Abstract
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.
Highlights
Genotyping in pharmacogenetics studies frequently becomes limited due to small DNA quantities
The Multiple Displacement Amplification Phi polymerase based amplification has been shown to accurately amplify DNA for a variety of genotyping assays; 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
Lymphocyte DNA samples from African Americans (n = 7), Caucasians (n = 8), and Asians (n = 9) were obtained directly from the Coriell DNA repository; second, Trial 1 DNA samples from whole blood collected in heparin vacutainers were obtained from a multisite trial carried out by the Consortium on Breast Cancer Pharmacogenomics (COBRA) of patients treated with tamoxifen (Clinicaltrials.gov # NCT00228930); and third, Trial 2 DNA samples from whole blood collected in EDTA vacutainers were obtained from a second multisite COBRA exemestane and letrozole pharmacogenetics (ELPh) clinical trial (Clinicaltrials.gov # NCT00228956)
Summary
Genotyping in pharmacogenetics studies frequently becomes limited due to small DNA quantities. DNA yields are low because of the methods of collection. DNA samples are depleted by extensive genotyping and DNA sequencing. Recontacting patients is often not feasible because the most informative patients have often had disease recurrences and died. Some genotyping techniques, including those directed at generation sequencing, genome wide association studies, and OpenArrayTM platforms, require high DNA concentrations that are higher than those obtained from the initial DNA extractions. DNA samples can be concentrated to achieve the necessary concentrations; sample recovery can be low and concentrating the samples often results in a parallel increase in the concentrations of contaminants
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