G169R mutation diminishes the metabolic activity of CYP2D6 in Chinese

The molecular basis of the reduced ability of a Chinese to metabolite debri soquine was explored by sequencing all of the nine exons of the CYP2D6 gene . The subject has T-188, A(1846), T-2938, and C-4268 (CYP2D6*14) instead of C-188, G(1846), C-2936, and G(4268) as in wild-type subjects. Xbal restric tion fragment length polymorphism indicated that the subject has a 29-kb al lele and a gene deletion (11.5 kb) in another allele (CYP2D6*5). A CYP2D6*1 4 allele together with a CYP2D6*5 allele may cause the poor metabolism of t he subject. T-188, T-2938, and C-4268 are common haplotypes in Chinese-exte nsive metabolizers. The effect of G(1846) to A mutation in CYP2D6 metabolis m has not been reported. A polymerase chain reaction-based endonuclease dig estion test was designed for the G/N-1846 polymorphism and 124 Chinese subj ects were screened. With DNA sequencing, two other subjects showed the hete rozygous G/A(1846) and have a relatively high metabolic ratio of debrisoqui ne hydroxylation. The site-directed mutagenesis was used to create recombin ant CYP2D6 cDNA with T-188, A(1846), or C-4268. The cDNA was then transfect ed into Rat-1 cells. The transfection was confirmed by Southern, Northern, and Western blots. Based on the same microsomal protein level, the bufuralo l 1'-hydroxylation activity of CYP2D6(T-188) or CYP2D6(A(1846)) was signifi cantly lower than that of the wild-type CYP2D6, P34S mutation (C-188 to T) significantly decreased CYP2D6 activity. G169R mutation (G(1846) to A) also decreased CYP2D6 activity and may further reduce the metabolic activity of CYP2D6 protein with P34S, R296C, and S486T mutations.