The disease mechanisms of skeletal dysplasia caused by two aggrecan mutations

Abstract

Skeletal dysplasias are a complex group of over 350 disorders of cartilage and bone with a combined incidence of 1/5000. Diagnosis is difficult and based on clinical and radiographic findings, with many cases having no known genetic cause. An allelic series of mutations has been identified in aggrecan that results in a broad phenotypic spectrum, including spondyloepimetaphyseal dysplasia (SEMD) and familial osteochondritis dissecans (OCD). Aggrecan, a large chondroitin sulfated proteoglycan, attracts ions and water molecules, allowing the cartilage to withstand the high mechanical load found in the skeletal joint. The SEMD (p.D2276N) and OCD (p.V2303M) causative mutations are found at highly conserved residues in the C-type lectin domain (CLD) which interacts with other extracellular matrix (ECM) molecules to provide stability. PolyGene Transgenetics introduced mutations into the C57Bl/6 mouse line using homologous gene targeting. The resultant mice were assessed with radiography, morphometry and growth measurements. Histological techniques, electron microscopy, RNA-sequencing and SDSPAGE Western blotting were used to analyse the underlying patho-molecular mechanisms. The homozygous V2019M Acan (OCD) mouse exhibits mild disproportionate short stature, whereas the homozygous D1983N Acan (SEMD) mouse has severe disproportionate short stature with associated skeletal abnormalities, including mid-face hypoplasia. OCD and SEMD mice also exhibit intervertebral disc degeneration, which has not previously been reported in human patients. OCD and SEMD growth plate cartilage is disorganised, with reduced aggrecan in the extracellular matrix, increased apoptosis and mis-expression of other extracellular matrix proteins. The unfolded protein response is not upregulated in either OCD or SEMD cartilage, although SEMD chondrocytes exhibit mild cellular stress. Comparative transcriptomic analysis indicates that autophagic flux, vesicular transport and regulation of chondrocyte differentiation are altered between these two models. The data presented in this thesis demonstrates that OCD and SEMD mice recapitulate the human short stature phenotypes and that altered matrix organisation may impair cellular differentiation, causing reduced bone growth. These mouse models could be used to further investigate the disease mechanisms of these two skeletal disorders.