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Identification and analysis of mutations in the TSC1 gene

Jeganathan, Dharini; (2000) Identification and analysis of mutations in the TSC1 gene. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Tuberous Sclerosis Complex (TSC) an autosomal dominant disorder which may cause many abnormalities including skin rashes, renal lesions, seizures, learning disability and behavioural problems is caused by mutations in either of two genes TSC1 or TSC2. This thesis focuses on the analysis of the recently cloned TSC1 gene. Mutation screening revealed that virtually all TSC1 mutations are predicted to truncate the protein product. This provided a model system for the investigation of nonsense mediated mRNA decay (NMD), an enigmatic surveillance mechanism that may offer the cell some protection against such "dominant-negative" nonsense mutations. Analysis of lymphoblastoid cell lines in seven TSC1 patients showed a striking reduction of the mutant transcript compared to the normal transcript in five cases. Coding and newly identified 3' UTR polymorphisms in TSC1 were utilised in the development of an assay to investigate imbalance between allelic transcripts in TSC patients. In doing so, it was demonstrated that the observed reduction of allele-specific RNA in TSC1 patients was not in general the result of exon skipping but the result of lower levels of the whole mutant transcript caused by the premature termination codon (PTC). In addition, the normal range of allelic imbalance in control samples was established. Based on these findings, the correct identification of six out of seven TSC1 patients tested blind from a panel of TSC1 and TSC2 patients was achieved with no false positives. Fresh blood samples were obtained from a panel of TSC patients to evaluate this approach as a diagnostic tool. Here, the discrimination between alleles was less sensitive with RNA obtained directly from blood samples than from lymphoblastoid lines. It was also found that an alternative transcript lacking exon 5 of TSC1 was a prominent feature of RNA from fresh lymphocytes. A further series of experiments on lymphoblastoid cell lines gave rise to three main findings of general interest. Firstly, the position of the mutation within the gene could not be used to predict the extent of NMD. Secondly, patients with the same mutation, whether or not known to be related, appeared to have comparable levels of NMD implying that the variation in severity seen within the same family is unlikely to be the result of variation in NMD. Thirdly, exposure of the cells in culture to puromycin, a translational inhibitor, reduced the imbalance between 'normal' and 'mutant' transcript. This suggested that NMD depends on translation or translational associated machinery, where its control may be determined directly by the mutation or by another factor yet to be identified. Overall, the findings suggest that from a mutation screening point of view, NMD may be a useful tool where cell-lines are available. Inspection of the literature shows that NMD is the rule rather than the exception for mutations which cause PTCs. Thus, in the future, with the advent of a dense SNP map of transcribed regions, it may be possible to take a similar approach to the assessment of candidate genes for diseases whose cause is still unknown.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Identification and analysis of mutations in the TSC1 gene
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Tuberous Sclerosis Complex
URI: https://discovery.ucl.ac.uk/id/eprint/10100389
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