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

等位基因表达失衡 (AER) 在 CYP3A4 活性个体差异中的作用及其临床意义。

基本信息

批准号：
17390043
负责人：
IEIRI Ichiro
金额：
$ 9.64万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

(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.

(1)等位基因表达比(AER)常规检测试剂盒的研制以人CYP3A4基因88742位T/T缺失的单核苷酸多态(SNP)为标记，DNA稀释标准样品(即每个等位基因纯合)用于校正等位基因比与荧光强度的相关性(VIC/FAM)很好。我们使用TaqMan引物和探针组研制了CYP3A4基因AER常规检测试剂盒。(2)利用该试剂盒，我们在日本健康志愿者中评估了个体AER与咪达唑仑和伊曲康唑代谢活性的关系。(3)甲基化分析在印迹控制中心有不同的CpG岛，当我们专注于位于82363到82893位的4个CG位点时，第14和16个CG位点的甲基化频率与CyP3A4mRNA的表达水平显著负相关。(4)组蛋白乙酰化5‘-上游和3’-下游区域，5‘-非编码区，以CYP3A4基因的启动子区和启动子区域为靶点，发现不同靶区的抗Ac-H3结合能力存在较大的个体差异，其5‘-上游区域的组蛋白乙酰化水平与基因表达水平显著相关。(5)上述4个CG位点的甲基化状态和组蛋白乙酰化频率与其上游约2000个碱基的组蛋白乙酰化水平显著相关。