Philips Santosh, Rae James M, Oesterreich Steffi, Hayes Daniel F, Stearns Vered, Henry N Lynn, Storniolo Anna M, Flockhart David A, Skaar Todd C
Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine Indianapolis, IN, USA.
Front Pharmacol. 2012 Mar 30;3:54. doi: 10.3389/fphar.2012.00054. eCollection 2012.
Whole genome amplification (WGA) technologies can be used to amplify genomic DNA when only small amounts of DNA are available. The Multiple Displacement Amplification Phi polymerase based amplification has been shown to accurately amplify DNA for a variety of genotyping assays; however, it has not been tested for genotyping many of the clinically relevant genes important for pharmacogenetic studies, such as the cytochrome P450 genes, that are typically difficult to genotype due to multiple pseudogenes, copy number variations, and high similarity to other related genes. We evaluated whole genome amplified samples for Taqman(™) genotyping of SNPs in a variety of pharmacogenetic genes. In 24 DNA samples from the Coriell human diversity panel, the call rates, and concordance between amplified (∼200-fold amplification) and unamplified samples was 100% for two SNPs in CYP2D6 and one in ESR1. In samples from a breast cancer clinical trial (Trial 1), we compared the genotyping results in samples before and after WGA for three SNPs in CYP2D6, one SNP in CYP2C19, one SNP in CYP19A1, two SNPs in ESR1, and two SNPs in ESR2. The concordance rates were all >97%. Finally, we compared the allele frequencies of 143 SNPs determined in Trial 1 (whole genome amplified DNA) to the allele frequencies determined in unamplified DNA samples from a separate trial (Trial 2) that enrolled a similar population. The call rates and allele frequencies between the two trials were 98 and 99.7%, respectively. We conclude that the whole genome amplified DNA is suitable for Taqman(™) genotyping for a wide variety of pharmacogenetically relevant SNPs.
当只有少量DNA可用时，全基因组扩增（WGA）技术可用于扩增基因组DNA。基于多重置换扩增Phi聚合酶的扩增已被证明能准确扩增DNA用于各种基因分型检测；然而，对于许多药物遗传学研究中重要的临床相关基因，如细胞色素P450基因，由于存在多个假基因、拷贝数变异以及与其他相关基因的高度相似性，其基因分型尚未经过测试，这些基因通常难以进行基因分型。我们评估了全基因组扩增样本用于多种药物遗传学基因中SNP的Taqman™基因分型。在来自Coriell人类多样性样本库的24个DNA样本中，CYP2D6中的两个SNP以及ESR1中的一个SNP在扩增样本（约200倍扩增）和未扩增样本之间的检出率和一致性均为100%。在一项乳腺癌临床试验（试验1）的样本中，我们比较了WGA前后样本中CYP2D6的三个SNP、CYP2C19的一个SNP、CYP19A1的一个SNP、ESR1的两个SNP以及ESR2的两个SNP的基因分型结果。一致性率均>97%。最后，我们将试验1（全基因组扩增DNA）中确定的143个SNP的等位基因频率与另一项招募了相似人群的独立试验（试验2）中未扩增DNA样本中确定的等位基因频率进行了比较。两项试验之间的检出率和等位基因频率分别为98%和99.7%。我们得出结论，全基因组扩增DNA适用于多种药物遗传学相关SNP的Taqman™基因分型。
