Forms Of Dystrophic Epidermolysis Bullosa Research Articles

Identification of the epitopes on human type VII collagen for monoclonal antibodies LH 7.2 and clone I, 185

Type VII collagen is the major component of anchoring fibrils, structures in human skin that mediate the adherence of the epidermis to the underlying dermis. Dystrophic forms of epidermolysis bullosa, a group of inherited mechanobullous disorders of the skin, are linked to the type VII collagen gene. Several mutations in the recessive form of this inherited disorder have been delineated. In this study, we mapped the epitopes of two commercially available monoclonal antibodies (clone I, 185 and LH 7.2) within the amino-terminal, non-collagenous domain of type VII (anchoring fibril) collagen. The precise localizations of the epitopes for these two monoclonal antibodies which are widely used to diagnose dystrophic epidermolysis bullosa, will be useful for the confirmation of gene mutations at the protein level.

Heterogeneity of mutations in the type VII collagen gene (COL7A1) in different forms of dystrophic epidermolysis bullosa

Heterogeneity of mutations in the type VII collagen gene (COL7A1) in different forms of dystrophic epidermolysis bullosa

Cloning of human type VII collagen. Complete primary sequence of the alpha 1(VII) chain and identification of intragenic polymorphisms.

Type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, attachment structures at the dermal-epidermal basement membrane zone. In this study, we have isolated overlapping cDNA clones which correspond to full-length human type VII collagen mRNA of approximately 9.2 kilobases. The full-length cDNA sequence contains an 8,833 nucleotide open reading frame encoding 2,944 amino acids. The deduced amino acid sequence revealed the presence of a central collagenous domain flanked by a large NH2-terminal non-collagenous (NC-1) domain which consists of submodules with homology to known adhesive proteins, including nine fibronectin type III-like segments (FNIII), and a smaller COOH-terminal non-collagenous (NC-2) domain. In addition, we report six intragenic polymorphisms in the type VII collagen gene (COL7A1) which can be detected by restriction enzyme digestion of polymerase chain reaction-amplified segments. The complete cDNA sequence and polymorphisms in COL7A1 will facilitate mutational analysis and prenatal diagnosis for patients with the dystrophic forms of epidermolysis bullosa, in which mutations in COL7A1 have been demonstrated.

Genetic skin diseases

Recent advances in molecular genetics have led to major breakthroughs in the understanding of two heterogeneous groups of inherited skin diseases, epidermolysis bullosa and the ichthyoses. Mutations in keratins K5 or K14 are found in epidermolysis bullosa simplex. The gravis (Herlitz) variety of junctional epidermolysis bullosa is characterized by defects in the anchoring filament protein kalinin. Both dominant and recessive forms of dystrophic epidermolysis bullosa appear to be due to mutations in the type VII collagen gene. Biochemical studies in patients with ichthyosis vulgaris reveal that the proteins profilaggrin and filaggrin are reduced or absent. Recessive X-linked ichthyosis is characterized by a deficiency of the enzyme steroid sulfatase. A type of lamellar ichthyosis may be explained on the basis of abnormal cornified cell envelope formation, and bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis) is caused by mutations in keratins K1 or K10.

Dominant dystrophic epidermolysis bullosa: identification of a Gly-->Ser substitution in the triple-helical domain of type VII collagen.

Epidermolysis bullosa (EB) represents a group of genodermatoses characterized by fragility and easy blistering of the skin. In the dystrophic forms of EB, blisters occur below the basement membrane of the skin, at the level of the anchoring fibrils. We have recently demonstrated tight genetic linkage between the type VII collagen gene (COL7A1) and both the dominant and recessive forms of dystrophic EB. We searched for mutations in dominant dystrophic EB by PCR amplification of genomic segments of COL7A1, followed by heteroduplex analysis. Examination of the PCR fragment corresponding to exon 73 of COL7A1 revealed a marked shift in the electrophoretic pattern in patients from a large Finnish dominant dystrophic EB family with genetic linkage to the COL7A1 locus (Z = 5.37, theta = 0). Sequence analysis revealed a G-->A transition at nucleotide 6118 in the triple helical domain of COL7A1, which converted a glycine residue to a serine (GGT-->AGT). This mutation occurs between interruptions 11 and 12 of the triple helix, in the seventh of a series of 24 uninterrupted Gly-Xaa-Yaa repeats. Pathogenetic glycine substitutions that disrupt the triple helix have been shown to exert a deleterious effect on the protein in several other disorders involving collagen genes. The clinical phenotype in this family probably arises due to a dominant negative mutation in type VII collagen, resulting in the formation of structurally abnormal anchoring fibrils.

Collagen VII in Severe Recessive Dystrophic Epidermolysis Bullosa: Expression of mRNA but Lack of Intact Protein Product in Skin and Cutaneous Cells In Vitro

The collagen VII gene, COL7A1, is the candidate gene for both the recessive and dominant forms of dystrophic epidermolysis bullosa (EBD). Collagen VII is a structural protein of the anchoring fibrils, which are rudimentary or altered in several subtypes of EBD. In severe recessive mutilating EBD, anchoring fibrils and collagen VII are not detectable in skin of most patients. To elucidate the underlying pathogenetic mechanisms, we analyzed collagen VII expression in cutaneous cells of six patients with this severe EBD subtype. Neither keratinocytes nor fibroblasts synthesized detectable amounts of collagen VII protein; however, Northern blot analysis revealed small amounts of normal-size collagen VII mRNA in both EBD and control fibroblasts. When the mRNA was amplified using reverse transcription-polymerase chain reaction, correct amplimers were present in all specimens. The results demonstrate that transcription of the COL7A1 gene occurs in these patients with severe mutilating EBD and suggest that post-transcriptional or post-translational events lead to absence of collagen VII protein from skin.

Characterization of mutations in the type VII collagen gene in patients with the dystrophic forms of epidermolysis bullosa

Characterization of mutations in the type VII collagen gene in patients with the dystrophic forms of epidermolysis bullosa

CDNA Cloning and Chromosomal Mapping of the Mouse Type VII Collagen Gene ( Col7a1): Evidence for Rapid Evolutionary Divergence of the Gene

Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study we have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of ∼9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5′ half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7A1. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome.

Structural Variations in Anchoring Fibrils in Dystrophic Epidermolysis Bullosa: Correlation with Type VII Collagen Expression

Dystrophic epidermolysis bullosa is characterized by various abnormalities of anchoring fibrils, which are mainly composed of type VII collagen, at the dermal-epidermal junction. To define these changes more clearly, we examined skin samples from 22 patients with different forms of dystrophic epidermolysis bullosa by pre-embedding immunoelectron microscopy using an antibody (LH 7:2) that binds to the NC-1 globular domain of type VII collagen, followed by 1 nm colloidal gold-labeled secondary antibodies and subsequent silver enhancement. In dominant dystrophic epidermolysis bullosa cases, there was only a slight but variable reduction in the immunolabeling density on anchoring fibrils and on the lamina densa, in parts similar to normal human skin. In localized recessive dystrophic epidermolysis bullosa skin, some fibrillar structures just below the lamina densa (and particularly subjacent to hemidesmosomes) had specific antibody labeling despite their lack of resemblance to definitive anchoring fibrils. Immunolabeling with LH 7:2 was also seen within basal keratinocyte endoplasmic reticulum and cytoplasmic vesicles in some dystrophic epidermolysis bullosa patients, usually with milder phenotypic features. Even in the most severe cases of generalized recessive dystrophic epidermolysis bullosa, occasional immunolabeling was found within the lamina densa and on scanty thin filamentous structures at sub-lamina densa sites usually occupied by anchoring fibrils. This study suggests that dystrophic epidermolysis bullosa patients express some type VII collagen NC-1 domain epitopes that may be variably reduced at the dermal-epidermal junction or retained within basal keratinocytes. The clinical heterogeneity in dystrophic epidermolysis bullosa is mirrored by a range of immunoelectron microscopy findings, indicating variability in completeness of anchoring fibril formation and a possible spectrum of underlying type VII collagen structural protein abnormalities.

Normal Molecular Weight of Type VII Collagen Produced by Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

Studies of the recessive dystrophic form of epidermolysis bullosa (RDEB) have suggested that an abnormality in type VII collagen may be involved in the pathogenesis of this disorder. Indirect immunofluorescence studies have shown that the staining for type VII collagen along the dermal-epidermal junction is markedly reduced or absent in all but rare cases of severe, generalized RDEB. These findings imply that the genetic defect may involve type VII collagen but do not exclude the possibility that the alterations demonstrated are secondary, for example, to nonspecific proteolysis of type VII collagen. To evaluate the ability of cells of affected patients to produce type VII collagen, we cultured keratinocytes from a severely affected patient and immunoprecipitated type VII collagen from the cells. Keratinocytes were metabolically labelled with 35S-methionine, and solubilized cell extracts were reacted with antibody to type VII collagen. The results indicate that the patient's keratinocytes synthesize type VII collagen and that the M(r) of the protein synthesized does not differ from that of an unaffected control. Because cultured cells from a patient severely affected with recessive dystrophic epidermolysis bullosa produce type VII collagen, the genetic defect, at least in this patient, is unlikely to reside in a major truncation of the type VII collagen molecule.

The carboxyl-terminal half of type VII collagen, including the non-collagenous NC-2 domain and intron/exon organization of the corresponding region of the COL7A1 gene.

The COL7A1 gene, which encodes type VII collagen, has been implicated as a candidate gene for dominantly and recessively inherited forms of dystrophic epidermolysis bullosa. In this study, hamster and human cDNA clones, which encode the previously uncharacterized carboxyl-terminal portion of type VII collagen, have been isolated and characterized. The previously uncharacterized carboxyl-terminal NC-2 non-collagenous domain is shown to be comprised of 161 amino acids in humans, 170 amino acids in hamster and to contain 8 conserved cysteines in each species. The 6 most carboxyl-terminal cysteines are contained in a conserved motif similar to domains found in Kunitz protease inhibitors, and most closely resembling a similar motif found in the carboxyl-terminal globular domain of the alpha 3 chain of type VI collagen. Also contained in the highly acidic NC-2 domain are a number of potential sites for phosphorylation by casein kinases. Human genomic clones containing 24 exons of COL7A1 were isolated and characterized. The NC-2 domain is encoded by 7 of these exons, which include a junctional exon encoding the end of the collagenous region and the beginning of the NC-2 domain and a final exon encoding the end of the NC-2 domain and 333 bp of 3' untranslated sequences. Comparison of hamster and human sequences shows the region surrounding the junction of the collagenous and NC-2 domains to be particularly conserved. This region is likely to contain residues involved in the proteolytic removal of the NC-2 domain and cysteines involved in formation of the disulfide linkages which stabilize type VII collagen dimers.

Collagens and their abnormalities in a wide spectrum of diseases.

Collagens are a family of extracellular matrix proteins that play a dominant role in maintaining the structural integrity of various tissues. Nineteen collagen types containing altogether more than 30 distinct polypeptide chains have now been identified, and their genes have been found to be dispersed among at least 12 chromosomes. Mutations in collagen genes or deficiencies in the activities of specific post-translational enzymes of collagen synthesis have been characterized in many heritable disorders such as osteogenesis imperfecta, several chondrodysplasias, several subtypes of the Ehlers-Danlos syndrome, the X-linked Alport syndrome and dystrophic forms of epidermolysis bullosa. In addition, collagen mutations have been found in certain common diseases, namely osteoporosis, osteoarthrosis and aortic aneurysms, and it is now evident that subsets of patients with these diseases have defects in types I, II or III collagen, respectively, as a predisposing factor. Mutations have so far been identified in only six of the more than 30 collagen genes, and thus research into collagen defects is only in its early stages. Transgenic mice have been shown to offer an excellent tool for investigating the consequences of mutations in collagen genes and identifying additional diseases caused by collagen defects. Excessive collagen accumulation also poses a common problem in medicine, leading to fibrosis with impairment of the normal functioning of the affected tissue. This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a particularly suitable target for antifibrotic therapy, and several compounds are now known that inhibit this enzyme. In particular, derivatives of pyridine 2,4-dicarboxylate have been shown to inhibit hepatic collagen accumulation in rats with two models of liver fibrosis.

Genetic Linkage Between the Collagen VII (COL7A1) Gene and the Autosomal Dominant Form of Dystrophic Epidermolysis Bullosa in Two Dutch Kindreds

Epidermolysis bullosa is a heterogeneous group of heritable blistering skin diseases affecting epidermis and the dermal-epidermal junction zone. Recently, genetic linkage to the type VII collagen gene (Z = 8.77; theta = 0.00) localized on chromosome 3p21 was shown in three Finnish families with the autosomal dominant form of dystrophic epidermolysis bullosa. Two Dutch kindreds with intrafamilial characteristics of both the Cockayne-Touraine type and Bart's syndrome of autosomal dominant dystrophic epidermolysis bullosa have been studied. Two-point linkage analysis in these two families with the COL7A1 marker revealed a combined lod score of Z = 6.08 at theta = 0.00. These data strongly suggest that the type VII collagen gene is the candidate gene in these Dutch pedigrees. At least two (Cockayne-Touraine and Bart) of the three subtypes of dominant dystrophic epidermolysis bullosa seem to represent different forms of expression of the same gene defect.

Epidermolysis bullosa: Recent advances in understanding pathogenetic mechanisms

Epidermolysis bullosa (EB) is a group of mechanobullous diseases of the skin and mucous membranes characterized by the development of blisters and erosions following minor trauma. Hereditary EB exists in four major patterns: (1) intraepidermal blistering in autosomal dominant EB simplex; (2) junctional blistering in autosomal recessive junctional EB; (3) dermal blistering in autosomal dominant dystrophic EB; and (4) dermal blistering in autosomal recessive dystrophic EB. Electron microscopic examination of the skin is the current standard for diagnosis, although a number of immunologic reagents have been developed that are diagnostic tools and probes to pathogenesis. Particularly important are the antibody probes that have been developed for use in junctional and dystrophic forms of EB, since several of the monoclonal antibody preparations also confer at least some information about the likely genetic pattern and prognosis. Antibody preparations have also been used to screen complementary DNA (cDNA) expression libraries for clones representing candidate genes in EB. In EB simplex, mutations in the keratin 14 gene have been discovered. Since keratin 14 combines with keratin 5 to form a heterodimer, the existence of these mutations results in poor keratin filament formation in vitro. In recessive junctional EB, antibody staining patterns suggest involvement of the proteins of the anchoring filaments, such as BM-800/nicein. A partial cDNA clone representing one of the subunits of BM-800/nicein has been developed, and this protein represents an isoform of laminin, suggesting the existence of a family of laminin-like molecules that are important for adhesion of epidermis in the basement membrane zone of the skin. In dominant dystrophic and recessive dystrophic EB, often there are decreased numbers and abnormal appearance of the anchoring fibrils suggesting involvement of type VII collagen. Genetic linkage has been established to the type VII collagen gene in dominant dystrophic EB families. In recessive dystrophic EB, abnormalities in anchoring fibrils may be due to mutation(s) in the type VII collagen gene and/or due to excessive degradation by increased amounts of collagenase. Spontaneous, transgenic, and xenograft models of EB exist. Among the spontaneous models is recessive dystrophic EB in white alpine sheep, which show no staining for type VII collagen in the skin. A new xenograft model of recessive dystrophic EB has also been developed using EB skin grafted onto severe combined immunodeficient mice. A transgenic model has been developed for the herpetiform type of EB simplex resulting from the insertion of a truncated form of the keratin 14 gene.

Molecular biology and pathology of type VII collagen.

Type VII collagen is a genetically distinct member of the collagen family of proteins. Type VII collagen has been shown to be the major component of anchoring fibrils, attachment complexes which secure the cutaneous basement membrane of the skin to the underlying dermis. Understanding of the structure of type VII collagen has been advanced by recent cloning of the corresponding gene. Chromosomal mapping of the gene to the short arm of chromosome 3 and identification of intragenic polymorphic markers have allowed demonstration of strong genetic linkage between the type VII collagen locus and the dystrophic forms of EB (epidermolysis bullosa). This overview summarizes the progress made in the molecular genetics of type VII collagen.

GB3 monoclonal antibody for the diagnosis of junctional epidermolysis bullosa: Results of a multicenter study

GB3 monoclonal antibody detects a normal basement membrane component (GB3 antigen) that is variably expressed in junctional epidermolysis bullosa. To assess the accuracy of GB3 in the diagnosis of junctional epidermolysis bullosa, we have reviewed its use in 250 cases of the major types of epidermolysis bullosa. In the majority of cases of the simplex and dystrophic forms of epidermolysis bullosa, GB3 antigen is normally expressed. In the Herlitz variant of junctional epidermolysis bullosa, GB3 antigen expression is consistently abnormal, but in the non-Herlitz and indeterminate forms of junctional epidermolysis bullosa, 40% of cases express GB3 antigen normally. We propose that GB3 monoclonal antibody is useful in the accurate identification of patients with Herlitz junctional epidermolysis bullosa and may prove equal to electron microscopy for the diagnosis of this disease. For the non-Herlitz variants, it should not be used as an alternative to electron microscopy but may be of special value in the determination of prognosis.

Patients with severe forms of inherited epidermolysis bullosa exhibit decreased lymphokine and monokine production

Peripheral blood mononuclear cells (PBMC) from patients with severe forms of inherited epidermolysis bullosa (EB) are deficient in functions governing cellular immunity. Very low levels of interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and interleukin-2 (IL-2) were produced in vitro by PBMC from patients with severe forms of EB (recessive dystrophic and dominant dystrophic) as compared to sex- and age-matched controls. Lymphokine production by PBMC from patients with junctional EB was somewhat greater than that from patients with dystrophic forms of EB but was significantly less than that from controls. The production of interferon-alpha was not found to be altered in the severe forms of EB. The PBMC from dystrophic types of EB were also deficient in production of tumor necrosis factors (TNF-alpha and TNF-beta). The degree of the reduction in immune functions was directly related to the severity of skin involvement, with recessive dystrophic EB having the lowest level of cytokine production. This reduced production of monokines and lymphokines may be partially responsible for the progression of cutaneous infections to septicemia and for the metastasis of cutaneous squamous cell carcinomas in patients with severe forms of dystrophic EB.

Intraepidermal type VII collagen: Evidence for abnormal intracytoplasmic processing of a major basement membrane protein in rare patients with dominant and possibly localized recessive forms of dystrophic epidermolysis bullosa

Of 84 patients with dystrophic forms of epidermolysis bullosa consecutively enrolled in the National Epidermolysis Bullosa Registry, four were noted by indirect immunofluorescence with the LH 7:2 monoclonal antibody to have granular basilar keratinocyte intracytoplasmic deposits, rather than exclusively linear basement membrane deposits, of type VII collagen. Indirect immunoelectron microscopy demonstrated that these deposits were primarily perinuclear, although lesser amounts were also detectable between tonofibril bundles, within hemidesmosomes, and within and beneath the lamina densa. In two patients the mode of transmission was autosomal dominant; in two others the inheritance pattern was unknown. Whereas widespread lesions were present at birth, in each case blistering ceased within the first year of life, reminiscent of the findings in transient bullous dermolysis of the newborn. We interpret these laboratory findings as indicative of the presence of a defect in the intracytoplasmic packaging or in the transport of type VII collagen within basilar keratinocytes.

Pretibial epidermolysis bullosa: Report of a case

Pretibial epidermolysis bullosa is a rare variant of hereditary epidermolysis bullosa that is characterized by the delayed onset of a blistering eruption limited to the pretibial area. We present a case in a 48-year-old woman. Immunofluorescence mapping demonstrated blister formation below the basement membrane. This was confirmed by ultrastructural investigations that also revealed rudimentary anchoring fibrils similar to those described in the dominant dystrophic forms of epidermolysis bullosa.

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.