The basic interest of my laboratory concerns proteins that promote the adhesion of keratinocytes to epidermal basement membrane. Our clinical interests focus on patients with autoimmune and genetic subepidermal blistering diseases. Several years ago we identified patients with a scarring and blistering disease of mucous membranes and skin who have IgG autoantibodies directed against a multiunit protein localized just beneath basal keratinocytes in epidermal basement membrane. Such patients, distinguished by their disease-specific autoantibodies, have now been identified throughout the World. Recently, we showed that the protein bound by these patients autoantibodies is the alpha subunit (and more specifically, the G domain of this subunit) of laminins 5 and 6, adhesion molecules thought to directly promote the adhesion of keratinocytes to epidermal basement membrane. To determine if anti- laminin 5 IgG can be pathogenic in vivo, we have developed a passive transfer animal model of this human blistering disease. These studies have shown that anti-laminin 5 IgG induces subepidermal blisters in the skin of neonatal and adult mice as well as human skin grafted onto immunodeficient mice. Such blisters develop independent of the activation of complement or the degranulation of dermal mast cells. Recently, we have shown that human anti-laminin alpha 3 (and G domain- specific) autoantibodies also produce subepidermal blisters in the latter in vivo model. These models should prove useful in further defining the pathophysiology and treatment of this human blistering disease as well as providing insights about which domains of laminin 5 promote keratinocyte adhesion. In other studies, we have shown that type XVII collagen, a constituent of hemidesmosomes in basal keratinocytes, is not present in the skin of patients with a distinctive inherited blistering disease (GABEB, OMIN 226650). Moreover, using intron-specific primers, PCR, heteroduplex analysis, and nucleotide sequencing, we have identified four different mutations in the COL17A1 gene of six such families. Affected individuals in three of these families are homozygous for the same 2 base pair deletion in the COL17A1 gene (4003delTC), while probands in two unrelated families carry one copy of this mutation as well as a nonsense mutation on their alternate allele. The other recently identified COL17A1 mutation is a splice site mutation. Interestingly, all of these mutations result in premature termination codons. Northern blot and immunoprecipitation studies of these patients keratinocytes have shown that these cells contain no detectable type XVII collagen mRNA or protein, findings supporting the idea that nonsense-mediated mRNA decay is responsible for the total lack of type XVII collagen protein in these patients skin. Using laser capture microdissection, we recently demonstrated that selected keratinocytes in one patient in the largest of these GABEB kindreds was homozygous for the 4003delTC germline deletion (like her affected siblings) yet mosaic for a second 2 base pair insertion on one COL17A1 allele. This second, post-zygotic mutation corrected the reading frame and allowed expression of partially corrected type XVII collagen in this patients epidermal basement membrane. This is the first demonstration of mosaic partial correction of a germline deletion by a second frame-restoring mutation in man - an observation of relevance to other diseases and an example of how the study of patients with dermatologic diseases has furthered our understanding of biology. - autoimmunity, extracellular matrix, genetics, keratinocytes, - Human Subjects
