Genexpressionsmuster von Epidermolysis bullosa

  • Krammer, Barbara, (Projektleitung)



    Epidermolysis bullosa (EB) covers a large group of inherited skin disorders characterized by skin fragility in the cutaneous basement membrane zone. Based on the location of disruption in the basement membrane three main types of EB can be determined. The different forms of EB have been linked to mutations in no less than ten distinct genes encoding the major structural basement membrane zone proteins involving keratins, collagens, laminin, plectin and integrins. Whereas the gene mutations of the various EB subtypes are well documented, a comprehensive gene expression profiling is still lacking. The aim of the present project is to study via a detailed and comprehensive approach using cDNA arrays covering 9738 ESTs and subsequent verification by real-time RT-PCR, which genes are differentially expressed as a consequence of the mutations present in junctional and dystrophic EB. This approach should provide the broad basis for a therapeutical utilization of gene expression regulation.

    For implementation of the research approach to screen a large number of genes for their expression, cDNA arrays shall be used. The verification of the results shall be carried out by real-time-RT-PCR.
    cDNA arrays with about 9738 human expressed sequence tags (ESTs) (Incyte Human UniGEM Microarray clone set) shall be constructed and hybridized with the isolated and radioactively labeled mRNA. After statistical evaluation of the hybridization spots, selected genes with an up- or down-regulation by at least a factor of two compared to “normal” samples will be verified by real-time-RT-PCR.
    Gene expression profiles shall be established of
    1) EB-related cell lines: type XVII collagen-deficient cell line (GABEB keratinocytes [12]), plectin-deficient cell line and a type VII collagen-deficient cell line (in cooperation with the head of the molecular biological labs of the EB-Center, SALK Salzburg, Doz. Dr. Johann Bauer) and of
    2) patient samples: still vital skin waste or part of biopsies, collected from the three different types of EB.
    As “normal” samples, the human keratinocyte cell line HaCat and waste skin samples from healthy patients (foreskin) shall serve.

    The aim of the project is therefore to investigate in a detailed and comprehensive approach using cDNA arrays and verification by real-time-PCR, which genes are up- and down-regulated as a consequence of mutated genes present in junctional or dystrophic EB. Differential expression of deficient cell lines, representative for EB mutations in plectin, type VII collagen or type XVII collagen and waste samples of patients representing different types of EB, related to the respective controls, shall be analyzed and compared to each other.

    Since it is conceivable that changed gene expression can be found which compensates for missense mutation or lack of genes, it shall be investigated in cooperation with the team of the EB-Center, SALK (see below), how this regulation can therapeutically be used or supported.
    KurztitelGenexpressionsmuster von EB
    Tatsächlicher Beginn/ -es Ende1/03/0828/02/10

    Systematik der Wissenschaftszweige 2002

    • 3211 Medizinische Molekularbiologie