TUFT1, a novel candidate gene for metatarsophalangeal osteoarthritis, plays a role in chondrogenesis on a calcium-related pathway

Osteoarthritis (OA) is the most common degenerative joint disorder and genetic factors have been shown to have a significant role in its etiology. The first metatarsophalangeal joint (MTP I) is highly susceptible to development of OA due to repetitive mechanical stress during walking. We used whole exome sequencing to study genetic defect(s) predisposing to familial early-onset bilateral MTP I OA inherited in an autosomal dominant manner. A nonsynonymous single nucleotide variant rs41310883 (c.524C>T, p.Thr175Met) in TUFT1 gene was found to co-segregate perfectly with MTP I OA. The role of TUFT1 and the relevance of the identified variant in pathogenesis of MTP I OA were further assessed using functional in vitro analyses. The variant reduced TUFT1 mRNA and tuftelin protein expression in HEK293 cells. ATDC5 cells overexpressing wild type (wt) or mutant TUFT1 were cultured in calcifying conditions and chondrogenic differentiation was found to be inhibited in both cell populations, as indicated by decreased marker gene expression when compared with the empty vector control cells. Also, the formation of cartilage nodules was diminished in both TUFT1 overexpressing ATDC5 cell populations. At the end of the culturing period the calcium content of the extracellular matrix was significantly increased in cells overexpressing mutant TUFT1 compared to cells overexpressing wt TUFT1 and control cells, while the proteoglycan content was reduced. These data imply that overexpression of TUFT1 in ATDC5 inhibits chondrogenic differentiation, and the identified variant may contribute to the pathogenesis of OA by increasing calcification and reducing amount of proteoglycans in the articular cartilage extracellular matrix thus making cartilage susceptible for degeneration and osteophyte formation.骨关节炎（Osteoarthritis, OA）是最常见的退行性关节疾病，遗传因素在其病因学中发挥着重要作用。第一跖趾关节（first metatarsophalangeal joint, MTP I）由于行走过程中反复承受机械应力，极易发生骨关节炎。本研究采用全外显子测序技术，探究以常染色体显性方式遗传的家族性早发性双侧第一跖趾关节骨关节炎的易感遗传缺陷。研究发现，TUFT1基因上的非同义单核苷酸变异rs41310883（c.524C>T, p.Thr175Met）与第一跖趾关节骨关节炎表型完美共分离。我们通过体外功能实验进一步评估了TUFT1的作用以及该鉴定出的变异在第一跖趾关节骨关节炎发病机制中的相关性。实验结果显示，该变异可降低HEK293细胞中TUFT1 mRNA及tuftelin蛋白的表达水平。将过表达野生型（wild type, wt）或突变型TUFT1的ATDC5细胞置于钙化培养条件中培养，与空载体对照细胞相比，两组细胞的标志物基因表达均出现下调，提示软骨分化受到抑制。同时，两种过表达TUFT1的ATDC5细胞群的软骨结节形成均受到削弱。培养周期结束后，与过表达野生型TUFT1的细胞及对照细胞相比，过表达突变型TUFT1的细胞外基质钙含量显著升高，而蛋白聚糖含量则有所降低。上述数据表明，在ATDC5细胞中过表达TUFT1可抑制软骨分化；所鉴定出的变异可能通过增加关节软骨细胞外基质的钙化程度、降低蛋白聚糖含量，使软骨更容易发生退行性变并形成骨赘，从而参与骨关节炎的发病过程。