STRUCTURALLY ABNORMAL TYPE-II COLLAGEN IN A SEVERE FORM OF KNIEST DYSPLASIA CAUSED BY AN EXON-24 SKIPPING MUTATION

Type II collagen mutations have been identified in a phenotypic contin uum of chondrodysplasias that range widely in clinical severity. They include achondrogenesis type II, hypochondrogenesis, spondyloepiphysea l dysplasia congenita, spondyloepimetaphyseal dysplasia, Kniest dyspla sia, and Stickler syndrome. We report here results that define the und erlying genetic defect and consequent altered structure of assembled t ype II collagen in a neonatal lethal form of Kniest dysplasia, Electro phoresis of a cyanogen bromide (CNBr) (CB) digest of sternal cartilage revealed an alpha 1(II)CB11 peptide doublet and a slightly retarded m obility for all major CB peptides, which implied post-translational ov ermodification. Further peptide mapping and sequence analysis of CB11 revealed equal amounts of a normal alpha 1(II) sequence and a chain la cking the 18 residues (361-378 of the triple helical domain) correspon ding to exon 24. Sequence analysis of an amplified genomic DNA fragmen t identified a G to A transition in the +5 position of the splice dono r consensus sequence of intron 24 in one allele. Cartilage matrix anal ysis showed that the short alpha 1(II) chain was present in collagen m olecules that had become cross-linked into fibrils. Trypsin digestion of the pepsin-extracted native type II collagen selectively cleaved th e normal length alpha 1(II) chains within the exon 24 domain, These fi ndings support a hypothesis that normal and short alpha-chains had com bined to form hetero trimeric molecules in which the chains were in re gister in both directions from the deletion site, accommodated effecti vely by a loop out of the normal chain exon 24 domain. Such an accommo dation, with potential overall shortening of the helical domain and he nce misalignment of intermolecular relationships within fibrils, offer s a common molecular mechanism by which a group of different mutations might act to produce the Kniest phenotype.