Mutation in the CCAL1 locus accounts for bidirectional process of human subchondral bone turnover and cartilage mineralization

OBJECTIVES: To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients. METHODS: OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation. RESULTS: Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively. CONCLUSIONS: The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humans via DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies.