CDA基因及其遗传变异在血压变化和CCB治疗中的作用

Hypertension is the most prevalent disease with high mortality and disability. Cytidine deaminase (CDA) is an important intracellular enzyme and contributes to the occurrence and development of many diseases. However, the role of CDA gene in blood pressure is not investigated. We determined the samples from a randomized, double-blind, multicenter clinical trial by Illumina iSelect HD chip, and found that the CDA genetic variations were significantly associated with antihypertensive response to calcium channel blockers (CCB), which were repeatedly validated in 4 years of follow-up. These findings indicated that CDA may be a novel mechanism for blood pressure. In the present study, we aims to elucidate the role of CDA gene and its genetic variations in blood pressure and antihypertensive response to CCB, by applying CRISPR/Cas9 technology, siRNA gene silencing, over expression, site-directed mutagenesis in the experiments of vascular smooth muscle cells, CDA knockout mice and clinical samples. Also, we will investigate the mechanism of CDA genetic mutations affecting gene function, by applying bioinformatics, chromatin immunoprecipitation, gel migration assay and luciferase reporter gene system in the experiments of HEK293 cells and clinical samples. Our study will not only shed light on the underlying molecular mechanisms of blood pressure, but also provide a novel biomarker for antihypertensive response to CCB. The study is of significance in prevention and treatment of hypertension.

高血压病是全球流行最广，致死率、致残率高的重大疾病。胞苷脱氨酶（CDA）是一种重要的细胞内酶，与多种疾病发生发展密切相关，但迄今尚未报道CDA基因与血压的关系。我们前期应用Illumina高通量芯片对随机双盲多中心的大型临床试验进行研究，发现多个CDA遗传变异显著影响钙通道拮抗剂（CCB）降压疗效，并在长达4年的随访中得到反复验证，提示CDA可能是影响血压变化的新机制。本项目拟在血管平滑肌细胞、基因敲除小鼠和临床样本三个层面应用CRISPR/Cas9技术、基因定点诱变、siRNA沉默、过表达等阐明CDA及其变异在血压变化和CCB治疗中的作用。同时，在HEK293细胞和临床样本两个层面应用生物信息学技术、染色质免疫共沉淀技术、凝胶迁移实验、荧光素酶报告基因系统等确证CDA变异影响基因功能的机制。本项目将为血压变化提供新机制，为预测CCB治疗提供分子标志物，对高血压个体化防治具有重要意义。

高血压是临床最常见的心血管疾病之一，是心脑血管疾病和全因死亡的最主要的单一危险因素。遗传药理学与药物基因组学的研究成果表明遗传因素在各类抗高血压药物降压疗效、长期临床事件及不良反应中起重要作用。本项目应用Illumina iSelect HD高通量芯片对随机、双盲、对照、多中心的持续随访周期长达4年的大型临床试验“非洛地平治疗高血压研究”(FEVER)样本进行了检测，发现CDA一号内含子的单核苷酸多态（SNP）位点rs818202和rs10916824遗传多态与非洛地平长期降压疗效强相关。生物信息学预测发现CDA遗传多态影响CDA基因表达，rs818202的遗传多态能在多个组织中导致CDA表达的显著改变；双荧光素酶报告基因实验结果发现，rs818202 A到G的突变会使得CDA基因启动子转录活性降低，这可能是由于突变之后G等位基因序列不能与转录因子CEBPβ结合导致。干扰CDA后的转录组学测序分析提示CDA可能影响钙信号通路；进一步研究发现干扰CDA的表达能够使血管平滑肌细胞内游离钙离子浓度降低，而使的L型钙通道CaV1.2表达增加；收集了6948例原发性高血压患者及健康对照人群，针对CDA的常见遗传多态进行了分型并探讨了二者之间的关系，在校正性别、年龄和合并疾病之后，发现rs818202突变杂合子AG基因型是高血压易感性的保护因素（OR=0.86，0.77-0.97），相比于野生型，杂合子个体患高血压的风险显著降低。