Collins Joseph M, Lester Hannah, Shabnaz Samia, Wang Danxin
Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, FL, United States.
Front Pharmacol. 2023 Jul 27;14:1186540. doi: 10.3389/fphar.2023.1186540. eCollection 2023.
CYP2D6 is one of the most polymorphic drug-metabolizing enzymes in the liver. While genetic variants serve as clinical biomarkers to predict CYP2D6 activity, large inter-person variability in CYP2D6 expression remains unaccounted for. Previous results suggest that there is variable expression of a CYP2D6 splice isoform with an in-frame deletion of exon 3 (CYP2D6ΔE3) encoding a protein lacking numerous active site residues. Here, using fragment analysis and RT-qPCR, we revealed that rs1058164 G (MAF = 27%-43%) is associated with increased formation of CYP2D6∆E3 in human liver samples (1.4-2.5-fold) and transfected cells. Furthermore, western blots showed that rs1058164 G was associated with a 50% decrease in full-length hepatic CYP2D6 protein expression. In addition, by studying a larger liver cohort, we confirmed our previous results that rs16947 (CYP2D6*2) reduces full-length CYP2D6 mRNA by increasing the production of an unstable splice isoform lacking exon 6 (CYP2D6ΔE6) and that the impact of CYP2D6ΔE6 is offset in carriers of the downstream enhancer variant rs5758550. The three frequent SNPs (rs1058164, rs16947, and rs5758550) form various 3-SNP-haplotypes, each with distinct CYP2D6 expression characteristics. Using an expression score (ES) system, we tested the impact of the 3-SNP-haplotype on improving the standard model to predict hepatic CYP2D6 protein expression based on genotype. A model that incorporates the 3-SNP-haplotype provided the best fit for CYP2D6 expression and also accounted for more variability in CYP2D6 protein levels (59%) than a model based on the accepted standard (36%) or one that only adds rs16947 and rs5758550 (42%). Clinical studies are needed to determine whether including the 3-SNP-haplotype alongside current standard CYP2D6 models improves the predictive value of CYP2D6 panels.
细胞色素P450 2D6（CYP2D6）是肝脏中多态性最高的药物代谢酶之一。虽然基因变异可作为预测CYP2D6活性的临床生物标志物，但CYP2D6表达的个体间差异仍未得到充分解释。先前的研究结果表明，存在一种CYP2D6剪接异构体，其编码的蛋白质缺乏许多活性位点残基，外显子3存在框内缺失（CYP2D6ΔE3），且表达存在差异。在此，我们通过片段分析和逆转录定量聚合酶链反应（RT-qPCR）发现，rs1058164 G（等位基因频率[MAF]=27%-43%）与人类肝脏样本（1.4-2.5倍）和转染细胞中CYP2D6∆E3的形成增加有关。此外，蛋白质免疫印迹法显示，rs1058164 G与全长肝脏CYP2D6蛋白表达降低50%有关。此外，通过对更大的肝脏队列进行研究，我们证实了之前的结果，即rs16947（CYP2D6*2）通过增加缺乏外显子6的不稳定剪接异构体（CYP2D6ΔE6）的产生来降低全长CYP2D6 mRNA，并且在下游增强子变异rs5758550的携带者中，CYP2D6ΔE6的影响被抵消。这三个常见的单核苷酸多态性（SNP，即rs1058164、rs16947和rs5758550）形成了各种3-SNP单倍型，每种单倍型都具有独特的CYP2D6表达特征。我们使用表达评分（ES）系统，测试了3-SNP单倍型对改进基于基因型预测肝脏CYP2D6蛋白表达的标准模型的影响。纳入3-SNP单倍型的模型对CYP-2D6表达的拟合度最佳，并且与基于公认标准的模型（36%）或仅添加rs16947和rs5758550的模型（42%）相比，该模型能解释更多CYP2D6蛋白水平的变异（59%）。需要开展临床研究来确定，在当前标准的CYP2D6模型中纳入3-SNP单倍型是否能提高CYP2D6检测板的预测价值。
