Chang Xiaotian, Zheng Yabing, Yang Qingrui, Wang Lin, Pan Jihong, Xia Yifang, Yan Xinfeng, Han Jinxiang
Arthritis Res Ther. 2012 Jul 27;14(4):R176. doi: 10.1186/ar3929.
Ankylosing spondylitis (AS) is characterized by abnormal bone formation in the spine and the sacroiliac joints. In vitro assays demonstrate that carbonic anhydrase I (CA1) promotes calcium precipitation. This study investigated the function of CA1 for bio-mineralization and determined if common polymorphisms in the CA1 gene might contribute to AS risk.
Calcification was induced in Saos-2 cells, a human osteosarcoma cell line, with ascorbic acid and β-glycerophosphate. Calcification was determined by Alizarin Red-S (AR-S) staining. Expressions of CA1, alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), osterix (OSX) and runt-related transcription factor-2 (Runx2) were determined by real-time PCR and western blotting. The cells were also treated with acetazolamide, an anti-carbonic anhydrase drug. Genotyping was performed using Illumina VeraCode microarray in a case-control study including 51 AS patients, 267 rheumatoid arthritis (RA) patients and 160 healthy controls. The result was confirmed by Taqman assay, including 258 AS patients, 288 RA patients and 288 healthy controls.
Following the induction of calcification, Saos-2 cells produced large amounts of calcium-rich deposits. Increased transcriptions of CA1, ALP, BSP, OCN, OSX and Runx2, essential genes for ossification, were detected in the cultured cells. Following treatmen with acetazolamide, the expression of CA1 obviously declined and mineralized nodule formation was also decreased. Illumina microarray indicates that SNP at rs7841425 also showed significant differences in allelic frequency (P = 0.01396) and genotypic frequency (P = 0.005902) between AS cases and controls. In addition, SNP at rs7827474 showed significant differences in allelic frequency (P = 5.83E-04) and genotypic frequency (P = 0.000186) between RA cases and controls (P values were adjusted to multiple comparisons). The Taqman assay revealed that rs725605 demonstrated statistically significant evidence of allele frequency (P = 0.022307) and gene frequency (P = 0.007731) for association with AS. This SNP did not show significant differences in allelic frequencies and gene frequencies between RA patients and controls.
CA1 may play an essential role in bio-mineralization and new bone formation. The gene encoding CA1 is susceptible to AS.
强直性脊柱炎（AS）的特征是脊柱和骶髂关节出现异常骨形成。体外实验表明，碳酸酐酶I（CA1）可促进钙沉淀。本研究调查了CA1在生物矿化中的作用，并确定CA1基因的常见多态性是否可能增加AS风险。
用抗坏血酸和β-甘油磷酸酯诱导人骨肉瘤细胞系Saos-2细胞钙化。通过茜素红S（AR-S）染色确定钙化情况。通过实时PCR和蛋白质免疫印迹法测定CA1、碱性磷酸酶（ALP）、骨唾液蛋白（BSP）、骨钙素（OCN）、osterix（OSX）和 runt相关转录因子2（Runx2）的表达。细胞还用乙酰唑胺（一种抗碳酸酐酶药物）处理。在一项病例对照研究中，使用Illumina VeraCode微阵列进行基因分型，该研究包括51例AS患者、267例类风湿性关节炎（RA）患者和160名健康对照。结果通过Taqman检测得到证实，该检测包括258例AS患者、288例RA患者和288名健康对照。
诱导钙化后，Saos-2细胞产生大量富含钙的沉积物。在培养细胞中检测到CA1、ALP、BSP、OCN、OSX和Runx2（骨化必需基因）的转录增加。用乙酰唑胺处理后，CA1的表达明显下降，矿化结节形成也减少。Illumina微阵列表明，rs7841425处的单核苷酸多态性（SNP）在AS病例和对照之间的等位基因频率（P = 0.01396）和基因型频率（P = 0.005902）也存在显著差异。此外，rs7827474处的SNP在RA病例和对照之间的等位基因频率（P = 5.83E-04）和基因型频率（P = 0.000186）存在显著差异（P值已针对多重比较进行调整）。Taqman检测显示，rs725605在等位基因频率（P = 0.022307）和基因频率（P = 0.007731）方面与AS关联具有统计学意义的证据。该SNP在RA患者和对照之间的等位基因频率和基因频率上没有显著差异。
CA1可能在生物矿化和新骨形成中起重要作用。编码CA1的基因易患AS。
