Novel mutations of COL3A1 resulting in Ehlers-Danlos syndrome type IV and their effect on the folding of type III procollagen

Abstract

Thesis (Ph. D.)--University of Washington, 1998Ehlers-Danlos syndrome type IV (EDS IV) is an inherited disease caused by mutation of COL3A1, the gene that encodes type III procollagen. Different mutations produce the disease: point mutations, exon skipping mutations and genomic insertations and deletions. The effect of these different lesions on type III procollagen, the cells that produce it, and the ultrastructural aggregates of which it is a component are not known.To better understand the consequences of COL3A1 mutation, protein secretion, protein folding, cell architecture, and dermal collagen fibril formation were examined following the characterization of novel EDS IV-causing mutations. I defined 15 mutations: skipping of exons 9, 14, 18, 22, 24, 34, and 38 and point mutations G373R, G769R, G829E, G904V, G1012R, G1015E, G1018V, G1021E all of which are in the triple helical portion of the protein. I determined the genomic origin of the exon 22 skip to be a 57 base pair deletion which removed 13 bases of intron 21 and 44 bases of exon 22. The remaining six exon skipping mutations were defined at the genomic level by another member of the lab.The proteins from affected individuals displayed a gradient of overmodification; proteins with mutations closest to amino terminal end of the triple helix displayed the least amount with the degree of overmodification increasing as the mutation moved toward the carboxyl-terminal end of the protein concomitant with increased intracellular retention. The mutations nearest the carboxyl-terminal end of the triple helix, G1012R, G1015E, G1018V, G1018D, G1021E, G1021R, interfere with disulfide bond formation and perturb triple helix formation at 37\sp\circC suggesting that the domain for nucleating folding of the triple helix is within the last 20-25 amino acids of this region.Electron microscopic examination of patient cells indicates that the ER is the site of retention and is increasingly dilated as mutation moves from N- to C-terminus. The fibrils from mutations in the first third of the triple helix display greater variability than normal and are also larger while those in the last third of the helix are much smaller than normal