CYP 2C19 genotype assessment

Genomic DNA was extracted from 2 ml EDTA anticoagulated peripheral venous blood obtained from fasting patients, using the Blood DNA TIANamp Kit (Tiangen Biotech Co., Ltd., Beijing, China). CYP2C19 (NCBI gene ID: 1557) genotype analysis was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology on a Bio-Rad S1000 PCR instrument, and the primer sequences are shown in Table 1. Three main alleles were found: wild-type (CYP2C19*1) and 2 mutants (mutations in exon 5 for CYP2C19*2 and in exon 4 for CYP2C19*3) (Supplementary Figure 1).

PCR primers.

DNA samples were examined for concentration and purity on an ultraviolet spectrophotometer and agarose gel electrophoresis, and qualified specimens were selected: concentration >50 ng/μL, OD260/OD280 ratio between 1.5 and 2.0, gel electrophoresis bands clear, and integrity >20 kb, ensuring DNA integrity and purity compatible with the PCR method.

Genomic DNA from patient blood samples was used as template with the specific primers described in Table 2. PCR was carried out with the Taq PCR Master Mix kit (TianGen Biotech Co., Ltd.) according to the manufacturer’s instructions. Briefly, a total reaction volume of 25 μl contained DNA template (5 μl), upstream and downstream primers (10 μM, 1 μl each), 2×Taq PCR Master Mix (12.5 μl), and ddH2O to 25 μl. The reaction conditions were: 94°C for 3 min (initial denaturation); 30 cycles of 94°C for 30 s (denaturation), 56 °C (exon 5) or 53 °C (exon 4) for 30 s (annealing), and 72°C for 1 min (extension); and final extension at 72°C for 5 min. PCR products of exon 5 carrying CYP2C19*2 mutation sites and exon 4 with CYP2C19*3 mutation sites were assessed by 2% agarose gel electrophoresis.

Baseline patient data.

Alcohol taking: Drink at least 4 times a week for the past 30 days.

Each PCR product (exon 5 and exon 4) was cut by restriction endonucleases SmaI and BamHI (NEB Co., Ltd.), respectively. Exons 5 and 4 were digested (37°C) for 15 min according to the manufacturer’s instructions.

Electrophoresis of enzyme digestion products was carried out on 2% agarose gel followed by band observation under ultraviolet light; the genotypes were assessed by standard DNA molecular techniques. Figure 1 shows polymorphism bands of CYP2C19 *2 and CYP2C19*3.

Flow chart of patient enrolment.