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

(1)等位基因表达率（AER）常规检测试剂盒的研制以人CYP 3A 4基因第88742位的单核苷酸多态性（SNP）T/T缺失为标志物，用DNA系列稀释标准样品（即，每个等位基因纯合）用于校准等位基因比率和荧光强度之间的相关性（维克/ FAM）检测结果良好。(2)临床应用使用该检测试剂盒，我们在日本健康志愿者中评价了个体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，未观察到明确的关系。由于成功估计了个体AER，因此需要使用更特异的CYP 3A 4底物药物进行更大样本量的研究。(3)甲基化分析印迹控制中心（ICR）内存在多个CpG岛，ICR位于人CYP 3A 4基因的上游。对82363 - 82893位的4个CG位点进行甲基化分析，发现第14和16位CG位点的甲基化频率与CYP 3A 4 mRNA表达水平呈显著负相关。(4)选择CYP 3A 4基因的5 '上游和3'下游区域、5 '-UTR和启动子区域作为靶点。在不同靶点区域观察到抗Ac-H3结合能力的较大个体间差异，CYP 3A 4基因5'上游区域的组蛋白乙酰化与mRNA表达显著相关。(5)上述4个CG位点的甲基化频率与CYP 3A 4基因上游约2000 bp处的组蛋白乙酰化水平显著相关。这些结果表明，ICR中DNA甲基化参与了CYP 3A 4基因上游组蛋白乙酰化，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.