PREMATURE TERMINATION CODON MUTATIONS IN THE TYPE-VII COLLAGEN GENE IN RECESSIVE DYSTROPHIC EPIDERMOLYSIS-BULLOSA RESULT IN NONSENSE-MEDIATED MESSENGER-RNA DECAY AND ABSENCE OF FUNCTIONAL PROTEIN

The severe mutilating Hallopeau-Siemens type of recessive dystrophic e pidermolysis bullosa (HS-RDEB) is characterized by the absence of anch oring fibrils that consist of type VII collagen. We have previously id entified premature termination codon (PTC) mutations in both alleles o f the type VII collagen gene (COL7A1) in HS-RDEB patients. In this stu dy 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 mut ant alleles by RT-PCR of parental RNA, The level of expression of the paternal mutant allele with a PTC in exon 2 was similar to 30% of that of the wild-type allele whereas that of the maternal mutant allele wi th a PTC in exon 104 was reduced to about 80% of the normal allele. Im munoprecipitation of newly synthesized type VII collagen with a monocl onal antibody revealed reduced quantities of alpha 1(VII) polypeptides in both parents' cells, whereas their synthesis was entirely absent i n 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 abs ence of anchoring fibrils in the proband. These results establish a me chanistic link between the presence of premature termination codon mut ations in both alleles of COL7A1 and the clinical phenotype of HS-RDEB .