GENETIC-ANALYSIS OF THE CHINESE CYTOCHROME P4502D LOCUS - CHARACTERIZATION OF VARIANT CYP2D6 GENES PRESENT IN SUBJECTS WITH DIMINISHED CAPACITY FOR DEBRISOQUINE HYDROXYLATION

Cytochrome P4502D6 (CYP2D6) catalyzes the oxidative metabolism of seve ral clinically important classes of drugs. Many of these have lower me tabolic clearance rates among Chinese, compared with Caucasians, and a re prescribed at lower doses for Asian patients. We have now evaluated the molecular genetic basis for this interethnic difference in drug m etabolism. The CYP2D loci from two Chinese subjects, one homozygous fo r the XbaI 44-kilobase haplotype and one homozygous for the XbaI 29-ki lobase haplotype, were cloned and characterized. Sequence analysis rev ealed two variant CYP2D6 genes, CYP2DGCh(1) and CYP2DGCh(2), having mu tations yielding two and eight amino acid substitutions, respectively. Exon 9 of the CYP2DGCh(2) gene contained a sequence of 49 bases origi nating from the pseudogene CYP2D7P. In addition, mutations in the 5' f lanking region common to both CYP2D6Ch genes were found. To evaluate t he origin of the detrimental mutation in the genes, parts of the 5' fl anking regions were introduced into a Hep G2/simian virus 40 expressio n system with chloramphenicol acetyltransferase as a reporter gene, an d transfected cells were analyzed for activity. The ability of the ups tream regions to bind nuclear factors was also evaluated using gel-shi ft analysis. Furthermore, several chimeric constructs of the CYP2D6wt and CYP2D6Ch genes were made, inserted into pCMV2 vectors, and express ed in COS-1 cells. A part of the upstream region of base pairs -1407 t o -1068 was found to constitute an enhancer element, but the CYP2D6Ch- specific mutations did not influence the chloramphenicol acetyltransfe rase activity in the expression system. In contrast, expression of the chimeric genes revealed that the detrimental mutation of the CYP2D6Ch genes was C-188 --> T, causing a Pro(34)-->Ser amino acid substitutio n in a region that is a highly conserved in cytochromes P450 belonging to gene families 1 and 2. This substitution caused expression of a mo re unstable gene product, as evident from comparison of the relative l evels of CYP2D6 mRNA, CYP2D6 protein, and bufuralol 1'-hydroxylase act ivities in pCMV2-CYP2D6-transfected COS-1 cells. Allele-specific polym erase chain reaction analysis of genomic DNA from 90 Chinese individua ls revealed that the CYP2D6Ch(1) allele was the most common one and it s distribution correlated well with a higher metabolic ratio for debri soquine. These data demonstrate that important interethnic differences exist in the structure of the CYP2D locus, and they suggest that the frequent distribution of the C-188-->T mutation among the CYP2D6Ch gen es explains the lower capacity among Chinese to metabolize drugs that are substrates of CYP2D6, such as antidepressants and neuroleptic agen ts.