COL5A1 exon 14 splice acceptor mutation causes a functional null allele, haploinsufficiency of α1(V) and abnormal heterotypic interstitial fibrils in Ehlers-Danlos syndrome II

We studied four affected individuals from a family of three generations with Ehlers-Danlos Syndrome II. Type V collagen transcripts of affected individuals were screened by reverse transcriptase-polymerase chain reaction. Amplification of the exon 9-28 region of alpha1(V) yielded normal and larger products from the proband, Sequencing of cDNA revealed a 100-base pair insertion from the 3'-end of intron 13 between exons 13 and 14 in one allele. The genomic defect was identified as an A(-2)-->G substitution at the exon 14 splice acceptor site. A cryptic acceptor site -100 nucleotide within intron 13 is used instead of the mutant splice site. The insertion shifts the reading frame +1 and results in a stop codon within exon 17. The mutant transcript was much less abundant than normal allele product in untreated cultured fibroblasts but was approximately equimolar in cycloheximide treated cells, suggesting that the mutation causes nonsense-mediated decay of mRNA. By RNase protection experiments, the level of mutant transcript was determined to be 8% that of the normal transcript in untreated proband fibroblasts, Relative to type I collagen, proband fibroblasts secreted only 65% of the amount of type V collagen secreted by normal controls. Selective salt precipitation of proband secreted collagen provided supportive evidence that the alpha chain composition of type V collagen remains alpha1(V)(2)alpha2(V) even in the context of alpha1(V) haploinsufficiency. Type V collagen incorporates into type I collagen fibrils in the extracellular matrix and is thought to regulate fibril diameter. Transmission electron micrographs of type I collagen fibrils in a proband dermal biopsy showed greater heterogeneity in fibril diameter than in a matched control. The proband had a greater proportion of both larger and smaller fibrils and occasional fibrils with a cauliflower configuration. Unlike the genotype/phenotype relationship seen for type I collagen defects and osteogenesis imperfecta, the null allele in this family appears to cause clinical features similar to those seen in cases with structural alterations in type V collagen.