396 Fifth International Conference on the Molecular Biology and Pathology of Matrix The Fibro-Reticular Network at the Dermal- Epidermal Junction in Recessive Dystrophic Epidermolysis Bullosa: An Immunogold Electron Microscopy Study Using Anti-Type VII Collagen and Anti-Fibrillin Antibodies John A. McGrath,* Lynn Y. Sakait and Robin A. J. Eady* *Department of Cell Pathology, St. John's Institute of Dermatology, St. Thomas's Hospital, London, U.K. and tDepartment of Biochemistry and Molecular Biology, Shriner's Hospital, Portland, Oregon, USA Recessive dystrophic epidermolysis bullosa (RDEB) encompasses a range of genetic skin disorders charac- terized by trauma-induced mucocutaneous blistering and scarring. Ultrastructurally, blisters are formed beneath the lamina densa and are associated with changes in anchoring fibrils at the dermal-epidermal junction. In many cases of RDEB, electron micro- scopy reveals no definite anchoring fibrils and only a variable number of morphologically indistinct sub- lamina densa wisp-like structures. The structural composition of these wisp-like fibrils is uncertain. We have attempted to" define the macromolecular nature of these structures using pre-embedding immunogold electron microscopy. An antibody to type VII collagen, the major component of anchoring fibrils, and an antibody to fibrillin, the main constituent of elastic microfibrils at the dermal-epidermal junction were used in six patients with the localized form of RDEB and 12 patients with generalized RDEB. In addition, eight non-affected controls and skin from a 15-week gestation fetus with generalized RDEB and one 14-week non-affected fetus were also studied. Specific immunolabelling for type VII collagen was seen on many of the previously ill-defined wisp-like fibrils. The number and width of these immunogold labelled fibrillar structures showed some inverse corre- lation with the degree of severity of the RDEB pheno- type. In intact skin, elastic microfibril labelling in both localized and generalized RDEB skin was similar to the normal controls. In sites of blistering, however, some of the sub-lamina densa wisp-like structures had definite elastic microfibril immunolabelling despite a lack of morphological distinction from many of the anchoring fibril epitope labelled fibrillar structures. In fetal skin, the ultrastructural appearances of the fibro- reticular network in normal and RDEB skin showed some similarities, but immunoelectron microscopy demonstrated that many of the ill-defined wisp-like structures at this gestational age constitute developing elastic microfibrils. This study suggests that the underlying abnormality in RDEB involves a range of primary structural abnormalities of anchoring fibrils, and that in the presence of such changes in anchoring fibrils, elastic microfibrils cannot sustain adhesion at the dermal-epidermal junction, are disrupted after trauma, and when fragmented have ultrastructural appearances similar to immature or defective anchor- ing fibrils. 8. Defects in Type X Collagen A Mutation in the Conserved NC1 Domain of Type X Collagen Prevents In Vitro Multimer Assembly Resulting in a Schmid- Type Metaphyseal Chondrodysplasia Danny Chan*, William G. Cole*, John Rogerst and John F. Bateman* *Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne; and tThe Murdoch Institute, Royal Children's Hospital, Parkville 3052, Australia Type X collagen is a homotrimer of 0tl(X) chains encoded by the COL10A1 gene. It is a highly special- ized extracellular matrix component and its synthesis is restricted to the zone of endochondral ossification, hypertrophic chondrocytes in the calcifying cartilage of the growth plate, and in zones of secondary ossifi- cation. Our studies on a family with the Schmid type of metaphyseal chondrodysplasia demonstrated that the affected individuals were heterozygous for a sin- gle-base substitution in the COL10A1 gene which changed the codon GGC for glycine 618 to GTC for valine in the highly conserved region of the NC1 domain and altered the amino-acid sequence in the putative oligosaccharide attachment site. The se- quence change was shown to segregate with the disor- der and was absent in a further 60 normal COL10A1 alleles. Since hypertrophic cartilage tissue or cell cul- tures were not available to assess the effect of the mutation, an in vitro cDNA expression system was used to study normal and mutant type X collagen biosynthesis and assembly. Full-length cDNA con- structs of the normal type X collagen sequence and also cDNA containing the specific Gly to Val NC1 mutation found in the patient were produced and expressed in vitro. While the control construct pro- duced type X collagen which formed trimeric collagen monomers which assembled into larger supramolecu- lar assemblies, the mutant collagen was unable to form these larger aggregates. These experiments directly demonstrated that the mutation disturbed type X collagen assembly in vitro, a finding consistent with the abnormal disorganized cartilage growth plate seen in the patient. These studies provide the first