冠心病患者低三碘甲状腺原氨酸的ADME基因甲基化调控机制及其对生存预后的影响

Coronary artery disease (CAD) is one of the most common reason for human mortality. Reported literatures and our studies indicated that 3,5,3'-triiodothyronine (T3) is an independent marker for predicting death risks of CAD patients. To date, the mechanism for the individual difference of T3 level in CAD patients remains unclear. In a state of illness, DNA methylation variations occur and could affect T3 level, which is known to be closely related to and regulated by metabolic enzymes and transporters (ADME). We therefore hypothesis that in CAD states, variations of ADME gene methylation could cause an excessive reduction of T3 level and therefor increase the death risk for patients. To clarify this hypothesis, we used epigenome-wide association study (EWAS) for 112 CHD patients to screen out 85 ADME gene methylation sites suggestively associated with plasma T3 level. In the proposal, ① a targeted bisulfite sequencing based on Padlock probe will be applied to detect the methylation level of 85 ADME gene CpG loci and verify their association with plasma T3 level in 1,600 CHD patients and a Mendelian randomization method will then be applied to reveal the potential causal impact of methylation on the T3 level; ②we will clarify the predictive value for low T3 level increasing the risks of major adverse cardiovascular events and construct a risk prediction model. ③we will further verify the impact of DNA methylations of metabolic enzyme genes, such as DIO2, on T3 metabolism and explore the regulatory mechanism on transcription of key metabolic enzyme genes.The results of this study will give us a novel insight into the epigenetic mechanism on the inter-individual difference of T3 in CAD patients and will help improve predictive ability of mortality risk of CAD.

冠心病是人类最主要的死亡危险因素。我们和他人研究表明，三碘甲腺原氨酸（T3）是独立预测冠心病患者死亡风险的代谢物。目前其个体差异的原因尚不清楚。疾病状态下，DNA甲基化发生改变，可影响T3水平，而T3水平与代谢酶和转运体（ADME）密切相关，因此我们提出假说：在冠心病疾病状态下，ADME基因甲基化变异可导致T3水平过低，增加死亡风险。为了阐明此假设，我们前期用EWAS筛选出85个与T3有关联的ADME基因甲基化位点。本研究拟进一步，①基于Padlock探针测序检测85个甲基化位点，明确其与冠心病患者T3水平的关联性，并用孟德尔随机化分析揭示其因果关系；②明确其对死亡的风险，并构建生存预后模型；③用人肝微粒体代谢模型和细胞模型，从功能上明确DIO2等关键代谢酶基因甲基化对T3代谢的影响，阐明其甲基化对转录的调节作用。研究结果有助于阐明T3个体差异的表观遗传机制和提高对冠心病死亡风险的预测性。

三碘甲腺原氨酸（T3）是已被本项目证实的独立预测冠心病患者死亡风险的代谢物，为了发现影响T3水平和介导其作用的基因甲基化并阐明其功能，为预测冠心病死亡风险提供新的预警标志物和潜在的干预靶点，本研究首先在5104例冠心病随访队列分析低T3水平对冠心病患者死亡风险的影响，结果显示低水平T3是死亡和MACE风险的独立预测因子（HR(95% CI)=0.577(0.454–0.734)，p=7.17 E-6; HR(95% CI)= 0.831(0.735–0.939)，p= 0.003）。然后通过461例单中心和507例多中心冠心病随访队列的EWAS发现并验证了与FT3（游离T3）最显著相关的CpG位点：SPATA4基因上cg02833127（P=3.47E-22)；cg02833127低甲基化水平是冠心病患者死亡和MACE风险的独立危险因素；中介分析显示cg02833127解释高达42.33%的FT3与死亡之间的关联，介导了42.38%的FT3对MACE事件的影响，cg02833127在FT3与血脂APOA水平中的介导比例为29.62%。另外用双向孟德尔因果关系分析发现DIP2C、SPTLC3和AHRR等10个基因甲基化对T3或FT3水平有潜在因果关系影响，DIP2C cg02614932高甲基化和SPTLC3 cg22939408去甲基化对增加FT3和T3水平均有潜在因果关系，AHRR cg03569073高甲基化对T3水平降低具有潜在因果关系。功能研究显示JAZF1 cg23597162和SPATA4 cg02833127甲基化水平与其所在基因表达成显著负相关，T3与5-Aza-CdR去甲基化协同抑制JAZF1和SPATA4基因表达、减少肝细胞脂质合成和TG水平、而增加APOA水平；在肝细胞和心肌细胞中，未观察到DIP2C与SPTLC3基因敲低对T4向T3代谢的影响；通过Ahrr-/-小鼠模型发现AHRR敲除可降低T3与T4的比率和增加Dio3表达，可能机制是AhRR敲除增加DIO3表达使T3脱碘增加。综上，本研究发现了2个潜在干预靶点SPATA4和AHRR，可通过抑制SPATA4表达增加T3对冠心病的保护作用，而增加AHRR表达可减少Dio3脱碘酶从而提高T3水平。