ABSTRACT
This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.
Subject(s)
Alleles , Genetic Testing/standards , Pharmacogenetics/standards , Terminology as Topic , Genes , Genetic Testing/trends , Genetic Variation , Humans , Pharmacogenetics/trends , Precision MedicineABSTRACT
Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.
Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism , Liver/metabolism , Organic Anion Transporters, Sodium-Independent/genetics , Pharmacogenomic Variants/genetics , Pravastatin/metabolism , Biological Transport , Gene Expression Regulation , HEK293 Cells , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Heterozygote , Humans , Kinetics , Mutation, Missense , Organic Anion Transporters, Sodium-Independent/metabolism , Phenotype , Transfection , White People/geneticsABSTRACT
Gene copy number variants (CNVs) of CYP2E1 have been described but not functionally characterized. Here we investigated effects of CNVs on hepatic and lymphoblastoid CYP2E1 expression. Using available single-nuleotide polymorphism microarray data and quantitative PCR, CYP2E1 gene duplication and deletion carriers were identified. CYP2E1 mRNA, protein and enzyme activity (chlorzoxazone-6-hydroxylation) phenotypes of CYP2E1 were not associated with gene copy number. Analysis of gene expression in lymphoblastoid cell lines in relation to CNV confirmed this finding in an extrahepatic tissue and for other ethnicities. Further analyses identified a linked haplotype cluster with possible influence on gene expression. In summary, our data suggest a homeostatic, gene dosage-insensitive regulation of CYP2E1 expression by unknown gene dosage compensation mechanisms. This is in striking contrast to well-known structural variations of CYP2A6 and CYP2D6 that have a strong impact on expression and activity. These findings are important in the context of pharmacogenetic prediction.
Subject(s)
Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , DNA Copy Number Variations , Gene Dosage , Hepatocytes/enzymology , Pharmacogenomic Variants , Cell Line , Chlorzoxazone/metabolism , Databases, Genetic , Gene Deletion , Gene Duplication , Gene Expression Profiling/methods , Gene Expression Regulation, Enzymologic , Haplotypes , Humans , Hydroxylation , Oligonucleotide Array Sequence Analysis , Phenotype , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , White People/geneticsSubject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Drug Discovery/methods , Epigenomics/trends , Genetics/trends , Drug Discovery/trends , Gene Expression Regulation, Enzymologic/genetics , Genetic Variation , Humans , Isoenzymes/genetics , Polymorphism, Single NucleotideABSTRACT
Expression quantitative trait loci (eQTL) analysis is a powerful approach toward identifying genetic loci associated with quantitative changes in gene expression. We applied genome-wide association analysis to a data set of >300 000 single-nucleotide polymorphisms and >48 000 mRNA expression phenotypes obtained by Illumina microarray profiling of 149 human surgical liver samples obtained from Caucasian donors with detailed medical documentation. Of 1226 significant associations, only 200 were validated when comparing with a previously published similar study. Potential explanations for low replication rate include differences in microarray platforms, statistical modeling, covariates considered and origin and collection procedures of tissues. Focused analysis revealed a subset of 95 associations related to absorption, distribution, metabolism and excretion of drugs. Of these, 21 were true replications and 74 were newly discovered associations in enzymes, transporters, transcriptional regulators and other genes. This study extends our knowledge about the genetics of inter-individual variability of gene expression with particular emphasis on pharmacogenomics.
Subject(s)
Liver/metabolism , Pharmacogenetics/methods , Pharmacokinetics , Quantitative Trait Loci , Absorption , Biological Transport , Chromosome Mapping/methods , Female , Gene Expression , Gene Expression Profiling/methods , Genetic Association Studies/methods , Genome-Wide Association Study/methods , Genotype , Humans , Inactivation, Metabolic , Male , Middle Aged , Phenotype , Polymorphism, Single Nucleotide , RNA, Messenger/genetics , Tissue DistributionABSTRACT
Pharmacogenomics follows the concept of individualized medicine via targeted and tailored therapy for each patient. This is aided by current progress in human genome research that provided the basis to link genetic markers with pathogenesis and progression for the definition of new therapy options. To achieve this it will be necessary to show the validity and clinical utility of pharmacogenomic tests prior to release. The implementation of pharmacogenomics into clinical practise is a momentous future challenge that requires the establishment of interdisciplinary networks and professional organizations.
Subject(s)
Genetic Predisposition to Disease/genetics , Genetic Testing/trends , Genetic Therapy/trends , Pharmacogenetics/trends , Precision Medicine/trends , HumansABSTRACT
The response to statins shows large interpatient variability. Atorvastatin delta-lactone is pharmacologically inactive but has been associated with toxicity. We investigated the role of UDP-glucuronosyltransferases (UGTs) in atorvastatin lactonization. In human liver microsomes, lactonization was correlated with UGT1A3 (r(s) = 0.61, P < 0.0001) but not with UGT1A1. Surprisingly, lactone formation was significantly higher in carriers of UGT1A1*28, an allele that is associated with lower UGT1A1 expression. We show that this inverse correlation is due to extensive linkage disequilibrium in the UGT1A locus and that several UGT1A3 haplotypes are associated with strong increases in UGT1A3 expression in vitro. Analyses of the pharmacokinetic parameters of atorvastatin and metabolites in genotyped volunteers confirmed that there is an increase in atorvastatin lactonization in carriers of UGT1A3*2 in vivo. The potential of UGT genotyping to identify patients who are at increased risk for failure of therapy and/or adverse effects of statins warrants further investigation.
Subject(s)
Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Heptanoic Acids/metabolism , Lactones/metabolism , Polymorphism, Genetic/genetics , Pyrroles/metabolism , Atorvastatin , Heptanoic Acids/adverse effects , Heptanoic Acids/pharmacology , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Pyrroles/adverse effects , Pyrroles/pharmacologyABSTRACT
The multidrug resistance protein 4 (MRP4) is an efflux transporter involved in the transport of endogenous substrates and xenobiotics. We measured MRP4 mRNA and protein expression in human livers and found a 38- and 45-fold variability, respectively. We sequenced 2 kb of the 5'-flanking region, all exons and intron/exon boundaries of the MRP4 gene in 95 patients and identified 74 genetic variants including 10 non-synonymous variations, seven of them being located in highly conserved regions. None of the detected polymorphisms was significantly associated with changes in the MRP4 mRNA or protein expression. Immunofluorescence microscopy indicated that none of the non-synonymous variations affected the cellular localization of MRP4. However, in cholestatic patients the MRP4 mRNA and protein expression both were significantly upregulated compared to non-cholestatic livers (protein: 299+/-138 vs 100+/-60a.u., P<0.001). Taken together, human hepatic MRP4 expression is highly variable. Genetic variations were not sufficient to explain this variability. In contrast, cholestasis is one major determinant of human hepatic MRP4 expression.
Subject(s)
Cholestasis/metabolism , Liver/metabolism , Multidrug Resistance-Associated Proteins/biosynthesis , Multidrug Resistance-Associated Proteins/genetics , Adult , DNA/genetics , DNA/isolation & purification , Female , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Genetic Variation , Genotype , Haplotypes , Humans , Immunohistochemistry , Introns , Liver/anatomy & histology , Liver/chemistry , Male , Microscopy, Fluorescence , Polymorphism, Genetic/genetics , Polymorphism, Single Nucleotide/genetics , Protein Conformation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Terminology as TopicABSTRACT
Pharmacogenetics holds promise in HIV treatment because of the complexity and potential toxicity of multidrug therapies that are prescribed for long periods. Thus far, few candidate genes have been examined for a limited number of allelic variants, but a number of confirmed associations have already emerged. A change in paradigm emerges from the availability of the HapMap, the wealth of data on less-common genetic polymorphisms, and new genotyping technology. This review presents a comprehensive analysis of the existing literature on pharmacogenetic determinants of antiretroviral drug exposure, drug toxicity, as well as genetic markers associated with the rate of disease progression. It is expected that larger-scale comprehensive genome approaches will profoundly change the landscape of knowledge in the future.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Infections/drug therapy , Pharmacogenetics , Animals , Anti-HIV Agents/adverse effects , Anti-HIV Agents/pharmacokinetics , Disease Progression , Genetic Markers , Genetic Predisposition to Disease , HIV Infections/genetics , Humans , Polymorphism, GeneticABSTRACT
The pyrazolone drug metamizole is a widely used analgesic. Analysis of liver microsomes from patients treated with metamizole revealed selectively higher expression of cytochromes P450, CYP2B6 and CYP3A4 (3.8- and 2.8-fold, respectively), and 2.9-fold higher bupropion hydroxylase activity compared with untreated subjects. Further investigation of metamizole and various derivatives on different potential target genes in human primary hepatocytes demonstrated time- and concentration-dependent induction by metamizole of CYP2B6 (7.8- and 3.1-fold for mRNA and protein, respectively, at 100 muM) and CYP3A4 (2.4- and 2.9-fold, respectively), whereas other genes (CYP2C9, CYP2C19, CYP2D6, NADPH:cytochrome P450 reductase, ABCB1, constitutive androstane receptor (CAR), pregnane X receptor (PXR)) were not substantially altered. Using reporter gene assays, we show that metamizole is not acting as a direct ligand to either PXR or CAR, suggesting a phenobarbital-like mechanism of induction. These data warrant further studies to elucidate the drug-interaction potential of metamizole, especially in patients with long-term treatment.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aryl Hydrocarbon Hydroxylases/biosynthesis , Cytochrome P-450 Enzyme System/biosynthesis , Dipyrone/pharmacology , Oxidoreductases, N-Demethylating/biosynthesis , Aged , Aryl Hydrocarbon Hydroxylases/genetics , Blotting, Western , Catalysis , Cells, Cultured , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B6 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , DNA/biosynthesis , DNA/genetics , Dipyrone/analogs & derivatives , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Female , Genotype , Hepatocytes/drug effects , Humans , In Vitro Techniques , Isoenzymes/metabolism , Liver/drug effects , Liver/enzymology , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Middle Aged , Oxidoreductases, N-Demethylating/genetics , Plasmids , Pregnane X Receptor , RNA/biosynthesis , RNA/genetics , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Steroid/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/drug effectsABSTRACT
To assess the association of CYP2B6 allelic diversity with efavirenz (EFV) pharmacokinetics, we performed extensive genotyping of 15 relevant single nucleotide polymorphism in 169 study participants, and full resequencing of CYP2B6 in individuals with abnormal EFV plasma levels. Seventy-seven (45.5%) individuals carried a known (CYP2B6*6, *11, *15, or *18) or new loss/diminished-function alleles. Resequencing defined two new loss-of-function alleles: allele *27 (marked by 593T>C [M198T]), that results in 85% decrease in enzyme activity and allele *28 (marked by 1132C>T), that results in protein truncation at arginine 378. Median AUC levels were 188.5 microg h/ml for individuals homozygous for a loss/diminished-function allele, 58.6 microg h/ml for carriers, and 43.7 microg h/ml for noncarriers (P<0.0001). Individuals with a poor metabolizer genotype had a likelihood ratio of 35 (95% CI, 11-110) of presenting very high EFV plasma levels. CYP2B6 poor metabolizer genotypes explain to a large extent EFV pharmacokinetics and identify individuals at risk of extremely elevated EFV plasma levels.
Subject(s)
Anti-HIV Agents/blood , Aryl Hydrocarbon Hydroxylases/genetics , HIV Infections/blood , HIV Infections/genetics , Oxazines/blood , Oxidoreductases, N-Demethylating/genetics , Adult , Alkynes , Alleles , Animals , Area Under Curve , Benzoxazines , Blotting, Western , COS Cells , Chlorocebus aethiops , Cyclopropanes , Cytochrome P-450 CYP2B6 , Exons/genetics , Female , Genotype , Humans , Male , Plasmids/genetics , Polymorphism, Single Nucleotide , Predictive Value of Tests , Prospective Studies , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.
Subject(s)
Cytokines/metabolism , Hepatocytes/metabolism , Models, Animal , Models, Biological , Multienzyme Complexes/metabolism , Signal Transduction/physiology , Systems Biology/standards , Animals , Computer Simulation , MiceABSTRACT
To evaluate the significance of inborn metabolic disorders of the pyrimidine degradation pathway, 450 children with unspecific neurological symptoms were comprehensively studied; 200 healthy children were recruited as controls. Uracil and thymine as well as their degradation products in urine were determined with an improved method based on reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry and detection by multiple-reaction monitoring using stable-isotope-labelled reference compounds as internal standards. From the results of the control group we established age-related reference ranges of all pyrimidine degradation products. In the patient group, two children with dihydropyrimidine dehydrogenase (DPYD) deficiency were identified; one of these was homozygous for the exon 14-skipping mutation of the DPYD gene. In addition, two patients with high uracil, dihydrouracil and beta-ureidopropionate were found to have ornithine transcarbamylase deficiency. In the urine of 9 patients, beta-alanine was markedly elevated owing to treatment with vigabatrin, an irreversible inhibitor of GABA transaminase, which interferes with beta-alanine breakdown. Four patients had exclusively high levels of beta-aminoisobutyrate (beta-AIB) due to a low activity of the D-beta-AIB-pyruvate aminotransferase, probably without clinical significance. In conclusion, quantitative investigation of pyrimidine metabolites in children with unexplained neurological symptoms, particularly epileptic seizures with or without psychomotor retardation, can be recommended as a helpful tool for diagnosis in clinical practice. Sensitive methods and age-related reference ranges enable the detection of partial enzyme deficiencies.
Subject(s)
Chromatography, High Pressure Liquid/methods , Pyrimidines/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Adolescent , Child , Child, Preschool , Dihydropyrimidine Dehydrogenase Deficiency , Dihydrouracil Dehydrogenase (NADP)/genetics , Epilepsy/diagnosis , Exons , Female , Genotype , Humans , Infant , Infant, Newborn , Ions , Male , Mass Spectrometry , Models, Statistical , Mutation , Normal Distribution , Ornithine Carbamoyltransferase Deficiency Disease , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Thymine/chemistry , Uracil/chemistry , beta-Alanine/analogs & derivatives , beta-Alanine/chemistryABSTRACT
The present study investigated the effect of cytochrome P450 2C9 (CYP2C9) genetic polymorphism on the biotransformation of valproic acid (VPA) to its hepatotoxic metabolite, 4-ene-VPA, and compared that to the formation of the inactive 4-OH-VPA and 5-OH-VPA. cDNA-expressed CYP2C9(*)2 and CYP2C9(*)3 variants were less efficient than the CYP2C9(*)1 wild type in catalyzing the formation of these metabolites, as assessed by the ratio of Vmax and apparent Km (in vitro intrinsic clearance). The reduced efficiency by CYP2C9(*)2 was due to a reduced Vmax, whereas, in the case of CYP2C9(*)3, it was the result of increased apparent Km. The formation rates of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in human liver microsomes were reduced by 29, 28, and 31%, respectively, in samples with one mutated CYP2C9 allele, and by 61, 73, and 58%, respectively, in samples with two mutated CYP2C9 alleles. Overall, the homozygote and heterozygote CYP2C9(*)2 and CYP2C9(*)3 genotypes may compromise hepatic VPA biotransformation.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Microsomes, Liver/drug effects , Valproic Acid/metabolism , Valproic Acid/toxicity , Analysis of Variance , Animals , Cytochrome P-450 CYP2C9 , Genotype , Humans , Insecta , Microsomes, Liver/enzymologySubject(s)
Methyltransferases/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Child, Preschool , Family Health , Humans , Male , Mercaptopurine/metabolism , Mercaptopurine/therapeutic use , Methyltransferases/deficiency , Mutation, Missense , Pedigree , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
Thiopurine S-methyltransferase (TPMT) deficient patients develop life threatening haematotoxicity (for example, pancytopenia) when treated with a standard dose of azathioprine (AZA) and 6-mercaptopurine (6-MP) due to excessive accumulation of cytotoxic metabolites. At present, it is generally recommended that these patients should not receive AZA or 6-MP treatment for inflammatory bowel disease. We report for the first time that Crohn's disease patients with TPMT deficiency can be successfully treated with AZA. We illustrate this with three cases where treatment has been successful and toxicity has been avoided by carefully titrating the drug dose. Thus very low TPMT activity demands pharmacogenetically guided dosing.
Subject(s)
Azathioprine/administration & dosage , Crohn Disease/drug therapy , Crohn Disease/enzymology , Immunosuppressive Agents/administration & dosage , Methyltransferases/deficiency , Adult , Drug Administration Schedule , Female , Humans , Male , Treatment OutcomeABSTRACT
CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Polymorphism, Single Nucleotide , Alternative Splicing , Blotting, Western , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Frameshift Mutation , Gene Expression , Gene Frequency , Genetic Markers , Germany , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Microsomes, Liver/enzymology , Phenotype , Sequence Alignment , Sequence Analysis, DNA , Switzerland , Transcription, Genetic , White People/geneticsABSTRACT
Variable expression and function of the cytochrome P4502D6 (CYP2D6) leads to distinct phenotypes termed ultrarapid (UM), extensive (EM), intermediate (IM) and poor metabolizer (PM). Whereas the PM phenotype is known to be caused by two null-alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remained largely unexplained. In this study, we systematically investigated 76 liver biopsies from individuals with known sparteine metabolic ratios (MRS) for the relationships between CYP2D6 genotype, microsomal protein expression, bufuralol 1'-hydroxylase activity and in-vivo phenotype. Average CYP2D6 protein levels ranged from undetectable in PMs (MRS > 20) to 2.6 +/- 2.7 pmol/mg microsomal protein in IMs (1.2 < MRS< 20), 7.6 +/- 4.7 in EMs (0.2 < MRS < 1.2) and 23.8 +/- 7.7 in UMs (MRS < 0.2), respectively. Analysis with respect to genotype demonstrated gradually increased expression and function for individuals with no, one, two or three functional gene copies per genome. The recently discovered -1584 C/G promoter polymorphism was identified as another major factor for expression and function with the mutant [-1584G] promoter type being consistently associated with significantly higher expression than [-1584C]. To investigate functional differences between the detected variant protein forms CYP2D6.1, 2D6.2, 2D6.9 and 2D6.10, we expressed them recombinantly in insect cells. The most significant difference was a decrease in the relative P450 holoprotein content of all allelic forms, including the common functional variant 2D6.2, in comparison to 2D6.1, whereas only modest Km changes were observed. Taken together, these data provide further insight into the complex mechanisms that govern the highly variable expression and function of CYP2D6.
Subject(s)
Cytochrome P-450 CYP2D6/biosynthesis , Cytochrome P-450 CYP2D6/genetics , Liver/enzymology , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP2D6/physiology , Gene Dosage , Genotype , Humans , Immunoblotting/methods , Isoenzymes/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , Isoenzymes/physiology , Phenotype , Polymorphism, Genetic/genetics , Promoter Regions, Genetic/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolismABSTRACT
OBJECTIVE: To investigate the influence of CYP2D6 genotype and medication on the reliability of phenotyping in a naturalistic setting of psychiatric inpatients. METHODS: The phenotype of 160 psychiatric inpatients was estimated by taking the urinary metabolic ratio (MR) of the concentrations of sparteine to 2- and 5-dehydrosparteine. Genotyping identified CYP2D6*1, *3, *4, *5 and *6 alleles as well as duplication of the CYP2D6 gene. All subjects underwent detailed drug history including drug dose and therapeutic drug monitoring to control compliance and abuse of other psychotropic drugs. These data were compared with those of 195 unmedicated healthy Germans. RESULTS: The cumulative distribution of the MR in patients showed a significant shift to higher MR when compared with that of healthy subjects (P < or = 0.001). Patients medicated either with selective serotonin reuptake inhibitors (SSRIs, P < or = 0.001), antipsychotic drugs (P= 0.002) or other drugs known to be substrates or inhibitors of CYP2D6 (P < or = 0.001) showed a significantly higher mean MR than unmedicated patients. However, there was no significant effect of tricyclic antidepressants on the MR. Healthy subjects with CYP2D6 deficiency were separated by a MR of greater than 20 from those who expressed functional CYP2D6. Seven patients carrying at least one functional CYP2D6 allele revealed a MR of greater than 20, indicating the occurrence of phenocopying. CONCLUSION: The results of phenotyping may be falsified by drugs known to be substrates or inhibitors of CYP2D6; thus, this method is not sufficiently reliable. However, since we observed the phenomenon of phenocopying only in patients treated with a SSRI such as fluoxetine, fluvoxamine or paroxetine, we conclude that sparteine phenotyping of medicated patients detects CYP2D6 deficiency correctly, provided that patients treated with these SSRIs are excluded.
Subject(s)
Antipsychotic Agents/pharmacology , Cytochrome P-450 CYP2D6/genetics , Selective Serotonin Reuptake Inhibitors/pharmacology , Sparteine/metabolism , Adult , Aged , Female , Genotype , Humans , Male , Middle Aged , Phenotype , Sparteine/administration & dosage , Sparteine/urineABSTRACT
The variability of P-glycoprotein expression between individuals is linked to a C3435T polymorphism of the human MDR1 gene. Concentration of P-glycoprotein in intestinal epithelial cells and in a subset of lymphoid cells is substantially lower in people with the T/T genotype than those with the C/C genotype. We compared allele frequencies of the C3435T polymorphism in random samples of west African, African American, white, and Japanese people. We recorded a significantly higher frequency of the C/C genotype in West Africans and African Americans (142 of 172 [83%] and 25 of 41 [61%], respectively), than in white people (139 of 537 [26%]) (p<0.0001). These findings could affect use of drugs that are P-glycoprotein substrates (such as HIV-1 protease inhibitors and ciclosporin) in African populations.