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Title: Molecular and functional characterisation of wildtype and mutant bestrophin-1 associated with retinal disease
Author: Davidson, Alice Elizabeth
ISNI:       0000 0004 2688 1918
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2010
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BEST1 encodes bestrophin-1, an integral membrane protein expressed in the basolateral membrane of retinal pigment epithelial (RPE) cells. Bestrophin-1 is hypothesised to function as a calcium-activated chloride channel (CaCC) (Sun et al. 2002) and/or a modulator of voltage-dependant calcium channels (VDCC) (Rosenthal et al. 2006) in the RPE. Mutations in BEST1 have previously been linked to a number of retinal dystrophies including Best disease (Marquardt et al. 1998; Petrukhin et al. 1998), ADVIRC (Yardley et al. 2004) and ARB (Burgess et al. 2008b). The aim of this work was to advance understanding of the spectrum of ocular disorders associated with BEST1 mutations. Novel BEST1 mutations were identified in Best disease and ARB patients. The identification of eleven ARB patients from eight separate families with either proven or presumed biallelic mutations in BEST1 supports previous data that ARB is caused by biallelic mutations in BEST1. The identification of homozygous nonsense mutation in BEST1 in a patient with ARB has greatly strengthened the hypothesis that ARB represents the null bestrophin-1 phenotype in humans. The work presented here also demonstrates that a greater range of retinal dystrophies, including concentric RP and severe early onset retinal dystrophies are associated with mutations in BEST1 expanding the clinical heterogeneity known to be associated with BEST1 mutations. Functional assays have been used to investigate the pathogenic effects of disease-associated BEST1 mutations. Whole-cell patch-clamp analysis has shown that ARB-associated BEST1 mutants inhibit the CaCC activity of wildtype bestrophin-1 to a lesser extent than Best disease-associated mutants, confirming the recessive and dominant inheritance patterns of the two diseases. The study of bestrophin-1 and disease-associated mutants in a polarised cell line reveals that many mutant isoforms have disrupted localisation and stability in vitro. The pathogeniCmechanisms contributing to bestrophinopathies therefore may, in many instances, be attributed to aberrant protein folding, stability and localisation Notably, ARB-associated bestrophin-1 isoforms were shown to be rapidly degraded via a proteasomaldependant mechanism. Rapid degradation of bestrophin-1 isoforms leading to reduced levels of functional bestrophin-1, unable to sustain normal physiological function may explain the loss of function phenotype displayed by ARB patients with biallelic missense mutations in BEST1. In conclusion the data presented here has advanced understanding about the spectrum of retinal dystrophies associated with mutations in BEST1 in addition to elucidating pathogenic mechanism that contribute to the bestrophinopathies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available