Abstract
Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence ofCYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A17776, designated asCYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6β-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6β-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4,CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic groups and its effect on the metabolic activity of CYP3A4 remain to be further evaluated.
Cytochrome P450s are heme-containing enzymes, which play important roles in the oxidative and reductive metabolism of a variety of endogenous and exogenous compounds (Nelson et al., 1996). There are three different cDNA clones (CYP3A4, CYP3A5, and CYP3A7), which have been isolated from human adult and fatal livers (Hashimoto et al., 1993) and were classified into the CYP3A subfamily. The cDNA homology between CYP3A4 and CYP3A5 is 90%, and the homology between CYP3A4 and CYP3A7 is 95% (Hashimoto et al., 1993; Watkins, 1994). The human CYP3A4 is a major P4501 enzyme in the liver and gastrointestinal tract (Watkins, 1994) that plays important roles in the metabolism of a wide variety of drugs, such as immunosuppressants, calcium channel blockers, cancer chemotherapeutic agents, antihistamines, sedatives, and synthetic estrogens. It can also metabolize some endogenous steroids, such as cortisol, testosterone, estradiol (Kitada et al., 1987; Waxman et al., 1988), and some harmful dietary contaminants. TheCYP3A4 gene was cloned and found to contain 13 exons (Hashimoto et al., 1993). It is located on chromosome 7q22.1 (Brooks et al., 1988; Spurr et al., 1989; Inoue et al., 1992; Scherer et al., 1993).
CYP3A4 exhibits remarkable interindividual variation in expression levels as assessed by direct analysis of liver microsome (Shimada and Guengerich, 1989) and through the use of in vivo probe drugs (Lown et al., 1995). The variation may be caused by genetic, environmental, pathological, hormonal, and dietary factors. Genetic polymorphism is a common source of interindividual difference in drug metabolism (Inaba et al., 1995). Genetic polymorphism of CYP3A4 was, however, unknown until recently. To date, several alleles of CYP3A4have been found.
The CYP3A4*1B has a point mutation in the 5′-flanking region (A-290 → G), which disrupts a so-called nifedipine-specific element. The allele frequency of this variant allele was estimated to be 9% in Caucasians, 53% in African Americans, and 0% in a Taiwanese population (Walker et al., 1998).Rebbeck et al. (1998) reported that CYP3A4*1B was associated with tumors of higher clinical stage and grade in men with prostate cancer. The allele was also shown to be under-represented in patients with treatment-related leukemia (Felix et al., 1998). It has not been established whether this mutation has any effect on levels of expression of CYP3A4 compared with the more common wild type. A recent study found that the allele does not influence the enzyme expression in liver to a significant degree (Westlind et al., 1999). In addition toCYP3A4*1B, Sata et al. (2000) reported aCYP3A4*2, with a Ser222Pro change, at a frequency of 2.7% in the white population and which was absent in the black and Chinese subjects. The CYP3A4*2 isozyme was found with a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6β-hydroxylation. The other rare allele,CYP3A4*3, was also found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450 (Sata et al., 2000).
Endogenous cortisol is also metabolized by hepatic cytochrome P450 to 6β-hydroxycortisol (6βOHF). The urinary output of 6βOHF and its ratio to free cortisol (6βOHF/F) (controlling for variation in endogenous cortisol production) reflected the activities of CYP3A4. Ged et al. (1989) found a high correlation between the urinary level of 6βOHF and liver microsomal cortisol 6β-hydroxylase activity and hepatic CYP3A activity. Thus 6βOHF excretion can be used as a marker of CYP3A4 activity. It has been reported that measurement of the morning spot urinary 6βOHF/F ratio is an efficient method for evaluating the potential of chemical agents to induce CYP3A4 (Tran et al., 1999). We have therefore collected morning spot urine in a preliminary test to detect the CYP3A4 activity in subjects with CYP3A4 mutations.
Materials and Methods
Genomic DNA Isolation.
Blood samples were obtained from 57 healthy unrelated subjects and 45 stroke patients from the Chinese (Han) population living in Taiwan. The samples were from two previous studies of CYP2D6 polymorphism. Both studies were approved by the Ethical Committee of National Cheng Kung University Medical Center. DNA was isolated from peripheral leukocytes using a DNA isolation kit (Puregene, Gentra Systems, Inc., Minneapolis, MN).
Determination of the Intron Sequences with PCR.
The intron sequences were obtained through direct sequencing of the genomic DNA from peripheral leukocytes. The introns 1 to 12 of CYP3A4 were sequenced with PCR using specific primers. The sequences of primers used for PCR amplification and sequencing are listed in Table1. Sequencing was done using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Applied Biosystems, Foster City, CA) and ABI PRISM 377-96 DNA sequencer (Perkin-Elmer).
PCR-SSCP and Sequencing Analysis.
All exons (including the exon-intron boundaries) and part of the 5′ upstream region from +174 to −1035 bp were amplified by PCR in each separate reaction. The primer sets and PCR conditions used are shown in Table 2. The PCR reaction was carried out in 50 μl of solution consisting of 5 μl of 10×Taq buffer, 0.27 μM dNTP, 0.1 μM each primer, 0.1 μg of genomic DNA as template, and 2.5 U of Taq polymerase (TaKaRa, Kyoto, Japan). To carry out SSCP gel electrophoresis, a GeneGel Excel 12.5/24 kit (Amersham Pharmacia Biotech, Uppsala, Sweden) was used. The PCR product (2.5–4.5 μl) was mixed with equal volume denatured dye, heated to 95°C for 10 min, cooled on ice for 5 min, and then spun at 4°C. Five microliters of heat-denatured PCR product was directly applied onto the gel. The gel was electrophoresed on a GenePhor unit (Amersham Pharmacia Biotech) at 600 V, 25 mA, 15W and kept at suitable running temperature. After a 2-h electrophoresis, the gel was transferred into a Hoefer automatic gel stainer (Amersham Pharmacia Biotech) and was stained with a PlusOne DNA Silver staining kit (Amersham Pharmacia Biotech) according to the instructions. PCR products showing differences on SSCP analysis were subjected to direct sequencing using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit and ABI PRISM 377-96 DNA Sequencer.
PCR-RFLP for Ile118Val (CYP3A4*4) in Exon 5.
A PCR-based test of Ile118Val was developed. The A13989 → G (GenBank no. AF209389) change in the sequence ATCTC creates the recognition site ofBsmAI (GTCTC). The PCR reaction was carried out in 50 μl of solution consisting of 5 μl of 10×Taq buffer, 0.27 μM dNTP, 0.1 μM primer exon 5(S) and exon 5(R) (Table 3), 0.1 μg of genomic DNA as template, and 2.5 U of Taq polymerase. After PCR amplification, the DNA fragments were digested with BsmAI before electrophoresis using a 12.5% polyacrylamide gel. Samples with A13989 gave 14-, 141-, and 94-bp bands, while samples with G13989 gave 14-, 47-, and 94-bp bands.
PCR-RFLP for Pro218Arg (CYP3A4*5) in Exon 7.
A PCR-based test of Pro218Arg was developed. The C15820 → G change in the sequence ATCCAT creates the recognition site of Cla I (ATCGAT). The PCR reaction was carried out in 50 μl of solution consisting of 5 μl of 10× Taq buffer, 0.2 μM dNTP, 0.27 μM primer exon 7(S) and exon 7(R) (listed in Table 3), 1.5 μM MgCl2, 0.1 μg of genomic DNA as template, and 2.5 U of Taq polymerase. After PCR amplification, the DNA fragments were digested with Cla I before electrophoresis using a 2.5% agarose gel. Samples with C15820 gave a single 450-bp band for the PCR product, while samples with G15820 gave 200- and 250-bp bands.
PCR-RFLP for A17776 Insertion (CYP3A4*6) in Exon 9.
A PCR-based test of A17776 insertion (GenBank no.AF209389) was developed. The A17776 insertion change in the sequence GA17775CTC blocks the recognition site of HinfI (GANTC). The PCR reaction was carried out in 50 μl of solution consisting of 5 μl of 10×Taq buffer, 0.2 μM dNTP, 0.27 μM primer exon 9(S) and exon 9(R) (Table 3), 0.1 μg of genomic DNA as template, and 2.5 U ofTaq polymerase. After PCR amplification, the DNA fragments were digested with HinfI before electrophoresis using a 4% agarose gel. Samples with G17776 gave 24-, 129-, and 137-bp bands for the PCR product, while samples with A17776 insertion gave 153- and 137-bp bands.
Urinary 6β-Hydroxycortisol and Free Cortisol Estimation.
Urine samples were collected from four normal volunteers (E5-30, E5-72, E7-37, and E9-35) and from two stroke patients (E5-U55 and E7-U86). The drug coadministration before and during the urine collection is listed in Table 4. Morning spot urine samples were collected, aliquoted, and frozen at −20°C until assayed for free cortisol and 6β-hydroxycortisol. 6β-Hydroxycortisol in urine was measured by a competitive enzyme immunoassay kit (Stabiligen S.A., Nancy, France). The kit consisted of specific 6βOHF antibodies, 6βOHF standards, a 6βOHF-peroxidase conjugate, and a peroxidase substrate,O-phenylenediamine/H2O2, for reading at 492 nm. Urinary free cortisol was measuring by VIDAS Cortisol kit (Biomérieux, Marcy l'Étoile, France) with an enzyme-linked fluorescent assay. The kit included specific cortisol antibodies, cortisol standards, cortisol-alkaline phosphatase conjugates, and a phosphatase substrate, 4-methyl-umbelliferyl-phosphate. The fluorescent reaction product was then measured for quantitation of cortisol.
Results
CYP3A4 Genomic DNA Sequences in Chinese.
The CYP3A4 gene exon 1 through 13 was directly sequenced to determine the exon-intron junctions and intron sequences. The sequence has been published in the GenBank database (accession no.AF209389). The length of human CYP3A4 gene from exon 1 (ATG start codon) to exon 13 (3′-untranslated sequences) was 26,502 base pairs. The length of some introns was longer than the previous estimation (Hashimoto et al., 1993). PCR were developed to specifically amplify each CYP3A4 fragment. The amplified fragments could each yield a clean sequence from leukocyte DNA sample. No evidence was found to indicate that CYP3A5 or CYP3A7 was amplified with these sequences.
Genetic Polymorphism in the CYP3A4 Gene in Chinese.
To detect the novel base change in the exons and 5′-flanking region, we carried out SSCP analysis of PCR products from 102 healthy unrelated subjects living in Taiwan. Three single base changes were detected by SSCP analysis of 11 exons and the 5′-flanking region. Typical results are shown in Fig. 1. The mutations were confirmed by using DNA sequencing. There were two missense and one insertion mutation found within the coding region of the gene. No base change was found in the 5′-flanking region in these Chinese subjects. In exon 5, a heterozygous A13989 → G base change (Ile118Val) was found in 3 of 102 subjects and designated as CYP3A4*4 allele. In exon 7, a heterozygous C15820 → G base change (Pro218Arg) was found in 2 of 102 subjects and designated asCYP3A4*5 allele. Another A17776insertion was found in exon 9 in 1 of 102 subjects, leading to frame shift and creating an early stop codon, which was designated asCYP3A4*6. We have developed PCR-RFLP methods to confirm the results in SSCP data. The primers for PCR-RFLP and enzyme recognition sites are summarized in Table 3. The same results were confirmed by BsmAI digestion test for exon 5 mutation as shown in Fig.2a, Cla I enzyme digestion test for exon 7 mutation (Fig. 2b), and a HinfI digestion test for exon 9 insertion as shown in Fig. 2c.
Determination of Urinary Ratios of 6β-Hydroxycortisol to Free Cortisol in Mutant Subjects.
Morning spot urine was collected from the three A/G13989 heterozygous subjects, the two C/G15820 heterozygous subjects, and the only subject with A17776 insertion. We determined the CYP3A4 activity in vivo by detecting the urinary ratios of 6β-hydroxycortisol to free cortisol. The results are shown in Table 4. All three mutations showed a diminished CYP3A4 activity compared with the literature control ratio of 5.92 ± 2.97 (mean ± S.D.), which was from 143 healthy Chinese volunteers in Hong Kong (Ng et al., 1996).
Discussion
In this study, the frequencies of the CYP3A4*1B allele that have an A-290G point mutation in the 5′-flanking region of the CYP3A4 gene were examined by SSCP assay (from −378 to −107). The result was the same with the published data of 0% in Chinese population (Walker et al., 1998; Sata et al., 2000).
A novel allele, CYP3A4*4, in exon 5 was point mutation A13989G, which encodes amino acid from Isoleucine118 to Valine. We found three heterozygous subjects. No homozygous were found in our samples. The urinary 6βOHF/F ratio in these three subjects was 2.53, 2.51, and 2.44. It has been reported that in the six highly conserved CYP3A residues of substrate recognition site 1, the Ser119 is a key determinant of CYP3A4 specificity and was shown to have an important role in the active site of steroid 6β-hydroxylation (Roussel et al., 2000). Although it is still uncertain whether this CYP3A4*4 mutation results in altered enzyme activity in vivo, there is a good possibility that the Ile118Val mutation may affect the substrate binding and cause some difference in CYP3A4 activity.
The allele CYP3A4*5 was C15820G point mutation in exon 7, which causes amino acid change from Pro to Arg at 218 site. In 102 subjects, we found two heterozygous subjects, with urinary 6βOHF/F ratios of 2.40 and 3.99. In the CYP3A4-erythromycin docking model (Szklarz and Halpert, 1997), the residues binding erythromycin are amino acids 101, 104, 105, 114, 119, 214, 215, 217,218, 301, 304, 305, 309, 370, 373, 374, 478, and 479. Erythromycin-CYP3A4 binding is stabilized mainly through hydrophobic interactions. The amino acid change from Pro to Arg in 218 site may affect the erythromycin recognition site due to a three-dimensional structure change of CYP3A4. The nitrogen atom of demethylation site in erythromycin molecule was at a distance of 4.7Å from the CYP3A4 heme atom. The distance allows for Van der Waals contacts between ferryl oxygen and the nitrogen atom. A change of the three-dimensional structure may alter the distance and therefore the demethylation efficiency.
The CYP3A4*6 allele was A17776insertion in exon 9. The frame shift due to insertion in exon 9 created an early TGA stop codon at 285 site. We found only one heterozygous A17776 insertion mutant in our samples, for which the urinary 6βOHF/F ratio was 0.88. The ratio was much lower than other mutant and control samples.
The data of urinary 6βOHF/F ratio were expected to provide preliminary information on the clinical significance of these point mutations and insertions. Several factors may affect the ratio, such as the coadministration of CYP3A4 inhibitors or inducers. Subject E5-30 took ketoconazole, a CYP3A4 inhibitor, 2 weeks before the study and was not taking any medication at the day of urine collection. A strong enzyme inhibition is not expected. Subject E5-72 also took drugs at the time of urine collection. The urinary 6βOHF/F ratios of all three subjects with the exon 5 mutation may be affected by drug coadministration to some extent. Similarly, the stroke patient, E7-U86, was taking a number of drugs. The 6βOHF/F ratio can only be considered as preliminary data. Furthermore, although most mutations in these six subjects have been screened out, there is also a possibility that other mutations in the CYP3A4 promoter may occur in these subjects. The significance of these mutations can only be revealed by the site-directed mutagenesis work, which is an on-going work in our laboratory and which we expect to report in the near future.
Acknowledgment
We thank Stabiligen for the assay of 6β-hydroxycortisol and cortisol in the study.
Footnotes
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Send reprint requests to: Jin-ding Huang, Ph.D., Dept. of Pharmacology, National Cheng Kung University, Medical College, 1 University Rd., Tainan 70101, Taiwan. E-mail:jinding{at}mail.ncku.edu.tw
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The work is supported by Grants NHRI-GT-EX89S831L and DOH88-HR-831 of the National Health Research Institute and Department of Health of Republic of China (Taipei).
- Abbreviations used are::
- P450
- cytochrome P450
- 6βOHF
- 6β-hydroxycortisol
- 6βOHF/F
- the ratio of 6βOHF to free cortisol
- dNTP
- deoxynucleotide triphosphates
- PCR
- polymerase chain reaction
- SSCP
- single-strand conformation polymorphism
- bp
- base pair
- RFLP
- restriction fragment length polymorphism
- Taq
- Thermus aquaticus
- Received August 17, 2000.
- Accepted October 31, 2000.
- The American Society for Pharmacology and Experimental Therapeutics