Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 53: 24-33, 2007. First published November 2, 2006; 10.1373/clinchem.2006.074856
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow 074856.Supplemental Data
Right arrow All Versions of this Article:
clinchem.2006.074856v1
53/1/24    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blievernicht, J. K.
Right arrow Articles by Zanger, U. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blievernicht, J. K.
Right arrow Articles by Zanger, U. M.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2007;53:24-33.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms

Julia K. Blievernicht1, Elke Schaeffeler1, Kathrin Klein1, Michel Eichelbaum1,2, Matthias Schwab1 and Ulrich M. Zanger1,a

1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
2 Division of Clinical Pharmacology, University Hospital Tuebingen, Tuebingen, Germany.

aAddress correspondence to this author at: Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany. Fax 49-(0)711-85-92-95; e-mail uli.zanger{at}ikp-stuttgart.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations.

Methods: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods.

Results: Five multiplex assays were developed, comprising the following 15 SNPs: –82T->C (*22); 86G->C (R29T, *17); 136A->G (M46V, *11); 296G->A (G99E, *12); 415A->G (K139E, *8, *13); 419G->A (R140Q, *14); 516G->T (Q172H, *6, *7, *9, *13, *19, *20), 547G->A (V183I); 769G->A (D257N); 785A->G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T->C (I328T, *16, *18); 1006C->T (R336C, *19); 1172T->A (I391N, *15); 1282C->A (P428T, *21); 1459C->T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing.

Conclusions: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pharmacogenetic diagnostics is a promising approach to predict the pharmacokinetics, as well as the effects and adverse drug reactions, of a drug in an individual patient (1)(2). The majority of all clinically used drugs are oxidatively metabolized by isoenzymes of the cytochrome P450 (CYP)1 superfamily, several of which are functionally highly variable, leading to different drug oxidation phenotypes within the population. Of established clinical importance are the genetic variations of CYP2D6,2 CYP2C19, and CYP2C9 (3)(4)(5). Compared with these well-studied genes, CYP2B6 has only recently been recognized as a gene that codes for a highly variable enzyme of potential clinical importance (6). Clinically relevant drug substrates include the antidepressant bupropion (7)(8)(9), the antineoplastic agent cyclophosphamide (10)(11), the anaesthetic propofol (12), the antimalarial artemisinine(13), the anti-Parkinsonian selegiline (14), and the nonnucleosidic reverse transcriptase inhibitor efavirenz (15).

The CYP2B6 gene is located, together with a closely related pseudogene CYP2B7P1, in a CYP2 gene cluster on chromosome 19 (16). In contrast to the above-mentioned CYP genes, variations in CYP2B6 have been discovered through reverse genetics studies, i.e., by initial sequencing and variation scanning studies and subsequent analysis of the functional consequences of the identified variations (17)(18)(19)(20)(21)(22)(23)(24)(25). More than 100 DNA variations, including numerous nonsynonymous variations, as well as silent, promoter, and intronic changes, were found within the CYP2B6 gene, many of them showing extensive linkage disequilibrium giving rise to distinct haplotypes (26). The spectrum of functional consequences of these variations is wide and includes null alleles with no detectable function and/or expression (alleles CYP2B6*8, *12, *15, *18, *21), alleles with partially reduced function/expression (CYP2B6*5, *6, *7, *11, *14, *19, *20, *21) (17)(19)(21)(23), and alleles with increased expression [CYP2B6*22; (24)]. Some of these variations are rare, but many are common, with allele frequencies between 10% and almost 50%, depending on the population studied (22)(23).

The clinical relevance of CYP2B6 variations has recently been demonstrated for the anti-HIV drug efavirenz. The common clinical practice of administering the same dose to all patients leads to profound differences in drug plasma concentrations, which are correlated with patient genotype (27)(28)(29). Patients with high drug concentrations are at risk of developing concentration-related central nervous system toxicity, including insomnia, fatigue, and headache and often leading to discontinuation of the therapy. Thus, for a drug such as efavirenz, dose adjustment based on CYP2B6 genotype could prevent administration of too-high doses, thus increasing the safety and efficacy of the therapy.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) proved to be a superior technology for the detection of single-nucleotide variations (SNPs). This method has several advantages over other methods, including high accuracy because of the direct measurement of molecular masses, high sensitivity in the detection of both homozygous and heterozygous base changes, and high-throughput and cost-effective multiplex capability (30)(31)(32)(33)(34)(35)(36)(37)(38). We developed a MALDI-TOF MS genotyping method to determine 15 SNPs in the CYP2B6 gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
dna samples
Anonymized blood samples were obtained from individuals of white European origin. All individuals gave written informed consent to genetic testing. The study was approved by the ethics committees of the Medical Faculties of the Charité, Humboldt-University Berlin, and of the University of Tübingen, and written informed consent was obtained from each patient. Genomic DNA was prepared by standard methods from whole blood samples as described previously (17)(21).

maldi-tof mass spectrometric cyp2b6 genotyping assay
The CYP2B6 promoter sequence (AC 24497632), gene sequence (NG_000008.5 and AC023172), and the CYP2B7 pseudogene sequence (AC008537) were used for specific CYP2B6 primer design with the AlignX program of the VectorNTI Suite 9 package (InforMax, Inc.). All reactions, including PCR amplification, shrimp alkaline phosphatase treatment, and base extension, were performed in 384 microtiter plates (ABgene, Epsom) with a Puredisk pipette robot (Cybio). PCR amplification and primer extension reaction were carried out in a DYADTM PCR thermal cycler (MJ Research), and no-template controls were carried along in every plate to exclude contaminations.

Multiplex PCR.
PCRs (final volume, 8 µL) contained 50 ng of DNA, 0.2 units of HotStarTaq polymerase, 1 mmol/L MgCl2 (Qiagen GmbH), and the desired primers (MWG Biotech AG) at their optimized concentrations (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue1). A tag (5'-ACGTTGGATG) was included in the primer sequence to equalize extreme relative percentage-GC contents (39). PCR conditions were 95 °C for 15 min followed by 45 cycles at 95 °C for 30 s, 60 °C or 61 °C (assay 5) for 1 min, and 72 °C for 1 min; and finally 72 °C for 10 min. After dephosphorylation of excess dNTPs with 0.3 units of shrimp alkaline phosphatase in 1x Rx-Buffer (Amersham) at 37 °C for 20 min, 85 °C for 10 min, and 20 °C for 1 s, the PCR products were used as templates for the primer extension reactions. CYP2B6-specific amplification was verified for all primer pairs by sequence analysis of products obtained in single reactions.


View this table:
[in this window]
[in a new window]

  		Table 1. Selected SNPs and their functional consequences.

Primer extension reactions.
Extension reactions (final volume, 16 µL) contained 1 µL of buffer C (500 mmol/L Tris-HCL, pH 9.5; 100 mmol/L MgCl2), 6 mmol/L MgCl2, 1 U of Termipol DNA polymerase I (all from Solis Biodyne), and extension primers at optimized concentrations (see Table 2 in the online Data Supplement). Assay-specific deoxynucleoside triphosphates (dATP, dGTP, dTTP; Amersham) and dideoxynucleoside triphosphates (ddA, ddC, ddG, ddT; BioLog Life Science Institute) were added at 0.12 mmol/L. In assays 1, 2, 3, and 5, dCTP (Amersham) and dGTP were added at 0.25 mmol/L. Extension reactions were performed at 94 °C for 4 min followed by 55 cycles at 94 °C for 30 s and 52 °C (assays 1 and 3), 56 °C (assay 2), or 60 °C (assay 4 and 5) for 30 s, and 72 °C for 30 s; and finally 72 °C for 2 min. The final nucleotide extension products were treated with a cationic exchange resin (AG® 50W-X8 Resin; Bio-Rad Laboratories, Inc.) for 30 min to remove salts.


View this table:
[in this window]
[in a new window]

  		Table 2. CYP2B6 SNP and genotype frequencies in 287 Caucasians.

MALDI-TOF MS measurement.
We spotted and air dried 1 µL of 3-hydroxypicolinic acid matrix (45 mmol/L and 3 mmol/L diammonium hydrogen citrate solution) onto a 384-format MTP AnchorChipTM 400/384TF target plate (Bruker Daltonik, spot size 400 µm). The extension reaction products (0.5 µL) were dispensed on the matrix and air dried. The target plate was then inserted into the Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonik), and analysis was performed with 180 nitrogen laser shots for each sample. The mass range of the MS instrument was set at 4000–9000 Da. Genotyping calls were made with the assistance of the GENOTOOLS software (Bruker Daltonik). DNA samples not automatically assigned were determined manually.

generation of control samples
Control samples are needed for each SNP in heterozygous and homozygous form to exclude artifacts such as pausing peaks or other DNA fragments. To generate a homozygous variant control sample from an available heterozygous sample, a PCR product was generated with the appropriate PCR primers (see Table 1 in the online Data Supplement). The 2 alleles were separated by subcloning the fragments in TOP10 cells by positive selection with a TOPO TA Cloning® Kit vector (Invitrogen) containing the F plasmid ccdB killer gene (40). DNA was isolated from bacteria and sequenced to verify the presence of the SNP. If no heterozygous sample was available, we used a previously developed touchdown PCR method to introduce the variation in a wild-type (wt) sample. A primer located within the amplified region and containing the variation of interest was designed with a melting temperature ~8 °C above that of the amplification primers (see Table 1 in the online Data Supplement), as described previously (41). The primer sequences are available on request. Sequence verification was performed with AB Big Dye® Terminator Mix v1.1. (Applied Biosystems) and the ABI Prism 310 Genetic Analyzer (Applied Biosystems).

allele nomenclature and statistics
Base numbering and allele definitions were according to the published recommendations of the CYPallele Nomenclature Committee (http://www.imm.ki.se/CYPalleles/criteria.htm). Numbering was based on the cDNA with the full-length cDNA sequence published by Yamano et al. (42) defined as the wt (CYP2B6*1). We used the DeFinetti program (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) to test genotype frequencies for conformance with Hardy–Weinberg equilibrium. Data were compiled according to the genotype and allele frequencies estimated from the observed numbers of each specific allele and with the assistance of the PHASE program v.2.0.2 (43)(44). The frequency of each SNP in our study participants is given with the 95% confidence interval (CI).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
selection of snps for genotyping
To choose relevant SNPs for CYP2B6 genotyping we considered more than 100 known and rare variations, most of which have been published and are publicly available in databases (CYPallele nomenclature: http://www.cypalleles.ki.se; dbSNP: http://www.ncbi.nlm.nih.gov/SNP/). Relevant data for the 15 SNPs finally selected are summarized in Table 1Up . They include the 2 most frequent amino acid variations, Q172H and K262R in exons 4 and 5, respectively, and the common R487C variation in exon 9, all of which are associated with lower expression in human liver (17). We also selected the more rare SNPs M46V, G99E, K139E, and I391N, described as phenotypic null alleles in Caucasians, and the R140Q SNP that strongly affects enzyme activity (21). Furthermore, the following amino acid variations reported in Ghanaian or African-American individuals were included: R29T, D257N, I328T, R336C, and P428T, some of which were found to result in small or undetectable amounts of residual protein (23). A novel SNP, V183I, found by sequence analysis in a Ghanaian individual (data not shown) was also included. Finally, we enclosed the –82T->C promoter SNP, which was recently shown to cause enhanced transcription and to result in increased expression (24).

The c.64C->T (R22T) variation, defining the CYP2B6*2 allele, was thus far not included because it was shown to be comparable to the wt in expression and function (17)(20).

development of maldi-tof ms assays
For the specific amplification of CYP2B6 gene fragments, it was important to (a) effectively discriminate between CYP2B6 and the pseudogene CYP2B7, and (b) minimize the possibility that nucleotide variations within the PCR- and extension-primer sequences interfered with amplification. To meet these requirements, the PCR primer pairs (see Table 1 in the online Data Supplement) were designed to bind to CYP2B6 in regions of low homology to CYP2B7, mostly found in intronic or exon/intron boundary regions. The only exon not included in the multiplex PCR assays is exon 6, for which no relevant variations had been described to date. All primer pairs were shown to be functional in various combinations up to a single-tube multiplex amplification reaction for all 8 amplicons. However, a lower degree of multiplexing was finally applied because not all extension reactions could be carried out simultaneously. The compatibility of the extension primers is strongly restricted by their sequence, molecular masses, and annealing temperatures. Individual extension primers were optimized with respect to primer length and concentration and tested with positive controls for all 3 genotypes (homozygous wt, homozygous variant, and heterozygous). The control DNAs were selected from previously genotyped genomic DNA samples or generated recombinantly as described in Materials and Methods. By testing various combinations, we arrived at a final design comprising 5 multiplex assays (assays 1–5; see Table 2 in the online Data Supplement). Representative MALDI-TOF MS spectra for all 5 assays are shown in Fig. 1 .


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Figure 1. MALDI-TOF MS–based analysis of 15 CYP2B6 SNPs.

Fragments of the CYP2B6 gene containing polymorphic nucleotides were amplified by 8 different specific PCRs. Purified amplification products were used as templates for polymorphism-specific primer extension reactions. The purified products were directly analyzed by MALDI-TOF MS. The molecular masses of the extension products are listed (see Table 2 in the online Data Supplement). Unextended primers are marked by an asterisk, and each product/extended primer is designated by the respective SNP. Typical examples of MALDI-TOF MS spectra representing the 5 multiplex assays (A–E) are shown.

evaluation of the maldi-tof ms genotyping assay
To evaluate the established CYP2B6 genotyping assay, we analyzed 287 genomic DNA samples from individuals of Caucasian origin who had previously been genotyped for some of the SNPs by various other methods. In total, this amounted to 4305 individual genotype determinations. Fig. 2 shows the relative intensities of the mass signals corresponding to the 2 primer extension products of a given CYP2B6 SNP, plotted against each other, for the 4 most frequent variations. As evident from this analysis, the mass spectral data were clearly structured into 3 groups, i.e., homozygous wt/wt and mutant (mut)/mut genotypes found along the axes, and wt/mut heterozygotes scattered in-between (no-template controls with relative signal intensities of 0 are indicated with white points and black from the middle). In total, 2300 genotypes have been previously determined by other methods, including sequencing, allele-specific 5'-nuclease assay (TaqMan), denaturing high-performance liquid chromatography, and/or PCR–restriction fragment length polymorphism (RFLP). In 9 cases (0.4%), MALDI-TOF MS analysis resulted in a genotype discrepant from that of the earlier studies. Retrospective inspection revealed that PCR-RFLP assays had been used for the earlier analyses of these samples and that the fragment profiles were ambiguous and difficult to interpret. After resequencing these samples, the MALDI-TOF MS outcomes were confirmed in all 9 cases.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		Figure 2. Genotype discrimination for the most frequent CYP2B6 polymorphisms.

For each polymorphism analyzed, the relative intensities of the 2 allele signals, automatically determined by the GENOTOOLS software, were plotted against each other. Data points on the x axis represent individuals with homozygous mut genotype (filled symbols), those on the y axis represent individuals with the wt genotype (open symbols), and heterozygotes are found in-between (gray symbols). No-template controls led to very low or undetectable signal intensities, indicated in the origin.

frequency analysis of cyp2b6 variations in caucasians
The allele and genotype frequencies observed in the analyzed population of 287 Caucasians are summarized in Table 2Up . All genotypes were shown to be in Hardy–Weinberg equilibrium. Five variations previously observed in persons of African or African-American origin were not detected in any Caucasian sample. The most frequent SNPs were the amino acid substitutions K262R (27.4%), Q172H (25.2%), and R487C (9.7%), followed by the promoter SNP –82C (2.1%). The variations M46V, G99E, K139E, R140Q, V183I, and I391N were present at a frequency of <1% (Table 2Up ). Results of haplotype analysis with the PHASE program indicated that the most frequent alleles were the wt allele (*1; 59%) and *6 (24.6%). The other CYP2B6 alleles and their frequencies were *4 (2.3%), *5(7.3%), *11 (0.35%), *12 (0.17%), *13 (0.52), *14 (0.52%), *15 (0.7%), and *22 (2.1%). The alleles *17, *18, *19, and *21 were not found in this population.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed a comprehensive SNP analysis method based on MALDI-TOF MS for the human drug metabolizing CYP2B6 gene and the application of this genotyping method to a study population of European Caucasian origin. The design of the assay consists of 5 multiplex subassays with carefully optimized protocols for 15 selected SNPs, thus allowing us to analyze SNP subsets and to incorporate additional SNPs for analysis if, for example, novel predictive SNPs are discovered. MALDI-TOF MS is a sensitive, accurate and reproducible method for the detection of oligonucleotides. In contrast to other genotyping technologies that rely on hybridization, MALDI-TOF MS directly measures the molecular weight of the oligonucleotide and therefore has several principal and methodologic advantages, which have been summarized in previous articles (31)(32)(33)(45). Further advantages of MALDI-TOF MS over some other methods are the option for highly automated processes and the relative ease of set up for multiplex assays, thus effectively reducing time and cost while increasing sample throughput.

Similar to other cytochrome P450 genes, CYP2B6 represents a particular challenge with respect to developing gene-specific assays because of its high homology to the pseudogene CYP2B7P1 (46). Because no variation analysis has been carried out for CYP2B7, the influence of contaminating CYP2B6 amplicons with CYP2B7 is unpredictable. We designed intronic primers such that each of them harbored at least 1 nucleotide difference from the pseudogene sequence, and we controlled each amplicon for its specificity by direct sequencing. Occasionally contamination was observed and PCR conditions were then modified to improve specificity. It is thus important that such controls are performed when setting up this or similar assays. The use of a common tagging sequence at the 5'-end of the amplification primers to level out differences in GC content proved to be advantageous for multiplexing (39). Under optimized conditions, the entire set of primer pairs gave satisfactory results in a single multiplex amplification reaction.

The combination of the various primer extension reactions into multiplex assays is another critical aspect, and support by suitable software is highly recommended to allow the optimization of several different parameters, i.e., GC content, molecular mass range, annealing temperatures, dimers, and hairpin loop formation. We tested the accuracy of conversion of the unextended primer into allele-specific analyte by use of either control DNAs from previously sequenced/genotyped samples or recombinantly generated controls. To achieve highest multiplexing levels, we tested many primer combinations, leading us to the final assay design consisting of 5 multiplex assays. More than 4 extension primers could not be combined into a single-multiplex extension reaction, mainly because of cross-binding problems and different extension mixes necessary for the 15 SNPs (see Table 2 in the online Data Supplement).

For evaluation, we analyzed all 15 SNPs in 287 genomic DNA samples of white European origin. As shown in Fig. 2Up for the most frequent CYP2B6 variations, the MALDI-TOF MS data obtained for the 3 possible genotypes (wt, mut, and heterozygous) were clearly structured into 3 clusters. On average, ~5% (equivalent to 14 samples) were not automatically identified and had to be assigned manually, resulting in an overall automated call rate of >95% for all 15 SNPs. Thus, with the assays presented, a reliable high-throughput genotyping of CYP2B6 variations is achievable. The comparison of the MALDI-TOF MS results with those obtained by other methods revealed a few discrepant results, which could be assigned to 2 distinct RFLP assays. These divergent results were retrospectively shown to be caused by erroneous data interpretation of the primary RFLP data, and the MALDI-TOF MS outcomes could be verified in all cases by sequencing, emphasizing the high accuracy of this method.

After completion of the mass spectrometric analysis, 9 genotypes (0.2%) of the 4305 total SNP analyses remained undetermined even after 3 repeated analyses. The reason for this is currently unclear. However, because only 4 different SNPs were affected (see Table 2Up ) and because PCR amplification was successful in each case, we speculate that unknown sequence variations within primer binding sites may have prevented the binding and/or extension of the respective primers. The overall genotyping success rate for the 287 samples was thus higher than 99% in 3 of the 5 assays, and even 100% in 2 assays, which confirms the high technical reliability of MALDI-TOF MS (47).

The SNP frequencies observed in this study confirm and extend current knowledge on CYP2B6 genetic variation in the middle European population. CYP2B6 variations have been discovered relatively recently, and thus their value for predicting drug response is largely unexplored. However, clinical applications of CYP2B6 genotyping so far include bupropion, used in smoking cessation (48); cyclophosphamide, in lupus nephritis (49) and in hematologic malignancies(50); and efavirenz, an anti-HIV agent (27)(28)(29). In particular, the consistent results of multiple studies on efavirenz pharmacogenetics emphasize the potential of CYP2B6 variations in predicting patient variability in drug side effects, response, and toxicity. To date, only the more common variations of CYP2B6 have been included in these studies, in part because comprehensive genotyping assays have not been available (51)(52)(53). In our assay, we have included numerous recently discovered, less frequent SNPs, which have pronounced effects on expression/function and which together account for 5% to 10% of functionally variant alleles, depending on the ethnic origin of the population studied (Table 1Up ) (19)(21)(23)(25). Their determination in the course of clinical studies should thus help to improve our understanding of the phenotype–genotype relationship and enhance the predictive value of CYP2B6 genotyping.


Figure 1
Continued.


   Acknowledgments
 
We are grateful to Igor Liebermann for technical assistance and to Dr. Werner Schroth (Stuttgart, Germany) for helpful discussions. This work was supported in part by the H.W. & J. Hector Foundation (Weinheim, Germany), the Robert Bosch Foundation (Stuttgart, Germany), and the German Federal Ministry of Education and Research (grant 0313080D).


   Footnotes
 
1 Nonstandard abbreviations: CYP, cytochrome P450; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; SNP, single-nucleotide polymorphism; wt, wild-type; mut, mutant; RFLP, restriction fragment length polymorphism.

2 Human genes: CYP2B6, cytochrome P450, family 2, subfamily B, polypeptide 6; CYP2B7P1, cytochrome P450, family 2, subfamily B, polypeptide 7 pseudogene 1; CYP2D6, cytochrome P450, family 2, subfamily D, polypeptide 6; CYP2C19, cytochrome P450, family 2, subfamily C, polypeptide 19; CYP2C9, cytochrome P450, family 2, subfamily C, polypeptide 9.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999;286:487-491.[Abstract/Free Full Text]
  2. Eichelbaum M, Ingelman-Sundberg M, Evans WE. Pharmacogenomics and individualized drug therapy. Annu Rev Med 2006;57:119-137.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  3. Meyer UA. Pharmacogenetics and adverse drug reactions. Lancet 2000;356:1667-1671.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  4. Zanger UM, Raimundo S, Eichelbaum M. Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry. Naunyn Schmiedebergs Arch Pharmacol 2004;369:23-37.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  5. Steimer W, Zopf K, von Amelunxen S, Pfeiffer H, Bachofer J, Popp J, et al. Allele-specific change of concentration and functional gene dose for the prediction of steady-state serum concentrations of amitriptyline and nortriptyline in CYP2C19 and CYP2D6 extensive and intermediate metabolizers. Clin Chem 2004;50:1623-1633.[Abstract/Free Full Text]
  6. Ekins S, Wrighton SA. The role of CYP2B6 in human xenobiotic metabolism. Drug Metab Rev 1999;31:719-754.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  7. Hesse LM, Venkatakrishnan K, Court MH, von Moltke LL, Duan SX, Shader RI, et al. CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab Dispos 2000;28:1176-1183.[Abstract/Free Full Text]
  8. Faucette SR, Hawke RL, LeCluyse EL, Shord SS, Yan B, Laethem RM, et al. Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 2000;28:1222-1230.[Abstract/Free Full Text]
  9. Kirchheiner J, Klein C, Meineke I, Sasse J, Zanger UM, Mürdter TE, et al. Bupropion and 4OH-bupropion pharmacokinetics in relation to genetic polymorphisms in CYP2B6. Pharmacogenetics 2003;13:619-626.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  10. Chang TK, Weber GF, Crespi CL, Waxman DJ. Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res 1993;53:5629-5637.[Abstract/Free Full Text]
  11. Roy P, Yu LJ, Crespi CL, Waxman DJ. Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab Dispos 1999;27:655-666.[Abstract/Free Full Text]
  12. Court MH, Duan SX, Hesse LM, Venkatakrishnan K, Greenblatt DJ. Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes. Anesthesiology 2001;94:110-119.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  13. Svensson US, Ashton M. Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin. Br J Clin Pharmacol 1999;48:528-535.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  14. Hidestrand M, Oscarson M, Salonen JS, Nyman L, Pelkonen O, Turpeinen M, et al. CYP2B6 and CYP2C19 as the major enzymes responsible for the metabolism of selegiline, a drug used in the treatment of Parkinson’s disease, as revealed from experiments with recombinant enzymes. Drug Metab Dispos 2001;29:1480-1484.[Abstract/Free Full Text]
  15. Ward BA, Gorski JC, Jones DR, Hall SD, Flockhart DA, Desta Z. The cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz as a substrate marker of CYP2B6 catalytic activity. J Pharmacol Exp Ther 2003;306:287-300.[Abstract/Free Full Text]
  16. Hoffman SM, Nelson DR, Keeney DS. Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 2001;11:687-698.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  17. Lang T, Klein K, Fischer J, Nüssler AK, Neuhaus P, Hofmann U, et al. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 2001;11:399-415.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  18. Ariyoshi N, Miyazaki M, Toide K, Sawamura Y, Kamataki T. A single nucleotide polymorphism of CYP2B6 found in Japanese enhances catalytic activity by autoactivation. Biochem Biophys Res Commun 2001;281:1256-1260.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  19. Lamba V, Lamba J, Yasuda K, Strom S, Davila J, Hancock ML, et al. Hepatic CYP2B6 expression: gender and ethnic differences and relationship to CYP2B6 genotype and CAR (constitutive androstane receptor) expression. J Pharmacol Exp Ther 2003;307:906-922.[Abstract/Free Full Text]
  20. Jinno H, Tanaka-Kagawa T, Ohno A, Makino Y, Matsushima E, Hanioka N, et al. Functional characterization of cytochrome P450 2B6 allelic variants. Drug Metab Dispos 2003;31:398-403.[Abstract/Free Full Text]
  21. Lang T, Klein K, Richter T, Zibat A, Kerb R, Eichelbaum M, et al. Multiple novel nonsynonymous CYP2B6 gene polymorphisms in Caucasians: demonstration of phenotypic null alleles. J Pharmacol Exp Ther 2004;311:34-43.[Abstract/Free Full Text]
  22. Solus JF, Arietta BJ, Harris JR, Sexton DP, Steward JQ, McMunn C, et al. Genetic variation in 11 phase I drug metabolism genes in an ethically diverse population. Pharmacogenomics 2004;5:895-931.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  23. Klein K, Lang T, Saussele T, Barbosa-Sicard E, Schunck WH, Eichelbaum M, et al. Genetic variability of CYP2B6 in populations of African and Asian origin, novel functional variants, and implications for anti-HIV therapy with efavirenz. Pharmacogenet Genomics 2005;15:861-873.[ISI][Medline] [Order article via Infotrieve]
  24. Zukunft J, Lang T, Richter T, Hirsch-Ernst KI, Nüssler AK, Klein K, et al. A natural CYP2B6 TATA box polymorphism (–82T->C) leading to enhanced transcription and relocation of the transcriptional start site. Mol Pharmacol 2005;67:1772-1782.[Abstract/Free Full Text]
  25. Wang J, Sonnerborg A, Rane A, Josephson F, Lundgren S, Stahle L, et al. Identification of a novel specific CYP2B6 allele in Africans causing impaired metabolism of the HIV drug efavirenz. Pharmacogenet Genomics 2006;16:191-198.[Medline] [Order article via Infotrieve]
  26. Ingelman-Sundberg M, Daly AK, Nebert DW. CYPallele nomenclature homepage. http://www.imm.ki.se/CYPalleles.htm (accessed June 2006)..
  27. Tsuchiya K, Gatanaga H, Tachikawa N, Teruya K, Kikuchi Y, Yoshino M, et al. Homozygous CYP2B6 *6 (Q172H and K262R) correlates with high plasma efavirenz concentrations in HIV-1 patients treated with standard efavirenz-containing regimens. Biochem Biophys Res Commun 2004;319:1322-1326.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  28. Haas DW, Ribaudo HJ, Kim RB, Tierney C, Wilkinson GR, Gulick RM, et al. Pharmacogenetics of efavirenz and central nervous system side effects: an Adult AIDS Clinical Trials Group study. AIDS 2004;18:2391-2400.[ISI][Medline] [Order article via Infotrieve]
  29. Rotger M, Colombo S, Furrer H, Bleiber G, Buclin T, Lee BL, et al. Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients. Pharmacogenet Genomics 2005;15:1-5.[Medline] [Order article via Infotrieve]
  30. Ross P, Hall L, Smirnov I, Haff L. High level multiplex genotyping by MALDI-TOF mass sepctrometry. Nat Biotechnol 1998;16:1347-1351.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  31. Pusch W, Wurmbach JH, Thiele H, Kostrzewa M. MALDI-TOF mass spectrometry-based SNP genotyping. Pharmacogenomics 2002;3:537-548.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  32. Gut IG. DNA analysis by MALDI-TOF mass spectrometry. Hum Mutat 2004;23:437-441.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  33. Tost J, Gut IG. Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications. Clin Biochem 2005;38:335-350.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  34. Bray MS, Boerwinkle E, Doris PA. High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise. Hum Mutat 2001;17:296-304.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  35. Buetow KH, Edmonson M, MacDonald R, Clifford R, Yip P, Kelley J, et al. High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proc Natl Acad Sci U S A 2001;98:581-584.[Abstract/Free Full Text]
  36. Nakai K, Habano W, Fujita T, Nakai K, Schnackenberg J, Kawazoe K, et al. Highly multiplexed genotyping of coronary disease-associated SNPs using MALDI-TOF mass spectrometry. Hum Mutat 2002;20:133-138.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  37. Stanssens P, Zabeau M, Meersseman G, Remes G, Gansemans Y, Storm N, et al. High-throughput MALDI-TOF discovery of genomic sequence polymorphisms. Genome Res 2004;14:126-133.[Abstract/Free Full Text]
  38. Ehrich M, Böcker S, van den Boom D. Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS. Nucleic Acids Res 2005;33:e38.[Abstract/Free Full Text]
  39. Ghebranious N, Ivacic L, Mallum J, Dokken C. Detection of ApoE E2, E3 and E4 alleles using MALDI-TOF mass spectrometry and the homogeneous mass-extend technology. Nucleic Acids Res 2005;33:e149.[Abstract/Free Full Text]
  40. Bernard P, Gabant P, Bahassi EM, Couturier M. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 1994;148:71-74.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  41. Fischer J, Schwab M, Eichelbaum M, Zanger UM. Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography. Genet Test 2003;7:97-105.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  42. Yamano S, Nhamburo PT, Aoyama T, Meyer UA, Inaba T, Kalow W, et al. cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 1989;28:7340-7348.[CrossRef][Medline] [Order article via Infotrieve]
  43. Stephens M, Smith HJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:978-989.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  44. Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction. Am J Hum Genet 2003;73:1162-1169.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  45. Wise CA, Paris M, Morar B, Wang W, Kalaydjive L, Bittles AH. A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2003;17:1195-1202.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  46. Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, Nebert DW. Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 2004;14:1-18.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  47. Meyer K, Fredriksen A, Ueland PM. High-level multiplex genotyping of polymorphisms involved in folate or homocysteine metabolism by matrix-assisted laser desorption/ionization mass spectrometry. Clin Chem 2004;50:391-402.[Abstract/Free Full Text]
  48. Lerman C, Shields PG, Wileyto EP, Audrain J, Pinto A, Hawk L, et al. Pharmacogenetic investigation of smoking cessation treatment. Pharmacogenetics 2002;12:627-634.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  49. Takada K, Arefayene M, Desta Z, Yarboro CH, Boumpas DT, Balow JE, et al. Cytochrome P450 pharmacogenetics as a predictor of toxicity and clinical response to pulse cyclophosphamide in lupus nephritis. Arthritis Rheum 2004;50:2202-2210.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  50. Xie H, Griskevicius L, Stahle L, Hassan Z, Yasar U, Rane A, et al. Pharmacogenetics of cyclophosphamide in patients with haematological malignancies. Eur J Pharm Sci 2006;27:54-61.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  51. Zanger UM, Fischer J, Klein K, Lang T. Detection of single nucleotide polymorphisms in CYP2B6 gene. Methods Enzymol 2002;357:45-53.[ISI][Medline] [Order article via Infotrieve]
  52. Jacob RM, Johnstone EC, Neville MJ, Walton RT. Identification of CYP2B6 sequence variants by use of multiplex PCR with allele-specific genotyping. Clin Chem 2004;50:1372-1377.[Abstract/Free Full Text]
  53. Futatsugawa Y, Kubota T, Ishiguro A, Suzuki H, Ishikawa H, Iga T. PCR-based haplotype determination to distinguish CYP2B6*1/*7 and *5/*6. Clin Chem 2004;50:1472-1473.[Free Full Text]



The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
E. Schaeffeler, U. M. Zanger, M. Eichelbaum, S. Asante-Poku, J.-G. Shin, and M. Schwab
Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups
Clin. Chem., October 1, 2008; 54(10): 1637 - 1647.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. H. Hofmann, J. K. Blievernicht, K. Klein, T. Saussele, E. Schaeffeler, M. Schwab, and U. M. Zanger
Aberrant Splicing Caused by Single Nucleotide Polymorphism c.516G>T [Q172H], a Marker of CYP2B6*6, Is Responsible for Decreased Expression and Activity of CYP2B6 in Liver
J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 284 - 292.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
J. C.H. Tsang, P. Charoenkwan, K. C.K. Chow, Y. Jin, C. Wanapirak, T. Sanguansermsri, Y.M. D. Lo, and R. W.K. Chiu
Mass Spectrometry Based Detection of Hemoglobin E Mutation by Allele-Specific Base Extension Reaction
Clin. Chem., December 1, 2007; 53(12): 2205 - 2209.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow 074856.Supplemental Data
Right arrow All Versions of this Article:
clinchem.2006.074856v1
53/1/24    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blievernicht, J. K.
Right arrow Articles by Zanger, U. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blievernicht, J. K.
Right arrow Articles by Zanger, U. M.
Related Collections
Right arrow Molecular Diagnostics and Genetics

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS