Epidermolysis bullosa simplex (EBS) belongs to a group of inherited disorders characterized by the high occurrence of bullous lesions after a certain degree of friction or trauma. It is most commonly caused by mutations in keratin 5 (KRT5) or keratin 14 (KRT14) (1,2). The protein products of these genes, keratin 5 and keratin 14, are paired intermediate filaments expressed in basal keratinocytes that contribute to mechanical stability of keratin filament networks (1,2). EBS is comprised of three major forms: i) Localized and mild subtype, called EBS, localized (EBS-loc; OMIM 131800) or EBS Weber-Cockayne; ii) generalized and severest subtype, EBS Dowling-Meara (EBS-DM; OMIM 131760); and iii) generalized but relatively milder subtype known as EBS, generalized intermediate (EBS-gen intermed; OMIM 131900) or EBS-Koebner (3). EBS, generalized severe, formerly known as EBS Dowling-Meara (EBS-gen sev; OMIM 131760), is the most severe form of EBS. Its cardinal features include large, generalized blisters, mucous membrane involvement, and dystrophic nails. Bullous lesions are usually most severe in the neonatal and infancy stages, diminishing in severity with age, especially during later childhood and adulthood (3). The ultrastructural pathogenesis of EBS-DM includes clumping or collapsing of keratin filaments in the basal epidermal cells, leading to basal cell cytolysis and sequent intraepidermal blister formation (4,5). In most cases, the clinical severity is related to the location of the mutations. Most of the mutations responsible for this subtype are located in the highly conserved α-helical end segments of helix 1A and 2B (including amino acid residue 125) of KRT5 and KRT14, which are critical for proper intermediate filament structure (6). The current study reported the distinct clinical features of three Chinese probands suspected of having EBS and diagnosed with the EBS-gen sev subtype by molecular analysis.

The proband in family 1 was a 10-month-old male. His mother first came to the Department of Dermatology, Xinhua Hospital in April 2014 complaining of generalized skin blisters on the baby's hands and feet since birth (Fig. 1A and B), especially after friction or trauma. Seven months later, the skin lesions over his extremities were slightly improved (Fig. 1C). His 26-year-old father with a similar history of blistering was also present at this visit. Dermatological investigation showed no scarring, but only mild, post-inflammatory pigmentation on skin regions previously covered in blisters (Fig. 1D), although he presented with widespread blisters as an early infant.

The second proband, a 19-year-old female, presented with diffuse pigmentation, scar formation after rupture of the blisters, and scattered new blistering, resembling dermatitis herpetiformis (Fig. 1E and F). With age, the blistering diminished.

The last proband was a 4-month-old infant, with clinical manifestations similar to those of proband 1 (Fig. 1G and H). His 25-year-old mother had a similar clinical history since birth, but currently showed only scattered blisters and mild, post-inflammatory pigmentation (Fig. 1I).

Pedigree charts of these three families are shown in Fig. 2. Mutation screening of KRT5 was negative, whereas heterozygous missense mutations c.374G>T (Arg125Leu), c.373C>T (Arg125Cys) and c.1231delG (p.Glu411Argfs*31) in KRT14 were identified in proband 1, proband 2 and proband 3, respectively, and were absent in unaffected family members (Fig. 3). Mutation delineation was based on comparisons with the reported cDNA reference sequence (GenBank accession number, NM_ 000526.4 for KRT14). Sequencing results were analyzed using Geneious, version 5.6.7 (Biomatters, Ltd.; http://www.geneious.com/).

The underlying pathological mechanism of EBS-gen sev is intraepidermal blister formation via basal cell cytolysis (or, rarely, acantholysis). Other subtypes can be distinguished by clumping or significant collapse of keratin filaments in the basal epidermal cells, which can be observed with immunofluorescence mapping (IFM) or electron microscopy (EM) (7). However, these primary methods may lead to discordance with the actual diagnosis (7). In addition, skin biopsy in infants is usually regarded as a somewhat unacceptable trauma for parents, and complementary genetic testing is required.

EBS-gen sev is inherited in an autosomal dominant pattern. Except for those rare cases caused by truncated mutations, including nonsense/in-frame deletion/frameshift/splicing mutations, most EBS-gen sev cases are attributed to missense mutations that exert a dominant negative effect on functional protein structure by altering inter-chain interactions (8). There is a close correlation between the mutational locus and the severity of EBS. Compared with other two major forms, EBS-loc and EBS-gen, most EBS-gen sev cases were associated with mutations in the highly conserved end segments of KRT5 or KRT14 rod domain (6), which highlights the importance of molecular diagnosis.

The site Arg125 (CGC) of KRT14 contains a CpG dinucleotide, making it a hotspot for EBS-causing mutations due to the disposition of a spontaneous mutant. Mutations in Arg125 (including Arg125His, Arg125Cys, Arg125Ser, Arg125Gly and Arg125Leu) have been shown to be responsible for at least 40% of EBS-gen sev cases. In particular, Arg125His and Arg125Cys accounted for the majority of the mutations in Arg125 (1,6,9–14). Arg125 is located in a highly conserved region and strongly perturbs keratin network formation and keratin filament assembly (15). Despite the fact that other identical sites of mutations, even in one pedigree, may lead to distinct subtypes (1,16), Arg125 mutations are confined mainly to the most severe subtype EBS-gen sev with similar clinical courses. To the best our knowledge, only three pathogenic mutations of EBS-gen sev in the Chinese population (Arg165Ser in KRT5, and Arg125His and Arg125Cys in KRT14) have been reported so far. Only Arg125Leu has been reported in Korean and Polish populations. Phenotypes of patients with Arg125Leu and Arg125Cys in the current study were in accordance with the previously reported phenotypes (1,6,9–14).

Novel mutation c.1231delG (p.Glu411Argfs*31) located near the highly conserved α-helical end segments of helix 2B could cause a stop codon in this highly conserved region, which may result in a more severe phenotype, such as EBS-gen sev. EBS harbors risk of death at an early age (17). Currently, there is no well-established cure, other than general care to prevent trauma, infection control, and good nutrition (18).

Gene therapy research of EBS is rare, although gene therapy and bone marrow transplantation for the relatively severe dystrophic or junctional epidermolysis bullosa, having the possibility of cure, have been well investigated (19,20). These studies highlight that a corrective gene therapy could be an ideal therapy for EBS, however more studies are required before it can be developed and used in daily clinical practice. Prenatal or preimplantation genetic diagnosis is another sensible option for families at high risk of EBS.

The present study provided valuable information and assistance in genetic counseling such that the symptoms of EBS-gen sev patients declined with age. In clinical dermatology, a diagnosis of EBS-gen sev should be considered for patients with widespread, herpetiform, clustered blistering, especially in the neonatal period, diminishing in late childhood. It is generally challenging to distinguish EBS subtypes clinically in newborns. Related effective methods such as IFM and EM on freshly induced blisters combined with molecular genetic testing can be conducted to make a clear diagnosis.