Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias belonging to the same bone dysplasia family. PSACH is characterized by generalized epi-metaphyseal dysplasia, short-limbed dwarfism, joint laxity and early onset osteoarthritis. MED is a milder disease with radiographic features often restricted to the epiphyses of the long bones. PSACH and some forms of MED result from mutations in cartilage oligomeric matrix protein (COMP), a pentameric glycoprotein found in cartilage, tendon, ligament and muscle. PSACH-MED patients often have a mild myopathy characterized by mildly increased plasma creatine kinase levels, a variation in myofibre size and/or small atrophic fibres. In some instances, patients are referred to neuromuscular clinics prior to the diagnosis of an underlying skeletal dysplasia; however, the myopathy associated with PSACH-MED has not previously been studied. In this study, we present a detailed study of skeletal muscle, tendon and ligament from a mouse model of mild PSACH harbouring a COMP mutation. Mutant mice exhibited a progressive muscle weakness associated with an increased number of muscle fibres with central nuclei at the perimysium and at the myotendinous junction. Furthermore, the distribution of collagen fibril diameters in the mutant tendons and ligaments was altered towards thicker collagen fibrils, and the tendons became more lax in cyclic strain tests. We hypothesize that the myopathy in PSACH-MED originates from an underlying tendon and ligament pathology that is a direct result of structural abnormalities to the collagen fibril architecture. This is the first comprehensive characterization of the musculoskeletal phenotype of PSACH-MED and is directly relevant to the clinical management of these patients.

The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was approximately 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.

Metaphyseal chondrodysplasia type Schmid (MCDS)is caused by mutations in COL10A1 that are clustered in the carboxyl-terminal non-collagenous (NC1) encoding domain. This domain is responsible for initiating trimerization of type X collagen during biosynthesis. We have built a molecular model of the NC1 domain trimer based on the crystal structure coordinates of the highly homologous trimeric domain of ACRP30 (adipocyte complement-related protein of 30 kDa or AdipoQ). Mapping of the MCDS mutations onto the structure reveals two specific clusters of residues as follows: one on the surface of the monomer which forms a tunnel through the center of the assembled trimer and the other on a patch exposed to solvent on the exterior surface of each monomeric unit within the assembled trimer. Biochemical studies on recombinant trimeric NC1 domain show that the trimer has an unusually high stability not exhibited by the closely related ACRP30. The high thermal stability of the trimeric NC1 domain, in comparison with ACRP30, appears to be the result of a number of factors including the 17% greater total buried solvent-accessible surface and the increased numbers of hydrophobic contacts formed upon trimerization. The 27 amino acid sequence present at the amino terminus of the NC1 domain, which has no counterpart in ACRP30, also contributes to the stability of the trimer. We have also shown that NC1 domains containing the MCDS mutations Y598D and S600P retain the ability to homotrimerize and heterotrimerize with wild type NC1 domain, although the trimeric complexes formed are less stable than those of the wild type molecule. These studies suggest strongly that the predominant mechanism causing MCDS involves a dominant interference of mutant chains on wild type chain assembly.Type X collagen is a short chain, homotrimeric collagen (␣1(X) 3 ) expressed specifically by hypertrophic chondrocytes in the endochondral growth plate (1). The expression of type X collagen is also re-activated during fracture repair and in osteoarthritis (2-4). The human ␣1(X) collagen chain consists of a short amino-terminal non-collagenous domain 2 of 37 amino acids followed by a triple helix-forming collagenous domain with 154 Gly-X-Y repeats and a carboxyl-terminal non-collagenous domain (NC1) 1 of 161 amino acids (5). The ␣1(X) 3 molecule is thought to assemble into a hexagonal lattice within the extracellular matrix in a fashion similar to that of type VIII collagen (6, 7). Type X collagen is part of a family of collagenlike proteins sharing a condensed gene structure, a collagen triple helical domain, and in particular, a highly conserved carboxyl-terminal non-collagenous (NC1-like) domain (8, 9). This family includes collagen types X and VIII, C1q component of complement, hibernation proteins (10), cerebellin (11, 12), and ACRP30, an abundant serum protein implicated in energy homeostasis and obesity (13).The precise function of type X collagen remains to be determined (14 -16), but mutations in the COL10A1 gene cause metaphyseal chondrodysplasia ...