Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable disorders of connective tissue. The type III variety is characterized by joint hypermobility and minor hyperextensibility and softness of the skin. While collagen fibril structure has been shown to be abnormal in such patients, the underlying molecular defect(s) has not been determined. Here we characterize the first mutation found in a family with EDS III. Analysis of cultured fibroblasts from the affected family revealed intracellular retention of type III collagen. This is usually a biochemical characteristic of EDS IV, caused by mutations of COL3A1. Analysis of the cDNA sequence in this EDS III family revealed a glycine to serine mutation at amino acid residue 637 of the type III collagen molecule. This was confirmed by allele-specific oligonucleotide hybridization against amplified genomic DNA. Thus mutations of type III collagen can cause either EDS IV or EDS III. Two affected family members had virtually normal skin and so more closely resembled the phenotype of articular hypermobility syndrome.

We have recently analysed by histological, protein and molecular DNA techniques 23 mutations of the collagen III gene (COL3A1), most of which cause premature arterial fragility, thin skin and variants of vascular Ehlers-Danlos syndrome. There were 14 glycine substitutions between residues 637 and 1021, eight exon skips between exons 7 and 45 and one small inframe deletion. The glycine substitutions produce a gradient of increasingly abnormal clinical phenotypes from exons 36 to 49 while the clinical severity of exon skips is much more variable. Each mutation is private for the affected family or individual concerned having the potential for early prenatal diagnosis and prevention.

Skin and temporal arterial biopsies were obtained from 17 patients undergoing surgery for ruptured cerebral aneurysm, and specimens were taken from six age- and sex-matched control surgical patients. Radioactively labeled and control tissue collagen patterns were studied by interrupted polyacrylamide gel electrophoresis (PAGE), using the trisborate buffer system or by carboxymethyl cellulose (CMC) chromatography. Type III/I collagen ratios were then measured from autoradiographs of the radioactively labeled samples using the Joyce Loebl gel scanner adapted for flat bed gels. In the case of the CMC labeled material, the ratios were measured by the ratios of the summed radioactively labeled alpha 1(III), alpha 2(II), and alpha 2(I) peaks. Eleven of the 17 patients were Type III collagen-deficient while all of the six control patients had normal collagen ratios. The implications of these findings are discussed.

We have studied a patient with Ehlers-Danlos syndrome type IV. Protein mapping studies of her type III collagen had indicated that cyanogen bromide fragment 9 contained the site of the mutation. Here we describe the mapping of this region for a single base mutation using a chemical modification and cleavage technique. Sequence analysis of cDNA showed a G to T mutation resulting in the substitution of glycine 910 by valine. This was confirmed by allele specific oligonucleotide hybridisation to the proband's genomic DNA.

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