Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly inherited, genetically heterogeneous connective-tissue disorder. Mutations in the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified in several individuals. Most mutations affect either the triple-helical domain of the protein or the expression of one COL5A1 allele. We identified a novel splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of COL5A1, in one patient with EDS type I. The outcome of this mutation was complex: In the major product, both exons 5 and 6 were skipped; other products included a small amount in which only exon 5 was skipped and an even smaller amount in which cryptic acceptor sites within exon 5 were used. All products were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted and were incorporated into extracellular matrix, and the mutation resulted in dramatic alterations in collagen fibril structure. The two-exon skip occurred in transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, leaving a large (270 nt) composite exon that can be skipped in its entirety. The transcripts in which only exon 5 was skipped were derived from those in which intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in exon 5 occurred in transcripts in which intron 4 was removed subsequent to introns 5 and 6. These findings suggest that the order of intron removal plays an important role in the outcome of splice-site mutations and provide a model that explains why multiple products derive from a mutation at a single splice site.

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Recessive dystrophic epidermolysis bullosa is a severe mutilating genodermatosis. Previous ultrastructural demonstrations of altered anchoring fibrils, and recent genetic linkage analyses have suggested that type VII collagen, the major component of anchoring fibrils, is a candidate gene. We have identified a homozygous methionine-to-lysine mutation in two affected siblings, while their unaffected mother and half-brother are heterozygous carriers. The mutation resides in a highly conserved region of the C-terminus of type VII collagen, strongly suggesting that it is the cause of the disease in this family.

Ehlers-Danlos syndrome (EDS) types I and II, which comprise the classical variety, are well characterized from the clinical perspective, but it has been difficult to identify the molecular basis of the disorder in the majority of affected individuals. Several explanations for this failure to detect mutations have been proposed, including genetic heterogeneity, failure of allele expression, and technical difficulties. Genetic heterogeneity has been confirmed as an explanation for such failure, since causative mutations have been identified in the COL5A1, COL5A2, and tenascin X genes and since they have been inferred in the COL1A2 gene. Nonetheless, in the majority of families with autosomal dominant inheritance of EDS, there appears to be linkage to loci that contain the COL5A1 or COL5A2 genes. To determine whether allele-product instability could explain failure to identify some mutations, we analyzed polymorphic variants in the COL5A1 gene in 16 individuals, and we examined mRNA for the expression of both alleles and for alterations in splicing. We found a splice-site mutation in a single individual, and we determined that, in six individuals, the mRNA from one COL5A1 allele either was not expressed or was very unstable. We identified small insertions or deletions in five of these cell strains, but we could not identify the mutation in the sixth individual. Thus, although as many as one-half of the mutations that give rise to EDS types I and II are likely to lie in the COL5A1 gene, a significant portion of them result in very low levels of mRNA from the mutant allele, as a consequence of nonsense-mediated mRNA decay.