Automatic DNA sequencing
All PCR products subjected to sequencing were purified with Qiagen PCR purification kit (Qiagen, Chatsworth, CA, USA) and sequenced using the ABI PRISM1 BigDyeTM Terminator Cycle Sequencing Kit (PE Biosystems). All primers used in sequencing are listed in Table 1. The CYP2D7-specific product, CYP-503/512, was sequenced using the CYP-503 primer. The CYP2D6 promoter region product, CYP-505/502, was sequenced using the primers CYP-502, CYP-505, CYP-506, CYP-507, CYP-508, CYP-509 and CYP-514. All reactions were analysed on an ABI 377 DNA Sequencer according to the manufacturer's instructions.
Detection of new polymorphisms
Polymorphisms were screened for by manually comparing all the ABI trace files from the same PCR products. We used both the ABI software Sequence Analysis version 3.2 (PE Biosystems, Foster City, CA, USA) and a shareware program called Chromas (http://www.technelysium.com.au/chromas.html) for viewing the traces.
Detection of DNA variants by means of restriction enzyme-based polymorphism assays
All primers for RFLP analysis are listed in Table 1. The following assays were set up: (i) The 31G > A (11V > M) polymorphism creates a Nla III restriction site, and a 341 bp fragment covering this single nucleotide polymorphism (SNP) was amplified using primers CYP-511 and CYP-518. Digestion of the wild-type product (31G) generated fragments of 36 bp + 305 bp, whereas the product with the 31A variant gave rise to fragments of 36 bp + 112 bp + 193 bp. (2) The 180 + 34G > C polymorphism creates a Bsu 36I restriction site, and a 557 bp fragment covering this SNP was amplified using primers CYP-503–2 and CYP-514. Digestion of the wild-type product (180 + 34G) generated one fragment of 557 bp, whereas the product with the 180 + 34C variant gave rise to fragments of 254 bp + 303 bp. (3) The 180 + 130G > T polymorphism destroys an Aat II restriction site, and a 557 bp fragment covering this SNP was amplified using primers CYP-503–2 and CYP-514. Digestion of the wild-type product (180 + 130G) generated fragments of 207 bp + 350 bp, whereas the product with the 180 + 130T variant remained uncut. (4) The 2850C > T polymorphism (CYP2D6*2-related) destroys a Hha I restriction site, and a 585 bp fragment covering this SNP was amplified using primers CYP-4 and CYP–7. Digestion of the wild-type product (2850C) generated fragments of 261 bp + 324 bp, whereas the product with the 2850T variant remained uncut. PCR amplification of all four RFLP assays was performed using the same reaction components as described above for the CYP-503/512 product. Amplification was performed in a GeneAmp PCR System 2400 or 9700 (PE Biosystems) with the following conditions: an initial denaturation at 96 °C for 1 min, followed by 35 cycles of 96 °C for 20 s, 55 °C (CYP-503–2/514) or 60 °C (CYP-4/7) or 63 °C (CYP-511/518) for 20 s and 72 °C for 45 s, and a final elongation at 72 °C for 7 min. The resulting PCR products and digestions were separated and detected in ethidium bromide-containing agarose gels.
Detection of DNA variants by means of automatic DNA sequencing
An approximately 900 bp fragment from the CYP2D6 promoter region was amplified from the CYP-505/502 product by nested PCR with the primers CYP-505 and CYP-508, using the PCR conditions described for amplification of the CYP-503/512 product. The CYP-505/508 product was then subjected to automatic DNA sequencing, and the −1770G > A and −1584C > G SNPs were determined by manual inspection of the trace files.
StatXact for Windows, version 4.0.1 (Cytel Software Corp., Cambridge, MA USA) and SPSS, version 9.0 (SPSS Inc., Chicago, IL, USA) were used for statistical analyses of the observed differences in allele, genotype and haplotype frequencies between the groups phenotyped with debrisoquine, and the differences observed in metabolic ratios between genotype groups. The samples studied consisted of unrelated individuals, and family data could therefore not be used to determine haplotypes in multiple heterozygous individuals. Instead, the software package Arlequin, version 2.0, for population genetic data analysis (Schneider et al., 2000; see http://lgb.unige.ch/arlequin) was used to determine maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data with unknown gametic phase using an expectation–maximization algorithm (Excoffier & Slatkin, 1995). Arlequin was also used to test the significance of the association between pairs of loci (linkage disequilibrium) when the gametic phase is not known and to test the hypothesis that the observed diploid genotypes are the product of a random union of gametes (Hardy–Weinberg equilibrium).
The positions of the polymorphisms were defined and described relative to the open reading frame as suggested in the nomenclature article by Antonarakis et al. (1998). CYP2D6 alleles were described according to the recommended CYP2D6 nomenclature (Daly et al., 1996a; see also http://www.imm.ki.se/CYPalleles/cyp2d6.htm).
A two-stage screening was used in this study to detect and analyse CYP2D variants of putative relevance to the ultrarapid metabolizer phenotype. DNA from 104 duplication-negative subjects phenotyped as extensive metabolizer or ultrarapid metabolizer, and homozygous or compound heterozygous for functional CYP2D6 alleles (CYP2D6*1 and *2) were collected. Initially, 17 samples (13 ultrarapid metabolizers and four extensive metabolizers) were randomly selected from the 104 DNA samples and subjected to polymorphism scanning by automated DNA sequencing. In the second stage, all 104 subjects were genotyped for selected variants from stage one, and statistical analyses of allele, genotype and estimated haplotype distributions were performed.
Analysis of the 5′-end of the CYP2D7 pseudogene
CYP2D7 has an insertion of a single T at position 137 in the first exon, leading to a disrupted reading frame, which classifies it as a pseudogene (Kimura et al., 1989). In light of the many gene-conversions and cross-over events that probably have occurred in the CYP2D locus, it is conceivable that the CYP2D7 exon 1 region could have converted back to the original CYP2D6 sequence, thereby giving rise to a full length CYP2D7 (CYP2D6-like) protein. We therefore amplified CYP2D7 exon 1 with flanking intronic sequences by a nested PCR reaction from the 17 selected subjects. Comparison of the ABI-sequence traces from these subjects revealed that all were homozygous for the T insertion in position 137, causing a frameshift and a subsequent stop codon in the mRNA. Three polymorphisms were detected in the 5′-end of the CYP2D7 pseudogene: A G > A transition was observed in 1 of 34 chromosomes in position −138 (counted from the A in the ATG start codon), a T > C transition was observed in 17 of 34 chromosomes in position −86, and a C > G transversion was observed in one of 34 chromosomes in position 109.
Analysis of the CYP2D6 promoter region
The 5′-end of CYP2D6 (approximately 2 kb putative promoter sequence plus exon 1 to intron 2, see Fig. 1b) was amplified and completely sequenced in the 17 subjects selected for the initial analysis. Compared with the previously published sequence of the CYP2D6 promoter region (Kimura et al., 1989; Genbank acc. no. M33388) we observed in all subjects five discrepancies consisting of five single nucleotide insertions, which most likely are due to sequencing errors in the original sequence. In addition, 29 different DNA variants were detected, resulting in a DNA variation present on average every 110 bp (Fig. 1b). The nature and position of the polymorphisms are described in Table 2 and Fig. 1. Thirteen variants were found in the promoter region, 12 in intron sequences and the remaining four in exon sequences. The three variants found in exon 1 predict amino acid changes, V11M (31G > A), R26H (77G > A) and P34S (100C > T), while the variant found in exon 2 is silent. All the exon SNPs and almost all the SNPs found in intron 1 have been identified and described previously (see Table 2). Fifteen of the polymorphisms were detected in three or more of the 34 chromosomes studied.
Selection of variants for further analysis
We investigated if any of the DNA variants detected could contribute to the observed ultrarapid metabolizer phenotype. Because all samples were sequenced on diploid DNA the genotype data obtained were unphased. However, manual distribution analysis of 13 of the most common variants found in the 17 selected subjects indicated that several of the polymorphisms were in allelic disequilibrium as complete homozygosity at all sites allowed unambiguous assignment of a haplotype and some variants always occurred together in the 17 subjects. The population genetic software package Arlequin was used to estimate the most likely haplotypes. From the 17 subjects with 34 potentially different haplotypes, the software suggested the presence of six haplotypes: three major haplotypes with frequencies 50%, 21% and 14%, and three less frequent haplotypes of 6%, 6% and 3% (data not shown). Based on the estimated haplotypes, five of the SNPs (−1770G > A, −1584C > G, 31G > A, 180 + 34G > C, 180 + 130G > T) representing most of the variation observed were selected for further analysis in the larger material of 104 individuals phenotyped with debrisoquine. To distinguish between the CYP2D6*1 and *2 alleles, we also genotyped all subjects for the 2850C > T SNP known to be present on the CYP2D6*2 allele (Johansson et al., 1993).
Frequency distribution of selected variants in subjects with the ultrarapid metabolizer or extensive metabolizer phenotype
The genotype distributions of the five SNPs (−1770G > A, −1584C > G, 31G > A, 180 + 34G > C, 180 + 130G > T) and the *1/*2-distinguishing polymorphism 2850C > T were determined by automatic sequencing and RFLP analysis of the DNA from 104 subjects. All subjects were previously phenotyped for debrisoquine, genotyped for known CYP2D6 mutations and known to be homozygous wild-type with regard to the Xba I 29 kb allele. We grouped samples having MR < 0.20 as ultrarapid metabolizers (n = 27) and samples with MR 0.20–5 as extensive metabolizers (n = 77). The allele and genotype distributions are given in Table 3. The genotype distributions of the SNPs were in accordance with a Hardy–Weinberg equilibrium, both in the total sample, and when split into ultrarapid metabolizer and extensive metabolizer groups (data not shown). The 31A allele (Met-11 protein variant) was significantly more frequent among ultrarapid metabolizer subjects, compared with extensive metabolizer subjects [14.8% versus 5.8%;P = 0.047 by two-sided Fisher exact probability test; odds ratio (OR) 2.8, 95% confidence interval (CI) 1.0–7.7]. Also, the genotype distribution of the 31G > A variant (all heterozygous) was significantly different in the ultrarapid metabolizer versus extensive metabolizer group (P = 0.039 by two-sided Fisher exact probability test; OR 3.2, 95% CI 1.1–9.4). The allele and genotype distributions of the five remaining SNPs were not significantly different in the ultrarapid metabolizer and extensive metabolizer groups, although the −1584G allele showed a trend towards over-representation in the ultrarapid metabolizer group compared with the extensive metabolizer group (36.5% versus 23%;P = 0.07 by two-sided Fisher exact probability test).
When the MR values were correlated with the various genotypes for the six SNPs (Table 4), we found that the subjects heterozygous for the 31G > A polymorphism (n = 17) had significantly lower MR values (median MR = 0.23, range 0.06–4.04;P = 0.02 by Mann–Whitney U-test) compared with the subjects homozygous for the 31G allele (n = 87, median MR = 0.39, range 0.02–4.15). With respect to the −1584C > G promoter polymorphism, of which the G-allele was non-significantly more frequent in the ultrarapid metabolizer group, subjects with the C/C (n = 56), C/G (n = 35) and G/G (n = 9) genotypes had median MR values of 0.43 (range 0.02–4.10), 0.30 (range 0.05–4.04) and 0.32 (range 0.07–4.15), respectively, and this difference was of borderline significance (P = 0.05 by Kruskal–Wallis H-test).
Haplotype analysis of the six SNPs
Of the 104 subjects included in the genotyping part of the study, four failed to give genotype data for all six SNPs examined. The genotype data from the remaining 100 individuals were analysed using the population genetic software package Arlequin to estimate the haplotypes based on the six SNPs. Eleven different haplotypes were suggested, 10 in the extensive metabolizer group and six in the ultrarapid metabolizer group (Table 5). While five of the SNPs were present on several of the haplotypes, the 31G > A SNP was mainly found on haplotype 11 in allelic disequilibrium with the other five SNPs. The overall distribution of haplotypes in the two groups was significantly different (P = 0.007 by two-sided Fisher-Freeman-Halton exact test). There were mainly two haplotypes contributing to the differences observed in the two groups: haplotype no. 7 constituted approximately 13.5% of the haplotypes in the extensive metabolizer group, but was not present in the ultrarapid metabolizer group (20/148 versus 0/52, P = 0.002 by two-sided Fisher exact test), whereas haplotype no. 11 constituted approximately 4.7% of the haplotypes in the extensive metabolizer group, but was present in 15.4% of the haplotypes in the ultrarapid metabolizer group (7/148 versus 8/52, P = 0.03 by two-sided Fisher exact test).
Subjects of African-American and North African origin (n = 11) present in our material could possibly possess haplotypes that might cause problems in the haplotype analysis. However, when these subjects were excluded from the statistical analyses, the results remained essentially the same (data not shown).
This study was undertaken to examine parts of the CYP2D locus for polymorphisms potentially related to the ultrarapid metabolizer phenotype in CYP2D6 duplication negative subjects. One speculative, but intriguing possibility could be the presence of an active CYP2D7 gene in some individuals. While CYP2D8 is definitely a pseudogene, containing several frame-disrupting mutations, CYP2D7 has only a single T insertion at position 137 in the first exon that leads to a disrupted reading frame (Kimura et al., 1989). Given the variety of gene-conversions and cross-over events reported in the CYP2D locus (Panserat et al., 1995;Steen et al., 1995;Daly et al., 1996b;Sachse et al., 1996;Lundqvist et al., 1999), there is a possibility that the CYP2D7 exon 1 region could have converted back to the original CYP2D6 sequence, resulting in a full-length CYP2D7 protein. In fact, Sachse et al. (1996) described an ‘inverse’ variant of such a gene-conversion. They detected a rare allele having a T insertion in position 137 in the CYP2D6 exon 1, and after analysis of the surrounding region hypothesized that this mutation most likely was caused by a gene-conversion event with the CYP2D7 pseudogene. Our initial hypothesis was therefore that expression of active enzyme from CYP2D7 could have a similar effect as a CYP2D6 duplication and explain the ultrarapid metabolizer phenotype. However, we found a T insertion in position 137 in CYP2D7 in all subjects examined, and concluded that in subjects from our sample the ultrarapid metabolizer phenotype is not caused by an additional functional enzyme from CYP2D7.
We therefore considered explanations directly related to CYP2D6 for the ultrarapid metabolizer phenotype in duplication negative subjects. First, DNA variants in the CYP2D6 coding sequence may cause amino acid changes which make the enzyme more effective. Second, polymorphisms in the CYP2D6 gene and nearby regions could affect mRNA processing and stability. Finally, sequence variants altering transcription factor binding sites in the CYP2D6 promoter region could have an effect on the expression level of the mRNA, explaining the ultrarapid metabolizer phenotype by over-expression of the enzyme.
By screening 17 subjects, we have identified 29 different variants in the 5′-end of the CYP2D6 locus (Table 2), several of which have not been previously described. Thirteen of the variants were detected in the promoter region. Using promoter deletion constructs, Johansson et al. (1994) and Cairns et al. (1996) examined parts of the CYP2D6 promoter for regulatory elements, and found both positively and negatively acting transcription factor binding sites. However, it is unclear if any of the polymorphisms observed in the present study are located in the regulatory elements or regions described in these studies. The four variants detected in CYP2D6 exon 1 and 2 (31G > A predicting Val11Met, 77G > A predicting Arg26His, 100C > T predicting Pro34Ser, and the silent 336C > T polymorphism) have all been described previously (see Table 2 for further references). The remaining 12 variants that we identified were located in intron sequences, 11 in intron 1 and one in intron 2. Six of the variants in intron 1 are the same as those reported by Johansson et al. (1993) to be present on the CYP2D6L allele (CYP2D6*2).
We found the A-allele of the 31G > A (Val11Met) polymorphism to be present at a significantly higher frequency in the ultrarapid metabolizer group compared with the extensive metabolizer subjects. Also, the average MR was significantly lower in subjects possessing the 31A allele compared with subjects homozygous for the wild-type allele. Even though these differences are of borderline significance and not corrected for multiple testing, the results indicate that the 31G > A variant could play a role in determining the ultrarapid metabolizer phenotype. In addition, estimated haplotype analysis of the genotype data shows that haplotype no. 11 with the 31A variant is significantly more frequent among the ultrarapid metabolizers compared with the extensive metabolizers. Furthermore, haplotype no. 7 seems to be associated with higher MR values as this haplotype is significantly more frequent in the extensive metabolizer group compared with the ultrarapid metabolizer group. Since the 0.20 limit value for ultrarapid metabolizer is arbitrarily defined, it should be noted that some studies refer to MR values below 0.15 to describe ultrarapid metabolizers (Johansson et al., 1996). When we regrouped our subjects with MR < 0.15 as criterion for inclusion in the ultrarapid metabolizer group, the 11 estimated haplotypes were distributed with 11 haplotypes in the extensive metabolizer group (n = 83) and four in the ultrarapid metabolizer group (n = 17) (data not shown). Haplotype no. 11 was the main haplotype contributing to the differences observed in the two groups, being almost six times more frequent in the ultrarapid metabolizer versus extensive metabolizer group. This estimated haplotype, having the 31G > A polymorphism, constituted about 4.2% of the haplotypes in the extensive metabolizer group and 23.5% of the haplotypes in the ultrarapid metabolizer group (7/166 versus 8/34, P = 0.0009 by two-sided Fisher exact test, odds ratio 7.0, 95% CI 2.3–20.9).
The 31G > A polymorphism causes a valine to methionine change in codon 11 in exon 1 of the CYP2D6 gene. This polymorphism was first described as CYP2D6*2B by Marez et al. (1997), who found 31G > A to be in allelic disequilibrium with the mutations constituting the CYP2D6*2 allele. Griese et al. (1998) and Legrand-Andreoletti et al. (1998) have also both detected and described this SNP with allele frequencies of 3.6% and 7.7%, respectively, in the general population. Their studies show that the 31A allele is found mainly in subjects having low MR values, but it is not possible from the literature to analyse if this SNP is associated with the ultrarapid metabolizer phenotype. According to the CYP2D6 nomenclature (http://www.imm.ki.se/CYPalleles/cyp2d6.htm) CYP2D6*35 is the new designation for the allele having the 31G > A variant present together with the other CYP2D6*2 allele-associated polymorphisms. There are as far as we know, no studies examining functional consequences of the CYP2D6*35 allele on the mRNA level or enzyme activity. Marez et al. (1997) suggested that the 31G > A polymorphism had no functional consequences, although in their study, the four subjects positive for this allele had metabolic ratios which were among the lowest seen in the extensive metabolizer groups for both substrates used in the phenotyping (sparteine or dextromethorphan).
Our results also indicate a functional role of the −1584 C > G promoter polymorphism, as there is a non-significant over-representation of the G-allele in the ultrarapid metabolizer group, as well as a possible correlation between low MR values and G-allele-containing genotypes. In addition, we note that the −1584 C > G SNP is located within an Alu repeat which could have functional relevance.
In conclusion, we have found preliminary evidence for a minor role of the 31G > A (Val11Met) variant and a −1584 C > G promoter polymorphism in determining the ultrarapid metabolizer phenotype. Since the results are based on a relatively low number of subjects and not adjusted for multiple testing, we cannot exclude the possibility that our findings could be due to spurious accumulation of some alleles in subjects with low MR values. Further studies on larger samples and functional analysis of the polymorphisms are therefore necessary to determine the role of these two SNPs in ultrarapid metabolizer subjects.
The work is supported by Dr Einar Martens’ Research Fund and the Research Council of Norway (NFR, Mental Health Program).
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Keywords:© 2001 Lippincott Williams & Wilkins, Inc.
CYP2D6; genotyping; debrisoquine 4-hydroxylase; ultrarapid metabolizer; cytochrome P450