CYP2E1、SOD2基因多态性与抗结核药物所致肝损伤易感性的相关性及其细胞分子机制的研究

Conventional therapy for Tuberculosis (TB) involves regimen containing isoniazid, rifampicin, ethamutol, and pyrazinamide, which may lead to adverse drug reactions (ADRs) in some patients. Anti-TB drug induced liver injury(ATLI) is the most common and serious ADR in clinics. Identifying the risk factors and understanding pathogenesis of ATLI may help prevent and manage potentially fatal hepatotoxicity. A number of studies have been conducted to assess the potential association between Drug Metabolizing Enzyme (DME) polymorphisms and ATLI risk and gave rise to inconsistent results, likely contributed by ethnicity difference or limited case volume among these studies. Our preliminary case-control study, including 316 ATLI patients and 337 control subjects, indicated an association between T/C or C/C genotype at rs4880 in SOD2 and ATLI risk in Chinese population. CYP2E1, an important DME, was indicated to be a potential ATLI risk factor by some studies. However, opposite conclusion about CYP2E1 was also reported by some studies with limited case volume. In order to unequivocally identify ATLI genetic risk factors, we proposed to expand the case-control study with a larger case volume (at least 600 cases and 600 controls) and explore the association of ATLI with multiple tagSNPs within the genomic regions of CYP2E1 and SOD2. Systematic bioinformatic analysis and functional studies were proposed to study whether CYP2E1 and SOD2 are the ATLI risk genes in Chinese population and to identify the ATLI susceptibility loci in these genes. More importantly, Establishing paired hepatocyte cell lines with same genetic background but distinct genotypes at each susceptible SNP loci using the CRISPR/Cas9 gene editing system was also proposed to facilitate extensive studies of cellular and molecular mechanisms underlying the association of these SNPs and ATLI. Studies proposed here will greatly improve our understanding the molecular genetic mechanisms of ATLI and help to reduce or prevent the incidences of ATLI during TB chemotherapy treatments.

异烟肼、利福平、乙胺丁醇和吡嗪酰胺组成的标准抗结核方案可能会导致患者严重的不良反应。抗结核药物所致肝损伤(ATLI)是临床上最常见、影响最大的不良反应。我们基于对316例ATLI患者与337例对照组的前期研究发现，Ⅱ相药物代谢酶SOD2基因rs4880位点T/C或C/C基因型与中国人群ATLI的易感性密切相关。在此基础上选择I相药物代谢酶CYP2E1作为另一个候选基因，选取CYP2E1和SOD2区段内tagSNPs，在扩大样本人群中进行基因分型。通过生物信息学分析结合功能研究，明确CYP2E1和SOD2是否是中国人群ATLI的易感基因及其致病易感SNP位点。针对每个易感SNP位点，利用CRISPR/Cas9基因编辑技术定点改变特定SNP位点，建立遗传背景一致但具有不同SNP基因型的两套肝细胞系，建立基本的细胞模型，开展深入的细胞分子机制研究，为临床正确认识和预测ATLI提供理论依据。

项目背景：抗结核药物所致肝损伤（ATDILI）是临床上最常见和影响最大的抗结核药物不良反应。随着精准医疗模式的发展，研究发现药物不良反应与患者自身遗传背景密切相关。本项目选取CYP2E1、SOD2、SLCO1B1、NOS2作为候选基因，通过大样本病例对照研究明确这些基因是否是中国汉族人群ATDILI易感基因以及阳性SNP位点对患者治疗结局的影响。.研究内容：通过生物信息统计学方法筛选出4个候选基因共49个tagSNPs（H-W平衡P＞0.001，最小等位基因频率＞0.1，r2＞0.8），采用SNPscanTM多重SNP分型技术对461例ATDILI患者和466例非ATDILI患者的DNA样本进行基因分型，分析这些位点基因型和单倍型在病例组和对照组之间的频率差异，分析在显性、隐性和加性遗传模型下各个SNP位点与ATDILI易感性的关联性。.研究结果：.1. 所有位点的等位基因频率分布均符合H-W平衡。NOS2基因rs9906835、rs944725、rs3794763、rs3794764以及rs6505469均可增加ATDILI易感性，在显性遗传模型下，rs9906835、rs944725、rs3794763、rs3794764以及rs6505469可增加患者抗结核治疗后发生肝损的风险；在加性遗传模型下，rs944725、rs3794763和rs3794764增加ATDILI发生的风险。SOD2基因rs732498位点在隐性模型下可显著增加ATDILI易感性。单倍型分析显示NOS2基因CGCATT、AC和AAA单倍型均可增加ATDILI发生的风险。未发现CYP2E1与SLCO1B1与ATDILI发生相关。.2. 携带NOS2 rs3794764 G/A+A/A基因型的患者治疗失败率显著高于携带G/G患者；携带NOS2 rs3794764 G/A+A/A患者痰菌阴转中位时间为2个月，痰菌阴转率92.4%，携带G/G患者阴转中位时间为1.5个月，阴转率95.2%，携带G/A+A/A患者显著低于G/G患者。.科学意义：. 本项目进一步补充了ATDILI分子遗传机制，发现了诊断和预后的分子标志物，是药物不良反应精准诊断的一种创新性探索。为将来以基因多态性检测为基础的个体化精准治疗运用于临床以减少药物不良反应并实现最佳治疗效果打下一定基础。