Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs

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Aus dem Institut für Tierzucht und Vererbungsforschung der Tierärztlichen Hochschule Hannover

Molecular genetic analysis of

canine congenital sensorineural deafness in Dalmatian dogs

INAUGURAL-DISSERTATION zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN

(Dr. med. vet.)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von Katharina Mieskes

aus Göttingen Hannover 2006

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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 Förderung Kynologischer Forschung (GKF) e.V., Bonn, Germany.

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To my family

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

1. Gene

2. Journal of Heredity 3. Animal Genetics

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Chapter 1 Introduction

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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.

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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 10q23.2 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.

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Chapter 2 A comparative overview of the molecular genetics of non-syndromic deafness

in dogs and humans

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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. Reissner´s 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 Reissner´s 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

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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 and/or 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

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Non-syndromic deafness in dogs and humans 9 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, 2003).

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:

http://webhost.ua.ac.be/hhh/).

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

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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 16.5 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.

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 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

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Non-syndromic deafness in dogs and humans 11 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 genes in carriers is urgently needed.

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 (Drögemüller 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 2.5 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

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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 (e.g. single nucleotide polymorphisms (SNPs), microsatellites) for high resolution mapping of candidate regions, and to examine each exon and exon/intron boundary for positional candidates. Availability of the second version of the dog genome assembly (build 2.1) of the NCBI database shortcut this effort and increase the investigators efficency.

The current RH map with 3,200 markers provides a good estimateof the order and physical spacing (i.e., in base pairs) of markersalong canine chromosomes (Guyon et al. 2003) and was recently complemented by the construction of a 4,249-marker integratedcanine genome RH map that consists of 900 genes, 1,589 microsatellites, and 1,760 BAC end markers (Breen et al. 2004), all included and available in the NCBI database. The second version 1 of the NCBI's 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 2.1 of the NCBI's 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 0.88 percent higher than the average identity of canine and mouse mRNA with 0.84 percent. Canine sequences that correspond to the human

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Non-syndromic deafness in dogs and humans 13 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.

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

Inheritance Gene Gene product Type of molecule Locus name^a ACTG1 γ-Actin cytoskeletal protein DFNA20,

DFNA26 COCH Cochlin extracellular matrix component DFNA9 COL11A2 Collagen XI

(α2-chain) extracellular matrix component DFNA13 CRYM µ-Cristallin thyroid hormone-binding protein DFNA^b 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, DFNA^b 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 I−–Cl− 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 DFNA22/DFNB37 MYO7A Myosin VIIA motor protein DFNB2/DFNA11 TECTA α-Tectorin extracellular matrix component DFNA8,

DFNA12/DFNB21 Autosomal

dominant and autosomal recessive

TMC1 TMC1 transmembrane channel-like

protein DFNB7,

DFNB11/DFNA36 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

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Non-syndromic deafness in dogs and humans 15 Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary defects in hair cells, non-sensory cells and the tectorial membrane or unknown cell type

Primary defect Gene

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 EDNRB endothelin

receptor type B receptor protein WS type IV⁴

MITF

microphthalmia- associated transcription factor

transcription factor WS type II²

PAX3 paired box 3 DNA-binding protein WS type I¹and III² 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)

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Length of contig (bp) 77990652 38210901 70772986 72515492 59897527 45337677 60938239 9498326 69610260 18595814 64211953

Score of contig 614 938 792 567 300 715 323 535 1208 1634 923

E-value of contig 4 E-172 0 0 2 E-158 1 e-78 0 2 e-85 7 E-149 0 0 0

Acc. No.of WGS contig* unknown NW_876311 NW_876295 NW_876327 NW_876254 NW_876321 NW_876292 NW_876258 NW_876315 NW_876269 NW_876278 NW_876259

Mb from..to* unknown 25.41..25.78 33.79..33.79 13.21..13.23 5.63..5.65 27.21..27.23 39.33..39.43 41.16..41.23 63.28..63.31 29.28..29.55 20.93..20.94 10.18..10.19

Gene location on CFA* unknown 4 31 8 12 6 2 14 5 1 25 15

Canine gene aliases* none none LOC487751 LOC490640 LOC481734 LOC479818 none LOC611223 LOC489631 EYA4 GJB2 LOC482486

Acc. No. human mRNA NM_001614 NM_022124 NM_144492 NM_004086 NM_080680 NM_001888 NM_005219 NM_004403 NM_031475 NM_172105 NM_004004 NM_024009

Gene location on HSA 17 10 21 14 6 16 5 7 1 6 13 1

Table 4 Localisation of human non-syndromic hereditary deafness genes in the canine genome, the canine gene localisation in megabases (Mb), the accession number (Acc.No.) of the whole genome shotgun (WGS) contig, containing the human homologous gene and the corresponding E-value, score and length of the contig Human deafness gene ACTG1 CDH23 CLDN14 COCH COL11A2 CRYM DIAPH1 DFNA5 ESPN EYA4 GJB2 GJB3 *derived from the NCBI's canine genome annotation version 2.1

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Length of contig 64211953 53004996 52942087 26073285 16545469 12499463 72515492 51024781 59897527 51591990 12847264 75215785

Score of contig 525 521 973 2256 348 567 1236 404 337 383 604 1715

E-value of contig 3 e-146 1 e-144 0 0 9 e-93 2 e-158 0 3 e-109 3 e-89 7 e-103 2 e-169 0

Acc. No.of WGS contig* unknown NW_876259 NW_876270 NW_876251 NW_876313 NW_876250 NW_876290 NW_876254 NW_876273 NW_876321 NW_876263 NW_876283 NW_879563

Mb from..to* unknown 5.21..5.23 109.24.. 109.35 31.13..31.19 44.36..44.41 4.15..4.17 10.34..10.56 40.41..40.50 24.54..24.60 26.13..26.19 23.50..23.59 37.14..37.69 67.48..67.48

Gene location on CFA* 25 15 1 10 5 10 2 12 21 6 17 26 X

Canine gene aliases* none LOC482451 none LOC481280 LOC479522 LOC474410 LOC487106 LOC481884 LOC485174 LOC608655 LOC607961 none LOC491988

Acc. No. human mRNA NM_006783 NM_004700 NM_024729 NM_002473 NM_016239 NM_005379 NM_017433 XM_376516 NM_000260 NM_144672 NM_194248 NM_033056 NM_000307

Gene location on HSA 13 1 19 22 17 12 10 6 11 16 2 10 X

Table 4 (continued) Human deafness gene GJB6 KCNQ4 MYH14 MYH9 MYO15 MYO1A MYO3A MYO6 MYO7A OTOA OTOF PCDH15 POU3F4 *derived from the NCBI's canine genome annotation version 2.1

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Length of contig 45337677 25320482 25320482 40206070 30029677 29689717 53004996 33097591 38210901 51024781 11048438 65355756

Score of contig 1404 283 283 1683 883 529 354 216 198 354 2238 689

E-value of contig 0 6 e-73 2 E-73 0 0 5 e-147 2 e-94 4 e-53 9 e-48 2 E-94 0 0

Acc. No.of WGS contig* NW_876292 NW_876265 NW_876265 NW_876294 NW_876312 NW_876255 NW_876270 NW_876272 NW_876295 NW_876273 NW_876256 NW_876253

Mb from..to* 43.61..43.61 15.86..15.92 19.79..19.82 13.43..13.45 15.88..15.95 6.22..6.37 88.08..88.22 45.05..45.05 39.03..39.05 43.24..43.28 41.49..41.51 71.64..71.72

Gene location on CFA* 2 18 18 30 5 13 1 20 31 21 13 11

Canine gene aliases* LOC487200 LOC483263 LOC483274 LOC478278 LOC489357 LOC481985 LOC484168 LOC609350 LOC610987 LOC610850 LOC482113 LOC612588

Acc. No. human mRNA NM_002700 NM_000441 NM_206883 NM_153700 NM_005422 NM_024915 NM_138691 NM_147196 NM_024022 NM_153676 NM_006005 NM_015404

Gene location on HSA 5 7 7 15 11 8 9 3 21 11 4 9

Table 4 (continued) Human deafness gene POU4F3 SLC26A4 SLC26A5 STRC TECTA TFCP2L3 TMC1 TMIE TMPRSS3 USH1C WFS1 WHRN *derived from the NCBI's canine genome annotation version 2.1

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Length of contig (bp) 47763139 55611003 25182130 30915115 52942087

Score of contig 262 721 2927 967 1179

E-value of contig 8e-67 0 0 0 0

Acc. No.of WGS contig* NW_876277 NW_876274 NW_876271 NW_876304 NW_87625 1

Mb from..to* 47.01..47.03 34.36..34.38 24.85..24.88 31.34..31.44 29.75..29.76

Gene location on CFA* 24 22 20 37 10

Canine gene aliases* EDN3 EDNRB MITF PAX3 LOC481258

Acc. No. human mRNA NM_207032 NM_000115 NM_198159 NM_181457 NM_006941

Gene location on HSA 20 13 3 2 22

Table 5 Localisation of genes involved in the human Waardenburg syndrome (WS) in the canine genome, the canine gene localisation in megabases (Mb), the accession number (Acc.No.) of the whole genome shotgun (WGS) contig, containing the human homologous gene and the corresponding E-value, score and length of the contig Human deafness gene EDN3 EDNRB MITF PAX3 SOX10 *derived from the NCBI's canine genome annotation version 2.1

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Table 6 Canine candidate genes for CCSD with their accession number (Acc.No.) 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 88.00% 86.90%

CLDN14 NC_006613 XM_544876 714 88.80% 84.60%

COCH NC_006590 XM_547762 3050 87.70% 81.70%

COL11A2 NC_006594 XM_857285 5202 89.70% 87.40%

CRYM NC_006588 XM_845361 1279 87.20% 78.90%

DIAPH1 none¹ none unknown 86.30% unknown DFNA5 NC_006596 XM_848863 1512 83.20% 76.80%

ESPN NC_006587 XM_546751 2565 89.30% 82.70%

EYA4 NC_006583 XM_541108 3969 92.40% 81.20%

GJB2 NC_006607 XM_543177 2208 78.60% 67.20%

GJB3 NC_006597 XM_539603 1305 85.50% 80.90%

GJB6 none none unknown unknown unknown

KCNQ4 NC_006597 XM_539568 1836 93.50% 87.60%

MYH14 none¹ none unknown 87.70% 82.20%

MYH9 NC_006592 XM_538401 6201 91.30% 89.40%

MYO15 NC_006587 XM_536660 10128 85.00% 79.60%

MYO1A NC_006592 XM_531642 3388 86.00% 83.50%

MYO3A NC_006584 XM_544234 5589 85.10% 89.10%

MYO6 NC_006594 XM_862465 3983 92.50% 86.10%

MYO7A NC_006603 XM_542292 6519 91.90% 88.80%

OTOA NC_006588 XM_845746 3558 87.40% 79.90%

OTOF NC_006599 XM_844665 5994 90.20% 87.80%

PCDH15 none¹ none unknown 82.10% 73.90%

POU3F4 NC_006621 XM_549108 1086 91.90% 90.60%

POU4F3 NC_006584 XM_544328 1017 93.80% 91.60%

SLC26A4 NC_006600 XM_540382 2382 79.70% 82.40%

SLC26A5 NC_006600 XM_540393 2235 92.50% 87.40%

STRC NC_006612 XM_535452 5301 89.80% 85.20%

TECTA NC_006587 XM_546475 7136 91.10% 85.10%

TFCP2L3 NC_006595 XM_539106 2127 90.30% 87.60%

TMC1 NC_006583 XM_541284 2580 88.60% 87.40%

TMIE NC_006602 XM_846596 396 91.10% 89.10%

TMPRSS3 NC_006613 XM_848589 1542 85.00% 83.40%

USH1C NC_006603 XM_860072 1730 90.10% 86.70%

WFS1 NC_006595 XM_539234 2667 85.10% 84.20%

WHRN NC_006593 XM_850321 2817 84.40% 81.10%

EDN3 NC_006606 NM_001002942 1976 74.7% 71.6%

EDNRB NC_006604 NM_001010943 1329 88.9% 82.7%

MITF NC_006602 XM_850501 1590 93.4% 86.2%

PAX3 NC_006619 XM_545664 1474 86.9% 86.1%

SOX10 NC_006592 XM_538379 1987 92.6% 90.0%

* derived from the NCBI's canine genome annotation version 2.1

1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs

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Non-syndromic deafness in dogs and humans 21 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

Number of gene- associated

SNPs

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

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Chapter 3 Linkage analysis of gene-associated microsatellite markers with

congenital sensorineural deafness

in Dalmatian dogs

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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 16.5- 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

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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 (Drögemüller 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

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Linkage analysis of gene-associated microsatellites 27 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 16.8 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 28.5%.

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 http://www-recomgen.univ-rennes1.fr/doggy.html.

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 (67.4%) of the 43 markers in the set was represented by dinucleotide repeats; 20.9% were tetranucleotide repeats, 4.7% trinucleotide repeats and 2.3%

pentanucleotide repeats. In addition, one marker (2.3%) was a compound di- tetranucleotide and another one (2.3%) was a compound tetra-pentanucleotide repeat. The average number of alleles was 3.5 with a minimum of 2 and a maximum of 8 different alleles per marker.

PCR reactions for microsatellites were carried out in 12 µl reaction mixtures containing 2 µl genomic DNA, 1.2 µl 10x PCR buffer (Qbiogene, Heidelberg, Germany), 0.24 µl dimethyl sulfoxide (DMSO), 0.2 µl dNTPs (100 µM), 0.1 µl Taq Polymerase (5U/µl) (Qbiogene), 0.6 µl (10 µM) 5’-IRD700 or IRD800 labelled forward primer, 0.6 µl (10 µM) unlabelled reverse primer. The PCR reactions were carried out in MJ Research thermocyclers with the following program: 4 min at 94 °C, followed by 32 cycles of 30 sec at 94°C, 30 sec at maximum annealing temperature and a final extension of 45 sec at 72°C. PCR-products were diluted with formamide loading buffer in ratios from 1:10 to 1:40 and then size-fractionated by gel electrophoresis on

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automated LI-COR 4200/4300 sequencers (LI-COR inc., Lincoln, NE, USA) using denaturing 4% and 6% polyacrylamide gels (Rotiphorese®Gel 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 2.1) of the NCBI GenBank by BLAST (Basic Local Alignment Search Tool) search (http://www.ncbi.nlm.nih.gov/BLAST/), using the human reference mRNA sequence (Table 3).

Linkage analysis

Multipoint linkage and haplotype analyses were performed using the MERLIN software, version 0.10.2 (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 SAS/Genetics (Statistical Analysis System, Version 9.1.3, 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 >1, 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

Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs