James, Victoria; (2012) Molecular insights into pathogenic mechanisms in startle disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study describes an in-depth investigation into the pathogenic mechanisms of inherited mutations that lead to disorders of inhibitory glycinergic transmission, primarily the rare human disorder known as startle disease/hyperekplexia. I also investigated mutations causing a similar startle phenotype in cows, mice and zebrafish. Using molecular genetics techniques, I identified pathogenic mutations in the genes that encode for proteins involved in glycinergic neurotransmission, specifically the postsynaptic glycine receptor (GlyR) subunits and the presynaptic glycine transporter GlyT2. Using homology modelling and other computational biology methods, I examined the structural and functional impacts of mutations on protein function, revealing key motifs and amino acids crucial for receptor and transporter activity. Using cDNA cloning and site-directed mutagenesis, I also generated expression constructs for wild-type and mutant proteins that were used in functional tests to measure the impact of pathogenic mutations on glycine receptor and transporter function. For certain animal models of startle disease, I was also able to develop diagnostic PCR tests for pathogenic mutations, which can be used to alleviate further animal suffering by preventing 'at risk matings' of carrier animals. Taken together, my findings reveal several new pathogenic mechanisms of GlyR and GlyT2 mutations in startle disease in humans and animals, revealing insights into receptor and transporter function that may be applicable to other neurological disorders.

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