Abstract

The molecular basis of the reduced ability of a Chinese to metabolize debrisoquine was explored by sequencing all of the nine exons of the CYP2D6 gene. The subject has T₁₈₈, A₁₈₄₆, T₂₉₃₈, and C₄₂₆₈ (CYP2D6*14) instead of C₁₈₈, G₁₈₄₆, C₂₉₃₈, and G₄₂₆₈ as in wild-type subjects. XbaI restriction fragment length polymorphism indicated that the subject has a 29-kb allele and a gene deletion (11.5 kb) in another allele (CYP2D6*5). A CYP2D6*14 allele together with a CYP2D6*5 allele may cause the poor metabolism of the subject. T₁₈₈, T₂₉₃₈, and C₄₂₆₈ are common haplotypes in Chinese-extensive metabolizers. The effect of G₁₈₄₆ to A mutation in CYP2D6 metabolism has not been reported. A polymerase chain reaction-based endonuclease digestion test was designed for the G/N₁₈₄₆polymorphism and 124 Chinese subjects were screened. With DNA sequencing, two other subjects showed the heterozygous G/A₁₈₄₆ and have a relatively high metabolic ratio of debrisoquine hydroxylation. The site-directed mutagenesis was used to create recombinant CYP2D6 cDNA with T₁₈₈, A₁₈₄₆, or C₄₂₆₈. The cDNA was then transfected into Rat-1 cells. The transfection was confirmed by Southern, Northern, and Western blots. Based on the same microsomal protein level, the bufuralol 1′-hydroxylation activity of CYP2D6(T₁₈₈) or CYP2D6(A₁₈₄₆) was significantly lower than that of the wild-type CYP2D6. P34S mutation (C₁₈₈ to T) significantly decreased CYP2D6 activity. G169R mutation (G₁₈₄₆ to A) also decreased CYP2D6 activity and may further reduce the metabolic activity of CYP2D6 protein with P34S, R296C, and S486T mutations.