Langaee Taimour, Hamadeh Issam, Chapman Arlene B, Gums John G, Johnson Julie A
Department of Pharmacotherapy and Translational Research and Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, Florida, United States of America.
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, United States of America.
PLoS One. 2015 Jan 27;10(1):e0113808. doi: 10.1371/journal.pone.0113808. eCollection 2015.
Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.
We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.
The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.
Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.
细胞色素P450 2D6（CYP2D6）基因重复和倍增可导致患者药物代谢超快，进而出现治疗失败或过度反应。长程聚合酶链反应（PCR）、限制性片段长度多态性（RFLP）和测序通常用于CYP2D6重复/倍增的基因分型及鉴定，但这些方法 labor intensive（人工操作繁琐）、耗时且成本高。
我们开发了一种基于等位基因定量的简单焦磷酸测序基因分型方法，该方法有助于进行CYP2D6拷贝数变异（CNV）基因分型，同时还能在杂合样本中鉴定等位基因特异性CYP2D6 CNV。大多数常规检测无法鉴定含有CNV的等位基因。对总共237份具有不同已知CYP2D6基因拷贝数的临床和科里尔DNA样本进行了CYP2D6 *2、*3、*4、*6、*10、*17、*41多态性基因分型及CNV测定。
CYP2D6基因等位基因定量/鉴定与CYP2D6*2、*3、*4、*6、*10、*17、*41基因分型同时进行。我们确定了确切的CYP2D6基因拷贝数，鉴定出哪个等位基因发生了重复或倍增，并为所有样本指定了正确的表型和活性评分。
我们的方法能够有效鉴定杂合样本中重复的CYP2D6等位基因，与传统方法相比，能在更短时间内确定其拷贝数，并防止错误的超快表型判定。它还大大降低了与CYP2D6 CNV基因分型相关的成本、工作量和时间。
