Abstract
Genetic variation in CYP3A activity may influence the rate of the metabolism and elimination of CYP3A substrates in humans. We previously reported four new CYP3A4 coding variants in three different racial groups. In the present study, we examined metabolism of nifedipine by the recombinant forms of these allelic variants. Metabolism of nifedipine by the L293P (CYP3A4*18), M445T (CYP3A4*3), and P467S (CYP3A4*19) allelic variants was not significantly different from wild-type CYP3A4*1. However, F189S (CYP3A4*17) exhibited a >99% decrease in both Vmax and CLmax of nifedipine compared with CYP3A4*1. Of 72 racially diverse individuals, CYP3A4*17 was identified in 1 of 24 Caucasian samples [1:5 Eastern European (Adygei ethnic group)]. Genotyping of an extended set of 276 genomic DNAs of Caucasians (100 from the Coriell Repository and an additional 176 from the United States) for CYP3A4*17 detected no additional individuals containing the CYP3A4*17 allele. However, additional genotyping of four more Adygei samples available from Coriell detected an additional individual carrying the CYP3A4*17 allele. New specific polymerase chain reaction-restriction fragment length polymorphism genotyping procedures were developed for the major splice variant of CYP3A5 (CYP3A5*3) and CYP3A4*17. Genotyping revealed that the two individuals carrying CYP3A4*17 were either homozygous or heterozygous for the more frequent CYP3A5*3 allele, suggesting that the two alleles may exist on the same chromosome as a new putative CYP3A poor metabolizer haplotype. We predict that individuals who are homozygous for defective alleles of both of these genes would metabolize CYP3A substrates poorly. The new genetic tests will be useful in future clinical studies to investigate genotype/phenotype associations.
The CYP3A subfamily of cytochrome P450 enzymes comprises approximately 30–60% of total cytochrome P450 in human liver (Shimada et al., 1994). The CYP3A enzymes are responsible for the metabolism of approximately half of all clinical drugs, steroids, and environmental chemicals (Li et al., 1995; Evans and Relling, 1999). Among the four human CYP3A genes, CYP3A4 is generally believed to be the most abundant in liver (Wrighton et al., 1990; Shimada et al., 1994; Hustert et al., 2001; Koch et al., 2002) and to metabolize the majority of clinically used drugs. CYP3A4 catalytic activity can be modulated by transcriptional induction by rifampin, phenobarbital, and the herbal antidepressant St. John's Wort (Lucey et al., 1990; Gibson et al., 2002) resulting in decreased drug concentrations in plasma. CYP3A4 activity can also be inhibited by ketoconazole, erythromycin, a component in grapefruit juice, and other compounds (Fukuda et al., 1997; Thummel and Wilkinson, 1998). A third possible source of variability is from genetic variations in CYP3A4 and CYP3A5. Current literature indicates that the major cause of variation in CYP3A5 activity is due to the frequency of the CYP3A5*3 splice variant (Kuehl et al., 2001) which affects protein expression in liver and other tissues. However, CYP3A4 and CYP3A5 have overlapping substrate specificity (Shimada et al., 1994; Wacher et al., 1995; Thummel and Wilkinson, 1998; Kuehl et al., 2001; Williams et al., 2002). Thus, active CYP3A4 largely compensates for defective CYP3A5 metabolism and vice versa. Therefore, the concomitant presence of defective alleles of both CYP3A4 and CYP3A5 would be much more likely to affect metabolism of CYP3A substrates in vivo than mutations in a single CYP3A gene.
Recently, we reported new coding alleles, CYP3A4*3 (M445T), CYP3A4*15 (R162Q), CYP3A4*17 (F189S), CYP3A4*18 (L293P), and CYP3A4*19 (P467S), after resequencing CYP3A4 in 72 racially diverse individuals (Dai et al., 2001). CYP3A5 was later resequenced in the same individuals (Lee et al., 2003). We originally expressed the coding variants of CYP3A4 in Escherichia coli, and preliminary studies compared the ability of these allelic variants to metabolize the hormone testosterone and the insecticide chlorpyrifos at only one substrate concentration due to its low activity in recombinant systems in vitro (Dai et al., 2001). CYP3A4*17 exhibited lower catalytic activity compared with wild type for both testosterone and chlorpyrifos (an organophosphate insecticide) metabolism, whereas CYP3A4*18 exhibited higher activity compared with wild type (Dai et al., 2001). CYP3A4 has multiple substrate binding sites, therefore, substrates may be metabolized differently (Gillam et al., 1995; Domanski et al., 1998; Wang et al., 1998; Williams et al., 2002). In the present studies, we examine the effect of the coding alleles of CYP3A4 on the metabolism and kinetics of the antihypertension drug nifedipine and provide strong evidence that CYP3A4*17 is a putative defective allele, with a >99% decrease in catalytic activity. Sequence analysis of CYP3A4 and CYP3A5, performed on the same 72 individuals from our two previous studies (Dai et al., 2001; Lee et al., 2003), showed that the CYP3A4*17 allele was present in a single Eastern European (Adygei, one of five individuals) who was homozygous for the CYP3A5*3 allele, suggesting these alleles may occur together on one haplotype. In this study, we have developed a genetic test for the CYP3A4*17 allele which may be useful in future clinical studies. Moreover, a previously published PCR-RFLP test for CYP3A5*3 was found to be unreliable in our hands because of a similarity in the primer sequences with multiple CYP3A genes (Fukuen et al., 2002; Balram et al., 2003). Therefore, we developed a new, more specific genotyping method for CYP3A5*3. In an attempt to verify whether CYP3A4*17 and CYP3A5*3 occur on a single haplotype in some Caucasians, DNA from two additional groups of Caucasians totaling 276 individuals was analyzed for the two alleles. In addition, DNA from nine Adygei, a Caucasian ethnic minority from Eastern Europe, was also analyzed. The CYP3A4*17 and CYP3A5*3 alleles were found together in two of nine Adygei, suggesting that these two alleles occur together as part of a defective CYP3A haplotype in certain Caucasian ethnic groups. Individuals homozygous (or heterozygous) for the defective CYP3A4*17/CYP3A5*3 haplotype would be predicted to metabolize CYP3A substrates more poorly than individuals carrying only a defective allele in one CYP3A gene.
Materials and Methods
Nifedipine, NADPH, δ-aminolevulinic acid, ampicillin, sodium cholate, l-α-dilauroyl-sn-glycero-3-phosphocholine and l-α-dioleoylsn-glycero-3-phosphocholine, bovine brain phosphatidylserine, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and lysozyme were purchased from Sigma-Aldrich (St. Louis, MO). E. coli DH5α competent cells, agarose-1000, and isopropyl β-d-thiogalactopyranoside were purchased from Invitrogen (Carlsbad, CA). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA). Oligonucleotide primers were obtained from Sigma-Genosys (The Woodlands, TX). Quick-change mutagenesis kit and proof-reading Pfu DNA polymerase were from Stratagene (La Jolla, CA). Ni-NTA affinity column was from QIAGEN (Valencia, CA). Imidazole was purchased from Calbiochem (San Diego, CA). Human NADPH-cytochrome P450 oxidoreductase (P450 reductase) and cytochrome b5 were purchased from Oxford Biomedical Research (Oxford, MI). All other chemicals and organic solvents for high-performance liquid chromatography were of the highest grade from commercial sources.
DNA Samples. Genomic DNAs from 72 racially diverse individuals for our earlier resequencing studies of CYP3A4 and CYP3A5 were obtained from lymphoblastoid cell lines selected from the Human Genetic Cell Repository sponsored by the National Institutes of Health housed at the Coriell Institute, Camden, NJ (Dai et al., 2001; Lee et al., 2003). From the present study, a second Human Variation Panel containing 100 Caucasian genomic DNAs and a third Human Variation Panel representing nine European Caucasians belonging to the Adygei ethnic minority were purchased from Coriell Institute, Camden, NJ. An additional set of 176 genomic DNAs were obtained from a community-based population of healthy, unrelated Caucasian volunteers from Durham and Chapel Hill, North Carolina. This study was approved by the National Institute of Environmental Health Sciences Institutional Review Board.
Construction of CYP3A4 Single Nucleotide Polymorphism cDNAs.CYP3A4 variant alleles constructed in pCW (Dai et al., 2001) were used for PCR templates to incorporate additional modifications. The N-terminal of CYP3A4*1 cDNA had been previously modified to MALLLAVF as described for bovine 17α-hydroxylase (Barnes et al., 1991). In the present study, an additional PCR was performed to add a 5× His tag to the C-terminal region of CYP3A4*1 to simplify purification. PCR primers included a forward primer, 5′-GGTGGTCATATGGCTCTGTTATTAGCAGTTTTTCTGGTGCTCCTCTATC-3′ and a reverse primer, 5′-GTGGCCTCTAGATCAGTGA TGGTGATGGTGGGCTCCACTTACGGTGCC-3′. The modified CYP3A4 cDNAs were recloned into the NdeI and XbaI sites of pCW. After construction of mutants, the entire single nucleotide polymorphism constructs were analyzed by sequencing in both directions before expression.
Expression and Purification of CYP3A4s. CYP3A4 wild-type and variant proteins were expressed in E. coli DH5α cells. Expression and purification of P450 proteins were performed as previously described (Lee et al., 2003). Optimal expression conditions for CYP3A4*1 were determined to be 0.5 mM isopropyl β-d-thiogalactopyranoside and 0.5 mM δ-aminolevulinic acid in Terrific broth for 72 h at 23°C with gentle shaking at 150 rpm. Cytochrome P450 expression was monitored by CO difference spectra measured using a DW-2000/OLIS spectrophotometer (Omura and Sato, 1964). Maximal expression varied from ∼200 nmol/l for wild type to 80 to 100 nmol/0.5 liter of culture for most mutant alleles. CYP3A4*17 exhibited low expression of ∼40 nmol. To minimize interexperimental variations in expression and purification, all five CYP3A4 cDNA constructs were simultaneously expressed, harvested, and purified under the same conditions. After solubilization of the membrane fraction, P450 was purified by a Ni-NTA affinity column utilizing the histidine tag. The eluted P450 was quantitated and diluted to 1 nmol of P450 per milliliter to avoid aggregation and precipitation during dialysis. It was then dialyzed twice for 48 h in two changes of dialysis buffer (100 mM potassium phosphate, pH 7.4, and 20% glycerol). A second set of all five purified proteins was generated for the purpose of comparison. Purification of P450s by Ni-NTA affinity column chromatography resulted in approximately 40% recovery.
Reconstitution and Nifedipine Oxidation. CYP3A4 (10 pmol), human reductase (40 pmol), cytochrome b5 (20 pmol), 0.05 μmol of sodium cholate, and 2 μg of lipid mix (l-α-dilauroyl-sn-glycero-3-phosphocholines and brain phosphatidyl serine) were preincubated at room temperature for 10 min and then 50 mM potassium buffer, pH 7.7 added to a final volume of 0.1 ml (Lee et al., 2003). The reaction was initiated with 1 mM NADPH and incubated at 37°C for 10 min. Nifedipine was protected from light during the experiment. Nifedipine concentrations for the kinetic analysis were 6.25, 12.5, 25, 50, 100, 200, and 400 μM. There was no catalytic activity in the absence of NADPH. The oxidized metabolite was analyzed by high-performance liquid chromatography with a Phenomenex Luna C18 column (5 μm, 250 × 4.6 mm; ANSYS Technologies, Inc., Lake Forest, CA) as described earlier (Lee et al., 2003).
Development of Genotyping Methods. Genomic DNA sequences for CYP3A4, CYP3A5, CYP3A7, and CYP3A43 were obtained from GenBank (accession number NG_000004.1). Before sequence alignment, all DNA sequences of CYP3A43 were changed to reverse complementary sequences using Vector NTI 8.0 due to its reverse direction of transcription. Sequence alignment was performed using BOXSHADE software version 3.21 in BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and analyzed to design a specific primer. Specific genotyping primers for the detection of CYP3A4*17 and CYP3A5*3 are described in Table 1. Detection of defective alleles was performed by PCR and restriction enzyme analysis. Genomic DNA (40 ng) was amplified by PCR containing 0.4 μM primers, 0.4 mM of each dNTP, and 2.5 units of AmpliTaq Gold (PerkinElmer Life and Analytical Sciences, Boston, MA) in 25-μl reactions. Amplification was performed for 38 cycles consisting of denaturation at 94°C for 30 s, annealing at 56°C for 20 s, and extension at 72°C for 30 s (CYP3A4*17) or 20 s (CYP3A5*3). An initial denaturation step at 95°C for 10 min and a final extension step at 72°C for 5 min were performed. Both PCR products were purified by QIAquick PCR Purification Kit (QIAGEN) and directly sequenced to check the specific amplification of the target DNA. Five-microliter aliquots of PCR product for CYP3A4*17 and CYP3A5*3 were directly digested with BpmI and BseMII (Fermentas Inc., Hanover, MD), respectively. Since we did not have DNA from an individual homozygous for CYP3A4*17, the efficiency of BpmI digestion in PCR buffer with additional 1× restriction enzyme buffer was examined using a PCR2.1 TOPO plasmid construct containing a PCR-amplified CYP3A4*17 fragment (using genotyping primers) as a positive control. Digested PCR products were analyzed on 3% agarose gels.
Analysis of Enzyme Kinetics. Nifedipine oxidation was analyzed by nonlinear regression using SigmaPlot 2001 (SPSS Inc., Chicago, IL) as described earlier (He et al., 2003; Lee et al., 2003). The goodness of fit was determined by the distribution of residual patterns, the residual sums of squares, and the standard error of the parameter estimates. Kinetics for nifedipine metabolism exhibited the best fit with the Hill equation as reported previously (Williams et al., 2002). The Hill eq. 1 was used to determine Vmax, S50, and the Hill coefficient (nH). CLmax, the maximum clearance resulting from positive cooperativity, was determined using eq. 2 (He et al., 2003; Lee et al., 2003).
Statistical Analysis. Two independently purified P450s for each allele were assayed in triplicate. The means of the triplicate assays were compared with the mean of the triplicate wild-type purified P450 using Dunnett's test. The assay was repeated on two separate days. A P value of <0.05 was considered statistically significant.
Results
The CYP3A4*17 Allele Is Defective in the Metabolism of Nifedipine. Purified CYP3A4*17 contained some denatured cytochrome P420 (Fig. 1), but none of the other variants contained P420 after purification. However, metabolic assays were normalized to P450 content and results should not be affected by the small amount of denatured P420. Since kinetic analysis requires large amounts of enzyme, we first compared the statistical differences in turnover numbers of CYP3A4 variants by assaying equivalent picomoles of each allelic variant in triplicate at two different concentrations of substrate: 30 μM which approximates the S50 value of wildtype CYP3A4 as determined in our laboratory and a high concentration of 200 μM which approaches the Vmax of the enzyme. Figure 2 shows the representative results of one of two independent sets of assays. CYP3A4*17 significantly decreased catalytic activity for nifedipine oxidation by 98 to 99% (P < 0.05) at the high concentration of nifedipine in both experiments. At the low substrate concentration, nifedipine oxidation by CYP3A4*17 was below detection levels. There was no statistically significant difference in nifedipine oxidation by the other recombinant allelic variants. Figure 3 and Table 2 compare kinetic parameters for nifedipine oxidation by the CYP3A4 recombinant mutant allelic proteins with that of wild type. Kinetic parameters for nifedipine metabolism by CYP3A4*1, CYP3A4*18, CYP3A4*3, and CYP3A4*19 allelic proteins were similar; however, CYP3A4*17 protein exhibited a 99.2 and 99.6% decrease in the Vmax and maximal clearance, respectively, for nifedipine compared with wild type.
New Genetic Test for CYP3A4*17. A PCR-RFLP genotyping test was developed to detect CYP3A4*17. Since there are four structurally similar CYP3A genes arranged in tandem comprising 230 kb on chromosome seven, PCR primers were designed to specifically amplify a 290-bp DNA fragment containing only CYP3A4 (Fig. 4C, lane 5). The T>C mutation present in CYP3A4*17 introduced a BpmI site (Fig. 4A) which was not present in wild-type CYP3A4. Sequencing of the amplified PCR product verified the specific amplification of CYP3A4. The DNA sample heterozygous for CYP3A4*17 produced bands at 290, 153, and 137 bp when digested with BpmI in PCR buffer (Fig. 4C, lane 3). Complete cutting of the BpmI site was observed in the presence of PCR buffer with additional 1× restriction enzyme buffer in the positive control of pCR2.1 TOPO plasmid construct containing a PCR-amplified CYP3A4*17 fragment (Fig. 4C, lane 2).
New CYP3A5*3 Genotyping Test. Initial trials of a PCR-RFLP for CYP3A5*3 based on literature methods (Fukuen et al., 2002; Balram et al., 2003) resulted in complex profiles due to incomplete digestion with DdeI (data not shown). Intron 3 sequences from CYP3A5, CYP3A4, CYP3A7, and CYP3A43 containing the corresponding CYP3A5*3 sequence were obtained from GenBank (accession number NG_000004.1) and aligned using Vector NTI 8.0. As shown in Fig. 5, there is a strong likelihood of nonspecific amplification of other CYP3A forms when using the published primers, since the reverse primer (shown with dashed box) does not differentiate between CYP3A4, CYP3A5, or CYP3A7 and the forward primer contains only one mismatch four bases from the 5′ end. In the present study, a new more specific PCR-RFLP for CYP3A5*3 was designed to differentiate the A>G change in genomic DNA samples. A mismatched nucleotide in a forward primer four bases from the 3′ end was used to introduce a BseMII recognition site specific for CYP3A5*3. DNA sequencing of the PCR product for CYP3A5*3 from the new pair of primers confirmed the specific amplification of CYP3A5. These primers produced a single band at 197 bp without nonspecific bands (Fig. 6C, lane 5). A DNA sample known to be heterozygous for CYP3A5*3 produced two fragments of 197 and 162 bp (Fig. 6C, lane 3). The DNA sample containing CYP3A5*1/*1 remained undigested by BseMII as shown in Fig. 6C, lane 4.
The Frequency of CYP3A4*17 and CYP3A5*3 in Caucasians. Only one individual of 72 from our original sequencing analysis (Dai et al., 2001) was found to carry the CYP3A4*17 allele (1:24 Caucasians), and this individual was also homozygous for CYP3A5*3 (Lee et al., 2003). This individual was of Eastern European descent. In the present study, further genotyping using a set of 100 Caucasian DNA samples and 176 North American DNA samples revealed no additional individuals carrying the CYP3A4*17 allele (0:552 alleles) (Table 3). In contrast, the frequency of the CYP3A5*3 allele was 94.2 ± 1.6 and 91.2 ± 1.8% (± standard error), respectively, in these two populations. Since the original finding of CYP3A4*17 was from an Adygei individual from the Coriell set, a total of nine DNA samples from the Adygei ethnic group available from Coriell was purchased and analyzed. Our PCR-RFLP test showed two of nine Adygei individuals carrying the CYP3A4*17 allele (11% frequency), including the one first identified. Of two individuals carrying the CYP3A4*17 allele, one was homozygous for the CYP3A5*3 allele and the other was heterozygous for CYP3A5*3. Five of the nine individuals were homozygous for CYP3A5*3 and the other four were heterozygous for this allele (14:18 alleles) (78% frequency), further suggesting that the CYP3A4*17 and more frequent CYP3A5*3 splice variant may be present on the same chromosome representing a defective CYP3A haplotype.
Discussion
It is well known that the metabolism of CYP3A substrates varies by 40 to 60% in vivo (Wrighton et al., 1990; Shimada et al., 1994; Hustert et al., 2001; Kuehl et al., 2001; Koch et al., 2002), but there is limited evidence linking individual underlying genetic mutations to variations in metabolism. CYP3A5*3, a splice variant responsible for decreased hepatic expression of CYP3A5 protein in some individuals, occurs with high frequency in many populations including Caucasians (Kuehl et al., 2001). A higher oral tacrolimus clearance has been found in patients with a CYP3A5*1 allele compared with homozygous CYP3A5*3 (Hesselink et al., 2003; Thervet et al., 2003; Zheng et al., 2003). Several studies have identified protein-coding variants of CYP3A4. Recombinant CYP3A4*2 (S222P) exhibits a lower Vmax for nifedipine metabolism (∼36% decrease) but not for that of testosterone when compared with CYP3A4 wild type (Sata et al., 2000). CYP3A4*4 (I118V), CYP3A4*5 (P218R), and CYP3A4*6 (a premature stop codon) were found in a Chinese population; the metabolite ratio of 6β-hydroxycortisol to free cortisol in the urine of mutant subjects was higher than those of wildtype subjects (Hsieh et al., 2001). However, the overlapping substrate specificity of CYP3A4 and CYP3A5 generally appears to partially mask phenotypic expression of a defect in only one gene.
The present study shows that recombinant CYP3A4*17 is markedly defective in nifedipine metabolism in vitro, exhibiting a >99% decrease in the Vmax and CLmax. In a previous study, CYP3A4*17 (with 2% allelic frequency in Caucasians) exhibited lower activity for both testosterone (70% decrease) and the insecticide chlorpyrifos than wild type (Dai et al., 2001). However, this study used a single 14C-testosterone concentration of 25 μM, which is below the Km and may have underestimated the magnitude of the defect. The complete kinetic analysis in the present study demonstrates more conclusively that the CYP3A4*17 allele is markedly defective. Moreover, the possible association of this allele on a single haplotype with the defective CYP3A5*3 splice variant in two individuals from Eastern Europe (Adygei) is reported in this study. This haplotype could result in defective metabolism of many CYP3A substrates including drugs and environmental chemicals such as organophosphate insecticides in vivo, since CYP3A5 and CPY3A4 would not compensate for each other. CYP3A4 and CYP3A5 are important in the metabolism of many insecticides including organophosphate insecticides such as chlorpyrifos, parathion, diazinon, and phorate (reviewed in Hodgson, 2003).
The mechanism of the effect of the F189S coding change on metabolism of the CYP3A4 substrates is not known. Examination of the crystal structure of human CYP3A4 (Yano et al., 2004) indicates that this amino acid is not located in the active site cavity of CYP3A4. It is located at a turn at the C-terminal end of the E helix and appears to be involved in packing with residues on helices G and H and the turn between helices G and H. The alignment of this amino acid with other CYP3As in different species indicates that the phenylalanine is conserved across species (data not shown). Its involvement with packing could result in influences on other parts of the protein, causing conformational changes that affect structure and indirectly affect activity.
The CYP3A4*3 (4% allelic frequency in Caucasians) and the CYP3A4*19 allelic proteins (2% frequency in Asians) exhibit normal metabolism of nifedipine, whereas recombinant CYP3A4*18 (2% allelic frequency in Asians) exhibited a 2-fold higher activity toward testosterone and chlorpyrifos in an earlier study (Dai et al., 2001). However, this variant showed no significant change in metabolism of nifedipine compared with wild type in the present study. Although this difference could reflect the existence of multiple substrate recognition sites on CYP3A4, the preliminary increase in activity reported in the earlier study was based on only one P450 purification and one substrate concentration. Therefore, earlier results may have reflected batch to batch variation in P450 preparations.
Although there are variations in estimates of the relative importance of CYP3A4 and CYP3A5 in the literature (Shimada et al., 1994; Evans and Relling, 1999; Kuehl et al., 2001), these P450s have overlapping substrate specificity (Wrighton et al., 1990; Li et al., 1995; Evans and Relling, 1999; Gibson et al., 2002; Koch et al., 2002). Since most CYP3A substrates are metabolized by both CYP3A4 and CYP3A5 in humans, individuals expressing defective alleles for both CYP3A4 and CYP3A5 would have a decreased drug metabolism for many important clinical drugs and environmental substrates. Therefore, PCR-RFLP genotyping tests were designed for the detection of CYP3A4*17 and CYP3A5*3. In our hands, a currently utilized PCR-RFLP for CYP3A5*3 (Fukuen et al., 2002; Balram et al., 2003) did not distinguish the hetero- and homozygous mutation of CYP3A5*3 due to complete homology of the reverse primer with CYP3A4, CYP3A5, and CYP3A7. We developed a new specific PCR-RFLP genotyping test for the CYP3A5*3 mutation. A second published PCR-RFLP genotyping method for CYP3A5*3 (King et al., 2003) also uses specific primers for CYP3A5 but requires the use of a 10% polyacrylamide gel and a longer duration of electrophoresis (∼16 h) to detect an 8-bp difference in PCR products. The present genetic tests will be useful for future clinical studies to investigate the effects of mutations in CYP3A4*17 and CYP3A5*3 on the metabolism of CYP3A substrates in vivo.
Genotyping of 276 Caucasian DNAs from the United States (North Carolina) and the Coriell repository for CYP3A4*17 revealed no individuals carrying this allele. However, two individuals with the CYP3A4*17 allele were found in nine Caucasians belonging to the Adygei ethnic minority. According to ALFRED Detailed Record information (http://alfred.med.yale.edu/alfred/recordinfo.asp), the Adygei, an ethnic group of the Russian Caucasus, can be traced back to at least the 6th century. Since they were conquered by the Russian Empire in the 19th century, much of the Adygei population was forced to immigrate to Turkey and other Middle Eastern countries (Chang et al., 1996; Popova et al., 2001). Of the original 1.2 million individuals, approximately 800,000 survived the exodus. A few also live in the United States in New Jersey (∼10,000). All of the nine Russian Adygei DNAs contained the CYP3A5*3 splice variant (14:18 alleles) which is known to have a high frequency in Caucasian populations. The CYP3A4*17 occurred at a frequency of 2:18 alleles suggesting a second haplotype containing both CYP3A4*17 and CYP3A5*3 on the same chromosome (one individual was homozygous for CYP3A5*3 and the other individual was heterozygous for CYP3A5*3). Therefore, we predict that a portion of Adygei populations (particularly those homozygous for poor metabolizer alleles of both CYP3A genes) could be at high risk to clinical drugs and to environmental chemicals such as organophosphate insecticides.
The present study describes a putative defective CYP3A4*17 allele was found with a moderate frequency (11%) with the defective CYP3A5*3 splice variant (frequency of 78%) in the Adygei, an ancient Caucasian ethnic group which originated in Russia, but migrated to Turkey, and several other Middle Eastern countries as well as certain regions of the United States and Germany. We predict that the coexistence of this allele in moderately frequent putative defective haplotype (2:18 individuals) with the CYP3A5*3 allele in these populations may result in lower CYP3A activity in vivo. The clinical effects and environmental risk of these two defective alleles should be addressed in future studies in man in the Adygei and related ethnic groups.
Acknowledgments
We thank Joyce A. Blaisdell for helpful discussions on the PCR-RFLP. We thank Drs. Stephen Ferguson and Gary S. Pittman for helpful comments during this project.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.078758.
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ABBREVIATIONS: PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; Ni-NTA, nickel-nitrilotriacetic acid; bp, base pair(s); P450, cytochrome P450.
- Received October 1, 2004.
- Accepted January 4, 2005.
- The American Society for Pharmacology and Experimental Therapeutics