Candidate genes for osteochondrosis

The whole genome scans were the first step towards the identification of genomic regions harbouring genes responsible for equine OC.

To select potential candidate genes, different information can be used.

Candidate genes are genes which code for 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).

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 (Table 3). 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 genes or MMP13 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.

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 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 interdependecy 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 target 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 2005b). Additional evidence for the crucial role of collagen was provided by the demonstration of differences in post-translational modifications of 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.

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-chondrocytes (Garvican et al. 2008, Kuroki et al.

2005, Mirams et al. 2008). 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 osteoarthritis joints.

The proteins encoded by the TIMP (tissue inhibitor of metalloproteinase) gene family are natural inhibitors of the matrix metalloproteinases (MMPs), and therefore vindicate 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 the 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

aetiopathogenesis of osteochondrosis further complicates the identification of candidate genes but leaves an ample scope. For this reason it is so much the better to delineate the QTL, so the number of potential candidate genes might be limited just by delimited genomic regions.