Biology of anchoring fibrils: lessons from dystrophic epidermolysis bullosa.

Anchoring fibrils are adhesive suprastructures that ensure the connection of the epidermal basement membrane with the dermal extracellular matrix. The fibrils represent polymers of collagen VII, the major structural fibril component, but may also contain other proteins. Remarkable progress has been made in the last few years in understanding the functions of skin basement membrane components including the anchoring fibrils. Novel insights into the biology of the anchoring fibrils have been gained from experimental studies on dystrophic epidermolysis bullosa (DEB), a group of inherited blistering disorders caused by mutations in the gene for collagen VII, COL7A1. Mutation analyses of DEB families have disclosed more than 100 COL7A1 gene defects so far, but the unusual complexity of the mutation constellations and their biological consequences are only beginning to emerge. In analogy to heritable disorders of other collagen genes, predictable phenotypes of COL7A1 mutations causing premature termination codons or dominant negative interference have been observed. However, collagen VII seems to represent a remarkable exception among collagens in that many mutations, including heterozygous glycine substitutions and deletions, lead to minimal phenotypes, or to no phenotype at all. In contrast to fibrillar collagens, structural abnormalities of collagen VII molecules in anchoring fibrils appear to be tolerated to a certain extent. However, the mild DEB phenotypes can be severely modulated by a second aberration in individuals compound heterozygous for two different COL7A1 mutations. Therefore, not only definition of mutation(s) but also cell biological, protein chemical and suprastructural studies of the mutated molecules yield novel insight into the molecular pathomechanisms underlying disease.

Transient bullous dermolysis of the newborn associated with compound heterozygosity for recessive and dominant COL7A1 mutations.

The neonatal skin blistering disorder transient bullous dermolysis of the newborn (TBDN) heals spontaneously or improves dramatically within the first months and years of life. TBDN is characterized by subepidermal blisters, reduced or abnormal anchoring fibrils at the dermo-epidermal junction, and electron-dense inclusions in keratinocytes. These features are partly similar to those in dystrophic epidermolysis bullosa, which is caused by defects in COL7A1 gene encoding collagen VII, the main anchoring fibril protein. TBDN has been grouped separately from dystrophic epidermolysis bullosa based on the pronounced morphologic features and the self-limiting course of the disorder; however, it remains unclear whether it represents a distinct clinical entity with a single etiology. We now report a TBDN patient who is compound heterozygous for a recessive and a dominant glycine substitution mutation in COL7A1. Two point mutations caused amino acid substitutions G1519D and G2251E in the triple helical domain of collagen VII. In the heterozygous state G1519D was silent, and G2251E led to nail dystrophy, but not to skin blistering. In the proband, compound heterozygosity for the mutations caused massive, transitory retention of collagen VII in the epidermis, its reduced deposition at the basement membrane zone, and extensive dermo-epidermal separation at birth. Accordingly, TBDN keratinocytes in vitro accumulated collagen VII intracellularly in the rough endoplasmic reticulum.

Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a soluble ectodomain.

The cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet. Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells. Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa alpha1(XVII) chains. The globular intracellular domain was disulfide-linked, and the N-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures. A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain. The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides. Northern blot analysis with probes spanning either the distal 5'or the distal 3' end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally. In concert, keratinocytes harboring a homozygous nonsense mutation in the COL17A1 gene synthesized neither the 180-kDa alpha1(XVII) chain nor the 120-kDa polypeptide. Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide. These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing. Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain.

Deletions within COL7A1 exons distant from consensus splice sites alter splicing and produce shortened polypeptides in dominant dystrophic epidermolysis bullosa.

We describe two familial cases of dominant dystrophic epidermolysis bullosa (DDEB) that are heterozygous for deletions in COL7A1 that alter splicing, despite intact consensus splice-site sequences. One patient shows a 28-bp genomic deletion (6081del28) in exon 73 associated with the activation of a cryptic donor splice site within this exon; the combination of both defects restores the phase and replaces the last 11 Gly-X-Y repeats of exon 73 by a noncollagenous sequence, Glu-Ser-Leu. The second patient demonstrates a 27-bp deletion in exon 87 (6847del27), causing in-frame skipping of this exon; consensus splice sites, putative branch sites, and introns flanking exons 73 and 87 showed a normal sequence. Keratinocytes from the probands synthesized normal and shortened type VII collagen polypeptides and showed intracellular accumulation of type VII procollagen molecules. This first report of genomic deletions in COL7A1 in DDEB suggests a role for exonic sequences in the control of splicing of COL7A1 pre-mRNA and provides evidence that shortened type VII collagen polypeptides can alter, in a dominant manner, anchoring-fibril formation and can cause DDEB of differing severity.

Some, but not all, glycine substitution mutations in COL7A1 result in intracellular accumulation of collagen VII, loss of anchoring fibrils, and skin blistering.

COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a skin blistering disorder. The phenotypes result from defects of collagen VII, the major component of the anchoring fibrils at the dermo-epidermal junction; however, the molecular mechanisms underlying the phenotypes remain elusive. We investigated naturally occurring COL7A1 mutations and showed that some, but not all, glycine substitutions in collagen VII interfered with biosynthesis of the protein in a dominant-negative manner. Three point mutations in exon 73 caused glycine substitutions G2006D, G2034R, and G2015E in the triple helical domain of collagen VII and interfered with its folding and secretion. Confocal laser scanning studies and semiquantitative immunoblotting determined that dystrophic epidermolysis bullosa keratinocytes retained up to 2.5-fold more procollagen VII within the rough endoplasmic reticulum than controls. Limited proteolytic digestions of mutant procollagen VII produced aberrant fragments and revealed reduced stability of the triple helix. In contrast, the glycine substitution G1519D in another segment of the triple helix affected neither procollagen VII secretion nor anchoring fibril function and remained phenotypically silent. These data demonstrate that collagen VII presents a remarkable exception among collagens in that not all glycine substitutions within the triple helix exert dominant-negative interference and that the biological consequences of the substitutions probably depend on their position within the triple helix.

A combination of a common splice site mutation and a frameshift mutation in the COL7A1 gene: absence of functional collagen VII in keratinocytes and skin.

We describe a patient with severe generalized dystrophic epidermolysis bullosa (EBD) and a novel combination of compound heterozygous mutations in the COL7A1 gene. The maternal mutation was an A-to-G transition (425-A --> G) at position -2 of the donor splice site within exon 3 that causes aberrant splicing of two abnormal transcripts. One includes intron 3, and one excludes both exon 3 and intron 3. Both splice variants contained a premature termination of the translation. The paternal mutation is a 25-bp deletion in exon 20 (2638de125) that leads to a frameshift and a premature termination codon 133 bp downstream from the site of deletion. This combination of mutations allowed expression of collagen VII mRNA. Immunofluorescence staining of the patient's skin and cultured keratinocytes with domain-specific collagen VII antibodies, however, demonstrated markedly reduced levels of alpha1(VII) polypeptides, and no stable collagen VII protein could be extracted from the patient's cells. Electron microscopy showed severely hypoplastic fibrils below the lamina densa, without evidence of normal anchoring fibrils. The clinically unaffected parents were heterozygous for the mutations, suggesting that both COL7A1 gene defects were recessively inherited disease-causing mutations that are "silent" in heterozygous carriers but in combination can severely interfere with the dermal-epidermal adhesion and lead to severe EBD.

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene.

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine-to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.

Three novel homozygous point mutations and a new polymorphism in the COL17A1 gene: relation to biological and clinical phenotypes of junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.