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Mutations in COL11A2 cause a spectrum of phenotypes affecting chondrogenic tissues. We analyzed this gene by conformation sensitive gel electrophoresis (CSGE) and sequencing in a family with non-ocular Stickler syndrome, and found a heterozygous C --> T mutation in exon 57 + 13 in affected members, resulting in Arg893Stop codon. Since heterozygous nonsense mutations in COL11A2 do not usually lead to any obvious phenotype, all exons and exon boundaries of COL11A2 in the sample of the propositus were sequenced. Because no disease-associated alterations were found, we performed RT-PCR analysis on the RNA. Analysis showed skipping of exon 57 in one allele, resulting in an inframe deletion of 54 bp or 18 amino acids, which would explain the phenotype observed in the family. Thus, the exon skipping resulted from a nonsense-associated altered splicing (NAS). This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299/suppmat/index.html.

The human COL11A2 gene was analyzed from two overlapping cosmid clones that were previously isolated in the course of searching the human major histocompatibility region (Janatipour, M., Naumov, Y., Ando, A., Sugimura, K., Okamoto, N., Tsuji, K., Abe, K., and Inoko, H. (1992) Immunogenetics 35, 272-278). Nucleotide sequencing defined over 28,000 base pairs of the gene. It was shown to contain 66 exons. As with most genes for fibrillar collagens, the first intron was among the largest, and the introns at the 5-end of the gene were in general larger than the introns at the 3-end. Analysis of the exons coding for the major triple helical domain indicated that the gene structure had not evolved with the genes for the major fibrillar collagens in that there were marked differences in the number of exons, the exon sizes, and codon usage. The gene was located close to the gene for the retinoic X receptor ␤ in a head-to-tail arrangement similar to that previously seen with the two mouse genes (P. Vandenberg and D. J. Prockop, submitted for publication). Also, there was marked interspecies homology in the intergenic sequences. The amino acid sequences and the pattern of charged amino acids in the major triple helix of the ␣2(XI) chain suggested that the chain can be incorporated into the same molecule as ␣1(XI) and ␣1(V) chains but not into the same molecule as the ␣3(XI)/␣1(II) chain. The structure of the carboxyl-terminal propeptide was similar to the carboxyl-terminal propeptides of the pro␣1(XI) chain and pro␣ chains of other fibrillar collagens, but it was shorter because of internal deletions of about 30 amino acids.Over 19 types of collagens are known, each with an apparently unique biological function (1-3). A major subclass is the fibrillar collagens that form ordered extracellular fibrils and that include type I, type II, type III, type V, and type XI collagens. Type I and type III collagens are found in most non-cartilaginous tissues. Type II is found primarily in cartilage where it is the most abundant protein, but it is also present in the vitreous humor and several other tissues in early embryonic development. Type XI collagen was originally recognized as a minor fibrillar collagen in cartilage that was similar to type II collagen. The protein was considered to consist of three ␣ chains referred to as ␣1(XI), ␣2(XI), and ␣3(XI) (4 -6). The ␣3(XI) chain was subsequently shown to be derived from the same gene as the ␣1(II) chain of type II collagen that, by an unknown mechanism, was assembled with the ␣1(XI) and ␣2(XI) chain to form a unique procollagen molecule (4 -9). In further analyses, type XI collagen was found to be closely related in structure to type V collagen, and both type V and type XI collagens were found in small amounts in a variety of cartilaginous and non-cartilaginous connective tissues (10, 11). Amino acid sequencing of fragments of collagen fibrils from mammalian vitreous humor demonstrated that fibrils were assembled from molecules containing ␣1(XI) and ␣2(V) chains (12). Also, during th...

As type IX collagen is a minor cartilage component, it is difficult to purify sufficient amounts of it from tissues or cultured cells to study its structure and function. Also, the conventional pepsin digestion used for fibrillar collagens cannot be utilized for purifying type IX collagen, because it contains several interruptions in its collagenous triple helix. A baculovirus expression system was used here to produce recombinant human type IX collagen by coinfecting insect cells with three viruses containing full-length cDNAs for the ␣1(IX), ␣2(IX), and ␣3(IX) collagen chains together with a double promoter virus for the ␣ and ␤ subunits of human prolyl 4-hydroxylase. Correctly folded recombinant type IX collagen was secreted, consisting of the three ␣ chains in a 1:1:1 ratio and showing the expected biphasic thermal melting profile. When the individual ␣ chains were expressed, disulfide-bonded homotrimers and homodimers of the ␣ chains were observed. When the cells were coinfected with the viruses for all three ␣ chains, heterotrimers of ␣1(IX), ␣2(IX), and ␣3(IX) were detected in cell culture medium, and the other possible combinations were less prominent. When any two of the ␣ chains were co-expressed, in addition to the homodimers and homotrimers, only ␣1(IX) and ␣3(IX) chains were disulfide-bonded. The results thus suggest that the most favored molecular species is an ␣1(IX)␣2(IX)␣3(IX) heterotrimer, but the chains are also able to form disulfide-bonded heterotrimers of ␣1(IX) and ␣3(IX) chains and (␣1(IX)) 3 , (␣2(IX)) 3 , and (␣3(IX)) 3 homotrimers.Type IX collagen, which belongs to the group of fibril-associated collagens with interrupted triple helices, is a component of hyaline cartilage, intervertebral discs, and the vitreous body. The molecule is a heterotrimer consisting of three genetically distinct chains, ␣1(IX), ␣2(IX), and ␣3(IX) (1) and possesses three collagenous domains (COL1 to COL3, numbered from the C terminus) flanked by four noncollagenous domains (NC1 to NC4) (2, 3). Type IX collagen is also a proteoglycan, because a glycosaminoglycan side chain is covalently attached to the NC3 domain of the ␣2(IX) chain (4).Hyaline cartilage contains mixed fibrils of types II, IX, and XI collagens, of which type II is the major component. Type XI collagen is an internal component of the fibril, whereas type IX collagen is located on the surface. Covalent lysine-derived cross-links between the central COL2 region of the ␣3(IX) chain and the C-telopeptide of type II collagen and between the N-terminal ends of the COL2 domains of all the type IX collagen ␣ chains and the N-telopeptide of type II collagen stabilize the interaction between type II and IX collagens (5-8). The flexibility of the NC3 domain of type IX collagen allows the COL3 and NC4 domains to project from the fibril surface, possibly to mediate interactions between cartilage collagens and noncollagenous proteins (6, 9, 10). The association of ␣ chains in proper stoichiometry and register is a prerequisite for the formation of a stable co...