Abstract 792 Mechanisms in Hereditary Disease Platform, Tuesday, 5/4

Ehlers-Danlos Syndrome (EDS) is a heterogeneous connective tissue disorder that affects skin, joints, ligaments, and blood vessels. Recently, defects in the α1 and α2 chains of type V collagen have been identified in several families with EDS I-II. We studied a family of three generations with EDS II. They show skin laxity, small joint hypermobility, and easy bruising. Cigarettes paper scars formed only at sites of extensive lacerations. The female proband brought four pregnancies to term without complications. We are using an RT-PCR based assay to screen EDS I and II patients for splicing defects in the α1 and α2 chains of type V collagen. Using primers located in exons 9 and 28 of α1(V) cDNA, we detected both a normal 1049 bp product and a larger 1149 bp product from the proband. Subcloning and sequencing revealed a 100 bp insertion of sequences from the 3′ end of intron 13 between exons 13 and 14. The genomic defect was identified as an A→G substitution at the -2 position of the 3′ splice (acceptor) site of exon 14. This mutation creates a novel Aci I restriction enzyme site that was used for mutation confirmation in family members. A cryptic acceptor site -100 bp within intron 13 is used as the new acceptor site. No additional alternatively spliced transcripts were detected by RT-PCR using a sense primer in exon 9 and an antisense primer at the 3′ end of intron 13. The insertion shifts the reading frame +1 and results in a stop codon within exon 17.

The insertion is located at the end of the N-propeptide, with the triple helical region beginning within exon 14. Since helix formation starts at the C-terminal end, the truncated propeptide can not be incorporated into helix and causes a null allele. We are investigating the effect of this defect on levels of type V collagen mRNA and protein. This is the first case in either the α1 or α2 chain of type V collagen in which the defect is located in the N-propeptide and causes a truncated chain. The only truncated α1(V) protein described previously was caused by a translocation with breakpoint in exon 24. The mild clinical features of this family may be attributable to the null allele. Most previously described type V collagen defects cause structural alterations in the α chains which permit incorporation into trimers and are associated with more severe connective tissue manifestations.