Discriminative quantification of cytochrome P4502D6 and 2D7/8 pseudogene expression by TaqMan real-time reverse transcriptase polymerase chain reaction.

The human drug oxidizing cytochrome P450, CYP2D6, is expressed at highly variable levels mainly due to a common genetic polymorphism which leads to the poor metabolizer phenotype in carriers of two nonfunctional alleles and to the extensive metabolizer phenotype in carriers of one or more functional alleles. Investigation of the role of CYP2D6 mRNA for expression and the possibility of using mRNA expression as a surrogate marker has been hampered by the presence of two pseudogenes, CYP2D7P and CYP2D8P. We therefore developed highly specific TaqMan real-time reverse transcriptase-PCR assays for the discriminative quantification of CYP2D6 and CYP2D7/8P transcripts. By in vitro transcription of plasmids containing the CYP2D6 cDNA or a hybrid CYP2D6/7 cDNA constructed by in vitro mutagenesis, authentic cRNAs were synthesized to be used for specificity testing and for absolute quantification. The method was used to determine CYP2D transcripts in a large number of human livers samples. CYP2D6 was not normally distributed with a median mRNA content of 3.2 transcripts per picogram of total RNA in all livers (range 0.32-14.8, N = 74). Expression in genetic poor metabolizers (1.81, N = 6) was significantly lower compared to extensive metabolizers (3.33, N = 68, P = 0.022). Similar expression levels were found for CYP2D7/8P (median 3.38 transcripts/pg, range 0.46-14.3), which were correlated to CYP2D6 mRNA (r(S) = 0.46, P < 0.0001) but did not depend on CYP2D6 genotype. These data demonstrate genotype-dependent mRNA expression for CYP2D6 and they emphasize the necessity of differentiating between the functional CYP2D6 and the CYP2D pseudogenes.