human candidate genes (Table 1)

This eight candidate genes were only recently identified for being responsible for

different form of human non-syndromic deafness In order to evaluate whether any of

this candidate genes is responsible for congenital sensorineural deafness in

Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES

TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four

families of Dalmatian dogs with frequent occurrence of CCSD

Material and methods Pedigree structure and sampling

For the linkage analysis we used blood samples from 39 Dalmatian dogs They

belong to four full-sib families segregating for CCSD At least two of the full sibs of

each family were unilaterally deaf The phenotype of the affected animals had been

confirmed by brainstem auditory evoked response (BAER) that detects electrical

activity in the cochlea and auditory pathways in the brain

The families consisted of eight to 12 individuals In two families a blood sample of the

sire and dam respectively was not available Screening for SNPs was performed by

comparative sequencing of genomic DNA from the parents of the families used for

linkage analyses

SNP and microsatellite marker identification for genotyping

The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene

sequences was derived from sequences deposited in the current dog genome

assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST

(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)

using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN

reference mRNA sequence

We compared the canine genomic DNA sequence from the eight candidate genes to

canine cDNA fragments in the canine EST database using the BLASTN program

For the localization of the exonintron boundaries canine or alternatively human

mRNA sequences were used for the mRNA-to-genomic alignment program Spidey

(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human

and canine mRNA sequences which were used to determine the exon organization of

the candidate genes are given in Table 2

For each of the eight candidate genes we designed intragenic primer pairs to amplifly

intronic sequences yielding products with a length of 560 to 670 bp PCR primers

were developed with the Primer3 program (httpfrodowimiteducgi-

binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl

containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by

the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After

a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and

80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch

Oldendorf Germany) The obtained PCR products were directly sequenced with the

DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a

MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR

primers as sequencing primers Sequence data were analyzed with Sequencher 42

(GeneCodes Ann Arbor MI)

In the first step sequence analyses were performed for PCR products of the parents

of four full-sib families If a heterozygous SNP was found for one or both parents all

progeny of the respective families were analyzed for that SNP Additionally to the

SNPs we used two microsatellite markers for linkage analyses We identified one

intragenic microsatelllite within the TMC1 gene As we could develop only one SNP

for the TMIE gene we additionally genotyped one microsatellite marker derived from

the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE

gene (Table 3)

SNP marker analysis

A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1

TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six

out of the observed 21 SNPs were polymorphic in all four examined families Out of

the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for

one or both parents in family 2 and 15 SNPs could be used for linkage analysis in

family 3 and 4 respectively (Table 4)

The most frequent form of SNPs with a frequency of 238 was the CT transition

motif The scarcest one was the CG transversion motif with a frequency of 48

respectively

Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version

0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics

University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean

and Lod score were used to test for the proportion of alleles shared by affected

individuals identical by descent (IBD) for the considered marker loci The data of the

genotypes was additionally computed using the software package SAS Genetics

(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)

to specify the observed heterozygocity values (HET) and the polymorphism

information content (PIC)

Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD

was tested using the procedures CASECONTROL and HAPLOTYPE of

SASGenetics (Statistical Analysis System version 913 Cary NC USA)

Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show

significant test statistics The highest Z-mean value was 046 the highest LOD Score

was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum

achievable Z-mean was 448 and the corresponding value for the LOD score was

160 Marker-trait association tests for haplotypes of the candidate gene markers

were not significant Obviously no haplotype was associated with CCSD in these

Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The

paternal and maternal haplotypes as far as they could be estimated were distributed

among the affected progeny closely to the expected proportion of 50 and thus no

excess of a certain haplotype could be observed in the affected dogs

Due to the fact that both animals with unilateral or bilaterally hearing loss and

bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN

MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the

pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A

PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for

further linkage studies in other Dalmatian dog populations and dog breeds other than

Dalmatians

Table 1 Details of the six selected human candidate genes

Gene symbol

Gene name Locus name1 Most important reference

ESPN espin DFNB36 Naz et al (2004)

MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)

MYO3A myosin IIIA DFNB30 Walsh et al (2002)

PRES solute carrier family 26

member 5 (prestin) DFNB61

Zheng et al (2000) Liu et

al (2003)

TMC1 transmembrane channel-like

gene 1

DFNA36 Kurima et al (2002)

TMIE transmembran inner ear DFNB6 Naz et al 2002

USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang

et al (2002)

WHRN whirlin DFNB31 Mburu et al (2003)

1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA

Table 2 Selected human candidate genes with their location on HSA and CFA and

corresponding accession numbers

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

Acc No 3 canine mRNA

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number

Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for

Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1

TMIE USH1C and WHRN with their corresponding primers the SNP motif the

product size and the annealing temperature the observed heterozygosity (HET) and

polymorphism information content (PIC)

SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)

Bp3 AT4 PIC ()

ESPN_SNP1

ACCAGCACCCTCTCCAACTA

AGGAATTCACAA (CT)CACACATACA

ACTCAAGCTCAGGGTGTGGT

565 60 9 10

ESPN_SNP2

ATGGCTGGCGCT (AG)GAGGCTGCCC

565 60 27 41

ESPN_SNP3

ACACTCTTCCCA (CT)GGCTGGCGCT

565 60 23 32

ESPN_SNP4

TGGGAAGAGGGA (AG)GGGGGAGCAT

565 60 23 32

ESPN_SNP5

GAGTGGGCCAGG (CT)TGGGAAGAGG

565 60 28 42

MYH14_SNP1

CTCTCCCCAACTCAGTTCCA

ACGTGTATTCGG (GT)CGCTTTTATT

GTGATAGGGACGAGCAGCAT

670 60 35 42

MYH14_SNP2

CATGGGACCGTTCCTACACT

AGCCTCGTTTAA (CT)CTAAAAGGAA

GCTCAATAGGCACGACATCA

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

AATGCTTGAGTTTGGGATGC

GGCAGTCCCATG (GT)CCCTTATAAG

ACCTAATTGCCCAGATGCAG

650 60 37 77

MYO3A_SNP2

GTGGAGAGCCAC (CG)TTGGGAGAGG

650 60 37 70

MYO3A_SNP3

AACCTCCTGGCGTAGTATTCC

CATTACCTATTT (AT)GATCCTTATA

TTTTCCACTTCAGGCACACA

650 60 25 36

PRES_SNP1

CCCTTACCCCATACCATTCC

GATAGACTTCCT (AG)CCCTCAGACT

TTCAGGACAGCATCATCTGC

560 60 37 64

PRES_SNP2

TGATGTCTGCTG (AT)TAACCCATTC

560 60 37 66

TMC1_SNP1

GCAACCTCTCGGTTTATCCA

CGTGAAGTGCCC (AT)TTGATGGAAA

AAGCTGGGGAAGTGGATATGT

610 60 57 37

TMC1_SNP2

GGAGACATTACC (AG)TGAAGTGCCC

610 60 47 29

TMC1_SNP3

GGAAGCAAGACTGAGGTTGG

AGGCTTTTTAAA (AG)CTGTTCTGGG

CTGCTGCATTTGCCTGTAAG

650 60 48 30

TMIE_SNP1

AGAACACCACCGTCTCCTTG

CAAGGCGACGCC (AT)GTGCTGTCCT

GCCTCTGGTCAGAAGAGGTG

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

GGCCTGGGGGGA (AC)AAGCGGACGG

ATGGCATCGACTTCTCCAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

GGTCTCAGACCG (AC)GGCAGGGAGA

560 60 37 37

WHRN_SNP1

TTCACCTCCAGGATCTGGTC

CCTGAGCCCGAG (CT)CCACGCTGCT

GGCTACTTTTCTTCCCCCTTT

600 60 25 37

WHRN_SNP2

GGTCACGGGGGC (CT)CCGGGAGGTT

600 59 24 33

WHRN_SNP3

GTCCGAGTCCCG (AG)CCCCAGCCTG

600 60 34 55

Microsatellite marker

Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()

TMC1_MS1

GCCCCCAGCTAAAAAGAGAA

TTCTCTTCCTCCCTCCTGTTC

220-220 60 76 57

FH2158 ATGGCCACATCACCCTAGTC

CTCTCTCTGCATCTCTCATGAA

274-302 58 57 66

Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)

Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the

candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN

with their nucleotide polymorphism allele and genotype frequencies

Gene symbol Fam1 Nucleotide polymorphism

Allele frequencies

Genotype frequencies2

ESPN_SNP1 4 CgtT 083017 840

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

WHRN_SNP1 2 CgtT 075025 360

WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1

second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)

Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two

microsatellite markers within the eight candidate genes regarding Zmean LOD score

and error probabilities (p-values)

Gene symbol Marker Zmean pz-value1 LOD score pL-value2

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score

ith th

llite-

Chapter 5

Molecular characterization of the canine myosin heavy polypeptide 9

non-muscle (MYH9) gene on dog chromosome 10q232

Canine MYH9 gene 65

Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which

encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural

deafness in a human family which was characterized by cochleosaccular

degeneration In the present study we evaluated whether MYH9 gene mutations are

responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs

As described in Chapter 3 two MYH9 gene-associated microsatellites were

genotyped in 25 Dalmatian dog families segregating for CCSD We could find

significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)

We used data deposited in the NCBI to assemble the canine MYH9 gene DNA

sequence Characterization of the canine MYH9 gene revealed that the canine gene

consists of 41 exons spanning approximately 90 kb

We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of

altogether 16 Dalmatian dogs from three families which showed significant linkage

between the deafness phenotype and the MYH9 gene-associated microsatellite

None of the SNPs affects the amino acid sequence of MYH9 We concluded that the

exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of

the microsatellite marker SNPs and DNA sequence reported in this study enhance

evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog

populations and other dog breeds affected by CCSD

Introduction Myosin is a functional protein associated with cellular movement cell division muscle

contraction and other functions Members of the myosin super-family are

distinguished from the myosin heavy chains that play crucial roles in cellular

processes The human MYH9 gene consists of 40 exons and spans 67959 bp

Canine MYH9 gene

Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human

chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene

to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-

The similarities between the autosomal dominant giant-platelet disorders May-

Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the

disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping

region of 480 kb on human chromosome 22 suggested that all these three disorders

may be allelic Among the identified candidate genes was the gene encoding

nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome

Consortium (2000) demonstrated that mutations in MYH9 result in one of the three

disorders mentioned above The same Consortium also speculated that mutations in

MYH9 may also play a role in another autosomal dominant disorder a form of

nonsyndromic deafness characterized by progressive hearing impairment and

cochleosaccular degeneration This autosomal dominant form of human

nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)

They studied a five-generation American family previously reported by Lalwani et al

(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the

most common histopathologic finding in cases of profound congenital deafness and

is estimated to occur in approximately 70 of cases in man

DFNA17 maps to the same region as MYH9 Because of the importance of myosins

in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and

demonstrated a missense mutation in the MYH9 gene in affected members of a

kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9

gene This missense mutation changed codon 705 from an invariant arginine to a

histidine within a highly conserved Src homology 1 (SH1) linker region Previous

studies had shown that modification of amino acid residues within the SH1 helix

causes dysfunction of the ATPase activity of the motor domain in myosin

a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential

candidate genes for sensorineural deafness in dogs by fluorescence in situ

hybridization and a radiation hybrid panel among them the MYH9 gene that was

assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness

has been reported for approximately 54 different breeds of dogs (Strain 1996)

Canine MYH9 gene 67

different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002

Juraschko et al 2003b)

In dog breeds the histological pattern is known as cochleosaccular degeneration

commonly known as Scheibe dysplasia as it is described in approximately 70 of

cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene

seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs

In this report we provide the genomic organization and the complete sequence of the

canine MYH9 gene A mutation analysis was performed to identify single nucleotide

polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is

responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed

the association of the MYH9 haplotypes with the CCSD phenotype in three families of

Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the

gene-associated microsatellite MYH9_MS3 (Chapter 3)

Materials and Methods

Cloning and sequencing of canine MYH9 cDNA

The canine MYH9 gene sequence was derived from sequences deposited in the

(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)

search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference

mRNA sequence (Genbank Acc No NM_002473)

The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb

whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9

The isolation of full length cDNAs was achieved by a modified rapid amplification of

cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle

(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE

products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion

Europe Huntingdon UK) according to the protocols of the manufacturer Reverse

transcription polymerase chain reaction (RT-PCR) using two pairs of nested

Canine MYH9 gene

gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the

complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using

two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-

specific primers Additionally RT-PCR using six pairs of gene-specific primers were

used to amplify the complete sequence of the investigated cDNAs

RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit

(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced

with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a

LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence

data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)

Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full

length canine cDNAs (as described above) the mRNA-to-genomic alignment program

Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was

used Repetitive elements were analyzed with Repeatmasker 2

(httprepeatmaskergenomewashingtonedu) The GC content was calculated with

the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)

Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking

regions were PCR amplified and sequenced from 16 Dalmatian dogs which

represent three families consisting of three to six full sibs and at the best of both

parents At least two of the full sibs of each family were unilaterally deaf The

phenotype of the affected animals had been confirmed by brainstem auditory evoked

response (BAER) that detects electrical activity in the cochlea and auditory pathways

in the brain PCR primers and conditions for the amplification of MYH9 exons with

flanking sequences and microsatellite flanking primers for PCR reactions are given in

Table 1 PCR primers were developed with the Primer3 program

(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for

exons and their flanking sequences were performed in a total of 50 microl containing 125

microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer

(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial

denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec

Canine MYH9 gene 69

at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch

Multipoint linkage and haplotype analysis were performed using MERLIN version

individuals identical by descent (IBD) for the considered marker loci

Linkage means that a haplotype characterized by microsatellites or SNPs is

significantly more often present in family members with the phenotype under study

than expected by random assortment For construction of haplotypes sets of closely

linked genetic markers on the same chromosome are needed which tend to be

inherited together as they are not likely to be separated by recombination

Association analysis can be carried out as a method of genetic analysis that

compares the frequency of alleles between affected and unaffected individuals

across all families A given allele is considered to be associated with the disease if

the presence of that allele explains a significant proportion of the phenotypic trait

variation

Results and Discussion

Analysis of the genomic organization and cDNA of the canine MYH9 gene

A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by

using the RACE protocol The obtained RT-PCR products were sequenced and the

generated sequence data were submitted to the EMBL nucleotide database (Acc No

AM086385)

Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected

that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and

exonintron boundaries that conform perfectly to the GTAG rule (Table 2)

The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that

the homologous ATG start codon as in man is used the canine MYH9 cDNA

Canine MYH9 gene

contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids

A canonical polyadenylation signal AAUAAA is located approximately 14 kb

downstream of the stop codon

The exon sizes range from 28 to 1556 bp the introns between these exons span

between 86 and 13493 bp the total size of the canine MYH9 gene is approximately

90 kb However the sequence homology between the human murine and canine

MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence

of canine MYH9 displays 926 and 899 similarities to the human and murine

MYH9 gene respectively In the untranslated regions the sequence similarity

between dog and human and mouse is rather low The canine MYH9 protein shows

982 and 968 identities to the orthologous human and murine protein

respectively (Fig 2)

The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of

the repetitive elements belong to the SINE family (892) followed by the fraction of

the LINEs (306) other repetitive elements constitute 165 respectively The

entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene

contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the

entire gene whereas by far the longest one with a length of 789 bp can be found in

the region of the second exon or in the first translated exon respectively (GC content

of 50 over 200 bp)

Mutation and haplotype analysis

All coding exons with flanking intronic regions of MYH9 could be amplified from the

examined 16 dogs and the sequences were compared to the Boxer genome

assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)

The search for sequence variations within the MYH9 gene revealed a total of 22

SNPs shown in Table 3 Most of the polymorphisms were found in the flanking

regions of exons only 3 were within exons Only five out of the observed 22 SNPs

were polymorphic in all three examined families

None of the observed polymorphism did alter the predicted amino acid sequence of

MYH9 nor the identified hapltypes showed an association with the CCSD phenotype

Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-

mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)

However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156

Canine MYH9 gene 71

(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this

result was heterogeneity among these three families For family 1 and 2 the test

statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)

whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=

Thus these silent point mutations found in affected and unaffected Dalmatian dogs

seem not responsible for the CCSD phenotype in these three families

Conclusions

The characterization of the transcript and genomic sequences of canine MYH9 gene

revealed a conserved organization with respect to the human orthologs In general

the gene size in dog is bigger compared to the human sequence due to the

untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the

canine MYH9 gene were identified However because of the fact that both animals

with unilateral hearing loss and bilateral hearing animals shared identical

haplotypes these polymorphisms are obviously not associated with CCSD in these

Dalmatian dog families Furthermore the present study revealed no functional

mutations of the complete coding region of MYH9 We can exclude the MYH9 gene

as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs

However the MYH9 gene sequence SNPs and microsatellite markers reported in

this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations

and dog breeds other than Dalmatians

Canine MYH9 gene

Table 1 PCR primers for the amplification of all MYH9 exons

Exon Primer Sequence (5rsquo ndash 3rsquo) Product

size (bp)

Annealing temperature

(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58

Canine MYH9 gene

Table 2 Exonintron boundaries of the canine MYH9 gene

3rsquo-Splice site Exon 5rsquo-Splice site

Intron phase

Intron size

-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc

gt30000 bp

-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc

4922 bp

+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt

13493 bp

+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc

803 bp

+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt

4077 bp

+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag

427 bp

+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat

738 bp

+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc

343 bp

+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc

749 bp

+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc

1041 bp

+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc

1877 bp

+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct

922 bp

+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct

1801 bp

+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt

2049 bp

+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg

1877 bp

+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc

343 bp

+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt

835 bp

+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct

1418 bp

+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg

851 bp

+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg

1428 bp

+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg

398 bp

+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg

971 bp

+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag

1603 bp

+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt

719 bp

+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc

270 bp

+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc

480 bp

+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg

232 bp

+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg

204 bp

+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg

1083 bp

+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc

1298 bp

+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca

150 bp

+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc

303 bp

+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg

1173 bp

+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca

941 bp

+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg

+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg

224 bp

+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc

563 bp

+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc

+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc

739 bp

+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA

Exon sequences are shown in uppercase letters and intron sequences in lowercase

letters Untranslated regions are shown in italics The conserved GTAG exonintron

junctions are shown in boldface type For the last exon the polyadenylation signal is

shown underlined instead of an exonintron junction Position +1 corresponds to the

adenine of the translation initiation codon ATG

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)

Canine MYH9 gene

Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine

MYH9 protein sequences The sequences where derived from GenBank entries with

the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)

Identical residues are indicated by asterisk beneath the alignment while dots and

colons represent similar or very similar amino acids respectively

dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597

Canine MYH9 gene 79

dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317

Canine MYH9 gene

dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960

Chapter 6

Identification of a 5 Mb region on canine chromosome 10

harbouring a causative gene responsible for congenital sensorineural deafness

in German Dalmatian dogs

Fine mapping of CFA10 83

Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs

Abstract

In the present study we evaluated whether the canine chromosome (CFA) 10

harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in

Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10

were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD

Significant linkage between the deafness phenotype and microsatellites located in a

region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the

causative canine congenital sensorineural deafness (CCSD) gene we used data

deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the

intronic sequences of the canine genes located on CFA10 in this region We

characterized 26 SNPs and used them for non-parametric linkage and association

analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We

could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb

and significant haplotype-trait association for SNPs in this region These results

enforce further evaluation of this 5 Mb region with the aim to detect the gene

responsible for CCSD in Dalmatian dogs

Introduction

Of the identified genes responsible for different forms of sensorineural non-syndromic

deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002

Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as

candidates for sensorineural deafness in dogs among them the MYH9 gene on

CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the

respective 24 candidate genes

As described in chapter 3 we could show significant linkage of CCSD with the MYH9

associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog

Fine mapping of CFA10

families However as described in Chapter 5 we already excluded MYH9 for being

responsible for the CCSD phenotype in German Dalmatian dog families segregating

for CCSD by comparative sequencing of genomic sequences from deaf and normal

hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA

The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a

closely linked gene involved in CCSD Thus the objective of the present study was to

perform a scan of canine chromosome 10 using microsatellite markers and single

nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a

gene responsible for the deafness phenotype in Dalmatian dogs Additionally we

analyzed the association of the CCSD phenotype with a large number of newly

developed SNPs located in the genomic deafness region on CFA10

For the linkage analysis of the microsatellite marker on CFA10 we used DNA from

altogether 176 animals belonging to 22 full-sib families and one large paternal half-

sib family of German Dalmatian dogs All families were segregating for CCSD The

genotyped families included all the affected dogs (unilaterally and bilaterally deaf)

their parents if available and one to four unaffected full-sibs At least two of the full

sibs of each family were unilaterally deaf The phenotype of the affected animals had

been confirmed by brainstem auditory evoked response (BAER) that detects

electrical activity in the cochlea and auditory pathways in the brain

Screening for SNPs was performed by comparative sequencing of DNA from parents

of five families with significant linkage of microsatellites located in the region

spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we

then used blood samples from 34 Dalmatian dogs consisting of the progeny and their

parents of the abovementioned five full-sib families of Dalmatian dogs with frequent

occurrence of CCSD The families consisted of five to nine individuals and their

parents

Microsatellite marker analysis

We used 27 microsatellite marker derived from the NCBI database

(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)

Germany)

Development of single nucleotide polymorphisms (SNPs)

We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously

proven to be linked to the CCSD phenotype The SNPs for this region were derived

from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited

in the current dog genome assembly (Boxer genome assembly 21) of the NCBI

GenBank The canine genomic sequences and mRNA of the genes that were used

for the analysis were also derived from sequences deposited in the current dog

genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)

In total 72 primer pairs were designed most of them located intragenic in intronic

sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective

gene yielding products with a length of around 600 bp PCR primers were developed

with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)

The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25

pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen

Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at

95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were

performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)

The obtained PCR products were directly sequenced with the DYEnamic ET

Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000

capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing

primers Sequence data were analyzed with Sequencher 42

(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both

parents all progeny of the respective families were analyzed for that SNP

We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic

sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with

one to four SNPs per gene (Table 3)

The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and

chosen for linkage analyses that were heterozygous for one or both parents of at

least two of the five families Of all SNPs only one was heterozygous in all families

(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG

transition motif The scarcest one with a frequency of 385 was the CG and the

AC transversion motif respectively

Linkage analysis

Multipoint non-parametric linkage and haplotype analysis were performed using the

MERLIN software version 0102 (multipoint engine for rapid likelihood inference

Center for Statistical Genetics University of Michigan MI USA Abecasis et al

2002) The test statistics Z-mean and Lod score were used to test for the proportion

of alleles shared by affected individuals identical by descent (IBD) for the considered

marker loci

In a first step linkage analysis was performed regarding the 27 marker covering the

whole CFA10 In a second step a linkage analysis was performed including the newly

added SNPs spanning the region 36 Mb to 48 Mb on CFA10

The observed heterozygosity (HET) and the polymorphism information content (PIC)

were calculated using the software package SASGenetics (Statistical Analysis

System Version 913 SAS Institute Inc Cary NC USA 2005)

Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested

using the procedures CASECONTROL and HAPLOTYPE of SASGenetics

(Statistical Analysis System version 913 Cary NC USA)

Results

A linkage analysis was at first carried out for the 176 animals that were analyzed with

27 microsatellite markers covering the whole CFA10

The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a

value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean

value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the

microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean

values and error probabilities of eight microsatellite markers in the interval from 3666

Mb up to 488 Mb were almost as high Consequently we screened the region

spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog

families five full-sib families were chosen to screen for SNPs because of their

significant contribution to the test statistics

Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were

developed and added to the linkage analysis These SNPs had PIC values ranging

from 013 to 037

Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on

CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-

values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow

the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and

Haplotype-trait association test statistics for the SNPs 16-23 located in the interval

from 39 Mb to 44 Mb were significant However the marker-trait association test

failed the 5 threshold of the error probability (p= 007) but was lowest for all

possible haplotype-trait combinations The χ2ndashtests of the procedure

CASECONTROL were not significant indicating that a SNP for the causative

deafness gene was not yet found

Discussion

We used 27 microsatellite marker and 26 newly developed SNP markers with the

intention to create a dense map for linkage analysis of CFA10 especially the region

spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP

markers were chosen due to their heterozygosity in one or both parents of at least

two families

The significant Zmeans on CFA10 reported for the chromosome scan using only

microsatellites was confirmed by adding the SNP markers Furthermore with the use

of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down

to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to

informative microsatellites appears highly recommendable

The identified CCSD region spanning 5 Mb might be participating in the development

of CCSD in the analyzed Dalmatian dog families

However genes that are known to be involved in the hearing process or known to be

expressed in the inner ear are not annotated at the syntenic human region on homo

sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-

defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb

does not exist This means that for all genes in this region informative SNPs have to

be developed and tested for linkage disequilibrium and association with CCSD

Despite the remarkable progress that has been made in the past few years in

identifying deafness genes in man and mouse it is suspected that only one-third of all

deafness causing genes have been identified so far Therefore more SNPs have to

be developed within the identified region on CFA10 to localize the deafness causing

gene or to find unambiguously associated SNP markers which could be used for a

population-wide genetic test for CCSD

Table 1 Canine genes where gene-associated SNPs could be developed with their

exact location on CFA10 and their corresponding accession numbers (Acc No)

Gene symbol Gene description Position in

Acc No canine mRNA

LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764

LOC474536 similar to KM-102-derived reductase-like factor

37023714 NC_006592 XM_531765

LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546

LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552

LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591

LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2

38353835 NC_006592 XM_848614

LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a

38503857 NC_006592 XM_531770

LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771

LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429

LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994

LOC609217 similar to family with sequence similarity 32 member A like

39453945 NC_006592 XM_858065

LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756

LOC481325 similar to ubiquitin-conjugating enzyme E2C

42564272 NC_006592 XM_538446

LOC481330 similar to Interleukin-1 receptor type II precursor

44084413 NC_006592 XM_538451

LOC481337 similar to DNA repair protein REV1

46264637 NC_006592 XM_538458

LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase

48494852 NC_006592 XM_849433

Predicted gene derived from the dog genome assembly (build 21) that used gene

prediction method GNOMON supported by EST evidence

Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region

spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif

the product size and the annealing temperature

description SNP

Location

(intron or

5rsquo3rsquo-end)

Primer F1 (5acute -gt 3acute)

SNP motif

Primer R2 (5acute -gt 3acute)

Bp3 AT4

LOC474535

intron

ACCCAAGCCTAACTGCAGAA

ACCCCAGTCT(CG)GCCAGAGCTGTT

590 60

SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT

TCATCTGTTAAAACAGGGGTGAT

LOC474536

intron

CCAGTTAATGATTGTTTCGTTGA

AAGCTGCTTT(AC)CACCCCCATCAG

TCATTCCTGCTGTTGTGCTC

590 60

LOC610953

intron

CTGTCTTGGGGACTGTTTGC

AAGGCAGACG(CT)AATGACTGAGGC

600 60

SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT

GCCATCACGATGAACTCAGA

LOC481302

3rsquo-end

AATTGAGGCCGAAGTCCAAT

CTTTTCCCCA(GT)GCCACCCCTCTG

GAGCACTATTTACGATACAAACAGGA

610 60

LOC610991

intron

CATGCATGATGCCCAGAGTA

CCCAAAGCAC(AG)CTGTGATTTAAT

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

CAGACCAACAGTGACCCAGA

TAGGCATACC(GT)TCAGTCCTAAAG

GCCTGTTGTGGGCAGAGTAT

480 60

LOC474541

intron

ACTGAGCCAAAGGTGGATTG

AGAGAATAGC(AG)CTGTGTTTTACA

ACCTGCACATCGGGATTTAG

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

CTTCCCCAGGAGAGAGTGAC

AATATGATCA(CT)ATTTAAAGAAAT

CTTTTGTCAACATCCCCTTCA

560 60

LOC481308

SNP_11

3rsquo-end

ACCCATTGTCTCTCCAGCAC

CCACATAACT(GT)AGCATCCCTAGC

600 60

TGATGATGTAAGTTGGCCTCA

LOC474543

SNP_12

intron

TTGAAGTTGTGTGAGTAAATGAAAGA

CAATATATAA(AG)CATTTGCTACAA 600 60

SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG

SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG

SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT

GGAAACCATGCAGTCTTTGG

LOC609217

SNP_16

5rsquo-end

TGGCCTCATTTTCCAGTATG

GAAGGAGTTA(CT)ACAGTGAAGATA

580 60

SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT

GGCAATTACCCTGAGTGGTG

LOC611115

SNP_18

3rsquo-end

GGGCTGTCTTAGAGGTGCTG

TGTGGTCTCA(CT)ACACTTCCTGAG

590 60

SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC

CTTCTTTGGGCAGGAAAGTG

LOC481325

SNP_20

intron

AAAATGATTGATCGCAAAAGAAA

AATTACTGTA(CT)AACAGTATCAGA

600 60

SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT

TTCTGTGATTGCACTGACCG

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

GGCAGGGAGG(AG)TCACCATCGTTC

AATTTCCCCAAATGCCTCAC

575 60

LOC611493

SNP_23

intron

GCATGAAGGAGCCCTATGTC

CCAAGAGTCC(AT)GCCCAACACCCT

GGAGGGATGGCATTCTATGA

590 60

LOC481337

SNP_24

intron

GGCTGAGGAGATTGTGTTTCA

GCTGATATTT(AG)GCCTTCTGAGAT

620 60

SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT

CAGCTCCCTGTAATGGGAAA

LOC611728

SNP_26

intron

TCCTACTCCCATCACTTCCAA

CCACACTGGG(GT)CCTGGGATGAGG

CACAGCTCCATGTAGGTCCA

620 60

hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)

Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their

nucleotide polymorphism allele and genotype frequencies observed heterozygosity

(HET) and polymorphism information content (PIC)

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

frequencies2 PIC HET

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous

homozygous for allele 2]

Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning

39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-

values)

Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score

Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10

harbouring a congenital sensorineural deafness region (number of families 23

number of genotyped dogs 176)

Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on

CFA10 harbouring a congenital sensorineural deafness region (number of families

23 number of genotyped dogs 176)

Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital

sensorineural deafness region (number of families 5 number of genotyped dogs 34)

Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the

region between 30 to 60 Mb harbouring a congenital sensorineural deafness region

(number of families 5 number of genotyped dogs 34)

CCSD region (SNP 16-23)

Chapter 7

Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene

in Dalmatian dogs segregating for congenital sensorineural deafness

CFA1 and the GJA1 gene 99

Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene

showed significant linkage with canine congenital deafness (CCSD) in a large French

half-sib family (Chapter 3) PCR products of this half-sib family were used to perform

a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As

the linkage could not be confirmed we used altogether 27 microsatellite markers for

a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating

for CCSD A non-parametric linkage analysis was performed to see whether

significant test statistics for other genomic regions on CFA1 and for more families can

be shown As a result we could not find linkage to any microsatellite in the analyzed

families

Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in

dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on

canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were

developed by Rak (2003) among them two microsatellite marker associated to the

GJA1 gene

GJA1 or connexin 43 is a member of the connexin gene family and a component of

gap junctions Mutations in 4 members of the connexin gene family have been shown

to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)

GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)

Liu et al (2001) reported mutations in GJA1 in association with sensorineural

recessive deafness in man However these mutations have recently been shown to

involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the

CFA1 and the GJA1 gene

GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the

senior author (W E Nance) of the paper by Liu et al 2001)

In previously performed studies (Chapter 3) one large French Dalmatian dog family

with frequent occurrence of CCSD showed linkage to microsatellites associated to

the GJA1 gene

In this report we performed a mutation analysis of the GJA1 gene sequence to

identify polymorphisms In order to evaluate whether the GJA1 gene is responsible

for congenital sensorineural deafness in Dalmatian dogs we analyzed the

association of the GJA1 haplotypes with the CCSD phenotype Furthermore we

employed 27 microsatellite markers covering the entire CFA1 and used them for a

non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176

animals with frequent occurrence of CCSD

For the linkage analysis of the microsatellite marker we used DNA from altogether

176 animals belonging to 22 full-sib families and one large paternal half-sib family of

German Dalmatian dogs All families were segregating for CCSD The genotyped

families included all affected dogs (unilaterally and bilaterally deaf) their parents if

available and one to four unaffected animals At least two of the full sibs of each

family were unilaterally or bilaterally deaf The phenotype of the affected animals had

Mutation analysis and screening for SNPs was performed by comparative

sequencing of DNA from 16 animals consisting of the parents and their progenies of

one large half-sib family of French Dalmatian dogs which showed significant linkage

to GJA1-associated microsatellites (Chapter 3)

Sequencing of canine genomic DNA and mutation analysis

The canine GJA1 gene sequence was derived from sequences deposited in the

(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence

to canine cDNA fragments in the canine EST database using the

BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine

ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the

assembly of the GJA1 gene

To identify polymorphisms within the canine GJA1 sequence the gene consisting of

one 1251 bp spanning exon were PCR amplified and sequenced from 16 French

Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer

pairs were designed yielding products with a length of around 600 bp PCR primers

Oldendorf Germany)

(GeneCodes Ann Arbor MI USA)

containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl

sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)

(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10

microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research

thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30

sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45

sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from

110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR

42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6

polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)

Linkage and haplotype analysis

Multipoint non-parametric linkage and haplotype analyses were performed using the

marker loci

A non-parametric linkage analysis was performed with 27 microsatellite markers in 23

Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to

the linkage analysis performed in Chapter 3

In previously performed studies (Chapter 3) one French Dalmatian dog family

reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286

(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the

GJA1 gene

By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-

UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of

the examined French half-sib family Neither of the observed polymorphism did alter

the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an

association with the CCSD phenotype (Figure 1)

There is no recombination of the haplotypes of the GJA1 gene in this family The

among the affected progeny closely to the expected proportion of 50 and therefore

no excess of a certain haplotype could be observed in the affected dogs

Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French

half-sib family Because of the fact that both animals with unilateral hearing loss and

bilateral hearing animals shared identical haplotypes these polymorphisms are

obviously not associated with CCSD in these Dalmatian dog families Furthermore

the present study revealed no functional mutations of the complete coding region of

GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of

CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and

GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in

CCSD among the half-sib family of French Dalmatian dogs or by a false positive

result of the microsatellite study performed in Chapter 3

To clarify whether significant test statistics for other genomic regions on CFA1 and

for more families can be shown we used 27 microsatellite markers derived from the

NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage

analysis was carried out after genotyping 176 German Dalmatian dogs with a set of

27 microsatellite markers The results of this linkage analysis were added to the

results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2

(Chapter 3)

As a result we could not find linkage to any microsatellite in the analyzed families

(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic

regions that are involved in the development of CCSD in the analyzed Dalmatian dog

families

With hindsight it was revealed that GJA1 is not responsible for sensorineural non-

syndromic deafness in humans as Liu et al (2001) has published GJA1 is

participating in a human syndrome called oculodentodigital dysplasia (ODDD) that

can be accompanied with hearing impairment (Paznekas et al 2003) But the type of

deafness in human ODDD differs from the typical hearing loss associated with other

connexin mutations because it is conductive rather than sensorineural

As deafness in dogs especially in Dalmatians is almost exclusively caused by

sensorineural non-syndromic forms also known as cochleosaccular degeneration

the GJA1 gene should not be considered as a candidate gene for CCSD anymore

Table 1 Three newly developed intragenic SNPs and two microsatellite markers for

Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the

SNP motif the product size and the annealing temperature

Primer F (5acute -gt 3acute)

SNP motif

Primer R (5acute -gt 3acute)

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

CACCTTAGGCGTTCATTTTG

CCGGGGAG(AG)AAAA(AG)AAAAATACTT

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

TCTGAAATGTAATCATGGATGC

CAGAACTTGTAT(AT)CTGTTAAGAG

AATCACACAGGATATAGAGGCTATC

600 58

Microsatellite

marker Primers (forward reverse ) 5acute -gt 3acute

Product

size (bp)

Annealing

temperatur

GJA1_MS1

ATGGCATGAAGAGGATACCG

AGGACAGGTGACGGCTCTAC

134 60

GJA1_MS2

GCTAGTACTCGATTGTGGTC

TCATGGGTTGTGAGATCCAG

190 60

Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of

families 23 number of genotyped dogs 176)

Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1

Chapter 8

Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene

CFA31 and the CLDN14 gene 109

Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of

microsatellite markers associated with the CLDN14 gene on canine chromosome

(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The

objective of the present study was to perform a sequence analysis in order to find

single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to

use a set of six microsatellite markers evenly distributed on CFA31 for non-

parametric linkage analysis with the aim to verify the significant test statistics shown

in Chapter 3

Introduction

Of the genes responsible for different forms of sensorineural non-syndromic deafness

in humans Rak (2003) considered 24 genes as candidates for sensorineural

deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major

components of tight juncions (TJ) The human CLDN14 gene consists of one

tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are

identified so far (Wilcox et al 2001 Wattenhofer et al 2005)

For the compartmentalization of perilymph and endolymph in the inner ear the

leakage of solutes through a paracellular pathway must be prevented by tight

junctions TJ are an intercellular junction found at the most apical region of polarised

epithelial and endothelial cells at which adjacent plasma membranes are joined

tightly together separating apical membranes and basolateral menbranes They are

specialised membrane domains containing branching strands of integral proteins and

create a primary barrier preventing paracellular transport of solutes and restricting

lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)

CFA31 and the CLDN14 gene

The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated

by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible

for a hereditary human deafness in families segregating for congenital recessive

deafness (DFNB29)

In previously performed studies one large half-sib family as well as several full-sib

families of German Dalmatian dog indicated linkage to CLDN14 gene-associated

markers (Chapter 3)

A sequence analysis was performed to identify single nucleotide polymorphisms

(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions

Furthermore we employed microsatellite markers covering CFA31 and used them for

a non-parametric linkage analysis with CCSD in a German Dalmatian dog population

of 176 animals with frequent occurrence of CCSD

families included all the affected dogs (unilaterally and bilaterally deaf) their parents

if available and one to four unaffected animals At least two of the full sibs of each

one half-sib family and four full-sib families of Dalmatian dogs which showed

significant linkage to a CLDN14-associated microsatellite (Chapter 3)

Sequencing of canine genomic DNA and development of single nucleotide

polymorphisms (SNPs)

The canine CLDN14 gene sequence was derived from sequences deposited in the

search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference

We compared the canine genomic DNA sequence to canine cDNA fragments in the

canine EST database using the BLASTN program As no ESTs could be found

human mRNA sequences were used for the localization of the exonintron

boundaries using the mRNA-to-genomic alignment program Spidey

(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)

We screened exon three for mutations as this exon is the only translated one in man

Additionally we screened large intronic sequences and sequences located in the 5rsquo-

and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs

were designed yielding products with a length of around 600 bp

PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-

Oldendorf Germany)

primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann

Arbor MI USA) The eight newly developed SNPs are shown in Table 1

In total six microsatellite marker were derived from the NCBI database

sec at 94degC 30 sec at maximum annealing temperature and a final extension of

45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios

from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-

COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4

and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)

Multipoint non-parametric linkage and haplotype analysis were performed using

MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for

by affected individuals identical by descent (IBD) for the considered marker loci A

linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs

The results were added to the linkage analysis performed in Chapter 3

Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of

candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of

four full-sib families and one half-sib family with the highest tests statistics lead to a

Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker

CLDN14_MS2

To substantiate the linkage to the CLDN14 gene we searched for sequence

variations within the CLDN14 gene in four full-sib families and one half-sib family with

the highest contribution to the test statistics shown in Chapter 3 Most of the identified

polymorphisms were found in intronic sequences none were within exon three None

of the observed polymorphism did alter the predicted amino acid sequence of exon

three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)

out of the identified eight SNPs were polymorphic in the examined families Both

unilaterally and bilaterally deaf animals as well as normal hearing animals showed

identical haplotypes for these two polymorphic SNPs and thus no co-segregation with

the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not

identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this

results a mutation outside of the translated genomic regions analyzed here may exist

that would affect CLDN14 expression

To clarify if other regions on CFA31 are responsible for the CCSD phenotype

additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian

dog families consisting of 176 individuals A non-parametric linkage analysis was

performed regarding these 23 families The results of this linkage analysis were

added to the results of the test statistics for the CLDN14 gene-associated

microsatellites (Chapter 3)

We could only find significant linkage with CCSD for CLDN14-associted

microsatellites in the abovementioned five families The two microsatellites located

most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a

Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not

indicating linkage

It can not be excluded that CLDN14 or genes in its flanking regions are involved in

the development of CCSD in the analyzed Dalmatian dog families Other genes than

the CLDN14 that are known to be involved in the hearing process or known to be

expressed in the inner ear are not annotated at the syntenic human region of homo

sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other

well-defined candidate gene for canine hereditary deafness exists in the linked

region Despite the remarkable progress that has been made in the past few years in

deafness causing genes have been identified so far and thus it seems possible that

other genes in the flanking region of the CLDN14 gene are involved in the

development of the disease Anyway to clarify the importance of CLDN14 in the

CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well

as in its flanking regions with the aim to find significant linkage disequilibrium of SNP

markers with CCSD

Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on

CFA31 with their corresponding primers the SNP motif the product size and the

annealing temperature

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CTTCCAGGGAAA(AT)TGTCGTAGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

GAAATTGTCGTA(AG)CCCGGCCGCT

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

CCAGAGGAATAA(CT)ATGATCGTGA

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

ACCACCGCACAC(CT)GTCACAGCTT

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP5 3rsquo-UTR

CATGCCTTTGTCCCAAACTT

GAGACCCTCTGG(CT)TCCTTTTGGC

GTACCTGTTGCCTGGGTTGT

610 60

CCTTCATCCTTTCTGGTTGA

GCTCACAGTCCC(AC)ATGGGGACAT

GGGGAGCATAATGTGGTCAT

585 60

TGAACTGGTCCCAAGGAAAG

GCACGACCGAGC(CT)CTGGCTTTAC

GGGATGAGAGGGAGGTTTTT

580 60

AATGCCTATCCCTTCTTTGGA

CACGTTACTGTG(AG)ACCTCTCTAC

GCAGGCTTTCCTCAAGTGTC

680 60

1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)

Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in

the analyzed four Dalmatian dog full-sib families

Chapter 9

General discussion

General discussion 119

General discussion

The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural

deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite

markers or alternatively by single nucleotide polymorphisms (SNPs)

The candidate genes for which a set of in total 43 microsatellites was available

included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5

DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A

MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3

(Rak 2003) These genes are known to be involved either in human non-syndromic

deafness or in the human Waardenburg syndrome The Waardenburg syndrome

(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and

skin WS is classified into four types depending on the presence or absence of

EDNRB MITF PAX3 and SOX respectively

For another eight recently identified genes responsible for different forms of human

non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES

WHRN and ESPN linkage and association analyses were performed using newly

developed SNPs

genes or expected to be located in candidate regions This was necessary to identify

new informative polymorphisms (eg SNPs microsatellites) for high resolution

mapping of candidate regions and to examine each exon and exonintron boundary

for positional candidates Availability of the second version of the dog genome

assembly (build 21) of the NCBI database shortcuts this effort and increases the

investigators efficiency Now either additional candidate genes for canine congenital

sensorineural deafness can be found directly by its gene symbol in the 21 of the

General discussion

NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST

(Basic Local Alignment Search Tool) search against the canine whole genome

shotgun (wgs) sequence resource can be used to obtain the sequence of the canine

genomic contigs containing the human homologous gene and thus intragenic

markers can be developed for subsequent linkage and association analyses

to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic

congenital hereditary deafness in humans seem to be appropriate candidate genes

for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate

gene approach combined with linkage analysis method using affected pedigree

members Once a significant linkage was found only the linked genes with the

required low error probability values were used for further molecular genetic analysis

The method of candidate gene approach using either gene-associated microsatellite

or alternatively SNP markers was applied for in total 32 candidate genes which

comprise nearly all of the identified mutated genes causing non-syndromic hereditary

hearing impairment in humans

Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a

relation between loci and association is a relation between alleles

Linkage means that a haplotype characterised by microsatellites or SNPs is

inherited together as they are not likely to be separated by recombination Linkage

creates associations within families but not among unrelated induviduals

Association is a statistical statement about the co-occurrence of alleles or

phenotypes Association analysis can be carried out as a method of genetic analysis

that compares the frequency of alleles between affected and unaffected individuals

across all families Thus for association family structures are not necessary A given

allele is considered to be associated with the disease if the presence of that allele

explains a significant proportion of the phenotypic trait variation For association

studies the developing of a marker set consisting of SNPs rather than microsatellites

is needed

In this thesis a total of 32 candidate genes for canine congenital deafness were

analyzed which showed an appropriate clinical and histological disease pattern in

comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43

microsatellites for in total 24 candidate genes among them the CLDN14 gene on

canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1

was also considered as a candidate gene for CCSD (Rak 2003) and therefore two

gene-associated microsatellites have been developed Recently it turned out that

GJA1 is not responsible for human sensorineural non-syndromic deafness but for a

human syndromic disorder that can be related with conductive deafness

By the use of a non-parametric linkage analysis using the existing set of 43

microsatellites associated to 24 candidate genes we found linkage to markers

associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1

For another another eight candidate genes it was possible to develop SNPs

Performing linkage analyses as well as association and haplotype studies it was

possible to exclude these eight candidate genes from being responsible for the

CCSD phenotype

Over the past ten years significant progress has been made in the identification of

deafness gene localisations Up to now approximately 120 loci have been reported

for both autosomal dominant and recessive forms of non-syndromic hereditary

deafness in humans and only for one third the responsible gene mutation could be

detected Thus it can be expected that additional potential human candidates for

CCSD in Dalmatian dogs will become available in future (Van Camp and Smith

clinical phenotypes In man genes that transport ions across membranes to

maintain appropriate solute concentration and pH as well as regulatory genes mainly

transcription factors and genes that play a part in structural integrity are essential

for the hearing process

General discussion

The results of this thesis indicate that the inheritance of hearing loss in Dalmatian

dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity

means that different mutations cause a similar phenotype the different mutations

can either be found at the same locus (allelic heterogeneity) or even at different loci

(non-allelic heterogeneity) As linkage was found for different candidate genes in

different families subsequently only the families indicating linkage were chosen for

further molecular analyses

GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions

are further analyzed with a combined approach using microsatellite and SNP

markers

CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog

family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value

of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence

analysis of the GJA1 gene using the above mentioned French Dalmatian dog family

was performed None of the observed polymorphism did alter the predicted amino

acid sequence of GJA1 nor showed the identified haplotypes an association with the

CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to

CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in

the pathogenesis of CCSD in Dalmatian dogs To see whether significant test

statistics for other genomic regions on CFA1 and for more families can be shown a

non-parametric linkage analysis was performed with 27 microsatellite markers

covering CFA1 completely In total 176 animals were genotyped We could not find

linkage to any microsatellite in the analyzed families Furthermore it was revealed

that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in

humans (WA Paznekas cited a personal communication from the senior author (W

E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human

syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with

hearing impairment (Paznekas et al 2003) But the type of deafness in human

ODDD is conductive rather than sensorineural As deafness in dogs especially in

Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also

known as cochleosaccular degeneration the GJA1 gene should not be considered

as a candidate gene for CCSD anymore

CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD

for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog

familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed

for exon three as this is the only translated one in man None of the observed

polymorphisms did alter the predicted amino acid sequence However to clarify the

importance of the CLDN14 gene and its flanking regions in the CCSD phenotype

more SNPs have to be developed within the CLDN14 gene as well as in its flanking

regions with the aim to find significant linkage disequilibrium of SNP markers

CFA10 A significant co-segregation of markers alleles and the phenotypic expression of

deafness in a large sample of German Dalmatian dog families was determined for

one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated

to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are

responsible for CCSD in these Dalmatian dog families An initial priority in defining

gene structure is to obtain a full-length cDNA sequence and identify translational

initiation and termination sites and polyadenylation site(s) Exonintron structure can

then be determined by referencing the cDNA sequence against sequences of

cognate genomic DNA One popular method of obtaining full-length cDNA sequences

is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor

PCR modification of RT-PCR The rationale is to amplify sequences between a single

previously characterised region in the mRNA (cDNA) and an anchor sequence that is

coupled to the 5 or the 3 end A primer is designed from the known internal

sequence and the second primer is selected from the relevant anchor sequence

To provide the genomic organization and the complete sequence of the canine

MYH9 gene the isolation of full length cDNAs was achieved with the help of a

modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was

performed to identify single nucleotide polymorphisms (SNPs) in this gene We

analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three

families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage

to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic

sequence comparisons we detected that the canine MYH9 gene is bigger compared

to the human sequence due to the untranlated first exon in the 5rsquo-UTR

General discussion

The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and

exonintron boundaries that conform perfectly to the GTAG rule

None of the observed polymorphisms did alter the predicted amino acid sequence of

MYH9 nor showed the identified haplotypes an association with the CCSD

phenotype

do not seem to be responsible for the CCSD phenotype in these three families

To clarify if other regions on CFA10 are responsible for the CCSD phenotype we

added in a second step 27 microsatellite markers derived from the NCBI database to

cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage

analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that

were genotyped with the marker set of 27 microsatellite markers

We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb

Consequently we screened this 12 Mb spanning region for SNPs Out of the 23

analyzed Dalmatian dog families five full-sib families were chosen to screen for

SNPs because of their obviously significant values at the above mentioned region

developed and added to the linkage analysis The significant Zmeans on CFA10 was

confirmed after adding the SNP markers Furthermore with the use of SNPs the

apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb

spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to

In further studies more SNPs have to be developed within the identified CCSD region

on CFA10 to localize the deafness causing gene or to find unambiguously associsted

SNP markers which could be used for a population-wide genetic test for CCSD

Chapter 10

Summary

Summary 127

Summary

Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs

Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is

involved in the development of canine congenital sensorineural deafness (CCSD) in

Dalmatian dogs

Dalmatian dog showing the highest incidence Many genetic disorders in humans

and domestic dogs (Canis familiaris) demonstrate a high level of clinical and

molecular similarity

Altogether 39 genes have already been found causative for sensorineural non-

syndromic hearing impairment in humans Out of this 39 deafness causing genes a

total of 32 candidate genes were selected for canine congenital deafness which

showed an appropriate clinical and histological disease pattern in comparison to

deafness in Dalmatians dogs

On the one hand an existing set of 43 microsatllite markers for in total 24 candidate

genes were used for a non-parametric linkage analysis among them the claudin-14

(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide

9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1

was also considered as a candidate gene for CCSD and thus GJA1-associated

microsatellites were part of the existing set Recently it turned out that GJA1 is not

responsible for human sensorineural non-syndromic deafness but for a human

syndromic disorder that can be related with conductive deafness In the last few

years more human deafness genes have been identified among them eight genes

that were considered as appropriate candidates for CCSD For these eight genes a

total of 21 SNPs were newly developed and used for non-parametric linkage and

association analyses

Summary

The used microsatellite and SNP markers derived either from a partial sequence

analysis of BAC clones each containing a different candidate gene or from

sequences deposited in the current dog genome assembly (Boxer genome assembly

21) of the NCBI GenBank We found significant linkage to markers associated to

CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the

linkage we searched for sequence variations within these three genes SNPs found

in intronic sequences of either gene were included in the linkage analyses Sequence

analysis neither revealed a causative mutation nor significant linkage disequilibrium

of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned

completely with microsatellite markers derived from the NCBI database with the

purpose to see if other regions on this three chromosomes harbour a gene that is

involved in the development of CCSD

The analyses of SNPs and more microsatellite markers on CFA1 revealed no

significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and

the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs

especially in Dalmatians is almost exclusively caused by sensorineural non-

syndromic forms the GJA1 gene should not be considered as a candidate gene for

CCSD anymore

On CFA10 we could exclude MYH9 for being causal for deafness but by adding

more microsatellites covering CFA10 completely we found significant linkage to the

CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine

mapping the region spanning 36 to 48 Mb were developed by sequence analyses of

different Dalmatian dogs The search for SNPs was carried out on genomic

sequences of genes located in the significantly linked region The sequences of

these genomic sequences were derived from the NCBI GenBank The SNPs

confirmed the linkage and narrowed the region harbouring a causative CCSD gene

down to 5 Mb spanning from 39 to 44 Mb

After scanning CFA31 we could not exclude CLDN14 for being responsible for the

CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage

However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs

have to be developed within the CLDN14 gene as well as in its flanking regions with

the aim to find linkage disequilibrium for SNP markers

Chapter 11

Erweiterte Zusammenfassung

Erweiterte Zusammenfassung 131

Katharina Mieskes

Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner

Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen

Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen

unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im

aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen

zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen

oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein

Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea

(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt

Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder

ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren

Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst

im Laufe der weiteren Lebenszeit auftreten

Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die

erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit

(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das

vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische

ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die

Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-

syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle

verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf

einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal

dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein

noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht

Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1

wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr

verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind

Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht

syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120

identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar

Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele

Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen

beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen

europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-

30 die houmlchste Taubheitsinzidenz auf

Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie

beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr

schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren

normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem

Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen

Houmlrverlust

Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt

sich auf die brainstem auditory evoked response (BAER in Deutschland

audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen

bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in

der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden

Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen

werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen

auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht

gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es

bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes

Gen zu identifizieren

Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer

Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer

polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten

Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu

Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe

signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit

Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten

Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen

Augen ist in Deutschland schon laumlnger verboten und es wird von den

Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa

6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt

tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige

Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen

wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu

rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner

als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube

Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch

oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit

beidseitigem Houmlrverlust euthanasiert

Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt

um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen

Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit

beim Dalmatiner molekulargenetisch zu untersuchen um so ein

molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die

Identifizierung von Anlagetraumlgern ermoumlglicht

Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten

Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene

zunehmend verdeutlicht

Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden

Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die

histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich

denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr

die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die

canine congenitale sensorineurale Taubheit zu sein

Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte

zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner

moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit

genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen

sensorineuralen Taubheit untersucht

Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen

Taubheit wurden weitergehend molekulargenetisch analysiert

Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern

Material und Methoden

Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das

Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23

CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2

GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4

SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie

wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte

Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die

genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder

bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw

Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-

oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen

audiometrischen Test bestimmt

Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese

ausgewertet

Kopplungsanalyse

Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software

MERLIN (multipoint engine for rapid likelihood inference Version 0102)

durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei

wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der

Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende

Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus

abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines

Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit

(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner

Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version

913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und

erwarteten Heterozygotiegrad und PIC (Polymorphism information content)

charakterisieren zu koumlnnen

Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei

Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine

Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen

Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die

Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft

Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere

Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden

Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr

die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im

Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien

gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren

Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert

sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den

Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei

annaumlhernd Null und waren nicht signifikant

Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-

syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er

sich beim Menschen darstellt

Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden

Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie

ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend

molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der

Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische

Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten

Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt

Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN

und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu

entwickelt

Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39

Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens

zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp

der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier

Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden

Elterntieren

Identifizierung von SNPs (Single Nucleotide Polymorphisms)

Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA

mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten

Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die

Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms

Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen

Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der

betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des

MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder

mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen

Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit

Hilfe des Sequencher 42 Programms

Kopplungsanalyse

Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms

MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical

Analysis System Version 913) bearbeitet um das Markerset bezuumlglich

Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism

information content) charakterisieren zu koumlnnen Des Weiteren wurde auf

Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels

des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical

Analysis System Version 913 Cary NC USA) getestet

Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt

Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken

Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen

Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und

bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen

verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14

MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner

beteiligt sind

Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232

Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA

Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus

veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen

cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid

amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung

der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42

Programms ausgewertet

Sequenzanalyse des caninen MYH9 Gens

Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden

die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms

Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm

Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport

ermittelt

Mutatiosanalyse

Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz

wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR

amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16

Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie

waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen

PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen

fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden

durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des

Sequencher 42 Programms ausgewertet

Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood

inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-

Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung

der sensorineuralen Taubheit beim Dalmatiner gesucht

Ergebnisse und Diskussion

Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens

Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber

die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank

eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich

einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG

Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432

bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen

Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963

Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das

gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen

zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die

kodierende Sequenz von Exon 2 bis 41

Mutations- und Haplotypenanalyse

Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9

Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden

mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen

Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei

die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden

wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die

Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine

Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von

IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei

annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten

zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien

erklaumlrt werden

Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen

MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das

humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist

das canine Gen groumlszliger als das humane Gen

Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische

Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen

Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese

Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region

von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als

Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner

ausschlieszligen

Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial

Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere

verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale

Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen

tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier

nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister

einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten

Tiere wurde durch einen audiometrischen Test bestimmt

Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen

Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs

wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu

der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10

wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur

Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier

Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie

verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus

einer groszligen Halbgeschwisterfamilie verwendet

Mikrosatellitenstudie

Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker

verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten

wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31

verwendet

Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und

wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet

Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden

zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10

und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten

genotypisiert

Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI

Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden

bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate

mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die

Auswertung erfolgte mit Hilfe des Sequencher 42 Programms

Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die

Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter

Verwendung des Programms MERLIN ausgewertet

Kopplungsanalyse

913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten

Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu

koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der

Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE

von SAS Genetics getestet

Ergebnisse und Diskussion CFA1

In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem

GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser

Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt

Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten

Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder

bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf

Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen

offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in

der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher

unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der

untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der

GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen

hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der

untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-

positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie

Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker

an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere

Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die

Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher

unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den

untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich

Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr

eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist

sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit

einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr

CCSD angesehen werden

Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von

Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der

sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich

zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen

translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte

Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der

identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien

Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten

identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern

festgestellt werden

Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14

gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14

Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit

beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner

naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu

einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden

konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in

seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den

untersuchten Dalmatinerfamilien verantwortlich ist

Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27

Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten

Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs

mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt

Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und

bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10

deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden

Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die

identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der

caninen kongenitalen Taubheit einwirkt

Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu

lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren

Chapter 12

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Chapter 13

Appendix

Appendix microsatellite marker III

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

Appendix microsatellite marker

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Appendix itemisation of alleles V

Table 2 Itemisation of alleles to bases for SNPs in the pedigrees

Code no 1 2 3 4

Base Adenine Cytosine Guanine Thymine

Appendix laboratory paraphernalia

Laboratory paraphernalia Equipment Thermocycler

PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)

PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)

Automated sequencers

LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)

MegaBACE 1000 (Amersham Biosciences Freiburg)

Centrifuges

Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)

Desk-centrifuge 5415D (Eppendorf Hamburg)

Biofuge stratos (Heraeus Osterode)

Centrifuge Centrikon H 401 (Kontron Gosheim)

Megafuge 1OR (Heraeus Osterode)

Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)

Agarose gel electrophoresis and pulsed field gel electrophoresis

Electrophoresis chambers OWL Separation Systems Portsmouth NH USA

Biometra Goumlttingen

BioRad Muumlnchen

Generators 2301 Macrodrive 1 (LKB Bromma Sweden)

Power Pac 3000 (BioRad Muumlnchen)

Gel documentation system BioDocAnalyze 312 nm Goumlttingen

Appendix laboratory paraphernalia VII

Others

Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)

Incubator VT 5042 (Heraeus Osterode)

UV-Illuminator 312 nm (Bachhofer Reutlingen)

Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)

Biophotometer (Eppendorf AG Hamburg)

DNA purification

Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)

Cloning

Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)

Isolation of DNA

QIAamp 96 DNA Blood Kit (QIAGEN Hilden)

Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)

Sequencing

ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)

DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg

Germany)

FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon

RNA Total RNA (Biocat Heidelberg Germany)

Size standards

100 bp Ladder (New England Biolabs Schwalbach Taunus)

1 kb Ladder (New England Biolabs Schwalbach Taunus)

IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)

Reagents and buffers APS solution (10 )

1 g APS

10 ml H2O

Bromophenol blue solution

05 g bromophenol blue

10 ml 05 M EDTA solution

H2O ad 50 ml

dNTP solution

100 microl dATP [100 mM]

100 microl dCTP [100 mM]

100 microl dGTP [100 mM]

100 microl dTTP [100 mM]

1600 microl H2O

the concentration of each dNTP in the ready-to-use solution is 5 mM

Gel solution

1275 ml UreaTBE solution (Roth Karlsruhe)

225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)

95 microl APS solution (10 )

95 microl TEMED

Appendix laboratory paraphernalia IX

Loading buffer for agarose gels

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

Bromophenol blue 025 (wv)

Xylencyanol 025 (wv)

Loading buffer for gel electrophoresis

2 ml bromophenol blue solution

20 ml formamide

TBE-buffer (1x)

100 ml TBE-buffer (10x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

55 g boric acid [6183 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

UreaTBE solution (6 )

425 g urea [6006 M]

250 ml H2O

solubilise in a water bath at 65degC

H2O ad 850 ml

Chemicals Agarose (Invitrogen Paisley UK)

Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)

Ampicillin (Serva Heidelberg)

Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)

Bromophenol blue (Merck KgaA Darmstadt)

dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)

Chloramphenicol (Serva Heidelberg)

DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)

dNTP-Mix (Qbiogene GmbH Heidelberg)

EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)

Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)

Ethyl alcohol (AppliChem Darmstadt)

Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)

LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)

Paraffin (Merck KgaA Darmstadt)

RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)

SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)

TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)

Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)

Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)

Water was taken from the water purification system Milli-Qreg

X-Gal (AppliChem Darmstadt)

Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)

Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)

Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)

The polymerase was always used in the presence of incubation Mix TPol 10x buffer

The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with

the adequate 10x encyme buffer

Appendix laboratory paraphernalia XI

Consumables

96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)

Combitipsreg plus (Eppendorf AG Hamburg)

Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)

Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)

Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)

Thermo-fast 96 well plate skirted (ABgene Hamburg)

Software BLAST trace archive httpwwwncbinlmnihgov

httpwwwensemblorg

EBI toolbox httpwwwebiacukToolssequencehtml

MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin

Order of primers MWG Biotech-AG Ebersberg (httpsecom

mwgdnacomregisterindextcl)

biomersnet GmbH Ulm (orderbiomersnet)

PED50 Dr H Plendl et al (2005) Institute for Human Genetics

Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi

Repeat Masker httpwwwrepeatmaskergenome washingtonedu

Sequencher 42 GeneCodes Ann Arbor MI USA

Spidey httpwwwncbinlmnihgovIEBResearch

OstellSpideyindexhtml

SUN Ultra Enterprise 450 Sun microsystems

List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular

characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)

gene on dog chromosome 10q232 Submitted for publication in Gene

2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide

polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness

in Dalmatian dogs Submitted for publication in Journal of Heredity

3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as

candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for

publication in Animal Genetics

Acknowledgements

First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my

doctoral thesis for his academic guidance and support of this work

I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical

expertise and assistance

I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his

help with computer problems

I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer

Forschung (GKF) eV Germany for funding this work with a grant

I am appreciative to all Dalmatian breeders and owners for providing me blood

samples and the results of the BAER tests

My special thanks go to all colleagues and friends of the Institute for Animal Breeding

and Genetics of the University of Veterinary Medicine Hannover for their support

humour and the friendly atmosphere in the laboratory

Last but not least I wish to thank my family for their support during the work on this

thesis

Contents

1 Introduction

2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans

3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs

4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs

5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232

6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs

7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene for congenital sensorineural deafness

8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness

9 General discussion

10 Summary

11 Erweiterte Zusammenfassung

12 References

13 Appendix

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 DAN 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 NLD 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 ESP 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 SUO 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 ITA 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 NOR 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 SVE ltFEFF0041006e007600e4006e00640020006400650020006800e4007200200069006e0073007400e4006c006c006e0069006e006700610072006e00610020006e00e40072002000640075002000760069006c006c00200073006b0061007000610020005000440046002d0064006f006b0075006d0065006e00740020006d006500640020006800f6006700720065002000620069006c0064007500700070006c00f60073006e0069006e00670020006f006300680020006400e40072006d006500640020006600e50020006200e400740074007200650020007500740073006b00720069006600740073006b00760061006c0069007400650074002e0020005000440046002d0064006f006b0075006d0065006e00740065006e0020006b0061006e002000f600700070006e006100730020006d006500640020004100630072006f0062006100740020006f00630068002000520065006100640065007200200035002e003000200065006c006c00650072002000730065006e006100720065002egt gtgtgtgt setdistillerparamsltlt HWResolution [2400 2400] PageSize [595000 842000]gtgt setpagedevice

Scientific supervisor Univ-Prof Dr Dr O Distl

Examiner Univ-Prof Dr Dr O Distl

Co-examiner Univ-Prof Dr H Y Naim

Oral examination 18 Mai 2006

This work was supported by a grant from the Gesellschaft zur Foumlrderung

Kynologischer Forschung (GKF) eV Bonn Germany

To my family

1 Gene

3 Animal Genetics

Contents

1 Introduction 1

Abstract 7

Deafness in man 8

Deafness in dogs 9

Abstract 25

Introduction 25

Abstract 43

Introduction 43

Abstract 65

Introduction 65

Abstract 83

Introduction 83

Results 86

Discussion 87

Abstract 99

Introduction 99

Abstract 109

Introduction 109

CFA1 122

CFA31 123

CFA10 123

10 Summary 125

12 References 145

Abbreviations

A adenine

ACTG1 actin gamma 1

bp base pair

C cytosine

CLDN14 claudin-14

cM centiMorgan

CRYM crystallin mu

CX connexin

Abbreviations

EDN3 endothelin 3

ESPN espin

F forward

G guanine

IRD infrared dye

Kb kilobase

M molar

Mb megabase

Abbreviations

MS microsatellite

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

OTOA Otoancorin

OTOF otoferlin

P error probability

R reverse

RH radiation-hybrid

Abbreviations

SH1 Src homology 1

STRC stereocilin

T thymine

TJ tight juncions

U unit

WHRN whirlin

Chapter 1

Introduction

Introduction 3

Introduction

Introduction 4

approaches

research context

Chapter 2

in dogs and humans

Abstract

been identified

exception

humans

Autosomal dominant

Autosomal recessive

DFNB7 DFNB11DFNA36

Primary defect

Non-sensory cells

Tectorial membrane

COL11A2 TECTA

Unknown

EDNRBendothelin

microphthalmia-

associated

transcription

factor

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Canine mRNA (bp)

Dalmatian dogs

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

Chapter 3

Introduction

TMPRSS3 (Table 2)

Germany)

(Table 3)

Linkage analysis

(Table 4 and 5)

the hearing process

2 Liu et al 2001

et al 1992

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

MS (bp) Location of

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS2 NW_876327 8 1321513232 13290 distal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Gene-associated

marker

family members

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

Chapter 4

in Dalmatian dogs

linkage analyses

gene (Table 3)

SNP marker analysis

respectively

Dalmatians

Gene symbol

al (2003)

gene 1

et al (2002)

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

Bp3 AT4 PIC ()

ESPN_SNP1

565 60 9 10

ESPN_SNP2

565 60 27 41

ESPN_SNP3

565 60 23 32

ESPN_SNP4

565 60 23 32

ESPN_SNP5

565 60 28 42

MYH14_SNP1

670 60 35 42

MYH14_SNP2

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

650 60 37 77

MYO3A_SNP2

650 60 37 70

MYO3A_SNP3

650 60 25 36

PRES_SNP1

560 60 37 64

PRES_SNP2

560 60 37 66

TMC1_SNP1

610 60 57 37

TMC1_SNP2

610 60 47 29

TMC1_SNP3

650 60 48 30

TMIE_SNP1

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

560 60 37 37

WHRN_SNP1

600 60 25 37

WHRN_SNP2

600 59 24 33

WHRN_SNP3

600 60 34 55

TMC1_MS1

220-220 60 76 57

274-302 58 57 66

Allele frequencies

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

ith th

llite-

Chapter 5

Canine MYH9 gene 65

Canine MYH9 gene

Canine MYH9 gene 67

Canine MYH9 gene

Canine MYH9 gene 69

variation

AM086385)

Canine MYH9 gene

of 50 over 200 bp)

Canine MYH9 gene 71

Conclusions

Canine MYH9 gene

size (bp)

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

Canine MYH9 gene

Intron phase

Intron size

gt30000 bp

4922 bp

13493 bp

803 bp

4077 bp

427 bp

738 bp

343 bp

749 bp

1041 bp

1877 bp

922 bp

1801 bp

2049 bp

1877 bp

343 bp

835 bp

1418 bp

851 bp

1428 bp

398 bp

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

971 bp

1603 bp

719 bp

270 bp

480 bp

232 bp

204 bp

1083 bp

1298 bp

150 bp

303 bp

1173 bp

941 bp

224 bp

563 bp

739 bp

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Canine MYH9 gene

Canine MYH9 gene 79

Canine MYH9 gene

Chapter 6

Abstract

Introduction

parents

Germany)

Linkage analysis

marker loci

Results

from 013 to 037

Discussion

two families

Acc No canine mRNA

37023714 NC_006592 XM_531765

38353835 NC_006592 XM_848614

38503857 NC_006592 XM_531770

39453945 NC_006592 XM_858065

42564272 NC_006592 XM_538446

44084413 NC_006592 XM_538451

46264637 NC_006592 XM_538458

48494852 NC_006592 XM_849433

description SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474535

intron

590 60

LOC474536

intron

590 60

LOC610953

intron

600 60

LOC481302

3rsquo-end

610 60

LOC610991

intron

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

480 60

LOC474541

intron

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

560 60

LOC481308

SNP_11

3rsquo-end

600 60

LOC474543

SNP_12

intron

LOC609217

SNP_16

5rsquo-end

580 60

LOC611115

SNP_18

3rsquo-end

590 60

LOC481325

SNP_20

intron

600 60

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

575 60

LOC611493

SNP_23

intron

590 60

LOC481337

SNP_24

intron

620 60

LOC611728

SNP_26

intron

620 60

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

values)

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

Chapter 7

families

GJA1 gene

the GJA1 gene

Oldendorf Germany)

marker loci

GJA1 gene

(Chapter 3)

families

SNP motif

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

600 58

Microsatellite

Product

size (bp)

Annealing

temperatur

GJA1_MS1

134 60

GJA1_MS2

190 60

Chapter 8

in Chapter 3

Introduction

deafness (DFNB29)

markers (Chapter 3)

Oldendorf Germany)

CLDN14_MS2

indicating linkage

markers with CCSD

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

610 60

585 60

580 60

680 60

Chapter 9

General discussion

developed SNPs

General discussion

is needed

CCSD phenotype

General discussion

markers

General discussion

phenotype

Chapter 10

Summary

Summary 127

Summary

Dalmatian dogs

Summary

CCSD anymore

Chapter 11

Katharina Mieskes

Houmlrverlust

ausgewertet

Kopplungsanalyse

entwickelt

Elterntieren

Kopplungsanalyse

beteiligt sind

ermittelt

Mutatiosanalyse

erklaumlrt werden

ausschlieszligen

verwendet

genotypisiert

Kopplungsanalyse

festgestellt werden

Chapter 12

References

References 147

References

3097-101

Genet 721315-1322

References

J Med 346243-249

74770-776

References 149

J Mol Biol 196261-282

2316-8

References

dogs Vet J 166164-169

References 151

Neurootol 2139-154

References

438803-819

References 153

26103-105

References

Genet 69635-640

Sci 63016-31

References 155

9109-119

41591-595

References

References 157

8251-255

References

Nat Genet 20194-197

Genet 1960-62

Genet 2651-55

References 159

Cell 104165-172

References

Chapter 13

Appendix

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Code no 1 2 3 4

Centrifuges

BioRad Muumlnchen

Others

DNA purification

Cloning

Isolation of DNA

Sequencing

Germany)

Size standards

1 g APS

10 ml H2O

H2O ad 50 ml

dNTP solution

1600 microl H2O

Gel solution

95 microl TEMED

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

20 ml formamide

TBE-buffer (1x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

425 g urea [6006 M]

250 ml H2O

H2O ad 850 ml

Consumables

httpwwwensemblorg

Acknowledgements

thesis

Contents

1 Introduction

10 Summary

12 References

13 Appendix

To my family

1 Gene

3 Animal Genetics

Contents

1 Introduction 1

Abstract 7

Deafness in man 8

Deafness in dogs 9

Abstract 25

Introduction 25

Abstract 43

Introduction 43

Abstract 65

Introduction 65

Abstract 83

Introduction 83

Results 86

Discussion 87

Abstract 99

Introduction 99

Abstract 109

Introduction 109

CFA1 122

CFA31 123

CFA10 123

10 Summary 125

12 References 145

Abbreviations

A adenine

ACTG1 actin gamma 1

bp base pair

C cytosine

CLDN14 claudin-14

cM centiMorgan

CRYM crystallin mu

CX connexin

Abbreviations

EDN3 endothelin 3

ESPN espin

F forward

G guanine

IRD infrared dye

Kb kilobase

M molar

Mb megabase

Abbreviations

MS microsatellite

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

OTOA Otoancorin

OTOF otoferlin

P error probability

R reverse

RH radiation-hybrid

Abbreviations

SH1 Src homology 1

STRC stereocilin

T thymine

TJ tight juncions

U unit

WHRN whirlin

Chapter 1

Introduction

Introduction 3

Introduction

Introduction 4

approaches

research context

Chapter 2

in dogs and humans

Abstract

been identified

exception

humans

Autosomal dominant

Autosomal recessive

DFNB7 DFNB11DFNA36

Primary defect

Non-sensory cells

Tectorial membrane

COL11A2 TECTA

Unknown

EDNRBendothelin

microphthalmia-

associated

transcription

factor

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Canine mRNA (bp)

Dalmatian dogs

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

Chapter 3

Introduction

TMPRSS3 (Table 2)

Germany)

(Table 3)

Linkage analysis

(Table 4 and 5)

the hearing process

2 Liu et al 2001

et al 1992

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

MS (bp) Location of

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS2 NW_876327 8 1321513232 13290 distal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Gene-associated

marker

family members

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

Chapter 4

in Dalmatian dogs

linkage analyses

gene (Table 3)

SNP marker analysis

respectively

Dalmatians

Gene symbol

al (2003)

gene 1

et al (2002)

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

Bp3 AT4 PIC ()

ESPN_SNP1

565 60 9 10

ESPN_SNP2

565 60 27 41

ESPN_SNP3

565 60 23 32

ESPN_SNP4

565 60 23 32

ESPN_SNP5

565 60 28 42

MYH14_SNP1

670 60 35 42

MYH14_SNP2

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

650 60 37 77

MYO3A_SNP2

650 60 37 70

MYO3A_SNP3

650 60 25 36

PRES_SNP1

560 60 37 64

PRES_SNP2

560 60 37 66

TMC1_SNP1

610 60 57 37

TMC1_SNP2

610 60 47 29

TMC1_SNP3

650 60 48 30

TMIE_SNP1

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

560 60 37 37

WHRN_SNP1

600 60 25 37

WHRN_SNP2

600 59 24 33

WHRN_SNP3

600 60 34 55

TMC1_MS1

220-220 60 76 57

274-302 58 57 66

Allele frequencies

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

ith th

llite-

Chapter 5

Canine MYH9 gene 65

Canine MYH9 gene

Canine MYH9 gene 67

Canine MYH9 gene

Canine MYH9 gene 69

variation

AM086385)

Canine MYH9 gene

of 50 over 200 bp)

Canine MYH9 gene 71

Conclusions

Canine MYH9 gene

size (bp)

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

Canine MYH9 gene

Intron phase

Intron size

gt30000 bp

4922 bp

13493 bp

803 bp

4077 bp

427 bp

738 bp

343 bp

749 bp

1041 bp

1877 bp

922 bp

1801 bp

2049 bp

1877 bp

343 bp

835 bp

1418 bp

851 bp

1428 bp

398 bp

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

971 bp

1603 bp

719 bp

270 bp

480 bp

232 bp

204 bp

1083 bp

1298 bp

150 bp

303 bp

1173 bp

941 bp

224 bp

563 bp

739 bp

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Canine MYH9 gene

Canine MYH9 gene 79

Canine MYH9 gene

Chapter 6

Abstract

Introduction

parents

Germany)

Linkage analysis

marker loci

Results

from 013 to 037

Discussion

two families

Acc No canine mRNA

37023714 NC_006592 XM_531765

38353835 NC_006592 XM_848614

38503857 NC_006592 XM_531770

39453945 NC_006592 XM_858065

42564272 NC_006592 XM_538446

44084413 NC_006592 XM_538451

46264637 NC_006592 XM_538458

48494852 NC_006592 XM_849433

description SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474535

intron

590 60

LOC474536

intron

590 60

LOC610953

intron

600 60

LOC481302

3rsquo-end

610 60

LOC610991

intron

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

480 60

LOC474541

intron

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

560 60

LOC481308

SNP_11

3rsquo-end

600 60

LOC474543

SNP_12

intron

LOC609217

SNP_16

5rsquo-end

580 60

LOC611115

SNP_18

3rsquo-end

590 60

LOC481325

SNP_20

intron

600 60

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

575 60

LOC611493

SNP_23

intron

590 60

LOC481337

SNP_24

intron

620 60

LOC611728

SNP_26

intron

620 60

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

values)

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

Chapter 7

families

GJA1 gene

the GJA1 gene

Oldendorf Germany)

marker loci

GJA1 gene

(Chapter 3)

families

SNP motif

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

600 58

Microsatellite

Product

size (bp)

Annealing

temperatur

GJA1_MS1

134 60

GJA1_MS2

190 60

Chapter 8

in Chapter 3

Introduction

deafness (DFNB29)

markers (Chapter 3)

Oldendorf Germany)

CLDN14_MS2

indicating linkage

markers with CCSD

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

610 60

585 60

580 60

680 60

Chapter 9

General discussion

developed SNPs

General discussion

is needed

CCSD phenotype

General discussion

markers

General discussion

phenotype

Chapter 10

Summary

Summary 127

Summary

Dalmatian dogs

Summary

CCSD anymore

Chapter 11

Katharina Mieskes

Houmlrverlust

ausgewertet

Kopplungsanalyse

entwickelt

Elterntieren

Kopplungsanalyse

beteiligt sind

ermittelt

Mutatiosanalyse

erklaumlrt werden

ausschlieszligen

verwendet

genotypisiert

Kopplungsanalyse

festgestellt werden

Chapter 12

References

References 147

References

3097-101

Genet 721315-1322

References

J Med 346243-249

74770-776

References 149

J Mol Biol 196261-282

2316-8

References

dogs Vet J 166164-169

References 151

Neurootol 2139-154

References

438803-819

References 153

26103-105

References

Genet 69635-640

Sci 63016-31

References 155

9109-119

41591-595

References

References 157

8251-255

References

Nat Genet 20194-197

Genet 1960-62

Genet 2651-55

References 159

Cell 104165-172

References

Chapter 13

Appendix

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Code no 1 2 3 4

Centrifuges

BioRad Muumlnchen

Others

DNA purification

Cloning

Isolation of DNA

Sequencing

Germany)

Size standards

1 g APS

10 ml H2O

H2O ad 50 ml

dNTP solution

1600 microl H2O

Gel solution

95 microl TEMED

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

20 ml formamide

TBE-buffer (1x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

425 g urea [6006 M]

250 ml H2O

H2O ad 850 ml

Consumables

httpwwwensemblorg

Acknowledgements

thesis

Contents

1 Introduction

10 Summary

12 References

13 Appendix

1 Gene

3 Animal Genetics

Contents

1 Introduction 1

Abstract 7

Deafness in man 8

Deafness in dogs 9

Abstract 25

Introduction 25

Abstract 43

Introduction 43

Abstract 65

Introduction 65

Abstract 83

Introduction 83

Results 86

Discussion 87

Abstract 99

Introduction 99

Abstract 109

Introduction 109

CFA1 122

CFA31 123

CFA10 123

10 Summary 125

12 References 145

Abbreviations

A adenine

ACTG1 actin gamma 1

bp base pair

C cytosine

CLDN14 claudin-14

cM centiMorgan

CRYM crystallin mu

CX connexin

Abbreviations

EDN3 endothelin 3

ESPN espin

F forward

G guanine

IRD infrared dye

Kb kilobase

M molar

Mb megabase

Abbreviations

MS microsatellite

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

OTOA Otoancorin

OTOF otoferlin

P error probability

R reverse

RH radiation-hybrid

Abbreviations

SH1 Src homology 1

STRC stereocilin

T thymine

TJ tight juncions

U unit

WHRN whirlin

Chapter 1

Introduction

Introduction 3

Introduction

Introduction 4

approaches

research context

Chapter 2

in dogs and humans

Abstract

been identified

exception

humans

Autosomal dominant

Autosomal recessive

DFNB7 DFNB11DFNA36

Primary defect

Non-sensory cells

Tectorial membrane

COL11A2 TECTA

Unknown

EDNRBendothelin

microphthalmia-

associated

transcription

factor

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Canine mRNA (bp)

Dalmatian dogs

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

Chapter 3

Introduction

TMPRSS3 (Table 2)

Germany)

(Table 3)

Linkage analysis

(Table 4 and 5)

the hearing process

2 Liu et al 2001

et al 1992

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

MS (bp) Location of

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS2 NW_876327 8 1321513232 13290 distal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Gene-associated

marker

family members

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

Chapter 4

in Dalmatian dogs

linkage analyses

gene (Table 3)

SNP marker analysis

respectively

Dalmatians

Gene symbol

al (2003)

gene 1

et al (2002)

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

Bp3 AT4 PIC ()

ESPN_SNP1

565 60 9 10

ESPN_SNP2

565 60 27 41

ESPN_SNP3

565 60 23 32

ESPN_SNP4

565 60 23 32

ESPN_SNP5

565 60 28 42

MYH14_SNP1

670 60 35 42

MYH14_SNP2

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

650 60 37 77

MYO3A_SNP2

650 60 37 70

MYO3A_SNP3

650 60 25 36

PRES_SNP1

560 60 37 64

PRES_SNP2

560 60 37 66

TMC1_SNP1

610 60 57 37

TMC1_SNP2

610 60 47 29

TMC1_SNP3

650 60 48 30

TMIE_SNP1

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

560 60 37 37

WHRN_SNP1

600 60 25 37

WHRN_SNP2

600 59 24 33

WHRN_SNP3

600 60 34 55

TMC1_MS1

220-220 60 76 57

274-302 58 57 66

Allele frequencies

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

ith th

llite-

Chapter 5

Canine MYH9 gene 65

Canine MYH9 gene

Canine MYH9 gene 67

Canine MYH9 gene

Canine MYH9 gene 69

variation

AM086385)

Canine MYH9 gene

of 50 over 200 bp)

Canine MYH9 gene 71

Conclusions

Canine MYH9 gene

size (bp)

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

Canine MYH9 gene

Intron phase

Intron size

gt30000 bp

4922 bp

13493 bp

803 bp

4077 bp

427 bp

738 bp

343 bp

749 bp

1041 bp

1877 bp

922 bp

1801 bp

2049 bp

1877 bp

343 bp

835 bp

1418 bp

851 bp

1428 bp

398 bp

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

971 bp

1603 bp

719 bp

270 bp

480 bp

232 bp

204 bp

1083 bp

1298 bp

150 bp

303 bp

1173 bp

941 bp

224 bp

563 bp

739 bp

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Canine MYH9 gene

Canine MYH9 gene 79

Canine MYH9 gene

Chapter 6

Abstract

Introduction

parents

Germany)

Linkage analysis

marker loci

Results

from 013 to 037

Discussion

two families

Acc No canine mRNA

37023714 NC_006592 XM_531765

38353835 NC_006592 XM_848614

38503857 NC_006592 XM_531770

39453945 NC_006592 XM_858065

42564272 NC_006592 XM_538446

44084413 NC_006592 XM_538451

46264637 NC_006592 XM_538458

48494852 NC_006592 XM_849433

description SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474535

intron

590 60

LOC474536

intron

590 60

LOC610953

intron

600 60

LOC481302

3rsquo-end

610 60

LOC610991

intron

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

480 60

LOC474541

intron

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

560 60

LOC481308

SNP_11

3rsquo-end

600 60

LOC474543

SNP_12

intron

LOC609217

SNP_16

5rsquo-end

580 60

LOC611115

SNP_18

3rsquo-end

590 60

LOC481325

SNP_20

intron

600 60

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

575 60

LOC611493

SNP_23

intron

590 60

LOC481337

SNP_24

intron

620 60

LOC611728

SNP_26

intron

620 60

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

values)

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

Chapter 7

families

GJA1 gene

the GJA1 gene

Oldendorf Germany)

marker loci

GJA1 gene

(Chapter 3)

families

SNP motif

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

600 58

Microsatellite

Product

size (bp)

Annealing

temperatur

GJA1_MS1

134 60

GJA1_MS2

190 60

Chapter 8

in Chapter 3

Introduction

deafness (DFNB29)

markers (Chapter 3)

Oldendorf Germany)

CLDN14_MS2

indicating linkage

markers with CCSD

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

610 60

585 60

580 60

680 60

Chapter 9

General discussion

developed SNPs

General discussion

is needed

CCSD phenotype

General discussion

markers

General discussion

phenotype

Chapter 10

Summary

Summary 127

Summary

Dalmatian dogs

Summary

CCSD anymore

Chapter 11

Katharina Mieskes

Houmlrverlust

ausgewertet

Kopplungsanalyse

entwickelt

Elterntieren

Kopplungsanalyse

beteiligt sind

ermittelt

Mutatiosanalyse

erklaumlrt werden

ausschlieszligen

verwendet

genotypisiert

Kopplungsanalyse

festgestellt werden

Chapter 12

References

References 147

References

3097-101

Genet 721315-1322

References

J Med 346243-249

74770-776

References 149

J Mol Biol 196261-282

2316-8

References

dogs Vet J 166164-169

References 151

Neurootol 2139-154

References

438803-819

References 153

26103-105

References

Genet 69635-640

Sci 63016-31

References 155

9109-119

41591-595

References

References 157

8251-255

References

Nat Genet 20194-197

Genet 1960-62

Genet 2651-55

References 159

Cell 104165-172

References

Chapter 13

Appendix

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Code no 1 2 3 4

Centrifuges

BioRad Muumlnchen

Others

DNA purification

Cloning

Isolation of DNA

Sequencing

Germany)

Size standards

1 g APS

10 ml H2O

H2O ad 50 ml

dNTP solution

1600 microl H2O

Gel solution

95 microl TEMED

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

20 ml formamide

TBE-buffer (1x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

425 g urea [6006 M]

250 ml H2O

H2O ad 850 ml

Consumables

httpwwwensemblorg

Acknowledgements

thesis

Contents

1 Introduction

10 Summary

12 References

13 Appendix

Contents

1 Introduction 1

Abstract 7

Deafness in man 8

Deafness in dogs 9

Abstract 25

Introduction 25

Abstract 43

Introduction 43

Abstract 65

Introduction 65

Abstract 83

Introduction 83

Results 86

Discussion 87

Abstract 99

Introduction 99

Abstract 109

Introduction 109

CFA1 122

CFA31 123

CFA10 123

10 Summary 125

12 References 145

Abbreviations

A adenine

ACTG1 actin gamma 1

bp base pair

C cytosine

CLDN14 claudin-14

cM centiMorgan

CRYM crystallin mu

CX connexin

Abbreviations

EDN3 endothelin 3

ESPN espin

F forward

G guanine

IRD infrared dye

Kb kilobase

M molar

Mb megabase

Abbreviations

MS microsatellite

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

OTOA Otoancorin

OTOF otoferlin

P error probability

R reverse

RH radiation-hybrid

Abbreviations

SH1 Src homology 1

STRC stereocilin

T thymine

TJ tight juncions

U unit

WHRN whirlin

Chapter 1

Introduction

Introduction 3

Introduction

Introduction 4

approaches

research context

Chapter 2

in dogs and humans

Abstract

been identified

exception

humans

Autosomal dominant

Autosomal recessive

DFNB7 DFNB11DFNA36

Primary defect

Non-sensory cells

Tectorial membrane

COL11A2 TECTA

Unknown

EDNRBendothelin

microphthalmia-

associated

transcription

factor

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Canine mRNA (bp)

Dalmatian dogs

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

Chapter 3

Introduction

TMPRSS3 (Table 2)

Germany)

(Table 3)

Linkage analysis

(Table 4 and 5)

the hearing process

2 Liu et al 2001

et al 1992

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

MS (bp) Location of

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS2 NW_876327 8 1321513232 13290 distal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Gene-associated

marker

family members

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

Chapter 4

in Dalmatian dogs

linkage analyses

gene (Table 3)

SNP marker analysis

respectively

Dalmatians

Gene symbol

al (2003)

gene 1

et al (2002)

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

Bp3 AT4 PIC ()

ESPN_SNP1

565 60 9 10

ESPN_SNP2

565 60 27 41

ESPN_SNP3

565 60 23 32

ESPN_SNP4

565 60 23 32

ESPN_SNP5

565 60 28 42

MYH14_SNP1

670 60 35 42

MYH14_SNP2

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

650 60 37 77

MYO3A_SNP2

650 60 37 70

MYO3A_SNP3

650 60 25 36

PRES_SNP1

560 60 37 64

PRES_SNP2

560 60 37 66

TMC1_SNP1

610 60 57 37

TMC1_SNP2

610 60 47 29

TMC1_SNP3

650 60 48 30

TMIE_SNP1

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

560 60 37 37

WHRN_SNP1

600 60 25 37

WHRN_SNP2

600 59 24 33

WHRN_SNP3

600 60 34 55

TMC1_MS1

220-220 60 76 57

274-302 58 57 66

Allele frequencies

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

ith th

llite-

Chapter 5

Canine MYH9 gene 65

Canine MYH9 gene

Canine MYH9 gene 67

Canine MYH9 gene

Canine MYH9 gene 69

variation

AM086385)

Canine MYH9 gene

of 50 over 200 bp)

Canine MYH9 gene 71

Conclusions

Canine MYH9 gene

size (bp)

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

Canine MYH9 gene

Intron phase

Intron size

gt30000 bp

4922 bp

13493 bp

803 bp

4077 bp

427 bp

738 bp

343 bp

749 bp

1041 bp

1877 bp

922 bp

1801 bp

2049 bp

1877 bp

343 bp

835 bp

1418 bp

851 bp

1428 bp

398 bp

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

971 bp

1603 bp

719 bp

270 bp

480 bp

232 bp

204 bp

1083 bp

1298 bp

150 bp

303 bp

1173 bp

941 bp

224 bp

563 bp

739 bp

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Canine MYH9 gene

Canine MYH9 gene 79

Canine MYH9 gene

Chapter 6

Abstract

Introduction

parents

Germany)

Linkage analysis

marker loci

Results

from 013 to 037

Discussion

two families

Acc No canine mRNA

37023714 NC_006592 XM_531765

38353835 NC_006592 XM_848614

38503857 NC_006592 XM_531770

39453945 NC_006592 XM_858065

42564272 NC_006592 XM_538446

44084413 NC_006592 XM_538451

46264637 NC_006592 XM_538458

48494852 NC_006592 XM_849433

description SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474535

intron

590 60

LOC474536

intron

590 60

LOC610953

intron

600 60

LOC481302

3rsquo-end

610 60

LOC610991

intron

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

480 60

LOC474541

intron

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

560 60

LOC481308

SNP_11

3rsquo-end

600 60

LOC474543

SNP_12

intron

LOC609217

SNP_16

5rsquo-end

580 60

LOC611115

SNP_18

3rsquo-end

590 60

LOC481325

SNP_20

intron

600 60

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

575 60

LOC611493

SNP_23

intron

590 60

LOC481337

SNP_24

intron

620 60

LOC611728

SNP_26

intron

620 60

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

values)

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

Chapter 7

families

GJA1 gene

the GJA1 gene

Oldendorf Germany)

marker loci

GJA1 gene

(Chapter 3)

families

SNP motif

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

600 58

Microsatellite

Product

size (bp)

Annealing

temperatur

GJA1_MS1

134 60

GJA1_MS2

190 60

Chapter 8

in Chapter 3

Introduction

deafness (DFNB29)

markers (Chapter 3)

Oldendorf Germany)

CLDN14_MS2

indicating linkage

markers with CCSD

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

610 60

585 60

580 60

680 60

Chapter 9

General discussion

developed SNPs

General discussion

is needed

CCSD phenotype

General discussion

markers

General discussion

phenotype

Chapter 10

Summary

Summary 127

Summary

Dalmatian dogs

Summary

CCSD anymore

Chapter 11

Katharina Mieskes

Houmlrverlust

ausgewertet

Kopplungsanalyse

entwickelt

Elterntieren

Kopplungsanalyse

beteiligt sind

ermittelt

Mutatiosanalyse

erklaumlrt werden

ausschlieszligen

verwendet

genotypisiert

Kopplungsanalyse

festgestellt werden

Chapter 12

References

References 147

References

3097-101

Genet 721315-1322

References

J Med 346243-249

74770-776

References 149

J Mol Biol 196261-282

2316-8

References

dogs Vet J 166164-169

References 151

Neurootol 2139-154

References

438803-819

References 153

26103-105

References

Genet 69635-640

Sci 63016-31

References 155

9109-119

41591-595

References

References 157

8251-255

References

Nat Genet 20194-197

Genet 1960-62

Genet 2651-55

References 159

Cell 104165-172

References

Chapter 13

Appendix

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Code no 1 2 3 4

Centrifuges

BioRad Muumlnchen

Others

DNA purification

Cloning

Isolation of DNA

Sequencing

Germany)

Size standards

1 g APS

10 ml H2O

H2O ad 50 ml

dNTP solution

1600 microl H2O

Gel solution

95 microl TEMED

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

20 ml formamide

TBE-buffer (1x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

425 g urea [6006 M]

250 ml H2O

H2O ad 850 ml

Consumables

httpwwwensemblorg

Acknowledgements

thesis

Contents

1 Introduction

10 Summary

12 References

13 Appendix

Abstract 83

Introduction 83

Results 86

Discussion 87

Abstract 99

Introduction 99

Abstract 109

Introduction 109

CFA1 122

CFA31 123

CFA10 123

10 Summary 125

12 References 145

Abbreviations

A adenine

ACTG1 actin gamma 1

bp base pair

C cytosine

CLDN14 claudin-14

cM centiMorgan

CRYM crystallin mu

CX connexin

Abbreviations

EDN3 endothelin 3

ESPN espin

F forward

G guanine

IRD infrared dye

Kb kilobase

M molar

Mb megabase

Abbreviations

MS microsatellite

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

OTOA Otoancorin

OTOF otoferlin

P error probability

R reverse

RH radiation-hybrid

Abbreviations

SH1 Src homology 1

STRC stereocilin

T thymine

TJ tight juncions

U unit

WHRN whirlin

Chapter 1

Introduction

Introduction 3

Introduction

Introduction 4

approaches

research context

Chapter 2

in dogs and humans

Abstract

been identified

exception

humans

Autosomal dominant

Autosomal recessive

DFNB7 DFNB11DFNA36

Primary defect

Non-sensory cells

Tectorial membrane

COL11A2 TECTA

Unknown

EDNRBendothelin

microphthalmia-

associated

transcription

factor

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Canine mRNA (bp)

Dalmatian dogs

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

Chapter 3

Introduction

TMPRSS3 (Table 2)

Germany)

(Table 3)

Linkage analysis

(Table 4 and 5)

the hearing process

2 Liu et al 2001

et al 1992

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

MS (bp) Location of

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS2 NW_876327 8 1321513232 13290 distal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Gene-associated

marker

family members

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

Chapter 4

in Dalmatian dogs

linkage analyses

gene (Table 3)

SNP marker analysis

respectively

Dalmatians

Gene symbol

al (2003)

gene 1

et al (2002)

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

Bp3 AT4 PIC ()

ESPN_SNP1

565 60 9 10

ESPN_SNP2

565 60 27 41

ESPN_SNP3

565 60 23 32

ESPN_SNP4

565 60 23 32

ESPN_SNP5

565 60 28 42

MYH14_SNP1

670 60 35 42

MYH14_SNP2

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

650 60 37 77

MYO3A_SNP2

650 60 37 70

MYO3A_SNP3

650 60 25 36

PRES_SNP1

560 60 37 64

PRES_SNP2

560 60 37 66

TMC1_SNP1

610 60 57 37

TMC1_SNP2

610 60 47 29

TMC1_SNP3

650 60 48 30

TMIE_SNP1

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

560 60 37 37

WHRN_SNP1

600 60 25 37

WHRN_SNP2

600 59 24 33

WHRN_SNP3

600 60 34 55

TMC1_MS1

220-220 60 76 57

274-302 58 57 66

Allele frequencies

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

ith th

llite-

Chapter 5

Canine MYH9 gene 65

Canine MYH9 gene

Canine MYH9 gene 67

Canine MYH9 gene

Canine MYH9 gene 69

variation

AM086385)

Canine MYH9 gene

of 50 over 200 bp)

Canine MYH9 gene 71

Conclusions

Canine MYH9 gene

size (bp)

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

Canine MYH9 gene

Intron phase

Intron size

gt30000 bp

4922 bp

13493 bp

803 bp

4077 bp

427 bp

738 bp

343 bp

749 bp

1041 bp

1877 bp

922 bp

1801 bp

2049 bp

1877 bp

343 bp

835 bp

1418 bp

851 bp

1428 bp

398 bp

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

971 bp

1603 bp

719 bp

270 bp

480 bp

232 bp

204 bp

1083 bp

1298 bp

150 bp

303 bp

1173 bp

941 bp

224 bp

563 bp

739 bp

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Canine MYH9 gene

Canine MYH9 gene 79

Canine MYH9 gene

Chapter 6

Abstract

Introduction

parents

Germany)

Linkage analysis

marker loci

Results

from 013 to 037

Discussion

two families

Acc No canine mRNA

37023714 NC_006592 XM_531765

38353835 NC_006592 XM_848614

38503857 NC_006592 XM_531770

39453945 NC_006592 XM_858065

42564272 NC_006592 XM_538446

44084413 NC_006592 XM_538451

46264637 NC_006592 XM_538458

48494852 NC_006592 XM_849433

description SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474535

intron

590 60

LOC474536

intron

590 60

LOC610953

intron

600 60

LOC481302

3rsquo-end

610 60

LOC610991

intron

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

480 60

LOC474541

intron

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

560 60

LOC481308

SNP_11

3rsquo-end

600 60

LOC474543

SNP_12

intron

LOC609217

SNP_16

5rsquo-end

580 60

LOC611115

SNP_18

3rsquo-end

590 60

LOC481325

SNP_20

intron

600 60

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

575 60

LOC611493

SNP_23

intron

590 60

LOC481337

SNP_24

intron

620 60

LOC611728

SNP_26

intron

620 60

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

values)

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

Chapter 7

families

GJA1 gene

the GJA1 gene

Oldendorf Germany)

marker loci

GJA1 gene

(Chapter 3)

families

SNP motif

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

600 58

Microsatellite

Product

size (bp)

Annealing

temperatur

GJA1_MS1

134 60

GJA1_MS2

190 60

Chapter 8

in Chapter 3

Introduction

deafness (DFNB29)

markers (Chapter 3)

Oldendorf Germany)

CLDN14_MS2

indicating linkage

markers with CCSD

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

610 60

585 60

580 60

680 60

Chapter 9

General discussion

developed SNPs

General discussion

is needed

CCSD phenotype

General discussion

markers

General discussion

phenotype

Chapter 10

Summary

Summary 127

Summary

Dalmatian dogs

Summary

CCSD anymore

Chapter 11

Katharina Mieskes

Houmlrverlust

ausgewertet

Kopplungsanalyse

entwickelt

Elterntieren

Kopplungsanalyse

beteiligt sind

ermittelt

Mutatiosanalyse

erklaumlrt werden

ausschlieszligen

verwendet

genotypisiert

Kopplungsanalyse

festgestellt werden

Chapter 12

References

References 147

References

3097-101

Genet 721315-1322

References

J Med 346243-249

74770-776

References 149

J Mol Biol 196261-282

2316-8

References

dogs Vet J 166164-169

References 151

Neurootol 2139-154

References

438803-819

References 153

26103-105

References

Genet 69635-640

Sci 63016-31

References 155

9109-119

41591-595

References

References 157

8251-255

References

Nat Genet 20194-197

Genet 1960-62

Genet 2651-55

References 159

Cell 104165-172

References

Chapter 13

Appendix

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Code no 1 2 3 4

Centrifuges

BioRad Muumlnchen

Others

DNA purification

Cloning

Isolation of DNA

Sequencing

Germany)

Size standards

1 g APS

10 ml H2O

H2O ad 50 ml

dNTP solution

1600 microl H2O

Gel solution

95 microl TEMED

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

20 ml formamide

TBE-buffer (1x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

425 g urea [6006 M]

250 ml H2O

H2O ad 850 ml

Consumables

httpwwwensemblorg

Acknowledgements

thesis

Contents

1 Introduction

10 Summary

12 References

13 Appendix

Abbreviations

A adenine

ACTG1 actin gamma 1

bp base pair

C cytosine

CLDN14 claudin-14

cM centiMorgan

CRYM crystallin mu

CX connexin

Abbreviations

EDN3 endothelin 3

ESPN espin

F forward

G guanine

IRD infrared dye

Kb kilobase

M molar

Mb megabase

Abbreviations

MS microsatellite

MYO1A myosin IA

MYO3A myosin IIIA

MYO6 myosin VI

MYO7A myosin VIIA

MYO15A myosin XVA

OTOA Otoancorin

OTOF otoferlin

P error probability

R reverse

RH radiation-hybrid

Abbreviations

SH1 Src homology 1

STRC stereocilin

T thymine

TJ tight juncions

U unit

WHRN whirlin

Chapter 1

Introduction

Introduction 3

Introduction

Introduction 4

approaches

research context

Chapter 2

in dogs and humans

Abstract

been identified

exception

humans

Autosomal dominant

Autosomal recessive

DFNB7 DFNB11DFNA36

Primary defect

Non-sensory cells

Tectorial membrane

COL11A2 TECTA

Unknown

EDNRBendothelin

microphthalmia-

associated

transcription

factor

6 2 14

5 1 25

5 7 1 6 13

5 7 7 15

8 9 3 21

0 0 0 0

3 2 22

Canine mRNA (bp)

Dalmatian dogs

microsatellites

CDH23 2 0 CLDN14 3 8 COCH 2 0

MYH14 0 2 MYH9 2 22

MYO15 2 0 MYO3A 0 3 MYO6 1 0

Chapter 3

Introduction

TMPRSS3 (Table 2)

Germany)

(Table 3)

Linkage analysis

(Table 4 and 5)

the hearing process

2 Liu et al 2001

et al 1992

6 4 4 4 9 8 6 6 6 13 5 11 5

62 60 56 60 60 62 58 60 62 58 58 60 58

(5rsquorarr

3rsquo)

llite-

14 6 7 6 9 5 9 6 13 13 4 6 4 14 6

164 96

227 94

60 58 58 58 60 60 58 62 58 58 62 56 58 58 62

(5rsquorarr

3rsquo)

5 4 6 13 7 8 5 3 10 11 8 8 8 10 11

56 60 56 62 62 62 60 60 58 58 56 58 58 62 60

(5rsquorarr

3rsquo)

362deg

2deg13

MS (bp) Location of

CLDN14_MS2 NW_876295 31 3379533796 33950 distal

COCH_MS2 NW_876327 8 1321513232 13290 distal

DFNA5_MS2 NW_876258 14 4116941237 41250 distal

EDN3_MS1 NW_876277 24 4701347032 47057 distal

GJA1_MS1 NW_876269 1 6399463996 64150 distal

GJA1_MS2 NW_876269 1 6399463996 64160 distal

GJB6_MS2 NW_876278 25 2090420906 20953 distal

Table 3 (continued)

MS (bp) Location of

MYH9_MS2 NW_876251 10 3113531193 31244 distal

MYH9_MS3 FH2293 10 3113531193 31696 distal

PAX3_MS2 NW_876304 37 3134831445 31481 distal

POU4F3_MS4 G2C02466 2 4361043612 - -

SLC26A4_MS2 NW_876265 18 1586715927 15960 distal

hO () 703 122 370 898

hE () 532 151 239 815

PIC () 667 130 336 889

No of alleles per

microsatellite

Number of marker

loci PIC ()

3 1 575

4 6 483

5 5 574

6 11 652

7 2 716

8 5 671

9 3 717

10 2 800

11 3 776

13 4 803

14 2 871

CLDN14_MS1 31q15 134 009 086 002

CLDN14_MS2 31q15 168 005 105 001

CLDN14_MS3 31q15 108 014 049 007

COL11A2_MS1 12q11-q12 166 005 085 002

COL11A2_MS3 12q11-q12 167 005 078 003

GJA1_MS1 1q24-q25 151 007 118 001

GJA1_MS2 1q24-q25 151 007 118 001

MITF_MS2 20q13 101 02 080 003

MITF_MS3 20q13 121 011 104 001

MYH9_MS2 10q232 080 02 018 02

MYH9_MS3 10q232 175 004 097 002

SOX10_MS2 10q21-q23 146 007 110 001

Gene-associated

marker

family members

CLDN14_MS1 51 40 278 0003 112 0011

CLDN14_MS2 383 000007 170 0003

CLDN14_MS3 281 0002 113 0011

MYH9_MS2 32 21 081 02 023 02

MYH9_MS3

(=FH2293) 156 006 058 005

GJA1_MS1 13 46 295 0002 052 006

Chapter 4

in Dalmatian dogs

linkage analyses

gene (Table 3)

SNP marker analysis

respectively

Dalmatians

Gene symbol

al (2003)

gene 1

et al (2002)

Gene symbol Gene

location on HSA1

Acc No 3 human mRNA

Gene location

on CFA2

Acc No 3 canine

genomic sequence

ESPN 1 NM_031475 5 NC_006587 XM_546751

MYH14 19 NM_024729 1 NW_876270 -

MYO3A 10 NM_017433 2 NC_006584 XM_544234

PRES 7 NM_206883 18 NC_006600 XM_540393

TMC1 9 NM_138691 1 NC_006583 XM_541284

TMIE 3 NM_147196 20 NC_006602 XM_846596

USH1C 11 NM_153676 21 NC_006603 XM_860072

WHRN 9 NM_015404 11 NC_006593 XM_850321

Bp3 AT4 PIC ()

ESPN_SNP1

565 60 9 10

ESPN_SNP2

565 60 27 41

ESPN_SNP3

565 60 23 32

ESPN_SNP4

565 60 23 32

ESPN_SNP5

565 60 28 42

MYH14_SNP1

670 60 35 42

MYH14_SNP2

640 60 34 39

Table 3 (continued)

Bp3 AT4 PIC ()

MYO3A_SNP1

650 60 37 77

MYO3A_SNP2

650 60 37 70

MYO3A_SNP3

650 60 25 36

PRES_SNP1

560 60 37 64

PRES_SNP2

560 60 37 66

TMC1_SNP1

610 60 57 37

TMC1_SNP2

610 60 47 29

TMC1_SNP3

650 60 48 30

TMIE_SNP1

625 60 59 36

Table 3 (continued)

Bp3 AT4 PIC()

USH1C_SNP2

CTCCCGGTCTGTCAGGAAC

560 60 37 35

USH1C_SNP4

CTCCCGGTCTGTCAGGAAC

560 60 37 37

WHRN_SNP1

600 60 25 37

WHRN_SNP2

600 59 24 33

WHRN_SNP3

600 60 34 55

TMC1_MS1

220-220 60 76 57

274-302 58 57 66

Allele frequencies

ESPN_SNP2 2 3 4 AgtG 074026 15160

ESPN_SNP3 2 3 TgtC 068032 07120

ESPN_SNP4 2 3 GgtA 068032 07120

ESPN_SNP5 2 3 4 CgtT 074026 15160

MYH14_SNP1 2 3 4 GgtT 058041 51610

MYH14_SNP2 2 3 4 CgtT 058041 51610

MYO3A_SNP1 1 2 GgtT 062038 5111

MYO3A_SNP2 1 2 CgtG 062038 5111

MYO3A_SNP3 1 2 4 TgtA 076024 15140

PRES_SNP1 1 2 3 4 AgtG 058042 10254

PRES_SNP2 1 2 3 4 TgtA 058042 10254

TMC1_SNP1 1 2 3 4 AgtT 056044 11226

TMC1_SNP2 1 2 3 4 AgtG 076024 20190

TMC1_SNP3 1 2 3 4 AgtG 074026 19200

TMIE_SNP1 1 2 3 4 AgtT 058042 12216

USH1C_SNP2 1 3 4 AgtC 053047 9147

USH1C_SNP4 1 3 4 AgtC 053047 9147

ESPN ESPN_SNP1 014 04 002 04

ESPN_SNP2 014 04 002 04

ESPN_SNP3 014 04 002 04

ESPN_SNP4 014 04 002 04

ESPN_SNP5 014 04 002 04

MYH14 MYH14_SNP1 -089 08 -019 08

MYH14_SNP1 -089 08 -019 08

MYO3A MYO3A_SNP1 -049 07 -011 08

MYO3A_SNP2 -049 07 -011 08

MYO3A_SNP3 -049 07 -011 08

PRES PRES_SNP1 -094 08 -019 08

PRES_SNP2 -094 08 -019 08

TMC1 TMC1_SNP1 -034 06 -008 07

TMC1_SNP2 -034 06 -008 07

TMC1_SNP3 -034 06 -008 07

TMC1_MS1 -035 06 -008 07

TMIE TMIE_SNP1 013 04 003 03

FH2158 -056 07 -013 08

USH1C USH1C_SNP2 018 04 04 03

USH1C_SNP4 018 04 04 03

WHRN WHRN_SNP1 046 03 008 03

WHRN_SNP2 046 03 008 03

ith th

llite-

Chapter 5

Canine MYH9 gene 65

Canine MYH9 gene

Canine MYH9 gene 67

Canine MYH9 gene

Canine MYH9 gene 69

variation

AM086385)

Canine MYH9 gene

of 50 over 200 bp)

Canine MYH9 gene 71

Conclusions

Canine MYH9 gene

size (bp)

Canine MYH9 gene 73

Table 1 (continued)

size (bp)

Canine MYH9 gene

Intron phase

Intron size

gt30000 bp

4922 bp

13493 bp

803 bp

4077 bp

427 bp

738 bp

343 bp

749 bp

1041 bp

1877 bp

922 bp

1801 bp

2049 bp

1877 bp

343 bp

835 bp

1418 bp

851 bp

1428 bp

398 bp

488 bp

Canine MYH9 gene 75

Table 2 (continued)

Intron phase

Intron size

971 bp

1603 bp

719 bp

270 bp

480 bp

232 bp

204 bp

1083 bp

1298 bp

150 bp

303 bp

1173 bp

941 bp

224 bp

563 bp

739 bp

Canine MYH9 gene

- - CC

237 Fa

257 Fa

A T A A T C

tellit

Canine MYH9 gene 77

Canine MYH9 gene

Canine MYH9 gene 79

Canine MYH9 gene

Chapter 6

Abstract

Introduction

parents

Germany)

Linkage analysis

marker loci

Results

from 013 to 037

Discussion

two families

Acc No canine mRNA

37023714 NC_006592 XM_531765

38353835 NC_006592 XM_848614

38503857 NC_006592 XM_531770

39453945 NC_006592 XM_858065

42564272 NC_006592 XM_538446

44084413 NC_006592 XM_538451

46264637 NC_006592 XM_538458

48494852 NC_006592 XM_849433

description SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474535

intron

590 60

LOC474536

intron

590 60

LOC610953

intron

600 60

LOC481302

3rsquo-end

610 60

LOC610991

intron

AGGGCTTCCTGGGAAAAGT

600 60

LOC611007

intron

480 60

LOC474541

intron

575 60

Table 2 (continued)

symbol SNP

Location

(intron or

5rsquo3rsquo-end)

SNP motif

Bp3 AT4

LOC474542

SNP_10

intron

560 60

LOC481308

SNP_11

3rsquo-end

600 60

LOC474543

SNP_12

intron

LOC609217

SNP_16

5rsquo-end

580 60

LOC611115

SNP_18

3rsquo-end

590 60

LOC481325

SNP_20

intron

600 60

Table 2 (continued)

symbol SNP

Location

(intron

or 5rsquo3rsquo-

SNP motif

Bp3 AT4

LOC481330

SNP_22

intron

GAAAGGCCTGGGTTCAAAA

575 60

LOC611493

SNP_23

intron

590 60

LOC481337

SNP_24

intron

620 60

LOC611728

SNP_26

intron

620 60

SNP Fam1 Nucleotide

polymorphism

Allele

frequencies

Genotype

SNP_1 125 CgtG 042054 3135 036 055

SNP_2 124 AgtG 064039 91013 037 044

SNP_3 2345 AgtC 041049 2156 033 047

SNP_4 345 CgtT 065035 6140 035 067

SNP_5 34 CgtT 065035 490 028 042

SNP_6 345 CgtT 066034 6130 035 039

SNP_7 1345 AgtG 054046 6174 037 053

SNP_8 145 GgtT 075025 01010 029 045

SNP_9 1345 AgtG 052054 6145 037 047

SNP_10 1234 CgtT 057043 7173 037 068

SNP_11 14 GgtT 065035 5120 035 062

SNP_12 15 AgtG 028072 097 030 044

SNP_13 45 AgtT 077023 870 017 021

SNP_14 5 AgtG 036064 052 013 015

SNP_15 134 AgtG 034066 0157 034 059

SNP_16 145 CgtT 030070 3812 033 036

SNP_17 245 AgtG 058042 6113 030 032

SNP_18 123 CgtT 037063 1126 027 035

SNP_19 123 GgtT 045055 3115 030 032

SNP_20 2345 CgtT 032068 1129 029 039

SNP_21 1235 AgtG 063037 883 037 052

SNP_22 1234 AgtG 068032 10102 030 034

SNP_23 12345 AgtT 030070 21313 033 046

SNP_24 124 AgtG 047053 667 033 019

SNP_25 234 AgtG 053047 3132 035 053

values)

SNP_16 39453 262 0004 123 0009

SNP_17 39455 262 0004 123 0009

SNP_18 39840 261 0004 123 0009

SNP_19 39843 261 0004 123 0009

SNP_20 4260 255 0005 118 0010

SNP_21 4270 255 0005 117 0010

SNP_22 4405 317 00008 131 0007

Chapter 7

families

GJA1 gene

the GJA1 gene

Oldendorf Germany)

marker loci

GJA1 gene

(Chapter 3)

families

SNP motif

Product

size (bp)

Annealing

temperatur

GJA1_SNP1+2

TGGCTTGATTCCCTGACTC

650 58

GJA1_SNP3

600 58

Microsatellite

Product

size (bp)

Annealing

temperatur

GJA1_MS1

134 60

GJA1_MS2

190 60

Chapter 8

in Chapter 3

Introduction

deafness (DFNB29)

markers (Chapter 3)

Oldendorf Germany)

CLDN14_MS2

indicating linkage

markers with CCSD

Location

(intron or

5rsquo3rsquo-UTR)

SNP motif

Bp3 AT4

CLDN14_SNP1

intron

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP2

GACCATATGTTTGTGGCC

CGTCAGGATGTTGGTGCC

580 60

CLDN14_SNP3

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

CLDN14_SNP4

3rsquo-UTR

CTGCCTTCAAGGACAACC

GATGAGTATCAGCCCAGC

550 60

610 60

585 60

580 60

680 60

Chapter 9

General discussion

developed SNPs

General discussion

is needed

CCSD phenotype

General discussion

markers

General discussion

phenotype

Chapter 10

Summary

Summary 127

Summary

Dalmatian dogs

Summary

CCSD anymore

Chapter 11

Katharina Mieskes

Houmlrverlust

ausgewertet

Kopplungsanalyse

entwickelt

Elterntieren

Kopplungsanalyse

beteiligt sind

ermittelt

Mutatiosanalyse

erklaumlrt werden

ausschlieszligen

verwendet

genotypisiert

Kopplungsanalyse

festgestellt werden

Chapter 12

References

References 147

References

3097-101

Genet 721315-1322

References

J Med 346243-249

74770-776

References 149

J Mol Biol 196261-282

2316-8

References

dogs Vet J 166164-169

References 151

Neurootol 2139-154

References

438803-819

References 153

26103-105

References

Genet 69635-640

Sci 63016-31

References 155

9109-119

41591-595

References

References 157

8251-255

References

Nat Genet 20194-197

Genet 1960-62

Genet 2651-55

References 159

Cell 104165-172

References

Chapter 13

Appendix

5acute -gt

3acute

5acute -

gt 3acute

8 4 7 11

3 6 8 7 2 2 3 4 2 3 5 8 12

7 8 7 14

9 7 3 3 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10

5acute -gt

3acute

5acute -

gt 3acute

3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11

5 6 2 4 7 4 8

b) c A

e obse

Code no 1 2 3 4

Centrifuges

BioRad Muumlnchen

Others

DNA purification

Cloning

Isolation of DNA

Sequencing

Germany)

Size standards

1 g APS

10 ml H2O

H2O ad 50 ml

dNTP solution

1600 microl H2O

Gel solution

95 microl TEMED

EDTA pH 8 100 mM

Ficoll 400 20 (wv)

20 ml formamide

TBE-buffer (1x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [12114 M]

744 g EDTA [37224 M]

H2O ad 1000 ml

425 g urea [6006 M]

250 ml H2O

H2O ad 850 ml

Consumables

httpwwwensemblorg

Acknowledgements

thesis

Contents

1 Introduction

10 Summary

12 References

13 Appendix

Documents

Molecular genetic analysis of canine congenital