CHARACTERIZATION OF 18 NEW MUTATIONS IN COL7A1 IN RECESSIVE DYSTROPHIC EPIDERMOLYSIS-BULLOSA PROVIDES EVIDENCE FOR DISTINCT MOLECULAR MECHANISMS UNDERLYING DEFECTIVE ANCHORING FIBRIL FORMATION

We have characterized 21 mutations in the type VII collagen gene (COL7 A1) encoding the anchoring fibrils, 18 of which were not previously re ported, in patients from 15 unrelated families with recessive dystroph ic epidermolysis bullosa (RDEB). COL7A1 mutations in both alleles were identified by screening the 118 exons of COL7A1 and flanking intron r egions. Fourteen mutations created premature termination codons (PTCs) and consisted of nonsense mutations, small insertions, deletions, and splice-site mutations. A further seven mutations predicted glycine or arginine substitutions in the collagenous domain of the molecule. Two mutations were found in more than one family reported in this study, and six of the seven missense mutations showed clustering within exons 72-74 next to the hinge region of the protein. Patients who were homo zygous or compound heterozygotes for mutations leading to PTCs display ed both absence or drastic reduction of COL7A1 transcripts and undetec table type VII collagen protein in skin. In contrast, missense mutatio ns were associated with clearly detectable COL7A1 transcripts and with normal or reduced expression of type VII collagen protein at the derm o/epidermal junction. Our results provide evidence for at least two di stinct molecular mechanisms underlying defective anchoring fibril form ation in RDEB: one involving PTCs leading to mRNA instability and abse nce of protein synthesis, the other implicating mis-sense mutations re sulting in the synthesis of type VII collagen polypeptide with decreas ed stability and/or altered function. Genotype-phenotype correlations suggested that the nature and location of these mutations are importan t determinants of the disease phenotype and showed evidence for interf amilial phenotypic variability.