ESPE Abstracts

Background: Aggrecan is the most abundantly expressed extracellular matrix proteoglycan in growth plate cartilage and is crucial for its normal functioning. Heterozygous, loss-of-function mutations in the aggrecan gene (ACAN) cause autosomal dominant short stature with advanced bone age, early-onset osteoarthritis and intervertebral disc disease (SSOAOD; OMIM#165800). ACAN mutations are a relatively frequent finding of approximately 2% in cohorts of idiopathic short stature (ISS).

Objective: To investigate the underlying cellular and molecular mechanisms of growth failure using a mouse model of SSOAOD.

Methods: Heterozygous cartilage matrix deficiency mouse (Acan^+/-) has a naturally occurring 7 bp micro-deletion in aggrecan gene. Wild-type and Acan^+/- male and female mice were assessed for skeletal and body growth, growth plate histomorphometry and cell proliferation at 1, 3, 6, 12, and 24 weeks of age. Single-cell RNA sequencing was carried out on chondrocytes isolated from 18-day-old WT and Acan^+/- female mice according to 3’ gene expression protocol. Raw data was processed, filtered and normalized using CellRanger/Seurat pipelines. A total of 6781 cells (Acan+/Col2a1+/Sox9+) were included for further analysis. Differential gene expression was validated by qPCR and immunofluorescence.

Results: Acan^+/- mice were born at a normal size and similar to humans with SSOAOD but showed decreased postnatal growth resulting in a gradually worsening dwarfism with reduced total body, tibial and femoral lengths (P <0.0001). In the growth plates, chondrocytes were found to be more tightly packed with reduced matrix area (P <0.0001) and increased column density in Acan^+/- mice compared to WT mice. Terminal hypertrophic cell height was significantly decreased in Acan^+/-mice across all ages. Single cell RNA sequencing analyses of 18-day old WT and Acan^+/- growth plates indicated impaired Akt signaling in Acan^+/- growth plate chondrocytes which was further confirmed by the finding of reduced levels of phospho-Akt1 (pAkt); thereby suggesting suppression of one of the main pathway regulating hypertrophic cell size, in pre-hypertrophic and hypertrophic zones (P <0.05).

Conclusion: Similar to children with heterozygous ACAN mutations, Acan^+/- mice have a growth pattern resulting in postnatal growth deficit. Our findings indicate that deficient matrix and impaired chondrocyte hypertrophy are the main drivers of linear growth failure and that suppression of PI3K-Akt signaling pathway in Acan^+/- chondrocytes contributes to the disease phenotype.