开发临床低量细胞的Tag-C技术研究遗传变异改变染色质构象的致病机制

解析遗传变异造成的染色质构象改变对于明确遗传变异的致病机制至关重要。常规用于捕获突变位点参与的染色质互作的方法是4C技术，但4C实验高度依赖观测位点是否存在合理的限制性内切酶酶切位点的组合，且实验流程复杂，所需的细胞量高，难以直接应用于细胞量有限的样本。基于4C技术固有的局限性，本项目拟开发的Tag-C技术适用于低细胞量样品，具有高灵敏性、高特异性和高分辨率的特点，可用于检测遗传变异导致的染色质结构的改变。我们的实验结果表明Tag-C方法具有技术可行性。本项目拟从简化实验流程、减少起始细胞量、拓展观测位点兼容性和提高信号特异性等方面优化Tag-C技术，并开发数据分析流程。将Tag-C技术用于解析系统性红斑狼疮相关遗传变异通过改变染色质构象致病的机制。Tag-C技术的成功开发有望填补可用于低量细胞的染色质构象捕获技术的空缺，为在临床标本中研究遗传变异改变染色质构象的致病机制提供技术支持。

It’s important to unravel the pathogenic mechanism of chromosome topological changes caused by genetic variations. The 4C-seq can capture the chromatin architecture interacting with the viewpoint. However, it has limitations that the selection of viewpoints and the captured signals are highly dependent on restriction endonuclease sites and the requirement of over 10 million input cells, making it difficult to apply to clinical specimens with limited sample sizes. This project aimed to develop the Tag-C technology based on the transposase Tn5, which can detect the chromatin loops with the viewpoint in 0.5 million cells. Specifically, we improved the Tag-C technology from the following aspects, simplifying the protocol, reducing the input cells, expanding the compatibility of viewpoints, and reducing the signal background. Also, we will develop a data analysis pipeline to determine the specificity and sensitivity of the Tag-C signal. The optimized Tag-C will be applied to the B cells of the patients with systemic lupus erythematosus to directly detect the chromatin conformation alteration caused by genetic variation, linking the alteration with disease susceptibility. Tag-C technology is expected to fill the gap in the chromatin conformation capture technology. It can directly detect the chromatin conformation changes caused by genetic variants in clinical specimens with limited sample sizes, providing technical support to study the pathogenic mechanism caused by clinical genetic variants.