Shafie, Intan Nur Fatiha
The establishment of potential cerebrospinal fluid biomarkers for canine degenerative myelopathy.
PhD thesis, University of Glasgow.
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Canine degenerative myelopathy(DM) is a late onset neurodegenerative disease that primarily affects German Shepherd dog (GSD), though a number of other specific breeds
are also affected. The underlying cause of the disorder remains elusive, though recent advances have implicated a mutation of superoxide dismutase 1(Sod1) in the aetiology, also implying DM is a potential orthologue of human amyotrophic lateral sclerosis. The identification of the Sod1 mutation raises the index of suspicion for an individual animal, however it is not specifically diagnostic as a proportion of dogs homozygous for the Sod1 mutation do not develop DM. Therefore, there is a clinical need for the development of specific biomarker(s) for DM to support genetic test. The aim of this study was to establish potential biomarkers for DM by exploring canine cerebrospinal fluid (CSF). A dual strategy was adopted;1) Evaluation of potential ALS biomarkers in DM CSF, 2) Identification of novel biomarker(s) in DM CSF. The cases selected in this project had a presumptive diagnosis of DM and were homozygous for Sod1 mutation. Preliminary characterisation by Western blot and mass spectrometry identified four protein candidates in DM CSF, comprised of cystatin C, transthyretin (dimeric and monomeric TTR), haptoglobin and clusterin. Since the validity of these putative biomarkers may be influenced by pre-analytical variables that may arise from the clinical environment, we therefore assessed the impact of three potential sample handling practices on these four proteins. The results from these experiments demonstrate that dimeric TTR and clusterin were affected by sample handling conditions. Therefore, an appropriate protocol for CSF sample handling was established. Western blot analyses indicated that clusterin is the most viable biomarker candidate for DM. Clusterin was significantly elevated in DM CSF when compared to a range of neurological conditions. The second potential candidate for DM biomarker is TTR, which is potentially reduced, an observation similar to those found in ALS CSF. The relationship of these proteins in the pathogenic mechanisms that underpin DM is unclear. However,
based on observations on ALS, it is reasonable to speculate that their alterations are associated with a toxic gain of function of the mutant SOD1 protein. The successful
characterisation of clusterin and TTR in DM CSF may therefore represent components of a panel of emerging biomarkers that may combine to distinguish DM in the clinic and provide further insights into the disease mechanisms.
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