Fukami Tatsuki, Nakajima Miki, Maruichi Taiga, Takahashi Shiori, Takamiya Masataka, Aoki Yasuhiro, McLeod Howard L, Yokoi Tsuyoshi
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
Pharmacogenet Genomics. 2008 Oct;18(10):911-20. doi: 10.1097/FPC.0b013e32830b0c5e.
Human carboxylesterase (CES) 1A1 gene (14 exons) and CES1A3 pseudogene (six exons) are inverted and duplicated genes in a reference sequence (NT_010498). In contrast, earlier studies reported the CES1A2 gene (14 exons) instead of the CES1A3 pseudogene. The sequences of the CES1A2 gene downstream and upstream of intron 1 are identical with those of the CES1A1 and CES1A3 genes, respectively. A CES1A1 variant of which exon 1 is converted with that of the CES1A3 gene (the transcript is CES1A2) has recently been identified. We sought to clarify the confusing gene structure of human CES1A.
A panel of 55 human liver as well as 318 blood samples (104 Caucasians, 107 African-Americans, and 107 Japanese) was used to clarify the gene structures of CES1A1, CES1A2, and CES1A3. Real-time reverse transcription-PCR and western blot analysis were carried out. Imidapril hydrolase activity in human liver microsomes and cytosol was determined by liquid chromatography-mass spectrometry (LC-MS)/MS.
By PCR analyses, we found that the CES1A2 gene is a variant of the CES1A3 gene. Four haplotypes, A (CES1A1 wild type and CES1A3), B (CES1A1 wild type and CES1A2), C (CES1A1 variant and CES1A3), and D (CES1A1 variant and CES1A2), were demonstrated. Ethnic differences were observed in allele frequencies of CES1A1 variant (17.3% in Caucasians and African-Americans and 25.2% in Japanese) and CES1A2 gene (14.4% in Caucasians, 5.1% in African-Americans, and 31.3% in Japanese). In human livers whose diplotype was A/A and C/C or C/D, no CES1A2 and CES1A1 mRNA was detected, respectively. In the other participants, the CES1A1 mRNA levels were higher than the CES1A2 mRNA levels. The CES1A proteins translated from CES1A1 mRNA and CES1A2 mRNA were detected in both human liver microsomes and cytosol fractions suggesting that the differences in exon 1 encoding a signal peptide did not affect the subcellular localization. Imidapril hydrolase activities reflected the CES1A protein levels.
We found that the CES1A2 gene is a variant of the CES1A3 pseudogene. The findings presented here significantly increase our understanding about the gene structure and expression properties of human CES1A.
人羧酸酯酶（CES）1A1基因（14个外显子）和CES1A3假基因（6个外显子）在参考序列（NT_010498）中是反向重复基因。相比之下，早期研究报道的是CES1A2基因（14个外显子）而非CES1A3假基因。CES1A2基因内含子1下游和上游的序列分别与CES1A1和CES1A3基因的序列相同。最近发现了一种CES1A1变体，其外显子1与CES1A3基因的外显子1发生了转换（转录本为CES1A2）。我们试图厘清人CES1A令人困惑的基因结构。
使用一组55份人肝脏样本以及318份血液样本（104名高加索人、107名非裔美国人、107名日本人）来厘清CES1A1、CES1A2和CES1A3的基因结构。进行了实时逆转录PCR和蛋白质免疫印迹分析。通过液相色谱 - 质谱联用（LC - MS）/ MS测定人肝微粒体和胞质溶胶中的咪达普利水解酶活性。
通过PCR分析，我们发现CES1A2基因是CES1A3基因的一个变体。证实了四种单倍型，A（CES1A1野生型和CES1A3）、B（CES1A1野生型和CES1A2）、C（CES1A1变体和CES1A3）和D（CES1A1变体和CES1A2）。观察到CES1A1变体（在高加索人和非裔美国人中为17.3%，在日本人中为25.2%）和CES1A2基因（在高加索人中为14.4%，在非裔美国人中为5.1%，在日本人中为31.3%）的等位基因频率存在种族差异。在双倍型为A/A和C/C或C/D的人肝脏中，分别未检测到CES1A2和CES1A1 mRNA。在其他参与者中，CES1A1 mRNA水平高于CES1A2 mRNA水平。在人肝微粒体和胞质溶胶组分中均检测到了从CES1A1 mRNA和CES1A2 mRNA翻译而来的CES1A蛋白，这表明编码信号肽的外显子1的差异并不影响亚细胞定位。咪达普利水解酶活性反映了CES1A蛋白水平。
我们发现CES1A2基因是CES1A3假基因的一个变体。此处呈现的研究结果显著增进了我们对人CES1A基因结构和表达特性的理解。
