Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.

BACKGROUND: Several new genes and clinical subtypes have been identified since the publication in 2014 of the report of the last International Consensus Meeting on Epidermolysis Bullosa (EB). OBJECTIVES: We sought to reclassify disorders with skin fragility, with a focus on EB, based on new clinical and molecular data. METHODS: This was a consensus expert review. RESULTS: In this latest consensus report, we introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Other disorders with skin fragility, where blisters are a minor part of the clinical picture or are not seen because skin cleavage is very superficial, are classified as separate categories. These include peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility. Because of the common manifestation of skin fragility, these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. CONCLUSIONS: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and genetic features of EB. What is already known about this topic? Epidermolysis bullosa (EB) is a group of genetic disorders with skin blistering. The last updated recommendations on diagnosis and classification were published in 2014. What does this study add? We introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Clinical and genetic aspects, genotype-phenotype correlations, disease-modifying factors and natural history of EB are reviewed. Other disorders with skin fragility, e.g. peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility are classified as separate categories; these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. Linked Comment: Pope. Br J Dermatol 2020; 183:603.

Management of cutaneous squamous cell carcinoma in patients with epidermolysis bullosa: best clinical practice guidelines.

This article summarizes recommendations reached following a systematic literature review and expert consensus on the diagnosis and management of cutaneous squamous cell carcinomas in people with epidermolysis bullosa. The guidelines are intended to help inform decision making by clinicians dealing with this complex complication of a devastating disease.

The risk of cardiomyopathy in inherited epidermolysis bullosa.

BACKGROUND: Case reports have suggested that cardiomyopathy may be a complication of recessive dystrophic epidermolysis bullosa (RDEB). OBJECTIVE: To determine the risk of congestive heart failure (CHF) or cardiomyopathy in each major EB subtype. METHODS: These data represent systematic case findings and data collection performed throughout the continental United States from 1986 through 2002, by the National Epidermolysis Bullosa Registry. Study design is cross-sectional (n = 3280) with a nested randomly sampled longitudinal subcohort (n = 450). Frequencies of CHF and cardiomyopathy were determined by patient self-reporting, medical histories and review of medical records. In those who died, death certificates were reviewed and histories obtained from surviving family. Cumulative risks were stratified by cause and EB subtype. RESULTS: Cardiomyopathy was reported as early as within the first year of life. In patients having no other known risk factors for CHF or cardiomyopathy, the highest risk of cardiomyopathy was seen among patients with Hallopeau-Siemens RDEB (RDEB-HS), with a cumulative risk of 4.51% on or after age 20 years. The cumulative risk of cardiomyopathy was only 1.14% and 0.40% in non-Herlitz junctional EB (JEB) and non-Hallopeau-Siemens RDEB, respectively, and was not observed in any other EB subtype. When patients with coexistent chronic renal failure were included, the cumulative risk for RDEB-HS rose to 18.86% by age 35 years. About 30% of our patients affected with RDEB-HS died of CHF or cardiomyopathy, even those with no other known risk factors. CONCLUSIONS: CHF and cardiomyopathy are uncommon complications in both major RDEB subtypes and non-Herlitz JEB, and may be fatal.

Pseudosyndactyly and musculoskeletal contractures in inherited epidermolysis bullosa: experience of the National Epidermolysis Bullosa Registry, 1986-2002.

Mitten deformities of the hands and feet occur in nearly every patient with the most severe subtype (Hallopeau-Siemens) of recessive dystrophic epidermolysis bullosa, and in at least 40-50% of all other recessive dystrophic epidermolysis bullosa patients. Smaller numbers of patients with dominant dystrophic, junctional, and simplex types of epidermolysis bullosa are also at risk of this complication. Surgical intervention is commonly performed to correct these deformities, but recurrence and the need for repeated surgery are common. Higher numbers of epidermolysis bullosa patients also develop musculoskeletal contractures in other anatomic sites, further impairing overall function. Lifetable analyses not only better project the cumulative risk of mitten deformities and other contractures but also emphasize the need for early surveillance and intervention, since both of these musculoskeletal complications may occur within the first year of life.

Epidermolysis bullosa: variability of expression of cicatricial pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita antigens in clinically uninvolved skin.

Indirect immunofluorescence was performed on skin from 13 patients with epidermolysis bullosa (EB) (simplex, 6; junctional, 2; dystrophic, 5) to compare the expression of 3 basement membrane components, bullous pemphigoid (BP) antigen, cicatricial pemphigoid (CP) antigen, and epidermolysis bullosa acquisita (EBA) antigen, in clinically uninvolved tissue. In addition, expression of laminin, type IV collagen, and KF-1 antigen was also evaluated. Whereas laminin, type IV collagen, and KF-1 antigen were each detectable in EB skin in a manner identical to that previously reported, marked variability was noted in the expression of BP, CP, and EBA antigens. However, no correlation was noted comparing lack of expression of any one of these latter antigens with either of the remaining two. Of these 3 antigens, BP antigen was the least often detectable, particularly in skin from patients with EB simplex. The lack of detectable BP antigen in EB simplex skin appeared to correlate with more extensive disease involvement and/or younger patient age. These findings may, therefore, limit the usefulness of BP serum in immunofluorescence mapping. Additionally, the disparity in expression of basement membrane antigens defined by BP and CP sera suggests that BP antigen and CP antigen are distinct entities.

International symposium on epidermolysis bullosa. The William and Ida Friday Continuing Education Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, April 25-26, 1994. Abstracts.

An international symposium on inherited epidermolysis bullosa was held at the University of North Carolina at Chapel Hill on April 25-26, 1994. All areas currently of clinical and research interest pertinent to this disease were discussed, ranging from basic epidemiologic issues to the definition of molecular defects in each of the three major types of epidermolysis bullosa and the potential for gene therapy. A major focus of this meeting was the presentation of data collected by the National Epidermolysis Bullosa Registry.

Chondroitin 6-sulfate proteoglycan but not heparan sulfate proteoglycan is abnormally expressed in skin basement membrane from patients with dominant and recessive dystrophic epidermolysis bullosa.

Two distinct groups of proteoglycans, chondroitin 6-sulfate (C6-S) proteoglycan and heparan sulfate proteoglycan (HSPG), have been recently shown to reside within the lamina densa of normal human skin basement membrane (BM). To determine whether either or both antigens are normally expressed in one or more forms of epidermolysis bullosa (EB), a disease known to have specific alterations in skin BM, we have examined by indirect immunofluorescence 31 specimens of clinically normal skin from 28 EB patients (simplex, 5; junctional, 8; dominant dystrophic [DDEB], 9; recessive dystrophic [RDEB], 9) with monoclonal antibodies to C6-S and HSPG. HSPG was normally expressed in all EB and control skin specimens, whereas C6-S was absent along the dermoepidermal junction of 9 of 9 RDEB and 7 of 9 DDEB, and reduced in 2 of 9 DDEB cases. In contrast, C6-S was normally expressed in 5 of 5 EB simplex, 5 of 6 junctional EB, and all control skin specimens. We have subsequently extracted a greater than 400 kD C6-S proteoglycan from normal skin BM and have found that the core protein may also contain heparan sulfate side chains. Our findings suggest that 3B3 monoclonal antibody recognizes a hybrid proteoglycan in human skin, and that its absent or reduced binding in dystrophic EB skin BM may reflect either absence of associated core protein or posttranslational alterations in the proteoglycan side chains.

Chondroitin-6-sulfate-containing proteoglycan: a new component of human skin dermoepidermal junction.

A murine monoclonal antibody (3B3) has been produced with specificity for chondroitin-6-sulfate (C-6-S) and proven binding to rodent basement membranes, presumably detecting a population of C-6-S-containing proteoglycans. Utilizing this antibody, we sought to determine whether a basement membrane chondroitin sulfate proteoglycan is present in adult, neonatal, and/or fetal skin, and if present, its ultrastructural localization. Indirect immunofluorescence was performed on human adult, neonatal, and fetal skin. To detect the antigen, specimens were pretreated with chondroitinase ABC; absence of enzyme treatment served as negative control. Chondroitin sulfate proteoglycan was detectable in linear homogeneous array along the dermoepidermal junction and within vascular (and when present, adnexal) basement membranes in both adult and neonatal skin. In fetal skin, basement membrane staining was noted as early as 54 gestational days. Indirect immunoelectron microscopy and NaCl-split skin studies were performed to ultrastructurally localize the antigen; immune deposits were detectable within the lamina densa in chondroitinase-treated skin. These findings demonstrate that chondroitin sulfate proteoglycan is present within all skin basement membranes; that it is present in the region of the lamina densa; and that similar to some other ubiquitous basement membrane antigens, it is present early in the developing fetus.

LDA-1: a ubiquitous noncollagenous lamina densa component of basement membrane detected by monoclonal antibody technique.

Using monoclonal antibody technology, a new basement membrane antigen, designated as LDA-1, has been identified. This antigen is expressed in all human tissues thus far examined; within skin it is detectable not only within the dermal-epidermal junction but also within dermal vascular and appendageal basement membranes. In addition to human skin, LDA-1 is also detectable within rabbit but not monkey, rat, mouse, guinea pig, or cow skin. This antigen has been ultrastructurally localized to the lamina densa and to a much lesser extent, the adjacent sublamina densa region. ELISA (enzyme-linked immunosorbent assay) revealed no cross-reactivity between LDA-1 and type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan. In vitro enzymatic studies suggest that LDA-1 is noncollagenous in nature.

Epitope mapping of the laminin molecule in murine skin basement membrane zone: demonstration of spatial differences in ultrastructural localization.

Results of studies performed to date with polyclonal antilaminin antibodies have been conflicting as to the ultrastructural localization of this glycoprotein in skin basement membrane zone (BMZ). Whereas initial reports suggested its presence solely within the lamina lucida (LL), others have suggested that laminin is instead an exclusive component of the lamina densa (LD). In an attempt to more critically address this issue, we have examined both intact and partially separated (via 1 M NaCl) murine skin BMZ by indirect immunoelectron microscopy via a two-step immunoperoxidase technique on unfixed cryopreserved tissue, utilizing nine well-characterized monoclonal antibodies with binding specificity for laminin. Localization of the sites of the epitopes recognized by these antibodies on isolated laminin molecules was previously determined by rotary shadowing and by biochemical analyses on enzymatic fragments of laminin. Whereas at least faint immunoreactants were detected in both regions with eight of nine antibodies, predominant staining was noted within the LL with three of eight and within (and even sparsely below) the LD in three of eight. One antibody bound solely to the LL; another bound equally within both regions. Although some overlap was noted, it appears that the epitope on the distal portion of the long arm of the laminin molecule resides primarily within the skin LD, whereas epitopes on more central portions of the short arms are present within the LL or within both LL and LD. The findings of stratification of laminin epitopes within skin BMZ supports a similar recent observation in mouse kidney and suggests that portions of the laminin molecule span both LD and LL, and that there may be a non-random spatial orientation for the laminin molecule within murine skin BMZ.

The presence of intra-lamina lucida blister formation in epidermolysis bullosa acquisita: possible role of leukocytes.

In evaluating patients we have noted disparity between the locations of bound immunoreactants and the level of blistering in epidermolysis bullosa acquisita (EBA). We examined 10 consecutive EBA patients by routine histology, direct (DIF) and indirect (IIF; intact and NaCl-split skin) immunofluorescence, immunofluorescence mapping (IM), and/or direct immunoelectron microscopy (DIEM). DIF was positive in each. IIF was positive in 3/8 and 6/7 patients when intact and split skin were used as substrates. DIEM revealed immunoreactants within the lamina densa (LD) in 6/10, sub-LD in 1/10, and both LD and sub-LD in 3/10 patients. In contrast, by DIEM and IM, blister formation was noted within the lamina lucida (LL) in 7/9 and 8/10, sub-LD in 1/9 and 1/10, and within both LL and sub-LD in 1/9 and 1/10, respectively. In the presence of neutrophils within the upper dermis (n = 6), cleavage occurred within the LL in 5 specimens; in one additional specimen containing predominantly neutrophils, cleavage occurred within both LL and sub-LD. In the presence of mononuclear cells (n = 2), intra-LL cleavage occurred. In the presence of eosinophils, cleavage occurred within both LL and sub-LD. In the one specimen lacking any infiltrate, the cleavage plane was exclusively sub-LD. Intra-LL cleavage planes are more common than sub-LD ones in at least early cases of EBA. These findings likely represent the intra-LL-separating effect of leukocyte-derived proteolytic enzymes, when such cells are chemoattracted to the dermoepidermal junction by bound immuno-reactants.

Genetic basis of dominantly inherited transient bullous dermolysis of the newborn: a splice site mutation in the type VII collagen gene.

Transient bullous dermolysis of the newborn (TBDN) is a blistering disease evident at birth or shortly thereafter, but the blistering tendency decreases with advancing age. The tissue separation in TBDN is below the lamina densa, and electron microscopy has revealed abnormalities in anchoring fibrils. Immunofluorescence staining demonstrates intracellular accumulation of type VII collagen. In this study, we report a G-to-C transversion mutation in the last nucleotide of intron 35 of the type VII collagen gene (COL7A1) in a family with autosomal dominant TBDN in three generations. This nucleotide substitution abolishes the obligatory consensus 3'-acceptor splice site, predicting in-frame skipping of exon 36. Thus, TBDN in this family is caused by a mutation in COL7A1, and is therefore allelic with other variants of dominant dystrophic epidermolysis bullosa.

Detection and partial characterization of a midlamina lucida-hemidesmosome-associated antigen (19-DEJ-1) present within human skin.

A murine anti-human monoclonal antibody (19-DEJ-1) has been produced that binds to basement membranes (BMs) of the dermoepidermal junction and arrector pili muscles but not to either dermal glandular or vascular BMs. 19-DEJ-1 also recognizes BMs underneath epithelia of buccal mucosa, tongue, esophagus, cervix, and cornea, and BMs surrounding smooth muscle in medium-sized vessels, placenta, uterus, and esophagus. When 16 human fetal skins (aged 54-142 gestational days) were examined, the antigen was first detected at 81 days. Using immunoperoxidase and immunogold staining techniques, indirect immunoelectron microscopy demonstrated localization of 19-DEJ-1 to the level of the midlamina lucida, directly underneath hemidesmosomes; absent staining was noted beneath melanocytes. 19-DEJ-1 antigen was detectable in unfixed A431 cells grown on coverslips. After radioincorporation of 35S-methionine into A431 cells, 19-DEJ-1 monoclonal antibody specifically precipitated 2.75% of the total radiolabeled proteoglycans produced in culture supernatant and isolated by anion exchange chromatography. On the basis of our present findings, we conclude that 19-DEJ-1 monoclonal antibody defines a unique primate-specific proteoglycan that is present within BMs along the epithelial-connective tissue interface and around smooth muscle in skin and other selected organs. Its unique ultrastructural localization suggests the possibility that 19-DEJ-1 may recognize an antigenic epitope of either anchoring filaments or alternatively, the subbasal dense plate.

KF-1 monoclonal antibody defines a specific basement membrane antigen defect in dystrophic forms of epidermolysis bullosa.

The monoclonal antibody, KF-1, identifies a noncollagenous constituent of the lamina densa of the basement membrane zone (BMZ) of skin. In order to determine whether this BMZ constituent is affected in epidermolysis bullosa (EB), a mechanobullous skin disease often resulting in marked disfigurement, we have examined skin from patients with various forms of this disease for binding by KF-1 as well as for binding by polyclonal antibodies to laminin, type IV collagen, and bullous pemphigoid antigen, three other known BMZ components of normal skin. In all specimens from patients with simplex and junctional forms of EB, all four antibodies bound normally. In contrast, absent or diminished KF-1 binding was noted in all skin specimens from patients with dystrophic EB; antibodies directed against the other BMZ constituents, however, bound normally. This suggests that KF-1 may play a role in the structural integrity of normal skin and its absence or diminution may be important in the pathogenesis of lesion formation in dystrophic EB.

Immunofluorescence and immunoelectron microscopic studies in cicatricial pemphigoid.

We have studied various tissues from 10 patients with cicatricial pemphigoid using direct and indirect immunofluorescence, mechanical suction blister induction, and immunoelectron microscopy. In 8 of the 10 patients, direct immunofluorescence of buccal mucosa showed a linear deposition of immunoreactants, IgG and C3 being those most commonly detected. Direct immunofluorescence of skin was positive in only 4 patients. Only 1 patient had a detectable circulating anti-basement membrane zone antibody. Substitution of normal human oral mucosa for adult skin as the tissue substrate for indirect immunofluorescence did not prove useful in the detection of circulating autoantibodies. Immunoelectron microscopy was performed in the skin or mucosa (buccal or ocular) of 6 patients, revealing lamina lucida localization of in vivo-bound immunoreactants. Indirect immunofluorescence studies on mechanically induced suction blisters in skin of 2 patients with in vivo-bound IgG suggest that the lamina lucida antigen involved in cicatricial pemphigoid may be distinct from the bullous pemphigoid antigen.

The National Epidermolysis Bullosa Registry.

Since its inception in 1986, the NEBR has proved to be an excellent example of how a relatively small allocation of federal research funds for the development of a registry of cases of a single rare disease can have a major impact on the rapid expansion in the depth of knowledge of not only the disease itself but of a number of associated biologic principles, including keratinization and epithelial cell-extracellular matrix interactions. At present, the NEBR is generating extensive clinical, laboratory, and demographic data, both from cross-sectional and longitudinal perspectives, as well as establishing a centralized cell and tissue bank that will serve the scientific community at large as a valuable resource for future basic research on this oftentimes devastating genetic disease.

The carboxyl-terminal domain of type VII collagen is present at the basement membrane in recessive dystrophic epidermolysis bullosa.

Recent studies showing that type VII collagen is a component of anchoring fibrils suggests that the absence of anchoring fibrils in recessive dystrophic epidermolysis bullosa may be due to a defect in the synthesis, secretion, and deposition of type VII collagen. That hypothesis is further supported by recent studies suggesting that monoclonal antibodies to type VII collagen do not react with the basement membrane in most patients. To investigate further, we examined skin from 12 patients by electron microscopy and by immunohistology and immunoelectron microscopy using a concentrated and purified monoclonal antibody to the carboxy-terminal domain of Type VII collagen. Although anchoring fibrils were not detected by electron microscopy, the results of immunohistology showed definite, but reduced, binding of the monoclonal antibody to type VII collagen at the basement membrane in a linear pattern in 11 of 12 patients. By immunoelectron microscopy, reduced deposition of anti-type VII collagen antibody was detected beneath the lamina densa. The results of this study show that the carboxyl-terminal domain of type VII collagen is synthesized, secreted, and deposited at the basement membrane zone in 11 of 12 patients with recessive dystrophic epidermolysis bullosa and suggest that the absence of anchoring fibrils may be due either to deposition of abnormal type VII collagen, reduced levels of normal type VII collagen, defective assembly of type VII collagen into anchoring fibrils, or destruction of the collagenous domain of type VII collagen.

The appearance of four basement membrane zone antigens in developing human fetal skin.

In order to study the ontogeny of various structural and antigenic components of the basement membrane zone of human skin, we have examined skin specimens from 20 aborted fetuses ranging in gestational ages from 6 to 25 weeks, utilizing light microscopy, transmission electron microscopy, and indirect immunofluorescence with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and to the antigen defined by KF-1 monoclonal antibody. Both laminin and type IV collagen were detectable as early as 6 weeks of gestational age. In contrast, bullous pemphigoid antigen and the antigen defined by KF-1 antibody were not detectable before 10 weeks and 16 weeks, respectively. The appearance of bullous pemphigoid antigen correlated with stratification of the epidermis and the formation of hemidesmosomes and anchoring fibrils at the basement membrane zone. KF-1 antigen is first expressed when the epidermis is further stratified, hemidesmosomes and anchoring fibrils are present in greater numbers and with increased frequency at the dermal-epidermal junction, and hair follicles have begun to bud downward from the basal layer of the epidermis. Our findings suggest an orderly sequence to the appearance of these basement membrane zone components within human skin.