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 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.
Bouma
P
P
Heritable Disorders Branch/NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cabral
W A
WA
Cole
W G
WG
Marini
J C
JC
D000293:Adolescent; D000328:Adult; D000483:Alleles; D017398:Alternative Splicing; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics* / Q000648:ultrastructure; D004535:Ehlers-Danlos Syndrome / Q000235:genetics* / Q000473:pathology; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D016368:Frameshift Mutation; D006801:Humans; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D010375:Pedigree; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure
D003094:Collagen