Manwaring, VJ; (2014) The identification of potential diagnostic biomarkers amd disease mechanisms in lysosomal storage disorders. Doctoral thesis , UCL (University College London).

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Abstract

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme alpha-galactosidase A. The resultant progressive intracellular accumulation of the glycosphingolipids globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are the source of a variety of clinical consequences. Biomarkers capable of detecting FD at an early stage of disease progression and that enable monitoring of response to treatment are required urgently. Using label-free quantitative proteomics two potential biomarkers, proactivator polypeptide and ganglioside GM2 activator protein, were identified in the urine of paediatric FD patients. An ultra-performance liquid chromatography-tandem mass spectrometry assay was then developed for validation purposes. Subsequently a second, larger multiplexed assay was developed to identify biomarkers capable of detecting and monitoring pre-symptomatic kidney disease in those patients most at risk. A complementary metabolomic approach was also used to identify and evaluate new Gb3-related biomarkers in the plasma of FD patients. This aspect of the study revealed five novel Gb3-related biomarkers as well as existing Gb3-related analogues and isoforms providing a metabolic profile in these patients. Potential disease mechanisms involved in FD were investigated by studying the interactions between proteins and those molecules involved in the glycosphingolipid pathway, with particular interest to Gb3. A number of mitochondrial proteins were found to interact with Gb3 resulting in the investigation of the effect of glycosphingolipids and their deacylated counterparts on ATP synthase activity. Increasing concentrations of Gb3 and lyso-Gb3 were shown to increase ATP synthase activity. Subsequently the activities of the enzymes preceding ATP synthase, the mitochondrial respiratory chain enzymes, were investigated in a Fabry mouse model. In this aspect of the study complex II/III activity was found to be significantly decreased in kidney tissues. Finally, assessment of pain thresholds in mice has demonstrated significant increases in sensitivity to applied mechanical stimuli following exposure to Gb3 and lyso-Gb3.

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