Genotype and phenotype correlation analysis in 27 families with multiple osteochondroma and validation by ATDC5 chondrocyte models

Aims: This study aimed to explore the genotype and phenotype correlation of patients with multiple osteochondroma (MO), and validate phenotypic differences in ATDC5 cell model with EXT1 or EXT2 gene disruption (EXT1^-/- or EXT2^-/-).

Methods: Mutation analysis was employed in 27 families with MO using polymerase chain reaction (PCR)-Sanger Sequencing and targeted next-generation Sequencing (t-NGS). ATDC5 cell model with EXT1^-/- or EXT2^-/- was established using CRISPR/Cas9 technology. Markers of chondrocyte proliferation and differentiation were assessed through quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and dyeing experiments at different timepoints.

Results: A total of 27 pathogenic mutations were identified in EXT1 and EXT2 genes, in which nine were novel mutations. In terms of mutation rate, onset age, medical treatment age, and numbers of moderate/severe (II/III) patients, the numbers with EXT1 gene mutations were more than those with EXT2 gene mutations. ATDC5 cell model with EXT1^-/- or EXT2^-/- was successfully established. The proliferation rate of ATDC5 chondrocytes in mutant groups (EXT1^-/- and EXT2^-/-) was higher than in WT group. The expressions of proliferation markers, including α-1 chain of type II Collagen (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), were almost all higher in mutant groups than that in WT group. Moreover, the expression of differentiation indicators, including α-1 chain of type X Collagen (COL10A1), Runt-related transcription factor 2 (RUNX2), and matrix metallopeptidase 13 (MMP13), was increased in mutant groups compared to WT group. The Alcian blue and Alizarin red staining obtained similar results.

Conclusion: Clinical research identified nine novel mutations in EXT1 and EXT2 genes from 27 families with MO, and patients with EXT1 mutations have more severe symptoms than with EXT2 mutations. Cellular experiments suggested that EXT1 and EXT2 gene mutations promote proliferation and differentiation of chondrocytes, which were consistent with clinical research.