Identification of Novel CYP2D7-2D6 Hybrids: Non-Functional and Functional Variants

Abstract

Polymorphic expression of CYP2D6 contributes to the wide range of activity observed for this clinically important drug metabolizing enzyme. In this report we describe novel CYP2D7/2D6 hybrid genes encoding non-functional and functional CYP2D6 protein and a CYP2D7 variant that mimics a CYP2D7/2D6 hybrid gene. Five-kilobyte-long PCR products encompassing the novel genes were entirely sequenced. A quantitative assay probing in different gene regions was employed to determine CYP2D6 and 2D7 copy number variations and the relative position of the hybrid genes within the locus was assessed by long-range PCR. In addition to the previously known CYP2D6*13 and *66 hybrids, we describe three novel non-functional CYP2D7-2D6 hybrids with gene switching in exon 2 (CYP2D6*79), intron 2 (CYP2D6*80), and intron 5 (CYP2D6*67). A CYP2D7-specific T-ins in exon 1 causes a detrimental frame shift. One subject revealed a CYP2D7 conversion in the 5'-flanking region of a CYP2D6*35 allele, was otherwise unaffected (designated CYP2D6*35B). Finally, three DNAs revealed a CYP2D7 gene with a CYP2D6-like region downstream of exon 9 (designated CYP2D7[REP6]). Quantitative copy number determination, sequence analyses, and long-range PCR mapping were in agreement and excluded the presence of additional gene units. Undetected hybrid genes may cause over-estimation of CYP2D6 activity (CYP2D6*1/*1 vs *1/hybrid, etc), but may also cause results that may interfere with the genotype determination. Detection of hybrid events, "single" and tandem, will contribute to more accurate phenotype prediction from genotype data.

Keywords: CYP2D6; CYP2D6 poor metabolizer; CYP2D6*35B; CYP2D6*67; CYP2D6*79; CYP2D6*80; hybrid genes.

Figure 1

Overview of the CYP2D7/2D6 hybrid genes. CYP2D6, CYP2D7, and CYP2D8 genes are shown in light gray, red, and dark gray boxes, respectively. The 600-bp repeat element immediately downstream of exon 9 is shown in blue. CYP2D6 and CYP2D7-derived repetitive elements (REP) are in red and light gray; REP-DEL indicates a fused repeat element generated by a large deletion involving parts of those elements from both genes. PCR fragments generated are represented as lines and their sizes are given in kilobyte. Red and black endpoints denote primer specificity to CYP2D6 and CYP2D7. (A) Graph represents the CYP2D reference locus. Areas affected by large deletions and implicated in CYP2D7/2D6 hybrid formation and the CYP2D6*5 gene deletion are as indicated. (B) Graphic display of the CYP2D6*5 gene deletion allele. XL-PCR amplicons utilized for detection are shown. (C) Graphic display of CYP2D7/2D6 hybrid genes and their detection by amplification of fragment H. Other depicted fragments are only amplified, if respective rearrangements are present in a sample. (D) Representation of an allele with a CYP2D7 gene lacking the 1.6-kb spacer. This CYP2D7 variant also supports formation of fragment H although the CYP2D7/2D6 switch occurs in the downstream region.

Figure 2

Long-range (XL) PCR products generated from hybrid genes and control samples. Fragment designation corresponds to that provided in Figure 1 and used throughout this manuscript. The genotypes of the cases are shown in Table 1. PCR product lengths are given in kilobyte to the right, marker sizes on the left. All PCRs were carried out on genomic DNA. (A) Triplex XL-PCR showing the formation of fragment A (entire CYP2D6 gene), fragment B (presence of gene duplication), and fragment H (indicating the presence of a CYP2D7/2D6 hybrid gene). (B) Fragment H was generated by itself. Note that this fragment is weak in the triplex reaction in panel A for case 9, but unequivocally present when amplified “solo”. (C) As expected from sequence data, cases 5, 6, and 7 generated a PCR product spanning CYP2D8 to CYP2D6 intron 6. CYP2D6*66 (index case) and case 9 serve as negative controls (see Figure 3 for hybrid details).

Figure 3

Graphic overview of hybrid alleles and their CYP2D7/2D6 switch region. CYP2D7 and CYP2D6 portions are shown in red and gray boxes, respectively. The CYP2D7 conversion in CYP2D6*35[variant] is shown as a red box upstream of exon 1. Switch regions are indicated in green. The red arrow notes a T-insertion in CYP2D7 exon 1 relative to CYP2D6. The hybrid sequences were aligned with CYP2D7 (M33387) and CYP2D6 (M33388) reference sequences to approximate the switch region. For completion, the graph also shows previously reported CYP2D6*76 + *1, *77 + *2, and *78 + *2 tandem hybrids (Gaedigk et al., 2010) as well as the CYP2D6*13 and *16 alleles (no reference sequences deposited in GenBank; graphs based on descriptions in (Panserat et al., , Daly et al., 1996).

Figure 4

Comparison of hybrid sequences upstream of exon 1 to CYP2D6 and CYP2D7 references. CYP2D6 sequences are highlighted in yellow, CYP2D7 in green and sequence variation deviating from both in orange. Position numbers are given according to M33388 (first column, 1 = first nucleotide in M33388; second column, −1 = first nucleotide upstream of ATG start codon. The forward primer binding site for fragment H is shown in darker green in the M33387 reference sequence and limits the sequence information obtained from the hybrids (X denotes no sequence). A commonly used CYP2D6-specific primer binding site is shown in darker yellow in the M33388 reference sequence. For CYP2D6*35B ∼1.5 kb of upstream region was sequenced.

Figure 5

Genotype analysis for CYP2D7 exon 9 conversion and CYP2D7[REP6]. M, 100-bp ladder. Cases, positive, and negative control DNAs and their respective genotypes are as indicated. Selected marker bands are given in kilobyte to the left; PCR product and restriction fragment lengths are shown to the right in base pair. The top panel (A) shows a duplex assay performed on genomic DNA that detects the CYP2D6 exon 9 conversion present in e.g., CYP2D6*36 and alleles carrying a CYP2D7[REP6]. The forward primer is specific for CYP2D7 exon 9 and the reverse primer for CYP2D6 binding to the REP6 junction (see Figure 1). A second primer pair served as internal control (IC). Samples positive for CYP2D6*36 or CYP2D7[REP6] produced a 597-bp long amplicon in addition to the 860-bp-long IC product. The bottom panel (B) shows a RFLP-based assay performed on the 6.6-kb long CYP2D6-specific genotyping template. Uncut PCR product (775 bp) is indicative of a CYP2D7 exon 9-derived sequence on a CYP2D6 background. The CYP2D6*2/*36 control DNA sample is positive for CYP2D7 exon 9 in both assays, while cases 10, 11 and 12 showed a CYP2D6-derived pattern in the RFLP assay (591 + 184 bp).