Genome-wide search for markers associated with osteochondrosis in Hanoverian warmblood horses
Chromosome 30. On ECA30 there was significant linkage between the microsatellite marker LEX025 at 0.00 cM and the trait OC fetlock
Discussion
The aim of this work was to detect microsatellite markers associated with osteochondrosis in Hanoverian warmblood horses. The high number of QTL on different chromosomes found for OC and OCD in this study suggest that several genes are possibly involved in the development of OC and OCD. However we cannot draw conclusions on the type of gene action and in which way the different genes may interact with each other. So it is not possible to decide if OC already develops in the case when at least one or both alleles of one responsive gene are mutated or only in this case when mutations at several gene loci are present.
We chose the location in the fetlock joints at sagittal ridge of third metacarpal and metatarsal bone for OC and OCD in our study due to the high number of animals with changes at this location. In the hock joints we included two locations in our study: at intermediate ridge of the distal tibia and at lateral trochlea of talus. Our definition for OC were in agreement with Kroll et al. (2001) who defined OC in German warmblood horses in fetlock joints as radiolucency, irregular bone margin, new bone formation or
osseous fragments at sagittal ridge of third metacarpal and metatarsal bone and in hock joints as general osteochondrotic findings or osseous fragments at medial or lateral malleolus, at intermediate ridge of distal tibia, at medial or lateral trochlear ridge of tibia or distal at lateral trochlea of talus. Philipsson et al. (1993) and Grøndahl and Dolvik (1993) defined OC in trotters in fetlock joints as palmar/plantar osteochondral fragments at attachment site of short sesamoidean ligaments, excluding bony fragments at dorsoproximal end of proximal phalanx and OC in hock joints as bony fragments at intermediate ridge of distal tibia and/or at trochlea of talus. Schougaard et al. (1990) investigated in their study about Danish trotters only osteochondral fragments with or without defect at different locations in hock joints, excluding defects without osteochondral fragments. Pieramati et al. (2003) included only osteochondral fragments in the different locations of fetlock and hock joints in a population of Maremmano horses.
The QTL for OC found in the present analysis were for the most part heterogeneously distributed between fetlock and hock joints. The QTL on ECA2 at 25.00-56.00 cM was detected for fetlock OC and OCD as well as for hock OC.
ECA16 is harbouring QTL at 0.00-49.00 cM for fetlock OC and OCD as well as for hock OC and OCD. All other QTL map to different chromosomes for the traits fetlock and hock OC. This may indicate that the genetic influences on the development of fetlock OC and hock OC are mostly due to different gene loci. This seems likely as the genetic correlations between fetlock OC and hock OC were close to zero in trotter horses (Grøndahl and Dolvik 1993), or even negative in Hanoverian warmblood horses (Stock et al. 2005b).
This genome scan was a first step towards the identification of genes responsible for OC in horses. In the future the positions of the QTL identified in this study have to be refined by increasing the density of the markers used in these specific genomic regions up to 1-2 cM. In this way, the QTL can be confirmed or excluded. At the moment, this is cost intensive due to the lack of high-density equine linkage maps.
So it is necessary to develop new markers (microsatellite markers or single nucleotide polymorphisms, SNPs) in the identified regions by sequencing those equine (BAC-) DNAs which contain the specific genomic QTL regions. The recently initiated horse genome project under the leadership of the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Germany, may
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greatly enhance the generation of new genomic equine sequences and markers as well as large improvements of the equine-human comparative map (Distl and Blöcker 2005, http://www.volkswagenstiftung.de/presse-news/presse05/08122005.pdf).
Candidate genes in the regions flanking QTLs may be chosen by means of comparative human-equine maps. The equine QTL positions can then be compared with the conserved chromosomal region(s) between horses and humans in order to identify positional candidate genes involved in cartilage maturation. Potential candidate genes have to code for hormones, enzymes, metabolic factors and/or their receptors involved in the complex of cartilage maturation and differentiation during enchondral ossification or in growth processes. Candidate genes may be also involved in osteoarthritis of other species. The Equine Articular Cartilage cDNA Library may be also helpful to select candidate genes. At the moment 13,966 equine articular ESTs (expressed sequence tag) can be found at the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=
nucleotide).
In a previous study, Andersson-Eklund et al. (2000) identified QTL for osteochondrosis in pigs on sus scrofa chromosomes (SSC) 5, 13 and 15. The QTL on SSC5 and SSC15 did not correspond to any QTL identified in horses here. A putative correspondence was identified between the QTL on SSC13 detected by Andersson-Eklund et al. (2000) and the QTL on ECA16 observed in the present study. The region containing the QTL on SSC13 was homologous to HSA3p, which contained the pituitary specific transcription factor 1 (POU1F1 at 87,391,473 bp, Homo sapiens genome Build 35.1) coding for a transcriptional factor of growth hormones and the gene coding for parathyroid hormone receptor 1 (PTHR1 at 46,894,240 bp, Homo sapiens genome view Build 35.1). These two genes were discussed to be responsible for OC in pigs in the study of Andersson-Eklund et al.
(2000). One QTL discovered by Andersson-Eklund et al. (2000) for bone dimensions on SSC17 might be the same as one QTL for OCD in all joints identified in this study on ECA22. This region is homologous to HSA20 harbouring two potential candidate genes: bone morphogenetic protein-2 (BMP2 at 6,697,207 bp, Homo sapiens genome view Build 35.1) and the gonadotropin-releasing hormone-2 (GNRH2 at 2,972,268 bp, Homo sapiens genome view Build 35.1).
Lee et al. (2003) also detected QTL associated with osteochondrosis related traits in
pigs. None of the QTL detected in the study of Lee et al. (2003) exceeded the chromosome-wide suggestive level. They found QTL on SSC7 and 16. Both QTL found in the study of Lee et al. (2003) did not correspond to any equine QTL identified in the present study.
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 be identified or resolved with the necessary accuracy. In Table 7 the equine chromosomes harbouring significant QTL are listed with human homology and possible candidate genes with a short summary of their function. Fine mapping using markers for all genes in the identified OC genomic regions should enable us to locate the responsible genes for OC. The causal mutations may be found by sequencing of at least the exons with their splice sites, the 5’- and 3’- untranslated regions of the candidate genes of a large sample with affected and non-affected horses from different families.
Acknowledgements
This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1). We wish to thank Prof. Dr. E. Bruns, Dr. L. Christmann, Prof. Dr. M.
Coenen and Prof. Dr. B. Hertsch for their commitment to the project. We are grateful to all further sponsors of this project. We also want to thank H. Klippert-Hasberg and S. Neander for their technical support during the work in the laboratory.
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Table 1 Number of families analyzed, their sizes and prevalences of osteochondrosis (OC), osteochondrosis dissecans (OCD), osteochondrosis in fetlock (OC-F) and hock (OC-H) joints and osteochondrosis dissecans in fetlock (OCD-F) and hock (OCD-H) joints by family and in total
Prevalences in % for
Half-sib family
Number of progeny
Male Female
OC OCD OC-F OCD-F OC-H OCD-H
1 4 1 3 100.0 100.0 50.0 50.0 75.5 75.0
2 9 4 5 44.4 33.3 11.1 0.0 33.3 33.3
3 7 5 2 71.4 42.9 71.4 42.9 28.6 14.3
4 15 9 6 53.3 33.3 26.7 6.7 26.7 26.7
5 8 1 7 100.0 25.0 75.0 12.5 37.5 12.5
6 4 2 2 100.0 25.0 50.0 25.0 50.0 0.0
7 5 3 2 100.0 40.0 80.0 40.0 20.0 0.0
8 6 2 4 100.0 66.7 83.3 50.0 66.7 33.3
9 20 12 8 75.0 60.0 50.0 20.0 50.0 45.0
10 8 4 4 62.5 25.0 50.0 12.5 12.5 12.5
11 5 3 2 40.0 20.0 20.0 0.0 20.0 20.0
12 5 2 3 100.0 80.0 100.0 80.0 0.0 0.0
13 5 3 2 40.0 40.0 40.0 40.0 0.0 0.0
14 3 1 2 100.0 66.7 100.0 66.7 0.0 0.0
Total 104 52 52 73.1 45.2 51.9 25.0 32.7 24.0
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Table 2 Distribution of the mean polymorphism information content (PIC in %), observed mean heterozygosity (HO in %) for all microsatellite markers (n=218) in the whole genome scan for each family and in total
Family number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total
HO 60.5 63.6 63.4 64.7 65.4 61.8 63.7 63.4 62.2 65.1 64.6 64.5 62.0 59.4 63.3 PIC 44.5 48.4 47.4 51.8 51.3 45.5 46.7 47.7 50.1 48.4 44.4 46.1 50.7 42.8 47.6
Table 3 Marker information per chromosome of the equine marker set used (218 microsatellite markers) in this study
Equine
average HET Average PIC
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Table 4 Test statistics Zmean and LODscore of the non-parametric multipoint linkage analysis for the traits OC (fetlock and/or hock) and OCD (fetlock and/or hock) and their error probabilities (pZ, pL) in Hanoverian warmblood horses
OC OCD
Table 5 Test statistics Zmean and LODscore of the non-parametric multipoint linkage analysis for the traits fetlock OC (OC-F) and fetlock OCD (OCD-F) and their error probabilities (pZ, pL) in Hanoverian warmblood horses
OC-F OCD-F POS: position on the horse maps (Swinburne et al. 2006, Penedo et al. 2005, Chowdhary et al. 2003)
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Table 6 Test statistics Zmean and LODscore of the non-parametric multipoint linkage analysis for the traits OC hock (OC-H) and OCD hock (OCD-H) and their error probabilities (pZ, pL) in Hanoverian warmblood horses
OC-H OCD-H
Table 7 Human homology to the QTL detected for osteochondrosis and osteochondrosis dissecans in Hanoverian warmblood horses and positional candidate genes with their function
QTL
Short summary of gene function3
collagen, type IX, alpha 2, mutations in this gene are associated with multiple
matrilin 1, a cartilage matrix protein, mutations of this gene have been associated with a variety of inherited
matrix metalloproteinase 15 is involved in the breakdown of extracellular matrix in normal physiological processes, as well as in disease processes, such as arthritis and metastasis 4, protein 10 interacts with insulin receptors and insulin-like B1 gene is down regulated by parathyroid hormone in
osteoblastic cells, is thought to be involved in parathyroid hormone action in bones 5,
collagen, type XXIV may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development
Whole genome scan for osteochondrosis
Short summary of gene function3
matrilin 3 is present in the
cartilage extracellular matrix and has a role in the development and homeostasis of cartilage and bone, mutations are associated with hand
osteoarthritis or one form of autosomal recessive helps to maintain a stable calcium concentration,
mutations that inactivate CASR cause familial hypocalciuric hypercalcemia, whereas mutations that activate CASR are the cause of autosomal dominant hypocalcemia
gene coding for parathyroid hormone receptor, defects in this receptor are known to be the cause of Jansen's
metaphyseal chondrodysplasia (JMC), chondrodysplasia Blomstrand type (BOCD) or enchondromatosis
gene coding for growth hormone secretagogue receptor which may play a role in energy homeostasis and regulation of body weight
ECA: Equus caballus autosome HSA: Homo sapiens autosome
1position in centiMorgan (cM) of microsatellite marker with highest test statistic Zmean
2human homology (Chowdhary et al. 2003, Swinburne et al. 2006) in base pairs (bp)
3NCBI gene information (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=gene), Homo sapiens genome view Build 35.1
Figure 1 Zmeans of the genomic region on ECA2 harbouring QTL for equine osteochondrosis. The maxima of the curves are marked with arrows indicating the genotyped microsatellite makers at the respective positions.
Figure 2 Zmeans of the genomic region on ECA4 harbouring QTL for equine osteochondrosis. The maxima of the curves are marked with arrows indicating the genotyped microsatellite makers at the respective positions.
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Figure 3 Zmeans of the genomic region on ECA5 harbouring QTL for equine osteochondrosis. The maxima of the curves are marked with arrows indicating the genotyped microsatellite makers at the respective positions.
Figure 4 Zmeans of the genomic region on ECA15 harbouring QTL for equine osteochondrosis. The maxima of the curves are marked with arrows indicating the genotyped microsatellite makers at the respective positions.
Figure 5 Zmeans of the genomic region on ECA16 harbouring QTL for equine osteochondrosis. The maxima of the curves are marked with arrows indicating the genotyped microsatellite makers at the respective positions.
Chapter 4
doi: 10.1111/j.1365-2052.2005.01273.x