Primers for Exon-Specific Amplification of the KRT5 Gene: Identification of Novel and Recurrent Mutations in Epidermolysis Bullosa Simplex Patients

The KRT5 and KRT14 genes encode the proteins keratin 5 and 14, respectively, which are the primary structural components of the 10-nm intermediate filaments of the mitotic epidermal basal cells. A single mutation in either gene can disrupt the keratin intermediate filament cytoskeleton, resulting in the skin fragility and blistering that is characteristic of the group of inherited disorders known as epidermolysis bullosa simplex. We have established a mutation detection system that facilitates KRT5 gene analysis from leukocyte genomic DNA, obviating the need for a skin sample or keratinocyte culture for cDNA synthesis. KRT5 intronic regions that flanked each exon were sequenced and sets of facing intronic primers were designed for specific amplification of each of the nine KRT5 exons. Direct sequencing of KRTSamplified exons identified three novel missense mutations. One mutation recurred in two unrelated patients with sporadic EBS. This glutamate to lysine substitution (E477K), located in the highly conserved KLLEGE motif at the end of the central rod domain, is the third recurrent mutation identified in dominant epidermolysis bullosa simplex disease. The corresponding glutamate in keratin 2e was previously reported to be frequently mutated in ichthyosis bullosa of Siemens, suggesting that this highly conserved residue may be a potential mutational hot spot in other type II keratins or nonkeratin intermediate filament proteins