Abstract The nucleotide sequence of the entire COL9A3 gene, coding for the human α3(IX) chain, was determined. The gene was approximately 23 kb in length and consisted of 32 exons. The polymerase chain reaction (PCR)-based procedure of conformation-sensitive gel electrophoresis (CSGE) was used to screen the gene for sequence variations and mutations in 83 unrelated patients with generalized primary osteoarthritis (OA), 31 with rheumatoid arthritis (RA), 171 with intervertebral disc disease (IDD), and 50 with various osteochondrodysplasias. The frequencies of certain sequence variations in healthy individuals were also determined. The COL9A3 gene was analyzed for mutations in two unrelated families with multiple epiphyseal dysplasia (MED). The analysis revealed a splice site mutation leading to skipping of exon 3 and an in-frame loss of 12 amino acid residues in the COL3 domain, the first diseasecausing mutation to be identified in the COL9A3 gene. Sequencing also indicated a 9 bp deletion in one allele in the second MED family that removed a Gly-Pro-Pro triplet. Surprisingly, the deletion did not co-segregate with the MED phenotype in the family. A similar 9 bp deletion, was also found in an unrelated family with no obvious phenotype, suggesting that the two 9 bp deletions represent neutral sequence variants. A construct with the deletion was then made in order to produce a recombinant protein, and the mutant type IX collagen was analyzed under reducing conditions by SDS-PAGE. The results indicated that the recombinant type IX collagen proteins consisted of three α chains, α1(IX), α2(JX), α3(IX), in a 1:1:1 ratio. To study the triple helix stability, pepsin treatment followed by SDS-PAGE was performed on normally folded and denatured recombinant type IX collagen samples. The results demonstrated that the recombinant type IX collagen containing the Gly-X-Y deletion in the a3(IX) chain is secreted as a correctly folded triple-helical molecule. CSGE analysis of exon 5 of the COL9A3 gene identified two nucleotide variations in the same codon, and thus three alleles: CGG (Arg), CAG (Gln), and TGG (Trp). The frequency of the Trp for Arg substitution, the Trp3 allele, was 0.244 among the probands with the IDD, while its overall frequency in the combined group of all non-IDD cases was 0.093. This difference was significant, with a p-value of 0.000013. The Trp3 allele increases the relative risk of IDD by a factor of 2.6 (95 percent confidence interval, 1.6 to 4.3). COL9A3 mutations are shown to be associated with mild cartilage and intervertebral disc diseases.

Abstract The role of sequence variations in genes encoding cartilage extracellular matrix (ECM) proteins were studied in osteoarthritis (OA) and multiple epiphyseal dysplasia (MED). The cartilage collagen genes COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, and COL11A2 were screened for sequence variations in 72 Finnish probands and one US family with primary early-onset hip and/or knee OA. Altogether 239 sequence variations were found, of which 16 were not present in the controls. Seven of the unique variations — four in COL11A1, two in COL11A2, and one in COL2A1 — were studied further, because they resulted in the substitution of conserved amino acids or were predicted to affect mRNA splicing. Association analysis was performed by genotyping 6–12 common polymorphisms from each gene in 72 OA patients and 103 controls; no common predisposing alleles were identified. The results, however, suggest that mutations in the minor cartilage collagen genes can be the cause of OA in a subgroup of OA patients. Two MED families with clinical and radiographic features suggestive of a collagen IX mutation were studied. Mutation screening of COL9A1, COL9A2, and COL9A3 yielded negative results. Instead, an R718W mutation in COMP was identified in both families. Clinical and radiographic overlap between patients with collagen IX mutations and patients with COMP mutations points to a common supramolecular complex pathogenesis. Clinical, radiological and molecular analyses of known MED genes were performed on a cohort of 29 consecutive MED patients. The DTDST mutation was identified in four patients (14%), the COMP mutation in three (10%), and the MATN3 mutation in three (10%). Two new distinct phenotypic entities were identified in patients in whom no mutation was found. The findings suggest that mutations in the above mentioned known MED genes are not the major cause of MED and are responsible for less than half of the cases. The existence of additional MED loci is supported by the exclusion of known loci and finding of the specific subgroups among these patients. The results suggest that genetic defects in ECM genes can predispose to OA and cause MED, even though the major genes involved in both disorders remain to be found.

Mutations in genes of importance for cartilage development may lead to skeletal malformations, chondroskeletal dysfunction and increased susceptibility to degenerative joint disease. Characterization of these mutations and identification of molecular pathways for the corresponding gene products have contributed to our understanding of mechanisms regulating skeletal patterning, endochondral ossification and joint formation. A five generation family segregating autosomal dominant osteochondritis dissecans (OCD) was identified. Affected family members presented with OCD in knees, hips and elbows, short stature, and early osteoarthritis. A genome wide scan and a multipoint linkage analysis identified aggrecan (ACAN) as a prime candidate gene. DNA sequence analysis of the ACAN-gene revealed heterozygosity for a missense mutation (c.6907G>A) in affected subjects, resulting in a p.V2303M substitution in the aggrecan G3 domain C-type lectin. This domain is important for the interaction with other proteins in the cartilage extracellular matrix. To determine the effect of the V2303M substitution on secretion and interaction, we performed binding studies with recombinant mutated and wild type G3 proteins. We found decreased affinity or complete loss of interaction between V2303M aggrecan and fibulin1, fibulin2 and tenascin-R. Analysis of articular cartilage from an affected family member confirmed that V2303M aggrecan is produced and present. In search for gene mutations associated with multiple epiphyseal dysplasia (MED) we considered the ACAN-gene a likely candidate. The ACAN-gene was analysed in 39 individuals with MED and screened negative for mutations in six previously known MED genes. Sequence analysis revealed a heterozygous missense mutation (c.1448G>T) in one adult male and compound heterozygous missense mutations (c.1366T>C and c.836G>A) in a five year old boy with healthy parents, each of them carrier for one of the mutations. A large family segregating autosomal dominant brachymesophalangia and OCD in finger joints was characterised. The clinical presentation in six affected family members was consistent with the diagnosis Brachydactyly type A1, in this family characterized by shortening of the middle phalanges, short ulnar styloid process, flattening of the metacarpal heads and mild osteoarthritis. The condition may be caused by mutations in the Indian hedgehog gene (IHH) or a yet unidentified gene on chromosome 5p13. Sequence analysis of the IHH-gene in affected individuals revealed a novel C to T transition (c.472C>T) leading to a p.158Arg>Cys substitution. Residue 158 in IHH is highly conserved throughout evolution and molecular structure modelling of IHH suggests that the R158C substitution leads to a conformational change at the site of interaction with the IHH-receptor. This supports that the substitution causes Brachydactyly type A1 in this family. In summary, we report on the clinical, radiological and molecular genetic characteristics of the three skeletal disorders OCD, MED and BDA1. Our results provide a novel molecular mechanism in the pathophysiology of familial osteochondritis dissecans confirming the importance of aggrecan C-type lectin for cartilage function. We also show that ACAN-gene mutations may be associated with MED extending the spectrum of skeletal dysplasias associated with the aggrecan gene. Finally, we report on a novel missense mutation in a conserved region of the IHH-gene associated with BDA1.

Cysteine rich with EGF-like domains 2 (Creld2) is a novel endoplasmic reticulum (ER) resident molecular chaperone that has been recently implicated in the ER stress signalling response (ERSS) and the unfolded protein response (UPR). Global transcriptomic data derived from in vivo mouse models of rare chondrodysplasias; Multiple Epiphyseal Dysplasia (MED Matn3 p.V194D) and Metaphyseal chondrodysplasia type Schmid (MCDS Col10a1 p.N617K), identified a significant upregulation in Creld2 expression in mutant chondrocytes. These chondrodysplasias share a common disease signature consisting of aberrant folding of a matrix component often as a result of inappropriate alignment of intramolecular disulphide bonds. This in turn culminates in toxic protein aggregation, intracellular retention mutant polypeptides and a classical ER stress response. The aim of this study was to further analyse the function of Creld2 in cartilage development and chondrodysplasias in which endochondral bone growth is perturbed. Protein disulphide isomerases (PDIAs) were amongst the most up-regulated genes in the MED and MCDS mouse models, consistent with the prolonged exposure of normally 'buried' cysteine residues. This led to the hypothesis that Creld2 was functioning as a novel PDI-like oxidoreductase to assist in the correct folding and maturation of aggregated misfolded polypeptide chains through REDOX regulated thiol disulphide exchange. A series of Creld2-CXXA substrate trapping mutants were generated in order to determine whether Creld2 possessed inherent isomerase activity. Here potential substrates interacting with Creld2 were 'trapped' as mixed disulphide intermediates, then isolated by immunoprecipitation and identified by mass spectrometry analysis. It was demonstrated that Creld2 possessed a catalytic active CXXC motif in its N-terminus that enabled the molecular chaperone to participate in REDOX regulated thiol disulphide exchange with at least 20 potential substrates including; laminin (alpha3,β3,γ2), thrombospondin 1, integrin alpha3 and type VI collagen. There was also numerous co-chaperones and foldases thought to be part of a specialised protein-protein interactome (PPI) for folding nascent polypeptides translocating the ER lumen. Moreover, co-immunoprecipitation experiments supported a protein-protein interaction between Creld2 and mutant matrilin-3, thereby inferring a potential chondro-protective role in resolving non-native disulphide bonded aggregates in MED. An established biochemical approach was employed to test the hypothesis that all MATN3-MED disease causing mutations have a generic cellular response to the β-sheet V194D mutation, consisting of intracellular retention, protein aggregation and ER stress induction. Several missense mutations were selected for analyses which encompassed a spectrum of disease severity and included examples of both β-sheet and alpha helical mutations. It was possible to define a reliable and reproducible assay for categorising MATN3 missense mutations into pathological or benign based on these basic parameters. This study was extended further to determine whether there were common pathological mechanisms behind MED and Bethlem myopathy (BM) caused by missense mutations in von Willebrand Factor A domain (vWF-A) containing proteins (matrilin-3 and type VI collagen respectively). We chose to compare and contrast the effects of an archetypal MATN3-MED causing mutation (R121W) with the equivalent COL6A2-BM causing mutation (R876H). These mutations compromised protein folding and maturation, resulting in the familiar disease profile of intracellular retention, protein aggregation and an ER stress response in an artificial overexpression system. However, the mutant C2 domain was efficiently targeted for degradation whilst mutant matrilin-3 vWF-A domain appeared to be resistant to these molecular processes.Molecular genetics was employed to study the role of Creld2 in vivo. Creld2-/- null mice (both global and conditional) were generated to directly examine the role of Creld2 in endochondral bone growth. Global knock-out mice were viable with no overt phenotype at birth. However, female Creld2-/- null mice showed a significant reduction in body weight and tibia bone length at 3 weeks of age. A cartilage specific knock-out was generated to determine whether these skeletal abnormalities were attributed to a systemic or a direct effect on cartilage development. [Creld2Flox/Flox Col2Cre (+)] demonstrated a severe chondrodysplasia with significantly reduced body weight and long bone growth compared to control littermates. Morphological and histochemical analysis of mutant growth plates revealed gross disorganisation of the chondrocyte columns with extensive regions of hypocellularity. These pathological features were confirmed to be the result of reduced chondrocyte proliferation and increased/spatially dysregulated apoptosis throughout all zones of differentiation. Taken together, these data provide evidence that Creld2 possesses isomerase activity and exhibits distinct substrate specificity. Furthermore, Creld2 has a fundamental role in post-natal cartilage development and chondrocyte differentiation in the growth plate.