QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.078758v1 313/1/302    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, S.-J. Articles by Goldstein, J. A. Search for Related Content PubMed PubMed Citation Articles by Lee, S.-J. Articles by Goldstein, J. A.

ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Recombinant CYP3A4^*17 Is Defective in Metabolizing the Hypertensive Drug Nifedipine, and the CYP3A4^*17 Allele May Occur on the Same Chromosome as CYP3A5^*3, Representing a New Putative Defective CYP3A Haplotype

Human Metabolism Section, Laboratory of Pharmacology and Chemistry (S.-J.L., S.J.C., J.A.G.), Environmental Genomics Section, Laboratory of Computational Biology and Risk Assessment (D.A.B.) and Metabolism and Exposure Markers Section, Laboratory of Pharmacology and Chemistry (B.G.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Received October 1, 2004; accepted January 4, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
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 V_max and CL_max 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.

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

Top Abstract Materials and Methods Results Discussion References

 
Nifedipine, NADPH, -aminolevulinic acid, ampicillin, sodium cholate, L--dilauroyl-sn-glycero-3-phosphocholine and L--dioleoylsn-glycero-3-phosphocholine, bovine brain phosphatidylserine, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and lysozyme were purchased from Sigma-Aldrich (St. Louis, MO). E. coli DH5 competent cells, agarose-1000, and isopropyl -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 17-hydroxylase (Barnes et al., 1991). In the present study, an additional PCR was performed to add a 5x 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 DH5 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 -D-thiogalactopyranoside and 0.5 mM -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--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 x 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 [GenBank] .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 1x 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 V_max, S₅₀, and the Hill coefficient (n_H). CL_max, the maximum clearance resulting from positive cooperativity, was determined using eq. 2 (He et al., 2003; Lee et al., 2003).

(1)

(2)

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

Top Abstract Materials and Methods Results Discussion References

 
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 V_max 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 V_max and maximal clearance, respectively, for nifedipine compared with wild type.

View larger version (10K): [in this window] [in a new window]   Fig. 1. CO difference spectrum of CYP3A4*1 wild type (A) and CYP3A4*17 allelic protein (B). DH5 E. coli competent cells were transformed with recombinant pCW vector containing each CYP3A4 allele. After 72 h expression, the P450 was purified as described earlier (Lee et al., 2003). The eluted P450 was measured by a DW-2000 spectrophotometer. CYP3A4*1 protein shows a single absorption at 450 nm. The CYP3A4*17 protein exhibited a major peak at 450 nm and a small peak at 420 nm.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Nifedipine metabolism by CYP3A allelic proteins. CYP3A4*1 and variant alleles were expressed in E. coli and purified as described under Materials and Methods. Purified P450 (10 pmol), human reductase (40 pmol), and cytochrome b5 (20 pmol) were reconstituted as described under Materials and Methods. To avoid intra-assay variations, P450s were expressed, purified, and assayed together as sets. Results are representative of two separate experiments with separately purified preparations of allelic proteins (the second set also exhibited the same pattern as shown). Nifedipine metabolism was measured at two concentrations, one near the S50 (30 µM) and the other near the Vmax (200 µM). All values are the mean ± S.E. of triplicate assays. *, significantly lower (P < 0.05) than CYP3A4*1 wild type using Dunnett's test.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Kinetic assessment of nifedipine oxidation by CYP3A4*1 and CYP3A4 allelic proteins. All reactions were performed with 10 pmol of P450, 40 pmol of human reductase, and 20 pmol of cytochrome b5 in 0.1 ml of reaction for 10 min. Details of reaction procedures are described under Materials and Methods. P450 alleles are indicated as CYP3A4*3 (), CYP3A4*18 (), CYP3A4*17 (), CYP3A4*19 (), and CYP3A4*1 (). Values plotted are the means ± S.E. of triplicate experiments, except for the P467S which was the mean of duplicate determinations. Details for kinetic equations are described under Materials and Methods. Data for CYP3A4 F189S is enlarged in the down inset.

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 1x restriction enzyme buffer in the positive control of pCR2.1 TOPO plasmid construct containing a PCR-amplified CYP3A4^*17 fragment (Fig. 4C, lane 2).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Diagram for the detection of CYP3A4*17 in human genomic DNA by PCR amplification of exon 7 followed by BpmI digestion. Detailed procedures are described under Materials and Methods. A, strategy for genotyping CYP3A4*17 by PCR-RFLP. B, the predicted sizes of the digested DNA fragments for the various genotypes. C, the actual sizes of the BpmI-digested PCR products. Lane 1, 100-bp ladder; lane2, CYP3A4*17/*17 plasmid positive control exhibiting two bands after BpmI digestion; lane 3, heterozygous CYP3A4*17/*1 individual exhibiting all three bands after BpmI digestion; lane 4, homozygous CYP3A4*1/*1 individual exhibiting a single band after BpmI digestion; lane 5, PCR product of heterozygous CYP3A4*17/*1 individual without BpmI digestion, showing a specific single band.

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 [GenBank] .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.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Design of CYP3A5*3 genotyping test. Genomic DNA sequences for CYP3A4, CYP3A5, CYP3A7, and CYP3A43 were obtained from GenBank (accession number NG_000004 [GenBank] .1). The DNA sequence for CYP3A43 was from the reverse complementary sequence. The sequence shaded in gray represents nonidentical nucleotides. DNA fragments flanking CYP3A5*3 regions were aligned as described under Materials and Methods. New specific genotyping primers for the detection of CYP3A5*3 are indicated with the black shaded background and solid line arrows. Dashed line arrows and boxes indicate CYP3A5*3 genotyping primers from a test reported in the literature (Fukuen et al., 2002; Balram et al., 2003). The A>G mutation indicated by an asterisk and a mismatched nucleotide (underlined) in the forward primer create a BseMII site. The location of the BseMII cut site is indicated by a vertical arrow.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Diagram for the detection of CYP3A5*3 in human genomic DNA by PCR amplification of intron 3 followed by BseMII digestion. Detailed procedures are described under Materials and Methods. A, strategy for genotyping CYP3A5*3 by PCR-RFLP. B, the predicted sizes of the digested DNA fragments for various genotypes. C, the actual sizes of BseMII-digested PCR products. Lane 1, 100-bp ladder; lane 2, homozygous CYP3A5*3/*3 individual showing one band of 162 bp after BseMII digestion indicating complete digestion (the 35-bp fragment is too small to be seen); lane 3, heterozygous CYP3A4*3/*1 individual showing two bands of 162 and 197 bp after BseMII digestion; lane 4, wild-type DNA after BseMII digestion; lane 5, PCR product of homozygous CYP3A5*3/*3 individual without BseMII digestion, showing an undigested band of 197 bp.

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

Top Abstract Materials and Methods Results Discussion References

 
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 V_max 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 6-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 V_max and CL_max. 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 K_m 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.

	Acknowledgements

 
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

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.078758.

ABBREVIATIONS: PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; Ni-NTA, nickel-nitrilotriacetic acid; bp, base pair(s); P450, cytochrome P450.

Address correspondence to: Dr. Joyce A. Goldstein, Human Metabolism Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. E-mail: goldste1{at}niehs.nih.gov

	References

Top Abstract Materials and Methods Results Discussion References

 

Balram C, Zhou Q, Cheung YB, and Lee EJ (2003) CYP3A5^*3 and ^*6 single nucleotide polymorphisms in three distinct Asian populations. Eur J Clin Pharmacol 59: 123-126.[Medline]

Barnes HJ, Arlotto MP, and Waterman MR (1991) Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli. Proc Natl Acad Sci USA 88: 5597-5601.[Abstract/Free Full Text]

Chang FM, Kidd JR, Livak KJ, Pakstis AJ, and Kidd KK (1996) The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus. Hum Genet 98: 91-101.[CrossRef][Medline]

Dai D, Tang J, Rose R, Hodgson E, Bienstock RJ, Mohrenweiser HW, and Goldstein JA (2001) Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos. J Pharmacol Exp Ther 299: 825-831.[Abstract/Free Full Text]

Domanski TL, Liu J, Harlow GR, and Halpert JR (1998) Analysis of four residues within substrate recognition site 4 of human cytochrome P450 3A4: role in steroid hydroxylase activity and alpha-naphthoflavone stimulation. Arch Biochem Biophys 350: 223-232.[CrossRef][Medline]

Evans WE and Relling MV (1999) Pharmacogenomics: translating functional genomics into rational therapeutics. Science (Wash DC) 286: 487-491.[Abstract/Free Full Text]

Fukuda K, Ohta T, Oshima Y, Ohashi N, Yoshikawa M, and Yamazoe Y (1997) Specific CYP3A4 inhibitors in grapefruit juice: furocoumarin dimers as components of drug interaction. Pharmacogenetics 7: 391-396.[Medline]

Fukuen S, Fukuda T, Maune H, Ikenaga Y, Yamamoto I, Inaba T, and Azuma J (2002) Novel detection assay by PCR-RFLP and frequency of the CYP3A5 SNPs, CYP3A5^*3 and ^*6, in a Japanese population. Pharmacogenetics 12: 331-334.[CrossRef][Medline]

Gibson GG, Plant NJ, Swales KE, Ayrton A, and El-Sankary W (2002) Receptor-dependent transcriptional activation of cytochrome P4503A genes: induction mechanisms, species differences and interindividual variation in man. Xenobiotica 32: 165-206.[CrossRef][Medline]

Gillam EM, Guo Z, Ueng YF, Yamazaki H, Cock I, Reilly PE, Hooper WD, and Guengerich FP (1995) Expression of cytochrome P450 3A5 in Escherichia coli: effects of 5' modification, purification, spectral characterization, reconstitution conditions and catalytic activities. Arch Biochem Biophys 317: 374-384.[CrossRef][Medline]

He YA, Roussel F, and Halpert JR (2003) Analysis of homotropic and heterotropic cooperativity of diazepam oxidation by CYP3A4 using site-directed mutagenesis and kinetic modeling. Arch Biochem Biophys 409: 92-101.[CrossRef][Medline]

Hesselink DA, van Schaik RH, van der Heiden IP, van der Werf M, Gregoor PJ, Lindemans J, Weimar W, and van Gelder T (2003) Genetic polymorphisms of the CYP3A4, CYP3A5 and MDR-1 genes and pharmacokinetics of the calcineurin inhibitors cyclosporine and tacrolimus. Clin Pharmacol Ther 74: 245-254.[CrossRef][Medline]

Hodgson E (2003) In vitro human phase I metabolism of xenobiotics I: pesticides and related compounds used in agriculture and public health. J Biochem Mol Toxicol 17: 201-206.[CrossRef][Medline]

Hsieh KP, Lin YY, Cheng CL, Lai ML, Lin MS, Siest JP, and Huang JD (2001) Novel mutations of CYP3A4 in Chinese. Drug Metab Dispos 29: 268-273.[Abstract/Free Full Text]

Hustert E, Haberl M, Burk O, Wolbold R, He YQ, Klein K, Nuessler AC, Neuhaus P, Klattig J, Eiselt R, et al. (2001) The genetic determinants of the CYP3A5 polymorphism. Pharmacogenetics 11: 773-779.[CrossRef][Medline]

King BP, Leathart JB, Mutch E, Williams FM, and Daly AK (2003) CYP3A5 phenotype-genotype correlations in a British population. Br J Clin Pharmacol 55: 625-629.[CrossRef][Medline]

Koch I, Weil R, Wolbold R, Brockmoller J, Hustert E, Burk O, Nuessler A, Neuhaus P, Eichelbaum M, Zanger U, et al. (2002) Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA. Drug Metab Dispos 30: 1108-1114.[Abstract/Free Full Text]

Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton SA, Hall SD, et al. (2001) Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet 27: 383-391.[CrossRef][Medline]

Lee SJ, Usmani KA, Chanas B, Ghanayem B, Xi T, Hodgson E, Mohrenweiser HW, and Goldstein JA (2003) Genetic findings and functional studies of human CYP3A5 single nucleotide polymorphisms in different ethnic groups. Pharmacogenetics 13: 461-472.[CrossRef][Medline]

Li AP, Kaminski DL, and Rasmussen A (1995) Substrates of human hepatic cytochrome P450 3A4. Toxicology 104: 1-8.[CrossRef][Medline]

Lucey MR, Kolars JC, Merion RM, Campbell DA, Aldrich M, and Watkins PB (1990) Cyclosporin toxicity at therapeutic blood levels and cytochrome P-450 IIIA. Lancet 335: 11-15.[CrossRef][Medline]

Omura T and Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification and properties. J Biol Chem 239: 2379-2385.[Free Full Text]

Popova SN, Slominsky PA, Pocheshnova EA, Balanovskaya EV, Tarskaya LA, Bebyakova NA, Bets LV, Ivanov VP, Livshits LA, Khusnutdinova EK, et al. (2001) Polymorphism of trinucleotide repeats in loci DM, DRPLA and SCA1 in East European populations. Eur J Hum Genet 9: 829-835.[Medline]

Sata F, Sapone A, Elizondo G, Stocker P, Miller VP, Zheng W, Raunio H, Crespi CL, and Gonzalez FJ (2000) CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity. Clin Pharmacol Ther 67: 48-56.[CrossRef][Medline]

Shimada T, Yamazaki H, Mimura M, Inui Y, and Guengerich FP (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270: 414-423.[Abstract/Free Full Text]

Thervet E, Anglicheau D, King B, Schlageter MH, Cassinat B, Beaune P, Legendre C, and Daly AK (2003) Impact of cytochrome p450 3A5 genetic polymorphism on tacrolimus doses and concentration-to-dose ratio in renal transplant recipients. Transplantation 76: 1233-1235.[CrossRef][Medline]

Thummel KE and Wilkinson GR (1998) In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol 38: 389-430.[CrossRef][Medline]

Wacher VJ, Wu CY, and Benet LZ (1995) Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy. Mol Carcinog 13: 129-134.[Medline]

Wang H, Dick R, Yin H, Licad-Coles E, Kroetz DL, Szklarz G, Harlow G, Halpert JR, and Correia MA (1998) Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe. Biochemistry 37: 12536-12545.[CrossRef][Medline]

Williams JA, Ring BJ, Cantrell VE, Jones DR, Eckstein J, Ruterbories K, Hamman MA, Hall SD, and Wrighton SA (2002) Comparative metabolic capabilities of CYP3A4, CYP3A5 and CYP3A7. Drug Metab Dispos 30: 883-891.[Abstract/Free Full Text]

Wrighton SA, Brian WR, Sari MA, Iwasaki M, Guengerich FP, Raucy JL, Molowa DT, and Vandenbranden M (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol Pharmacol 38: 207-213.[Abstract]

Yano JK, Wester MR, Schoch GA, Griffin KJ, Stout CD, and Johnson EF (2004) The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-Å resolution. J Biol Chem 279: 38091-38094.[Abstract/Free Full Text]

Zheng H, Webber S, Zeevi A, Schuetz E, Zhang J, Bowman P, Boyle G, Law Y, Miller S, Lamba J, et al. (2003) Tacrolimus dosing in pediatric heart transplant patients is related to CYP3A5 and MDR1 gene polymorphisms. Am J Transplant 3: 477-483.[CrossRef][Medline]

This article has been cited by other articles:

				M. Miyazaki, K. Nakamura, Y. Fujita, F. P. Guengerich, R. Horiuchi, and K. Yamamoto Defective Activity of Recombinant Cytochromes P450 3A4.2 and 3A4.16 in Oxidation of Midazolam, Nifedipine, and Testosterone Drug Metab. Dispos., November 1, 2008; 36(11): 2287 - 2291. [Abstract] [Full Text] [PDF]

				S.-J. Lee, S. S. Lee, H.-E. Jeong, J.-H. Shon, J.-Y. Ryu, Y. E. Sunwoo, K.-H. Liu, W. Kang, Y.-J. Park, C.-M. Shin, et al. The CYP3A4*18 Allele, the Most Frequent Coding Variant in Asian Populations, Does Not Significantly Affect the Midazolam Disposition in Heterozygous Individuals Drug Metab. Dispos., November 1, 2007; 35(11): 2095 - 2101. [Abstract] [Full Text] [PDF]

				S.-J. Lee, I. P. van der Heiden, J. A. Goldstein, and R. H. N. van Schaik A New CYP3A5 Variant, CYP3A5*11, Is Shown to Be Defective in Nifedipine Metabolism in a Recombinant cDNA Expression System Drug Metab. Dispos., January 1, 2007; 35(1): 67 - 71. [Abstract] [Full Text] [PDF]

				S.-J. Lee, W. J. Jusko, C. G. Salaita, K. A. Calis, M. W. Jann, V. E. Spratlin, J. A. Goldstein, and Y. Y. Hon Reduced Methylprednisolone Clearance Causing Prolonged Pharmacodynamics in a Healthy Subject Was Not Associated With CYP3A5*3 Allele or a Change in Diet Composition. J. Clin. Pharmacol., May 1, 2006; 46(5): 515 - 526. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.078758v1 313/1/302    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, S.-J. Articles by Goldstein, J. A. Search for Related Content PubMed PubMed Citation Articles by Lee, S.-J. Articles by Goldstein, J. A.