CYP2D6 genotyping analysis was commissioned to SPMED Co. Ltd (Busan, South Korea). Patient DNA was isolated and purified from blood samples using SPMED™ Genotyping Kit: CYP2D6 (KFDA -IVD 20–297 and CE-IVD, SPMED Co. Ltd) according to the manufacturer’s instructions and CYP2D6 *2, *3, *4, *5 (deletion), *6, *9, *10, *14, *17, *18, *21, *29, *41, *49, *52, *60, *XN (duplicate) were analysed. These 17-star alleles cover most (99.3%) of the major mutation in Asian races based on previous results [10, 11].
Previously, full sequencing data from Korea have shown the *10 promoter and intron SNPs to be linked, such that the *10 alleles detected above is the CYP2D6*10b allele [12] and remains a reduced function allele.
Briefly, genomic DNA was extracted according to the manufacturer’s instructions of QIAgen Mini Kit (QIAamp Company’s DNA Mini Kit, Germany). The extracted DNA was stored at − 20 °C. PCR reactions were performed with 0.5 µl genomic DNA, 10 µl of PCR amplification primer mix, 4 µl of each CYP2D6 amplification primer mix. The total volume was adjusted to 20 µl with nuclease-free water. The PCR reaction protocol was controlled with an initial denaturation step at 94 °C for 5 min, followed by 35 cycles with first set at 98 °C for 20 s, 64 °C for 30 s, and 72 °C for 30 s, and a final extension reaction at 72 °C for 5 min. The amplified products were separated based on 1% agarose gel electrophoresis and visualized by ethidium bromide under a UV transilluminator. Only the sequences that showed identical size and 90% or more similar thickness with the wild type DNA were used for subsequent analysis.
Specific primers were used for single-base extension using SNaPshot kit (Applied Biosystems, Mannheim, Germany), according to manufacturer’s instructions. Reactions were carried out in a final volume of 13.5 µl, containing 1 µl of the SNaPshot Multiplex Reagent, 1 µl of the primer mix, 7.5 µl of purified product and 4 µl of 1/2 term buffer. Purified products underwent capillary electrophoresis on 3500 DX Series Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the standard fragment analysis protocol.
GeneMapper software (version 4.0 Thermo Scientific, MA, USA) was used for allele-specific gene expression analysis and calculated the peak color of CYP2D6 single-nucleotide variant (SNV) to the peak density of the wild type DNA. The CYP2D6*1 allele was set when no nucleotide change was observed in all genotyped SNPs.
CYP2D6 genotype identified levels of enzymatic activity qualitatively by the activity score model, where a score of 0.0, 0.25–1.0, 1.25–2.25, and > 2.25 each indicate a poor metabolizer (PM), intermediate metabolizer (IM), normal metabolizer (NM), and ultrarapid metabolizer (UM), respectively [13].
Statistical analysis
Descriptive statistics are expressed as means, medians or proportions. The associations between categorical data were tested using the χ-squared test or Mann-Whitney U test. For analysing the trends between categorical variables, the Cochran-Armitage test was used. All tests of significance were two-tailed, and differences were considered statistically significant at p < 0.05. All statistical analyses were performed using SPSS Statistics version 23 (IBM Corp., Armonk, NY, USA; https://www.ibm.com).
Ethics statement
This study was approved by the ethical committees of each hospital (Asan Medical Centre: 2018-0650, Armed Forces Capital Hospital: AFCH 19-IRB-018, Dongguk University Ilsan Hospital: 2019-08-004-002, Inje University Ilsan Paik Hospital: 2019-07-020-001, National Health Insurance Service Ilsan Hospital: 2019-07-022-001, Severance Hospital: 4-2019-1240, Catholic Kwandong University International St. Mary’s Hospital: 19-IRB-059-1, Ajoo University Hospital: BMR-SMP-19-302). Signed written informed consents were obtained from prospectively registered patients or their guardians before the study. A waiver of patient consent was granted for data that were retrospectively collected.