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.
Footnotes
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Send reprint requests to: Jin-ding Huang, Ph.D., Department of Pharmacology, National Cheng Kung University Medical College, Tainan 70101, Taiwan. E-mail: jinding{at}mail.ncku.edu.tw
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This study was supported by Grant NSC85-2331-B006-041 from the National Sciences Council of the Republic of China (Taipei, Taiwan).
- Abbreviations used are::
- CYP
- cytochrome P-450
- MR
- metabolic ratio
- PCR
- polymerase chain reaction, RFLP, restriction fragment length polymorphism
- kb
- kilobases
- bp
- base pairs
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- Received June 22, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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