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

Abstract

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).

Novel and recurrent mutations in Keratin 5 and 14 in Korean patients with Epidermolysis bullosa simplex

“…Overall, a single amino acid substitution was the predominant mutational event, which is consistent with previous reports [23]. Previous studies have shown that the frequencies of K5 and K14 mutations in EBS are approximately similar [16]. In this study, nine and six mutations were on K5 and K14, respectively, making the frequency of K5 mutations slightly higher than K14 mutations (60% vs 40%, respectively), although the significance of this difference was not determined.…”

“…Total DNA was used as a template for amplification of the genomic sequences of KRT5 and KRT14. KRT5 segments including nine exons and all exon-intron borders and KRT14 segments including eight exons and all exon-intron borders were amplified as previously described [16,17]. Sequence analyses were performed using Big Dye terminator technology (ABI 3100 PerkinElmer, Warrington, UK).…”

Epidermolysis bullosa simplex due toKRT5mutations: mutation-related differences in cellular fragility and the protective effects of trimethylamineN-oxide in cultured primary keratinocytes

“…Amino acid substitutions are more common at codon 475 in patients with EBS-DM and both p.E475G and p.E475K substitutions have been reported. [20][21][22] Our analysis of cultured EBS-derived keratinocytes demonstrated some common features as well as divergence related to the phenotypic variation seen in the patients. We observed that KIF organization appeared surprisingly normal in cultures of keratinocytes isolated from these patients with EBS.…”

Gene expression analysis of an epidermolysis bullosa simplex Dowling‐Meara cell line by subtractive hybridization: recapitulation of cellular differentiation, migration and wound healing

“…In particular, the helix initiation peptide in region 1A and the helix termination peptide in the 2B domain are important for heterodimerization of compatible type 1 and type 2 keratins (18,19). The phenotypes of the three major subtypes of EBS, EBS Dowling-Meara (EBS-DM), localized EBS (EBS-loc) and generalized other EBS are the consequence of mutations at different residues in K5 or K14 genes; mutations in the critical 1A and 2B regions lead to the most severe form, EBS-DM (20).…”