COL5A1 exon 14 splice acceptor mutation causes a functional null allele, haploinsufficiency of alpha 1(V) and abnormal heterotypic interstitial fibrils in Ehlers-Danlos syndrome II

We studied four affected individuals from a family of three generations wit h Ehlers-Danlos Syndrome II. Type V collagen transcripts of affected indivi duals were screened by reverse transcriptase-polymerase chain reaction. Amp lification of the exon 9-28 region of alpha1(V) yielded normal and larger p roducts from the proband, Sequencing of cDNA revealed a 100-base pair inser tion from the 3'-end of intron 13 between exons 13 and 14 in one allele. Th e genomic defect was identified as an A(-2)-->G substitution at the exon 14 splice acceptor site. A cryptic acceptor site -100 nucleotide within intro n 13 is used instead of the mutant splice site. The insertion shifts the re ading frame +1 and results in a stop codon within exon 17. The mutant trans cript was much less abundant than normal allele product in untreated cultur ed fibroblasts but was approximately equimolar in cycloheximide treated cel ls, suggesting that the mutation causes nonsense-mediated decay of mRNA. By RNase protection experiments, the level of mutant transcript was determine d to be 8% that of the normal transcript in untreated proband fibroblasts, Relative to type I collagen, proband fibroblasts secreted only 65% of the a mount of type V collagen secreted by normal controls. Selective salt precip itation of proband secreted collagen provided supportive evidence that the alpha chain composition of type V collagen remains alpha1(V)(2)alpha2(V) ev en in the context of alpha1(V) haploinsufficiency. Type V collagen incorpor ates into type I collagen fibrils in the extracellular matrix and is though t 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 propo rtion of both larger and smaller fibrils and occasional fibrils with a caul iflower configuration. Unlike the genotype/phenotype relationship seen for type I collagen defects and osteogenesis imperfecta, the null allele in thi s family appears to cause clinical features similar to those seen in cases with structural alterations in type V collagen.