The CYP2C19 Intron 2 Branch Point SNP is the Ancestral Polymorphism Contributing to the Poor Metabolizer Phenotype in Livers with CYP2C19*35 and CYP2C19*2 Alleles.

CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.

Databases in the area of pharmacogenetics.

In the area of pharmacogenetics and personalized health care it is obvious that databases, providing important information of the occurrence and consequences of variant genes encoding drug metabolizing enzymes, drug transporters, drug targets, and other proteins of importance for drug response or toxicity, are of critical value for scientists, physicians, and industry. The primary outcome of the pharmacogenomic field is the identification of biomarkers that can predict drug toxicity and drug response, thereby individualizing and improving drug treatment of patients. The drug in question and the polymorphic gene exerting the impact are the main issues to be searched for in the databases. Here, we review the databases that provide useful information in this respect, of benefit for the development of the pharmacogenomic field.

Intronic polymorphisms of cytochromes P450.

The cytochrome P450 enzymes active in drug metabolism are highly polymorphic. Most allelic variants have been described for enzymes encoded by the cytochrome P450 family 2 (CYP2) gene family, which has 252 different alleles. The intronic polymorphisms in the cytochrome P450 genes account for only a small number of the important variant alleles; however, the most important ones are CYP2D6*4 and CYP2D6*41 , which cause abolished and reduced CYP2D6 activity, respectively, and CYP3A5* 3 and CYP3A5*5 , common in Caucasian populations, which cause almost null activity. Their discoveries have been based on phenotypic alterations within individuals in a population, and their identification has, in several cases, been difficult and taken a long time. In light of the next-generation sequencing projects, it is anticipated that further alleles with intronic mutations will be identified that can explain the hitherto unidentified genetic basis of inter-individual differences in cytochrome P450-mediated drug and steroid metabolism.

The Human Cytochrome P450 (CYP) Allele Nomenclature website: a peer-reviewed database of CYP variants and their associated effects.

Pharmacogenetics affects both pharmacokinetics and pharmacodynamics, thereby influencing an individual's response to drugs, both in terms of response and adverse reactions. Within the area of pharmacogenetics, findings of genetic variation influencing drug levels have been more prevalent, and variation in the cytochrome P450 (CYP) enzymes is one of the most common causes. Much of the work concerning sequence variations in CYPs aims at finding biomarkers of use for individualised treatment, thereby increasing the treatment response, lowering the number of side effects and decreasing the overall cost of treatment regimens. For over ten years, the Human Cytochrome P450 Allele Nomenclature (CYP-allele) website (http://www.cypalleles.ki.se/) has offered a database of genetic information on CYP variants, along with effects at the molecular as well as clinical level. Thus, this database serves as an assembly of past, current and soon-to-be published information on CYP alleles and their outcome effects. The website is used by academic researchers and companies (eg as a tool in drug development and for outlining new research projects). By providing peer-reviewed genetic information on CYP enzymes, the CYP-allele website has become increasingly popular and widely used. Recently, NADPH cytochrome P450 oxidoreductase (POR), the electron donor for CYP enzymes, was included on the website, which already contains 29 CYP genes, hence POR alleles are now also designated using the star allele (POR*) nomenclature. Although most CYPs on the CYP-allele website are involved in the metabolism of xenobiotics, polymorphic enzymes with endogenous functions are also included. Each gene on the CYP-allele website has its own webpage that lists the different alleles with their nucleotide changes, their functional consequences and links to publications in which the allele has been identified and/or characterised. Thus, the CYP-allele website offers a rapid online publication of new alleles, as well as providing an overview of peer-reviewed data.

Molecular genetics and epigenetics of the cytochrome P450 gene family and its relevance for cancer risk and treatment.

The cytochromes P450 (CYPs) are very efficient catalysts of foreign compound metabolism and are responsible for the major part of metabolism of clinically important drugs. The enzymes are important in cancer since they (a) activate dietary and environmental components to ultimate carcinogens, (b) activate or inactivate drugs used for cancer treatment, and (c) are potential targets for anticancer therapy. The genes encoding the CYP enzymes active in drug metabolism are highly polymorphic, whereas those encoding metabolism of precarcinogens are relatively conserved. A vast amount of literature is present where investigators have tried to link genetic polymorphism in CYPs to cancer susceptibility, although not much conclusive data have hitherto been obtained, with exception of CYP2A6 polymorphism and tobacco induced cancer, to a great extent because of lack of important functional polymorphisms in the genes studied. With respect to anticancer treatment, the genetic CYP polymorphism is of greater importance, where treatment with tamoxifen, but also with cyclophosphamide and maybe thalidomide is influenced by CYP genetic variants. In the present review we present updates on CYP genetics, cancer risk and treatment and also epigenetic aspects of interindividual variability in CYP expression and the use of these enzymes as targets for cancer therapy. We conclude that the CYP polymorphism does not predict cancer susceptibility to any large extent but that this polymorphism might be an important factor for optimal cancer therapy using selected anticancer agents.

Pharmacogenetic biomarkers as tools for improved drug therapy; emphasis on the cytochrome P450 system.

Important interindividual differences in drug pharmacokinetics cause absence of drug response or adverse drug reactions in significant fractions of the populations. The identification of the major enzymes participating, and the elucidation of the genetic basis for this variation in particular among cytochromes P450, provide tools for a personalized medicine treatment, which can make drug therapy much more effective at a lower cost. Much of the pioneering work linking drug metabolizing phenotype to genetic polymorphism among the P450 enzymes has been carried out at Karolinska Institutet. In this review we give a background and description of this work as well as the important implications for future medicine.

Linkage disequilibrium between the CYP2C19*17 allele and wildtype CYP2C8 and CYP2C9 alleles: identification of CYP2C haplotypes in healthy Nordic populations.

PURPOSE: To determine the distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations with special focus on linkage disequilibrium. METHODS: A total of 896 healthy subjects from three Nordic populations (Danish, Faroese, and Norwegian) were genotyped for five frequent and clinically important CYP2C allelic variants: the defective CYP2C8*3, CYP2C9*2, CYP2C9*3, and CYP2C19*2 alleles, and the CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire population. RESULTS: Ten CYP2C haplotypes were inferred, the most frequent of which (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1, CYP2C9*1, and CYP2C19*1. The second most frequent haplotype (19%) is composed of CYP2C19*17, CYP2C8*1, and CYP2C9*1. This predicted haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896 subjects. CONCLUSION: CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wildtype CYP2C8*1 and CYP2C9*1 alleles, which effectively makes it a determinant for a haplotype exhibiting an efficient CYP2C substrate metabolism.

Association between CYP2C19 polymorphism and depressive symptoms.

Cytochrome P450 2C19 (CYP2C19) is a polymorphic enzyme active in the metabolism of for example diazepam and the antidepressants sertraline, citalopram, and escitalopram, whereby allelic variants cause increased (CYP2C19*17) or abolished (mainly CYP2C19*2) enzymatic activity in drug metabolism. In light of the importance of CYP2C19 in the metabolism of psychoactive substances we considered it of interest to investigate the relationship between CYP2C19 polymorphisms and depressive symptoms in 1,472 subjects of European ancestry (45-98 years old) from the Swedish Twin Registry. Depressive symptoms were assessed using the Center of Epidemiologic Studies Depression (CES-D) scale. We found that poor metabolizers lacking CYP2C19 activity (PMs, CYP2C19*2/*2) had significantly lower levels of depressive symptoms than extensive metabolizers (EMs, CYP2C19*1/*1) (P = 0.0018). The size of this difference was in the same range as that between subjects reported taking antidepressants (n = 104) and those without antidepressant treatment (P < 0.0001). Our results suggest for the first time that the CYP2C19 polymorphism might be of importance for depressive symptoms, as here shown for older European adults.

Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects.

The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies significantly, and the cause has been suggested to be mainly of genetic origin but the exact molecular basis remains unknown. We present a review of the major polymorphic CYP alleles and conclude that this variability is of greatest importance for treatment with several antidepressants, antipsychotics, antiulcer drugs, anti-HIV drugs, anticoagulants, antidiabetics and the anticancer drug tamoxifen. We also present tables illustrating the relative importance of specific common CYP alleles for the extent of enzyme functionality. The field of pharmacoepigenetics has just opened, and we present recent examples wherein gene methylation influences the expression of CYP. In addition microRNA (miRNA) regulation of P450 has been described. Furthermore, this review updates the field with respect to regulatory initiatives and experience of predictive pharmacogenetic investigations in the clinics. It is concluded that the pharmacogenetic knowledge regarding CYP polymorphism now developed to a stage where it can be implemented in drug development and in clinical routine for specific drug treatments, thereby improving the drug response and reducing costs for drug treatment.

Generation of mice transgenic for human CYP2C18 and CYP2C19: characterization of the sexually dimorphic gene and enzyme expression.

CYP2C19 is an important enzyme for human drug metabolism, and it also participates in the metabolism of endogenous substrates, whereas the CYP2C18 enzyme is not expressed in human liver despite high mRNA expression. Mice transgenic for the human CYP2C18 and CYP2C19 genes were generated. Quantitative mRNA analysis showed CYP2C18 and CYP2C19 transcripts in liver, kidneys, and heart to be expressed in a sexually dimorphic manner, with male mice having 2- to 100-fold higher levels. Transcript levels in the small intestine were somewhat higher than liver but were similar in both sexes. Transgene mRNA expression was much lower in lung and brain and least in the heart. Immunoblotting using an antipeptide antiserum, reactive with human CYP2Cs and mouse CYP2C70, revealed increased immunoreactive protein in liver microsomes from heterozygous transgenic male mice and a concomitant increase in 5'-hydroxylation of R-omeprazole and S-mephenytoin intrinsic clearance, consistent with CYP2C19 overexpression. A CYP2C18-specific antiserum showed that this enzyme was not expressed in livers or kidneys from heterozygous transgenic mice, but the antiserum had high affinity for recombinant CYP2C18 expressed in COS-7 cells. It is concluded that 1) both the CYP2C18 and CYP2C19 genes are subject to sexually dimorphic regulation in murine liver, kidney, and heart; 2) the CYP2C18 protein is not expressed in murine liver or kidney despite high levels of the corresponding mRNA; and 3) this transgenic model may be suitable for studying sex-dependent regulation of the human CYP2C genes and possibly serve as an in vivo model for CYP2C19-dependent drug metabolism.

Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects.

PURPOSE: Ultrarapid drug metabolism of antidepressants has been associated with therapeutic failures. The CYP2C19*17 allele has been associated with higher levels of CYP2C19 gene transcription and increased rates of omeprazole and mephenytoin metabolism. The aim of this study was to compare the impact of the CYP2C19*17 allele on omeprazole single-dose kinetics with escitalopram exposure at steady state in volunteers genotyped as either CYP2C19*17/*17 or CYP2C19*1/*1. METHODS: Sixteen healthy volunteers participated in both study parts, five homozygous for CYP2C19*17 and 11 homozygous for CYP2C19*1. Individual pharmacokinetic parameters were determined after single-dose omeprazole of 40 mg and after 1 week on escitalopram 5 mg b.i.d. RESULTS: Escitalopram area under the concentration time curve from zero to 12 h (AUC(0-12h)) was 21% lower in homozygous carriers of CYP2C19*17 compared with CYP2C19*1 (p = 0.08). There was a significant correlation between escitalopram exposure at steady state and the single-dose kinetics of omeprazole (Spearman correlation coefficient of 0.67; p = 0.006). CONCLUSION: Based on our investigation using two different CYP2C19 substrates, we concluded that a clinically significant difference in escitalopram or omeprazole kinetics between the genotypes appears unlikely.

A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants.

BACKGROUND AND OBJECTIVE: Many drugs, including proton pump inhibitors and certain antidepressants, are metabolized by the polymorphic cytochrome P450 (CYP) 2C19 enzyme. A significant portion of extensive metabolizers do not reach appropriate drug levels, and our objective was to investigate any genetic background. METHODS: The 5'-flanking region of the CYP2C19 gene from subjects with rapid omeprazole metabolism was sequenced, and CYP2C19 phenotype-genotype associations were analyzed in Swedish (n = 107) and Ethiopian (n = 126) extensive metabolizers. The relationship of the metabolic ratio of omeprazole (omeprazole/5-hydroxyomeprazole in plasma 3 hours after drug intake) with the area under the plasma concentration-time curve was used for prediction studies. Electrophoretic mobility shift assays were conducted by use of human nuclear protein extracts. Hepatic reporter vector transfections were carried out in CD1 mice. RESULTS: We identified a novel allele (CYP2C19*17) carrying -806C>T and -3402C>T, with a frequency of 18% in both Swedes and Ethiopians and 4% in Chinese subjects. In Swedes the metabolic ratio of omeprazole was higher in subjects homozygous for CYP2C19*1 (median, 0.50 [interquartile range, 0.37-0.73]) than in those homozygous for CYP2C19*17 (median, 0.25 [interquartile range, 0.15-0.33]) (P = .010). In Ethiopians a similar difference in the S/R-mephenytoin ratio was observed between individuals homozygous for CYP2C19*1 (median, 0.20 [interquartile range, 0.12-0.37]) and those homozygous for CYP2C19*17 (median, 0.05 [interquartile range, 0.03-0.06]) (P = .013). Electrophoretic mobility shift assays showed specific binding of human hepatic nuclear proteins to an element carrying -806T but not -806C. Reporter vector experiments showed an increased transcriptional activity of the CYP2C19*17 allele in vivo in mice. Predictions revealed that CYP2C19*17 homozygotes would attain 35% to 40% lower omeprazole area under the plasma concentration-time curve values than subjects homozygous for CYP2C19*1 taking standard doses of omeprazole. CONCLUSION: CYP2C19*17 is likely to cause therapeutic failures in drug treatment with, for example, proton pump inhibitors and antidepressants.

The human cytochrome P450 Allele Nomenclature Committee Web site: submission criteria, procedures, and objectives.

Interindividual variability in xenobiotic metabolism and drug response is extensive. Genetic factors are predicted to account for 15-30% of this variability in general, but for certain drugs the genetic factor is the major determinant for outcome of drug therapy. Of particular importance for drug metabolism, drug response, and adverse drug reactions are the cytochrome P450 (CYP) enzymes, many of which are polymorphic. An essential basis for research and applications regarding interindividual variability in xenobiotic metabolism and toxicity by polymorphic CYPs is to have a common nomenclature for genetic variants and a system that allows researchers to be rapidly updated within the field. Since 1999 this has been achieved by the operation of the Human Cytochrome P450 Allele Nomenclature Committee Web site (www.imm.ki.se/CYPalleles/), where novel allelic variants are published after peer review. Currently, this Web site covers the nomenclature for polymorphic alleles of 22 CYP isoforms including more than 200 functionally different variants. Each CYP has its own Web page, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications where the allele has been identified and characterized. The CYP allele Web site offers a rapid on-line publication of new alleles, provides an overview of peer-reviewed data, and serves as a form of quality control on research on new alleles.

New approaches and applications in chemical biology.

The 15th International Symposium on Microsomes and Drug Oxidations: Chemical Biology in the Postgenomic Era--New Approaches and Applications in Mainz, Germany, July 4-9, 2004, had 570 attendees and was nicely organized by Franz and Barbara Oesch and colleagues. At the meeting, 73 different lectures were presented and about 240 posters were shown. This review discusses most of the lectures presented at the Symposium.

Update on allele nomenclature for human cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database.

Interindividual variability in xenobiotic metabolism and drug response is extensive and genetic factors play an important role in this variation. A majority of clinically used drugs are substrates for the cytochrome P450 (CYP) enzyme system and interindividual variability in expression and function of these enzymes is a major factor for explaining individual susceptibility for adverse drug reactions and drug response. Because of the existence of many polymorphic CYP genes, for many of which the number of allelic variants is continually increasing, a universal and official nomenclature system is important. Since 1999, all functionally relevant polymorphic CYP alleles are named and published on the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Web site (http://www.cypalleles.ki.se). Currently, the database covers nomenclature of more than 660 alleles in a total of 30 genes that includes 29 CYPs as well as the cytochrome P450 oxidoreductase (POR) gene. On the CYP-allele Web site, each gene has its own Webpage, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications identifying or characterizing the alleles. CYP2D6, CYP2C9, CYP2C19, and CYP3A4 are the most important CYPs in terms of drug metabolism, which is also reflected in their corresponding highest number of Webpage hits at the CYP-allele Web site.The main advantage of the CYP-allele database is that it offers a rapid online publication of CYP-alleles and their effects and provides an overview of peer-reviewed data to the scientific community. Here, we provide an update of the CYP-allele database and the associated nomenclature.

Strong effects of environmental factors on prevalence and course of major depressive disorder are not moderated by 5-HTTLPR polymorphisms in a large Dutch sample.

BACKGROUND: There is ongoing interest in the possible interaction of the serotonin-transporter-linked polymorphic region (5-HTTLPR) with environmental factors in determining Major Depressive Disorder (MDD). The current study contributes to this research area by comprehensively examining the interaction-effects and direct-effects of 5-HTTLPR and five environmental factors on MDD prevalence and course in a well-characterized longitudinal sample. METHODS: The sample consisted of 1625 patients with a CIDI-confirmed diagnosis of MDD and 1698 screened controls from the Netherlands. Four MDD outcomes were studied as dependent variables: one main MDD prevalence-outcome (all MDD), two more severe MDD prevalence-outcomes (suicidal and chronic MDD), and one MDD course outcome (chronic versus non-chronic MDD). Because SNP rs25531 modifies the effect of 5-HTTLPR, haplotypes of 5-HTTLPR and rs25531 were measured. For the four MDD outcome measures, we examined the direct effects of 5-HTTLPR/rs25531-haplotypes, five environmental factors (lifetime and recent stressful life-events, sexual abuse, low educational attainment, and childhood trauma) and their interaction in logistic regression models. RESULTS: The environmental factors had large and consistent effects on all four MDD outcomes, including course of MDD. The 5-HTTLPR/rs25531-haplotype had a suggestive effect on course of MDD, but not on presence of MDD. Gene-by-environment interaction was significant (<0.05) for one of the 20 tests performed, which is not more than expected by chance. LIMITATIONS: Environmental factors were not assessed before the onset of MDD. CONCLUSIONS: Environmental factors had a strong impact on the presence and course of MDD, but no evidence for gene-by-environment interaction was found.

Institutional profile: Karolinska Institutet.

Research in pharmacogenomics has been intensive at Karolinska Institutet (KI) for approximately 25 years. Initial initiatives were focused on the identification and characterization of novel CYP2D6 alleles causing ultrarapid or defective drug metabolism. Such discoveries were possible owing to the early implementation of therapeutic drug monitoring and the access to individuals phenotyped with respect to drug metabolism. The translational work at KI has been of utmost importance for successful research, including functional characterization and clinical validation of allelic variants in drug metabolism, as well as discoveries of novel polymorphisms, recent examples being the CYP2C19 and UGT2B17 genes. The clinical pharmacology laboratory at KI campus Huddinge is one of the leading sites for therapeutic drug monitoring in northern Europe and obtains an increasing number of clinical requests, also important for pharmacogenetic research. Furthermore, the recently opened Center for Hematology and Regenerative Medicine, with a clear translational emphasis, offers an opportunity for studying drug metabolism and toxicity in vitro by use of human hepatocytes.