CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction

Abstract

Aims: Allelic variants of cytochrome P450 CYP2D6 (CYP2D6), such as gene deletion, duplication, multiplication and conversion, contribute to the wide range of CYP2D6 activity. Novel gene arrangements were discovered and characterized.

Materials & methods: DNA from 32 Caucasian and 59 African-American duplication-positive subjects were analyzed by long-range PCR and genotyping to detect CYP2D7-2D6 hybrid tandem alleles. Novel allelic variants were sequenced and a strategy for the detection and analysis of hybrid genes was refined.

Results: CYP2D7-2D6 hybrid tandem alleles were identified in one African-American and four Caucasian subjects. Three novel hybrid genes were found on CYP2D6*1 and CYP2D6*2 duplication backgrounds and designated CYP2D6*76, *77 and *78. CYP2D7 to 2D6 conversion occurred in introns 1 and 4, and exon 9. All carried a T-insertion in exon 1 abolishing activity. In Caucasians, four out of 33 (12%) of the duplication-positive alleles were hybrid tandems, three CYP2D6*77 + *2 and one CYP2D6*78 + *2. By contrast, in African-Americans only one of 60 duplication-positive alleles was identified as a hybrid tandem. This allele was designated CYP2D6*76 + *1.

Conclusion: Hybrid tandem alleles occur infrequently (<0.25%) in Caucasians, but may explain why not every subject with a CYP2D6 duplication presents with an ultrarapid metabolizer phenotype.

Conflict of interest statement

Financial & competing interests disclosure

Sequences have been submitted to GenBank and accession numbers assigned. Sequences will be released upon acceptance of the manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.. Overview of CYP2D6 gene duplication structures.

CYP2D6, 2D7 and 2D8 gene sequences are shown as pink, green and gray boxes. Repeat elements located downstream of CYP2D6 (REP-6), CYP2D7 (REP-7) and CYP2D6-derived gene duplications (REP- DUP) are shown in open boxes in respective colors. PCR fragments generated are represented as lines and their sizes are given in kb. Pink, green and gray end points indicate primer specificity to CYP2D6, 2D7 and 2D8 sequences. (A) Normal CYP2D6 gene locus. (B) Most common duplication structure that entails two (or more) genes of the same kind back-to-back, such as CYP2D6*1xN, *2xN, *4xN and so on. Primers utilized for duplication detection take advantage of sequences at the REP-DUP junction and within respective genes. (C) Summary of the novel duplication structures carrying a functional CYP2D6*1 or *2 at the 3’-end of the gene locus and a hybrid gene (CYP2D6*76, *77 or *78) upstream in the duplication position. The intergenic region between the two genes supports amplification of widely used long-range PCR products such as fragment B and C. Depending on where the CYP2D7–2D6 switch occurred, primer pairs will generate allele-specific PCR fragments. The formation of PCR products with CYP2D8 and 2D6 primer combinations demonstrates that the hybrid is situated downstream of CYP2D8. (D) For completion, the structure of the CYP2D6*36 + *10 tandem is provided. Due to the presence of CYP2D7 sequences in CYP2D6*36 - that is, the exon 9 conversion - the spacer sequence and REP-7, fragments B, C and D fail to amplify. Fragment A, however, is generated and when genotyped indicates a CYP2D6*10 allele assignment. REP: Repetitive element.

Figure 2.. Long-range PCR products generated from the hybrid tandems and control samples.

Fragment designation corresponds to that shown in Figure 1 and used throughout his manuscript. The genotypes of positive and negative control DNAs are as indicated. PCR product lengths are given in kb pairs to the right, marker sizes on the left. All PCR reactions were carried out on genomic DNA. (A) Fragments A, B and H were generated in separate reactions and in a triplex PCR. The five cases consistently produced all three amplicons indicating the presence of a ‘normal’ CYP2D6 gene, a gene duplication and a hybrid gene. (B) A duplication event was further demonstrated by amplifying the entire intergenic region (fragment C). While cases 1–4 produced fragment E in addition to an internal control amplicon, this fragment failed to amplify from the CYP2D6*76 case because the forward primer used cannot bind to the CYP2D7 sequence present in intron 6.

Figure 3.. Alignment of selected regions of hybrid sequences to CYP2D6 and 2D7 references.

Position numbers are given according to M33388, with −1 referring to the first nucleotide 5’ of the ATG start codon. CYP2D6 sequences are shown in yellow, CYP2D7 in green. Blue highlights deviating nucleotides or SNPs within the novel alleles. Known CYP2D6 SNPs that correspond to nucleotides found in CYP2D7 are as indicated (e.g., G>C at position 1833). Note that other known SNPs within CYP2D6 are not shown. Bold boxes indicate the switch regions. In CYP2D6*77, two nucleotides in the switch region (orange) could not unequivocally be assigned to CYP2D6 or 2D7. Regions referred to as CYP2D7 intron 1 and exon 9 conversions are as indicated. GenBank accession numbers are as shown.

Figure 4.. Further characterization of the tandem hybrid gene locus using long-range PCR.

Fragment designations correspond to those in Figure 1. The genotypes of positive and negative control DNAs are as indicated. PCR product lengths are given in kb to the right, marker sizes on the left. All PCR reactions were carried out on genomic DNA. (A) Series of PCR reactions carried out with CYP2D8 forward and CYP2D6 reverse primers binding to intron 1, intron 6 and exon 9 sequences, demonstrate that each hybrid is located immediately downstream of CYP2D8. According to their respective hybrid composition, CYP2D6*77 produced all three amplicons, while CYP2D6*78 and CYP2D6*76 produced two and one, respectively. (B) In this series of reactions, the forward primer is CYP2D7 specific (also used for fragment H). As in (A), the hybrid tandems amplified fragments according to their hybrid structure. These shorter fragments readily amplify in less than 3 h and provide a convenient way to further analyze hybrids. Note that amplicon sizes vary slightly (middle and bottom panels) due to sequence differences between the hybrids.

Figure 5.. Strategy to genotype hybrid-derived templates.

The graph underneath each gel print provides an overview of the hybrid tandem, the long-range PCR fragment(s) present in each genotyping reaction and the sequence variation interrogated. For RFLP genotyping, a PCR fragment was amplified, cut with a restriction enzyme and resulting fragments resolved by agarose gel electrophoresis. Fragment lengths are given in bp to the right, marker sizes on the left. (A) Genotyping assay detecting the T-insertion in exon 1 on fragment H. DNA samples with hybrid tandems, CYP2D7 only (control plasmid) and a single hybrid (CYP2D6*66) were completely cut, indicating the presence of the T-insertion. The CYP2D6 control plasmid remained uncut due to the absence of the insertion. (B) Genotyping assay detecting the exon 9 conversion on fragment H. DNA samples with hybrid tandems, a single hybrid (CYP2D6*66) and CYP2D6 only (plasmid control) were cut, indicating that exon 9 is derived from CYP2D6. The CYP2D7 plasmid control remained uncut due to the presence of the exon 9 conversion. (C) Assay as in (A), but performed on triplex long-range PCR products. Since the primers will bind to fragments A (from both alleles) and fragment H (from the tandem hybrid) the resulting band pattern appears heterozygous. A similar (heterozygous) result is obtained when genotyping is performed for the exon 9 conversion (not shown).