A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans

Abstract

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.

Figure 1

Radiographic Phenotype of Familial Osteochondritis Dissecans and Gene Linkage with Chromosome 15q (A–D) MRI (A) and radiographs (B–D) from a 14-year-old male patient with familial osteochondritis dissecans. The image shows osteochondritic lesions (arrows) at the lateral aspect of the medial femoral chondyle in the knee (A and B) and at the humeral capitellum in the elbow (C and D). (E) Genome-wide scan showing linkage to chromosome 15q in a family displaying autosomal-dominant familial osteochondritis dissecans over five generations. (F) Multipoint analysis, with the polymorphic markers D15S128, D15S1002, D15S165, D15S1007, D15S1012, D15S994, D15S978, D15S117, D15S153, D15S131, D15S205, D15S127, D15S130, and D15S120, resulting in a maximal lod score of 6.36 at marker locus D15S127. (G) Haplotype showing polymorphic marker alleles from chromosome 15q in familial osteochondritis dissecans. The markers shown are (from top to bottom): D15S117, D15S153, D15S131, D15S205, D15S127, D15S130, and D15S120. The disease haplotype is highlighted by the open boxes within the haplotypes below the symbols. Filled symbols represent affected individuals, the arrow indicates the proband, and asterisks denote individuals examined (by E.-L. S.). In addition, the diagnoses of individuals 6, 8, and 11 in generation III have been verified through archived medical journals and radiographs.

Figure 2

A Missense Mutation in the Aggrecan C-type Lectin Repeat Is Present in Patients with Familial Osteochondritis Dissecans (A) Organization of the ACAN gene (top), domain structure of the aggrecan core protein (middle), and proteoglycan (bottom). (B) Sequence analysis from individuals affected by familial osteochondritis dissecans (middle and bottom traces), but not from an unaffected family member (top trace), show heterozygosity for a G-to-A transition in the ACAN gene. (C) The c.6907G > A mutation results in the replacement of the highly conserved Val2303 residue in the aggrecan C-type lectin (CLD) repeat with a Met residue. (D) The Val2303 residue (arrow) is located in a beta strand underlying the CLD ligand-binding site, and its side chain is buried in the tightly packed hydrophobic core of the CLD repeat. The location of the CLD ligand-binding surface is indicated by an arrowhead.

Figure 3

The V2303M Mutation Affects Aggrecan CLD Interactions (A) Domain organization of three different splice variants of the wild-type aggrecan G3 domain, and the corresponding V2303M variants, produced as recombinant proteins in human embryonic kidney 293 cells. (B) Ni²⁺-nitriloacetic acid (NiNTA)-agarose precipitation of His-tagged recombinant proteins from conditioned medium containing fetal calf serum. Bovine fibulin-1, a known aggrecan CLD ligand present in serum, copurified with the wild-type recombinant G3 variants, but not with the V2303M proteins. The identity of fibulin-1 and the recombinant proteins was verified by MALDI-TOF mass spectrometry (not shown). (C) Solid-phase competition binding assay. Microtiter plate wells were coated with the FnIII 3-5 fragment of the CLD ligand tenascin-R, and alkaline phosphatase-tagged aggrecan CLD was allowed to bind in the presence of varying concentrations (0.3–1000 nM) of competitor proteins (open circles, wild-type E1E2LCt; filled triangles, V2303M E1E2LCt). Each data point represents the average of five measurements; error bars show standard deviation. (D) Surface plasmon resonance interaction analysis. The sensorgrams show overlays of duplicate samples of the different recombinant aggrecan G3 variants. The lowest traces in each sensorgram show injections of running buffer alone. Samples (at 1.5 to 50 nM in HEPES-buffered saline containing 2 mM CaCl₂) were injected for 4 min at 50 μl/min over a BIAcore sensor chip coupled with the CLD ligand fibulin-2, followed by injection of running buffer. The binding and dissociation of the G3 variants was registered in a BIAcore 2000 instrument. All wild-type G3 proteins showed strong binding and slow dissociation, whereas no or insignificant binding of the V2303M variants was observed.

Figure 4

Aggrecan Containing the V2303M Mutation Is Produced and Present in Patient Cartilage (A) Schematic representation of the extraction, purification, and analysis of aggrecan from articular cartilage obtained after knee-replacement surgery in a patient with familial osteochondritis dissecans. (B) Top: MALDI-TOF mass spectrometry of trypsin digests identified peptides from both wild-type (DCVVMIWHE, 1188.51 Da) and Val > Met (DCMVMIWHE, 1220.48 Da) aggrecan in C2/C18 fraction 17 from the patient cartilage extract. Middle: Patient cartilage extract with added wild-type recombinant protein. Bottom: Patient cartilage sample with added recombinant mutant protein. (C and D) Ion trap MSMS analysis of C2/C18 fraction 17 from the patient sample confirmed the presence and sequence of both wild-type (C) and V2303M (D) aggrecan in patient cartilage. Wild-type DCVVMIWHE, m/z = 594.76 Da (+2); mutant DCMVMIWHE, m/z = 610.75 Da (+2).