CYP2C8 pharmacogenetics and functional characterization of promoter polymorphic variants in Asian population

A59

The objective of the present study was (1) to determine polymorphic variations in the CYP2C8 gene in three distinct healthy Asian subjects (Chinese: N=101, Malay: N=91 and Indian: N=90, populations), and (2) to functionally characterize high frequency CYP2C8 promoter polymorphisms. Screening for genetic variations in the promoter, exons and exon-intron junctions of CYP2C8 gene was performed by PCR followed by direct DNA sequencing. Seven polymorphisms were identified and their allelic frequencies were as follows: 5’-UTR: g.-411C>T (C:0.33;T:0.67), g.-370T>G (T:0.72;G:0.28) and g.-271C>A (C:0.88;A:0.12); intron 2: I.-64A>G (A:0.50;G:0.50), -13insT (Wt:0.87;insT:0.13); intron 7: +49T>A (T:0.47;A:0.53) and 3’-UTR: 24C>T (C:0.62;T:0.38). Haplotype analysis revealed fourteen different haplotypes in Chinese, eighteen in Malays, and twenty one in the Indian population. Two haplotype blocks were inferred in each ethnic group based on the solid-spline algorithm. The functional influence of CYP2C8 promoter polymorphisms were studied by using various combinations of plasmid constructs containing the three polymorphic variants. The different constructs were cloned in pGL3 expression vector and investigated for their activity in driving reporter gene expression in transfected HepG2 cells under optimized conditions. The promoter construct harboring the single g.-411C>T variant showed approximately 2-fold higher luciferase activity compared with the reference construct (P=0.002). The construct harboring the combined g.-411C>T and g.-271C>A polymorphic variants showed a severe reduction (44-fold) in luciferase activity compared with the construct containing the reference sequence (P=0.006). Further studies to establish the mechanistic basis for the altered promoter activity due to the promoter polymorphic variants are ongoing.