Supplementary Material for: Allele Drop Out Conferred by a Frequent CYP2D6 Genetic Variation For Commonly Used CYP2D6*3 Genotyping Assays

<b><i>Background/Aim:</i></b> Accurate genotyping of <i>CYP2D6</i> is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (∼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. <b><i>Methods:</i></b> A cohort of 365 patient samples was genotyped for <i>CYP2D6</i> using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. <b><i>Results:</i></b> A discrepant result between the three genotyping methods for the loss of function <i>CYP2D6*3</i> (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the <i>CYP2D6</i> g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the <i>CYP2D6*3</i> pyrosequencing and hydrolysis probe assays to avoid <i>CYP2D6</i> g.2470 corrected the anomaly. <b><i>Conclusion:</i></b> To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered.