2.3. CYP2C19 genotyping

CYP2C19 genotyping was performed for each of the treated patients. Briefly, blood sample (2 mL) was collected into a vacuum tube with EDTA. DNA was isolated from the blood using the RelaxGene Blood DNA System (Tiangen Biotech Co, Ltd, Beijing, China). Then, PCR restriction fragment-length polymorphism (RFLP)-based analysis was carried out with following primers designed by the Hua Gene Biotech Company (Beijing, China). The forward and reverse primers of mutation1 (m1) were 5′-CAACCAGAGCTTGGCATATTG-3′ and 5′-CACAAATACGCAAGCAGTCAC-3′, respectively. The forward and reverse primers of mutation 2 (m2) were 5′-CACCCTGTGATCCCACTTTC-3′ and 5′-CTAATGGGCTTAGAAGCCTG-3′, respectively. The amplified PCR products, which were 301 and 376 bp gene fragments for m1 and m2, respectively, were digested with enzyme, SmaI for m1 and BamHI for m2 (New England Biolabs, Beverly, MA). Then, the digested PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide. Because CYP2C19 m1 lacks SmaI site and CYP2C19 m2 lacks BamHI site, the mutant alleles are resistant to endonuclease digestion. Meantime, we chose 70 samples for direct sequencing (Hua Gene Biotech Company) to verify the accuracy of PCR-RFLP. Studied patients were categorized into 3 groups, based on the existence of m1 or m2 on CYP2C19 genotyping: homozygous extensive metabolizers (wild type, wt/wt); heterozygous extensive metabolizers (wt/m1 or wt/m2), and poor metabolizers (PM) (m1/m1, m1/m2, or m2/m2).