The role of allelic expression imbalance (AER) in interindividual differences in CYP3A4 activity and it's clinical implications.

(1)开发常规的等位基因表达比（AER）检测试剂盒采用人CYP3A4基因88742位点的单核苷酸多态性（SNP）， T/T-缺失作为标记，采用dna（即每个等位基因的纯合子）进行系列稀释标准样品进行校准，等位基因比例与荧光强度（VIC / FAM）之间的相关性非常好，我们使用TaqMan引物和探针集开发了CYP3A4基因AER常规检测试剂盒。使用该检测试剂盒，我们评估了日本健康志愿者个体AER与咪达唑仑和伊曲康唑代谢活性之间的关系。不幸的是，由于样本量小，没有观察到明确的关系。由于个体AER被成功估计，因此需要更大的样本量研究，使用更特异性的CYP3A4底物药物。(3)甲基化分析印迹控制中心（ICR）中存在多个CpG岛，ICR位于人类CYP3A4基因的上游。我们对位于82363 ~ 82893的4个CG位点进行分析，发现第14和第16个CG位点的甲基化频率与CYP3A4 m RNA表达水平呈显著负相关。(4)以CYP3A4基因5′-上游和3′-下游、5′-UTR和启动子区域的组蛋白乙酰化为靶点，各靶点的抗ac - h3结合能力存在较大的个体间差异，CYP3A4基因5′-上游的组蛋白乙酰化与mRNA表达显著相关。(5)上述4个CG位点的甲基化频率与CYP3A4基因上游约2000 bp的组蛋白乙酰化显著相关，提示ICR DNA甲基化参与了CYP3A4基因上游的组蛋白乙酰化，导致顺式作用现象，等位基因表达失衡。

(1) Development of conventional assay kit for the allelic expression ratio (AER) A single nucleotide polymorphism (SNP), T/T-deletion, position at 88742 in human CYP3A4 gene is used for the marker Serial dilution standard samples using DNAs (i e, homozygous for each allele) were used for the calibration Correlation between allelic ratio and fluorescence intensity (VIC / FAM) was excellent We developed conventional assay kit for AER in the CYP3A4 gene using TaqMan primers and probe sets.(2) Clinical Application Using this assay kit, we evaluated relationship between individual AER and metabolic activities of midazolam and itraconazole in Japanese healthy volunteers Unfortunately, due to the small sample sizes, clear relationships were not observed Since individual AER was estimated successfully, a larger sample size study using more specific substrate drugs for CYP3A4 is warranted.(3) Methylation analysis There are various CpG islands in the imprinting control center (ICR), ICR is located on the upstream of human CYP3A4 gene When we focused on 4 CG sites at position 82363 to 82893, methylation frequencies at 14th and 16th CG sites negatively correlated with the CYP3A4 m RNA expression levels at significantly.(4) Histone acetylation 5'-upstream and 3'-downstream regions, 5'-UTR, and promoter regions of the CYP3A4 gene were selected as targets Large interindividual differences in the anti-Ac-H3 binding capacity were observed in various target regions, histone acetylation in 5'-upstream regions of CYP3A4 was significantly correlated with mRNA expression.(5) Methylation status and Histone acetylation Methylation frequencies in above mentioned 4 CG sites were significantly correlated with histone acetylation at approximately 2000 bp upstream of the CYP3A4 gene These results suggest that DNA methylation in the ICR involved in the histone acetylation at the upstream of the CYP3A4 gene, leading to cis-acting phenomenon, allelic expression imbalance.