Cytochrome P4502D6 (CYP2D6) Gene Locus Heterogeneity: Characterization of Gene Duplication Events

Abstract

Duplications and multiplications of active CYP2D6 genes can cause ultrarapid drug metabolism and lead to therapeutic failure. Multiple functional and non-functional duplication alleles have been further characterized. Duplications were detected by long-range polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and sequence analysis. A PCR fragment encompassing the entire duplicated gene was utilized for detailed characterization. Duplications occurred at 1.3, 5.75, and 2.0% in Caucasian, African American, and racially mixed populations, respectively (n=887 total). Of those 28, 47, and 17% were non-functional CYP2D6*4 x N. Twelve unique duplication alleles were detected: *1 x N, *2 x N, *4 x N, *6 x N, *10 x N, *17 x N, *17 x N[spacer], *29 x N, *35 x N, *43 x N, *45 x N, and a novel non-functional tandem arrangement of a chimeric 2D7/2D6 and *1 gene. All novel duplications except *35 x N were found in African Americans. Accurate identification of gene duplication events is essential to avoid false-positive ultrarapid metabolism assignments and thus, overestimation of predicted activity and increased risk for unwanted adverse events.

Impact of variable CYP genotypes on breast cancer relapse in patients undergoing adjuvant tamoxifen therapy

“…Genotypes of CYP2D6 were assigned to the expected corresponding metabolizer phenotypes according to the evaluation system by Gaedigk et al [8]. The genotype/phenotype assignments are summarized in table 2.…”

CYP2D6 Haplotype Determination Using Long Range Allele-Specific Amplification

“…We designed a pair of primers for these SNP positions that differed at their 3 0 ends to match to either the wild-type or variant nucleotide, as shown in Table 1. Each AS forward primer was paired with a CYP2D6-specific universal reverse primer that we routinely use for amplifying XL-PCR fragment A 21 and tested for specificity with a series of amplification reactions at increasing annealing temperatures, ranging from 63 C to 70 C. A universal fragment A forward primer was utilized for amplifying both alleles simultaneously.…”

“…The primer specific for the SNP at -740 was also successfully paired with the reverse primer amplifying duplicated genes (fragment D 21 ) to discriminate between duplicated genes located on both chromosomes, or potentially in tandem.…”

“…In subjects carrying a gene duplication or a multiplication event, an 8-Kb XL-PCR product encompassing the duplicated gene was generated to determine which of the two alleles carries the copy number variation. 21 This fragment was genotyped for the presence of sequence variations as described above (see CYP2D6 Genotype Analysis). Alleles are defined according to the Nomenclature Database.…”

“…This assignment was also supported by the finding that fragment D was about 1.6 Kb longer compared with that amplified from a CYP2D6*2x2 (the length difference is due to CYP2D7 extending downstream and containing a so-called spacer region that is absent in CYP2D6). 21 When fragment D was amplified in a non-AS manner, the shorter CYP2D6*2x2-derived product was preferentially amplified, leading to a dropout of the CYP2D6*4N-derived amplicon, which explains why the latter was initially not detected. Because the exon 9 conversion was not found on fragment A, the duplication was subtyped as a CYP2D6*4Nþ*4 tandem structure, as depicted in Figure 5.…”