Leung Edward Ki Yun, Agolini Emanuele, Pei Xun, Melis Roberta, McMillin Gwendolyn A, Friedman Paula N, Peterson Patrick, Danahey Keith, O'Donnell Peter H, Yeo Kiang-Teck J
Department of Pathology, UChicago Advanced Technology Clinical Laboratory, The University of Chicago, Chicago, IL.
ARUP Laboratories, Salt Lake City, UT.
J Appl Lab Med. 2017 Mar 1;1(5):471-482. doi: 10.1373/jalm.2016.021923.
CYP2D6 is involved in the oxidative metabolism of approximately 20% of all clinically used medications. Genotyping cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), is a challenge because of the high complexity of the locus.
Twenty-nine CYP2D6 sequence variants were genotyped in 50 deidentified patient samples and 29 Coriell DNAs by Invader assay, and results were compared with Infiniti assay and Sanger sequencing. To determine CYP2D6 copy number, 3 TaqMan real-time hydrolysis probes were used and results were compared with long-range PCR. Discrimination of the duplicated alleles was done on 17 DNA samples with 3 copies of CYP2D6 by long-range PCR followed by Invader genotyping and single nucleotide extension for the comparison.
Complete concordance was observed for all samples between platforms except for 2 samples due to the lack of the *45 allele in the Infiniti panel. Reproducibility with the Invader assay and TaqMan copy number was 100%. Analytical sensitivity using DNA with 2 copies was determined to be 10 ng DNA for the Invader assay and 1 ng/μL DNA for the TaqMan assay, respectively. Complete concordance and reproducibility were observed for duplicated allele discrimination with the exception of 1 sample, determined to be *29/*43X2 by the Invader test and *1X2/*29 by the Infiniti method, which did not test for *43.
This validation study showed that Invader and TaqMan assay combined panel provides an attractive, valid, highly accurate, and reproducible approach for CYP2D6 genotyping for clinical implementation.
CYP2D6参与了约20%的临床常用药物的氧化代谢。细胞色素P450 2D6（CYP2D6）基因分型具有挑战性，因为该基因座高度复杂。
采用Invader检测法对50份身份不明的患者样本和29份Coriell DNA样本中的29个CYP2D6序列变异进行基因分型，并将结果与Infiniti检测法和桑格测序法进行比较。为确定CYP2D6拷贝数，使用了3种TaqMan实时水解探针，并将结果与长片段PCR进行比较。通过长片段PCR对17份含有3个CYP2D6拷贝的DNA样本进行重复等位基因鉴别，随后进行Invader基因分型和单核苷酸延伸以作比较。
除2份样本外，各平台间所有样本的结果完全一致，这2份样本是因为Infiniti检测组中缺乏45等位基因。Invader检测法和TaqMan拷贝数检测法的重复性均为100%。使用2拷贝DNA时，Invader检测法的分析灵敏度确定为10 ng DNA，TaqMan检测法为1 ng/μL DNA。重复等位基因鉴别结果完全一致且具有重复性，只有1份样本例外，Invader检测法判定为29/(43)X2，Infiniti检测法判定为(1)X2/29，后者未检测(43)。
这项验证研究表明，Invader检测法和TaqMan检测法联合使用为临床实施的CYP2D6基因分型提供了一种有吸引力、有效、高度准确且可重复的方法。
