Browsing by Subject "Retinitis pigmentosa"

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    Open Access
    An investigation into the molecular mechanisms underlying retinitis pigmentosa 17 with the view to developing novel gene- based therapies
    (2012) Pandor, Aisha
    Retinitis pigmentosa (RP) is a highly heterogeneous form of inherited blindness that affects more than 1.3 million individuals worldwide. The RP17 form of the disease is caused by an arginine to tryptophan (R14W) mutation in the signal sequence of carbonic anhydrase IV (CAIV). In an effort to elucidate the molecular mechanisms underlying RP17, three cell types were transfected with the wild type (WT ) and the R14W mutant form of the protein. We show using immunocytochemistry that unlike transfected WT CAIV which is transported to the plasma membrane of transfected COS-7 and HT-1080 cells, R14W mutant CAIV is retained in the endoplasmic reticulum when transfected into the same cell type. Further analyses of these cells by western blotting reveal that whereas the WT CAIV is processed to its mature form in both these cell lines, significant levels of the R14W mutant protein remain in its immature form. Importantly, flow cytometry experiments demonstrate that compared to WT CAIV protein, expression of specifically the R14W CAIV results in an S and G2/M cell cycle block, followed by apoptosis. Interestingly, when the above experiments were repeated in the human embryonic kidney cell line, HEK-293, strikingly different results were obtained. These cells were unaffected by the expression of the R14W mutant CAIV and were able to process the mutant and WT protein equally effectively. These findings regarding cell type specificity were used as a basis to explore methods of therapy for RP17. In particular, allele-specific small hairpin RNA was used to silence expression of R14W mutant CAIV, and to rescue cells from undergoing cell cycle arrest and apoptosis. A study of specific chaperones involved in protein folding, as well as gene and protein expression studies (microarray and mass spectrometry analysis), were also carried out to determine which proteins that were expressed in HEK-293 cells play a part in the ability to fold, process and transport R14W mutant CAIV. The results of this study have important implications for our understanding of the RP17 phenotype, and in investigating gene and protein therapy for the prevention and treatment of RP17.
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    Open Access
    A mutation in a splicing factor that causes retinitis pigmentosa has a transcriptome-wide effect on mRNA splicing
    (BioMed Central, 2014-06-27) Korir, Paul K; Roberts, Lisa; Ramesar, Raj; Seoighe, Cathal
    Background: Substantial progress has been made in the identification of sequence elements that control mRNA splicing and the genetic variants in these elements that alter mRNA splicing (referred to as splicing quantitative trait loci – sQTLs). Genetic variants that affect mRNA splicing in trans are harder to identify because their effects can be more subtle and diffuse, and the variants are not co-located with their targets. We carried out a transcriptome-wide analysis of the effects of a mutation in a ubiquitous splicing factor that causes retinitis pigmentosa (RP) on mRNA splicing, using exon microarrays. Results: Exon microarray data was generated from whole blood samples obtained from four individuals with a mutation in the splicing factor PRPF8 and four sibling controls. Although the mutation has no known phenotype in blood, there was evidence of widespread differences in splicing between cases and controls (affecting approximately 20% of exons). Most probesets with significantly different inclusion (defined as the expression intensity of the exon divided by the expression of the corresponding transcript) between cases and controls had higher inclusion in cases and corresponded to exons that were shorter than average, AT rich, located towards the 5’ end of the gene and flanked by long introns. Introns flanking affected probesets were particularly depleted for the shortest category of introns, associated with splicing via intron definition. Conclusions: Our results show that a mutation in a splicing factor, with a phenotype that is restricted to retinal tissue, acts as a trans-sQTL cluster in whole blood samples. Characteristics of the affected exons suggest that they are spliced co-transcriptionally and via exon definition. However, due to the small sample size available for this study, further studies are required to confirm the widespread impact of this PRPF8 mutation on mRNA splicing outside the retina.