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Technical Briefs |
Departments of1 Health Risk Analysis and Toxicology and2 Population Genetics, Genomics and Bioinformatics, University of Maastricht, Maastricht, The Netherlands;
aaddress correspondence to this author at: Department of Health Risk Analysis and Toxicology, University of Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands; fax 31-43-3884146, e-mail j.vandelft{at}grat.unimaas.nl
Studies have reported a large interindividual variation in susceptibility to health effects caused by exposure to xenobiotic compounds such as drugs or chemical carcinogens. There is evidence that this can be partly explained by the existence of genetic polymorphisms in metabolic enzymes, such as cytochrome P450 (CYP450), N-acetyltransferases (NATs), and glutathione S-transferases (GSTM, GSTT, GSTP) [see, e.g., Refs. (1)(2)(3)(4)]. However, studies investigating associations between genetic polymorphisms and disease have reported conflicting results, probably caused by insufficient statistical power (5). Moreover, the majority of these studies focused on single polymorphisms. Regarding the number of genes implicated in the metabolism of xenobiotics and the large number of polymorphisms present in the human genome (6), these approaches fail to fully determine the role of genetic variation in an individual’s susceptibility to xenobiotic exposures. Such observations underline the need for methodologies that allow for high-throughput, low-cost genotyping of multiple polymorphisms in large populations (7)(8). In this study we describe the development, validation, and application of a cost-effective and rapid method for simultaneous genotyping of nine polymorphisms in five key enzymes involved in metabolism of xenobiotics: CYP1A2, GSTM1, GSTP1, GSTT1, and NAT2.
The fragments containing the nine single-nucleotide polymorphisms (SNPs) were amplified in one sevenplex and one duplex PCR reaction (Table 1
). Primers were obtained from Qiagen. For the sevenplex PCR, a 50-µL reaction mixture was prepared containing PCR buffer, 0.2 mM deoxynucleotide triphosphates, 0.5 mM MgCl2, 1.25 U of Platinum® Taq Polymerase (Invitrogen), and 200 ng of template DNA. The final primer concentrations were 0.22 µM (for GSTP1*3, GSTT1, NAT2*6, and NAT2*7), 0.45 µM (for CYP1A2*1F and NAT2*5), and 0.16 µM (for GSTP1*2). PCR was conducted as follows: denaturation at 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. For the duplex PCR, the final primer concentration was 0.1 µM, and PCR was performed with denaturation at 95 °C for 5 min; 40 cycles of 95 °C for 30 s, 59 °C for 60 s, and 72 °C for 90 s; and a final extension at 72 °C for 5 min (Fig. 1
). In later experiments, the reaction volumes were reduced to 10 µL, and all fragments are amplified from 80 ng of DNA as starting material. After PCR amplification, the products were pooled (5 µL of the sevenplex and 4 µL of the duplex PCR product) and incubated (37 °C for 45 min) with 4 µL of ExoSAP-IT (Amersham) to digest contaminating deoxynucleotide triphosphates and primers. Enzymes were deactivated at 75 °C (15 min).
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The primers for single base extension (SBE) were designed to bind immediately adjacent 5' to the SNP. During thermal cycling, the primers are extended at their 3' end with a single dideoxyribonucleoside triphosphate labeled with a distinct fluorophore, revealing the genotype of the SNP. For GSTT1 and GSTM1 (deletions), the extension primers were designed to hybridize in the middle of the amplified fragment. All primers were designed to anneal to the antisense strand, except for the CYP1A2*1C primer, which annealed to the sense strand. To facilitate detection of nine polymorphisms in a single run, the length of the extension primers was adjusted to a distinct size by addition of a nonhomologous tail to their 5' site (Table 1
). These tails were taken from the 5' site of the random sequence 5'-AACTGACTAAACTAGGTGCCACGTCGTGAAAGTCTGACAA-3' (9).
Multiplex SBE was performed using SNaPshotTM as described by the manufacturer (Applied Biosystems). Briefly, 4 µL of purified PCR product (containing nine fragments) was mixed with 3.5 µL of SNaPshot reaction mixture, 1 µL of pooled SBE primers, and 1.5 µL of water. The final concentration for all SBE primers was 0.2 µM, except for NAT2*5 and GSTP1*3 (1 µM), and NAT2*6 (0.04 µM). SBE was performed using 25 cycles of 96 °C for 10 s and 60 °C for 30 s. Subsequently, the samples were incubated at 37 °C for 1 h with 1 U of shrimp alkaline phosphatase (Amersham), followed by enzyme deactivation at 75 °C for 15 min. The SBE products were finally analyzed by capillary electrophoresis, for which 1 µL of the (fivefold-diluted) SBE product was mixed with 13 µL of deionized formamide and 0.4 µL of Genescan-120 LIZTM size marker. Samples were denatured at 95 °C and run on an ABI-Prism® 3100 genetic analyzer using a 36-cm capillary array and POP-6 polymer. Analyses were performed with GenescanTM software (Ver. 3.7; Fig. 1C
).
For validation, 67 lymphocyte DNA samples from healthy Caucasian volunteers were used (10). Informed consent was obtained from all individuals. All 67 samples were genotyped by the SBE assay. Accurate and complete genotypes were obtained from all samples, and frequencies were as expected. To test for interassay variation, SBE genotyping of 20 different individuals was performed twice, and no differences were found. Further validation was performed by use of conventional methods (restriction fragment length polymorphism analysis and sequencing) in at least 10 different samples (67 samples for GSTM1, GSTT1, and NAT2*5), and concordance was 100% between the SBE assay and the conventional methods. For NAT2*5 genotyping, we evaluated the SNP at position 341 because this covers all possible NAT2*5 subtypes. Our data showed that this approach is more accurate than evaluating NAT2*5 by the conventionally used restriction fragment length polymorphism analysis for detection of 481C>T variation. Moreover, haplotype analysis of defective NAT2 alleles has been shown to be a good predictor of acetylation phenotype. By analyzing variation at positions 341, 590, and 857, we expect the phenotype to be correctly predicted in 90–95% of the population (11)(12).
Although SBE technology was originally designed to genotype SNPs, our data demonstrate that it can be applied for analysis of gene deletions, such as GSTM1-null and GSTT1-null. Heterozygous deletions cannot be distinguished because peak height does not quantitatively reflect gene copy number. However, because GSTM1 and GSTT1 enzyme activity is completely eliminated only through homozygous gene deletions, distinction between heterozygous and wild-type carriers is of less importance. Earlier studies that used comparable SBE genotyping technologies started with pools of simplex PCR products (13), pooled DNA samples from cases or controls (14), or with single long-PCR fragments (15). Others have used PCR multiplexing of five segments in a single gene (16). To fully utilize the high-throughput capabilities of the SNaPshot technology, we designed a multiplex PCR-based amplification of nine alleles in five different genes. This approach reduces costs, avoids the use of large amounts of DNA, and increases the speed of the assay. All PCR reactions and purification steps can be performed in 96-well plates, at a speed of 2 plates/day. Additionally, apart from the fact that the presented method is quick, accurate, and inexpensive, its versatility adds another major advantage: the protocol can be easily adapted for new sets of polymorphisms.
In conclusion, we developed a rapid, accurate, and thoroughly validated method for simultaneous genotyping of nine polymorphisms in five xenobiotic-metabolizing enzymes. The selected polymorphisms are located in genes involved in activation (CYP1A2) as well as detoxification (GSTM1, GSTP1, GSTT1, and NAT2) of xenobiotics such as chemical carcinogens. Although some of these genes may contain additional polymorphisms, those included in our assay were specifically selected based on known effects on enzyme activity as well as on health effects, including (chemical) carcinogenesis (Table 1
). Therefore, our assay is well suited for large-scale studies that aim to identify xenobiotic-related health effects by studying polymorphisms in multiple susceptibility genes, their mutual interactions, and their effect on phenotypic characteristics such protein/DNA-adduct formation. Such studies could help to develop and specify individual health-risk profiles.
Acknowledgments
This study was supported by the research program "Environment and Health" of the Flemish Government (Belgium), and by the Trans-national University Limburg.
References
A polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine. Br J Clin Pharmacol 1999;47:445-449.[CrossRef][ISI][Medline]
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