Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.

BACKGROUND: Several new targeted genes and clinical subtypes have been identified since publication in 2008 of the report of the last international consensus meeting on diagnosis and classification of epidermolysis bullosa (EB). As a correlate, new clinical manifestations have been seen in several subtypes previously described. OBJECTIVE: We sought to arrive at an updated consensus on the classification of EB subtypes, based on newer data, both clinical and molecular. RESULTS: In this latest consensus report, we introduce a new approach to classification ("onion skinning") that takes into account sequentially the major EB type present (based on identification of the level of skin cleavage), phenotypic characteristics (distribution and severity of disease activity; specific extracutaneous features; other), mode of inheritance, targeted protein and its relative expression in skin, gene involved and type(s) of mutation present, and--when possible--specific mutation(s) and their location(s). LIMITATIONS: This classification scheme critically takes into account all published data through June 2013. Further modifications are likely in the future, as more is learned about this group of diseases. CONCLUSION: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and molecular features of each EB subtype, and has sufficient flexibility incorporated in its structure to permit further modifications in the future.

The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB.

BACKGROUND: Since publication in 2000 of the Second International Consensus Report on Diagnosis and Classification of Epidermolysis Bullosa, many advances have been made to our understanding of this group of diseases, both clinically and molecularly. At the same time, new epidermolysis bullosa (EB) subtypes have been described and similarities with some other diseases have been identified. OBJECTIVE: We sought to arrive at a new consensus of the classification of EB subtypes. RESULTS: We now present a revised classification system that takes into account the new advances, as well as encompassing other inherited diseases that should also be included within the EB spectrum, based on the presence of blistering and mechanical fragility. Current recommendations are made on the use of specific diagnostic tests, with updates on the findings known to occur within each of the major EB subtypes. Electronic links are also provided to informational and laboratory resources of particular benefit to clinicians and their patients. LIMITATIONS: As more becomes known about this disease, future modifications may be needed. The classification system has been designed with sufficient flexibility for these modifications. CONCLUSION: This revised classification system should assist clinicians in accurately diagnosing and subclassifying patients with EB.

Survival is not enough: reflections of a long-term renal patient.

A kidney patient recalls his experience of almost 45 years of renal replacement therapy covering nearly 25 years of dialysis and 20 years with a transplant. At the beginning, patient or graft survival was a major goal and symbol of successful treatment. But for the patient, what really matters is the quality of his life, assuming he can survive.

Reduced expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in Squamous cell carcinoma complicating recessive dystrophic epidermolysis bullosa.

Squamous cell carcinoma (SCC) is a common complication in individuals with recessive dystrophic epidermolysis bullosa (RDEB). For the severe Hallopeau-Siemens subtype, the mortality rate from SCC is over 55% by the age of 40 y. Currently, little is known about the molecular pathology or cell biology of SCC in RDEB. In this study, we compared gene expression in RDEB SCC (n=3) and non-EB SCC (n=3) with corresponding RDEB and non-EB peri-tumoral skin, with microarray analysis using DermArray membranes as well as semi-quantitative and real-time RT-PCR. Both tumor sets showed downregulation of epidermal differentiation markers (e.g., profilaggrin, keratins 1 and 10) as well as certain pro-apoptotic genes (e.g., death-associated kinase-3 or ZIP kinase). Likewise, in both groups there was upregulation of matrix metalloproteinase 1 and laminin 5 in the tumors. But we found that the expression of insulin-like growth factor-binding protein-3 (IGFBP-3) was lower (mean of 5.8-fold) in RDEB SCC compared with non-EB SCC. These data were verified by immunohistochemistry. IGFBP-3 has an important role in cancer cell apoptosis mediated via the nuclear retinoid X receptor alpha (RXRalpha). Reduced expression of IGFBP-3 in RDEB SCC may provide a partial explanation for the aggressive behavior and poor prognosis of these tumors in this genodermatosis.

Alterations in desmosome size and number coincide with the loss of keratinocyte cohesion in skin with homozygous and heterozygous defects in the desmosomal protein plakophilin 1.

Recessive mutations in the desmosomal plaque protein plakophilin 1 (PkP1) underlie ectodermal dysplasia/skin fragility syndrome (MIM 604536). We undertook an immunohistochemical and quantitative electron microscopic examination of suprabasal desmosomes from 4 skin samples from 3 PkP1 deficient patients, an unaffected carrier with a PKP1 heterozygous acceptor splice site mutation and 5 healthy control subjects. Desmosomal plaque size (>50 desmosomes per individual) and frequency (>20 high power fields, HPF) were assessed. Compared with controls, desmosomes were reduced dramatically both in size (49%) and frequency (61%) in the lower suprabasal layers (LSB) in PkP1 null patients (P<0.01). In the LSB compartment of the heterozygous carrier, corresponding reductions were 37% and 20%, respectively (P<0.01). Surprisingly, the PkP1 null patient's upper suprabasal layer, (USB), desmosome size was larger (59%, P<0.01) than the control value, and showed increased desmoglein 1 and PkP2 USB staining. The USB desmosome frequency in PKP1 null patients was similar to the LSB compartment (but reduced by 43% compared to USB controls). The carrier showed no difference in the USB desmosome size and frequency compared with the controls (P>0.05). The PKP1 null patients showed poorly developed inner and outer desmosomal plaques. Thus, both the patients and unaffected carrier showed reductions in the LSB desmosome size and number; despite only PkP1 null patients exhibiting any phenotype. These findings attest to the molecular recruiting and stabilizing roles of PkP1 in desmosome formation, particularly in the LSB compartment.

Sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and keratin 1 deimination.

The final step of keratinocyte differentiation, transition from the granular cells to the cornified cells, involves various post-translational modifications that include deimination of arginine residues. Major deiminated epidermal proteins are derived from K1. Two preferred deimination sites were identified in mouse K1, one in the V1 and the other in the V2 subdomains. An antibody against the deiminated peptide sequence in the V2 subdomain recognized not only deiminated mouse K1 but also deiminated human K1. In this study we analyzed distribution of deiminated K1 in normal human skin and in bullous congenital ichthyosiform erythroderma at light and electron microscopic levels. In normal skin the first few (1-3) cornified cell layers were positive for filaggrin and negative for the antibody against deiminated mouse K1 peptide, whereas the more superficial cells were negative for filaggrin and strongly positive for the antibody against deiminated mouse K1 peptide, indicating slightly delayed onset of K1 deimination at the initial stage of cornification. The clumped keratin in bullous congenital ichthyosiform erythroderma that was not properly compacted with filaggrin was poorly positive to the antibody against deiminated mouse K1 peptide. In addition, K1 derivatives in bullous congenital ichthyosiform erythroderma reacted poorly with the antibody against deiminated mouse K1 peptide compared with the normal control in immunoblot analyses. Our results suggest sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and K1 deimination. Abnormal keratin aggregation in bullous congenital ichthyosiform erythroderma is likely to disturb the normal deimination of K1.

Compound heterozygosity for non-sense and mis-sense mutations in desmoplakin underlies skin fragility/woolly hair syndrome.

The constitutive desmosomal plaque protein desmoplakin plays a vital part in keratinocyte adhesion in linking the transmembranous desmosomal cadherins to the cytoplasmic keratin filament network. Recently, mutations in desmoplakin have been shown to underlie some cases of the autosomal dominant disorder, striate palmoplantar keratoderma, as well as an autosomal recessive condition characterized by dilated cardiomyopathy, woolly hair, and keratoderma. Here, we describe two unrelated individuals with a new autosomal recessive genodermatosis characterized by focal and diffuse palmoplantar keratoderma, hyperkeratotic plaques on the trunk and limbs, varying degrees of alopecia, but no apparent cardiac anomalies. Mutation screening of desmoplakin demonstrated compound heterozygosity for a non-sense/mis-sense combination of mutations in both cases, C809X/N287K and Q664X/R2366C, respectively. Heterozygous carriers of any of these mutations displayed no phenotypic abnormalities. Immunohistochemistry of skin biopsies from both affected individuals revealed that desmoplakin was not just located at the cell periphery but there was also cytoplasmic staining. In addition, electron microscopy demonstrated acantholysis throughout all layers of the skin, focal detachment of desmosomes into the intercellular spaces, and perinuclear condensation of the suprabasal keratin intermediate filament network. Clinicopathologic and mutational analyses therefore demonstrate that desmoplakin haploinsufficiency can be tolerated in some cases, but that in combination with a mis-sense mutation on the other allele, the consequences are a severe genodermatosis with specific clinical manifestations.

Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome.

Kindler syndrome (OMIM 173650) is a rare autosomal recessive disorder characterized by trauma-induced blister formation (especially in childhood) and photosensitivity. Other features include mucocutaneous scarring and progressive poikiloderma. There is also an increased risk of skin and mucous membrane malignancy. The disorder was recently mapped to 20p12.3 and pathogenic mutations were identified in a new gene, KIND1. This gene encodes a 677 amino acid protein, kindlin-1, a component of focal contacts in keratinocytes. In this study, we identified four new recurrent mutations in KIND1 in 16 individuals with Kindler syndrome from 13 families of Pakistani (676insC), UK Caucasian (E304X), Omani (W616X), or Italian (958-1G > A) origins. Haplotype analysis demonstrated common ancestral mutant alleles for each mutation, apart from one of the six Pakistani families in which the mutation 676insC (which occurs in a repeat of seven cytosines) was present on a different genetic background. All mutations were homozygous, apart from the three UK Caucasian cases that were all compound heterozygotes (second allele mutations: L302X, 1161delA, 1909delA). All mutations were associated with markedly reduced or absent skin immunostaining with an antikindlin-1 antibody. These loss-of-function KIND1 mutations demonstrate the importance of kindlin-1 in maintaining epithelial integrity, although the mechanism linking this mutant protein to photosensitivity and poikiloderma remains to be determined. Delineation of these recurrent mutations is also relevant to optimizing mutation detection strategies in Kindler syndrome patients from particular ethnic backgrounds.

Changes in gap junction distribution and connexin expression pattern during human fetal skin development.

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.

Transmission electron microscopy for the diagnosis of epidermolysis bullosa.

Transmission electron microscopy (TEM) has long been the best available method for the diagnosis of epidermolysis bullosa. Today, TEM is largely superseded by immunofluorescence microscopy mapping, which is generally more available. This article discusses its continuing role in confirming or refining results obtained by other methods, or in establishing the diagnosis where other techniques have been unsuitable or have failed. It covers key steps for optimizing tissue preparation, features of analysis, recently classified epidermolysis bullosa disorders, and strengths and weaknesses of TEM.

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8).

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.

Mutations in ABCA12 underlie the severe congenital skin disease harlequin ichthyosis.

Harlequin ichthyosis (HI) is the most severe and frequently lethal form of recessive congenital ichthyosis. Although defects in lipid transport, protein phosphatase activity, and differentiation have been described, the genetic basis underlying the clinical and cellular phenotypes of HI has yet to be determined. By use of single-nucleotide-polymorphism chip technology and homozygosity mapping, a common region of homozygosity was observed in five patients with HI in the chromosomal region 2q35. Sequencing of the ABCA12 gene, which maps within the minimal region defined by homozygosity mapping, revealed disease-associated mutations, including large intragenic deletions and frameshift deletions in 11 of the 12 screened individuals with HI. Since HI epidermis displays abnormal lamellar granule formation, ABCA12 may play a critical role in the formation of lamellar granules and the discharge of lipids into the intercellular spaces, which would explain the epidermal barrier defect seen in this disorder. This finding paves the way for early prenatal diagnosis. In addition, functional studies of ABCA12 will lead to a better understanding of epidermal differentiation and barrier formation.

Desmosomes exhibit site-specific features in human palm skin.

Hereditary skin disorders resulting from desmosome gene pathology may preferentially involve the palms and soles. Why this is so is not clear. Moreover, even in normal control skin it is unknown whether there are differences in desmosome number, size or structural organization in palmoplantar sites compared with skin from other body regions. Therefore, we sought evidence for such differences by examining desmosome expression in relation to epidermal differentiation in both epidermis and cultured keratinocytes from normal human palm and breast skin samples. Confocal microscopy of skin biopsy material showed relative differences in the expression profiles of several desmosomal proteins (desmogleins, desmocollins, desmoplakin, plakoglobin and plakophilin 1) between the two sites. Western blotting revealed a higher expression level of all five proteins in palm compared with breastcultured keratinocytes. Staining for the differentiation-associated component, involucrin, suggested an earlier onset of synthesis of this protein in palm epidermis, and a suspension-induced differentiation assay showed that involucrin synthesis began earlier in palm keratinocytes than in breast cells. At 4-8 h, the number of involucrin-positive cells in palm keratinocytes was almost twice that in breast. Morphometric analysis showed that, overall, desmosomes were larger but of similar population density in the palm compared with breast skin. These findings demonstrate differences in desmosome structure and protein expression between the two sites, possibly reflecting the needs of palms and soles to withstand constant mechanical stress. They may also help to explain the preferential involvement of this region in certain hereditary disorders (palmoplantar keratodermas), associated with mutations in desmoplakin or desmoglein 1.

Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes.

No single method has been universally adopted for identifying and isolating epidermal stem/progenitor cells, and the emergence of new markers of stem cell populations is worth exploring. Here we report, for the first time, that clusters of basal keratinocytes at the tips of the rete ridges in human palm, previously recognised as a major repository of stem cells, had very low levels of desmoplakin protein and mRNA expression, compared with cells at the sides of the ridges or above the dermal papillae. We found that in populations of palm keratinocytes, selected by their ability to adhere rapidly to type IV collagen, there were significantly reduced levels of desmoplakin and other major desmosome proteins. We then showed that a low desmoglein 3 (Dsg3) expression on the cell surface could be used to enrich for a cell population with high clonogenecity, colony forming efficiency and enhanced proliferative potential, but with a low ability to form the abortive clones, compared with populations with a higher Dsg3 expression. Moreover, stringent sorting of populations showing both beta1 integrin-bright and Dsg3-dull expression enabled even further enrichment of a population containing the putative epidermal stem cells. These findings provide the basis for a new strategy for epidermal stem/progenitor cell enrichment, and encourage further study of the role of desmosomes in stem cell biology.

Efficacy and safety of endoscopic dilation of esophageal strictures in epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa is a rare genetically determined disorder of the stratified squamous epithelium. Patients with the most severe forms develop scarring of the esophagus after ingestion of food. This results in dysphagia, which severely compromises the ability to eat. Maintenance of adequate nutritional intake is a central aim, but the most appropriate method is unknown. METHODS: The results of endoscopic through-the-scope balloon dilation under propofol anesthesia in 53 patients with epidermolysis bullosa and esophageal strictures are reported. RESULTS: Seventy-five percent of patients had a single stricture (range 1 to 6 strictures), most often in the proximal esophagus (median 20 cm from incisors). A total of 182 dilations were performed (median two per patient) over a median follow-up period of 3.5 years. For all but 3 patients, there was an improvement in the dysphagia score. There was a mean increase in weight after the procedure of 2.9 kg: 95% CI[2.0, 3.8]; p<0.001, over a median 29 days. There was no significant post-procedure morbidity. CONCLUSIONS: Endoscopic balloon dilation is a safe and effective treatment for the esophageal strictures of epidermolysis bullosa. In the majority of patients, dilation relieves dysphagia and improves nutritional status.

Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability.

Ablation of the desmosomal plaque component plakophilin 1 underlies the autosomal recessive genodermatosis, skin fragility-ectodermal dysplasia syndrome (OMIM 604536). Skin from affected patients is thickened with increased scale, and there is loss of adhesion between adjacent keratinocytes, which exhibit few small, poorly formed desmosomes. To investigate further the influence of plakophilin 1 on keratinocyte adhesion and desmosome morphology, we compared plakophilin 1-deficient keratinocytes (vector controls) with those expressing recombinant plakophilin 1 introduced by retroviral transduction. We found that plakophilin 1 increases desmosomal protein content within the cell rather than enhancing transcriptional levels of desmosomal genes. Re-expression of plakophilin 1 in null cells retards cell migration but does not alter keratinocyte cell growth. Confluent sheets of plakophilin 1-deficient keratinocytes display fewer calcium-independent desmosomes than do plakophilin 1-deficient keratinocytes expressing recombinant plakophilin 1 or keratinocytes expressing endogenous plakophilin 1. In addition electron microscopy studies show that re-expression of plakophilin 1 affects desmosome size and number. Collectively, these results demonstrate that restoration of plakophilin 1 function in our culture system influences the transition of desmosomes from a calcium-dependent to a calcium-independent state and this correlates with altered keratinocyte migration in response to wounding. Thus, plakophilin 1 has a key role in increasing desmosomal protein content, in desmosome assembly, and in regulating cell migration.

An unusual N-terminal deletion of the laminin alpha3a isoform leads to the chronic granulation tissue disorder laryngo-onycho-cutaneous syndrome.

Laryngo-onycho-cutaneous (LOC or Shabbir) syndrome (OMIM 245660) is an autosomal recessive epithelial disorder confined to the Punjabi Muslim population. The condition is characterized by cutaneous erosions, nail dystrophy and exuberant vascular granulation tissue in certain epithelia, especially conjunctiva and larynx. Genome-wide homozygosity mapping localized the gene to a 2 Mb region on chromosome 18q11.2 with an LOD score of 19.8 at theta=0. This region includes the laminin alpha3 gene (LAMA3), in which loss-of-expression mutations cause the lethal skin blistering disorder Herlitz junctional epidermolysis bullosa. Detailed investigation showed that this gene possesses a further 38 exons (76 exons in total) spanning 318 kb of genomic DNA, and encodes three distinct proteins, designated laminin alpha3a, alpha3b1 and alpha3b2. The causative mutation in 15 families was a frameshift mutation 151insG predicting a stop codon 7 bp downstream in an exon that is specific to laminin alpha3a. This protein is secreted only by the basal keratinocytes of stratified epithelia, implying that LOC is caused by dysfunction of keratinocyte-mesenchymal communication. Surprisingly, the 151insG mutation does not result in nonsense-mediated mRNA decay due to rescue of the transcript by an alternative translation start site 6 exons downstream. The resultant N-terminal deletion of laminin alpha3a was confirmed by immunoprecipitation of secreted proteins from LOC keratinocytes. These studies show that the laminin alpha3a N-terminal domain is a key regulator of the granulation tissue response, with important implications not only in LOC but in a range of other clinical conditions associated with abnormal wound healing.

Lipoid proteinosis maps to 1q21 and is caused by mutations in the extracellular matrix protein 1 gene (ECM1).

Lipoid proteinosis (LP), also known as hyalinosis cutis et mucosae or Urbach-Wiethe disease (OMIM 247100) is a rare, autosomal recessive disorder typified by generalized thickening of skin, mucosae and certain viscera. Classical features include beaded eyelid papules and laryngeal infiltration leading to hoarseness. Histologically, there is widespread deposition of hyaline (glycoprotein) material and disruption/reduplication of basement membrane. The aetiology of LP is currently unknown. Using DNA from three affected siblings in a consanguineous Saudi Arabian family we performed genome-wide linkage and mapped the disorder to 1q21 (marker D1S498) with a two-point LOD score of 3.45 at theta = 0. A further 28 affected individuals from five other unrelated consanguineous family groups from different geographical regions also showed complete linkage and resulted in a maximum two-point LOD score of 21.85 at theta = 0. Using available markers in the interval between D1S442 and D1S305, the observed recombinants placed the gene in a 2.3 cM critical interval between D1S2344 and D1S2343 (Marshfield genetic map) corresponding to an approximately 6.5 Mb region on the UCSC physical map. Using a candidate gene approach (comparison of control versus LP gene expression in cultured fibroblasts) and subsequent direct sequencing of genomic DNA, we identified six different homozygous loss-of-function mutations in the extracellular matrix protein 1 gene (ECM1). Although the precise function of ECM1 is not known, our findings provide the first clinical indication of its relevance to skin adhesion, epidermal differentiation, wound healing, scarring, angiogenesis/angiopathy and basement membrane physiology, as well as defining the molecular basis of this inherited disorder.