Sanford Jonathan C, Wang Xinwen, Shi Jian, Barrie Elizabeth S, Wang Danxin, Zhu Hao-Jie, Sadee Wolfgang
aCenter for Pharmacogenomics, College of Medicine, The Ohio State University, Columbus, Ohio bDepartment of Clinical Pharmacy, University of Michigan, Ann Arbor, Michigan, USA.
Pharmacogenet Genomics. 2016 May;26(5):197-207. doi: 10.1097/FPC.0000000000000206.
CES1 encodes carboxylesterase-1, an important drug-metabolizing enzyme with high expression in the liver. Previous studies have reported a genomic translocation of the 5' region from the poorly expressed pseudogene CES1P1, to CES1, yielding the structural variant CES1VAR. The aim of this study was to characterize this translocation and its effect on CES1 expression in the human liver.
Experiments were conducted in human liver tissues and cell culture (HepG2). The promoter and exon 1 of CES1 were sequenced by Sanger and Ion Torrent sequencing to identify gene translocations. The effects of CES1 5'UTRs on mRNA and protein expression were assessed by quantitative real-time PCR, allelic ratio mRNA analysis by primer extension (SNaPshot), quantitative targeted proteomics, and luciferase reporter gene assays.
Sequencing of CES1 identified two translocations: first, CES1VAR (17% minor allele frequency) comprising the 5'UTR, exon 1, and part of intron 1. A second shorter translocation, CES1SVAR, was observed excluding exon 1 and intron 1 regions (<0.01% minor allele frequency). CES1VAR is associated with 2.6-fold decreased CES1 mRNA and ∼1.35-fold lower allelic mRNA. Luciferase reporter constructs showed that CES1VAR decreases luciferase activity 1.5-fold, whereas CES1SVAR slightly increases activity. CES1VAR was not associated with CES1 protein expression or metabolism of the CES1 substrates enalapril, clopidogrel, or methylphenidate in the liver.
The frequent translocation variant CES1VAR reduces mRNA expression of CES1 in the liver by ∼30%, but protein expression and metabolizing activity in the liver were not detectably altered - possibly because of variable CES1 expression masking small allelic effects. Whether drug therapies are affected by CES1VAR will require further in-vivo studies.
CES1编码羧酸酯酶-1，这是一种重要的药物代谢酶，在肝脏中高表达。先前的研究报道了5'区域从低表达的假基因CES1P1向CES1的基因组易位，产生了结构变体CES1VAR。本研究的目的是表征这种易位及其对人肝脏中CES1表达的影响。
在人肝脏组织和细胞培养（HepG2）中进行实验。通过桑格测序和离子激流测序对CES1的启动子和外显子1进行测序，以鉴定基因易位。通过定量实时PCR、引物延伸等位基因比率mRNA分析（SNaPshot）、定量靶向蛋白质组学和荧光素酶报告基因测定评估CES1 5'UTR对mRNA和蛋白质表达的影响。
CES1测序鉴定出两种易位：第一种是CES1VAR（次要等位基因频率为17%），包含5'UTR、外显子1和部分内含子1。观察到第二种较短的易位CES1SVAR，不包括外显子1和内含子1区域（次要等位基因频率<0.01%）。CES1VAR与CES1 mRNA降低2.6倍和等位基因mRNA降低约1.35倍相关。荧光素酶报告构建体显示CES1VAR使荧光素酶活性降低1.5倍，而CES1SVAR则略微增加活性。CES1VAR与肝脏中CES1蛋白表达或CES1底物依那普利、氯吡格雷或哌醋甲酯的代谢无关。
常见的易位变体CES1VAR使肝脏中CES1的mRNA表达降低约30%，但肝脏中的蛋白质表达和代谢活性未检测到改变——可能是因为CES1表达的变异性掩盖了小的等位基因效应。CES1VAR是否影响药物治疗将需要进一步的体内研究。
