Advertisement

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

      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 M.G.
      • Chung L.C.
      • Ryynänen J.
      • Woodley D.T.
      • Wynn K.C.
      • Bauer E.A.
      • et al.
      Human type VII collagen: cDNA cloning and chromosomal mapping of the gene.
      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 N-to 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.
      Figure thumbnail gr1
      Figure 1The skin basement membrane zone and associated genetic diseases. Center: schematic representation showing the dermal-epidermal adhesion complex consisting of a number of structural proteins. Intracellular keratin filaments (keratin 5/14) bind to BPAG1 and plectin, which are ligands of hemidesmosomal transmembrane proteins collagen XVII and a6p4 integrin. These have binding sites for components of the basement membrane, such as laminin 5, laminin 5/6, laminin 10, nidogen, perlecan and collagen IV. Collagen VII is the major protein of the anchoring fibrils, which extend into the dermis and entrap dermal collagen and elastin fibers. Adherence of the basement membrane with the anchoring fibrils is secured by protein-protein interactions between collagen VII and laminin 5 and collagen IV. Different forms of hereditary EB are associated with defects in distinct components of the adhesion complex. (a) Electron microscopy shows intracellular disruption of basal keratinocytes in EB simplex (EBS). (b) Mechanically induced palmar blistering in EBS. (c) X-ray of pyloric atresia in a patient with JEB associated with pyloric atresia. (d) Trauma-induced skin blister in junctional EB (JEB). (e) Electron microscopy of intracellular disruption in basal keratinocytes in EBS. Note that in this case the cleavage plane is close to the hemidesmosomes. This form, often caused by defects in a6p4 integrin or plectin, has also been called hemidesmosomal EB (EBHD). (f) Non-scarring alopecia in JEB. (g) Scarring of the knees in mild dystrophic EB (DEB). (h) Electron microscopy shows tissue separation along the lamina lucida of the basement membrane in JEB. (i) Electron microscopy demonstrates sub-lamina densa blister formation in DEB. Note residual anchoring fibrils attached to the lamina densa in the roof of the blister. (j) Severe scarring and pseudosyndactyly of the toes in DEB. (k) Severe skin blistering and erosions in a newborn. On the basis of clinical appearance alone, it is not possible to recognize the subtype at this stage. Molecular tests, such as antigen mapping and mutation analysis, are required for precise diagnosis. EB, epidermolysis bullosa; JEB, junctional epidermolysis bullosa.
      In 1994, two important studies appeared back-to-back in Nature Genetics.
      • Pulkkinen L.
      • Christiano A.M.
      • AirenneT Haakana H.
      • Tryggvason K.
      • Uitto J.
      Mutations in the g2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.
      and
      • Aberdam D.
      • Galliano M.F.
      • Vailly J.
      • Pulkkinen L.
      • Bonifas J.
      • Christiano A.M.
      • et al.
      Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the g2 subunit of nicein/kalinin (LAMININ-5).
      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 D.
      • Galliano M.F.
      • Vailly J.
      • Pulkkinen L.
      • Bonifas J.
      • Christiano A.M.
      • et al.
      Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the g2 subunit of nicein/kalinin (LAMININ-5).
      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 L.
      • Christiano A.M.
      • AirenneT Haakana H.
      • Tryggvason K.
      • Uitto J.
      Mutations in the g2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.
      proceeded by reverse transcriptase PCR of mRNA isolated from JEB patients’ keratino-cytes, 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.

      REFERENCES

        • Aberdam D.
        • Galliano M.F.
        • Vailly J.
        • Pulkkinen L.
        • Bonifas J.
        • Christiano A.M.
        • et al.
        Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the g2 subunit of nicein/kalinin (LAMININ-5).
        Nat Genet. 1994; 6: 299-304
        • Parente M.G.
        • Chung L.C.
        • Ryynänen J.
        • Woodley D.T.
        • Wynn K.C.
        • Bauer E.A.
        • et al.
        Human type VII collagen: cDNA cloning and chromosomal mapping of the gene.
        Proc Natl Acad Sci USA. 1991; 88: 6931-6935
        • Pulkkinen L.
        • Christiano A.M.
        • AirenneT Haakana H.
        • Tryggvason K.
        • Uitto J.
        Mutations in the g2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.
        Nat Genet. 1994; 6: 293-298