CYP2D6 genotype analysis

CYP2D6 genotyping was carried out according to procedures described in detail elsewhere (Brown et al., 2016). Briefly, a 6.6 kb long-range (XL) PCR fragment was amplified with CYP2D6-specific primers. Formation of the PCR product was verified by agarose gel electrophoresis before it was diluted and used as a template to detect SNPs and other sequence variations including nucleotide deletions and insertions. TaqMan (Applied Biosystems, Foster City, CA, USA) or RFLP assays were employed for the detection of sequence variations. Genotyping included the following single nucleotide variations (SNVs): 100C>T (rs1065852), 1023C>T (rs28371706), 1707delT (rs5030655), 1846G>A (rs1800716), 1862ins18bp (no rs), 2549delA (rs35742686), 2615delAAG (rs5030656), 2850C>T (rs16947), 2935A>C (rs5030867), 2988G>A (rs28371725), 3183 G>A (rs59421388), 3201C>T (rs147960066; tested only in the presence of a CYP2D6^*10 allele) and the exon 9 conversion (no rs). The CYP2D6^*5 gene deletion, the presence of gene duplications/multiplications and CYP2D7/2D6 hybrid genes were assayed by XL-PCR. An XL-PCR product of approximately 8 kb in length encompassing the duplicated gene was generated and subsequently genotyped (Gaedigk et al., 2007) for selected SNPs to unequivocally determine the allelic variation of gene duplications (to discriminate, for example, between CYP2D6^*2x2/^*4 and ^*2/^*4x2). Samples positive for a duplications/multiplication were also tested by a quantitative gene copy number variation (CNV) assay to determine the number of gene copies present in each sample (Gaedigk et al., 2012). The following CYP2D6 allelic variants were determined using the aforementioned tests: CYP2D6^*2, ^*3, ^*4, ^*5 (gene deletion), ^*6, ^*7, ^*9, ^*10, ^*13 (2D6/2D7 hybrid genes), ^*17, ^*29, ^*41, ^*56 and gene duplications/multiplications. Alleles were assigned according to the Human Cytochrome P450 Nomenclature database at www.cypalleles.ki.se/cyp2d6.htm. Alleles carrying no SNVs were defaulted to CYP2D6^*1 and those carrying only 2850C>T to CYP2D6^*2 assignments, respectively. CYP2D6 genotypes were translated into phenotype according to the guidelines published by the Clinical Pharmacogenetics Implementation Consortium (CPIC) (Hicks et al., 2015). Table S3 summarizes the main active and inactive alleles associated with different phenotypes. Genotyping was done at Children's Mercy Kansas City, Missouri. Ultrarapid metabolizer (UM), normal metabolizer (NM), intermediate metabolize (IM), and poor metabolizer (PM) categories were described according to Gaedigk et al. (2008) Tables S4, S5 summarizes antidepressants metabolism and excretion.