A multiplex pharmacogenetics assay using the MinION nanopore sequencing device Objectives The MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. As a proof of principle, we developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, the two commonly used drugs that show response variability due to genetic polymorphisms. Methods Six reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting 27 key variants. These products were then barcoded to enable sample multiplexing in one sequencing run. Four variant calling tools (marginCaller, VarScan 2, nanopolish, Clairvoyante) were used to compare genotyping accuracy. Results In our cohort, 81 out of 84 samples were successfully sequenced and genotyped. Using nanopolish as the variant calling tool achieved accuracy >95% for all except two variants. A known single base deletion (CYP2C9*6) was successfully detected. Conclusion While minor misgenotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays using small PCR amplicons are possible on the MinION sequencing device.

The relevance of the individual screening for genetic variants in predicting ovarian response Objective To investigate if polymorphisms of some genes involved in folliculogenesis predict ovarian response. Methods This prospective randomized study includes 124 egg donors genotyped for six SNPs ESR1 (rs2234693), AMHR2 (rs2002555), GDF-9 (rs10491279 and rs254286), AMH (rs10407022) and LHCBR (rs229327) genes and four STRs in ESR1 rs3138774), SHBG (rs6761), CYP19A1 (rs60271534) and AR genes (CAG repeats in exon 1). All donors followed standard ovarian stimulation protocol using a daily dose of 225 UI. The genotypes obtained were compared with the ovarian stimulation outcome. Results Regarding the number of retrieved oocytes, we found statistical differences for the ESR1 SNP and STR (19.3 ± 8.9 for TT vs 15.3 ± 6.2 for CC/CT, P = 0.027; 19.1 ± 8.3 for <17repeats vs 14.7 ± 6.2 for >17repeats, P = 0.020). Moreover, women carrying TT in the ESR1 at position c.-397T>C with ESR1 (TA)n=17 retrieved the highest number of oocytes (20.4 ± 9.3) (P = 0.001). Concerning AMHR2, we observed an association with the length of stimulation (9.1 ± 1.4 d for AA vs 9.7 ± 1.3 d for AG/GG, P = 0.021) and gonadotropin received (2050 ± 319 for AA vs 2188 ± 299 for AG/GG, P = 0.017). No significant differences were observed for the other polymorphisms (P > 0.05). Conclusion The polymorphisms in ESR1 and AMHR2 genes showed a clear association with the number of retrieved oocytes and the stimulation data, respectively. Our results suggest that polymorphisms in the genes for key reproductive hormones receptors could be used to predict the ovarian response and to personalize the stimulation prior the treatment.

PharmGKB summary: very important pharmacogene information for CYP1A2: Erratum No abstract available