肾移植受者嘌呤类药物不良反应缘由：NDPK/TGTP/Rac1代谢通路关键基因变异相关研究

Thiopurines have been widely used in a variety of clinical conditions, however, their clinical use is frequently associated with a relatively high incidence of adverse reactions. Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) can catalyze the conversion of 6-thioguanosine diphosphate (TGDP) into the active metabolite 6-thioguanosine triphosphate (TGTP). Furthermore, the molecular mechanism of immunosuppression by thiopurines is due to the specific end-metabolite 6-TGTP that binds and inhibits the function of the small guanosine triphosphatase (GTPase) Rac1 in activated T-lymphocytes leading to apoptosis. Therefore, NDPK/TGTP/Rac1 metabolite pathway plays a pivotal role in the drug response of thiopurines. In the present study, a "two-stage analysis" method is employed to investigate the association. Firstly, we will determine and analyze the activity and protein expression of NDPK and Rac1 in human erythrocytes, the TGTP concentration, as well as their relationships with thiopurine-induced adverse drug reactions in Chinese kidney transplant recipients. Secondly, we will carry out a comprehensive investigation to ascertain the association between genetic variations in their encoded NME and RAC1 genes and the thiopurine-induced adverse drug reactions, particularly the influence of gene-gene interplays. Furthermore, we will investigate the underlying biological functions of genetic variations associated with adverse drug reactions via molecular biology, cell biology techniques. Thus, a deep insight into genetic variations in key genes in NDPK/TGTP/Rac1 metabolic pathway and their underlying mechanisms in thiopurine-induced adverse drug reactions in Chinese kidney transplant recipients will be illuminated, which can provide new ideas and experimental foundations for improving drug safety of thiopurines therapy.

嘌呤类药物临床应用广泛，但其不良反应发生率高是面临的最大挑战。核苷二磷酸激酶（NDPK）在体内催化产生嘌呤类药物活性代谢物硫鸟嘌呤核苷三磷酸（TGTP），而TGTP直接作用于分子靶标小G蛋白Rac1发挥作用，因此NDPK/TGTP/Rac1代谢通路在该类药物反应中起关键作用。本课题采用"两阶段分析"关联研究策略，以服用嘌呤类药物硫唑嘌呤的肾移植受者为主要研究对象，首先检测分析红细胞中NDPK和Rac1活性、蛋白含量，TGTP浓度，药物不良反应间相关性。其次，对编码NDPK和Rac1的关键基因NME和RAC1遗传变异与不良反应关联性进行全面分析，重点探讨基因-基因交互作用的影响。同时结合分子和细胞生物学技术手段，深入探讨相关联基因变异的生物学功能。本研究将系统阐明NDPK/TGTP/Rac1代谢通路关键基因变异在嘌呤类药物不良反应中的作用机制，可为提高其临床应用安全性提供新思路和实验基础。

嘌呤类药物临床应用广泛，但其不良反应（ADR）发生率高是面临的最大挑战。本项目以1000例中国汉族健康人群和602例移植患者为研究对象，针对NDPK/TGTP/Rac1代谢通路上关键基因，开展基因遗传变异新靶标Nm23/RAC1与嘌呤类免疫抑制剂疗效及ADR间的相关性分析，并进行机制研究。.（1）、系统研究Nm23基因多态性-mRNA转录-NDPK活性-TGTP代谢物浓度-ADR相关性，首次报道如下结果：①Nm23基因启动子及内含子区域共10个单核苷酸多态性（SNPs）在中国健康人群和移植患者人群的分布特征，并筛选出5个标签SNPs（tag-SNPs）：rs16949649、rs2302254、rs34214448、rs11868380、rs2318785。②红细胞（RBC）中的NDPK活性呈正偏态分布，且在两部分人群间存在显著差异。③对mRNA转录表达有影响的SNPs位点：rs2302254、rs2318785、rs34214448。对NDPK活性有影响的SNPs位点：rs16949649、rs11868380、rs2318785、rs7207370、rs34214448。④发现NDPK活性与6-TGTP稳态浓度呈正相关。⑤肾移植受者rs2302254突变纯合子基因型与骨髓毒性有关，rs2318785突变纯合子基因型与肝脏损害有关；出现骨髓毒性或肝脏损害的患者中均呈现NDPK高活性及6-TGTP高稳态浓度。.（2）、系统研究RAC1基因多态性-mRNA转录-Rac1蛋白含量-TGTP代谢物浓度-ADR相关性，首次报道如下结果：①RAC1基因多态性的分布呈正偏态分布；并筛选出4个tag-SNPs：rs702482、rs10951982、rs702483、rs6954996。②Rac1总蛋白和Rac1-GTP活性蛋白表达呈正偏态分布，且在两部分人群间存在显著差异。③对mRNA转录和Rac1蛋白表达有影响的SNPs位点：rs702482、rs10951982、rs702483、rs6954996。④肾移植受者rs702482突变纯合子基因型与骨髓毒性有关，rs10951982突变纯合子基因型与胃肠道紊乱有关。.本研究取得了多项创新性成果，并进行了临床推广应用，将为提高嘌呤类药物临床应用安全性提供新思路和实验基础。