Recessive epidermolysis bullosa simplex phenotype reproduced in vitro: ablation of keratin 14 is partially compensated by keratin 17

Recessive epidermolysis bullosa simplex (REBS) is characterized by generalized cutaneous blistering in response to mechanical trauma. This results from fragility of the basal keratinocytes that lack keratin tonofilaments because of homozygote null mutation in the keratin 14 gene. REBS patients display in addition focal dyskeratotic skin lesions with histology of epidermolytic hyperkeratosis (EHK) and tonofilament clumping in the suprabasal layers of the epidermis. In this study we examined whether it is possible to mimic in vitro the bullous and dyskeratotic cellular phenotype. For this purpose, fibroblasts from nondyskeratotic (K14-/-) and dyskeratotic (K14-/-) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor were placed onto the collagen matrices and cultured at the air-liquid interface. Epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a series of keratins. The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitro but not the EHK. Fibroblasts seem to play an important regulatory role in establishing the REBS phenotype. These findings suggest that wild-type fibroblasts may enhance the stability of K14-/- keratinocytes in vitro.     

Cytofluorometric study of lectin binding to the keratinocytes of epidermolysis bullosa simplex

Epidermolysis bullosa (EB) simplex is a congenital disease that has blister formation following minor mechanical trauma to the skin. The least amount of information concerning the pathogenesis is known in this disease. One possibility is that there are structural abnormalities in keratinocytes. In the present study, we report the binding of lectin (Ricinus communis agglutinin, Peanut agglutinin, and Soybean agglutinin) to keratinocytes using cytofluorometry. Biopsy skin specimens were taken from patients with simplex, junctional, and dystrophic forms of EB, and normal volunteers. Free keratinocytes were obtained by the treatment of EDTA and trypsin, and fractionated by centrifugation on a continuous colloidal silica (Percoll) density gradient. Fractionated basal cells were stained with biotinyl lectins and avidin-FITC, and measured by cytofluorometry. In all lectins examined, the intensity was low in the basal cells of EB simplex, as compared with normal controls. However, there were no differences among the other forms of EB and normal controls. This results suggest the presence of structural abnormalities in epidermis of EB simplex.     

Novel and recurrent mutations in keratin KRT5 and KRT14 genes in epidermolysis bullosa simplex: implications for disease phenotype and keratin filament assembly

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant genetic skin disorders caused by mutations of the keratin genes KRT5 and KRT14. It is characterised by lysis of basal keratinocytes leading to the development of intraepidermal blisters upon minor mechanical trauma. We investigated 27 EBS patients and families of mainly German origin by sequence analysis of the entire coding sequences of KRT5 and KRT14 and identified 12 novel and seven previously reported mutations within the KRT5 and KRT14 genes. The study discusses possible implications of the novel mutations on protein structure, keratin intermediate filament (KIF) formation and the corresponding phenotype, and summarises the spectrum of mutations reported so far in EBS. Detailed knowledge of the spectrum of EBS mutations and their genotype-phenotype correlation is essential for accurate genetic counselling and prenatal diagnosis.     

A mutation (met→arg) in the type I keratin (K14) gene responsible for autosomal dominant epidermolysis bullosa simplex

We have identified a single base change in exon 4 of the type I keratin gene which results in the replacement of a methionine for an arginine residue at codon 272 in an Irish family displaying an autosomal dominant simplex (Koebner) form of epidermolysis bullosa (EB). This family had previously provided tentative evidence for linkage to genetic markers on chromosome 1q. The mutation cosegregates with the disease, producing a lod score of 4.8 at θ = 0. © 1993 Wiley-Liss, Inc.     

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).     

Plectin expression patterns determine two distinct subtypes of epidermolysis bullosa simplex

Plectin is a cytoskeletal linker protein that has a dumbbell‐like structure with a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS‐MD), and EB simplex with pyloric atresia (EBS‐PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full‐length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS‐MD and three EBS‐PA patients. In EBS‐PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS‐MD, the expression of the N‐ and C‐terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full‐length plectin isoform with residual expression of the rodless plectin isoform leads to EBS‐MD, and that complete loss or marked attenuation of full‐length and rodless plectin expression underlies the more severe EBS‐PA phenotype. These results also clearly account for the majority of EBS‐MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS‐PA PLEC1 mutations are generally outside exon 31. Hum Mutat 30:1–9, 2010. © 2010 Wiley‐Liss, Inc.     

Plectin deficiency leads to both muscular dystrophy and pyloric atresia in epidermolysis bullosa simplex

Plectin is a cytoskeletal linker protein which has a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS‐MD), and EBS with pyloric atresia (EBS‐PA). Previous studies have demonstrated that loss of full‐length plectin with residual expression of the rodless isoform leads to EBS‐MD, whereas complete loss or marked attenuation of expression of full‐length and rodless plectin underlies the more severe EBS‐PA phenotype. However, muscular dystrophy has never been identified in EBS‐PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon‐causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient. © 2010 Wiley‐Liss, Inc.     

Abnormal binding of lectin to the epidermal cell membranes in the skin of epidermolysis bullosa simplex

Epidermolysis bullosa (EB) simplex is a congenital disease that has blister formation following minor mechanical trauma to the skin. However, the least amount of information concerning the pathogenesis is known in this condition. One possibility is that there are structural abnormalities in epidermal cell membranes. In the present study, we examined the binding of lectins to epidermal cell membranes by ABC method. Biopsy skin specimens were obtained from patients with simplex, junctional and dystrophic forms of EB, and normal volunteers. In the case of EB simplex, the cell membranes of keratinocytes in the basal and spinous cell layers did not bind to soybean agglutinin in specimens from blister edges or mechanical traumatized areas. No differences were found in binding pattern between the other forms of EB and normal controls. This results suggest the presence of structural abnormalities in epidermis of EB simplex.     

A novel autosomal partially dominant mutation designated G476D in the keratin 5 gene causing epidermolysis bullosa simplex Weber-Cockayne type: a family study with a genetic twist

Epidermolysis bullosa simplex Weber-Cockayne type (EBS-WC) is a genetically inherited skin disease characterized by blistering restricted to the palms and soles. Its inheritance in nearly all kindreds is caused by a dominant-negative mutation in either KRT5 or KRT14, the genes encoding keratin 5 and keratin 14 proteins, respectively. Rarely, recessive mutations have also been found. We described a family with EBS-WC caused by a novel autosomal dominant mutation (G476D) in the keratin 5 gene. One family member was first seen with mucosal erosions and generalized blisters localized on the anogenital area, trunk, face and sites of mechanical trauma. Molecular analysis in this patient showed the presence of an additional mutation, an autosomal recessive (G183E) one, in the same gene. This observation suggests an additional effect of a recessively inherited mutation modulating the phenotypic expression of EBS caused by a partially dominant mutation and is important for accurate genetic counseling.     

Loss of interaction between plectin and type XVII collagen results in epidermolysis bullosa simplex

Plectin is a linker protein that interacts with intermediate filaments and β4 integrin in hemidesmosomes of the epidermal basement membrane zone (BMZ). Type XVII collagen (COL17) has been suggested as another candidate plectin binding partner in hemidesmosomes. Here, we demonstrate that plectin–COL17 binding helps to maintain epidermal BMZ organization. We identified an epidermolysis bullosa (EB) simplex patient as having markedly diminished expression of plectin and COL17 in skin. The patient is compound heterozygous for sequence variants in the plectin gene (PLEC); one is a truncation and the other is a small in‐frame deletion sequence variant. The in‐frame deletion is located in the putative COL17‐binding domain of plectin and abolishes the plectin–COL17 interaction in vitro. These results imply that disrupted interaction between plectin and COL17 is involved in the development of EB. Our study suggests that protein–protein binding defects may underlie EB in patients with unidentified disease‐causing sequence variants.     

A compound heterozygous one amino-acid insertion/nonsense mutation in the plectin gene causes epidermolysis bullosa simplex with plectin deficiency

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.     

Homozygous nonsense mutation in helix 2 of K14 causes severe recessive epidermolysis bullosa simplex

We have studied a consanguineous family containing two children with severe, generalized epidermolysis bullosa simplex (EBS). Electron microscopy of skin biopsies from the affected individuals showed that basal keratinocytes were devoid of tonofilament bundles, although some single intermediate filament were visible. Genetic linkage analysis with the microsatellite probe D12S96 excluded the type II keratin gene cluster in this family. However, homozygosity by descent was observed with the polymorphic probes KRT9, KRT10 Ava II, and D17S1787 in both affected children, consistent with a recessive defect in a type I keratin. Immunoreactivity to keratin K5 and K15 was normal, but monoclonal antibodies LL001 and RCK107 against K14 showed no staining, suggesting a deficiency of K14 in these individuals. MRNA extracted from biopsy material was amplified by RT-PCR to obtain full-length K14 cDNA. Direct automated sequencing identified a homozygous nonsense mutation, W305X. A Hinf I restriction enzyme site is created by this nucleotide transition, which was used to confirm the presence of the mutation in this kindred and exclude it from 100 normal chromosomes. This is the fourth kindred with severe recessive EBS for whom a mutation has been found in the K14 gene. In this instance, the premature termination codon is the farthest downstream of the reported cases, occurring in the helix 2 domain and so giving a much longer translation product. Nevertheless, the heterozygous carriers are unaffected by the disease and display no epidermal fragility. We postulate that translation of the potentially dominant-negative truncated K14 might be down-regulated due to instability of the mutant mRNA, as observed in previous cases with similar mutations. Hum Mutat 11:279–285, 1998.© 1998 Wiley-Liss, Inc.     

Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.