| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Drug Monitoring and Toxicology |
1 Cancer Research UK General Practice Research Group, Department of Clinical Pharmacology, University of Oxford, Oxford, United Kingdom.
aAddress correspondence to this author at: General Practice Research Group, Cancer Research UK, Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, United Kingdom. Fax 44-1865-224989; e-mail robert.walton{at}clinpharm.ox.ac.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We used a two-stage procedure. An initial multiplex PCR reaction amplified the relevant gene fragments in exonic and regulatory regions to ensure isolation of CYP2B6 from its similar pseudogene (CYP2B7). This product was then genotyped by allele-specific PCR.
Results: The assay detected the following published single-nucleotide polymorphisms: C64T (Arg22Cys), C78T, G216C, G516T (Gln172His), C777A (Ser259Arg), A785G (Lys262Arg), and C1459T (Arg487Cys), as well as additional loci found within the single-nucleotide polymorphism (SNP) databases: A1190G, C1268A, C1330T, A1382G, A1402T, and an A/T SNP in intron 2 (A12917T). This approach detected all common, previously reported alleles and identified a new allele (CYP2B6*4C) present in 2.2% of a Caucasian population. Genotypic frequencies obtained were consistent with previously published results.
Conclusions: This method is simple, reliable, rapid, and amenable to automation and could facilitate the large-scale genotypic analysis of CYP2B6.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The CYP2 gene family is large, having numerous subfamilies that are physically clustered together in the genome. CYP2B is located in such a cluster containing six subfamilies (CYP2A, -2B, -2F, -2G, -2S, and -2T) on human chromosome 19 (18). There are two known human CYP2B loci: the functional CYP2B6 and its pseudogene, CYP2B7 (Fig. 1A
). These are located inside a block of 
112 kb in the middle of the CYP2A18P locus, between 19q12 and 19q13.2 (18)(19)(20). Like most other CYP2 family members, CYP2B6 (Fig. 1B
) contains nine exons, which encode a protein of 491 amino acids (20). In addition to the wild-type allele, CYP2B6*1, eight CYP2B6 alleles have been identified, CYP2B6*2 (C64T), *3 (C777A), *4 (A785G), *5 (C1459T), *6 (G516T and A785G), *7 (G516T, A785G, and C1459T) (21), and more recently, *8 (A415G) and *9 (G516T) (17). To develop a comprehensive genotyping assay, we included all CYP2B6 single-nucleotide polymorphisms (SNPs) published at the start of the project in addition to several unpublished SNPs reported in public databases.
|
At present, CYP2B6 can be genotyped by an assay combining PCR with restriction fragment length polymorphism analysis (21). We aimed to develop a rapid, robust multiplex PCR method for the simultaneous detection of CYP2B6 polymorphisms that does not rely on the use of restriction enzymes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
sequence alignments and primer design
To design an assay for CYP2B6, we used genomic sequences for CYP2B6 and CYP2B7 (NG_000008) and the mRNA sequence for CYP2B6 (NM_000767), retrieved from the online National Center for Biotechnology Information database. The sequences were aligned by use of CLUSTAL W, and PCR primers were designed based on this alignment.
multiplex amplification of cyp2b6
The multiplex PCR reaction was performed in a total volume of 25 µL with 20–500 ng of genomic DNA, multiplex primer pairs (see Table 1
in the Data Supplement that accompanies this article at http://www.clinchem.org/content/vol50/issue8/), 1x ammonium buffer [67 mM Tris-HCl (pH 8.8), 16 mM (NH4)2SO4, 0.1 mL/L Tween 20], 1.5 mM MgCl2, 80 µM deoxynucleotide triphosphates (Sigma-Aldrich, UK), and 2 U of Taq DNA polymerase (Bioline, UK).
|
For the multiplex reaction, we used an initial denaturation step of 94 °C for 4 min, followed by 20 cycles of 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 2 min, with a final extension cycle at 68 °C for 10 min. To verify the presence of each amplicon, we subjected 10 µL of the PCR product to electrophoresis on a 1% agarose gel stained with ethidium bromide.
specificity of multiplex amplicons
The CYP2B6 specificity of first-round product was initially tested against CYP2B7 positive primers. The PCR reaction was set up in a volume of 8 µL containing 1x ammonium buffer, 1.9 mM magnesium chloride, 400 µM deoxynucleotide triphosphates, and 0.125 U of Taq DNA polymerase (23). We added 1 µL of a 1:100 dilution of PCR product from the multiplex round to 60 µL of this buffer and then added 5 µL of the DNA–buffer mixture to each well, which contained 10 µL of mineral oil and 3 µL of allele-specific primer mixture (see Table 2
in the online Data Supplement).
|
The reaction was subjected to the following PCR conditions: an initial denaturation step of 96 °C for 1 min; 5 cycles of 96 °C for 20 s, 70 °C for 45 s, and 72 °C for 25 s; 21 cycles of 96 °C for 25 s, 65 °C for 50 s, and 72 °C for 30 s; and a final round of 4 cycles of 96 °C for 30 s, 55 °C for 60 s, and 72 °C for 30 s. We added 5 µL of loading buffer to the post-PCR reaction mixture and subjected it to electrophoresis on a 1% agarose gel stained with ethidium bromide at 200 V for 25 min.
To further verify specificity, we purified amplicons by use of shrimp alkaline phosphatase (Promega UK Ltd.) and exonuclease 1 (New England BioLabs, UK) and performed DNA sequencing with the original amplification primers used as sequencing primers.
genotyping by allele-specific pcr
Assays were developed for the following published polymorphisms: C64T (Arg22Cys), C78T, G216C, G516T (Gln172His), C777A (Ser259Arg), A785G (Lys262Arg), and C1459T (Arg487Cys); and for the following variations found within the SNP databases: A1190G, C1268A, C1330T, A1382G, A1402T, and the intronic A12917T.
We genotyped the samples by use of PCR with sequence-specific primers (PCR-SSP), using conditions and components identical to those described above for testing the specificity of the multiplex amplicons. The template DNA used was 1 µL of a 1:100 dilution of our initial multiplex-PCR product. The sense, antisense, and consensus primers used to detect each SNP as well as the control primers used in each reaction are listed in Table 3
of the online Data Supplement.
|
haplotyping
The SNPs investigated in this study were reconstructed into inferred haplotypes by use of the statistical software PHASE v2.0 (24)(25), which implements Bayesian statistical methods to reconstruct haplotypes from genotypic data. PHASE can be downloaded from the web site listed in Ref. ((26)).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in the online Data Supplement) were designed over regions where the two genomic sequences differed and were chosen with the intention of amplifying regions of differing sizes for ease of resolution when viewed on an agarose gel stained with ethidium bromide. Primers were designed with similar melting temperatures so as not to hybridize with each other, thus preventing problems with primer-dimers. The melting temperature of each primer was calculated by use of the online "Oligonucleotide Properties Calculator" (27). To test for possible primer-primer interactions, we used "Oligos 9.9", now replaced by "Fast PCR" (28). To test that the PCR products generated from the original amplification were specific for CYP2B6 at all amplicons, we designed primers to test for coamplification of homologous CYP2B7 genomic regions. We designed this step using SSPs to detect differences between the CYP2B6 and CYP2B7 genomic sequences. If an amplicon was found not to be specific for CYP2B6, i.e., a product was generated in the CYP2B7-specific reaction, the initial amplification primers for that amplicon were redesigned. Once the amplicon appeared to be specific for CYP2B6, it was verified by sequencing.
Initially, each amplicon was amplified individually, and once the cycling conditions and reaction components had been optimized for each reaction, the primers were combined, at a concentration of 0.4 µM each, into a multiplex mixture. Using multiplex PCR guidelines (29), we optimized the cycling conditions and reaction components to generate the best amplification of all products. The relative amplicon yields were increased or decreased by adjusting primer concentrations, thus generating a more balanced multiplex reaction (Fig. 2
). Once the multiplex mixture was optimized, it was retested for CYP2B6 specificity with the described previously primers (Table 2
in the online Data Supplement).
|
pcr-ssp
The polymorphisms chosen for analysis included all published SNPs up to June 2003 and a selection found on SNP databases to cover the length of the gene. The location of each SNP investigated is illustrated in Fig. 1
.
PCR reactions were carried out for both the common and variant alleles. Each reaction contained internal control primers, which amplified a nonpolymorphic region within the multiplex template (Fig. 3
). A choice of four controls were used, depending on the location and size of product. Other than primer concentration and choice of control primer, the PCR conditions and components were kept the same for each genotyping assay.
|
genotypic frequencies
The genotyping assays were tested on a Caucasian panel for comparison with previously published allele frequencies (21). Of those SNPs published previously, C64T, C78T, G216C, G516T, A785G, and C1459T were found to vary in this cohort. C777A was observed only as the wild-type C allele. Of the SNPs identified in SNP databases, the intronic A12917T was the only one to vary in our population. All loci were in Hardy–Weinberg equilibrium (Table 1
), and allele frequencies were similar to those previously published (21), although a recent publication (17) has reported lower allele frequencies for A785G.
haplotypic data
CYP2B6 alleles *1, *2, *4, *5, and *6 (Table 2
) were identified by use of the program PHASE. An additional intronic SNP (A12917T) is present in the *4 haplotype; we have tentatively termed this allele *4C. Haplotype frequencies were similar to those published previously (21). CYP2B6*6, which has two coding mutations, was the most frequent variant allele. The most frequent haplotypic combinations were *1/*6, *1/*1, *1/*5, *6/*6, *5/*6, and *1/*2B at frequencies of 31.8%, 29.6%, 9.6%, 7.4%, 6.7%, and 6.0%, respectively (Table 3
). The nine other haplotypic combinations identified all occurred at frequencies <3%.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Several research groups have reported highly variable activity of hepatic CYP2B6 (13)(16)(21). This interindividual variability may have important clinical implications affecting the metabolism of a wide range of exogenous and endogenous substances. The precise effects of genetic variation on protein enzyme function have not yet been fully elucidated. It has been reported, however, that the exon 9 polymorphism C1459T (R487C) leads to significantly reduced protein concentrations (21) and in females leads to significantly lower CYP2B6 activity (17), whereas the exon 4 polymorphism G516T (Q172H) (30) and exon 5 polymorphism A785G (K262R) (31) enhance the catalytic activity of the enzyme. This variation may also affect clinical response to CYP2B6 substrates by modification of the pharmacokinetics. A recent study suggested that total clearance of bupropion did not differ for individuals with the *1, *2, and *6 alleles but that enzyme kinetics were significantly different for individuals with the *4 allele (32), potentially impacting on dosing of bupropion. Because more data on the expression and function of CYP2B6 are being collected, it is important to have a high-resolution rapid genotyping procedure to correlate genomic and phenotypic data in large-scale studies.
The current method for the genotypic analysis of the CYP2B6 gene uses an assay combining PCR with restriction fragment length polymorphism analysis (21). Although this published method covers all exonic regions and exon/intron boundaries and is thus suitable for the determination of most genotypes, each fragment is amplified individually. The disadvantage of this is that in a large genotyping study of multiple polymorphisms, many template gene fragments would be generated for each individual. We have designed a novel genotyping technique for CYP2B6 that does not rely on restriction fragment length polymorphism analysis and reduces the number of PCR reactions substantially without loss of coverage of the genomic region. The method successfully separates CYP2B6 from the similar CYP2B7 by means of a first-round amplification step, verified by sequencing. The initial step is multiplexed, allowing seven gene fragments to be amplified simultaneously. An advantage of this approach is that additional regions of interest can be added to the multiplex mixture if required.
An alternative approach would be to use long-range PCR to amplify the entire gene; however, because of its size this would be unlikely to give reliable results in routine use. Our method of multiplexing shorter fragments avoids problems with low DNA concentrations or poor-quality DNA. The amplification product sizes varied from 582 to 1865 bp, and no products failed to amplify. The method was tested on five individual DNA samples from a separate test panel over a range of concentrations (20–500 ng/µL) and still retained its specificity (data not shown). The assay was not tested with DNA <20 ng/µL, but no loss of sensitivity was observed at this concentration. Acceptable performance was assured by the repeated clear visualization of the internal controls and allele-specific products. Because this method uses a two-step PCR process, the amount of template DNA necessary for the genotyping analysis is greatly reduced. Thus, samples with low DNA concentrations can easily be genotyped because a small amount of template DNA generates ample second-round PCR product.
The genotyping method using SSPs (23) is robust and simple. It has been optimized so that the buffer and PCR conditions suit a variety of simple SSP assays. The buffer mixture can be made up in bulk and stored (without Taq DNA polymerase) for several months at –20 °C. Each assay uses the same template DNA with only the primer mixture changing, facilitating ease of use.
The method was tested on a population panel of 135 healthy British Caucasians, and the results compared favorably with genotypic and allelic frequencies published previously for a Caucasian population of 215 individuals (21). Five of the seven currently published alleles were found in our population (*1, *2, *4, *5, and *6). Although we did not find the rare C777A polymorphism in our Caucasian population, we have since identified individuals heterozygous for this polymorphism in another cohort. The new allele that we identified, defined by the intronic SNP 12917, may not itself be functionally relevant, and any effects on phenotype will therefore need to be investigated in future studies. Our method successfully types the coding region of CYP2B6 and also includes a 5' upstream region amplicon for future identification of variation in this region. The initial multiplex reaction allows for easy analysis of new polymorphisms (and thus alleles) by the design of additional allele-sequence-specific primers and genotyping on the same PCR-product template. Sequencing of regions of interest by use of this first-round product may also identify new polymorphisms, and the method has be shown to be capable of detecting new CYP2B6 alleles.
In conclusion, this multiplex PCR assay is simple and robust and likely to be useful in large-scale studies of the effects of genetic variation in CYP2B6 on response to drug therapy.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  J. K. Blievernicht, E. Schaeffeler, K. Klein, M. Eichelbaum, M. Schwab, and U. M. Zanger MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms Clin. Chem., January 1, 2007; 53(1): 24 - 33. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
