TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6 (*) 15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6 (*) 15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6 (*) 35) which is also located in exon 1. Although alternative CYP2D6 (*) 15 and (*) 35 assays resolved the issue, we discovered a novel CYP2D6 (*) 15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6 (*) 15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6 (*) 43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe regions can impact the performance of PCR-based genotype assays, including TaqMan. Regardless of the test platform used, it is prudent to confirm rare allele calls by an independent method.
Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.
e13018 Background: Approx 20% of patients receiving FP chemotherapy experience grade 3-5 toxicity. Known deleterious DPYD mutations account for 20% of these toxicities. We set out to examine whether polymorphisms/mutations in additional candidate genes involved in FP metabolism might account for severe toxicity in DPYD wild type (WT) individuals Methods: All patients receiving FP chemotherapy in our centre are prospectively screened for 4 common DPYD polymorphisms. Colorectal cancer patients treated between 2012-2015 were eligible if they had previously tested DPYD WT. Patients were identified using an institutional database and medical record review. DPYD WT patients who experienced grade 3/4 toxicity were defined as the case population and those without severe toxicity were categorised as the control population. Following informed consent, buccal swabs were taken to obtain DNA samples in order to genotype candidate genes (TPMT, TYMS and MTHFR). Statistical analysis was carried out on the data set returned. Results: 49 patients were recruited, 24 cases and 25 controls. Repeat testing confirmed DPYD WT status in all patients in concordance with previous genotyping. Polymorphisms in other candidate genes were found in both cases and controls. TPMT *1/*3A or *3C polymorphism was present in 6/24 cases and not found in controls (χ2 p = 0.007). Compound heterozygote status (polymorphisms in both the 5’ promoter and 3’ untranslated regions) of the TYMS gene were present in 21/24 cases and 12/25 controls (χ2 p = 0.003). Conclusions: In patients who are DPYD WT, TPMT *1/*3A or *3C may predict severe toxicity. TYMS compound heterozygote status is also associated with severe toxicity. This remained predictive when patients with concurrent TPMT mutations were excluded. These findings should be replicated in larger data sets, ideally in a prospective trial
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