CYP1A2 polymorphism has been well studied in white persons and Asians but not in Africans. We performed CYP1A2 genotype and phenotype analysis using caffeine in Ethiopians living in Ethiopia (n ϭ 100) or in Sweden (n ϭ 73). We sequenced the CYP1A2 gene using genomic DNA from 12 subjects, which revealed a novel intron 1 single-nucleotide polymorphism (SNP), Ϫ730CϾT. We developed SNP-specific polymerase chain reaction-restriction fragment length polymorphism genotyping and molecular haplotyping methods for the intron 1 SNPs, and four different haplotypes were identified: CYP1A2*1A (wild-type for all SNPs), CYP1A2*1F (Ϫ164A), CYP1A2*1J (Ϫ740G and Ϫ164A), and CYP1A2*1K (Ϫ730T, Ϫ740G, and Ϫ164A), having frequencies of 39.9, 49.6, 7.5, and 3.0%, respectively. The frequency of CYP1A2*1J and CYP1A2*1K among Saudi Arabians (n ϭ 136) was 5.9% and 3.6%, and among Spaniards (n ϭ 117) 1.3% and 0.5%, respectively. Functional significance of the different intron 1 haplotypes was analyzed. Subjects with CYP1A2*1K had significantly decreased CYP1A2 activity in vivo, and reporter constructs with this haplotype had significantly less inducibility with 2,3,7,8-tetrachlorodibenzo-p-dioxin in human B16A2 hepatoma cells. Electrophoretic mobility shift assay using nuclear extracts from B16A2 cells revealed a specific DNA binding protein complex to an Ets element. Efficient competition was obtained using oligonucleotide probes carrying the wt sequence and Ets consensus probe, whereas competition was abolished using probes with the Ϫ730CϾT SNP alone or in combination with Ϫ740TϾG (CYP1A2*1K). The results indicate a novel polymorphism in intron 1 of importance for Ets-dependent CYP1A2 expression in vivo and inducibility of the enzyme, which might be of critical importance for determination of interindividual differences in drug metabolism and sensitivity to carcinogens activated by CYP1A2.
Nicotine C-oxidation is primarily catalyzed by CYP2A6 in humans. This enzymatic activity exhibits a large interindividual variability, which to a great extent is caused by genetic polymorphisms in the CYP2A6 gene. There are large interindividual differences in CYP2A6 mRNA and protein levels, but little is known about the transcriptional regulation of CYP2A6, which can, e.g., explain such differences. Using transient transfections of 5Ј-deleted CYP2A6 promoter constructs in human hepatoma B16A2 cells, we show that maximal promoter activity was harbored in the sequence spanning from Ϫ112 to Ϫ61. Putative response elements for the transcription factors hepatocyte nuclear factor-4 (HNF-4)␣, CCAAT-box/enhancer binding protein (C/EBP)␣, C/EBP, and octamer transcription factor-1 (Oct-1) were identified in this region, and electrophoretic mobility shift assays showed that these transcription factors bind to the predicted elements. To determine the relevance of these sites, expression vectors for these transcription factors were cotransfected with CYP2A6 promoter constructs in HepG2 cells. HNF-4␣, C/EBP␣, and Oct-1 exerted an activating effect, whereas overexpression of C/EBP reduced CYP2A6 promoter activity. To confirm the importance of these sites in vivo, mutated CYP2A6 reporter constructs were injected into mouse liver. Mutation of either HNF-4 or C/EBP-Oct-1 motifs significantly decreased promoter activity, 52 and 26% of wildtype, respectively, whereas when both motifs were mutated the activity in mice decreased to 14% of wild type. In conclusion, the data indicate that the constitutive hepatic expression of CYP2A6 is governed by an interplay between the transcription factors HNF-4␣, C/EBP␣, C/EBP, and Oct-1. These results will be important for the identification of new polymorphisms affecting CYP2A6 gene expression.
In humans, several polymorphic variants have been described for the gene encoding the major nicotine C-oxidase, cytochrome P450 2A6 (CYP2A6), which is to a great extent responsible for the large interindividual differences seen at the enzymatic and activity levels. Hitherto, mainly polymorphic variants in the open reading frame have been identified. In the present study, we identified a novel single nucleotide polymorphism (SNP) located in the 5' flanking region of the CYP2A6 gene. Sequencing of 1.4 kb of the 5'-upstream region of the CYP2A6 gene from eight individuals revealed a c.-1013A>G polymorphism defining two new alleles, CYP2A6*1D and CYP2A6*1E, lacking or having also the CYP2A7 3'-UTR. Analysis of genomic DNA from 32 Swedish and 109 Turkish subjects by dynamic allele-specific hybridization (DASH) showed that, in both groups, the variants carrying the c.-1013A>G SNP represent approximately 70% of the total number of alleles. Transfection of HepG2 cells with luciferase reporter constructs containing 1019 bp of the CYP2A6 5'-regulatory sequence showed that the region between c.-1005 and c.-1019 elicited a strong enhancer effect and that the CYP2A6*1D promoter had significantly reduced expression as compared to CYP2A6*1A carrying c.-1013A. Electrophoretic mobility shift assays (EMSA) showed that nuclear proteins from HepG2 and B16A2 cells exhibited a higher binding affinity to the probe harboring c.-1013A as compared to the c.-1013G probe, although the transcription factor(s) responsible for this binding could not be identified. In conclusion, our results indicate the presence of a strong enhancer or promoter responsive element between c.-1005 and c.-1019 in the CYP2A6 gene and that a c.-1013A>G polymorphism in this region affects CYP2A6 transcription.
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