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Allele-Specific Expression and Gene Methylation in the Control of CYP1A2 Mrna Level in Human Livers

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Allele-Specific Expression and Gene Methylation in the Control of CYP1A2 Mrna Level in Human Livers The Pharmacogenomics Journal (2009) 9, 208–217 & 2009 Nature Publishing Group All rights reserved 1470-269X/09 $32.00 www.nature.com/tpj ORIGINAL ARTICLE Allele-specific expression and gene methylation in the control of CYP1A2 mRNA level in human livers Roza Ghotbi1, Alvin Gomez2, The basis for interindividual variation in the CYP1A2 gene expression is not 3 1 fully understood and the known genetic polymorphisms in the gene provide Lili Milani , Gunnel Tybring , no explanation. We investigated whether the CYP1A2 gene expression is 3 Ann-Christine Syva¨nen , regulated by DNA methylation and displays allele-specific expression (ASE) Leif Bertilsson1, Magnus using 65 human livers. Forty-eight percent of the livers displayed ASE not Ingelman-Sundberg2 and associated to the CYP1A2 mRNA levels. The extent of DNA methylation of a 1 CpG island including 17 CpG sites, close to the translation start site, inversely Eleni Aklillu correlated with hepatic CYP1A2 mRNA levels (P ¼ 0.018). The methylation of 1Division of Clinical Pharmacology, Department two separate core CpG sites was strongly associated with the CYP1A2 mRNA of Laboratory Medicine, Karolinska University levels (P ¼ 0.005) and ASE phenotype (P ¼ 0.01), respectively. The CYP1A2 Hospital Huddinge, Karolinska Institutet, expression in hepatoma B16A2 cells was strongly induced by treatment with 2 Stockholm, Sweden; Section of 5-aza-20-deoxycytidine. In conclusion, the CYP1A2 gene expression is Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, influenced by the extent of DNA methylation and displays ASE, mechanisms Stockholm, Sweden and 3Molecular Medicine, contributing to the large interindividual differences in CYP1A2 gene Department of Medical Sciences, Uppsala expression. University, Uppsala, Sweden The Pharmacogenomics Journal (2009) 9, 208–217; doi:10.1038/tpj.2009.4; published online 10 March 2009 Correspondence: Dr E Aklillu, Division of Clinical Pharmacology, Department of Laboratory of Medicine, Keywords: CYP1A2; drug-metabolizing enzyme; gene expression; allele-specific expression; Karolinska Institutet, Karolinska University epigenetics; DNA methylation Hospital Huddinge, C-168, SE-141 86 Stockholm, Sweden. E-mail: [email protected] Introduction The human cytochrome P450 1A2 (CYP1A2) enzyme is expressed mainly in the liver and accounts for about 13% of the total P450 content in the liver.1 It has an important function in the metabolism of caffeine as well as of several clinically important drugs such as clozapine,2 phenacetin,3 verapmil,4 endogenous substrates such as melatonin,5 estradiol,6 and procarcinogenic substances including heterocyclic amines, arylamines and aflatoxin B1.7,8 The CYP1A2 gene shows large interindividual and interethnic differences in both mRNA expression level and enzyme activity.8–10 The underlying causes for this variation are not known and could be caused by genetic, epigenetic or environmental factors. Indeed, CYP1A2 is genetically polymorphic with more than 16 variant alleles described so far (http://www.cypalleles.ki.se/cyp1a2.htm). However, the low frequency of the functionally different variant alleles in the populations and the results of several genotype–phenotype association studies Received 7 October 2008; revised 13 January 2009; accepted 3 February 2009; published indicate a limited function of CYP1A2 genetic polymorphism as a cause of online 10 March 2009 interindividual variation in CYP1A2 gene expression or activity.11,12 In line of ASE and gene methylation of CYP1A2 R Ghotbi et al 209 this research, we have found one allele CYP1A2*1K causing genes26 causing transcriptional repression by inhibition of lower expression of the gene but it is present in Ethiopians13 binding of transcription factor. and almost absent in whites and Asians.11,12 The interethnic While appreciating the existence of the wide interindivi- differences in CYP1A2 activity is important and by control- dual variation in the constitutive CYP1A2 mRNA expression ling for the effects of smoking, oral contraceptive use and and enzyme activity, the underlying causes remain unclear. genotype, we recently reported the presence of significant It is generally accepted that most of the known SNPs in the differences in enzyme activity between Swedish, Korean and CYP1A2 gene and 50-flanking regions do not determine the Serbian populations, differences not explained by the observed large interindividual variability in constitutive known CYP1A2 single nucleotide polymorphisms (SNPs)/ hepatic CYP1A2 mRNA expression and enzyme activity.11 haplotypes.12,14 Identification of the molecular mechanism behind variation Apart from genetic polymorphisms, phenotypic variations in CYP1A2 enzyme activity may have important implica- are governed by other factors affecting gene expression, tions for drug safety/efficacy and cancer susceptibility. such as gene cis- and trans-acting transcriptional compo- Because the epigenetic factors and ASE can lead to nents, alternative splicing, RNA stability, expression of phenotypic variability in the pharmacodynamic and phar- regulatory RNAs and epigenetics such as histone modifica- macokinetic outcomes of drug therapy, we considered it of tions and DNA methylation.15 Imprinted genes (parent of interest to investigate the function of DNA methylation and origin-specific gene expression in somatic cells) and genes differential allelic expression in CYP1A2 gene regulation as subject to X chromosome inactivation display monoallelic determinants of interindividual variation in total CYP1A2 expression. Differential gene expression between the two mRNA expression and enzyme activity. The results indicate alleles or allele-specific expression (ASE) in nonimprinted the importance of two core methylation CpG sites located genes is also common in the human genome.16–19 The near the transcription start site to be associated with degree of differences in the expression between the two variations in the total CYP1A2 transcript level and differ- alleles varies from individual to individual causing pheno- ential allelic expression phenotype (Figure 1). typic variability. ASE reflects the presence of putative allele- specific cis-acting factors of either genetic or epigenetic Results origin. Consequently, ASE can be used as a promising quantitative phenotyping method for identifying cis-acting Detection of ASE in CYP1A2 factors as well as epigenetic variation influencing gene A total of 65 human liver samples were genotyped for the expression because individuals heterozygous for cis-acting marker SNP rs2470890 and of those 23 were found to be polymorphism show a different level of mRNA expression heterozygous for this SNP. The regions encompassing the 16,17,20 originating from one allele compared to the other. rs2470890 were PCR amplified from both genomic DNA The two major mechanisms for epigenetic modifications (gDNA) and mRNA (after conversion to cDNA), followed by are DNA methylation and histone deacetylation, and minisequencing analysis of each allele. PCR testing for interindividual variations in the degree of these mechan- gDNA and cDNA samples was carried out using the same isms could form a basis for interindividual differences in conditions for all fragments, and the amplicon size was gene expression. Epigenetic modifications and ASE in verified by agarose gel electrophoresis. The averages of the pharmacogenetic-related genes have been shown to affect signal fraction (SAllele1/(SAllele1 þ SAllele2)) from the cDNA expression and activity. ASE for the MDR1, CYP2D6 and were compared with the signal fraction from the respective 21 22 CYP2C9 genes has been reported recently. Hirota et al. gDNA as a reference to determine the presence of reported the occurrence of ASE in the CYP3A4 gene differential allelic expression. Of the 23 heterozygous liver expression that correlated with the total CYP3A4 mRNA samples, 11 (47.8%) displayed significant differences in their levels in liver but no clear association between gene allelic expression (Po0.05, Figure 2). There was no correla- methylation and total mRNA levels was found. Nakajima tion in these samples between the total mRNA expression 23 et al. indicated a function of histone deacetylation and level and allelic expression ratio or ASE phenotype. DNA methylation in cell-specific inducibility of the human CYP1 family. Tissue-specific variation in the degree DNA methylation frequency in human livers and hepatic CYP1A2 of gene methylation in CYP2E1 gene is associated with mRNA expression CYP2E1 mRNA levels and enzyme activity that has been The region covering the CpG island (Figure 3) was PCR reported previously.24 Furthermore, Hammons et al.25 amplified from gDNA after bisulfite treatment. DNA methy- showed an association between interindividual variation in lation frequency was determined by pyrosequencing using human hepatic CYP1A2 expression and degree of methyla- six internal sequencing primers that cover overlapping tion of a CpG site (bp À2759) in the 50-flanking region of the regions. Complete data concerning the DNA methylation CYP1A2 gene, with hypermethylation correlated with frequencies and mRNA quantification were obtained from decreased CYP1A2 expression. However, the CpG site 48 human livers. Analysis of the methylation pattern investigated by Hammons et al. is not located within a revealed two domains with region-specific differences in CYP1A2 CpG island and is located far away from the the distribution and level of CpG methylation. The first transcription start
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