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Technical Briefs |
1 Department of Dermatology and Allergology, University Hospital of the RWTH, Aachen, Aachen, Germany;2 Department of Ecotoxicology/Toxicology, University Trier, Trier, Germany
aaddress correspondence to this author at: Department of Ecotoxicology/Toxicology, University Trier, Science Park, Bldg. 24, Sickingenstrasse 96, D-54290 Trier, Germany; fax 49-651-201-3780, e-mail BLOEMEKE{at}UNI-TRIER.DE
N-Acetyltransferase 2 (NAT2; EC 2.3.1.5) is involved in the detoxification of numerous xenobiotics. The human NAT2 gene is highly polymorphic and represents one of the best studied examples of the large interindividual variability of genetic control of drug or xenobiotic metabolism. In addition to the antituberculosis agent isoniazid (1), the first NAT2 substrate discovered, numerous other chemicals possessing a primary aromatic amine or hydrazine group, such as sulfamethazine, procainamide, hydralazine (2), dapsone, nitrazepam, and caffeine, are metabolized by this enzyme. Arylamine chemicals such as benzidine, ß-naphthylamine, and 2-aminofluorene can also be acetylated by NAT2. As early as 1970, Reidenberg et al.(3) reported clinical problems associated with polymorphic acetylation of procainamide. The determination of NAT2 genotype or NAT2 phenotype has been proposed as a way to predict adverse reactions in patients with tuberculosis and before the concomitant administration of procainamide and phenytoin (4).
The proportions of rapid and slow phenotypes vary in different ethnic groups. In Caucasians, 40–70% of individuals have the slow acetylator phenotype, whereas Asian populations have only 10–30% slow acetylators (5). Several single-nucleotide polymorphisms have been identified in the human NAT2 coding region, which are responsible for the observed phenotypes [see Ref. (6); accession no. X14672]. The presence of the NAT2*4 (wild-type) allele defines the NAT2 genotype as rapid, and combinations of the frequently occurring mutant alleles NAT2*5B, *5C, *6A, *7B, and *14 cluster as slow. Allele frequencies in Caucasians are 23% for NAT2*4 (wild type), 1.3% for *5A, 35% for *5B, 5.0% for *5C, 30.5% for *6A, 4.5% for *7B, and 0.2% for *12A. The ability to simultaneously detect these mutations enhances opportunities to validate their impact on adverse risk in large-scale studies and allows physicians to avoid individual variations in drug response (7). Current options for NAT2 genotyping include sequencing, multiple PCRs, PCR with restriction fragment length polymorphism analysis, and PCR in combination with hybridization probes.
Recently, a commercial assay (LightCyclerTM Roche Diagnostics) has been introduced for the rapid detection of four common mutations (G191A, C481T, G590A, G857A) in the NAT2 gene based on hybridization probes labeled with fluorescent dyes. Although this assay allows for easy, unambiguous, and rapid detection of the major mutations, it has some limitations; for example, it cannot detect the slow NAT2*5C (341C, 803G) allele. To overcome these drawbacks, we developed a novel assay for the detection of the T341C mutation based on hybridization probes.
Genotyping for the T341C mutation was performed by PCR using specific fluorescently labeled hybridization probes. The reaction was performed in 10 µL containing 10–50 ng of DNA, 1 µL (10-fold) of reaction mixture, 3 mM MgCl2, 13 pmol of each primer (forward, 5'-CTT GAG CAC CAG ATC CGG G-3'; reverse, 5'-TAA TTC TAG AGG CTG CCA CAT CT-3'), 2 pmol of sensor and anchor (5'-LC Red 640-TCG ATG CTG GGT CTG GAA GCT CCT CCC p-3' and 5'-GAC CAC TGA CGG CAG GAA TTA C X-3'; designed and synthesized by TIB MOLBIOL). After initial denaturation (95 °C for 10 min), a total of 50 cycles were performed consisting of denaturation at 95 °C, annealing at 58 °C for 10 s, and extension at 72 °C for 15 s. A sample was classified as TT or CC according to the melting curves. For detection of the C481T, G590A, G191A, and G857A polymorphisms in the NAT2 gene, we used the NAT2 Dicolor Mutation Detection Kit (Roche Diagnostics) as recommended by the manufacturer.
The results for the three different T341C genotypes in the NAT2 gene are shown in Fig. 1
. Fig. 1A
shows the melting curves; the corresponding melting peaks (Tm) derived from these data are shown in Fig. 1B
. Samples homozygous for the T mutation had a Tm of 57.3 ± 0.2 °C, whereas the melting peak for samples homozygous for the C was at 65.2 ± 0.2 °C (n = 30). Heterozygous samples exhibited two distinct Tms. No-template controls did not produce a signal. The melting peaks for the C481T, G590A, G191A, and G857A mutations were as follows. For C481T, the Tm for the C481 variant was at 49.1 ± 1.0 °C and that for the T481 variant was at 56.9 ± 0.8 °C. For G590A, the Tm for the A590 variant was 62.0 ± 1.0 °C and that for the G590 variant was 67.4 ± 1.0 °C. For G191A, the Tm for the A191 variant was at 47.7 ± 0.4 °C and that for the G191 variant was at 55.1 ± 0.5 °C. Finally, for G857A, the Tm for the A857 variant was at 61.5 ± 0.5 °C and that for the G857 variant was at 66.6 ± 0.8 °C (data not shown).
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The NAT2 alleles with mutations detected by this approach are summarized in Table 1
. After validation of the assay, we also used the PCR cycling conditions of the NAT2 Dicolor Mutation Detection Kit to detect the T341C mutation, which allowed us to detect five polymorphisms in a single run. Using just this assay, we could detect the alleles NAT2*5A/B/F, NAT2*5E, NAT2*6A/B/C/D, NAT2*7A/B, NAT2*11, NAT2*12C, and NAT2*14A/B/C/D/E/F/G, and the assay for the T341C polymorphism detected the alleles NAT2*5A/B/C/D/E/F and NAT2*14C/F. The combination of these two assays allowed additional detection of the NAT2*5C/D allele and separation of the fast allele NAT2*12A (803A) from the slow *5C allele. This significantly decreased the chances for misclassification (8). Moreover, the NAT2*11 and NAT2*12C alleles could be differentiated from the NAT2*5A/B/F allele and the NAT2*14F allele from the NAT2*14A/B/C/D/E/G alleles. On the basis of all mutations known in these alleles, we completely typed 7 alleles, whereas typing of the other 14 alleles remained incomplete with the combination of these two assays.
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To validate the method, we used this novel approach to genotype 155 DNA samples from Caucasians with known mutations (9) and found complete agreement. The allele frequencies for NAT2*4 (wild type), *5A/B/F, *5C/D,*6A/B/C/D, and *7A/B were similar to published frequencies (5). A G191A mutation was not detected. The concordance rate was 100% for each polymorphic site with DNA from whole blood, whereas the sensitivity was 70% and the specificity was 100% for a second set of DNA samples (n = 400) extracted from plasma or serum. Overall, we noticed some lower absolute fluorescence for C481T, but intensities were still high enough to allow unambiguous results. However, it must be noted that >30 different NAT2 alleles are currently known (http://www.louisville.edu/medschool/pharmacology/NAT2.html). Several single-nucleotide polymorphisms have been reported in the 5'- and 3'-flanking regions of the gene, which also might affect phenotypes through endogenous or exogenous regulation or other interactions. In addition, striking ethnic differences in the frequencies of the slow acetylator alleles and phenotypes (10)(11) must be considered. Therefore, analysis of additional mutations (C282T and A803G) should be performed when mixed Hispanic or Orientals individuals are studied to minimize chances for misclassification (12).
In conclusion, the procedure is very simple, results are robust, and repeats on 20% of the samples were in complete agreement. This approach allows the detection of five major NAT2 mutations (G191A, T341C, C481T, G590A, and G857A) and prediction of the resulting phenotype in just 1 h, making it suitable for clinical applications (13).
Acknowledgments
This project was supported by a grant from the "Deutsche Forschungsgemeinschaft".
Footnotes
1 current affiliation: Grünenthal GmbH, Aachen, Germany 
References
G) codes for rapid arylamine n-acetylation in humans. Pharmacogenetics 1996;6:257-259.[CrossRef][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  J. A. G. Agundez, K. Golka, C. Martinez, S. Selinski, M. Blaszkewicz, and E. Garcia-Martin Unraveling Ambiguous NAT2 Genotyping Data Clin. Chem., August 1, 2008; 54(8): 1390 - 1394. [Abstract] [Full Text] [PDF]
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