Identification and in vitro functional assessment of 16 CYP2A6 variants detected in the Chinese Han population

CYP2A6 is a hepatic enzyme involved in the metabolism of approximately 3% of clinically used drugs and various xenobiotics. Genetic polymorphisms in CYP2A6 contribute to interindividual differences in drug clearance and susceptibility to smoking-related diseases, yet the allelic landscape and functional impact in Chinese Han population remain incompletely characterized. A high-throughput CYP2A6 genotyping method by combining multiplex PCR with next-generation sequencing (NGS) was developed to characterize CYP2A6 genetic profiles in Chinese Han individuals. Novel CYP2A6 variants were recombinantly expressed using a yeast microsomal expression system, and coumarin was used as a probe substrate to evaluate their drug-metabolizing activity in vitro by UPLC-MS/MS. A total of 12 previously reported CYP2A6 allelic variants and 16 novel missense variants were identified in 1163 Chinese Han individuals. Functional assays revealed that five variants (I71F, Q239K, V334E, V334A, and P402S) exhibited significantly increased intrinsic clearance, while five others (G100R, E103K, T212M, R372H, and Q409R) showed significantly decreased activity; the remaining six variants retained wild-type-comparable catalytic function. This study presents a cost-effective genotyping strategy for large-scale CYP2A6 screening and provides in vitro functional characterization of newly identified variants, contributing clinically relevant pharmacogenetic evidence for personalized drug therapy in the Chinese Han population.