Nielsen Kim, Heegaard Steffen, Vorum Henrik, Birkenkamp-Demtröder Karin, Ehlers Niels, Orntoft Torben Falck
Department of Ophthalmology, Aarhus University Hospital, Denmark.
Cornea. 2005 Aug;24(6):661-8. doi: 10.1097/01.ico.0000153556.59407.69.
This investigation was designed to determine whether the five genes, CLC, DSG3, EMP3, S100A2 and SLPI, are differentially expressed in keratoconus, as indicated from another study.
Gene expression was monitored using quantitative real-time PCR on 14 keratoconus samples and 16 controls, and normalized to GAPDH and B2M. The DSG3, S100A2, and SLPI proteins were quantified by Western blotting, and the cellular localization was determined by immunohistochemistry. One of the genes, CLC, was reduced in gene expression and its four exons were sequenced.
The five genes were all differentially expressed in keratoconus (P < 0.04) and so were at least three of the encoded proteins (P = 0.009). DSG3 was expressed in association with the cell membrane of the basal and suprabasal epithelial cells, and S100A2 was expressed in the nucleus and cytoplasm, often as intracellular granules. Two SNPs (rs374185 and rs384138) were observed in the CLC gene, each with an allele frequency of 68%. No other mutations were detected.
The five genes, and three of the encoded proteins, were shown differentially expressed between a group of keratoconus patients and a reference group using different techniques. These alterations, in combination with earlier findings, strongly demonstrate the genes to be involved in the corneal disease. We suggest the unambiguously expressed DSG3 protein to be used as a marker for keratoconus.
本研究旨在确定如另一项研究所表明的，CLC、DSG3、EMP3、S100A2和SLPI这五个基因在圆锥角膜中是否存在差异表达。
使用定量实时PCR对14例圆锥角膜样本和16例对照样本的基因表达进行监测，并以GAPDH和B2M作为标准化对照。通过蛋白质印迹法定量分析DSG3、S100A2和SLPI蛋白，并通过免疫组织化学确定其细胞定位。对其中一个基因CLC进行基因表达下调检测，并对其四个外显子进行测序。
这五个基因在圆锥角膜中均存在差异表达（P < 0.04），其编码的至少三种蛋白质也存在差异表达（P = 0.009）。DSG3与基底及基底上层上皮细胞的细胞膜相关表达，S100A2在细胞核和细胞质中表达，常呈细胞内颗粒状。在CLC基因中观察到两个单核苷酸多态性（SNP，rs374185和rs384138），每个等位基因频率均为68%。未检测到其他突变。
使用不同技术表明，在一组圆锥角膜患者和参照组之间，这五个基因及其编码的三种蛋白质存在差异表达。这些改变与早期研究结果相结合，有力地证明了这些基因与角膜疾病有关。我们建议将明确表达的DSG3蛋白用作圆锥角膜的标志物。
