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Ehlers-Danlos syndrome type IV, an inherited connective tissue disease, is usually caused by mutations in the gene for type III collagen. Here, we describe a glycine to glutamic acid substitution in a patient with this syndrome. Previous studies had shown that fibroblasts from the patient, his mother and brother secreted a reduced amount of type III collagen and also produced an overmodified form of the protein that was preferentially retained intracellularly. Peptide mapping experiments indicated that the mutation was located within cyanogen bromide peptide 9. This was supported by chemical cleavage analysis and sequencing of cDNA encoding this region. Allele-specific oligonucleotide hybridisation of genomic DNA confirmed that a G to A mutation converted Gly 847 to Glu. The mutation was present in two other affected family members and also in a third, who was clinically unaffected. Further analysis of this unaffected individual revealed reduced mutant:normal ratios in DNA obtained from both blood and hair samples, showing that she was mosaic for the mutation.

We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some alpha 2(I) chains. Peptide mapping localized this to the CB peptide alpha 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified a heterozygous deletion of the 54 bp comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5' splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.

A large family with Ehlers-Danlos syndrome type IV (EDS IV) has previously been described. Unlike most cases of EDS IV, fibroblasts from affected members secreted near normal amounts of type III collagen. We have localized the mutation in this family to the CB5 peptide of type III collagen, by using both protein and cDNA mapping techniques. Sequence analysis of cDNA revealed a 27-bp deletion within exon 37, a deletion that removed nine amino acids and maintained the Gly-X-Y repeat of the collagen helix. Further sequencing of genomic DNA confirmed its location, and amplification of DNA from family members showed that it was absent in unaffected individuals but present in all the affected individuals tested. This deletion is flanked by two short direct repeats of CTCC; it may have arisen by slipped mispairing, and has subsequently been transmitted to all affected family members.

We have examined the procollagens and collagens produced by skin fibroblasts from a patient with Ehlers-Danlos syndrome type VII. The patient was heterozygous for an abnormal alpha 2(I) chain migrating with the approximate size of pN alpha 2(I) chains after pepsin digestion. Peptide mapping suggested that the abnormality was located at the amino-terminus of the alpha 2(I) chain. Quantitative analysis of the alpha 2(I) mRNA indicated loss of the exon 6 sequences, and subsequent polymerase chain reaction amplification of cDNA demonstrated a deletion of the 54 bp of exon 6 from some of the alpha 2(I) mRNA. Analysis of genomic DNA from the patient revealed a single base change in one COL1A2 allele, substituting an A for a G as the first base of intron 6. This change mutates the obligate GT-dinulceotide splicing signal to AT and leads to exon skipping with splicing from exon 5 to exon 7. Loss of exon 6 sequences results in the loss of the procollagen-N-propeptidase cleavage site and a lysine residue that normally participates in covalent intermolecular crosslinking within collagen fibres.

We have examined a large family in which eleven members have a form of autosomal dominant Ehlers-Danlos syndrome type IV. Analysis of fibroblast cultures from affected individuals showed a partial deficiency of type III collagen production. The protein produced was, however, normal in all aspects examined. Using a restriction site polymorphism associated with the structural gene for human type III collagen (COL3A1), we have found tight linkage between the low frequency polymorphic allele and the clinical expression of the disease (lod = 3.86 at 0 = 0), identifying the type III collagen gene as the disease locus.