Cloning of Genes for Basement Membrane Proteins and Discovery of Mutations in Epidermolysis Bullosa

Abstract

In the 1980s, the specialized basement membrane zone proteins collagen VII and laminin 5 were discovered as components of the anchoring fibrils and anchoring filaments, respectively. The next milestones were reached with cloning of the cDNAs for these proteins. In 1991, Parente et al. (1991) reported the cloning of a partial cDNA for collagen VII. Logically, this cDNA was used to determine the size of the mRNA of collagen VII, 8.5 kb, and to determine the chromosomal localization of the COL7A1 gene in the locus 3p21. However, the cloning also had an unexpected impact: the cDNA sequence demonstrated that the Nto C-terminal orientation of the collagen VII molecule was the opposite of what had previously been assumed. Protein chemical analysis yielded a collagen with a large non-collagenous NC1 and a small non-collagenous NC2 domain. In analogy to procollagens, it was assumed that the large globulus was the propeptide, located in the C-terminus. The cDNA sequence clearly showed that this assumption was not correct: the NC-1 domain was the N-terminal globulus and the small NC-2 domain represented the C-terminal propeptide. This finding was immensely important for understanding the aggregation and polymerization of collagen VII to the anchoring fibrils. It also had an impact on the understanding of genotype–phenotype correlations in dystrophic epidermolysis bullosa (EB), because soon after the paper by Parente and co-workers, the full-length cDNA was cloned, and the first COL7A1 mutations were reported in dystrophic epidermolysis bullosa (Figure 1). Moreover, the cDNA cloning allowed recombinant expression of the N-terminal NC-1 domain of collagen VII and its use in enzyme-linked immunosorbent assay type assays for detection of autoantibodies in epidermolysis bullosa acquisita. In 1994, two important studies appeared back-to-back in Nature Genetics. Pulkkinen et al. (1994) and Aberdam et al. (1994) reported the first laminin 5 mutations in junctional EB (JEB). Both used the candidate gene approach, based on the knowledge that antibodies to laminin 5 (at that time known as kalinin or nicein) produced reduced/negative staining patterns in Herlitz JEB skin, indicating that this protein is abnormal in this disease. Aberdam et al. (1994) employed linkage analysis in four large families using microsatellites associated with the LAMC2 gene, which encodes the laminin g2 chain; they found linkage of Herlitz JEB to LAMC2, with a high logarithm of ODDS (LOD) score of 5.3. Subsequent mutation screening revealed a homozygous LAMC2 point mutation leading to a premature stop codon in one of the families. Pulkkinen et al. (1994) proceeded by reverse transcriptase PCR of mRNA isolated from JEB patients’ keratinocytes, followed by mutation detection using heteroduplex analysis. The heteroduplexes, which indicated mutations, were sequenced. In one patient, a homozygous point mutation was found and predicted to cause aberrant splicing. In another patient, a heterozygous deletion–insertion mutation led to a premature termination codon. While the molecular consequences of the mutations remained elusive in part, these two papers established laminin 5 as the Herlitz JEB gene. Consequently, mutations in the LAMA3 and LAMB3 genes encoding the other chains of laminin 5, a3 and b3 were disclosed soon thereafter. These studies opened the avenue for mutation detection in at least 11 different genes encoding proteins of the dermal–epidermal junction. To date, many hundreds of mutations have been disclosed in different EB subtypes, certain genotype–phenotype correlations have emerged and we are starting to understand the molecular rationales for novel, biologically valid therapeutic strategies.