Molecular genetic analysis of quantitative trait loci (QTL) for osteochondrosis in Hanoverian warmblood horses

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Aus dem Institut für Tierzucht und Vererbungsforschung der Tierärztlichen Hochschule Hannover

Molecular genetic analysis of

quantitative trait loci (QTL) for osteochondrosis in Hanoverian warmblood horses

INAUGURAL-DISSERTATION zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN

(Dr. med. vet.)

durch die Tierärztliche Hochschule Hannover

Vorgelegt von

Claudia Dierks geb. Böneker aus Hildesheim

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.-J. Hedrich

Oral examination: 24.05.2006

This work was supported by grants from the German Research Council, DFG, Bonn, Germany (DI 333/12-1).

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To my family

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Parts of this work have been accepted for publication or have been published in the following journals:

1. Animal Genetics

2. Cytogenetic and Genome Research

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Table of contents

Chapter 1 Introduction

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Introduction

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Introduction

Osteochondrosis (OC) is a developmental orthopaedic disorder, which is found in growing animals of many domestic species. Abnormale chondrocyte differentiation and maturation is believed to lead to altered enchondral ossification of the joints. This focal failure in the growing cartilage causes further damage and secondary lesions of the cartilage in the joints like subchondral fractures, subchondral cysts, wear lines, chondromalacia, cartilage flaps and joint mice or free joint bodies (chips).

Osteochondral fragments are characteristics of osteochondrosis dissecans (OCD).

Affected articulations in horses are the fetlock, hock, carpal, stifle, elbow, hip and vertebral joints. These lesions can further develop into chronic degenerative joint diseases such as osteoarthrosis or arthropathy in the cervical spine, which can lead to vertebral stenosis and wobbler syndrome. The prevalence of OC in Warmblood and trotter horses ranges between 10 and 79.5%. The pathogenesis of OC remains still unknown. The disease appears to be multifactorial in origin, including the factors skeletal growth rates, nutrition, endocrinological factors and biomechanical trauma.

Furthermore, hereditary dispositions play an important role in the aetiology of the osteochondrosis syndrome, but no responsible genes have as yet been identified.

The objective of the present study is to identify the genomic regions harbouring the gene loci responsible for OC. In order to achieve this goal, a whole genome scan was performed and after that, for the genomic regions significantly linked to OC, a comparative human-equine map with high resolution was constructed. Using this comparative map and existing comparative maps in horses, single nucleotide polymorphisms (SNPs) were developed for fine mapping the identified OC regions in horses. The complete sequence of a positional and functional candidate gene was analyzed in order to develop single nucleotide polymorphisms. Finally, the development of genetic tests based on gene-associated markers is shown.

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Overview of chapter contents

Chapter 2 reviews the literature for OC in Hanoverian warmblood horses and other horse breeds, including pathogenesis, clinical signs, prevalences and heritability estimates. Candidate genes for osteoarthritis in man are listed, and the advantages of comparative genomics and different molecular genetic approaches are discussed.

Chapter 3 contains the whole genome scan performed on 14 paternal half-sib families and the linkage analysis to determine the genomic regions responsible for OC in Hanoverian warmblood horses.

In Chapter 4 and 5 the mapping of two positional candidate genes for OC are described.

Chapter 6 contains the molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene localized on equine chromosome 2.

In Chapter 7 a high resolution human-equine comparative map for a large genomic region on horse chromosome 4 harbouring a quantitative trait locus for osteochondrosis was established.

Chapter 8 and 9 show the development of new SNP markers and the development of two genetic tests for OC-genes from two different genomic regions.

Chapter 10 provides a general discussion and conclusions referring to Chapters 1-9.

Chapter 11 is a concise English summary of this thesis, while Chapter 12 is an expanded, detailed German summary which takes into consideration the overall research context.

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Chapter 2 Osteochondrosis (OC) in horses: a molecular genetic approach

Claudia Dierks and Ottmar Distl

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Germany

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Osteochondrosis in horses: a molecular genetic approach

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Osteochondrosis (OC) in horses: a molecular genetic approach

Abstract

Osteochondrosis (OC) is a developmental orthopaedic disorder frequently observed in young horses. Signs of osteochondrosis are lesions of the cartilage in the joints like subchondral fractures, subchondral cysts, wear lines, chondromalacia, cartilage flaps and joint mice or free joint bodies (chips). The age for permanent OC in individual horses is difficult to define. However, signs of OC in foals at an age of between four and nine months appeared as well suited genetic indicators for OC in two-year old horses. Hereditary factors play an important part in the pathogenesis of OC. An optimized microsatellite marker set for complete genome scans in horses was developed including 155 highly polymorphic markers equally distributed at a distance of about 20 cM. BLAST (basic local alignment search tool) searches against the human genome were used to identify equine expressed sequence tags (ESTs) corresponding to human candidate genes involved in osteoarthritis. These ESTs can be considered as candidate genes for equine OC and can be used for the development of new informative single nucleotide polymorphisms to refine putative quantitative trait loci (QTL) for OC or to detect genes responsible for OC. The advances in horse genomics and comparative human-equine mapping enable now molecular genetic approaches to identify the genetic components involved in OC.

Introduction

Osteochondrosis (OC) is a developmental orthopaedic disorder, which is found in growing animals of many domestic species (Olsson 1978). The disease belongs to the complex of developmental orthopaedic diseases (DOD), which includes all skeletal problems associated with growth and development in foals. It is defined as a disturbance in the process of endochondral ossification of growing cartilage of the growth plates and/or the articular/epiphyseal complex (Van de Lest et al. 1999).

Osteochondrosis can be associated with reduced performance, which may ultimately

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lead to loss of the animal according to the severity of clinical signs (e.g. joint swelling, pain, recurrent lameness) (Stock and Distl 2006). The prevalence of OC in warmblood and trotter horses ranges between 10 and 79.5% (Stock et al. 2005a, Willms et al. 1999, Winter et al. 1996, Philipsson et al. 1993). The disease appears to be multifactorial in origin, including the factors skeletal growth rates, nutrition, endocrinological factors, biomechanical trauma and hereditary dispositions (Jeffcott 1991). The objective of this paper is to review pathogenesis, age of onset and heritability estimates of osteochondrosis. Furthermore, molecular genetic approaches for identifying the responsive genes are discussed. Whole genome scans and candidate gene approaches might here be useful for further research work.

Pathogenesis and age of onset

Osteochondrosis (OC) develops due to a defect in the process of endochondral ossification in the growing horse (Jeffcott 1996). Endochondral ossification is an ordered process, including cartilage proliferation, maturation and calcification followed by osseous replacement, and is responsible for longitudinal bone growth and enlargement of the epiphysis. In osteochondrosis, cartilage cells appear to proliferate normally, but maturation and differentiation are abnormal (Trotter and McIlwraith 1981). The altered process of endochondral ossification leads to retention and thickening of cartilage, resulting in the development of a cartilage core, which is the primary lesion. This focal failure in the growing cartilage causes further damage and secondary lesions of the cartilage in the joints like subchondral fractures, subchondral cysts, wear lines, chondromalacia, cartilage flaps and joint mice or free joint bodies (chips) as the characteristics of osteochondrosis dissecans (OCD) (Jeffcott and Henson 1998, Trotter and McIlwraith 1981). Affected articulations are primarily fetlock, hock, carpal, stifle, elbow, hip and vertebral joints. Finally, these lesions can further develop into chronic degenerative joint diseases such as osteoarthrosis or arthropathy in the cervical spine, which can lead to vertebral stenosis and wobbler syndrome (Jeffcott 1996).

OC is usually detectable in animals less than 6 months old by radiographic examination (Hoppe 1984, Grøndahl 1991, Carlsten et al. 1993, Dik et al. 1999, Kroll et al. 2001). As growing cartilage is a tissue undergoing complex processes of

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proliferation and maturation especially during the first months of life, it is very important to distinguish between the time of the first detection of an osteochondrotic lesion and the time from which it is definitively permanent. Most of abnormalities detected very early disappear during the first months of life. The point of no return can be defined as the time at which no resolution of abnormal findings can be detected and when shifts from normal to abnormal findings become rare.

Several authors have reported on the age of onset of osteochondrotic lesions in different articulations of young horses. Stromberg (1979) mentioned a three-day-old Standardbred colt with a dissecting lesion. Yovich et al. (1986) described a six- month-old male Quarter horse foal with OCD lesions in the fetlock joints. Smallwood and Kelly (1991) found radiological signs of OCD in the fetlock joints of a ten-week- old Quarter Horse foal which was examined radiologically at regular intervals since its birth. The trotters in the study of Carlsten et al. (1993) were classified as having permanent hock or fetlock OC at the age of 12 months although all permanent lesions had already been visible earlier. Dik et al. (1999) concluded in a study about Dutch Warmblood foals that hock OC is permanent at the age of five months. In the stifle joints the lesions were considered to be permanent at the age of eight months.

Kroll et al. (2001) found that fetlock and hock OC became manifest by the age of four months. Arnan and Hertsch (2005) described in their study the development of osteochondrosis in four to nine months old foals and when these animals were two years old. They examined the prevalence in four to nine months old foals and when these animals were two years old in the fetlock joints at five locations: at sagittal ridge of third metacarpal bone, at palmar rim of proximal phalanx, proximal of proximal sesamoids, distal of proximal sesamoids and at plantar rim of proximal phalanx. No significant changes were found regarding the changes at sagittal ridge of third metacarpal bone. For the remaining four locations in the fetlock joints a distinct increase in osteochondrotic fragments was observed. In hock joints the osteochondrotic lesions in foals developed to osteochondrotic fragments in the two- years-old horses in many cases. In the majority of cases, osteochondrotic lesions in stifle joints in foals disappeared until the horses were two years old and some of these lesions developed to osteochondrotic fragments. A genetic analysis of the same data set used by Arnan and Hertsch (2005) was performed by Schober (2003).

In contrast to the phenotypic relationships reported by Arnan and Hertsch (2005),

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genetic correlations close to one were demonstrated among signs for osteochondrosis in four to nine months old Hanoverian warmblood foals and osteochondrosis dissecans in the same horses at an age of two years. The estimates were rg = 0.84 for fetlock joints and rg = 0.99 for hock joints (Schober 2003). The corresponding phenotypic correlations were rp = 0.66 (fetlock joints) and rp = 0.68 (hock joints) indicating a significant consistency of the radiological findings at the different age classes.

Clinical signs, prevalences and heritability estimates

The clinical signs of osteochondrosis (OC) are difficult to characterize in horses because of the wide range of lesions and sites involved (Jeffcott 1996). Most commonly affected are the fetlock, hock and stifle joints. Not all lesions of OC produce clinical signs and horses often exhibit very severe pathological changes without showing much pain or distress. The main signs in yearlings or older animals are stiffness of joints, positive responses to flexion tests and in some cases varying degrees of lameness. Thus, OC can be associated with reduced performance, which may ultimately lead to loss of the animal.

Additive genetic correlations between the numbers of annual entries and placings in tournament competitions (dressage, show-jumping and basic build up) and osseous fragments in fetlock and hock joints were analyzed by Stock et al. (2006) in warmblood riding horses. There were some indications for significantly negative, but few indications for significantly positive additive genetic correlations, so the authors concluded that breeding measures, that allow for orthopaedic health traits could therefore contribute to maximize the breeding progress in terms of sport performance.

The diagnosis of OC is usually based on the results of radiographic examinations.

Radiographic signs of OC are detected by examination of the outline of the bone contour, the density of the subchondral bone and the existence and size of visible fragments at the predilection sites (Dik et al. 1999). Radiographic findings indicative of OC are signs of irregular bone with variable radiopacity and changes of the regular bone contour such as irregular bone margins and flattened or concave outlining or osseous fragments visible as isolated radiodense areas. Radiographic appearances

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in the sense of osteochondrosis dissecans (OCD) are osseous fragments (joint mice) in the joint space with or without a defect in the underlying bone in the joint space (Carlsten et al. 1993; Kroll et al. 2001).

The predilection sites in hock joints are at intermediate ridge of the distal tibia, at lateral/medial trochlear ridge of the talus and at lateral/medial malleolus of the tibia (McIlwraith 1993). Additionally, Kroll et al. (2001) regarded the lateral trochlea of talus as a further predilection site for OC in warmblood horses. Philipsson et al. (1993) and Grøndahl and Dolvik (1993) only regarded bony fragments at intermediate ridge of distal tibia and/or at trochlea of talus as osteochondrosis. In the stifle joints, the predilection sites are at lateral/medial trochlear ridge of the femur, at trochlear groove of the femur and the patella. The intervertebral articular processes are most commonly affected in the vertebral joints (Van Weeren and Barnefeld 1999). In trotters signs of OC in the fetlock joints are often characterized as POF (palmar/plantar fragment(s) and/or bone defects at the site of attachment of the short sesamoidean ligaments to the proximal phalanx), UPE (ununited palmar/plantar eminence of the proximal phalanx, including intra- and extraarticular fragments) or DF (dorsal fragment(s) at the dorsoproximal rim of the proximal phalanx and/or fragments or defects at dorsal part of sagittal ridge of third metacarpal bone) (Carlsten et al. 1993). According to their findings Carlsten et al. (1993) regard fetlock POF and UPE as one entity. The affiliation of fetlock POF and UPE to the OC complex has been questioned by several authors (e.g. Dalin et al. 1993; Sandgren et al. 1993; Nixon and Pool 1995). After histological examination of osteochondral fragments from the proximoplantar/proximopalmar region of the proximal phalanx of 30 horses Nixon and Pool (1995) suggested that these fragments may be a result of fracture rather than a manifestation of OC. Dalin et al. (1993) assumed that fetlock POF are the result of outwardly rotated hindlimb axes and subsequent point loading in the medial fetlock area, and concluded that the origin of these lesions was traumatic. Sandgren et al. (1993) stated that the differences in body weights, body measurements and clinical signs between horses affected by tarsocrural OC and horses affected with fetlock POF or UPE in their study are indications of the different pathogeneses of these lesions.

Another question is whether fragments in the dorsal recessus of the fetlock joints belong to the OC complex. Grøndahl (1992) reported that opinions differ as to

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whether bony fragments located at the dorsoproximal end of the proximal phalanx are traumatic in origin or a manifestation of OC. Schube et al. (1991) found free joint bodies in seven fetlock joints, two of which were considered to be chip fractures of the phalangeal dorsal margins; the remaining five fragments obviously developed in the squamous ruptures of the hyperplastical chondroblastema. Kroll et al. (2001) defined OC in German warmblood horses in fetlock joints as radiolucency, irregular bone margin, new bone formation or osseous fragments at dorsal sagittal ridge.

Yovich et al. (1986) classified osteocartilaginous bodies associated with the dorsal sagittal ridge in young horses as a form of OC.

The prevalence of OC in warmblood and trotter horses ranged between 10 and 79.5% (Table 1). The previously published heritabilities estimated in threshold models for OC in warmblood horses ranged between h² = 0.10-0.34 (KWPN 1994, Pieramati et al. 2003, Willms et al. 1999, Stock et al. 2005b). The threshold model heritability estimates for trotters were in the similar range between h² = 0.17-0.52 (Schougaard et al. 1987, Grøndahl and Dolvik 1993, Philipsson et al. 1993). In a recent study, analysis of mode of inheritance of osteochondral lesions (osteochondrosis, OC) in pigs showed clear evidence for Mendelian segregation of a major gene affecting osteochondrosis (Kadarmideen and Janss 2005).

Whole genome scan to identify quantitative trait loci for osteochondrosis Many traits of economic interest in animals are of a quantitative genetic nature, which means that the influences of many genes combine to contribute to a particular phenotype. According to Geldermann et al. (1985) a quantitative trait locus (QTL) is a single gene locus, or a marked DNA region that contains the gene, with a measurable effect on the genetic variance of a trait. Such a QTL or so-called major gene should determine more than 10% of the phenotypic variance of the targeted trait. Two basic approaches or a combination of them are used in molecular biology to detect quantitative trait loci: the candidate gene approach (Rothschild et al. 1994), linkage analysis using molecular markers (Andersson et al. 1994), or a combined approach using microsatellites of the candidate genes and the genomic regions surrounding them. A whole genome scan can be used to detect markers significantly linked to the OC phenotype. This approach is based on highly informative

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microsatellites evenly spread over all chromosomes with an average distance of approximately 20 cM.

The equine genome is estimated to contain approximately 3000 megabases (Mb) of DNA distributed on 31 pairs of autosomes and the X and Y chromosomes (Murray and Bowling 2000). At present, 792 genes and 1525 microsatellite markers are registered at the INRA horsemap Database (http://dga.jouy.inra.fr/cgi- bin/lgbc/loci_micro.operl?BASE=horse). Out of these 1525 microsatellites, 1273 are assigned to an equine chromosome. At the NCBI nucleotide database, 6139 CoreNucleotide, 37,135 horse ESTs (expressed sequence tag) and 9,974 horse GSS (genome survey sequence) can be found (http://www.ncbi.nlm.nih.gov/entrez/query.

fcgi?db=nucleotide&cmd=search&term=equus+caballus). Gene mapping in horses has become increasingly important because gene maps will aid in the study of, and in the establishment of tests for inherited defects and quantitative traits of the horse (Swinburne et al. 2000). This development started eleven years ago with the publication of the first equine microsatellite markers by Ellegren et al. (1992).

Subsequently, Marklund et al. (1994) described initial linkage observations, and Breen et al. (1997) and Godard et al. (1997) reported physical position assignments for a limited number of markers.

The first genetic linkage map of Lindgren et al. (1998) contained 140 markers covering 679 cM with a mean marker distance of 12.6 cM. The linkage map created by Guerin et al. (1999) contained 161 markers which were assigned to 26 autosomes. Out of these 161 markers, 124 could be arranged in 29 linkage groups with an average interval between loci of 14.2 cM and a total map distance of 936.5 cM. The linkage map published by Swinburne et al. (2000) contained 359 equine markers that have been assigned to all 31 autosomes and the X chromosome. A total of 334 markers (93%) were significantly linked to at least one other marker, so that 42 linkage groups have been formed. In addition, the physical location of 85 markers was known, which allowed the anchoring of 37 linkage groups to the physical map.

The average distance between the markers was 10.5 cM and this map covered 2450 cM. The low-density linkage map published by Guerin et al. (2003) included 310 markers on all 31 autosomes arranged in 34 linkage groups. The map spanned 2262 cM with an average interval between the loci of 10.1 cM. The comprehensive male linkage map of Penedo et al. (2005) was constructed from a total of 766 markers

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distributed on the 31 equine autosomes. The map spanned 3740 cM with an average distance of 6.3 cM between the markers. Recently, Swinburne et al. (2006) constructed a sex-averaged genetic likage map consisting of 742 markers distributed over 31 autosomes and the X chromosome. This map spanned 2772 cM with an average distance of 3.7 cM between the markers.

Milenkovic et al. (2002) used fluorescence in situ hybridization (FISH) to map 136 genes in the horse. Raudsepp et al. (1999), Godard et al. (2000), Lear et al. (2001), Mariat et al. (2001), Lindgren et al. (2001), Böneker et al. (2005a, 2005b), Müller et al. (2005a, 2005b, 2005c, 2006), Musilova et al. (2005) and Perrocheau et al. (2005) mapped further genes to equine chromosomes using FISH. Zoo-FISH was carried out by Raudsepp et al. (1996), Chowdhary et al. (1998), Caetano et al. (1999a) and Yang et al. (2004), who identifed conserved chromosomal segments between the horse and human genome.

With the development of a horse-mouse somatic cell hybrid panel it was possible to assign large numbers of markers to horse chromosomes by synteny analysis (Caetano et al. 1999b, Shiue et al. 1999).

Kiguwa et al. (2000) established preliminary radiation hybrid (RH) maps for chromosomes ECA1 and ECA10, and these were followed by RH and comparative maps for equine chromosomes 11, 17, 22, X and Y (Chowdhary et al. 2002, Raudsepp et al. 2002, 2004a and 2004b, Gustafson-Seabury et al. 2005, Lee et al.

2004). Brinkmeyer-Langford et al. (2005) generated preliminary radiation hybrid (RH) maps for chromosome HSA19 in comparison to ECA7, 21 and 10. A first-generation whole-genome radiation hybrid map in the horse was published by Chowdhary et al.

(2003). That map was established using 92 horse x hamster hybrid cell lines and 730 equine markers. The 730 loci (258 type I and 472 type II) were clustered in 101 RH groups distributed over all equine autosomes and the X chromosome, on average every 19 cR. Comparison of the 447 equine genes (256 linearly ordered RH-mapped and additional 191 FISH-mapped) with the location of draft sequences of their human and mouse orthologs provides the most extensive horse-human and horse-mouse comparative map to date.

A medium-density map of the horse genome was constructed by Perrocheau et al.

(2006). Overall, 165 genes were assigned to the equine genomic map by RH and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse

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genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified.

Development of an optimized microsatellite marker set for complete genome scans in horses

An optimized microsatellite marker set for horses was developed containing 155 microsatellite markers. The microsatellite markers were chosen from the equine linkage and RH5,000 maps (Swinburne et al. 2000 and Chowdhary et al. 2003) and from the HORSEMAP database at the INRA Biotechnology Laboratories Home Page (http://dga.jouy.inra.fr/cgi-bin/lgbc/loci_micro.operl?BASE=horse). Information on all 155 microsatellite markers including the position on the respective chromosome, annealing temperature, primer sequences and allele size is given in the Supplemental table. The marker set was tested on a reference group consisting of 71 Hanoverian warmblood horses, including 36 (50.7%) females and 35 (49.3%) males.

The relative informativeness of the marker set was evaluated by means of the observed heterozygosity (HO) and the polymorphism information content (PIC). Only female animals were involved in the calculation for the X chromosome. The observed heterozygosity indicates the proportion of heterozygote individuals in regard to the number of genotyped individuals in a given population. The polymorphism information content is defined as the probability that the marker genotype of a given offspring will, in the absence of crossing-over, make it possible to deduce which of the two marker alleles of the affected parent the offspring received. The PIC value is thus the most suitable value for describing the usefulness of a marker for linkage analysis. The characteristics of the whole marker set including 155 microsatellite markers are given in Table 2. The characteristics established for each marker were the number of alleles, observed heterozygosity (HO) and PIC. Furthermore, the distances between the markers are given according to the linkage map of Swinburne et al. (2006). Some microsatellites were not mapped on this linkage map and for these markers the linkage map of Penedo et al. (2005) and the radiation hybrid map of Chowdhary et al. (2003) were used. Due to the fact that the marker set was developed before the maps of Penedo et al. (2005) and Swinburne et al. (2006) were

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available, the current marker set shows large gaps on ECA14 and ECA28 which can now be closed by adding new informative markers on these chromosomes. The estimated length of ECA14 increased to 152.8 cM (Swinburne et al. 2006) and 168.4 cM (Penedo et al. 2005) whereas only 46 cM were covered by markers in the previous linkage map reported by Swinburne et al. (2000). For ECA28 also a significant higher marker density led to higher estimates for the length. The estimated length of ECA28 was 74.9 cM in the map of Penedo et al. (2005) and 63.1 cM in the map of Swinburne et al. (2006) whereas previous maps included only very few markers on ECA28 (Swinburne et al. 2000) or only one linkage group of approximately 180 cR (Chowdhary et al. 2003).

Candidate genes for osteochondrosis

It has become apparent that there are extensive genetic homologies between the human and even distantly related species. Candidate genes in the regions flanking QTL may be chosen by means of comparative human-equine maps. Great progress has been made in the comparative gene map between humans and horse. Although, it is difficult to locate positional candidate genes for the QTL regions by using the existing equine-human comparative maps (Chowdhary et al. 2003, Milenkovic et al.

2002, Swinburne et al. 2006) because the syntenic human regions to some of the QTL cannot identified or resolved with the necessary accuracy. Positional candidate genes should code for hormones, enzymes, metabolic factors and/or their receptors involved in the complex of cartilage differentiation and maturation during enchondral ossification or in growth processes. Candidate genes may be also involved in osteoarthritis of other species.

Andersson-Eklund et al. (2000) identified three QTL for osteochondrosis on pig chromosomes SSC5, 13 and 15. Possible candidate genes derived from QTL positions might be pituitary specific transcription factor 1 (POU1F1), which codes for a transcriptional factor of growth hormone and genes coding for parathyroid hormone receptors (PTHR) for the chromosome-wide QTL on SSC13, and insulin-like growth factor I (IGF-I) and cartilage homeoprotein1 (CART1) for the genome-wide QTL on SSC5, respectively. These genes were selected on the one hand for their indicated role in the development of OC or cartilage growth and on the other hand due to their

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locations in the vicinity of an identified QTL or in the homologous region of the human genome.

Lee et al. (2003) also detected QTL associated with osteochondrosis related traits in pigs on SSC7 and SSC16 but none of these QTL exceeded the chromosome-wide suggestive level.

The Equine Articular Cartilage cDNA Library may be also helpfully to select candidate genes. At the moment 13,966 equine articular ESTs are available in the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=

search&DB=nucleotide).

Genes causing osteoarthritis that have already been identified in man can be used as candidate genes for the molecular genetic analysis of OC in horses. These genes can be taken from the Online Mendelian Inheritance in Man (OMIM) database (Table 3). This database is a catalog of human genes and genetic disorders developed for the World Wide Web by NCBI, the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=OMIM).

The corresponding equine ESTs to the human candidate genes were identified by BLAST (basic local alignment search tool) of all equine ESTs against the human genome Builds 35.1 (http://www.ncbi.nlm.nih.gov/genome/seq/HsBlast.html). The corresponding equine ESTs with significant hits (Score > 50 or E-value < 10^-5) are listed in table 4. Only those equine ESTs are displayed which were unique and not part of another EST of the same gene. Most of the ESTs matched to the 3’ end of the homologous human genes. For the following genes no corresponding equine ESTs have been identified: ADAM17, ATP7B, BFHD, CASR, CCAL1, CLCN7, COL10A1, CYP2C9, ESR1, FLNA, IL2RB, LEP, MATN3, MEFV, MMP13, OAP, OADIP, OPTA2, PHEX, PRDX5, TP53, TRAPPC2 and VDR. The COMP gene was mapped to equine chromosome 21q12–q14 by FISH and radiation hybrid mapping (Müller et al. 2005a).

One gene of the matrix metalloproteinase (MMP) family, MMP19 was mapped to ECA6 near to MAN1 by radiation hybrid mapping (Wittwer et al. 2005). The COL9A2 gene was characterized and mapped to ECA2p15-p16 by Dierks et al. (2006). For the following genes, mapping results in horses can be retrieved from the comparative cytogenetic and RH5,000 map of Chowdhary et al. (2003): COL1A1, COL1A2, COL3A1, COL9A1, COL10A1, CTSK, ESR1, MMP13 and PRG4.

Localisation of QTL for osteochondrosis needs the refinement of its position through

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single nucleotide polymorphisms (SNPs) as known equine mirosatellites are too sparse on the equine maps. Development of SNPs can then be preferentially performed using ESTs from these candidate genes associated with human osteoarthritis and further equine ESTs located in the QTL regions. Once a genomic QTL region for OC has been successfully narrowed down to 1-2 Mb, further positional candidate genes from the conserved chromosomal region in man can be identified for testing cosegregation with OC. If a cosegregation with OC was found, the causal mutations may be discovered by sequencing of at least the exons with their splice sites and the 5’ and 3’- untranslated regions of the candidate genes of a large sample with affected and non-affected horses from different families.

Differential cDNA (complementary deoxyribonucleic acid) expression analysis of the cartilage or synovial fluid of affected and non-affected horses by means of human high-density-cDNA-microarrays may help to elucidate the causal gene. Semevolos et al. (2005) determined changes in the expression of different genes in horses affected with osteochondrosis by using cartilage, harvested from OC affected joints and real- time quantitative polymerase chain reaction.

Conclusions

Osteochondrosis (OC) is widespread in many breeds of horses. The disease is important for horse keeping, breeding and sports. Hereditary dispositions play an important role in the aetiology of the osteochondrosis syndrome. Major advances in mapping the equine genome allow performing whole genome scans in order to detect quantitative trait loci for the OC phenotype. Equine ESTs corresponding to human positional candidate genes help for further refinement of the QTL region. As a prerequisite for whole genome scans in horses an optimized microsatellite marker set has been developed. The first steps to identify QTL for equine osteochondrosis can now be done.

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19 References

Andersson L, Haley CS, Ellegren H, Knott SA, Johansson M, Andersson K, Andersson-Eklund L, Edfors-LIlja I, Fredholm M, Hansson I, Hakansson J and Lundström K (1994) Genetic mapping of quantitative trait loci for growth and fatness in pigs. Science 263, 1771-1774

Andersson-Eklund L, Uhlhorn H, Lundeheim N, Dalin G, Andersson L (2000) Mapping quantitative trait loci for principal components of bone measurements

and osteochondrosis scores in a wild boar x large white intercross. Genet Res 75, 223-230

Arnan P, Hertsch B (2005) OCD des Fessel-, Sprung- und Kniegelenks im Vergleich vom Fohlen zum Zweijährigen. Pferdeheilkunde 21, 322-326

Böneker C, Kuiper H, Wöhlke A, Drögemüller C, Chowdhary BP, Distl O (2005a) Assignment of the COL16A1 gene to equine chromosome 2p15.1-p15.3 by FISH and confirmation by RH mapping. Anim Genet 36, 262-263

Böneker C, Müller D, Kuiper H, Drögemüller C, Chowdhary BP, Distl O (2005b) Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH mapping. Anim Genet 36, 444-445

Breen M, Lindgren G, Binns MM, Norman J, Irvin Z, Bell K, Sandberg K and Ellegren H (1997) Genetical and physical assignments of equine microsatellites - first integration of anchored markers in horse genome mapping. Mamm Genome 8, 267-273

Brinkmeyer-Langford C, Raudsepp T, Lee EJ, Goh G, Schaffer AA, Agarwala R, Wagner ML, Tozaki T, Skow LC, Womack JE, Mickelson JR, Chowdhary BP (2005) A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals. Mamm Genome 16, 631- 649

Caetano AR, Shiue YL, Lyons LA, O'Brien SJ, Laughlin TF, Bowling AT, Murray JD (1999a) A comparative gene map of the horse (Equus caballus). Genome Res 9, 1239-1249

Caetano AR, Lyons LA, Laughlin TF, O'Brien SJ, Murray JD, Bowling AT (1999b) Equine synteny mapping of comparative anchor tagged sequences (CATS) from human Chromosome 5. Mamm Genome 10, 1082-1084

(32)

Carlsten J, Sandgren B, Dalin G (1993) Development of osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints of Standardbred trotters. I. A radiological survey. Equine vet J, Suppl 16, 42-47 Chowdhary BP, Raudsepp T, Fronicke L, Scherthan H (1998) Emerging patterns of

comparative genome organization in some mammalian species as revealed by Zoo-FISH. Genome Res 8, 577-589

Chowdhary BP, Raudsepp T, Honeycutt D, Owens EK, Piumi F, Guerin G, Matise TC, Kata SR, Womack JE, Skow LC (2002) Construction of a 5,000(rad) whole-genome radiation hybrid panel in the horse and generation of a comprehensive and comparative map for ECA11. Mamm Genome 13, 89-94 Chowdhary BP, Raudsepp T, Kata SR, Goh G, Millon LV, Allan V, Piumi F, Guerin G,

Swinburne J, Binns M, Lear TL, Mickelson J, Murray J, Antczak DF, Womack JE, Skow LC (2003) The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes.

Genome Res 13, 742-751

Dalin G, Sandgren B and Carlsten J (1993) Plantar osteochondral fragments in the metatarsophalangeal joints in Standardbred trotters; result of osteochondrosis or trauma? Equine vet J, Suppl 16, 62-65

Dierks C, Drögemüller C, Kuiper H, Chowdhary BP, Distl O (2006) Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p15-p16. submitted

Dik KJ, Enzerink E, Van Weeren PR (1999) Radiographic development of osteochondral abnomalities, in the hock and stifle of Dutch Warmblood foals, from age 1 to 11 months. Equine vet J, Suppl 31, 9-15

Ellegren H, Johansson M, Sandberg K and Andersson L (1992) Cloning of highly polymorphic microsatellites in the horse. Anim Genet 23, 133-142

Geldermann H, Pieper U and Roth B (1985) Effects of marker chromosome sections on milk performances in cattle. Theor Appl Genet 70, 138-146

Godard S, Vaiman D, Oustry A, Nocart M, Bertaud M, Guzylack S, Meriaux JC, Cribiu EP, Guerin G (1997) Characterization, genetic and physical mapping analysis of 36 horse plasmid and cosmid-derived microsatellites. Mamm Genome 8, 745-750

(33)

21

Godard S, Vaiman A, Schibler L, Mariat D, Vaiman D, Cribiu EP, Guerin G (2000) Cytogenetic localization of 44 new coding sequences in the horse. Mamm Genome 11, 1093-1097

Grøndahl AM (1991) The incidence of osteochondrosis in the tibiotarsal joint of Norwegian Standardbred trotters. A radiographic study. J Equine vet Sci 11, 272-274

Grøndahl AM (1992) The incidence of bony fragments and osteochondrosis in the metacarpo- and metatarsophalangeal joints of standardbred trotters. J Equine vet Sci 12, 81-85

Grøndahl AM, Dolvik NI (1993) Heritability estimations of osteochondrosis in the tibiotarsal joint and of bony fragments in the palmar/plantar portion of the metacarpo- and metatarsophalangeal joints of horses. J Am Vet Med Assoc 203, 101-104

Guerin G, Bailey E, Bernoco D, Anderson I, Antczak DF, Bell K, Binns MM, Bowling AT, Brandon R, Cholewinski G, Cothran EG, Ellegren H, Forster M, Godard S, Horin P, Ketchum M, Lindgren G, McPartlan H, Meriaux JC, Mickelson JR, Millon LV, Murray J, Neau A, Roed K, Ziegle J, et al. (1999) Report of the International Equine Gene Mapping Workshop: male linkage map. Anim Genet 30, 341-354

Guerin G, Bailey E, Bernoco D, Anderson I, Antczak DF, Bell K, Biros I, Bjornstad G, Bowling AT, Brandon R, Caetano AR, Cholewinski G, Colling D, Eggleston M, Ellis N, Flynn J, Gralak B, Hasegawa T, Ketchum M, Lindgren G, Lyons LA, Millon LV, Mariat D, Murray J, Neau A, Roed K, Sandberg K, Skow LC, Tammen I, Tozaki T, Van Dyk E, Weiss B, Young A, Ziegle J. (2003) The second generation of the International Equine Gene Mapping Workshop half- sibling linkage map. Anim Genet 34, 161-168

Gustafson-Seabury A, Raudsepp T, Goh G, Kata SR, Wagner ML, Tozaki T, Mickelson JR, Womack JE, Skow LC, Chowdhary BP (2005) High-resolution RH map of horse chromosome 22 reveals a putative ancestral vertebrate chromosome. Genomics 85, 188-200

Hoppe F (1984) Radiological investigations of osteochondrosis dissecans in Standardbred trotters and Swedish Warmblood horses. Equine vet J 16, 425- 429

(34)

Jeffcott LB (1991) Osteochondosis in the horse - searching for the key to pathogenesis. Equine vet J 23, 331-338

Jeffcott LB (1996) Osteochondrosis – an international problem for the horse industry.

J Equine vet Sci 16, 32-37

Jeffcott LB, Henson FMD (1998) Studies on growth cartilage in the horse and their application to aetiopathogenesis of dyschondroplasia (osteochondrosis). The vet J 156, 177-192

Kadarmideen HN and Janss LL (2005) Evidence of a major gene from bayesian segregation analyses of liability to osteochondral diseases in pigs. Genetics 171, 1195-1206

Kiguwa SL, Hextall P, Smith AL, Critcher R, Swinburne J, Millon L, Binns MM, Goodfellow PN, McCarthy LC, Farr CJ, Oakenfull EA (2000) A horse whole- genome-radiation hybrid panel: chromosome 1 and 10 preliminary maps.

Mamm Genome 11, 803-805

Kroll A, Hertsch B, Höppner S (2001) Entwicklung osteochondraler Veränderungen in den Fessel- und Talokruralgelenken im Röntgenbild beim Fohlen.

Pferdeheilkunde 17, 489-500

KWPN (1994) The frequency and heredity of navicular disease, sesamoidosis, fetlock joint arthrosis, bone spavin, osteochondrosis of the hock. A radiographic progeny study. (KWPN: Koninklijke Vereniging Warmbloed Paardenstamboek Nederland, Zeist)

Lear TL, Brandon R, Piumi F, Terry RR, Guerin G, Thomas S, Bailey E (2001) Mapping of 31 horse genes in BACs by FISH. Chromosome Res 9, 261-262 Lee EJ, Raudsepp T, Kata SR, Adelson D, Womack JE, Skow LC and Chowdhary

BP (2004) A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues. Genomics 83, 203- 215

Lee GJ, Archibald AL, Garth GB, Law AS, Nicholsen D, Barr A, Haley CS (2003) Detection of quantitative trait loci for locomotion and osteochondrosis-related traits in Large White X Meishan pigs. Animal Science 76, 155-165

Lindgren G, Sandberg K, Persson H, Marklund S, Breen M, Sandgren B, Carlsten J, Ellegren H (1998) A primary male autosomal linkage map of the horse genome. Genome Res 8, 951-966

(35)

23

Lindgren G, Breen M, Godard S, Bowling A, Murray J, Scavone M, Skow L, Sandberg K, Guerin G, Binns M, Ellegren H (2001) Mapping of 13 horse genes by fluorescence in-situ hybridization (FISH) and somatic cell hybrid analysis. Chromosome Res 9, 53-59

Mariat D, Oustry-Vaiman A, Cribiu EP, Raudsepp T, Chowdhary BP, Guerin G (2001) Isolation, characterization and FISH assignments of horse BAC clones containing type I and II markers. Cytogenet Cell Genet 92, 144-148

Marklund S, Ellegren H, Eriksson S, Sandberg K and Andersson L (1994) Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites. Anim Genet 25, 19-23

McIlwraith CW (1993) Interferences from referred clinical cases of osteochondritis dissecans. Equine vet J, Suppl 16, 27-30

Milenkovic D, Oustry-Vaiman A, Lear TL, Billault A, Mariat D, Piumi F, Schibler L, Cribiu E, Guerin G (2002) Cytogenetic localization of 136 genes in the horse:

comparative mapping with the human genome. Mamm Genome 13, 524-534 Müller D, Kuiper H, Mömke S, Böneker C, Drögemüller C, Chowdhary BP, Distl O

(2005a) Assignment of the COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping. Anim Genet 36, 277-279

Müller D, Kuiper H, Böneker C, Mömke S, Drögemüller C, Chowdhary BP, Distl O (2005b) Physical mapping of the PTHR1 gene to equine chromosome 16q21.2. Anim Genet 36, 282-284

Müller D, Kuiper H, Böneker C, Mömke S, Drögemüller C, Chowdhary BP, Distl O (2005c) Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping. Anim Genet 36, 457-461

Müller D, Kuiper H, Mömke S, Böneker C, Drögemüller C, Swinburne JE, Binns M, Chowdhary BP, Distl O (2006) Physical mapping of the ATP2A2 gene to equine chromosome 8p12-p14 by FISH and confirmation by linkage and RH mapping. Cytogenet Genome Res 114, 94

Murray JD and Bowling AT (2000) Linkage and comparative maps for the horse (equus caballus). In: The genetics of the horse, edited by Bowling AT and Ruvinsky A, CABI Publishing, Wallingford, UK

(36)

Musilova P, Kubickova S, Vychodilova-Krenkova L, Kralik P, Matiasovic J, Hubertova D, Rubes J, Horin P (2005) Cytogenetic mapping of immunity-related genes in the domestic horse. Anim Genet 36, 507-510

Nixon AJ and Pool RR (1995) Histologic appearance of axial osteochondral fragments from the proximoplantar/proximopalmar aspect of the proximal phalanx in horses. J Am Vet Med Assoc 207, 1076-1080

Olsson SE (1978) Osteochondrosis in domestic mammals. Introduction. Acta Radiol, Suppl 358, 9-14

Penedo MC, Millon LV, Bernoco D, Bailey E, Binns M, Cholewinski G, Ellis N, Flynn J, Gralak B, Guthrie A, Hasegawa T, Lindgren G, Lyons LA, Roed KH, Swinburne JE, Tozaki T (2005) International Equine Gene Mapping Workshop Report: a comprehensive linkage map constructed with data from new markers and by merging four mapping resources. Cytogenet Genome Res 111, 5-15

Perrocheau M, Boutreux V, Chadi-Taourit S, Di Meo GP, Perucatti A, Incarnato D, Cribiu EP, Guerin G, Iannuzzi L (2005) Equine FISH mapping of 36 genes known to locate on human chromosome ends. Cytogenet Genome Res 111, 46-50

Perrocheau M, Boutreux V, Chadi S, Mata X, Decaunes P, Raudsepp T, Durkin K, Incarnato D, Iannuzzi L, Lear TL, Hirota K, Hasegawa T, Zhu B, de Jong P, Cribiu EP, Chowdhary BP and Guérin G (2006) Construction of a medium- density horse gene map. Anim Genet 37, 145-155

Philipsson J, Andreasson E, Sandgren B, Dalin G, Carlsten J (1993) Osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints in Standardbred trotters. II. Heritability. Equine vet J, Suppl 16, 38-41

Pieramati C, Pepe M, Silvestrelli M, Bolla A (2003) Heritability estimation of osteochondrosis dissecans in Maremmano horses. Livest Prod Sci 79, 249- 255

Raudsepp T, Fronicke L, Scherthan H, Gustavsson I, Chowdhary BP (1996) Zoo- FISH delineates conserved chromosomal segments in horse and man.

Chromosome Res 4, 218-225

(37)

25

Raudsepp T, Kijas J, Godard S, Guerin G, Andersson L, Chowdhary BP (1999) Comparison of horse chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping. Mamm Genome 10, 277-282

Raudsepp T, Kata SR, Piumi F, Swinburne J, Womack JE, Skow LC, Chowdhary BP (2002) Conservation of gene order between horse and human X chromosomes as evidenced through radiation hybrid mapping. Genomics 79, 451-457, Erratum in: Genomics 2003 82, 501

Raudsepp T, Santani A, Wallner B, Kata SR, Ren C, Zhang HB, Womack JE, Skow LC, Chowdhary BP (2004a) A detailed physical map of the horse Y chromosome. Proc Natl Acad Sci U S A 101, 9321-9326

Raudsepp T, Lee EJ, Kata SR, Brinkmeyer C, Mickelson JR, Skow LC, Womack JE, Chowdhary BP (2004b) Exceptional conservation of horse-human gene order on X chromosome revealed by high-resolution radiation hybrid mapping. Proc Natl Acad Sci U S A 101, 2386-91

Rothschild M.F, Jacobson C, Vaske DA, Tuggle CK, Short TH et al. (1994) A major gene for litter size in pigs. 5^th World Congr Genet Appl Livest Prod 21, 225-228 Sandgren B, Dalin G, Carlsten J and Lundeheim N (1993) Development of

osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints of Standardbred trotters. II. Body measurements and clinical findings. Equine vet J, Suppl 16, 48-53

Schober M (2003) Schätzung der genetischen Effekte beim Auftreten von OCD.

Dissertation, Georg-August-Universität-Göttingen

Schougaard H, Ronne JF, Philipsson J (1987) Incidence and inheritance of osteochondrosis in the sport horse. 38^th Annual Meeting EAAP, 28 September to 1 October 1987, Lisboa, Portugal

Schougaard H, Ronne JF, Philipsson J (1990) A radiographic survey of tibiotarsal osteochondrosis in a selected population of trotting horses in Denmark and its possible genetic significance. Equine vet J 22, 288-289

Schube S, Dämmrich K, Lauk HD and Von Plocki KA (1991) Untersuchungen zur Pathogenese der Arthropathia deformans und der Entstehung von „Chips“ im Fesselgelenk der Pferde. Pferdeheilkunde 7, 69-77

(38)

Semevolos SA, Strassheim ML, Haupt JL, Nixon AJ (2005) Expression patterns of hedgehog signaling peptides in naturally acquired equine osteochondrosis. J Orthop Res 23, 1152-1159.

Shiue YL, Bickel LA, Caetano AR, Millon LV, Clark RS, Eggleston ML, Michelmore R, Bailey E, Guerin G, Godard S, Mickelson JR, Valberg SJ, Murray JD, Bowling AT (1999) A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers. Anim Genet 30, 1-9

Smallwood JE and Kelly EJ (1991) A xeroradiographic study of osteochondrosis in the metacarpophalangeal region of two foals. Vet Radiol Ultrasound 32, 26-34 Stock KF, Distl O (2006) Correlations between sport performance and different

radiographic findings in the limbs of Hanoverian Warmblood horses Anim Sci 82, 83-93

Stock KF, Hamann H, Distl O (2005a) Prevalence of osseous fragments in distal and proximal interphalangeal, metacarpo- and metatarsophalangeal and tarsocrural joints of Hanoverian Warmblood horses. J Vet Med A 52, 388-394 Stock KF, Hamann H, Distl O (2005b) Estimation of genetic parameters for the

prevalence of osseous fragments in limb joints of Hanoverian Warmblood horses. J Anim Breed Genet 122, 271-280

Stromberg B (1979) A review of the salient features of osteochondrosis in the horse.

Equine vet J 11, 211-214

Swinburne J, Gerstenberg C, Breen M, Aldrige V, Lockhart L, Marti E, Antczak D, Eggleston-Stott M, Bailey E, Mickelson J, Roed K, Lindgren G, Von Haeringen W, Guerin G, Bjarnason J, Allen T, Binns M (2000) First comprehensive low- density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families. Genomics 66, 123-134

Swinburne JE, Boursnell M, Hill G, Pettitt L, Allen T, Chowdhary B, Hasegawa T, Kurosawa M, Leeb T, Mashima S, Mickelson JR, Raudsepp T, Tozaki T, Binns M (2006) Single linkage group per chromosome genetic linkage map for the horse, based on two three-generation, full-sibling, crossbred horse reference families. Genomics 87, 1-29

Trotter GW, McIlwraith CW (1981) Osteochondrosis in horses: pathogenesis and clinical syndromes. Am Ass equine Pract 27, 141-160

(39)

27

Van de Lest CHA, Van Den Hoogen BM, Van Weeren PR, Brouwers JFHM, Van golde LMG and Barneveld A (1999) Changes in bone morphogenic enzymes and lipid composition of equine osteochondrotic subchondral bone. Equine vet J, Suppl 31, 31-37

Van Weeren PR and Barneveld A (1999) The effect of exercise on the distribution and manifestation of osteochondrotic lesions in the Warmblood foal. Equine vet J, Suppl 31, 16-25

Willms F, Röhe R, Kalm E (1999) Genetische Analyse von Merkmalskomplexen in der Reitpferdezucht unter Berücksichtigung von Gliedmaßenveränderungen.

Züchtungskunde 71, 330-345

Winter D, Bruns E, Glodek P, Hertsch B (1996) Genetische Disposition von Gliedmaßenerkrankungen beim Reitpferd. Züchtungskunde 68, 92-108

Wittwer C, Chowdhary BP, Distl O (2005) Radiation hybrid mapping of equine CDK2, DGKA, DNAJC14, MMP19, CTSL and GAS1. Anim Genet 36, 536-537

Yang F, Fu B, O'Brien PCM; Nie W, Ryder OA, Ferguson-Smith MA (2004) Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules. Chromosome Res 12, 65–76

Yovich JV, McIlwraith CW and Stashak TS (1986) Osteochondrosis dissecans am sagittalen Rollkamm von Mc III und Mt III beim Pferd. Pferdeheilkunde 2, 109- 116

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Table 1 Prevalences of osteochondrosis in different horse populations

Horse

population/Age

Affected joint / Osteochondral findings % Affected horses

Authors Danish Trotters

(n = 325) 18 months to 2 years

Hock: Osteochondral fragments at

intermediate ridge of distal tibia 12.0% Schougaard et al., 1990

Dutch Warmblood Horses (n = 590) 3 years

Hock: No specification 13.7% KWPN, 1994

Swedish Standardbred Trotters (foals, n = 77) 1-16 months

Fetlock: POF and UPE Fetlock: DF

Fetlock: Abnormal enchondral ossification

Hock: Bony fragment with or without defect in underlying bone or

subchondral bone defect >5mm

14.3%

2.6%

24.7%

10.4%

Carlsten et al., 1993

Swedish Standardbred Trotters (n = 674) 11 to 24 months

Fetlock: POF Fetlock: UPE Fetlock: DF

subchondral bone defect >5mm

21.5%

4.2%

4.4%

10.5%

Philipsson et al., 1993

Italian Maremmano Horses (n = 350) 2 to 3 years

Fetlock: OF

Fetlock: OF at sagittal ridge of McIII Fetlock: OF at dorsal margin of foreleg proximal phalanx

Hock: Osteochondral fragments (OF) Hock: OF at distal intermediate ridge of tibia

Hock: OF at medial malleolus Hock: OF at lateral malleolus

2.8%

0.8%

2.0%

9.2%

8.3%

0.3%

0.6%

Pieramati et al., 2003

Norwegian Trotters (n = 753)

6 to 21 months

Fetlock: POF Fetlock: UPE Fetlock: DF

Hock: OCD in tibiotarsal joint

subchondral bone defect >5mm Hock: DBT

Hock: defect(s) at lateral trochlea talus

11.8%

2.4%

4.8%

14.3%

12.5%

1.7%

1.1%

Grøndahl and Dolvik, 1993

German Holstein Horses

(foals, n = 220) 5 to 9 months

Hock: OCD tarsi (no specification), minor and extensive changes without loss of performance

22.2% Willms et al., 1999

German Holstein Horses (n = 190) 1 year

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29 Table 1 continued

Horse

population/Age

Affected joint / Osteochondral findings % Affected horses

Authors German Holstein

Horses

(foals, n = 220) 5 to 9 months

German Holstein Horses (n = 190) 1 year

German Holstein Horses (n = 151) 2 years

German

warmblood Horses (n = 3566)

3 to 8 years

Fetlock, Hock: Osteochondrosis dissecans (no specification)

11.0% Winter et al., 1996

Hanoverian

warmblood Horses (n = 3749)

3 to 7 years

Fetlock: Isolated radiopaque area Hock: Isolated radiopaque area in talocrural joint

20.7%

9.6% Stock et al., 2005

McIII: third metacarpal bone.

POF: Palmar/plantar fragment(s) and/or bone defect >5mm at attachment site of short sesamoidean ligament

UPE: ununited palmar/plantar eminence of proximal phalanx (including intra- and extra-articular fragments)

DF: dorsal fragment(s) at dorsoproximal rim of proximal phalanx and/or fragment(s) or defects >5mm at dorsal part of sagittal ridge of distal third metacarpal bone

DBT: defect(s) in bone tissue at cranial edge of intermediate ridge of distal tibia

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Table 2 Marker information of the equine marker set per chromosome developed for complete genome scans in horses

Equine chromosome

Average allele number

Observed average

HET

Average PIC

Average distance

(cM)

Chromosome size (cM)

Number of markers

ECA1 5.1 66.2 57.6 19.4 193.5 10

ECA2 5.9 65.1 57.4 16.1 128.8 8

ECA3 5.8 54.3 49.6 20.0 120.2 6

ECA4 7.8 62.4 58.2 15.3 122.5 8

ECA5 4.4 56.9 53.9 20.0 100.1 5

ECA6 6.0 72.2 69.9 18.1 126.8 7

ECA7 4.4 55.4 50.0 20.4 102.1 5

ECA8 6.2 73.3 62.9 21.8 108.8 5

ECA9 6.0 66.9 59.2 21.0 104.9 5

ECA10 6.6 67.4 64.1 21.2 105.8 5

ECA11 7.3 71.7 66.7 16.2 64.9 4

ECA12 7.3 66.1 64.6 14.5 58.0 4

ECA13 5.0 72.3 61.9 14.5 58.0 4

ECA14 4.3 49.9 44.1 38.2 152.8 4

ECA15 6.8 74.7 68.9 16.1 96.7 6

ECA16 4.6 58.4 52.0 13.9 111.2 8

ECA17 4.5 61.3 54.5 17.8 71.3 4

ECA18 6.3 69.5 63.7 14.6 87.6 6

ECA19 6.2 69.5 59.7 11.1 55.7 5

ECA20 6.0 67.2 59.2 16.2 80.9 5

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Equine chromosome

Average allele number

Observed average

HET

Average PIC

Average distance

(cM)

Chromosome size (cM)

Number of markers

ECA21 4.8 59.1 50.6 15.2 75.8 5

ECA22 5.2 48.6 46.7 15.9 79.7 5

ECA23 5.8 63.4 54.6 14.1 56.2 4

ECA24 5.3 62.0 56.2 11.8 47.2 4

ECA25 7.0 57.8 60.3 16.2 48.7 3

ECA26 8.0 75.4 73.5 12.1 24.4 2

ECA27 6.5 72.2 63.7 23.3 93.0 4

ECA28 4.5 60.6 50.8 31.6 63.1 2

ECA29 6.7 68.2 58.3 25.0 75.0 3

ECA30 6.7 73.1 66.1 16.6 49.7 3

ECA31 7.5 75.2 67.0 20.6 41.1 2

ECAX 9.3 73.1 76.6 16.3 65.2 4

Average 6.1 65.3 59.5 18.3 86.6 4.8

HET: Heterozygosity (%)

PIC: Polymorphism information content (%) cM: centiMorgan

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Table 3 Description, their cytogenetic locations and their start of the genomic sequence in bp on the respective human chromosome (Homo sapiens genome view, Build 35.1) for genes responsible for osteoarthritis in man

Gene name

Gene description Human

cytogenetic location

Start of sequence in bp on the

respective human chromosome ADAM17 ADAM metallopeptidase domain 17

(tumor necrosis factor, alpha, converting enzyme)

2p25 9,580,011

ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5 (aggrecanase-2)

21q21.3 27,215,657

AGC1 aggrecan 1 (chondroitin sulfate proteoglycan 1, large aggregating proteoglycan, antigen identified by monoclonal antibody A0122)

15q26.1 87,147,994

ANKH ankylosis, progressive homolog (mouse)

5p15.1 14,762,019

ASPN asporin (LRR class 1) 9q21.3-q22 92,298,046 ATP7B ATPase, Cu++ transporting, beta

polypeptide

13q14.3 51,334,147

BFHD¹ Beukes familial hip dysplasia 4q35 CASR calcium-sensing receptor

(hypocalciuric hypercalcemia 1, severe neonatal

hyperparathyroidism)

3q13 123,385,220

CCAL1¹ chondrocalcinosis 1 (calcium pyrophosphate-deposition disease, early onset osteoarthritis)

8q

CCL2 chemokine (C-C motif) ligand 2 17q11.2-q12 29,606,411 CLCN7 chloride channel 7 16p13 1,435,346

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Gene name

Gene description Human

cytogenetic location

Start of sequence in bp on the respective human chromosome COL1A1 collagen, type I, alpha 1 17q21.3-

q22.1

45,616,456

COL1A2 collagen, type I, alpha 2 7q22.1 93,668,858 COL2A1 collagen, type II, alpha 1 (primary

osteoarthritis, spondyloepiphyseal dysplasia, congenital)

12q13.11- q13.2

46,653,018

COL3A1 collagen, type III, alpha 1 (Ehlers- Danlos syndrome type IV,

autosomal dominant)

2q31 189,664,605

COL5A1 collagen, type V, alpha 1 9q34.2-q34.3 134,759,597 COL5A2 collagen, type V, alpha 2 2q31 189,722,748

COL9A1 collagen, type IX, alpha 1 6q12-q14 70,982,948 COL9A2 collagen, type IX, alpha 2 1p33-p32 40,435,256 COL9A3 collagen, type IX, alpha 3 20q13.3 60,918,832 COL10A1 collagen, type X, alpha 1(Schmid

metaphyseal chondrodysplasia)

6q21-q22 116,546,814

COL10A1 collagen, type X, alpha 1(Schmid metaphyseal chondrodysplasia)

6q21-q22 116,546,814

COL11A1 collagen, type XI, alpha 1 1p21 103,055,016 COL11A2 collagen, type XI, alpha 2 6p21.3 33,238,447 COMP cartilage oligomeric matrix protein 19p13.1 18,754,584 CTSK cathepsin K (pycnodysostosis) 1q21 147,581,760 CYP2C9 cytochrome P450, family 2,

subfamily C, polypeptide 9

10q24 96,688,430

ESR1 estrogen receptor 1 6q25.1 152,220,800 FLNA filamin A, alpha (actin binding

protein 280)

Xq28 153,019,779

FRZB frizzled-related protein 2q31-q33 183,524,243