Dermal-epidermal Basement Membrane Zone Research Articles

A Clinical Profile and Co-morbidities of Pemphigus Vulgaris Patients: A Study of 35 Cases

Background: Pemphigus vulgaris (PV), an autoimmune blistering disease involving the skin and mucosa. PV frequently begins with oral lesions and progresses to skin lesions. Autoimmune bullous skin disorders are associated with IgG or IgA auto- antibodies against distinct adhesion molecules of the epidermis and dermal epidermal basement membrane zone, respectively. These auto- antibodies lead to a loss of skin adhesion which shows up clinically as the formation of blisters or erosions.
 Objectives: To characterize the clinical parameters and co-morbidities of PV patients from a single tertiary medical centre in Bangladesh.
 Material and Methods: This observational study was done including 35 PV patients attending in the department of Dermatology and Venereology, Khwaja Yunus Ali Medical College Khawja Eunus Ali Medical College from 2010 to 2014. Thirty patients of pemphigus diagnosed clinically confirmed and treated over a 4-year period (2010-2014).
 Results: Majority of the patients 45.7% belongs to age group 41-50 years. Mean age 47.12±11.13. The male to female ratio in our study sample was 1:1.5. The youngest patient was 17 years old and the oldest 68. For both genders, the risk of onset peaked during the fifth and sixth decade of life. Out of 35 patients, 15(42.9%) was presented with mucosal lesions only, while 13 patients 37.1% had mucocutaneous lesions and 7 patients 20% had only cutaneous lesions. The most common comorbidies were hypertension 20.0%, osteoporosis 17.1%, and diabetes 8.6%, thyroid disease 8.6%, psoriasis 5.7%, rheumatoid arthritis 2.7%, rheumatic fever 2.9%, autoimmune hepatitis 2.9%, and myasthenia gravis 2.9%.
 Conclusion: The associated comorbidities of PV emphasize the need for dermatologists to keep a high index of suspicion and actively evaluate patients to determine their presence.
 KYAMC Journal Vol. 10, No.-2, July 2019, Page 106-109

Intrafamilial Diversity of Clinical Severity in Dominant Dystrophic Epidermolysis Bullosa: Case Series of Three Generations

Background: Epidermolysis bullosa, a group of inheritable blistering diseases with considerable clinical and genetic diversity, is divided into distinct subtypes depending on the level of tissue separation in the dermal–epidermal basement membrane zone. The dystrophic form of epidermolysis bullosa (DEB) is characterized by tense blisters and erosions which heals with extensive scarring. The fact that DEB can be inherited in either autosomal dominant (DDEB) or autosomal recessive (RDEB) pattern adds to its clinical diversity. The cause of marked clinical diversity in mild to severe DDEB is still unidentified. Main Observation: We report an intrafamilial diversity of clinical severity in dominant dystrophic epidermolysis bullosa (DDEB) cases within three generations. Conclusion: We emphasize the variety of clinical severity in DDEB cases within three generations which might be caused by unknown gene modifiers and environmental factors.

A systematic review with pooled analysis of clinical presentation and immunodiagnostic testing in mucous membrane pemphigoid: association of anti-laminin-332 IgG with oropharyngeal involvement and the usefulness of ELISA.

Mucous membrane pemphigoid (MMP) is characterized by subepithelial blistering due to IgG autoantibodies targeting various components of the dermal-epidermal basement membrane zone. Immunodiagnostics play an important role in making a precise diagnosis. Measures of test sensitivity and specificity, however, typically come from studies in diseases such as bullous pemphigoid, where the exact antigenic site may not be the same. Additionally, the association of clinical phenotype and autoantibody profiles has been an area of debate. We evaluated the sensitivity and specificity of ELISA in MMP for the known target antigens BP180 and laminin-332, as well as characterized the frequency of IgG antibodies against each laminin-332 subdomain. Lastly, we analysed whether IgG auto-antibody profiles were associated with clinical phenotype. We performed a systematic review of Medline/PubMed to compile MMP patient demographics, clinical manifestations and immunodiagnostic results. ELISA sensitivities and specificities for autoantigens were calculated in patients with positive immunoblot or immunoprecipitationstudies. IgG reactivity for each laminin-332 subunit was recorded. Associations between positive immunological tests and clinical presentation were evaluated using chi-squared test tests or Fisher's exact test when appropriate. For patients with a positive immunoblot or immunoprecipitation to NC16a, ELISA using both the NC16a and C-terminal portion of BP180 demonstrated a sensitivity and specificity of 73% and 93%. However, for individuals with IgG against the C-terminal domain of BP180, the sensitivity and specificity was 43% and 56%, respectively. LN-332 ELISA demonstrated 75% sensitivity, but only for patients with IgG reactivity against the α3 subunit. In patients with laminin-332 MMP, 86.4% demonstrated IgG against the α3 subunit. IgG autoantibodies against laminin-332 are significantly associated with pharyngo-laryngeal (P < 0.0001) and oro-pharyngo-laryngeal (P = 0.006) involvement. Immunodiagnostics play a major role in diagnosing MMP, though limited sensitivity may necessitate several forms of testing for confirmation.

Épidermolyse bulleuse acquise et réaction du greffon contre l’hôte

Epidermolysis bullosa acquisita (EBA) is a rare acquired blistering disorder caused by production of auto-antibody directed against type-VII collagen. Autoimmune disorders can occur after allogenic bone marrow transplantation as manifestations associated with chronic graft-versus-host disease (GVHD). To date, there have been 10 cases reported in the literature concerning autoimmune blistering diseases following allogenic stem-cell transplants. Herein we describe a new case involving EBA. A 46-year-old woman developed EBA 4 years after allogenic cord blood transplantation for non-Hodgkin T-cell lymphoma complicated by acute digestive and cutaneous GVHD. At physical examination, she had some cutaneous blisters on the abdomen, arms and face, as well as numerous erosions in the buccal cavity. Direct immunofluorescence microscopy revealed linear IgG and C3 deposits along the dermal-epidermal basement membrane zone. Indirect immunofluorescence showed weak IgG G4 anti-basement membrane zone antibodies, which reacted with the dermal side of 1M NaCl-split skin; the autoantibodies were directed against type-VII collagen. This second case of EBA was evocative of a GVHD blistering disease. After the therapeutic failure of dapsone and of combined mycophenolate-prednisone, treatment with rituximab proved effective. EBA may form part of the autoimmune signs associated with chronic GVH. The destruction of basement membrane and of epidermal basal cells that occurs in GVH could give rise to autoimmune bullous disease. However, in our patient, in whom manifestation of chronic GVH was restricted to the lungs, it is difficult to rule out the fortuitous onset of EBA, which presented at a sizeable interval after acute GVH.

Molecular Heterogeneity of Blistering Disorders: The Paradigm of Epidermolysis Bullosa

Molecular Heterogeneity of Blistering Disorders: The Paradigm of Epidermolysis Bullosa

Bullöse Autoimmundermatosen

Autoimmune bullous skin disorders are rare, potentially fatal disorders of skin and mucous membranes which are associated with IgG or IgA autoantibodies against distinct adhesion molecules of the epidermis and dermal epidermal basement membrane zone, respectively. These autoantibodies lead to a loss of skin adhesion which shows up clinically as the formation of blisters or erosions. In pemphigus, loss of adhesion occurs within the epidermis while in the pemphigoids, linear IgA dermatosis, epidermolysis bullosa acquisita and dermatitis herpetiformis, loss of adhesion takes place within or underneath the basement membrane zone. The autoantigens of these disorders are largely identified and characterized. Making the diagnosis of autoimmune bullous skin diseases is based on histology and direct immunofluorescence of perilesional skin and the serological detection of autoantibodides by indirect immunofluorescence and recombinant autoantigens. Therapeutically, systemic treatment with glucocorticoids is combined with immunosuppressive adjuvants which allow for the fast reduction of systemic steroids. A prospective trial in pemphigus showed that adjuvant treatment with azathioprine, mycophenolate mofetil and cyclophosphamide, respectively, led to a significant reduction of the cummulative dose of systemic steroids until complete clinical remission was achieved. In bullous pemphigoid, topical treatment with clobetasol led to complete clinical remissions without major side effects seen when glucocorticoids were applied systemically. Therapeutic depletion of B cells by rituximab as a second line therapy has significantly improved the overall prognosis of pemphigus. Comparable controlled therapeutic trials have not yet been performed in dermatitis herpetiformis and epidermolysis bullosa acquisita.

Role of Dermal-Epidermal Basement Membrane Zone in Skin, Cancer, and Developmental Disorders

The dermal-epidermal basement membrane zone is an important epithelial and stromal interface, consisting of an intricately organized collection of intracellular, transmembrane, and extracellular matrix proteins. The basement membrane zone has several main functions including acting as a permeability barrier, forming an adhesive interface between epithelial cells and the underlying matrix, and controlling cellular organization and differentiation. This article identifies key molecular players of the dermal-epidermal membrane zone, and highlights recent research studies that have identified structural and functional roles of these components in the context of various blistering, neoplastic, and developmental syndromes.

Linear immunoglobulin A bullous dermatosis

Linear immunoglobulin A bullous dermatosis (LABD) is a rare acquired, heterogeneous, subepidermal blistering disease characterized by linear IgA deposits at the dermal–epidermal basement membrane zone, often with circulating IgA antibodies in the basement membrane zone. Linear IgA disease shares many characteristics with other subepidermal bullous diseases, mainly bullous pemphigoid and dermatitis herpetiformis. However, there are clinical and immunopathologic distinctions that have led to the recognition of LABD as a separate entity. This review will focus on the clinical manifestations, the etiopathogenesis, as well as the management of LABD, based on the best data available at the time of preparation.

Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR

Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid (gelatin-C6S-HA) biomatrices were used as the scaffold to investigate the phenotypic and molecular expression of basement membrane (BM) and extracellular matrix (ECM) proteins in vitro. The cells were cultured in three different culture conditions: keratinocytes (K) monoculture, or dermal fibroblasts (FB) monoculture, or organotypic keratinocytes and dermal fibroblasts (K&FB) coculture model. The deposition of BM proteins and ECM proteins secreted by these two kinds of cells was quantitatively characterized by real time RT-PCR and examined by immunohistochemistry. The results showed that K expressed specific keratin and E-cadherin proteins, while type I collagen was secreted by FB. FB were shown to synthesize and deposit laminin 5, type IV collagen, and type VII collagen, whereas K dominantly produced integrin alpha 6 and integrin beta 4 as well as laminin 5. Interestingly, the integrin beta 4 was expressed neither in K monoculture nor in FB monoculture, but was seen in organotypic K&FB coculture model in the early culture stage. The histology studies revealed numerous features of epidermalization including a well organized basal layer of distinct cylindrical cells, granular and a horny layer, as well as complete BM formation. These results indicated that K and FB not only kept their phenotype when culturing on 3D scaffold, but also worked together to reconstruct dermal-epidermal basement membrane zone. In brief, our results directly provide the quantification in the expression of BM and ECM proteins by using real time RT-PCR in mRNA level and morphological appearance by immunostain in protein level.

Real-Time PCR Quantification of Protein Expression in Skin Equivalent Monoculture and Coculture Model

Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid biomatrix was used as the scaffold to investigate the phenotypic and molecular expression in human keratinocytes (K) and dermal fibroblasts (FB) in three different culture conditions in vitro. The cells were cultured in either monolayer (K or FB only) or coculture (K&amp;FB) model. The deposition of basement membrane proteins secreted by these two kinds of cells was quantitatively characterized by real-time PCR. In the results, dermal fibroblasts were shown to synthesize and deposit laminin 5, type IV and type VII collagen, whereas keratinocytes produced integrin alpha 6 and beta 4 as well as laminin 5 and collagen type IV, VII. Interestingly, the integrin beta 4 subunit was not expressed either in keratinocytes or dermal fibroblasts monoculture but was seen in organotypic coculture model in the early culture period. Furthermore, we found that the expression of those marker compounds was reciprocally regulated when keratinocytes and dermal fibroblasts were cultured together. These results indicated that keratinocyes and dermal fibroblasts worked together to reconstruct dermal-epidermal basement membrane (BM) zone. In brief, our data provide the first time in directly quantifying the expression of BM proteins by using real-time PCR, and also demonstrate that BM proteins were regulated by cell-cell interaction.

Nephrotic Syndrome and Aberrant Expression of Laminin Isoforms in Glomerular Basement Membranes for an Infant With Herlitz Junctional Epidermolysis Bullosa

Herlitz junctional epidermolysis bullosa (H-JEB) is a hereditary bullous disease caused by absent expression of laminin-5, a component of anchoring filaments within the dermal-epidermal basement membrane zone. Affected individuals usually die during the first 1 year of life. We studied an infant with H-JEB who presented with nephrotic syndrome, a previously unreported complication that may contribute to early death in this disease. DNA analysis revealed a compound heterozygote for mutations 2379delG and Q995X in the LAMB3 gene. The patient had massive albuminuria, attributable to failure of the glomerular filtration barrier, and high urinary N-acetylglucosaminidase levels, indicating renal tubular involvement. Electron-microscopic examination of the renal tissue revealed diffuse fusion of the foot processes, irregular swelling of the lamina rara interna, and disappearance of endothelial cell fenestrations. Immunohistopathologic analysis of the patient's renal tissue revealed compositional changes in laminin isoforms of the glomerular basement membrane and no detectable laminin-5 in the renal tubular basement membrane, which suggests that laminin-5 may play an important role in renal function. Our findings strongly suggest that H-JEB should be considered in the spectrum of congenital nephrotic syndromes. Combination therapy with meticulous skin care and treatment strategies established for congenital nephrotic syndromes may rescue patients with this disease.

Dystroglycan in skin and cutaneous cells: beta-subunit is shed from the cell surface.

In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. In this study, we characterized the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. We show that DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the alpha- and beta-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous beta-subunit is shed from the cell surface by metalloproteinases. Shedding of the beta-subunit releases the alpha-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1beta and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of alpha-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing.

In this Issue

Sentinel lymph node biopsy has been recently advocated as a way to determine whether lymph node dissection would be necessary as part of the surgical treatment of melanoma. This concept is based on the notion that the “sentinel lymph node”, the first lymph node of the draining site of the primary tumor, is never bypassed in malignant melanoma. Thus, lack of metastases in the sentinel lymph node has been considered a good prognostic finding. In this issue, Lukowsky et al. (p. 554) have evaluated this concept by examining the regional lymph nodes, sentinel and nonsentinel, in 24 patients with malignant melanoma by histopathology and immunohistochemistry, and compared these results for the presence of tyrosinase mRNA as determined by RT-PCR. Because the tyrosinase gene is expressed exclusively in melanocytic cells, detection of the corresponding transcripts would signify the presence of metastases in the tissue. As expected, 10 of 20 patients with histologic evidence of metastasis in the sentinel lymph node were also positive for tyrosinase mRNA, as were three additional patients without morphologic evidence, attesting to the sensitivity of the RT-PCR approach. Surprisingly, however, in seven patients RT-PCR was clearly positive for tyrosinase mRNA in nonsentinel lymph nodes, whereas the sentinel lymph node was negative. Assuming the specificity of the PCR for tyrosinase, it appears that the sentinel lymph node biopsy may miss micrometastases that have spread beyond the primary tumor and somehow bypassed the sentinel lymph node. These results clearly warrant further studies to validate the concept of sentinel lymph node biopsy. They also suggest that incorporation of RT-PCR into the armamentoire to examine regional lymph nodes in patients with malignant melanoma increases the sensitivity for detection of metastases Collagens comprise a superfamily of proteins, and as many as 20 different mammalian collagens have been studied thus far to the extent that they have been designated by a Roman numeral. Among these 20 collagens, at least a dozen have been demonstrated in the skin. In this issue, Peltonen et al. (p. 635) have examined the localization of another collagen, type XIII, which has recently been shown to be a transmembrane protein. Using a variety of immunologic and molecular biologic techniques, the authors demonstrate that type XIII collagen is expressed in the epidermis in various contact sites. Specifically, colocalization studies suggested that type XIII collagen is not a member of the desmosome, but is likely to be associated with adherens type junctions. Previously, another collagen, type XVII collagen, has been shown to be a keratinocyte gene product, in type 2 transmembrane orientation and a component of hemidesmosomes. Type XVII collagen clearly confers stability to the dermal–epidermal basement membrane zone, as mutations in the corresponding gene (COL17A1) underlie a non-Herlitz type of junctional epidermolysis bullosa, known as generalized atrophic benign EB (GABEB). The functional role of type XIII collagen within the epidermis is currently unknown, but the authors speculate that, in analogy with type XVII collagen, aberrations in this collagen may be associated with heritable and/or acquired blistering skin diseases. The spectacular progress recently made in understanding the molecular basis of various heritable skin diseases is exemplified by dystrophic epidermolysis bullosa (DEB). At the beginning of this decade nothing was known of the specific mutations resulting in various forms of DEB, and the prevailing notion suggested that somehow the expression of proteolytic enzymes, primarily collagenase, was altered, resulting in dissolution of anchoring fibrils and other collagenous structures. Following the cloning of type VII collagen as the candidate gene for DEB, a number of reports have detailed specific mutations in COL7A1 in different variants of DEB. In this issue, Whittock et al. (p. 673) compare the efficiency of three different mutation detection strategies, i.e., protein truncation test, fluorescence chemical cleavage of mismatch, and conformation sensitive gel electrophoresis, for their sensitivity to detect mutations in COL7A1 in patients with DEB. The results indicate that none of the these methods alone is able to detect all putative mutations, but combined use of these techniques yields an overall sensitivity of 87%. In fact, the authors were able to detect 147 of 169 mutations in a total of 93 patients with DEB, 57 of the mutations being previously unreported. These novel mutations, together with previously published ones, brings the total number of COL7A1 mutations in DEB to over 200. Examination of the mutation database reveals that the majority of the genetic lesions are family specific with a few recurrent mutations being detected. There is thus far no evidence for locus heterogeneity, and the gene for collagenase has been excluded from DEB locus by genetic linkage analyses. These observations have diagnostic importance with prognostic implications, and they form the basis for DNA-based prenatal testing and preimplantation genetic diagnosis, as well as for gene therapy in the future.

Novel ITGB4 Mutations in Lethal and Nonlethal Variants of Epidermolysis Bullosa with Pyloric Atresia: Missense versus Nonsense

Epidermolysis bullosa with pyloric atresia (EB-PA), an autosomal recessive genodermatosis, manifests with neonatal cutaneous blistering associated with congenital pyloric atresia. The disease is frequently lethal, but nonlethal cases have also been reported. Expression of the alpha6 beta4 integrin is altered at the dermal-epidermal basement-membrane zone; recently, mutations in the corresponding genes (ITGA6 and ITGB4) have been disclosed in a limited number of patients, premature termination codons in both alleles being characteristic of lethal variants. In this study, we have examined the molecular basis of EB-PA in five families, two of them with lethal and three of them with nonlethal variants of the disease. Mutation analysis disclosed novel lesions in both ITGB4 alleles of each proband. One of the patients with lethal EB-PA was a compound heterozygote for premature termination-codon mutations (C738X/4791delCA), whereas the other patient with a lethal variant was homozygous for a missense mutation involving a cysteine residue (C61Y). The three nonlethal cases had missense mutations in both alleles (C562R/C562R, R1281W/R252C, and R1281W/R1281W). Immunofluorescence staining of skin in two of the nonlethal patients and in one of the lethal cases was positive, yet attenuated, for alpha6 and beta4 integrins. These results confirm that ITGB4 mutations underlie EB-PA and show that missense mutations may lead to nonlethal phenotypes.

Strategy for identification of sequence variants in COL7A1 and a novel 2‐bp deletion mutation in recessive dystrophic epidermolysis bullosa

The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal-epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon-intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence. Hum Mutat 10:408–414, 1997. © 1997 Wiley-Liss, Inc.

Strategy for identification of sequence variants in COL7A1 and a novel 2-bp deletion mutation in recessive dystrophic epidermolysis bullosa.

The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal-epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon-intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence.

Premature termination codons on both alleles of the type VII collagen gene (COL7A1) in three brothers with recessive dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. In the most severe, dystrophic (scarring) forms of EB, blisters form below the cutaneous basement membrane at the level of the anchoring fibrils, which are composed of type VII collagen. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the type VII collagen locus (COL7A1) have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and the gene for human COL7A1. In this study, we describe distinct mutations in both COL7A1 alleles in three brothers with severe, mutilating recessive dystrophic EB (the Hallopeau-Siemens type, HS-RDEB). The patients are compound heterozygotes for two different mutations, both of which result in a premature termination codon in COL7A1, and the parents were shown to be clinically heterozygous carries of the respective mutations. Premature termination codons in both alleles of COL7A1 appear to be the underlying cause of severe, recessive dystrophic EB in this family.

Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories, the simplex, junctional, and dystrophic forms, on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. Approaches of molecular biology have demonstrated that these three different forms of EB result from mutations in distinct genes: the simplex forms are due to mutations in the genes encoding keratins 5 and 14 expressed in basal keratinocytes; the junctional forms are associated with mutations in the kalinin/laminin 5 genes; and the dystrophic forms result from mutations in the type VII collagen gene (COL7A1). In this overview, we summarize our recent discoveries of pathogenic mutations in COL7A1, including premature termination codons that result in the severe, mutilating (Hallopeau-Siemens) type of recessive dystrophic EB and a glycine substitution in the collagenous region resulting in dominant dystrophic EB. Furthermore, we present evidence that implicates mutations in the kalinin/laminin 5 gamma 2 chain gene (LAMC2) in some forms of junctional EB. This information has provided the basis for DNA-based prenatal diagnosis during the first trimester of gestation, and sets the stage for the application of gene therapy to these devastating skin diseases in the future.

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.

Premature Termination Codons in the Type VII Collagen Gene (COL7A1) Underlie Severe, Mutilating Recessive Dystrophic Epidermolysis Bullosa

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. The most severe, dystrophic (scarring) forms of EB demonstrate blister formation below the cutaneous basement membrane at the level of the anchoring fibrils. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the gene encoding type VII collagen (COL7A1), the major component of anchoring fibrils, have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and gene for human COL7A1, which has been mapped to 3p21. In this study, we describe mutations in four COL7A1 alleles in three patients with severe, mutilating recessive dystrophic EB (Hallopeau-Siemens type, HS-RDEB). Each of these mutations resulted in a premature termination codon (PTC) in the amino-terminal portion of COL7A1. One of the patients was a compound heterozygote for two different mutations. The heterozygous carriers showed an ∼50% reduction in anchoring fibrils, yet were clinically unaffected. Premature termination codons in both alleles of COL7A1 may thus be a major underlying cause of the severe, recessive dystrophic forms of EB.