Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19
Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19
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DOI：
10.1124/dmd.118.084269
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发表时间：
2019-04-01
影响因子：
3.9
通讯作者：
Reid, Joel M.
中科院分区：
文献类型：
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作者：
Devarajan, Sandhya;Moon, Irene;Reid, Joel M.
CYP2C9和CYP2C19是高度多态的药物基因；然而，由于这些基因导致的药物代谢的临床可操作遗传变异性仅限于几个常见的等位基因。不常见的开放阅读框序列变异的鉴定和功能表征可能有助于个体化药物治疗，药物是这些基因编码的酶的底物。本研究使用1013名受试者的下一代序列数据确定了CYP2C9和CYP2C19中每一个的七个未表征的变体，并对编码的蛋白质进行了功能表征。构建并在COS-1细胞中瞬时表达载体，以甲苯丁胺和S-美苯妥因为原型底物，利用荧光底物和液相色谱-串联质谱法测定蛋白质浓度和酶活性。将结果与SIFT、Polyphen和Provean功能预测软件程序进行比较。测定细胞色素P450氧化还原酶(CPR)活性。蛋白质含量与荧光酶活性呈显著正相关(P<；0.0005)。然而，根据蛋白质含量预测，细胞色素P450 C9 709G>；C和CYP2C19 65A>；G活性明显低于预测。CYP2C9 218C>；T、343A>；C和CYP2C19 337G>；A、518C>；T、556C>；T和557G>；A的底物本征清除值均低于野生型等位酶的25%。所有变种的CPR活动水平都是相似的。总而言之，对1013名受试者进行的CYP2C9和CYP2C19的测序发现了以前没有功能特征的低频变异。在电子计算机中，预测结果并不总是与功能分析结果一致。这些观察结果强调了高通量方法用于药物基因变异突变和功能鉴定的必要性。
CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes; however, clinically actionable genetic variability in drug metabolism due to these genes has been limited to a few common alleles. The identification and functional characterization of less-common open reading frame sequence variation might help to individualize therapy with drugs that are substrates for the enzymes encoded by these genes. The present study identified seven uncharacterized variants each in CYP2C9 and CYP2C19 using next-generation sequence data for 1013 subjects, and functionally characterized the encoded proteins. Constructs were created and transiently expressed in COS-1 cells for the assay of protein concentration and enzyme activities using fluorometric substrates and liquid chromatography-tandem mass spectrometry with tolbutamide (CYP2C9) and (S)-mephenytoin (CYP2C19) as prototypic substrates. The results were compared with the SIFT, Polyphen, and Provean functional prediction software programs. Cytochrome P450 oxidoreductase (CPR) activities were also determined. Positive correlations were observed between protein content and fluorometric enzyme activity for variants of CYP2C9 (P < 0.05) and CYP2C19 (P < 0.0005). However, CYP2C9 709G>C and CYP2C19 65A>G activities were much lower than predicted based on protein content. Substrate intrinsic clearance values for CYP2C9 218C>T, 343A>C, and CYP2C19 337G>A, 518C>T, 556C>T, and 557G>A were less than 25% of wild-type allozymes. CPR activity levels were similar for all variants. In summary, sequencing of CYP2C9 and CYP2C19 in 1013 subjects identified low-frequency variants that had not previously been functionally characterized. In silico predictions were not always consistent with functional assay results. These observations emphasize the need for high-throughput methods for pharmacogene variant mutagenesis and functional characterization.