CHARACTERIZATION AND PCR-BASED DETECTION OF 2 DIFFERENT HYBRID CYP2D7P CYP2D6 ALLELES ASSOCIATED WITH THE POOR METABOLIZER PHENOTYPES/

The majority of humans deficient in the cytochrome P450 CYP2D6 enzyme, so-called poor metabolizers (PMs), can now be identified by genotypin g for several different PM-associated mutations, However, additional n ull alleles remain to be identified as demonstrated by subjects with t he PM phenotype in the absence of a corresponding genotype, The rare 1 1 kb band on Xba I RFLP analysis, which is distinct from the 13 kb CYP 2D6D (CYP2D65) allele, has been proposed to constitute such a unique non-functional allele, Here we demonstrate that the 11 kb band represe nts at least two different nine exon CYP2D7P/CYP2D6 hybrids generated by large deletions in the CYP2D gene cluster due to unequal cross-oner or looping-out mechanisms, The total allele frequency was approximate ly 0.001-0.01 in European and North American Caucasians, The most comm on variant (CYP2D616) had breakpoints lying between the end of exon 7 and the start of exon 9 of the respective genes, The 'CPP2D7-like' pa rt of the gene was most homologous to the previously described CYP2D7A P and CYP2D7(44/11.5) sequences, The other chimeric allele consisted o f exon 1 of CYP2D7 and exons 2-9 from CYP2D6, and may be similar to a hybrid gene termed CYP2D613 recently described in a French individual , Two different routine PCR assays were developed for rapid and sensit ive detection of these alleles, namely amplification of a 8 kb fragmen t from both CYP2D613 and CYP2D6*16, together with a CYP2D6*16-specifi c method which gave a 1.4 kb PCR product, The 8 kb assay for the CYP2D 613 and CYP2D6*16 alleles also produced a 9.5 kb fragment in samples positive for the 13 kb CYP2D65 allele, Therefore, it is now possible to screen for the large CYP2D gene deletions by a single long PCR meth od.