PRIMERS FOR EXON-SPECIFIC AMPLIFICATION OF THE KRT5 GENE - IDENTIFICATION OF NOVEL AND RECURRENT MUTATIONS IN EPIDERMOLYSIS-BULLOSA SIMPLEXPATIENTS

The KRT5 and KRT14 genes encode the proteins keratin 5 and 14, respect ively, which are the primary structural components of the 10-nm interm ediate filaments of the mitotic epidermal basal cells, A single mutati on in either gene can disrupt the keratin intermediate filament cytosk eleton, resulting in the skin fragility and blistering that is charact eristic of the group ofinherited disorders known as epidermolysis bull osa simplex. We have established a mutation detection system that faci litates KRT5 gene analysis from leukocyte genomic DNA, obviating the n eed for a skin sample or keratinocyte culture for cDNA synthesis, KRT5 intronic regions that flanked each exon were sequenced and sets of fa cing intronic primers were designed for specific amplification of each of the nine KRT5 exons, Direct sequencing of KRT5-amplified exons ide ntified three novel missense mutations. One mutation recurred in two u nrelated patients with sporadic EBS. This glutamate to lysine substitu tion (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 g lutamate in keratin 2e was previously reported to be frequently mutate d in ichthyosis bullosa of Siemens, suggesting that this highly conser ved residue may be a potential mutational hot spot in other type II ke ratins or nonkeratin intermediate filament proteins.