Patients affected by DEB suffer from loss of adhesion between the epidermis (outer layer of the skin) and the dermis (inner layer). This results in severe blistering of the skin and mucosa after mild trauma and is evident from birth. The extreme fragility of the skin results in traumatic blistering, wounds and scarring. These lead to increasing disfigurement, deformity and disability.

DEB is caused by abnormalities in the type VII collagen gene (COL7A1) encoding anchoring fibrils which form attachment structures playing a key role in the adhesion of the epidermis to the dermis. The majority of patients do not produce type VII collagen protein because they have inherited a deficient COL7A1 gene from each of their parents. The identification of COL7A1 as the defective gene in 1991 has allowed searches for mutations in patients and their family members to be carried out.

Identification of the COL7A1 defect in a given family has important implications for genetic counselling eg the detection of carriers, assessment of the mode of inheritance and early prenatal diagnosis (11 weeks of gestation) in affected families. It is also the first step towards genetic correction of the defect, since it enables the presence of a normal copy of the gene, following gene transfer, to be distinguished from the mutated copy.

The research will also look at some of the control mechanisms that may act as accelerators or brakes in controlling the expression of genes in EB skin. The project is expected to improve the understanding of how the inherited gene abnormalities lead to skin fragility as well as providing useful information for genetic counselling, antenatal diagnosis and a baseline for planning newer forms of treatment, including gene therapy.

Recent years have seen considerable progress in determining the precise molecular basis for the different forms of EB. In both the dominant and recessive forms of dystrophic EB mutations occur in the type VII collagen gene, COL7A1. More than 100 pathogenetic COL7A1 mutations have been reported and some specific genotype-phenotype correlations have emerged.

Dr McGrath’s laboratory has been involved in mutation detection in the dystrophic and junctional forms of EB since 1996. In that time they have characterised more than 80 different COL7A1 mutations in the various forms of dystrophic EB and over 40 different mutations in junctional EB. Some of these mutations have proved to be recurrent mutations in several individuals, and if these are taken into account, collectively they have delineated the molecular basis of EB in more than 160 sufferers.

Although the world literature now contains a substantial number of mutations in COL7A1, LAMA3, LAMB3, LAMC2, COL17A1, ITGA6 and ITGB4, the reasons for searching for mutations in these genes which will be of continued benefit to dystrophic and junctional EB sufferers and their families are:

• New cases of dystrophic and junctional EB will continue to be born or referred for clinical assessment. Molecular analysis can provide clinically relevant information about diagnosis, prognosis and genetic counselling.
• Although paradigms have been established for genotype-phenotype correlations, it is clear that unusual mutations that provide exceptions to the rules continue to occur. Studies of such mutations may provide further information about gene expression and protein interactions at the cutaneous basement membrane zone.
• DNA-based prenatal diagnosis has become a major advance for families at risk for recurrence of the severe forms of dystrophic or junctional EB. In many of these cases, and as a mandatory requirement in junctional EB, mutation detection prior to testing is a prerequisite for undertaking the procedure.

• As strategies for gene manipulation are developed for dystrophic and junctional forms of EB, it is probable that there will be several different approaches to restoring function of the defective gene. Knowledge of the precise gene mutation in a given individual will be helpful in determining the most appropriate strategy to pursue.

The aim of this project is to be able to continue to undertake mutation analysis of the candidate genes for dystrophic and junctional EB. The plan is to extend this work by looking at the consequences of certain splice site mutations, missense mutations or silent polymorphisms on RNA processing to try to provide a fuller understanding between genotype and phenotype. Finally, their extensive COL7A1 mutation screening has shown that using a combination of mutation detection strategies (protein truncation, chemical cleavage of mismatch and heteroduplex analysis) they are able to detect about 87% of all mutations. Clearly, further mutations exist that have not yet been identified, either because of the sensitivity of the screening methods or because the mutations lie elsewhere.