Future studies

The next stage of research into chondrodysplasia of Texel sheep would likely involve sequencing of CHST11, the gene encoding chondroitin 4- sulphotransferase-1, in chondrodysplastic and unaffected Texel sheep in an attempt to find a causative mutation and therefore allow development of a diagnostic test. The histological and biochemical studies in this thesis have indicated that the pathology of chondrodysplasia in Texel sheep is consistent with a potential defect in the function of chondroitin 4-sulphotransferase, while current and earlier genetic studies combined with biochemical findings and fibroblast sulphate uptake in vitro suggest that genes involved in earlier steps of the cartilage proteoglycan sulphation pathway are less likely to be involved.

Should sequencing of CHST11 fail to find a mutation causing chondrodysplasia of Texel sheep, another chromosome or genome scanning experiment could be conducted. This would require a new breeding trial designed to provide an increased number of samples from a new pedigree containing a large number of unaffected relations to affected animals, and a greater marker density than that used in the initial linkage disequilibrium study. Ongoing genomics research will result in more markers being identified within the ovine genome, including both microsatellites and single-nucleotide polymorphisms (SNPs) (Cockett, 2006; Pariset et al., 2006). This, combined with the increasing availability and cost- effectiveness of large-scale scanning techniques such as SNP-chips, will increase the potential sensitivity of future scanning studies within the ovine genome. A more cost-effective alternative may be to work closely with Texel breeders known to have had affected animals in their flocks. A major difficulty would lie in farmer compliance, since the identification of a genetic disease on a stud farm generates significant stigma. Additionally, pedigree accuracy may not be assured, although genotyping of parents and offspring would be able to reinforce the pedigree information. A scanning study could then be followed up by candidate gene analysis, preferably supported by knowledge of gene function and its potential involvement with the pathogenesis of the disease. Once the causative mutation has been identified, a genetic test to detect phenotypically normal carrier sheep may be developed, enabling eradication of the mutant gene from farms if the allele frequency is low, or control of the disease by breeding only from tested sires if the allele frequency is very high. A genetic test may also be used to determine whether the gene for chondrodysplasia in Texel sheep can be found in Texel populations in countries such as Denmark and Finland, from which the founding embryos for the New Zealand Texels were sourced.

If a new breeding trial is not feasible, and an appropriate collection of samples cannot be obtained from farms, the difference in chondroitin sulphate disaccharide ratios between chondrodysplastic and unaffected sheep could be used to increase the number of usable samples from the original back-cross trial by providing phenotypic information about the tissue collected from newborn lambs produced in that trial. This would increase the power of genetic analyses even though the pedigree structure was not ideal. To do this, the capillary electrophoresis experiment would ideally be repeated on a larger number of samples from animals of known phenotype in order to adequately assess the normal variation in the ratio

of ∆di-mono4S to ∆di-mono6S between animals. This would determine whether the ratio itself can be used as a diagnostic tool, or if it would be more properly used as a quantitative trait in scanning genetic studies designed to test for quantitative trait linkage (QTL testing). Unfortunately, the generation of newborn lambs known to be affected by chondrodysplasia is difficult since it requires both parents to be affected by the disease, and many chondrodysplastic Texel sheep do not survive to maturity. Because of this, it would be difficult to gain enough samples to thoroughly test the diagnostic power of the ∆di-mono4S to ∆di-mono6S ratio in newborn lambs.

Further biochemical characterisation of chondrodysplasia of Texel sheep in order to help identify candidate genes, either alone or in conjunction with genetic scanning studies, may be of use in ongoing research. These could include an assessment of PAPSS function in vitro using radiolabelled sulphate to quantify PAPS (Rossi et al., 1996a). This experiment would test the integrity of the proteoglycan sulphation pathway up to and including PAPSS function. Alternatively, mRNA analysis could be used to measure the level of expression of various enzymes and transporters involved in the sulphation pathway.

A potential future study on the development of chondrodysplasia in Texel sheep could consist of serial capillary electrophoresis studies using cartilage biopsies to determine the way in which the ratio of ∆di-mono4S to ∆di-mono6S changes with age in chondrodysplastic Texel sheep, and whether the deviation from age-matched control cartilage is influenced by the severity of the disease. This would provide more information about the pathogenesis of the lesions in chondrodysplasia of Texel sheep, and may help to elucidate the relationship between the altered ratio in other diseases and their development.