Epidermolysis bullosa (EB) is a group of inherited blistering diseases that share the common feature of blister formation subsequent to normal mechanical insult of the epidermis. Despite two decades of investigation at both epidemiological and genetic levels, there remains much yet to be uncovered about the pathophysiology of this disease. This research had dual aims. Firstly, by enrolling patients in Australia and New Zealand with the simplex type of EB (EBS) in a screening programme in conjunction with a highly detailed review of the EB and intermediate filament literatures, we hoped to gain a better understanding of the correlation between genotype and phenotype in EBS. Secondly, we attempted to evaluate the use of baculoviral vectors as a means of introducing the type VII collagen cDNA into human dermal fibroblasts and primary human epidermal keratinocytes. Among the EBS patients screened we identified a novel multi residue deletion in keratin 5 which led to a surprisingly mild form of epidermolysis bullosa Dowling ??? Meara. The quantitative polymerase chain reaction analysis of epidermal tissue from the affected individual showed that the mutant transcript was present at levels 2.7 fold higher than the wild type transcript. We demonstrated a family in which the presence of a previously reported keratin 14 mutation A413T appeared to induce disease expression in only one of the three individuals found to carry the mutations. We also identified two EBS families with no mutations in either keratin 5 or keratin 14. This is the first study to analyse EB patients from New Zealand and the first to report the presence of a deletion mutation in the 2B domain of keratin 5. The results of our type VII collagen transfer work showed that baculoviruses can be engineered to contain insert sequences in excess of 10kb and are able to gain entry to both human dermal fibroblasts and primary human epidermal keratinocytes. Electron microscopy and analyses of the baculoviral vectors showed that the lack of protein expression subsequent to infection of human dermal fibroblasts and primary human epidermal keratinocytes is likely due to the inability of the vector to escape cytoplasmic endosomes.
| Identifer | oai:union.ndltd.org:ADTP/258844 |
| Date | January 2005 |
| Creators | Kemp, Matthew W, St. George Clinical School, UNSW |
| Publisher | Awarded by:University of New South Wales. St. George Clinical School |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Matthew W Kemp, http://unsworks.unsw.edu.au/copyright |
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