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Human Molecular Genetics Advance Access originally published online on September 30, 2004
Human Molecular Genetics 2004 13(23):2959-2969; doi:10.1093/hmg/ddh313
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Human Molecular Genetics, Vol. 13, No. 23 © Oxford University Press 2004; all rights reserved

Allelic expression imbalance of the human CYP3A4 gene and individual phenotypic status

Takeshi Hirota1, Ichiro Ieiri2,*, Hiroshi Takane2, Shinji Maegawa3, Masakiyo Hosokawa5, Kaoru Kobayashi5, Kan Chiba5, Eiji Nanba3, Mitsuo Oshimura4, Tetsuo Sato5, Shun Higuchi1 and Kenji Otsubo2

1Clinical Pharmacokinetics, Division of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 8128582, Japan, 2Department of Hospital Pharmacy, Faculty of Medicine, 3Division of Functional Genomics, Research Center for Bioscience and Technology and 4Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago 6838504, Japan and 5Laboratory of Biochemical Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 2740816, Japan

Received August 6, 2004; Accepted September 20, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human cytochrome P450 3A4 (CYP3A4) plays a dominant role in the metabolism of numerous clinically useful drugs. Alterations in the activity or expression of this enzyme may account for a major part of the variation in drug responsiveness and toxicity. However, it is generally accepted that most of the known single nucleotide polymorphisms in the coding and 5'-flanking regions are not the main determinants for the large inter-individual variability of CYP3A4 expression and activity. We show that the allelic variation is critically involved in determining the individual total hepatic CYP3A4 mRNA level and metabolic capability. There exists a definite correlation between the total CYP3A4 mRNA level and allelic expression ratio, the relative transcript level ratio derived from the two alleles. Individuals with a low expression ratio, exhibiting a large difference of transcript level between the two alleles, revealed extremely low levels of total hepatic CYP3A4 mRNA, and thus low metabolic capability as assessed by testosterone 6ß-hydroxylation. These results present a new insight into the individualized CYP3A4-dependent pharmacotherapy and the importance of expression imbalance to human phenotypic diversity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Among the human cytochrome P450 (CYP) proteins, the members of the CYP3A subfamily occupy an important position owing to their abundance in liver and gut and to their collective large substrate spectrum (1). CYP3A proteins account for up to 50% of total CYP activity in the liver, and they metabolize up to 60% of all drugs currently in use (2,3). Cytochrome P450 3A4 (CYP3A4) is the major form of CYP in human liver; accounting for 30% of total CYP protein content (4). A wide inter-individual variation exists in CYP3A4 activity as assessed by direct analysis of liver microsomes (4) and through the use of in vivo probe drugs (5). The basis of this variation is not yet understood but may be due to genetic factors. A clinical study with CYP3A4 substrates suggested that ~60–90% of the inter-individual variability in hepatic CYP3A4 activity is genetically determined (6). The coding and 5'-flanking regions of the CYP3A4 gene have been isolated and sequenced, and some single nucleotide polymorphisms (SNPs) have been identified; however, their allelic frequencies and/or the available functional experiments indicate a limited role for these variants in the inter-individual variability of CYP3A4 expression and activity (7–12).

In addition to SNPs, various gene expression mechanisms have recently been reported to determine phenotypic variability; these patterns include genomic imprinting (13,14), X-chromosome inactivation (15) and other mechanisms (16,17). Among them, genomic imprinting is an epigenetic phenomenon where parental alleles are genetically marked, leading to the differential expression of paternal and maternal alleles in somatic cells (13,14). Imprinting genes are often clustered in chromosomal domains and are thought to be coordinately regulated by imprinting control centers (13,18). Interestingly, three members of the CYP3A subfamily, CYP3A4, CYP3A5 and CYP3A7, are localized in tandem on the long arm of chromosome 7 at q21–q22.1 (19,20), where several imprinted genes are clustered (21). In addition, differences in expression levels between the two alleles of the same gene (i.e. preferential expression of one of the two alleles), which were not consistent with parental imprinting, have been reported in various genes such as PKD2, p73 and Calpain-10 (16,22). Some investigators indicated that such allelic variation in gene expression was common in the human genome (17,22). The individual allelic expression status may result in a change in the expression level of the gene (23,24), leading to phenotypic variability in the pharmacokinetic and pharmacodynamic outcomes of drug therapy. On screening the entire CYP3A4 gene, we found two common non-functional and racially independent intronic SNPs, C78013T (C->T at nt 78013) and C78649T (C->T at nt 78649), and used them as markers in subsequent gene expression analyses. Using these SNPs, we elucidated that the allelic variation is critically involved in the hepatic CYP3A4 mRNA expression. Individuals with a low expression ratio, exhibiting a large difference of transcriptional level between the two alleles, revealed extremely low hepatic CYP3A4 levels and thereby reduced metabolic activity. These findings explain the substantial inter-individual differences in CYP3A4 expression and activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Screening of the marker SNPs for assessment of allelic variation
To screen the marker SNPs for assessment of allelic variation and to evaluate the presence of functional CYP3A4 gene variants, we amplified and sequenced the whole CYP3A4 gene, CYP3A4 5'-flanking region and 3'-untranslated region (UTR), spanning ~60 kb, using genomic DNAs extracted from eight Caucasian liver samples with high (HHL-6, -7, -14 and -26) or low (HHL-12, -17, -19 and -20) levels of total CYP3A4 mRNA (Fig. 1). The former and latter samples also indicated high and low testosterone 6ß-hydroxylation activity, respectively. In the present study, testosterone 6ß-hydroxylation capability was used as an index for individual CYP3A4 activity. We identified 4, 35 and 17 polymorphisms in the 5'-flanking region, 3'-UTR and intronic region, respectively, but did not find any coding SNPs. Although three samples, HHL-6, HHL-7 and HHL-17, had many SNPs in the regions analyzed, the other samples had only 2–4 SNPs. Among them, frequencies of the C78013T (8/8) and C78649T (4/8) polymorphisms were relatively high. Thus, we used these two intronic SNPs as markers in the subsequent gene expression study.



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  		Figure 1. Relationship between CYP3A4 phenotypes and CYP3A4 genotype in Caucasian livers. Open, partially filled and closed squares correspond to liver homozygous for the reference sequence (R), and heterozygous and homozygous for the variant sequence (V).

 
We next examined the association of the allelic pattern with total hepatic CYP3A4 mRNA levels. Although the CYP3A4 level was comparable between HHL-6 and HHL-26, the SNP patterns were clearly different. In contrast, there were remarkable differences in the CYP3A4 levels between HHL-7 and HHL-17 even though the SNP pattern was similar. In addition to the total mRNA levels, a similar trend was observed in the testosterone 6ß-hydroxylation activity. As has previously been demonstrated (7–12), these results suggest that SNPs in these regions are not involved in the large variability in the CYP3A4 gene expression and metabolic activity.

Correlation between total hepatic CYP3A4 mRNA levels and CYP3A4 hnRNA levels
We next examined whether tissue levels of heterogeneous nuclear RNA (hnRNA), the unprocessed precursor of the mature, functional mRNA, can be used as a surrogate for gene transcription. CYP3A4 hnRNA and total mRNA levels were determined in a total of 18 hepatic samples. Quantification of total CYP3A4 mRNA and hnRNA was performed by the real-time PCR method. As shown in Figure 2, CYP3A4 mRNA showed significant regression (r=0.775; P<0.001). These results validated the use of CYP3A4 hnRNA as an estimate of CYP3A4 gene activity in human liver samples.



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  		Figure 2. Relationship of total CYP3A4 mRNA and hnRNA levels in human liver samples. A simple linear regression analysis was performed.

 
Allelic variations in human liver samples
Prior to evaluating the functional significance of allelic variation in total hepatic CYP3A4 mRNA levels and metabolic activity, we estimated allelic expression ratios, defined as a measure of the expression of the less-abundant allele divided by that of the more-abundant allele, in order to confirm inter-individual variation in the allelic expression of CYP3A4. As described in more detail in the Materials and Methods, allelic variation was determined on the basis of difference in band intensities between the two alleles using a fluorescence image analyzer. When an individual is heterozygous for a marker SNP, it is possible to detect the relative abundance of allelic transcripts. Generally, both copies of human autosomal genes are assumed to be co-dominantly expressed in equal proportions. The detection of allelic variation is based on a quantitative analysis of RNA transcripts in order to detect deviations from the expected equimolar ratio between two alleles in a heterozygous sample. After the screening of genomic DNA from all 40 Caucasian liver samples, it was possible to identify 18 individuals who were heterozygous for either the MnlI (C78013T) or AciI (C78649T) site. The difference in expression between the two alleles varied among samples (Fig. 3), and some of the 18 individuals had fractional allelic expression values lower than the 95% confidence interval for the mean (0.70±0.20; 95% confidence interval, 0.60–0.80). Notably, the values in HHL-12 (0.28), HHL-16 (0.45), HHL-20 (0.46) and HHL-36 (0.49) were extremely low, being well outside the intervals, indicating monoallelic expression. Preferentially expressed allele in an individual sample was also shown in Figure 3. Predominantly expressed allele was different among samples, suggesting that both marker SNPs have no significant effects on the expression of the CYP3A4 gene.



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  		Figure 3. Allelic expression ratio of the CYP3A4 gene in Caucasian livers. The expression ratio (y-axis) was estimated on the basis of the average less-/more-abundant ratios (replicated data-points for each sample) at either the C78013T or the C78649T polymorphism, and corrected using the average genomic ratio. The shaded box represents ~95% confidence interval and the red bars indicate individuals displaying significant variation.

 
Allelic expression pattern in informative lymphoblasts
To determine whether the two alleles of the human CYP3A4 gene are differentially expressed according to parental origin, we used reverse transcription–polymerase chain reaction (RT–PCR) of total RNA extracted from Epstein–Barr (EB) virus-transformed lymphoblasts, followed by PCR–RFLP. The parental origin of alleles expressed in children was identified by RFLP analysis. Lymphoblasts were obtained from a panel of 22 healthy Japanese individuals who were members of six distinct families. These samples allowed the precise determination of the parental origin of alleles in the heterozygous children. Of all the cases, two siblings were heterozygous for a polymorphism at either the MnlI site (C78013T in intron 7) or the AfaI site (G82266A in intron 10) (Fig. 4A). As the SNPs used here are in intronic regions of CYP3A4, the cDNA analyzed represents unspliced, hnRNA (25). Thus, all RT reactions in the present study included a negative control to ensure that genomic DNA did not contaminate the subsequent PCR. We firstly determined the parent's genotypes using genomic DNA samples. At the MnlI site (for C1 in Fig. 4B and C), although the maternal genotype was homozygous for the T78013 allele, the paternal genotype was heterozygous for the C78013 allele. At the AfaI site (for C2), the corresponding genotypes were homozygous for the G82266 allele and heterozygous for the A82266 allele, respectively. In the RT–PCR products, in contrast to C1, who showed a monoallelic maternal (T78013) expression, the sibling C2 showed a biallelic expression, but the paternal allele (A82266) was preferentially expressed.



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  		Figure 4. Identification of allelic variation of the CYP3A4 gene. (A) Schematic of the PCR–RFLP for the three polymorphisms. Black squares denote CYP3A4 exons. Locations of PCR primers are indicated by arrows. Predicted RFLP products of each polymorphism are drawn below the schematic. (B) Actual expression patterns in lymphoblasts obtained from two informative siblings. (C) Validation of the allelic variation by sequencing.

 
It is interesting to know whether allelic variation is inherited. In order to address this issue, we further analyzed allelic expression patterns using paternal RT–PCR products, because paternal genotype was heterozygous for both polymorphic sites (at genomic DNA-based genotypes). As shown in Figure 4B, paternal alleles were inherited by the two siblings; the paternal inactive allele (i.e. unexpressed C78013 allele at the MnlI site) and active allele (i.e. expressed A82266 allele at the AfaI site) were inherited by siblings C1 and C2, respectively. These results suggest that allelic variation is inherited, at least in B virus-transformed lymphoblasts.

Methylation status in the most proximal 5'-CpG island of the CYP3A4 gene
We focused on the most proximal 5'-CpG island (covering ~450 bp), which is ~30 kb upstream of the translational start codon, and examined the association of the methylation status with total CYP3A4 mRNA levels using six liver samples; samples HHL-6, -7 and -14 had high and HHL-12, -19 and -20 had low mRNA levels (Fig. 1). The percent methylation of the 31 CpG sites analyzed in the CYP3A4 CpG island was calculated. Although differentially methylated CpG sites were found, most of the sites analyzed were largely methylated (Fig. 5). No clear association between the methylation status and total mRNA levels was observed.



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  		Figure 5. Methylation analysis of the CpG island of the CYP3A4 gene. Bisulfite PCR products were subcloned and sequenced. The degree of methylation on each CpG dinucleotide was obtained from 20 individual clones. Open and closed areas represent unmethylated and methylated CpG dinucleotides, respectively.

 
Allelic variation and CYP3A4 phenotypes
Correlations between the allelic expression ratio and phenotype indexes are shown in Figure 6. The total hepatic CYP3A4 mRNA level correlated strongly with the allelic ratio (Fig. 6A; r2=0.786, P<0.001). Of these 18 samples, we could measure CYP3A4 activity using testosterone as an enzyme-specific substrate in 10. As expected, 6ß-hydroxylation capability also correlated strongly with the allelic ratio (Fig. 6B; r2=0.541, P<0.05). These results indicate that the individuals with a low ratio, who exhibited a large difference in hnRNA expression level between the two alleles, have extremely low total hepatic CYP3A4 mRNA levels, and consequently poor metabolic capability.



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  		Figure 6. Allelic expression ratio and CYP3A4 phenotypes in Caucasian livers. (A) Relationship between total mRNA level (y-axis) and allelic ratio. The ratio was estimated based on the average less-/more-abundant ratios at either the C78013T or the C78649T polymorphism. (B) Relationship between testosterone 6ß-hydroxylation capability and allelic ratio. The numbers are identical with the sample numbers in Figures 1 and 3. (C) Actual expression patterns in the livers.

 
Frequency of the two marker SNPs in different racial populations
We determined frequencies of heterozygous carriers for C78013T and C78649T polymorphisms, which were relatively common among the eight liver samples, using genomic DNA from three racial populations (Table 1). We found that the frequency of heterozygous carriers of the C78013T allele in Caucasians was 0.35; in Japanese, 0.39; and in African Americans, 0.56. The corresponding values for the C78649T allele were 0.21, 0.16 and 0.36, respectively. When excluding individuals having two SNPs simultaneously, 50.0% of Caucasians, 41.7% of Japanese and 61.5% of African Americans were heterozygous for either SNP.


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  		Table 1. Heterogeneous carriers for CYP3A4 mutations in different racial populations
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Variations in gene sequence and expression underlie much of human variability. To decide individual gene expression status, we first sequenced the whole CYP3A4 gene, CYP3A4 5'-flanking region and 3'-UTR using liver tissues from eight Caucasians, and then examined the association of SNP patterns with total CYP3A4 mRNA levels and testosterone 6ß-hydroxylation capability. Although the observed nucleotide diversity in the CYP3A4 promoter was 1 in 7246 bp in Caucasians (26), two recent reports provided evidence for the existence of a specific cis-acting element, 8000 bp distal to the transcription start point, which plays an important role in the transcriptional induction of CYP3A4 (27,28). Thus, we analyzed the CYP3A4 5'-flanking region, spanning 8 kb. As has been expected, none of the SNPs in these regions was clearly associated with differences in CYP3A4 levels and metabolic capability. These findings raise the possibility that other mechanisms such as an epigenetic gene alteration affect CYP3A4 levels more frequently than SNPs (7–12).

In the present study, we used unspliced hnRNA as the template and used intronic SNPs as the marker to assess the allelic variation, because there were no exonic SNPs that can be used to test for allelic variation in mRNA levels. Thus, we secondarily examined whether tissue levels of hnRNA, the unprocessed precursor of the mature, functional mRNA, can be used as a surrogate for gene transcription. To ensure precise determinations for hnRNA and mRNA levels separately, we have designed intron- and exon-specific oligonucleotide primers, respectively. As shown in Figure 2, the two exhibited a significant correlation. These results validated the use of CYP3A4 hnRNA as an estimate of CYP3A4 gene activity in human liver samples, and suggest that total CYP3A4 mRNA levels are controlled at a point upstream of the precursor by the transcriptional generation of hnRNA (29,30).

Recently, in order to determine allelic variation, an allele-specific quantitative PCR method with allele-specific probes has been developed. Such an analytical method provides the direct expression level of each allele separately by measuring the fluorescent intensity (22,31). In the present study, we had also tried to develop a real-time quantitative PCR method; however, we were unsuccessful owing to a technical limitation, low expression levels of CYP3A4 hnRNA, especially in samples indicating monoallelic expression. Thus, we determined allelic variations on the basis of difference of band intensities between the two alleles using a fluorescence image analyzer. With such an analytical method, in addition to DNA contamination, splicing variants are drawbacks for the accurate estimation of allelic variation. In the CYP3A4 gene, one variant, CYP3A4*6, with an insertion of adenine at position 17776, has been reported to create premature mRNA because the variant causes a frame shift and an early stop codon in exon 9 (32). However, the CYP3A4*6 variant was not observed in our liver samples (data not shown), and this finding was in keeping with previous reports that it has not been observed in Caucasian populations (33,34).

Differential allelic gene expression resulting from genomic imprinting has been a focus of cancer research. Among known imprinted genes, the Wilms' tumor suppressor gene (WT1) has been reported to exhibit a unique allele-specific expression profile (35); cultured human fibroblasts and lymphocytes showed a paternal or biallelic expression of WT1 in some cases, whereas a maternal or biallelic expression was observed in human placental villi and fetal brain tissue (36,37). These results suggest that the allele-specific expression profile (e.g. allele switching) of certain genes depends on the tissue source. Indeed, although somatic allele switching is not a common feature of imprinted genes, this unusual phenomenon is also observed in human H19 (38) and IMPT1 (39) genes. However, in the present study, the degree of difference in the expression between the two alleles varied among samples, and large variations were observed in only a minority of samples (Fig. 3). In addition, informative lymphoblast samples indicated opposite-directional expression; both paternal and maternal preferential expressions were observed in the two siblings (Fig. 4). Thus, taking these findings into consideration, it is feasible that, at least in human liver, CYP3A4 is not an imprinting gene.

Although allelic variation in gene expression is common in the human genome (17,22), its pharmacokinetic and pharmacodynamic significance has not been reported. One study has demonstrated that the allelic variation in APC gene expression plays a critical role in colon cancer (16). The present study, however, is the first to demonstrate that variations in CYP3A4 phenotypes are caused by changes in allelic expression levels. The strong correlation between the allelic ratio and phenotypic indexes (e.g. total hepatic mRNA level and testosterone 6ß-hydroxylation activity) indicates that individuals having low ratios, who exhibit a large difference in hnRNA expression levels between the two alleles, have extremely low levels of total CYP3A4 mRNA and thereby reduced metabolic activity. Human CYP3A activity reflects the heterogeneous expression of at least two CYP3A family members, CYP3A4 and CYP3A5. Although the role of CYP3A5 in testosterone 6ß-hydroxylation in vivo has not been defined (40), this heterogeneity is a possible reason for the lower correlation observed between the allelic ratio and hydroxylation activity.

Although not much is known about mechanisms regulating the constitutive basal expression of the CYP3A4 gene in human tissues, currently available data indicate that numerous transcriptional factors such as hepatocyte nuclear factor-1 (HNF-1), HNF-2, HNF-4 and CCAAT/enhancer-binding protein (C/EBP) regulate the constitutive expression of CYP genes (41). Liver-enriched transcription factors C/EBP and HNF-3{gamma}, which are involved in the regulation of numerous liver-specific genes (42), trans-activate and cooperatively regulate hepatic-specific CYP3A4 gene expression (43,44). Because these liver-enriched trans-activating factors play an important roles in the constitutive expression of CYP3A4, variations in their expression could ultimately be responsible for the different expression levels of CYP3A4 found in various human tissues; CYP3A4 is expressed primarily in liver and intestine and at very low and physiologically insignificant levels in lymphoblasts (45). Although transcriptional elements in lymphoblasts are suggestive of a limited expression (low or absent), allelic variation was clearly observed in lymphoblasts as well as in liver samples (Figs 4 and 6). Thus, these results suggest that it is unlikely that the transcriptional elements described earlier are involved in the allelic imbalance.

Initially in the present study, we could not find any functional SNPs in the CYP3A4 gene. These results suggest that the post-transcriptional regulation of CYP3A4 gene expression is likely to be similar for both alleles. As a majority of the differentially expressed genes have a virtually identical sequence in their mRNAs (46), transcriptional initiation by cis-acting components is one of the most important controls in regulating the variation in allelic gene expression. The deviation of allelic variation varied among samples (Fig. 3), which also suggests that the variation could result from the allelic heterogeneity of one or more cis-acting regulatory polymorphisms (47) or from epigenetic factors such as DNA methylation (48) and the existence of non-coding RNAs (49). In this regard, we examined methylation status in the CpG island, which is ~30 kb upstream of the translational start codon, using six Caucasian liver samples with high and low levels of total CYP3A4 mRNA by cloning and sequencing bisulfite-treated DNA. However, unfortunately, we did not observe allele-specific differential methylation (Fig. 5).

Recently, Lo et al. (22) reported that 326 of 602 genes showed a preferential expression of one allele, and 170 of those showed greater than a 4-fold difference between the two alleles. Interestingly, some of the genes that showed a skewed allelic expression were located next to each other (i.e. clustered), and a subset of these genes is located in known imprinting domains. ASB4, DLX5, SGCE and PEG10, which have previously been reported to be imprinted, are located in the imprinting domain at 7q21–q31 (21). The human CYP3A genes, CYP3A43, CYP3A4, CYP3A7 and CYP3A5, consist of a cluster spanning 231 kb within this domain, at 7q21–q22.1 (19,20). In addition, two CYP3A pseudogenes are formed in two intergenic regions (CYP3A4–CYP3A7 and CYP3A7–CYP3A5) (50). These unique gene-structural features suggest that the allelic variation in hepatic CYP3A4 expression can be attributed to unknown non-coding RNA(s). The paternally expressed non-coding RNA (Air RNA) overlapping one of three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been reported to play an important role in repression of all three genes on the paternal expression (49). Nevertheless, we cannot exclude the possibility that the cis-acting regulatory polymorphisms (47) responsible for the change in CYP3A4 expression reside far up- and downstream of the gene of the affected allele. Indeed, Wojnowski and Brockmoller (51) have recently indicated a hepatic transcriptional imbalance of the CYP3A5 gene in CYP3A5*1A/*3 heterozygous samples, and the cis-acting *1A variant, which increases the expression of the CYP3A5 gene transcript from the allele carrying the variant, is a possible mechanism for the imbalance.

One report provided evidence that allelic variation can be transmitted by Mendelian inheritance (16). As shown in Fig. 4B, our results indicated that the allelic variation of CYP3A4 expression was also inherited, as has been observed in the genes Calpain-10 and PKD2 (16). Yan et al. (16) identified three informative families and found that altered expression of the genes CAPN10 and PKD2 was consistently inherited with a single haplotype defined by at least two adjacent microsatellite markers. In contrast to the findings by Yan et al. (16), although the allelic variation of CYP3A4 expression was consistent with Mendelian inheritance, allelic expression ratio appeared to be independent from genotypes. In the present study, we had tried to find the useful haplotypes within the regions we had analyzed; however, we were unsuccessful owing to large inter-individual variability in the SNP pattern (Fig. 1). The mechanism that generates allelic variation between two alleles remains unclear. However, as the expression of CYP3A4 is unlikely to be regulated by imprinting, these results also suggest the existence of unknown, unidentified cis-acting inherited variations influencing gene expression. If cis-acting inherited variations in gene expression are common among normal populations, an insight into how this occurs in individuals may help us to understand the large variability in CYP3A4 phenotypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Population samples
We examined allelic frequencies of C78013T and C78649T mutations in genomic DNA samples from unrelated Caucasian, Japanese and African American volunteers (96 subjects each) (Tennessee Blood Services, Memphis, TN, USA). We also obtained samples from the following sources: 22 lymphoblast samples for which the parental origin of the CYP3A4 alleles was determined (35); 18 livers (selected from 40 Caucasian donors, National Disease Research Interchange, Philadelphia, PA, USA) for which allelic expression variations and total hepatic CYP3A4 mRNA and hnRNA levels were determined, and 10 of the 18 liver samples in which testosterone 6ß-hydroxylation capabilities had been analyzed. We sequenced the CYP3A4 gene, 5'-flanking region and 3'-UTR in eight of the 18 liver samples whose total CYP3A4 mRNA and hnRNA levels had been determined. We also determined methylation status in six of the 18 liver samples. EB virus-transformed lymphoblast cultures were obtained using standard procedures. This study was approved by the Ethical Board of the Faculty of Medicine, Tottori University and informed consent was obtained from all individuals.

Primers and sequencing
We designed 72 primer sets based on a published sequence (GenBank accession no. AF280107.1) to amplify the CYP3A4 gene, CYP3A4 5'-flanking region and 3'-UTR; the amplicons were ~900 bp long (sequence available on request). Primer pairs were used for 30–40 cycles to amplify genomic DNA. The following conditions were used in each cycle: 95°C for 40 s, 52°C for 45 s, and 72°C for 1 min. PCR products were sequenced either directly or after subcloning using BigDye Terminator (Applied Biosystems, Foster City, CA, USA) sequencing. The sequencing primers were those used in the PCR amplifications. The sequence of both strands was analyzed for products from at least two independent PCR amplifications to ensure that the identified mutations were not PCR-induced artifacts.

cDNA synthesis
Total RNA was extracted with an RNAeasy Kit (Qiagen, Hilden, Germany) from EB virus-transformed lymphoblasts and liver samples. Prior to RT, total RNA samples were first treated with RNase-free DNase I, and then digested with HaeIII and MboII (Takara, Kyoto, Japan). Hae III and MboII digest the potential DNA template which would lead to the amplification of both alleles and thus mask allelic variation. The RNA samples were then reverse-transcribed into first strand cDNA with 1 µg of total RNA, 4 µl of 5xfirst strand buffer, 4 µl of 0.1 mM DTT, 1 µl of 500 µg/ml random primer (Promega, Madison, WI, USA), 4 µl of 10 mM dNTP mixture and 200 U of SuperScript II RNase H– reverse transcriptase (Life Technologies, Rockville, MD, USA). The reaction was incubated at 42°C for 60 min. RT reactions were always carried out in the presence or absence of reverse transcriptase to ensure that genomic DNA did not contaminate the subsequent PCR. In all experimental procedures, no amplification was detected in the absence of RT, excluding DNA contamination.

Assessment of allelic variation (estimation of allelic expression ratio)
To assess allele-specific expression of CYP3A4, an MnlI RFLP and an AciI RFLP in intron 7 (for liver samples), and an MnlI RFLP in intron 7 and an AfaI RFLP in intron 10 (for lymphoblast samples) were analyzed. Primer sequences for cDNA amplification were as follows: MnlI RFLP, 5'-TATCAGCCCCCTGTCACAAAC-3' (forward) and 5'-TTCATGCCACAACATAGTAAA-3' (reverse); AciI RFLP, 5'-CAATAGATAAAGGCAAAGAGA-3' (forward) and 5'-GAAAGACTGCTGTAGGAAAAA-3' (reverse); AfaI RFLP, 5'-GCAGTGTTCTCTCCTTCATTATGTA-3' (forward) and 5'-CTATGTTTCTTTCTTTTTCTTTTCA-3' (reverse). All PCR products contain either a HaeIII or MboII restriction site. PCR was carried out under the same conditions for the screening of the variants, but only for 24–30 cycles. RFLP products were electrophoresed on a 3% agarose gel, then stained with SYBR green I (Takara). The relative expression of each allele was quantified on the basis of the difference in band intensities between the two alleles with a fluorescence image analyzer (Hitachi, Tokyo, Japan) using Analysis Version 6.0 software. As a control, genomic DNA PCR–RFLP products were included and ratios of the allele-specific band intensities were taken as a 1 : 1 allelic representation. In order to eliminate sampling or measurement error, we conducted the experiment for each sample with three replicates.

Methylation analysis
The methylation status of the CpG island which is ~30 kb upstream of the translational start codon (nt 36441–36896, GenBank accession no. AF280107), was confirmed by the bisulfite sequencing method (52). DNA was treated with sodium bisulfite using a CpGenome DNA modification kit (Intergen, Purchase, NY, USA) according to the manufacturer's instructions. PCR was performed in a total volume of 25 µl consisting of 50 ng of bisulfite modified genomic DNA, 0.625 U of DNA polymerase and 0.25 µM of each primer; 5'-GGGTTTTATTTAGTTTGAGTTT-3' (forward) and 5'-TAACCCCTCCTCTACATTCTAT-3' (reverse). After an initial denaturation at 95°C for 9 min, 43 cycles of 40 s at 95°C, 45 s at 52°C and 1 min at 72°C, as well as a final extension for 5 min at 72°C, were performed. The PCR product was cloned into the pGEM-T easy vector (Promega, Madison, WI, USA) then transformed into JM-109 (Promega), and plasmid DNA was collected by QIAprep Spin Mini-prep kit (Qiagen,). The CpG methylation status of individual DNA strands was determined on the basis of a comparison with the sequence obtained from the genomic DNA without the addition of bisulfite modifications. Percent methylation of each site was determined by dividing the number of methylated CpGs at a specific site by the total number of clones analyzed (n=20 in all cases).

Quantitative real-time PCR
Quantification of total hepatic CYP3A4 mRNA and hnRNA was performed by real-time PCR detection using an ABI PRISM 7700 sequence detector (Applied Biosystems) with SYBR green detection of amplification products. Amplification mixtures contained 12.5 µl of 2xSYBR green I Universal PCR Mix (Applied Biosystems), 0.5 µl of cDNA synthesis mixture, 5 pmol each of the forward and reverse primers and distilled water in a total volume of 25 µl. All primers were designed using the PrimerExpress program (Applied Biosystems). Primers for CYP3A4 mRNA were directed to a sequence that spans the junction of exons 12 and 13, corresponding to open reading frame 1405–1465; 5'-AAAGAAACACAGATCCCCCTGAA-3' (forward) and 5'-CGGGTTTTTCTGGTTGAAGAAGT-3' (reverse). CYP3A4 hnRNA primers were directed to a sequence located at bases +2253 to +2351 within intron 12 of the CYP3A4 gene sequence; 5'-CACAGGTTTCCATGAATTTGTCT-3' (forward) and 5'-AAGATTGGACAGTGAGAGCATTC-3' (reverse). The copy number of the transcript was measured against a copy-number standard curve of cloned target templates consisting of serial 10-fold dilution points. ß2-Microglobulin mRNA was used as the reference message for both CYP3A4 mRNA and hnRNA.

Testosterone 6ß-hydroxylation capability in human liver samples
All incubations were performed in duplicate in solutions containing potassium phosphate (0.1 M, pH 7.4) and human liver microsomes (0.05 mg). Testosterone (final concentration, 30 µM) was added to the incubation mixture at a final methanol concentration of 1%, and the mixtures were incubated at 37°C for 10 min. We added a NADPH-generating system to initiate the reaction. Reactions were terminated by the addition of 100 µl of ice-CH3CN. We used high-performance liquid chromatography (HPLC) to measure the quantities of extracted compounds (53).


   ACKNOWLEDGEMENTS
 
This study was supported by RR2002, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports and Technology, and Health and Labour Science Research Grants (Research on Advanced Medical Technology) from The Ministry of Health, Labour and Welfare, Tokyo, Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Hospital Pharmacy, Faculty of Medicine, Tottori University, Nishi-machi 36-1, Yonago 6838504, Japan. Tel: +81 859348385; Fax: +81 859348087; Email: ieiri{at}grape.med.tottori-u.ac.jp


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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