Severe Epidermolysis Bullosa Simplex Research Articles

188 Profiling of the transcriptome in hidradenitis suppurativa: A case-control sample

188 Profiling of the transcriptome in hidradenitis suppurativa: A case-control sample A Vossen, A Stubbs, M van Doorn, K van Straalen, H van der Zee and E Prens 1 Dermatology, Erasmus MC, Rotterdam the Netherlands, Rotterdam, Netherlands and 2 Bioinformatics, Erasmus MC, Rotterdam the Netherlands, Rotterdam, Netherlands Hidradenitis suppurativa (HS) is a chronic, inflammatory skin disease. The pathogenesis is poorly understood and is thought to originate in the follicular infundibulum due to unknown etiology. To increase our understanding of HS, we used RNA-sequencing to investigate the transcriptome of lesional HS skin. Polyadenylated RNA-derived cDNAs from HS subjects (n1⁄422) and skin samples from healthy volunteers (n1⁄410) were sequenced on a Affymetrix sequencing platform. Data analysis identified 1,827 genes (of which 1,279 unique known genes) as being differentially expressed with an at least 2-fold change and FDR <0.01. Comparison of the 32 samples revealed marked differences in genes associated with agranulocyte adhesion and diapedesis, the IL-17 A and F, IL-21, and the antimicrobial peptides especially S100A7, S100A8 and HBD-2. Furthermore inflammasome pathway activation in keratinocytes was demonstrated by upregulation of caspases (CASP 1, CASP 5) and members of the NLR-family. Consistent with the hypothesis, IL-1 family cytokines (IL-1a, IL-17, IL-36A) were significantly elevated in lesional skin homogenates. This was further confirmed by immunohistochemistry for IL-1. Most interestingly, a dominant plasma cell and B cell signatures were most evident in lesional skin. This study confirms, now at the RNA-seq level, previous findings on the expression of abovementioned cytokines in HS lesions. In addition, our RNA-seq data were compared with two existing data sets from psoriasis and revealed similarities in a total of 47 genes, including UBIAD1, SPRR2B/3, IL-19 and several AMPs. This study for the first time demonstrates, at the RNA-seq level, activation of inflammatory pathways and genes associated with comorbidities in HS. We postulate that IL-1a originating from keratinocytes of the infundibulum initiate an inflammatory cascade that further activates IL-1b, IL-8, and IL-17A, leading to a predominant neutrophil influx causing a significant disease burden. 189 A novel PLEC isoform modifies the phenotype in epidermolysis bullosa simplex KB Gostynska, H Lemmink, J Bremer, H Pas, M Nijenhuis, P van den Akker, R Sinke, M Jonkman and M Pasmooij 1 Dermatology, Univeristy of Groningen, University Medical Centre Groningen, Groningen, Netherlands and 2 Genetics, University of Groningen, University Medical Centre Groningen, Groningen, Netherlands Epidermolysis bullosa simplex (EBS) is caused by autosomal recessive and dominant mutations in PLEC in 8% of all cases. In humans, eight distinct plectin isoforms have been identified arising from tissue specific translation, differing in the first exon. Additionally, alternative splicing of exon 31 results in a rodless plectin variant, seen in patients affected with EBS with muscular dystrophy (EBS-MD). Complete absence of plectin leads to a severe phenotype with generalized blistering and pyloric atresia (EBS-PA), and early mortality. Here we studied the phenotype-genotype correlation in a patient with clinical EBS. A 17-year old girl had mild acral blistering present since early childhood with progressive muscular dystrophy and cardiomyopathy. Immunofluorescence antigen mapping and electron microscopy pointed both to a plectin deficiency. Plectin expression was reduced in skin and DNA analysis identified a homozygous deletion of 22 base pairs in intron 8 of PLEC. RT-PCR revealed intron retention ending in a premature termination codon predicting complete loss of plectin expression. However, an alternative splice site upstream was identified in patient and control samples. The mRNA transcript produced by the use of the alternative splice site resulted in an internally truncated protein and could explain plectin skin expression and lack of pyloric atresia in our patient. This transcript was also present in samples of healthy control keratinocytes, myocardium and striated muscle. We describe physiological alternative splicing of exon 8, resulting in a novel in frame plectin isoform. Our data demonstrate that plectin is not only alternatively spliced in exon 1 and 31, but also in exon 8 in healthy individuals. A severe EBS phenotype in our patient was most likely averted due to the production of the slightly shorter, functional protein.

The p.Glu477Lys Mutation in Keratin 5 Is Strongly Associated with Mortality in Generalized Severe Epidermolysis Bullosa Simplex

The p.Glu477Lys Mutation in Keratin 5 Is Strongly Associated with Mortality in Generalized Severe Epidermolysis Bullosa Simplex

Distinct Impact of Two Keratin Mutations Causing Epidermolysis Bullosa Simplex on Keratinocyte Adhesion and Stiffness

Keratin filaments constitute the major component of the epidermal cytoskeleton from heterodimers of type I and type II keratin subunits. Missense mutations in keratin 5 or keratin 14, highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS) in humans by rendering the keratin cytoskeleton sensitive to mechanical stress; yet, the mechanisms by which individual mutations cause cell fragility are incompletely understood. Here, we compared the K14p.Arg125Pro with the K5p.Glu477Asp mutation, both giving rise to severe generalized EBS, by stable expression in keratin-free keratinocytes. This revealed distinctly different effects on keratin cytoskeletal organization, in agreement with in vivo observations, thus validating the cell system. Although the K14p.Arg125Pro mutation led to impaired desmosomes, downregulation of desmosomal proteins, and weakened epithelial sheet integrity upon shear stress, the K5p.Glu477Asp mutation did not impair these functions, although causing EBS with squamous cell carcinoma in vivo. Atomic force microscopy demonstrated that K14 mutant cells were even less resistant against deformation compared with keratin-free keratinocytes. Thus, a keratin mutation causing EBS compromises cell stiffness to a greater extent than the lack of keratins. Finally, re-expression of K14 in K14 mutant cells did not rescue the above defects. Collectively, our findings have implications for EBS therapy approaches.

Malignant Melanoma Arising in the Setting of Epidermolysis Bullosa Simplex

Patients with epidermolysis bullosa (EB) do not carry a significantly increased risk of melanoma but are prone to developing large, markedly atypical melanocytic nevi (EB nevi), which may mimic melanoma clinically and histologically. Many authors now favor a conservative approach in managing atypical pigmented lesions in patients with EB. We present the case of a 30-year-old woman with severe EB simplex who sought care for a large red and black ulcerated plaque. The clinical differential diagnosis included EB nevus and melanoma. An incisional punch biopsy specimen revealed an atypical melanocytic proliferation with focal florid pagetoid spread and involving elongated rete ridges, consistent with invasive acral lentiginous melanoma. The subsequent amputation was confirmatory. Micrometastasis was detected in 1 of 5 sentinel lymph nodes. To our knowledge, this is the first reported case of melanoma arising in EB simplex-affected skin. It highlights the difficulty in differentiating melanoma from an EB nevus. Despite the increasing awareness of EB nevi, a high index of suspicion for melanoma should be maintained, and early biopsy is recommended when evaluating large pigmented lesions in patients with EB.

Expression signature of epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a skin disorder resulting from a weakened cytoskeleton of the proliferative compartment of the epidermis, leading to cell fragility and blistering. Although many mutations have been identified in intermediate filament keratins KRT5 and KRT14, detailed pathogenic mechanisms and the way these mutations affect cell metabolism are unclear. Therefore, we performed genomic and transcriptomic study in six Canadian EBS patients and six healthy subjects. We first characterized these patients at the genetic level and identified six pathogenic mutations of which two were novel. Then, we performed an expression microarray analysis of the EBS epidermis tissue to identify potential regulatory pathways altered in this disease. Expression profiling comparisons show that 28 genes are differentially expressed in EBS patients compared to control subjects and 41 genes in severe phenotype patients (EBS-DM) compared to their paired controls. Nine genes involved in fatty acid metabolism and two genes in epidermal keratinization are common altered expressed genes (up regulated) between the two subgroups. These two biological pathways contribute both to the formation of the cell envelope barrier and seem to be defective in the severe EBS phenotype. This study identifies, for the first time, the fatty acid metabolism disruption in EBS.

Chemical Chaperones Protect Epidermolysis Bullosa Simplex Keratinocytes from Heat Stress–Induced Keratin Aggregation: Involvement of Heat Shock Proteins and MAP Kinases

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.

ERK Involvement in Resistance to Apoptosis in Keratinocytes with Mutant Keratin

The consequences of cell stress induced by misfolded proteins are an important contributor to many human diseases. One such disease is epidermolysis bullosa simplex (EBS), caused by mutations in the structural proteins (keratins K5 or K14) of the proliferative compartment of the epidermis (basal keratinocyte layer), leading to cell fragility and blistering. In severe EBS, the mutation is associated with aggregates of nonfilamentous keratin protein, and cell lines carrying such mutations show a constitutively activated stress response. Analysis of the cellular mechanisms leading to cell breakdown on physical stress may point the way to mutation-independent therapeutic approaches to these incurable genetic disorders. We therefore subjected EBS cell lines, immortalized from patients with EBS, to an oscillating mechanical stress in assays designed to mimic the physical trauma that leads to cell breakdown in vivo. These experiments show that mechanical stress activates extracellular signal-regulated kinase (ERK) signaling in these cells, and that the keratin mutant cells also show a resistance to apoptosis following mechanical stress that is reversed by inhibiting ERK. The consequences of constitutive expression of large amounts of defective structural protein in a tissue cell must be properly understood for the development of safe and effective therapies for these disorders.

Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations.

Epidermolysis bullosa simplex (EBS) is an inherited skin fragility disorder caused by mutations in keratin intermediate filament proteins. While discoveries of these mutations have increased understanding of the role of keratins and other intermediate filaments in epithelial tissues, progress towards the development of therapy for these disorders is much slower. Cell culture model systems that display these structural defects are needed for analysis of the cellular consequences of the mutations and to enable possible therapeutic strategies to be developed. Our aim was to generate immortalized cell lines as such model systems for the study of EBS. We generated a series of stable cell lines expressing EBS-associated keratin mutations, by immortalizing keratinocytes from EBS-affected skin biopsies with either simian virus 40 (SV40) T antigen or human papillomavirus 16 (HPV16) E6/E7, and assessed their keratin expression (by immunofluorescence), proliferation rates and migratory behaviour (in outgrowth and scratch wound assays). Clonal immortalized keratinocyte cell lines KEB-1, KEB-2, KEB-3 (using SV40 T antigen) and KEB-4, KEB-7 and NEB-1 (using HPV16 E6/E7) were established. These include two lines from a single individual with Weber-Cockayne EBS (i.e. KEB-3 and KEB-4, mutation K14 V270M), and three cell lines from a second family, two from siblings carrying the same mutation (KEB-1, KEB-2 lines from Dowling-Meara EBS, mutation K5 E475G) and one from an unaffected relative (NEB-1). The sixth cell line (KEB-7), with a previously unreported severe mutation (K14 R125P), was the only one to show keratin aggregates in resting conditions. Despite variations in the immortalization procedure, there was no significant difference between cell lines in keratin expression, outgrowth capabilities or response to transient heat shock. However, cell migration, as measured by speed of scratch wound closure, was significantly faster in cells with severe EBS mutations. These cell lines provide useful culture systems in which to assess aspects of EBS-induced cell changes. The faster migration after scratch wounding of the EBS keratinocytes may be a consequence of the known upregulation of stress-activated kinase pathways in these cells.