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

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Fine mapping of quantitative trait loci (QTL) for osteochondrosis

in Hanoverian warmblood horses

Thesis

Submitted in partial fulfilment of the requirements for the degree -Doctor of Veterinary Medicine-

Doctor medicinae veterinariae ( Dr. med. vet. )

by

Karina Komm Verl

Hannover 2010

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Academic supervision: Univ.-Prof. Dr. Ottmar Distl

Institute for Animal Breeding and Genetics University of Veterinary Medicine Hannover

1. Referee: Univ.-Prof. Dr. Ottmar Distl 2. Referee: Univ.-Prof. Dr. Karsten Feige Day of the oral examination: 20.05.2010

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Dedicated to my family.

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List of abbreviations

A adenine Acc. No Accession number

APS ammonium persulphate ATM animal threshold model

BAC bacterial artificial chromosome BLAST basic local alignment search tool

bp base pairs

BSA bovine serum albumin C cytosine

cDNA complementary deoxyribonucleic acid chrM mitochondrial sequence

chrUn chromosome unknown cM centiMorgan

df degrees of freedom

DL Dempster Lerner transformation

DFG Deutsche Forschungsgemeinschaft (German Research Council) DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTP deoxy nucleoside 5’triphosphates (N is A, C, G or T) DOD developmental orthopaedic diseases

ECA Equus caballus autosome

EDTA ethylenediamine tetraacetic acid EquCab2 Equus caballus assembly 2 EST expressed sequence tag F forward

FISH fluorescence in situ hybridisation G guanine

GS Gibbs sampling

h² heritability

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HET heterozygosity HSA Homo sapiens autosome

HW Hanoverian warmblood

HWE Hardy Weinberg equilibrium IBD identical by descent

IRD infrared dye

kb kilobase LAM linear animal model

LD linkage disequilibrium LOD logarithm of the odds

LSM linear sire model MAF minor allele frequency MAS marker assisted selection Mb megabase

MC III metacarpal III MT III metatarsal III n number

NCBI National Center for Biotechnology Information ng nanogram

NPL non-parametric linkage OC osteochondrosis

OC-F osteochondrosis in fetlock joints OC-H osteochondrosis in hock joints OCD osteochondrosis dissecans

OCD-F osteochondrosis dissecans in fetlock joints OCD-H osteochondrosis dissecans in hock joints OMIM Online Mendelian Inheritance in Man P or p error probability

PL error probability of LOD score PZ error probabilityof Zmean PCR polymerase chain reaction

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PIC polymorphism information content pmol picomol

POF palmar/plantar osseous fragment POS position

QTL quantitative trait locus R reverse

REML residual maximum likelihood

RFLP restriction fragment length polymorphism

RH radiation hybrid

SAS statistical analysis system SGC South German coldblood

SNP single nucleotide polymorphism

SSAHA2 Sequence Search and Alignment by Hashing Algorithm combined with the cross-match sequence alignment program developed by Phil Green at the University of Washington

SSC sus scrofa chromosome STM sire threshold model

T thymine

Ta annealing temperature

TBE tris-borate-ethylenediamine tetraacetic acid TEMED N,N,N’,N’-tetrametylenediamine

U unit

UCSC University of California Santa Cruz WB warmblood

WGS whole genome shotgun sequence X² ChiSquare

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CHAPTER 1

Introduction

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1 Introduction

“A horse doesn't care how much you know until he knows how much you care” (Pat Parelli).

Horses play an important role for human beings since the early domestication 5.000 years ago. In the modern age until the agricultural industrialization beginning in the thirties, we basically used horses as farm animals. Today, this species is our partner in spare time and sports. Phenotypic traits have changed according to our requirements. The ideological and economical value of these animals is immeasurable these days and last but not least this is the reason why health and fitness have first priority.

One of the most frequent diseases of the locomotory system is Osteochondrosis (OC). OC is defined as a focal disturbance of enchondral ossification in young horses. Multifactorial factors may lead to this disease, specific causes are still unknown, but it is proven, that genetic aspects play an important role. The heritability is in the range of h² = 0.1 - 0.34 for warmblood horses and between h² = 0.17 and 0.52 for trotters.

Further influences for the development of OC are environmental factors like anatomic constitutions, traumata, nutritition, growth rate, mineral imbalances and dysfunctions of endocrinology. Recent studies strongly support a failure of blood supply to the growth cartilage as the triggering factor in the pathogenesis. However, in this study we concentrate on genetic factors having influence for OC.

OC describes a wide range of different lesions in human beings and in many animal species, especially fast growing animals like dogs, pigs and horses. Different clinical manifestations like subchondral fractures, wearlines, chondromalazia, cartilage flaps or synovitis often cause lameness or other performance deficits.

Osteochondrosis dissecans (OCD) is a special description of OC and is characterised by free joint fragments (chip, joint mice, corpora libera).

Articulations most commonly affected are fetlock, hock and stifle joints.

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One further aspect for the rising appearence of this complex disease may be common practice of radiographic examinations these days. Clinical and radiographic examinations are more or less conventional practice in horse-dealing. Furthermore, the use of digital radiographic system leads to more exact diagnosis of even minimal pathological changes.

In Hanoverian warmblood horses, a whole genome scan was performed in order to clarify genetic aspects of this joint disorder. Qantitative trait loci (QTL) could be detected on horse chromosomes (ECA) 2, 4, 5, 16, 18 and 21.

The aim of the present study was fine mapping of identified QTL on ECA2 and 4 using dense markersets. In order to develop genetic tests based on gene-associated markers, we employed more microsatellites and single nucleotide polymorphisms (SNPs).

Furthermore, a whole genome scan with more than 50.000 SNPs was carried out in order to approve published QTL, to define new potential QTL and, consequently, to identify putative regions for positional and functional candidate genes of OC.

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Overview of chapter contents

The contents of the present thesis are presented in single papers as allowed by §4(4) of the Rules of Graduation (Promotionsordnung) of the University of Veterinary Medicine in Hannover.

Chapter 2 reviews the literature for OC in horses, including pathogenesis, clinical signs and proposed etiologies. Furthermore, the results of whole genome scans for osteochondrosis in Hanoverian warmblood and South German coldblood are presented, as well as new developments in horse genomics and potential candidate genes for osteochondrosis.

Chapter 3 contains refinement of a quantitative trait locus for fetlock osteochondrosis on equine chromosome 2.

In Chapter 4 the refinement of a quantitative trait locus responsible for osteochondrosis on equine chromosome 4 is described.

Chapter 5 describes the performance of a whole genome single nucleotide polymorphism assay using the Equine SNP50 chip.

Chapter 6 provides a general discussion and conclusions referring to chapters 2-4.

Chapter 7 is a concise English discussion and conclusions of this thesis and Chapter 8 is an expanded, detailed German summary which shows current developments in molecular genetic research.

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CHAPTER 2

Genetics of osteochondrosis in horses

Karina Komm, Virginie Lampe, Ottmar Distl

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

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2 Genetics of osteochondrosis in horses

2.1 Abstract

Osteochondrosis (OC) is a crucial disorder concerning the locomotory system in horses and belongs to the great complex of developmental orthopaedic diseases (DOD). OC is defined as a focal disturbance of enchondral ossification in growth cartilage and presents a complex disease of a multifactorial etiology, in which genetic aspects play a major role. To substantiate the inheritance of OC, linkage and association analyses with the help of genetic markers were performed. In previous studies, quantitative trait loci (QTL) for OC could be identified for Hanoverian warmblood and South German Coldblood horses. Novel equine microarray technologies enable a whole genome association and gene expression analyses in matters of common and severe horse diseases. This paper summarizes currently used terms of orthopaedic joint diseases related to OC and gives an overview over recent developments concerning molecular genetic and biochemical analyses of these conditions in horses.

2.2 Introduction

In the horse industry, much importance is attached to joint diseases as a common cause of lameness. Osteochondrosis (OC), frequently diagnosed radiographically in young horses (Arnan and Hertsch 2005, Carlsten et al. 1993, Hoppe 1984a, b), belongs to the complex of developmental osteochondral diseases (DOD) which generally describes all orthopaedic problems in growing horses (Pool 1986, McIllwraith 2004). Advanced digital x-raying techniques enable the detection of even minimal alterations in affected joints. High prevalences and incidences across different horse breeds and potential economical losses attracted notice to the OC affection status for owners and breeders.

OC results from a failure of enchondral ossification in growth cartilage (Olsson and Reiland 1978, Ytrehus et al. 2007). Affected joints may show different kinds of osteochondral lesions including clinically obvious synovial effusions, rough and

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flattened bone contours, and cartilage flaps (Jeffcott and Henson 1998, Trotter and Mcllwraith 1981). When detached cartilage flaps or osteochondral fragments are present, the condition is more specifically termed osteochondrosis dissecans (OCD).

Disturbed differentiation and maturation of growing cartilage, primarily seen at specific predilection sites of articulations, characterize OC in the horse (Jeffcott and Henson 1998). Main affection sites are fetlock, hock and stifle joints.

The aim of the present paper is to review current develepments in genetic and expression analyses to understand the mechanisms underlying OC. Furthermore, this paper presents latest perspectives of gene expression analyses and shows ways of early diagnosis of initial cartilage lesions in young horses.

2.3 Developmental osteochondral disorders concerning the locomotory system

Osteochondrosis (OC) and Osteochondrosis dissecans (OCD)

Osteochondrosis (OC) appertains to developmental orthopaedic diseases (DOD) and causes different forms of joint alterations (Van Grevenhof et al. 2009). Many authors prefer the term DOD to describe those focal disturbances (Olsson and Reiland 1978, Ytrehus et al. 2007). Because of the uncertain etiopathological homogeneity of DOD, the use of this broad term has often been discussed controversially. Therefore, studies were usually limited to more specific terms like OC and OCD (Sandgren et al.

1993a, Grondahl and Engeland 1995, Jorgensen et al. 1997, Dierks et al. 2006, Wittwer et al. 2006). Based on the unknown relationship of the different forms of DOD, McIlwraith (2004) accented the importance not to use the terms DOD and OC synonymously.

Because epiphyseal growth cartilage is absent in adult, OC can only develop in growing animals (Ekman and Carlson 1998). Enchondral ossification is one of the two essential processes during fetal development of the mammalian skeletal system resulting in bone tissue. It is a process of chondrocyte proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralisation and vascular invasion (Lefebvre and Smits 2005). Continuous addition of cartilage followed by bony replacement leads to growth and elongation of bone. A failure of blood supply of the

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growth cartilage may be the first step towards the development of osteochondral lesions (Ekman and Carlson 1998, Ytrehus et al. 2007). This may result in focal necrosis leading to fissures, cartilage flaps or subchondral bone cysts (Van Weeren 2005). Furthermore, the collagen metabolism in affected joints seems to play an important role in the etiopathogenesis of OC. So far, there exists no evidence that biochemical changes of collagen modification cause osteochondral disturbances (Van de Lest et al. 2002). However, the development of OC is regarded as a dynamic process. In the early horse life, lesions may become visible and either those findings develop into clinical OC with characteristic joint swelling and lameness or cartilage lesions decay (McIlwraith 2002, Dik et al. 1999). Equine OC can affect different parts of the skeleton (Van Weeren 2006), whereas fetlock, hock and stifle joints are primarily affected. Previous studies in Standardbred and Dutch Warmblood horses showed the early development of osteochondral alterations. Findings could be detected in the neonate and in X-rays of one to three months old foals (McIlwraith 2004).

OC is regarded as being multifactorial in origin (Jeffcott 1991, Philipsson et al. 1993, Wittwer et al. 2006), but a genetic predisposition appears to be the main cause (Schougaard et al. 1990, Grøndahl and Dolvik 1993, Philipsson et al. 1993, Stock and Distl 2005, Wittwer et al. 2007b). Rapid growth and body conformation are further enhancing factors (Sandgren et al. 1993b, Pagan and Jackson 1996, Van Weeren et al. 1999, Kadarmideen et al. 2004, Donabedian et al. 2006, Stock et al.

2006). Other influences like anatomic characteristics (Grøndalen 1974, Schenck and Goodnight 1996, Olsson and Reiland 1978), trauma (Ekmann and Carlson 1998), feeding practice (Glade and Belling 1986, Savage et al. 1993a) and dietary imbalances (Bridges and Harris 1988, Knight et al. 1990, Savage et al. 1993b, Pearce et al. 1998) may also have an effect on the development of OC.

In summary, cartilage damage may cause predisposition to the formation of osteochondral lesions of any kind in growing horses without any primary inflammation or considerable trauma to the joint (Ytrehus et al. 2007). The cartilage damage may then cause secondary changes in bone. However, there is no clear

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understanding of the contribution of each factor and the exact etiopathogenesis is still unknown (Jeffcott 1991, 2005, Van Weeren 2006).

Radiographically detectable osteochondral fragments (chips, joint mice, corpora libera) characterize a specific form of OC, the osteochondrosis dissecans (OCD).

Biochemical influences on disturbed joint cartilage may cause further damage to the abnormal tissue (Byron 2008). This may lead to several problems which are recognized as OCD, including local detachment and fracture of cartilage and bone.

These detached areas may remain partially attached to the surrounding area, which then exist as loose flaps of tissue, or may become completely detached, resulting in free joint bodies which can travel throughout the joint (Schober 2003). OCD can cause inflammation in affected articulations, and afterwards the disease may cause damage to healthy cartilage.

Palmar/plantar osteochondral fragments (POFs)

Palmar/plantar osteochondral fragments (POFs) of the third metacarpal and metatarsal (MC/MTIII) condyles are regarded as having a traumatic etiology (Pool 1996). Biomechanical forces, especially for high-performance horses like thoroughbred racehorses result in a repetitive overload (Pool 1996). Barr et al. (2009) considered POFs being a manifestation of traumatic overload arthrosis. Pool et al.

(1996) characterised these lesions as small, ovoid defects in the palmar/plantar joint surface. Barr et al. (2009) presumed that there exists an association between grade of POFs and other findings of the third metacarpal/metatarsal condyles like wear lines or linear fissures. Further investigations should declare the role of subchondral bone adaption in this context. The main problem in diagnosing POFs is that only severe lesions are visible on routine radiography of fetlock joints (Richardson 2003).

Thus, many lesions remain undetected until advanced stages which may lead to chronic joint disease (Barr et al. 2009).

Jönsson et al. (2009) investigated heritabilities and prevalences of OC and POFs in Swedish Warmblood horses. The genetic influence on the development of POFs in fetlock joints is still not totally explained and there are still opposed views. Several authors suggested combining the terms fetlock POF and OC (Dalin et al. 1993,

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Sandgren et al. 1993b, Nixon and Pool 1995), but came to the conclusion, that these findings result from traumatic genesis, finally.

2.4 Prevalence and heritabilities

Previous studies showed prevalences mostly between 10-30% in trotters, warmblood horses, thoroughbreds and coldblood horses (Jeffcott 1991, Philipsson et al. 1993, Schober 2003, Stock et al. 2005, Wittwer et al. 2006). The large differences of affection frequencies probably result from different definitions of clinical and radiographic findings. Clinical prevalence of OCD is usually between 5 and 25% in a given horse population, but radiographic signs of abnormal development can be as high as 60% in certain groups (Wittwer et al. 2006).

However, prevalence and incidence of this disease are severe (Jeffcott 1993, van Weeren and Barneveld 1999) and OC affected horses usually implicate high financial expenses for owners and breeders (Jeffcott 1991, 1996, Olivier et al. 1997, Rossdale et al. 1985, Todhunter and Lust 1990). Van der Harst et al. (2005) even constituted that OC and OCD contributed three times more to economic losses compared to any other equine disease. In order to prevent OC occurrence in horses, detailed knowledge of etiopathological factors is required. The objectives concerning research in OC seem to be to clarify influencing factors, to diagnose early osteochondral lesions and, to minimize genetic predispositions. Heritabilities vary widely between 10 and 52% (Schoughaard et al. 1990, Philipsson et al. 1993, Schober 2003, van Weeren 2006, Wittwer et al. 2007b), probably depending on differences in selected horse material and statistical methods used in the respective study (Ricard et al.

2002, Stock et al. 2005). 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, Schober 2003, Stock et al. 2005b, van Grevenhof et al. 2009). The threshold model heritability estimated 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 Dutch warmblood horses, van Grevenhof et al. (2009) estimated heritabilities between h² = 0.05 and 0.36 using the continious scale.

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In South German Coldblood horses, heritabilities were estimated between h² = 0.16- 0.48 after Dempster-Lerner transformation (Wittwer et al. 2007b). Prevalence and heritabilities of OC in different horse breeds of previously published studies are shown in Table 1.

2.5 Quantitative trait loci (QTL) for osteochondrosis Linkage analyses

Research of inherited equine diseases was given an amazing boost since the first assembly of the horse genome sequence in January 2007. In March 2008, the second assembly was released with more than 20,000 annotated protein-coding genes. Using microsatellites and single nucleotide polymorphisms (SNPs), whole genome scans, fine mappings and even geonome-wide association studies could be accomplished. Quantitative trait loci (QTL) were detected for different traits of osteochondrosis (OC) in Hanoverian warmblood horses (Dierks et al. 2007) and in South German Coldblood horses (Wittwer et al. 2007a). Analyses were done for six phenotypic traits: (1) OC in fetlock and/or hock joints, (2) OCD in fetlock and/or hock joints, (3) fetlock OC, (4) hock OC, (5) fetlock OCD and (6) hock OCD.

Dierks et al. (2007) employed a marker set containing 260 microsatellites, equally distributed over all chromosomes with an average distance of 16.9 cM. Linkage and association analyses were performed in 211 Hanoverian warmblood horses from 14 paternal half-sib families. Genome-wide significant QTL were located on ECA2, 4, 5, and 16. In addition, chromosome-wide significance levels were reached on ECA3, 15, 19, and 21. In order to refine those identified QTL, an extended marker set containing published as well as newly developed microsatellites and SNPs was used.

Fine mapping confirmed QTL on ECA2, 4, 5, 16, and 21 (Dierks et al. 2010a, 2010b, Felicetti et al. 2010, Lampe et al. 2009b, 2009c). On ECA18, a new QTL could be identified in Hanoverian warmblood horses (Lampe et al. 2009a). Dierks et al.

(2010a, 2010b) selected positional candidate genes on ECA2 and 4 in order to design new single nucleotide polymorphisms (SNPs) for linkage and association analyses. On ECA2, one SNP of the neurochondrin (NCDN) gene was significant associated for every trait of OC, except for OCD in hock joints. The expression of the

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Neurochondrin/norbin protein could be associated to neural, bone and chondral functions. On ECA4, Dierks et al. (2010) found three significantly associated SNPs within the genome-wide significant QTL. Two SNPs were located in intergenic regions, one SNP was found in intron 2 of the HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 (HECW1). HECW1 is involved in encoding a cytoplasmic phosphoprotein that regulates cell proliferation. Further mutation and expression analyses may help to investigate the role of these putative candidate genes in the etiopathogenesis of OC.

In South German Coldblood horses, a whole genome scan was performed for the same traits as for Hanoverian warmblood horses and additionally for palmar/plantar osseous fragments (POFs). Heritability of POFs implicated a genetic component in the variation of the development of osteochondrosis (Wittwer et al. 2007a). The genome scan included 250 polymorphic microsatellites equally distributed over all chromosomes with a mean distance of 17.7 cM with an enhanced use of microsatellites in putative QTL regions. Chromosome-wide significant QTL were found on 10 chromosomes, including seven QTL for fetlock OC and one QTL on ECA18 for hock OC and fetlock OC. Significant QTL for POFs in fetlock joints were located on equine chromosomes 1, 4, 8, 12 and 18. On ECA4, the QTL for POFs reached the genome-wide significance level (Wittwer et al. 2007b). On ECA4, significantly associated SNPs in the acyloxyacyl hydrolase (AOAH) gene could be identified (Wittwer et al. 2008). On ECA18, Wittwer et al. (2009) found significantly associated intronic SNPs in the xin actin-binding repeat containing 2 (XIRP2) gene, which was significantly upregulated in mice with cardiomyopathy (Duka et al. 2006).

Mutation and expression analyses may explain the role of these genes in the development of OC in South German Coldblood horses.

QTL of Hanoverian warmblood and South German Coldblood horses reaching the genome-wide and chromosome-wide significance levels are shown in Table 2.

Most genetic research on OC and related conditions provided evidence that many genes are involved in the development of these complex diseases. Kadarmideen et al. (2005) used statistical models to show a major gene with Mendelian transmission significantly determining porcine OC. Christensen et al. (2010) revealed many QTL

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on different chromosomes exceeding the 5% genome-wide threshold which is consistent with the opinion that many QTL affect osteochondrosis. Till now, refinement studies provide an indispensable basis towards unravelling the genes underlying QTL for equine OC in horses.

Genome-wide association analyses

Since 2008, a commercially available SNP microarray containing more than 50,000 SNPs enables genome-wide association analyses, quantitative trait loci (QTL) identification and additionally QTL validiation. The equine SNP50 Genotyping BeadChip was developed by Illumina in collaboration with the International Equine Genome Mapping Workshop and the Morris Animal Foundation’s Equine Genome Consortium. Researchers around the globe use the equine SNP chip to investigate common single gene defects and multigenic diseases including musculoskeletal, neuromuscular, cardiovascular and respiratory disorders (Swinburne 2009). In order to approve detected QTL of OC in Hanoverian warmblood horses, this whole genome scan with SNPs was performed. Association and variance analyses enabled the validation of known QTL on ECA2, 4, 16 and furthermore, the detection of new potential QTL on ECA3, 7, 19, 20, 22, 26 and 29. Consequently, new potential candidate genes could be revealed. Using such microarray technologies implies a great improvement towards unravelling genes underlying QTL for equine OC and therefore, the development of a marker test for OC might get available.

2.6 Candidate genes and gene expression analyses

Candidate genes for osteochondrosis encode hormones, enzymes, metabolic factors and/or their receptors involved in the complex of cartilage differentiation and maturation during enchondral ossification, in growth processes, or vascularisation. It can be helpful to use the Equine Articular Cartilage cDNA Library to select candidate genes which are at least expressed in cartilage. At the moment a total of 13,964 equine articular ESTs (expressed sequence tag) can be found at the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/sites/entrez).

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Genes causing osteoarthritis in other species can also be used as candidate genes for the molecular genetic analysis of OC in horses.

Andersson-Eklund et al. (2000) identified three QTL for OC in pigs on Sus scrofa chromosomes SSC5, 13 and 15. Possible candidate genes derived from these QTL might be pituitary specific transcription factor 1 (POU1F1), insulin-like growth factor (IGF-I), cartilage homeoprotein 1 (CART1) because of their indicated role in the development of OC and their location in the homologous region of the human genome.

For man, these genes can be taken from the Online Mendelian Inheritance in Man (OMIM) database (Supplementary Table 1). This database is a catalog of human genes and genetic disorders developed by NCBI (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim). Some of these genes, for example a part of the collagen or matrix metalloproteinase genes could be confirmed by various expression studies in horses with osteochondrosis.

Studies on the variation in gene expression of key chondrogenic genes and genes associated with cartilage pathology between normal and OC chondrocytes may also help to identify candidate genes and their potential role in the pathogenesis of osteochondrosis. Changed gene expression is an elementary biological mechanism and represents physiological process in growth homeostasis.

The endocrinological procedures of skeletal development and growth are controlled by hormones that are most likely to participate in enchondral ossification, such as insulin, thyroxine, growth hormone, parathyroid hormone and calcitonin (Glade 1986, Jeffcott 1997). Of the regulating proteins involved in enchondral ossification, the transforming growth factor ß (TGF-ß) plays an important role in growth cartilage metabolism, particulary in the control of chondrocyte differentiation and hypertrophy (Glade 1986, Henson et al. 1997, Jeffcott and Henson 1998). Henson et al. (1997) described a reduced expression and immunoreactivity in focal lesions compared to normal cartilage but strong expression of TGFß1 in the chondrocyte clusters immediately surrounding a lesion and therefore a possible involvement in the pathogenesis of OC. Semevolos et al. (2001) found a higher expression of TGF-ß in affected tissue, but not significantly so, and suggested a healing response to the OC

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lesion. Hypertrophic differentiation and enchondral ossification of growth cartilage are regulated by a complex array of signaling peptides, including parathyroid hormone related protein (PTHrP), Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs). A negative feedback loop between PTHrP and Ihh, controlling the rate of hypertrophic differentiation has been well described (Chung et al. 1998, Juppner et al. 2000, Vortkamp et al. 1996). Hedgehog signaling occurs through the transmembrane receptor, Patched (Ptc), which upon binding of Ihh, releases its inhibition of a second transmembrane recteptor, Smoothened (Smo). Smo activation then results in stimulation of transcription factors, Gli1, Gli2 and Gli3, which translocate into the nucleus to bind the DNA. While a significant increase of PTHrP and Ihh expression in chondrocytes from OC-affected cartilage and a decrease of Gli1 expression could be observed, no different expression patterns were identified for BMP, Gli2, Gli3, Ptc and Smo (Semevolos et al. 2002, 2004, 2005).

Insulin like growth factors (IGFs) play an important role in cartilage metabolism and growth, including the introduction of increasing cellular proliferation and the synthesis of cartilage aggrecan and collagen (Semevolos et al. 2001). There has been ascertained an interdependency of OC in hock joints and plasma IGF-I levels (Sloet van Oldruitenborgh-Oosterbaan 1999). Foals with osteochondrotic findings showed significantly lower IGF-I levels than unaffected foals. It is suggested that reduction in chondrocyte differentiation, as caused by lower plasma IGF-I concentrations, may contribute to the development of osteochondrosis. The significantly higher expression of IGF-I in cartilage obtained from osteochondrotic lesions (Semevolos et al. 2001) in eight to twelve month old horses, reflects a healing response to injured tissue rather than a primary alteration.

The composition of the extracellular matrix has been targeted as another molecular mechanism involved in the development of OC. Various collagen types that are represented in the extracellular cartilage matrix are known to play a role in the development and maturation of cartilage. It is well known that the extracellular matrix of the articular cartilage goes through a phase of rapid remodelling in the neonatal animal (Van Weeren 2005). Additional evidence for the crucial role of collagen was provided by the demonstration of differences in post-translational modifications of

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collagen type II in samples from early osteochondrotic lesions (Van de Lest et al.

2004). The expression of Coll-I, -II and –X in chondrocytes from OC cartilage was significantly higher than in normal cartilage (Garvican et al. 2008). These results could partly be confirmed by Mirams et al. (2008) who found a significantly higher expression of Coll-I and –X in the lesions, but no differences in the expression patterns of Coll-II. Also Semevolos et al. (2001) could not find any significant differences in expression of Coll-I, -II and -X between OC and normal joints. Gläser et al. (2009) found cartilage specific matrix genes (COL2A1, COL3A1, COL11A1, COL1A2) abundantly expressed in physiological articular cartilage.

Mienaltowski et al. (2008) presented upregulated cartilage matrix proteins (COL2A1, COL9A1, COL9A2, COL9A3, TNC) and collagen-modifying enzymes (P4HA1, LOXL2) in neonatal cartilage and validated microarray results for selected genes by real-time quantitative polymerase chain reaction (COL2A1, COMP, P4HA1, TGFB1, TGFBR3, TNC). COL9A2 encodes a collagen component of hyaline cartilage and seems to be a suitable candidate gene for osteochondral diseases. COL9A2 is located on ECA2 within the QTL for fetlock OC of Hanoverian warmblood horses (15.65 to 30.94 Mb) (Dierks et al. 2010) at 17.80 to 17.81 Mb, but Böneker et al.

(2006) could not show association of this gene with the affection status. Miyaki et al.

(2009) investigated human articular chondrocyte expression and constituted an increased COL2A1, SOX9 and AGGRECAN expression during chondrogenesis. In osteoarthritic cartilage they showed a down-regulated COL2A1 and SOX9 while cartilage-degrading enzymes (ADAMTS5 and MMP-13) were up-regulated.

Peansukmanee et al. (2009) found a reduced GLUT1 (glucose transporter 1) gene expression in pathologic cartilage. Glucose is taken up by glucose transporters (GLUTs) and this carbohydrate illustrates the main source of metabolic energy. The equine GLUT1 gene is located on ECA2 at 15.56 to 15.57 Mb and, consequently close to detected QTL for OC in fetlock joints of Hanoverian warmblood horses (15.65 to 30.94 Mb). Peansukmanee et al. (2009) analysed effects of hypoxia on glucose transport in equine chondrocytes and compared expression of the hypoxia responsive GLUT1 gene in affected and in unaffected cartilage. They suggested that reduced GLUT1 might contribute to degenerative cartilage defects.

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Furthermore, they showed immature cartilage being influenced by a large spectrum of growth factors. The most expressed growth factors were CTGF (connective tissue growth factor), WISP3 (WNT1 inducible signalling pathway protein 3), and FGF-2 (fibroblast growth factor-2). Besides, FGF receptor-2 expression tended to dominate over FGF ligand expression. CTGF could be found in cartilage (Mirams et al. 2008) and seems to play an important role in matrix synthesis and integrin formation (Nishida et al. 2007, Oka et al. 2007). Nishida et al. (2007) suggested CTGF being involved in cartilage repair, among other functions.

The ADAM metallopeptidase with thrombospondin type 1 motif, 4 (ADAMTS4) gene encodes for an enzyme, which is responsible for the degradation of aggrecan, a major proteoglycan of cartilage. Aggrecan degradation is an important factor in the erosion of articular cartilage in arthritic diseases, which is also reflected in a significantly higher expression of ADAMTS-4 in OC cartilage than in chondrocytes from normal cartilage (Garvican et al. 2008). However, aggrecan itself was not differently expressed (Garvican et al. 2008, Mirams et al. 2008, Semevolos et al.

2001).

Metalloproteinases are considered to be a key feature in the loss of articular cartilage seen in many joint diseases. Different studies on the expression of matrix metalloproteinases MMP-1, -3 and -13 revealed the same results as there was no significant difference in the expression of either MMP-1 or MMP-3 but a significant upregulation of MMP-13 in OC-affected chondrocytes (Garvican et al. 2008, Kuroki et al. 2005, Mirams et al. 2008). Furthermore, Mirams et al. (2008) showed greatest differences of RUNX2 (runt-related transcription factor 2) gene expression between lesions and physiological cartilage. MMP13 and RUNX2 are normally expressed by hypertrophic chondrocytes (Inada et al. 2004, Enomoto et al. 2000) and that might have consequences for the regular removal of cartilage during enchondral ossification. Brama et al. (2000) investigated the role of MMP-3 activity in synovial fluid in common joint disorders in the horse and concluded that MMP-3 activity in OC joints appears not to be different from normal joints but was four times higher in osteoarthritic joints.

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Proteins encoded by the TIMP (tissue inhibitor of metalloproteinase) gene family are natural inhibitors of the matrix metalloproteinases (MMPs), and therefore indicate further observation. While TIMP-1 showed a significant increase of expression in chondrocytes from OC cartilage in comparison to normal cartilage, the expression of TIMP-2 and TIMP-3 in OC chondrocytes was significantly less (Garvican et al. 2008).

The fact that nearly all mentioned genes are not located in identified QTL regions leads to the assumption that the hitherto definition of a candidate gene for osteochondrosis leaves a lot to be desired. Maybe one has to detach from the cascade of ossification, maturation and vascularisation, but rather for example focus on secondary responses or repair processes. The unsatisfying knowledge of the etiopathogenesis of osteochondrosis further complicates the identification of candidate genes. For this reason, it is more auxiliary to delineate QTL, so the number of potential candidate genes might be limited just by delimited genomic regions.

However, gene expression analyses, especially with the help of modern microarray technologies, provide an insight into several pathogenesis of extensive diseases.

Once we fathom the genesis of complex developmental disorders like OC, we may be able to avoid them as possible.

All mentioned candidate genes of latest gene expression analyses, a short description and their location on the equine genome are shown in Table 3.

2.7 Alternative ways of diagnosing early osteochondral lesions

Increasing use of radiography and advanced digital x-raying in equine medicine has brought to light the great meaning of joint diseases. In order to clarify the etiopathogenesis, especially in the early beginning of cartilage or bone alterations, current studies investigated associations of biochemical markers and gene polymorphisms with osteochondral diseases and production traits in pigs (Kadarmideen 2008) and the relationship between biochemical markers and osteoarticular findings in horses (Verwilghen 2009a). Donabedian et al. (2008) determined the relationship between serum concentrations of biomarkers that reflect changes in cartilage and bone turnover and age, feeding level, growth and the occurrence of OC in juvenile horses. Osteocalcin, released by osteoblasts and

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responsible for forming and mineralizing the bone, could be used as a serum marker predicting radiographically evident OC. Billinghorst et al. (2004) showed significantly correlations between osteocalcin concentrations and the severity of OC in Dutch Warmblood foals. Vervuert et al. (2008) disproved osteocalcin to be a useful serum marker. In their study, concentrations did not differ between healthy and affected foals.

Verwilghen et al. (2009b) investigated the relationship between the radiological status and levels of four biochemical markers. They showed the nitrated form of type II collagen degradation (COL2-1NO2) to be a potentially useful predictor of the osteoarticular status of horses.

2.8 Conclusions

Osteochondrosis (OC) is an important and common joint disease in many horse breeds. The etiopathogenesis of OC depends on multifactorial factors. Genetic parameters were the most constistent and largest factors contributing to the pathogenesis of OC. Genetic influences have been greatly substantiated through identification of quantitative trait loci (QTL) in Hanoverian warmblood and South German Coldblood horses. These studies enabled the wise search of potential and functional candidate genes in equine chromosomes. In South German Coldblood horses, further analyses showed associations between the acyloxyacyl hydrolase (AOAH) gene on ECA4 and the xin actin-binding repeat containing 2 (XIRP2) gene on ECA18 and the OC affection status in horses. The equine 50k Beadchip constitutes an amazing progress in molecular genetic research and allows genome- wide association analyses. Furthermore, novel equine microarrays illustrate further possibilities of quantitative and qualitative gene expression analyses as an important biological mechanism. The COL9A2 and the GLUT1 gene on ECA2 were located within identified QTL in Hanoverian warmblood horses and approve previous linkage studies. Regarding complex diseases, exact and consequent trait classification is an indispensible condition. Identification of causal gene mutations would imply a progress for moleculargenetic research and subsequent selection could reduce the OC incidence in horses.

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Table 1 Prevalence and heritabilities (h²) of osteochondrosis (OC) and osteochondrosis dissecans (OCD) in different limb joints by horse breeds

Population Number of

animals

Radiographic finding

Prevalence Heritability estimate

Method of analysis

Reference

Dutch WB n=811 OC (stifle) OC (hock) OC (fetlock) OCD (stifle) OCD (hock) OCD (fetlock)

0.07±0.06 0.15±0.08

0.08±0.10 0.02±0.04 0.26±0.09 0.06±0.07

LAM⁵ (REML²) Van Grevenhof et al. 2009

Dutch WB stallions

n=1965 OC (stifle) OC (hock)

11.5%

16.0%

0.09 0.11

ATM¹ (REML², DL³)

Der Kinderen 2005

Dutch WB mares

n=590 OC (hock) 13.7% 0.01±0.06 0.14±0.17

LSM⁴ (REML²) LAM⁵(REML²)

KWPN 1994 Italian WB n=350 OCD (stifle)

OCD (all) 16.6%

0.09±0.24 0.14±0.23

ATM¹ (AIREML)

Pieramati et al. 2003 Hanoverian

WB

n=624 OC (hock) OC (fetlock) OCD (hock) OCD (fetlock)

5.9%

9.1%

7.2%

11.9%

0.08±0.05 0.07±0.08 0.10±0.05 0.15±0.07

LAM⁵ (REML²) Schober 2003

Hanoverian WB

n=3725 OCD (hock) OCD (fetlock)

9.6%

20.8%

0.37±0.06 0.19±0.03

LAM⁵ (REML², DL³)

Stock et al.

2005 Hanoverian

WB

n=5231 OCD (hock) OCD (fetlock) OCD (hock) OCD (fetlock) OCD (hock) OCD (fetlock)

9.2%

23.5%

0.28±0.04 0.17±0.03 0.27±0.04 0.17±0.03 0.17±0.07 0.12±0.05

LSM⁴ (REML², DL³)

STM⁶ (GS⁷)

Stock and Distl 2006

Danish trotters

n=325 OCD (hock) 12.0% 0.26±0.14 STM⁶ Schougaard et al. 1990

South German Coldblood

n=167 OC (hock) OC (fetlock) OCD (fetlock)

40.1%

53.9%

26.4%

0.04±0.07 0.16±0.16 0.08±0.09

Wittwer et al.

2007b

WB=warmblood

1 Animal threshold model

2 Residual Maximum Likelihood

3 Dempster Lerner transformation onto the liability model

4 Linear sire model

5 Linear animal model

6 Sire threshold model

7 Gibbs sampling

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

Table of contents

List of abbreviations