Abstract
Despite a wide interindividual variation of cytochrome P-450 1A2 (CYP1A2) activity, genetic polymorphism of CYP1A2 has not been reported. By amplification of exons of CYP1A2 by polymerase chain reaction in eight Chinese subjects, the polymerase chain reaction products were directly sequenced. One subject showed heterozygous C2866→G (Phe21→Leu) polymorphism. DNA from 157 Chinese subjects (104 polychlorinated biphenyl-exposed subjects and 53 control subjects) was screened for polymorphism by single-strand conformation polymorphism method and MboII endonuclease digestion. Only 1 of 157 samples showed another heterozygous C2866→G mutation. The subject was previously exposed to polychlorinated biphenyl and showed a value of 3.5% in the caffeine breath test. The value is not significantly higher than the mean value of polychlorinated biphenyl-exposed subjects (3.12 ± 0.29%, mean ± S.E.M.). The incidence of the point mutation in these Chinese subjects is less than 1%. The prevalence of the F21L mutation in other ethnic groups and its effect on the metabolic activity of CYP1A2 remain to be further evaluated.
Cytochrome P-450 1A2 (CYP1A2)1is a constitutively expressed enzyme in the human liver. The enzyme is well known for its role in the metabolic activation of environmental and food-borne carcinogens, including arylamines and heterocyclic amines (Boobis et al., 1994; Eaton et al., 1995; Hammons et al., 1997). To measure the metabolic activity of CYP1A2, caffeine has been suggested to be used as the CYP1A2 phenotyping probe in vivo (Kalow and Tang, 1991; Fuhr and Rost, 1994; Denaro et al., 1996; Fuhr et al., 1996;Miners and Birkett, 1996). Recently, CYP1A2 was found to metabolize a large number of drugs or chemicals, such as 2-amino-α-carboline (Raza et al., 1996), antipyrine (Sharer and Wrighton, 1996), estrone (Shou et al., 1997), 7-ethoxycoumarin (Yamazaki et al., 1996), flutamide (Shet et al., 1997), imipramine (Koyama et al., 1997), mianserin (Koyama et al., 1996), naproxen (Tracy et al., 1997), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (Smith et al., 1996), nortriptyline (Olesen and Linnet, 1997), olanzapine (Ring et al., 1996), tacrine (Benoit et al., 1997), toluene (Nakajima et al., 1997), and R-warfarin (Zhang et al., 1995) in the cDNA-expressed human CYP1A2 enzyme. There is an increasing understanding of the importance of this enzyme in therapeutics no less than in the toxicological consideration.
There is a wide interindividual variation of CYP1A2 activity among human subjects (Kalow and Tang, 1991; Butler et al., 1992; Fuhr and Rost, 1994). The variation may be due to the enzyme induction and inhibition by other drugs or environmental exposures to a large extent. Smoking status and coffee and alcohol consumption in daily life contribute to variability. Other than induction and inhibition, ethnic variation was also reported (Butler et al., 1992). Moreover, there is also a possibility of genetic polymorphism of CYP1A2 as frequently shown in other cytochrome P-450 isozymes. Human CYP1A2 cDNA has been cloned and sequenced (Ikeya et al., 1989; Jaiswal et al., 1986; Quattrochi et al. 1986). No genetic polymorphism resulting in an altered protein sequence for CYP1A2 has been reported. Because the genetic polymorphism is often ethnic-specific, we examined the exon sequences of CYP1A2 in Chinese by polymerase chain reactions (PCRs).
Materials and Methods
Human Subjects and DNA Isolation.
We recruited eight healthy unrelated subjects from the Chinese (Han) population living in Taiwan. Whole blood (12 ml) was obtained from each of the subjects and DNA was isolated from peripheral leukocytes according to the method by Blin and Stafford (1976).
DNA Sequencing with PCR.
The exons 2 to 7 ofCYP1A2 were sequenced with PCR using specific primers. The primers used for PCR amplification and sequences are listed in Table 1. The exons were divided into fragments of 2B, 2C, 2D, 3, 4, 5, 6, 7A, and 7B for sequencing. The specific primers were synthesized with an Applied Biosystems model 380B and were used without further purification. The PCR reaction was carried out in a 100-μl solution consisting of 10 μl of 10×Taq buffer, 0.25 mM 4 dNTPs, 100 pmol of each primer, 10 ng of genomic DNA as template, and 2.5 units of Taqpolymerase. After 20 cycles of 95°C for 30 s, 55°C for 30s, and 70°C for 1 min, another 10 cycles of 30 s at 95°C, 1 min at 70°C were repeated. The double-stranded PCR product was purified by agarose gel electrophoresis and BIO101 (Viata, CA) Geneclean II kit . DNA sequencing was then performed with a double-stranded-DNA cycle sequencing kit (BRL, Gaithersburg, MD) according to the dideoxy chain termination method developed by Sanger et al. (1977).
Sequence and location of primers used in PCR reaction and sequencing
Single-Strand Conformation Polymorphism (SSCP) in Exon 2.
A PCR-based SSCP test of C2866G polymorphism was developed. The oligonucleotide fragment containing the position (2A as listed in Table1) was amplified by PCR at the experimental condition as described above. The PCR product was mixed with 5 volumes of 0.1% SDS/10 mM EDTA and then mixed with equal volume of 95% formamide/20 mM EDTA/0.05% xylene cyanol. After heating at 95°C for 5 min, the sample was electrophoresed with 10% polyacrylamide gel at 4°C with 80 V for 22 h. Genomic DNA samples (157 samples) from an epidemiological study were analyzed. CYP1A2 activity in these subjects was measured by the caffeine breath test (manuscript in preparation). One hundred four polychlorinated biphenyl-exposed subjects have an average metabolic ratio of 3.12 ± 0.29% (mean ± S.E.M.), whereas the 53 control subjects have an average metabolic ratio of 1.47 ± 0.13% (unpublished data).
Diagnostic Test of C/G2866 Polymorphism.
A PCR-based test of C/G2866 polymorphism was also developed. The PCR product 2A containing C2866 in the CTTCT sequence (2863–2867) can be digested by MboII endonuclease (recognizing the antisense GAA/GA). When C2866 was substituted with G2866, the endonuclease site disappears. After PCR, the DNA fragments were digested with MboII before electrophoresis with a 10% polyacrylamide gel. Samples with homozygous C2866 gave 79- and 122-bp bands, whereas samples with heterozygous C/G2866 gave the 79- and122-bp bands in addition to the intact 201-bp band (Fig.1).
Analysis of PCR-extended fragments from exon 2 of CYP1A2.
Lanes 1 and 2 [without (−) and with (+) MboII digestion] are from a sample of homozygous C2866. Lanes 3 and 4 [without (−) and with (+) MboII digestion)] are from a sample of heterozygous C/G2866.
Results
Detection of CYP1A2 Mutation in Chinese Subjects.
We detected a heterozygous C/G2866 site in exon 2 in blood from one of eight volunteers. The sequence of PCR products is shown in Fig.2. The mutation causes an amino acid change of Phe21 to Leu. In addition, we confirmed the two variation sites in exon 7 as reported by Nakajima et al. (1994). One is three bases (CTG) insertion after the nucleotide 749 in all eight subjects. The insertion causes one addition amino acid Leu in Chinese CYP1A2 (Quattrochi et al. 1986; Nakajima et al., 1994). The other is C/T763 polymorphism, which does not change amino sequence (Asn). Six subjects showed homozygous C763 and two subjects showed heterozygous T/C763.
Sequencing gel of the PCR product containing heterozygous C/G2866 in exon 2.
SSCP and Endonuclease Tests of Samples of Known CYP1A2 Activity.
We have developed an SSCP method to detect the C/G2866polymorphism in CYP1A2 exon 2. In 157 samples from an epidemiological study including subjects with caffeine breath test data, we found only one heterozygous C/G2866 mutation (Fig.3). The same results were confirmed byMboII digestion test (Fig. 1). The subject showing C/G2866 was previously exposed to polychlorinated biphenyl has a metabolic ratio of 3.5%. The value is not significantly different from the mean value of the PCR-exposed subjects (3.12 ± 0.29%, mean ± S.E.M.), but it is significantly higher than that of the healthy controls (1.47 ± 0.13%).
SSCP analysis of genomic PCR products from exon 2 of CYP1A2.
Lanes 1 and 3 to 5 show the samples of homozygous C2866, whereas lane 2 shows the sample of heterozygous C/G2866 samples. Arrow 1, the single-stranded band of altered migration. Arrow 2, the location of double-stranded DNA.
Discussion
No genetic polymorphism resulting in an altered protein sequence for CYP1A2 has been reported. In this study, we reported the detection methods of a C2866G mutation in CYP1A2 exon 2, which causes a Phe21 to Leu change. The mutation is actually rare in the Chinese population. In samples from the caffeine breath test study, only 1 of 314 alleles showed the mutation. If the eight volunteers are included in the calculation, only 2 of 330 alleles showed the mutation. The possibility to detect the homozygous G2866 subjects in Chinese is very low. Different ethnic groups often show different polymorphism characteristics. For example, the CYP2D6*4 allele is the most predominant in Caucasian-poor metabolizers of CYP2D6 (Marez et al., 1997), whereas the mutation was found rare in oriental subjects (Johansson et al., 1991;Wang et al., 1993; Roh et al., 1996). There is a possibility that C/G2866 polymorphism may be important in some other ethnic groups.
If the mutation increased the metabolic activity of CYP1A2, it can be of high toxicological interest because of the role of CYP1A2 in the metabolic activation of environmental and food-borne carcinogens. Based on the P-450 membrane topology, Phe21 is located in a hydrophobic domain ( Nelson and Strobel, 1988; Edwards et al., 1989). The Phe21 to Leu mutation might have little impact in CYP1A2 topology as well as the catalytic activity. A cDNA expression study will help to reveal the significance of this mutation. Before the site-directed mutagenesis studies, data of caffeine breath test were expected to provide preliminary information on the clinical significance of this mutation as well as the effect of the C2866G mutation on CYP1A2 activity. However, the number of mutant allele in this study was limited. Only one subject with caffeine breath test datum showed heterozygous C/G2866. No conclusion in the CYP1A2 metabolic activity could be made from this work. The data are however interesting.
The incidence of the mutation in the Chinese is low. Studies in other ethnic groups are worth of performing. The detection methods of the point mutation in this report in combination with some phenotyping study may divulge the significance of this CYP1A2 polymorphism. A cDNA expression study may then be pursued.
Footnotes
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Send reprint requests to: Jin-ding Huang, Doctor of Philosophy, Department of Pharmacology, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan. E-mail:jinding{at}mail.ncku.edu.tw
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This work was supported by Grant NSC87 -2314-B006-103 from the National Sciences Council of the Republic of China. The study was part of the Masters thesis of W.-C.K. The abstract was presented in XIIIth International Congress of Pharmacology, 1988, Munich, Germany.
- Abbreviations used are::
- CPY1A2
- cytochrome P-450 1A2
- PCR
- polymerase chain reaction
- SSPC
- single-strand conformation polymorphism
- Received April 22, 1998.
- Accepted July 13, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
