Human aromatase: Gene resequencing and functional genomics
Human aromatase: Gene resequencing and functional genomics
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DOI：
10.1158/0008-5472.can-05-1218
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发表时间：
2005-12-01
期刊：
影响因子：
11.2
通讯作者：
Weinshilboum, RM
中科院分区：
文献类型：
--
作者：
Ma, CX;Adjei, AA;Weinshilboum, RM
芳香酶[细胞色素P450 19]是雌激素生物合成的关键酶，芳香酶抑制剂在乳腺癌的治疗中发挥着越来越重要的作用。我们开始鉴定和表征芳香酶基因CYP19的遗传多态，作为芳香酶抑制剂的药物基因组学研究的一步。具体地说，我们使用来自四个种族的240个DNA样本对所有编码外显子、所有上游未翻译的外显子及其推测的核心启动子区域、所有外显子-内含子剪接连接以及CYP19的一部分3‘-非翻译区进行了重新测序。共检测到88个多态性，共产生44种单倍型。对我们观察到的四个非同义编码单核苷酸多态(CSNP)进行了功能基因组学研究，其中两个是新发现的。这些cSNP改变了以下氨基酸：Trp(39)Arg、Thr(201)Met、(264)Cys和Met(364)Thr。在COS-1细胞中瞬时表达后，与野生型(WT)酶相比，CyS264、Thr(364)和双变Arg(39)Cys(264)等位基因的活性和活性蛋白水平显著降低。与WT酶相比，Arg(39)等位酶的蛋白质水平也略有下降，而Met(201)的活性或蛋白质水平没有显著变化。以雄烯二酮为底物，苏氨酸的表观K值也增加了4倍(364)。在重组等位酶中，只有双突变(Arg(39)Cys(264))对芳香酶抑制剂依西美坦和来曲唑的抑制常数与WT酶有显著差异。这些观察表明，CYP19的遗传变异可能是雌激素依赖性疾病的病理生理学变异的原因之一。
Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we "resequenced" all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exon-intron splice junctions, and a portion of the 3'-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. Those cSNPs altered the following amino acids: Trp(39)Arg, Thr(201)Met, Are(264)Cys, and Met(364)Thr. The CYS264, Thr(364), and double variant Arg(39)CYS(264) allozymes showed significant decreases in levels of activity and inummoreactive protein when compared with the wild-type (WT) enzyme after transient expression in COS-1 cells. A slight decrease in protein level was also observed for the Arg(39) allozyme, whereas Met(201) displayed no significant changes in either activity or protein level when compared with the WT enzyme. There was also a 4-fold increase in apparent K., value for Thr(364) with androstenedione as substrate. Of the recombinant allozymes, only the double mutant (Arg(39)CYS(264)) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease.