Mutation detection and correction experiments in epidermolysis bullosa simplex

This thesis deals with DNA diagnosis of epidermolysis bullosa simplex (EBS) and a potential gene therapeutic approach of this disease

Partial revertant mosaicism of keratin 14 in a patient with recessive epidermolysis bullosa simplex

A patient with recessive epidermolysis bullosa simplex due to a previously described homozygous KRT14 1842-2A-->C splice-site mutation was re-examined, because we unexpectedly found signs of revertant mosaicism. The germline mutation resulted in different aberrant transcripts containing premature termination codons, all leading to truncated keratin 14 proteins. Basal keratinocytes in skin and in culture completely lacked keratin 14 and intermediate filaments. From this keratin 14-/- patient we started cultures from a new skin biopsy and here, we serendipitously found keratinocytes that spontaneously expressed keratin 14. This biopsy had been taken from an area of skin that was clinically affected, because blisters could simply be evoked by gentle rubbing. Immunofluorescence and electron microscopy of additional biopsies from this skin area revealed a mosaic expression of keratin 14 and reappearance of intermediate filaments in basal keratinocytes. Immunoblotting showed a revertant keratin 14 polypeptide with seemingly normal molecular weight. DNA analysis of exon 2 and its flanking intron borders showed no additional mutations in the genomic KRT14 sequence. Analysis of mRNA isolated from mosaic skin keratinocytes revealed an additional in-frame transcript (1844T-->G, 1845Delta6) that codes for an abnormal keratin 14 polypeptide with a two residue deletion and one amino acid change. The re-expression of a revertant, albeit abnormal, keratin 14 polypeptide, so-called partial revertant mosaicism, accounts for the antibody staining pattern and for the reappearance of intermediate filaments, which however, are semifunctional and not able to revert the clinical phenotype. The combination of a keratin 14-positive and a keratin 14-negative cell population in epidermis as well as in cultured keratinocytes suggests that the cellular reversion might be caused by an endogenous factor. We hypothesize that a second somatic modulating factor in the genome that affects the processing of the mutant KRT14 pre-mRNA may underlie this phenomenon

Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations.

Epidermolysis bullosa simplex is a group of blistering skin disorders caused by defects in one of the keratin genes, KRT5 and KRT14. Previously reported KRT5 and KRT14 mutations are clustered in several hotspots, namely the rod ends of the 1A and 2B domains and in the non-helical linker region L12. Therefore, genomic KRT5 and KRT14 mutation analysis was initially limited to these hotspots. In this study we describe the screening of nine EBS patients for mutations in the hotspots. In two patients, with the Koebner and the Weber-Cockayne subtypes of epidermolysis bullosa simplex respectively, we could, however, not identify any mutation in one of the hotspot domains of KRT5 and KRT14. Therefore, it appeared to be necessary to screen the entire genes for mutations. For KRT5, a complete genomic mutation detection system was previously described. We now developed a complete genomic mutation detection system for KRT14. For the amplification of the KRT14 genes, we make use of restriction sites to exempt the keratin 14 pseudogene sequence from polymerase chain reaction amplification. Using the complete genomic mutation detection system for both KRT5 and KRT14, we identified four novel KRT5 mutations (IVS1-1G>C, K404E, A438D, E475K), two of which are outside the KRT5 hotspot domains, and three novel KRT14 mutations (IVS4+1G>A, L408M, L130P)