The Validation and Application of a Novel CYP2C19 Genotyping Approach Based on Capillary Electrophoresis in Chinese Han population

Background: CYP2C19 gene polymorphisms have been described to have an important influence on the drug me-tabolism observed in human populations. A series of PCR-based molecule detection methods are applied to identi-fy CYP2C19 genotype. The aim of the study is to validate the novel CYP2C19 genotyping approach with other methods and reveal the allele frequency distribution of CYP2C19 in Chinese Han population. Methods: We applied a novel genotyping approach for CYP2C19 gene which was combining direct PCR and capil-lary electrophoresis (CE) technique. A series of fluorescent labeled primers were designed to amplify the particu-lar DNA fragments which indicated the wild type of CYP2C19 genotype. The variants consist of CYP2C19*2, CYP2C19*3 and CYP2C19*17 alleles. Both the novel PCR-based CE method and real-time quantitative PCR (RT-qPCR) method were used to identify the CYP2C19 genotypes in 324 whole blood samples originated from Chinese Han population. According to the different criterions for judgement of two methods, we can obtain the CYP2C19 alleles and genotypes of the same participants. Kappa statistics was used to evaluate the consistency of the two re-sults and the frequencies of CYP2C19 alleles. The genotypes in Chinese Han population were calculated using EXCEL. Furthermore, to ensure the accuracy and reliability of the CYP2C19 genotypes obtained by using the novel ap-proach, Sanger sequencing was conducted to validate the CYP2C19 genotypes *1/*17 and *2/*3. Results: Among the 324 specimens, 111 were *1/*1, 141 were *1/*2, 10 were *1/*3, 4 were *1/*17, 46 were *2/*2, 10 were *2*/3, 1 was *2/*17, and 1 was *3/*17. Allele distributions for CYP2C19 were *1, *2, *3, and *17 at 58.18%, 37.65%, 3.24%, and 0.93%, respectively. Both PCR-based CE method and RT-qPCR methods had good consistency in the genotypes of CYP2C19 polymorphism (Kappa value = 1.000, p < 0.05). The DNA sequences of CYP2C19 genotype *1/*17 were composed of c.681 G/G, c.636 G/G, and c.-806 C > T. In the same way, the DNA sequences of CYP2C19 genotype *2/*3 were composed of c.681 G > A, c.636 G > A, and c.-806 C/C. Conclusions: The variants including the CYP2C19*2 allele were the most common mutations in Chinese Han unrelated individuals. Both PCR-based CE method and RT-qPCR method had good consistency in the genotypes of CYP2C19 polymorphism. Nevertheless, because of more convenience and higher throughput, the novel PCR-based capillary electrophoresis approach showed to be more suitable for clinical gene screening. (Clin. Lab. 2022;68:2325-2332. DOI: 10.7754/Clin.Lab.2021.220212)