Key Laboratory of Trace Elements and Endemic Diseases, Collaborative Innovation Center of Endemic Disease and Health Promotion for Silk Road Region, School of Public Health, Health Science Center, Xi'an Jiaotong University, 710061, China.
Department of Joint Surgery, Xi'an Honghui Hospital, Xi'an Jiaotong University Health Science Center, Xi'an, China.
Bone. 2021 Sep;150:115997. doi: 10.1016/j.bone.2021.115997. Epub 2021 May 6.
Kashin-Beck disease (KBD) is an endemic chronic osteochondropathy. The clinical manifestations and radiographic features of adult KBD were similar to those of osteoarthritis (OA).
We first performed a genetic association scan of 32 OA susceptibility genes with KBD in 898 Han Chinese subjects. The MassARRAY genotyping system (Agena) was used for SNP genotyping. PLINK 1.9 was used for quality control and association testing. Using articular cartilage specimens from 7 adult KBD patients and 4 control subjects, lentivirus-mediated RNA interference (RNAi), qRT-PCR, Western blot and immunohistochemistry were employed to explore the functional relevance of TP63 to KBD chondrocyte.
SNP genotyping and association analysis identified TP63 (rs12107036, P = 0.005, OR = 0.71) and OARD1 (rs11280, P = 0.004, OR = 1.51) were significantly associated with KBD. It was also found that TP63 was significantly up-regulated in KBD articular cartilage in both mRNA and protein level compared with the controls (P < 0.05). TP63 suppression by lentivirus-mediated RNAi notably decreased the abundance of Caspase3 and SOX9 in chondrocytes. Most importantly, compared with the scrambled sequence (shControl) group, the protein level of ACAN was increased in the shTP63 group. The mRNA expression of chondrocyte marker genes (COL2A1 and ACAN) was not significantly changed after TP63 knockdown relative to shControl group.
Our study identifies TP63 as a novel susceptibility gene for KBD, and demonstrates that the inhibition of TP63 suppresses chondrocyte apoptosis and partly facilitates chondrogenesis. The combination of SNP genotyping and molecular biology techniques provides a useful tool for understanding the biological mechanism and differential diagnosis studies of KBD and OA.
大骨节病（KBD）是一种地方性慢性骨软骨病。成人 KBD 的临床表现和影像学特征与骨关节炎（OA）相似。
我们首先在 898 名汉族受试者中对 32 个 OA 易感基因与 KBD 进行了遗传关联扫描。SNP 基因分型采用 MassARRAY 基因分型系统（Agena）。PLINK 1.9 用于质量控制和关联测试。使用 7 例成人 KBD 患者和 4 例对照的关节软骨标本，采用慢病毒介导的 RNA 干扰（RNAi）、qRT-PCR、Western blot 和免疫组化技术，探讨 TP63 与 KBD 软骨细胞的功能相关性。
SNP 基因分型和关联分析发现 TP63（rs12107036，P=0.005，OR=0.71）和 OARD1（rs11280，P=0.004，OR=1.51）与 KBD 显著相关。还发现与对照组相比，KBD 关节软骨中 TP63 在 mRNA 和蛋白水平上均显著上调（P<0.05）。慢病毒介导的 RNAi 抑制 TP63 显著降低了软骨细胞中 Caspase3 和 SOX9 的丰度。最重要的是，与 scrambled sequence（shControl）组相比，shTP63 组的 ACAN 蛋白水平升高。与 shControl 组相比，TP63 敲低后软骨细胞标志物基因（COL2A1 和 ACAN）的 mRNA 表达没有显著变化。
我们的研究将 TP63 鉴定为 KBD 的一个新的易感基因，并证明抑制 TP63 可抑制软骨细胞凋亡，并在一定程度上促进软骨形成。SNP 基因分型和分子生物学技术的结合为理解 KBD 和 OA 的生物学机制和鉴别诊断研究提供了有用的工具。
