The dermal-epidermal junction.

Recent insights into the structure and function of the dermal-epidermal junction have resulted from two converging lines of experimental evidence, namely, the study of inherited blistering disorders of the skin, in which mutations in genes encoding proteins of this region have been discovered, and the targeted ablation of the same genes in knockout mouse models. In addition to these studies, elegant analyses of the cell biology of the hemidesmosome/anchoring filament complex have revealed not only functionally important interactions between structural protein components, but also the role of certain of these proteins in mediating cell adhesion, migration, and signal transduction of messages from the extracellular matrix into the keratinocyte. Our current understanding of the dermal-epidermal junction forms a new model encapsulating the nature both of the hemidesmosomal attachment structures and of the interhemidesmosomal attachments that are mediated by differential cell type specific expression of proteins of the cutaneous adhesion zone.

Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides.

Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo.

Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.

Basal keratinocytes attach to the underlying dermal stroma through an ultrastructurally unique and complex basement membrane zone. Electron-dense plaques along the basal surface plasma membrane, termed hemidesmosomes, appear to attach directly to the lamina densa of the basement membrane through fine strands, called anchoring filaments. The lamina densa is secured to the stroma through a complex of type VII collagen containing anchoring fibrils and anchoring plaques. We have identified what we believe is a novel antigen unique to this tissue region. The mAbs to this antigen localize to the anchoring filaments, just below the basal-dense plate of the hemidesmosomes. In cell culture, the antigen is deposited upon the culture substate by growing and migrating human keratinocytes. Addition of mAb to the cultures causes the cells to round and detach, but does not impair them metabolically. Skin fragments incubated with antibody extensively de-epithelialize. These findings strongly suggest that this antigen is intimately involved in attachment of keratinocytes to the basement membrane. This antigen was isolated from keratinocyte cultures by immunoaffinity chromatography. Two molecules are observed. The most intact species contains three nonidentical chains, 165, 155, and 140 kD linked by interchain disulfide bonds. The second and more abundant species contains the 165- and 140-kD chains, but the 155-kD chain has been proteolytically cleaved to 105 kD. Likewise, two rotary-shadowed images are observed. The larger of the two, presumably corresponding to the most intact form, appears as an asymmetric 107-nm-long rod, with a single globule at one end and two smaller globules at the other. The more abundant species, presumably the proteolytically cleaved form, lacks the distal small globule. We propose the name "kalinin" for this new molecule.

Type III collagen can be present on banded collagen fibrils regardless of fibril diameter.

Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibody applied to adult human cornea localizes to isolated fibrils, which appear randomly throughout the matrix. These studies indicate that type III collagen remains associated with collagen fibrils after removal of the amino and carboxyl propeptides, and suggests that fibrils of skin, tendon, and amnion (and presumably many other tissues that contain both types I and III collagens) are copolymers of at least types I and III collagens.

Type VII collagen is a major structural component of anchoring fibrils.

Anchoring fibrils are specialized fibrous structures found in the subbasal lamina underlying epithelia of several external tissues. Based upon their sensitivity to collagenase and the similarity in banding pattern to artificially created segment-long spacing crystallites (SLS) of collagens, several authors have suggested that anchoring fibrils are lateral aggregates of collagenous macromolecules. We recently reported the similarity in length and banding pattern of anchoring fibrils to type VII collagen SLS crystallites. We now report the construction and characterization of a murine monoclonal antibody specific for type VII collagen. The epitope identified by this antibody has been mapped to the carboxyl terminus of the major helical domain of this molecule. The presence of type VII collagen as detected by indirect immunofluorescence in a variety of tissues corresponds exactly with ultrastructural observations of anchoring fibrils. Ultrastructural immunolocalization of type VII collagen using a 5-nm colloidal gold-conjugated second antibody demonstrates metal deposition upon anchoring fibrils at both ends of these structures, as predicted by the location of the epitope on type VII collagen. Type VII collagen is synthesized by primary cultures of amniotic epithelial cells. It is also produced by KB cells (an epidermoid carcinoma cell line) and WISH (a transformed amniotic cell line).

The dermal-epidermal junction of human skin contains a novel laminin variant.

We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.

Identification and partial characterization of two type XII-like collagen molecules.

We have identified two distinct collagenous macromolecules in extracts of fetal bovine skin. Each of the molecules appears to contain three identical alpha-chains with short triple-helical domains of approximately 25 kD, and nontriple-helical domains of approximately 190 kD. Consistent with these observations, extracted molecules contain a relatively short triple-helical domain (75 nm) and a large globular domain comprised of three similar arms. Despite these similarities, the purified collagenase-resistant domains are distinguished by a number of criteria. The globular domains can be chromatographically separated on the basis of charge distribution. Peptide profiles generated by V8 protease digestion are dissimilar. These molecules are immunologically unique and have distinct distributions in tissue. Finally, rotary shadow analysis of purified domains identifies size and conformation differences. Structurally, the molecules are very similar to type XII collagen, but differ in tissue distribution, since both these molecules are present in cartilage, while type XII is reported to be absent from that tissue.

Two type XII-like collagens localize to the surface of banded collagen fibrils.

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.

Type VII collagen forms an extended network of anchoring fibrils.

Type VII collagen is one of the newly identified members of the collagen family. A variety of evidence, including ultrastructural immunolocalization, has previously shown that type VII collagen is a major structural component of anchoring fibrils, found immediately beneath the lamina densa of many epithelia. In the present study, ultrastructural immunolocalization with monoclonal and monospecific polyclonal antibodies to type VII collagen and with a monoclonal antibody to type IV collagen indicates that amorphous electron-dense structures which we term "anchoring plaques" are normal features of the basement membrane zone of skin and cornea. These plaques contain type IV collagen and the carboxyl-terminal domain of type VII collagen. Banded anchoring fibrils extend from both the lamina densa and from these plaques, and can be seen bridging the plaques with the lamina densa and with other anchoring plaques. These observations lead to the postulation of a multilayered network of anchoring fibrils and anchoring plaques which underlies the basal lamina of several anchoring fibril-containing tissues. This extended network is capable of entrapping a large number of banded collagen fibers, microfibrils, and other stromal matrix components. These observations support the hypothesis that anchoring fibrils provide additional adhesion of the lamina densa to its underlying stroma.

Laminin 5 binds the NC-1 domain of type VII collagen.

Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment.

Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.

A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization.

The netrins are a family of laminin-related molecules. Here, we characterize a new member of the family, beta-netrin. beta-Netrin is homologous to the NH(2) terminus of laminin chain short arms; it contains a laminin-like domain VI and 3.5 laminin EGF repeats and a netrin C domain. Unlike other netrins, this new netrin is more related to the laminin beta chains, thus, its name beta-netrin. An initial analysis of the tissue distribution revealed that kidney, heart, ovary, retina, and the olfactory bulb were tissues of high expression. We have expressed the molecule in a eukaryotic cell expression system and made antibodies to the expressed product. Both in situ hybridization and immunohistochemistry were used to describe the cellular source of beta-netrin and where beta-netrin is deposited. beta-Netrin is a basement membrane component; it is present in the basement membranes of the vasculature, kidney, and ovaries. In addition, beta-netrin is expressed in a limited set of fiber tracts within the brain, including the lateral olfactory tract and the vomeronasal nerve. Functional studies were performed and show that beta-netrin promotes neurite elongation from olfactory bulb explants. Together, these data suggest that beta-netrin is important in neural, kidney, and vascular development.

Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain.

Laminins are heterotrimeric molecules composed of an alpha, a beta, and a gamma chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known alpha and beta chains but containing a novel gamma chain, gamma3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that gamma3 contains all the expected domains of a gamma chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that gamma3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of gamma3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, gamma3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The gamma3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of gamma3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.

Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen.

Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.

Type VII collagen, anchoring fibrils, and epidermolysis bullosa.

The anchoring fibrils at the dermal-epidermal junction have been well characterized as ultrastructural entities. From their appearance, it was proposed that they fortified the attachment of the epidermis to the dermis. This hypothesized function was strengthened by observations indicating that the anchoring fibrils were abnormal, diminished, or absent from individuals with dystrophic epidermolysis bullosa. Therefore, characterization of the molecular constituents of the anchoring fibrils and their interactions with other basement membrane and dermal components might lead to identification of the gene defects underlying at least some forms of epidermolysis bullosa. Type VII collagen was identified as the protein component of anchoring fibrils in 1986. Since then, the major characteristics of the molecule have been described. These are consistent with a model wherein secreted type VII collagen molecules form disulfide-bond stabilized antiparallel dimers. The dimers then condense laterally into unstaggered arrays that are the anchoring fibrils. This arrangement allows for the protrusion of large globular domains (NC-1) from both ends of the fibrils. The aggregated triple-helical domains extend into the papillary dermis and entrap fibrous dermal components. The NC-1 domains are believed to interact with components of the basement membrane and thus to mediate the attachment of the basement membrane to the dermis. This model predicts that mutations in the type VII collagen gene that prevent the secretion of the molecule will be the most devastating, whereas mutations in the regions encoding the globular domains may show more variable phenotype. Ultimately, understanding the function of type VII collagen at the molecular level will be the key to devising strategies to moderate the pathophysiology of dystrophic epidermolysis bullosa.

Genetic heterogeneity of collagens.

The term collagen has recently been expanded to include at least 7 genetically distinct structural elements of mammalian connective tissues. It is assumed that differences in the primary structure specify the interactions of these different collagens with one another and with noncollagen connective tissue elements. The specific biomechanical properties of individual connective tissues result from the relative content of the different collagen types. Four major classes of collagens can be defined on the basis of compositional and structural characteristics. The first class includes collagens Type I, II, and III which form classically described compact banded film structures resulting from crosslink stabilized side-by-side interactions of the triple helical structural domains. Type IV, or basement membrane collagen, comprises the second class. These molecules form open fiber structures by disulfide stabilized end-to-end interactions of the nonhelical amino and carboxyterminal structural domains. The third class of collagens contains the Type V collagens and the molecules containing the E and F chains found in cartilage. These molecules may interact by a combination of side-by-side and end-to-end aggregation. Still a fourth class is suggested by recent descriptions of several collagens which appear to contain extensive regions of unstable triple helix within the triple helical structural domain. It has been suggested that these collagens may serve as links between collagen and noncollagen structural elements. These concepts suggest that the specificity of interactions directed by the different collagen types result from (1) the extent of removal of the nonhelical collagen domains and (2) the integrity of the triple helical structure within the triple helical domain. These 2 properties of the different collagens are directly specified by genetically determined amino acid sequence differences.

Anti-basement membrane autoantibodies in patients with anti-epiligrin cicatricial pemphigoid bind the alpha subunit of laminin 5.

Recent studies have identified a group of cicatricial pemphigoid patients who have IgG anti-basement membrane autoantibodies that recognize epiligrin, a set of disulfide-linked polypeptides closely related if not identical to laminin 5 (formerly called kalinin, nicein, or BM600). To further understand the pathophysiology of blister formation in these patients, we have sought to identify the specific polypeptide(s) targeted by their autoantibodies. Comparative studies show that sera from these patients (nine of nine), P1E1 monoclonal anti-epiligrin antibody, and polyclonal as well as monoclonal anti-laminin 5 antibodies immunoprecipitate the same set of disulfide-linked polypeptides from media of biosynthetically radiolabeled human keratinocytes. Moreover, sera from eight of nine patients with anti-epiligrin cicatricial pemphigoid immunoblot the alpha subunit of laminin 5 but show no reactivity to its beta or gamma subunits. In addition, circulating IgG from a representative patient was affinity-purified against the alpha subunit of laminin 5 and shown to bind the dermal side of 1 M NaC1 split skin in the same manner as autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid. Sera from patients with bullous pemphigoid (n = 5), other forms of cicatricial pemphigoid (n = 5), epidermolysis bullosa acquisita (n = 4), or bullous systemic lupus erythematosus (n = 1) show no reactivity against any subunit of this laminin isoform in immunoprecipitation or immunoblot experiments. These findings correlate with prior reports showing that a monoclonal antibody directed against the alpha subunit of laminin 5 (i.e., laminin subunit alpha 3) induces detachment of human keratinocytes from extracellular matrix in vitro as well as epidermis from human skin in situ. Together, these studies suggest that laminin subunit alpha 3 mediates attachment of basal keratinocytes to epidermal basement membrane and that autoantibodies directed against it may be pathogenic.

Ontogeny of structural components at the dermal-epidermal junction in human embryonic and fetal skin: the appearance of anchoring fibrils and type VII collagen.

The ontogeny and composition of the dermal-epidermal junction (DEJ) in developing human embryonic and fetal skin was studied at progressive stages of gestation by immunofluorescence microscopy and immunocytochemistry using transmission electron microscopy (TEM). The DEJ of embryonic skin at 5 weeks estimated gestational age (EGA) was a simple basement membrane zone limited to the basal cell plasma membrane, lamina lucida, and lamina densa. A network of reticular collagen fibrils (reticular lamina) was deposited beneath the lamina densa by 6 weeks. Coincident with the onset of increased complexity in epidermal and dermal structure, at the time of the embryonic to fetal transition, the DEJ displayed additional components that were markers of maturation. At 7-8 weeks EGA, fine filamentous structures extended from the DEJ into the reticular lamina. By 9 weeks EGA hemidesmosomes and banded anchoring fibrils were recognizable, although distributed sparsely at the DEJ. With increasing gestational age, these structures displayed greater electron density and structural completeness. By the end of the first trimester, the DEJ appeared ultrastructurally similar to that of mature skin. Weak immunofluorescent labeling demonstrated the presence of type VII collagen at the DEJ by 8 weeks EGA. From 10-12 weeks EGA immunofluorescent labeling of the DEJ for type VII collagen was distinctly punctate, while immunoperoxidase labeling observed by TEM was linear, continuous, and sublamina densa in position. With ongoing gestation the immunofluorescent labeling became increasingly stronger at the DEJ. Thus, type VII collagen was present at the DEJ in the zone immediately beneath the lamina densa, before the appearance of mature anchoring fibrils but coordinate with the appearance of fine filamentous, unbanded structures, and appeared to increase with the development and accumulation of anchoring fibrils.

Structure of basement membranes in malignant melanoma and nevocytic nevi.

Basement membranes found around tumor cells in nevocytic nevi, Spitz's nevi, and malignant melanomas were analyzed by electron microscopy and antibody staining for several basement membrane proteins. Nevocytic nevi and Spitz's nevi showed a distinct, occasionally discontinuous lamina densa regardless of whether they were located in junctional zones of the epidermis or within the dermis. All basement membranes around nests of aggregated nevus cells, however, lacked anchoring fibrils. This correlated with the absence of type VII collagen. In contrast, type IV collagen, laminin, and nidogen were present at the periphery of the nevus cell clusters in agreement with the presence of an intact lamina densa. Aggregated tumor cells in malignant melanomas were bordered by a lamina densa when located in a junctional position and lacked this structure when they had migrated into the dermis. This process was accompanied by a drastically reduced staining for collagen type IV and nidogen, whereas laminin was still detectable. Anchoring fibrils and their molecular correlate, type VII collagen, were consistently absent. These observations demonstrate major alterations in the composition of basement membranes around malignant melanomas, which can be an important factor for the invasive growth and formation of metastases of these tumors.

Premature termination codon mutations in the type VII collagen gene in recessive dystrophic epidermolysis bullosa result in nonsense-mediated mRNA decay and absence of functional protein.

The severe mutilating Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is characterized by the absence of anchoring fibrils that consist of type VII collagen. We have previously identified premature termination codon (PTC) mutations in both alleles of the type VII collagen gene (COL7A1) in HS-RDEB patients. In this study we have defined the mechanism by which these mutations elicit their phenotypic consequences in a family. The extent of nonsense-mediated mRNA decay induced by these mutations was assessed by quantitation of the level of expression of the corresponding mRNA from each of the mutant alleles by RT-PCR of parental RNA. The level of expression of the paternal mutant allele with a PTC in exon 2 was approximately 30% of that of the wild-type allele whereas that of the maternal mutant allele with a PTC in exon 104 was reduced to about 80% of the normal allele. Immunoprecipitation of newly synthesized type VII collagen with a monoclonal antibody revealed reduced quantities of alpha1(VII) polypeptides in both parents' cells, whereas their synthesis was entirely absent in the proband's keratinocytes. Thus, a consequence of these premature termination codon mutations in COL7A1 is nonsense-mediated mRNA decay, with a dramatic reduction in type VII collagen synthesis, and the absence of anchoring fibrils in the proband. These results establish a mechanistic link between the presence of premature termination codon mutations in both alleles of COL7A1 and the clinical phenotype of HS-RDEB.