Identification of four novel COL10A1 missense mutations in schmid metaphyseal chondrodysplasia: further evidence that collagen X NC1 mutations impair trimer assembly

Schmid metaphyseal chondrodysplasia (SMCD) is an autosomal dominant disorder affecting the growth plate cartilage of long bones caused by heterozygous mutations in the gene for collagen X (COL10A1), a short-chain collagen expressed by hypertrophic chondrocytes of growth plate cartilage. In this paper we analyzed six unrelated patients clinically determined as affected by SMCD, and characterized four missense mutations, c.52G>A (p.G18R), c.1744T>G (p.Y582D), c.1792T>G (p.Y598D) and c.1958A>C (p.Q653P). These mutations were clustered in the two regions of the collagen X protein shown to contain all previous SMCD mutations; the signal sequence cleavage site (p.G18R), or the C-terminal NC1 trimerization domain (p.Y582D, p.Y598D and p.Q653P). To determine the functional effect of the mutations we produced engineered p.Y582D, p.Y598D and p.Q653P cDNA and expressed these in vitro. Our data showed that while the wild-type collagen X assembled in vitro into trimers that were stable to SDS-PAGE analysis, p.Y582D (the most N-terminal of the SMCD NC1 mutations described), p.Q653P, and the previously analyzed p.Y598D impair collagen X trimerization. However, in two patients no mutations were detected despite complete sequence analysis of the COL10A1 coding region, the exon-intron splice consensus sequences and the 500bp gene promoter region. Heterozygosity for known polymorphisms ruled out major COL10A1 gene deletions and Southern analysis excluded major rearrangements. The data suggest that in these two patients, SMCD results from mutations at another gene locus. No mutations were detected in RMRP, the gene for cartilage-hair hypoplasia that has phenotypic overlap with SMCD.