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Title: The molecular characterisation of TIMP3 mutations responsible for Sorsby's Fundus Dystrophy : is there a link to Age-related Macular Degeneration?
Author: Alsaffar, Fatimah
ISNI:       0000 0004 6347 5046
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Sorsby's fundus dystrophy (SFD) is a rare autosomal, dominantly inherited, degenerative disease of the retina that results in a loss of central vision in middle age. The disease is a single-gene disorder caused by specific mutations in the tissue inhibitor of metalloproteinase3 (TIMP3) gene on chromosome 22. The SFD phenotype is very similar to age-related macular degeneration (AMD), a very common cause of blindness in the elderly. These phenotypic similarities include drusen, thickened Bruch's membrane, atrophy of choroid and photoreceptors and neovascularisation. While AMD is a multifactorial disorder that is not associated with mutations in TIMP3, increased levels of TIMP3 protein are observed in the retina of both diseases indicating TIMP3 could still play a role in this disease. Although the disease mechanism responsible for the SFD phenotype is still uncertain, the evidence implies that the phenotype results from a toxic effect of the mutant TIMP3, rather than haploinsufficiency. At the start of this project, ten of the known mutations were localised to the coding sequence of exon 5 comprising most of the C-terminal domain of the molecule and demonstrated to result in dimeric TIMP3. There were also two exceptional mutations, including a mutation in exon 1 that encodes part of the N-terminal domain (Ser15Cys), and a splice site mutation caused by a single adenosine insertion at the intron4/exon5 junction. Recently, three additional mutations, including Tyr128Cys, Tyr154Cys and Tyr159Cys, were also identified in exon 5 but their effects on TIMP3 had not been examined. Thus, the consequences of these exceptional mutations and the newly identified C-terminus mutations on TIMP3 were still unknown. We hypothesised that both SFD and ageing result in cross-linking of TIMP3 leading to formation of a toxic molecule that may play a role in both diseases. The initial aim of this project was to investigate the consequence of the TIMP3 splice site mutation and these other recently described mutations on the TIMP3 protein. The second aim was to examine the effects of SFD mutation on a number of potential target molecules and downstream pathways. These included the interaction with tumour necrosis factor a converting enzyme (TACE) and its consequence for retinal pigment epithelial cell apoptosis; with vascular endothelial growth factor receptor 2 (VEGFR2) and its role in angiogenesis; with epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1), mutations in which also cause an inherited retinopathy, and finally to examine potential activation of the receptor for advanced glycation end-products (RAGE). This research project confirmed that all examined TIMP3 mutations expressed by human retinal cells formed dimers, including the novel splice site mutant, which resulted in the formation of several abnormally spliced truncated products. Dimerisation, therefore, almost certainly plays a crucial role in the SFD disease process. Retinal pigment epithelial cells expressing these mutant proteins were more sensitive to Fasinduced apoptosis than those expressing the normal protein, although we were unable to confirm if this was mediated by increased avidity for TACE. Moreover, mutant TIMP3 proteins had a pro-inflammatory effect relative to the wild-type molecule, as determined by NF-?B activation, likely mediated by RAGE, although this still needs confirmation. Mutant proteins were, however, less effective at inhibiting VEGF-induced endothelial cell invasion than their wild-type counterpart, almost certainly due to decreased ability to inhibit VEGFR2, and this could explain the choroidal neovascularisation that accompanies SFD, despite the high levels of TIMP3 protein present in the retina. Mutations in TIMP3, responsible for SFD and in EFEMP1, responsible for Malattia Leventinese, did not appear to impair their mutual interaction and we speculate that increased deposition of TIMP3/EFEMP1 complexes are a common feature of SFD, Malattia Leventinese and age-related macular degeneration, and this may in turn lead to sequestration of complement factor H, a known binding partner for EFEMP1, impairing regulation of complement activation in the retina. If this proves to be the case, targeting the interaction between these three molecules may provide a novel therapeutic strategy for treating these currently intractable diseases.
Supervisor: Barker, Mike ; Nicklin, Martin Sponsor: Ministry of Higher Education, Saudi Arabia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available