Alternative splicing of exon 12 of the COL2A1 gene interrupts the triple helix of type‐II collagen in the kniest form of spondyloepiphyseal dysplasia

An autosomal dominant mutation in the COL2A1 gene was identified in a child with the Kniest form of spondyloepiphyseal dysplasia. A C to T transition at nucleotide 35 of exon 12 changed the codon GCG for alanine 102 of the triple helical domain of α1 (II) chains of type‐II collagen to GTG for valine. The transition also introduced a GT dinucleotide into exon 12. Analysis of cDNA prepared from Kniest cartilage showed that in vivo the transition resulted in an alternatively spliced mRNA that lacked the 21 3′ nucleotides from exon 12. The cartilage cDNA contained approximately equal amounts of normal cDNA and shortened mutant cDNA. The deletion of 21 nucleotides from the mutant cDNA maintained the translational reading frame but resulted in the loss of alanine 102 to lysine 108, which interrupted the repetitive glycine‐X‐Y triplet sequence required for formation of the triple helix. Type‐II collagen molecules containing one or more mutant chains were expected, therefore, to contain interrupted triple helices with a short amino‐terminal helical domain A and a large carboxy‐terminal helical domain B. Kniest cartilage contained a reduced amount of pepsin‐solubilized type‐II collagen that consisted of overmodified α1 (II) chains. Peptide mapping showed that the overmodifications extended to the carboxy terminus of the α1(II) chains. Pepsin digestion also yielded shortened α1 (II) chains corresponding to helical domain B, Kniest chondrocytes cultured in alginate beads produced type‐II collagen that was not stably incorporated into the pericellular matrix. This study highlights the importance of dominant negative mutations of COL2A1 in producing Kniest dysplasia.