In order to substantiate the genetic influence on the development of equine osteochondrosis (OC), a whole genome scan for the purpose of quantitative trait loci (QTL) detection was performed in Hanoverian warmblood horses (Dierks et al. 2007) and South German Coldblood horses (Wittwer et al. 2007a). In Hanoverian warmblood horses, adjacent fine mapping confirmed and narrowed down significant regions on equine chromosomes (ECA) 2, 4, 5, 16, 18 and 21 (Dierks et al. 2010, Lampe et al. 2009a, 2009b, 2009c, Felicetti et al. 2010).
The development of equine OC implies a complex disorder of multifactorial etiology (Jeffcott 1991, Philipsson et al 1993, Wittwer et al. 2006). Previous studies suggested several genes probably being involved in the genesis of osteochondral lesions. Up to the present, there exists no detailed knowledge about the etiopathology of complex diseases like OC and the exact way of gene functions or interactions are unenlightened. Linkage and association studies were the first step towards the identification of significant regions harbouring putative candidate genes for equine OC. Functional candidate genes have to code for hormones, enzymes, metabolic factors and/or their ligands which have a share in the great complex of enchondral ossification. This might be processes like growth, differentiation, vascularisation, regeneration, degeneration or even indirectly involved metabolic pathways. Additionally, it might be auxiliary to search for genes which cause osteochondral diseases like osteoarthritis in other species. Particularly human genetics with great progresses in research and an improved moleculargenetic intelligence allows a wise search for functional candidate genes. Current studies on gene expression analyses of genes associated with cartilage and bone development may also help to explain their respective roles in the etiopathology of OC.
Furthermore, the relationship between biochemical markers and osteochondral lesions might deliver insight into pathologic processes during enchondral ossification.
Present researchers investigate levels of bloodparameters to find useful predictors of the osteoarticular status.
However, purposeful search for potential candidate genes implied linkage analyses and adjacent fine mapping in order to refine significant regions. For fine mapping of
ECA2 and 4, genome-wide significant QTL were chosen (Dierks et al. 2010). The release of the second horse genome assembly (EquCab2) in 2008 enabled linkage studies with marker distances less than 1 Mb in the significant region. All detected QTL of the previous genome scan could successfully be approved.
On ECA2, a total of 62 microsatellites and 43 SNPs were genotyped. EquCab2 enabled the development of 27 new microsatellites. The QTL for OC (in fetlock and/or hock joints) could be narrowed down at 20.08 to 30.94 Mb, for OCD (in fetlock and/or hock joints) at 26.89 to 29.47 Mb, for fetlock OC at 15.65 to 30.94 Mb, for fetlock OCD at 21.15 to 31.91 Mb and for hock OC at 26.89 to 33.05 Mb. Due to their location within these mostly overlapping QTL, a total of 16 positional candidate genes could be identified. Among other genes, collagen type IX, alpha 2 (COL9A2) gene at 17.78 Mb, the major collagen component of hyaline cartilage, seemed to be a promising candidate gene for OC. Mutations in this gene are associated with multiple epiphyseal dysplasia. Previous studies could not show an association of COL9A2 with equine osteochondrosis (Böneker et al. 2006). Furthermore, the neurochondrin (NCDN) gene, located at 21.73 Mb may be involved in the development of OC. One SNP of this gene was significant associated for every phenotypic trait, except for hock OCD. This neurochondrin is probably involved in signal transduction in the nervous system and might play a role in bone metabolism and chondrocyte differentiation (Mochizuki et al. 2003). Due to its function, the NCDN gene could be regarded as a functional candidate gene. The exostoses-like 1 (EXTL1) gene, at 29.43 Mb, a candidate gene for the development of exostoses in human, might also play a major role in the etiopathogenesis of osteochondral lesions. Mutation and expression analyses of these genes would be helpful in order to clarify the role of the NCDN and EXTL1 genes in the genesis of developmental osteochondral diseases in horses.
The QTL on ECA4 could be delimited to an interval between 4.92 and 39.76 Mb for OC (fetlock and/or hock joints), between 7.42 and 13.10 Mb, at 27.15 and 28.79 Mb and between 56.15 and 59.84 Mb for fetlock OC and between 3.62 and 6.34 Mb for hock OC by the use of 74 microsatellites and 26 SNPs in total. In addition, three significantly associated SNPs could be identified at 13.10 Mb, 14.08 Mb, and 15.99
Mb. The GLI-Kruppel family member 3 (GLI3), the HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 (HECW1) gene and receptor (G protein-coupled) activity modifying protein 3 (RAMP3) genes are in close proximity and might play a role in the development of equine OC. GLI3 is a candidate gene for different kinds of polydactyly (Radhakrishna et al. 1999). Wang et al. (2000) demonstrated that cAMP-dependent protein kinase cAMP-dependent processing of vertebrate GLI3 in developing limb generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. Mutations of the GLI3 gene may produce a range of limb patterning malformations. Previous expression analyses showed no significant difference in expression of GLI3 in affected joints compared with unaffected cartilage (Semevolos et al. 2005). The HECW1 gene encodes a cytoplasmic phosphoprotein that regulates cell proliferation and acts as a transducer molecule for developmental processes, including segmentation and neuroblast specification. Till know, no associations between mutations of this gene and equine OC could be found. The RAMP3 gene interacts with human and porcine calcitonin receptor-like receptor (CRLR) in HEK-293 cells (Aiyar et al. 2001).
Calcitonin is involved in control of bone metabolism and therefore, RAMP3 seems to be a suitable functional candidate gene for OC. Further studies are necessary to evaluate their effective role in this complex disorder.
Nowadays, SNPs are the preferred genetic marker for large-scale genetic mapping projects and have successfully been used to identify chromosome regions associated with complex human diseases. Since 2008, a commercially available equine SNP microarray containing more than 50,000 SNPs enables genome-wide association analyses with SNPs in horses. The object using such a powerful platform was to verify detected QTL of previous genome scan and fine mapping studies and to identify new genomic regions harbouring potential candidate genes for the development of OC in Hanovarian warmblood horses. The analysis revealed a large number of significant associated SNPs distributed over the whole genome. Further statistical calculations were required in order to localize significant regions. The BeadChip implies an enormous progress in moleculargenetic research, but complexity and problems of interpretation may not be underestimated. Furthermore,
a distuingished trait classification is an indispensable assumption regarding such complex diseases like OC. However, high density of informative markers and further statistical calculations allowed refined QTL localization and consequently an improved candidate gene detection. Performed variance analyses enabled the conformation of known significant regions on chromosomes 2, 4 and 16. In addition, we could detect new QTL on chromosomes 3, 5, 7, 16, 19, 20, 22, 26 and 29.
Previously unknown potential candidate genes could be taken into consideration being involved in the development of equine OC. Following studies have to validate these results with the help of an expanded sample composition and with further breeds in addition to Hanoverian warmblood horses.
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CHAPTER 7
Summary
7 Summary