■CYP2D6检测越来越多地用于指导药物治疗,因此,需要可靠的方法来测试这个复杂和多态的基因位点。一个特殊的挑战来自于结构变体(SV)的检测和解释,包括基因缺失,重复,和与CYP2D7假基因的杂种。本研究通过将结果与使用QX200平台使用先前建立的方法获得的结果进行比较,验证了AbsoluteQ™平台用于基于数字PCR的CYP2D6拷贝数变异(CNV)测定。此外,建立并验证了精简CYP2D6CNV测试的方案,包括“一锅”单步限制性内切酶消化和同时靶向CYP2D65UTR的多重测定,内含子6和外显子9区域。
■来自Coriell(n=13)和血液的基因组DNA(gDNA)样本,唾液,在绝对Q和QX200平台上测试代表0-6个拷贝的肝组织(n=17)。定制TaqMan™拷贝数(CN)检测针对CYP2D6的5'UTR,内含子6和外显子9区域以及参考基因分析(TERT或RNaseP)被组合用于通过光学通道进行多路复用。此外,评估了两种消化方法(一锅消化和传统消化)。使用替代的参考基因和/或稀释的gDNA解析绝对值Q上的不确定的CN值。
■总的来说,两个平台和消化方法之间的结果是一致的。“一锅”消化方法和多达三个CYP2D6区域的光学多路复用在DNA样品类型和不同的SV之间产生了一致的结果,可靠地检测多达6个基因拷贝。发现参考基因的罕见变异会干扰结果和解释,这是通过使用不同的参考解决。
■绝对Q产生准确和可靠的CYP2D6拷贝数结果,允许使用一锅消化和多路复用三个目标区域的简化和经济的方案。目前正在将方案扩展到其他具有SVs/CNVs的药物源。
UNASSIGNED: CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the \"One-pot\" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5\'UTR, intron 6, and exon 9 regions.
UNASSIGNED: Genomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5\'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA.
UNASSIGNED: Overall, results between the two platforms and digestions methods were consistent. The \"One-pot\" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference.
UNASSIGNED: The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.