Identification and Functional Assessment of Eight CYP3A4 Allelic Variants *39-*46 Detected in the Chinese Han Population.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to the severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1,163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex PCR amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising 6 previously reported alleles and 8 new non-synonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was utilized as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, while the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. Significance Statement In this study, we established a multiplex PCR amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these 8 novel CYP3A4 allele variants in vitro This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.