Carta, Eloisa; (2008) The glycine transporter GlyT2: Mutations, interactors and diseases. Doctoral thesis (Ph.D.), University College London (United Kingdom).

Text The_glycine_transporter_GlyT2.pdf Download (23MB)

Abstract

Hyperekplexia or startle disease (OMIM: 149400) is caused by defects in mammalian glycinergic neurotransmission, resulting in a complex motor disorder characterised by neonatal hypertonia and an exaggerated startle reflex. This affects newborn children and is characterised by noise or touch-induced seizures which result in muscle stiffness and breath-holding episodes that can lead to brain damage and/or sudden infant death. Hyperekplexia is caused by mutations in the glycine receptor al and p subunit genes. However, a significant proportion of hyperekplexia patients do not have mutations in these genes. Mutations in the presynaptic glycine transporter 2 (GlyT2) gene, SLC6A5, are a recently discovered cause of hyperekplexia (Rees et al, 2006). This work aims to study the functional consequences of novel SLC6A5 mutations, to characterise new GlyT2 interacting proteins as candidates for genetic studies in hyperekplexia, and to determine whether any animal disorders linked to glycinergic dysfunction might have defects in SLC6A5. The functional consequences of hyperekplexia mutations in SLC6A5 were assessed using [3H]-glycine uptake assays and immunocytochemistry and revealed defective glycine uptake compared to controls, often resulting from aberrant trafficking of the transporter to the cell membrane. Using the GAL4 yeast two-hybrid (YTH) system, seven new proteins potentially involved in the localisation and trafficking of GlyT2 were identified, of which the cytoskeletal protein FMNL2 appears to be the most promising candidate for genetic analysis in hyperekplexia. Lastly, a new example of GlyT2 dysfunction in animals was characterised: congenital muscular dystonia 2 (CMD2) in Belgian blue cattle. Whole-genome screening and DNA sequencing of candidate genes revealed a missense mutation in SLC6A5, resulting in a L270P substitution in the third membrane-spanning domain of GlyT2. Functional studies showed that this alteration abolishes [3H]-glycine uptake in recombinant systems. Identification of this mutation had an immediate translation into breeding practice, allowing marker assisted selection against CMD2 by avoiding 'at risk' matings.

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