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Clinical Chemistry 52: 1033-1039, 2006. First published March 23, 2006; 10.1373/clinchem.2005.063198
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(Clinical Chemistry. 2006;52:1033-1039.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Simultaneous Determination of 7 N-Acetyltransferase-2 Single-Nucleotide Variations by Allele-Specific Primer Extension Assay

Yusheng Zhu1, David W. Hein2, Mark A. Doll2, Kristen K. Reynolds1, Ntei Abudu1, Roland Valdes, Jr1 and Mark W. Linder1,a

Departments of1 Pathology and Laboratory Medicine and 2 Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY.

aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202. Fax 502-852-1177; e-mail mwlind01{at}gwise.louisville.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Genotyping of N-acetyltransferase-2 (NAT2) is useful in predicting the risk for toxicity of NAT2 substrates. Current methods cannot detect the 7 most important single-nucleotide variations in NAT2 simultaneously in 1 tube.

Methods: We developed an assay that uses allele-specific primer extension (ASPE) and microsphere hybridization for the simultaneous detection of 7 single-nucleotide variations in NAT2. Using 12 samples previously genotyped by a TaqMan-based assay for method development and as positive controls, we amplified the genetic locus of NAT2 comprising the single-nucleotide variations of interest by PCR and then performed ASPE with allele-specific primers and biotinylated dCTP followed by bead hybridization and streptavidin–R-phycoerythrin binding. Genotypes were determined according to the allele-specific fluorescent signal ratios.

Results: The mean (SD) allelic ratios for homozygous common, heterozygous variant, and homozygous variant NAT2 genotypes were 0.0394 (0.0113) (n = 80), 0.4372 (0.0270) (n = 148), and 0.9331 (0.0127) (n = 325). The assay had 100% (95% confidence interval, 99%–100%) within-run reproducibility for 12 samples repeated 6 times and 100% (98%–100%) between-run reproducibility for a 5-sample subset run on 6 different days. NAT2 genotypes of 30 blinded samples determined by this assay were 100% (98%–100%) concordant with results obtained using the TaqMan method.

Conclusions: The developed assay can simultaneously determine single-nucleotide variations in NAT2. The assay demonstrates no overlap in allele-specific signal ratios between homozygous common, heterozygous, and homozygous variant and shows agreement with a reference method and reproducibility of genotype identification.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
N-Acetyltransferase 2 (NAT2) 1 catalyzes the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Individual variations in the activity of this enzyme (rapid, intermediate, and slow acetylator) are broadly distributed among populations. Intersubject variability in NAT2 can be attributed to genetic variation of the NAT2 gene2 (1)(2). Sequence variations in the NAT2 gene have been associated with increased risks for a variety of cancers and drug-induced toxicities(3)(4)(5)(6)(7). NAT2 is encoded by an 870-bp gene (NAT2), and 36 human NAT2 alleles have been identified (http://www.louisville.edu/medschool/pharmacology/NAT2.html). The NAT2*4 allele encodes for a fully active enzyme and is traditionally considered as the reference rapid acetylator allele. Additional alleles that encode a fully functioning enzyme include the NAT2*12 and NAT2*13 alleles. There are primarily 4 nucleotide substitutions at positions 341, 590, 857, and 191 that are associated with low enzyme activity. These nucleotide substitutions are found within the NAT2*5, NAT2*6, NAT2*7, and NAT2*14 allele clusters, and several studies have shown that the members of these clusters are responsible for the slow acetylator phenotype(8). Most variant alleles include one or more of the following 7 most frequent nucleotide substitutions: 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, and 857G>A(2)(9)(10)(11). In the majority of reported studies investigating the relationship between NAT2 genotype and disease risk, NAT2 acetylation status was investigated by use of PCR-based assays that detected only 3 single-nucleotide variations (481C>T, 590G>A, and 857G>A)(12), but several reports described determination of more single-nucleotide variations(12)(13)(14)(15). Although several NAT2 single-nucleotide variations are in linkage disequilibrium, assessment of only these 3 variations leads to the misclassification of some NAT2 alleles(12). The detection of 11 single-nucleotide variations (111T>C, 191G>A, 282C>T, 341T>C, 434A>C, 481C>T, 590G>A, 759C>T, 803A>G, 845A>C, and 857G>A) can determine most allelic variants and is considered the gold standard approach(16). Compared with the assay for 11 single-nucleotide variations, the sensitivity and specificity of the assay for 3 single-nucleotide variations are 94% and 100%(12). After using the assay for 11 single-nucleotide variations to screen 950 alleles, Deitz et al.(12) recommended that 7 single-nucleotide variations be screened in Caucasian and African-American populations. One of the characteristics of NAT2 sequence variations is that multiple single-nucleotide variations are frequently linked together as haplotypes. The common haplotypes of NAT2 are listed in Table 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue6/.

Investigations have shown both a bimodal distribution of NAT2 phenotypes in human populations (rapid and slow acetylators) (1)(10)(13)(17) and a trimodal distribution (rapid, intermediate, and slow acetylators)(11)(18). Some common NAT2 alleles, along with corresponding nucleotide changes and phenotypes, are listed in Table 2 of the online Data Supplement. Although there is minor controversy regarding the NAT2*13 allele, the consensus is that it is associated with rapid acetylation(5).

Slow acetylator variants of the NAT2 gene have been reported to be associated with adverse reactions to a variety of drugs, such as isoniazid (19)(20), sulfasalazine(8)(21)(22), and co-trimoxazole(23)(24). Therefore, analysis of sequence variations in the NAT2 gene for patients on these drugs is useful to predict the risk for drug-induced toxicity.

Reported methods to determine NAT2 single-nucleotide variations include restriction fragment-length polymorphism (RFLP) analysis after PCR, allele-specific amplification, allele-specific hybridization, and oligonucleotide ligation assays, which are labor-intensive and time-consuming (10)(13)(25)(26)(27)(28)(29)(30), and the TaqMan real-time PCR(31) and microarray(32) methods. None of these reported methods can detect all 7 single-nucleotide variations simultaneously in a single tube; therefore, multiple-tube PCR must be used, which is more complicated and requires more DNA. As a result, samples with insufficient DNA cannot be analyzed. To overcome these limitations, we sought to develop an assay to simultaneously detect all 7 single-nucleotide variations in the NAT2 gene by use of allele-specific primer extension (ASPE) in a single tube with 7 pairs of tag-labeled allele-specific primers on the Luminex® 100TM IS System.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
dna samples for assay development and verification
We used 10 human genomic DNA and 2 plasmid DNA samples in which the NAT2 genotype or haplotype was determined by TaqMan real-time PCR with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as described previously (31). Genomic DNA was extracted with QIAamp® DNA Blood Mini Kits (Qiagen). The final DNA concentrations were 5.4 to 21.9 ng/µL, as determined by the NanoDrop® ND-1000 Spectrometer (NanoDrop Technologies). DNA samples were stored frozen until use.

assay design
The NAT2 assay is illustrated in Fig. 1 .


Figure 1
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  		Figure 1. Illustration of the NAT2 assay.

(Step 1. PCR and enzyme treatment), the genetic locus of NAT2 comprising the 7 single-nucleotide variations of interest is amplified, and the PCR product is treated with SAP and EXO I. (Step 2. Multiplex ASPE), a common allele-specific primer containing T with tag 1 anneals with a common allele containing A, and a variant allele-specific primer containing C anneals with a variant allele containing G. Allele-specific primers are extended by DNA polymerase, and biotinylated dCMP is incorporated into the extended primers. (Step 3. Hybridization and reporter binding), extended allele-specific primers with specific tags are hybridized with specific beads with anti-tags. Streptavidin–R-phycoerythrin binds the biotin in the extended allele-specific primers. (Step 4. Data analysis), beads are sorted on the Luminex 100 IS System, and the fluorescent signal of phycoerythrin is measured. The MFI in arbitrary units is given on the y axis.

Step 1. PCR amplification of the NAT2 fragment spanning the location of 7 single-nucleotide variations in the NAT2 coding region.
To detect 7 single-nucleotide variations at nucleotide positions 191, 282, 341, 481, 590, 803, and 857 in the NAT2 coding region, we amplified an 866-bp fragment of NAT2 DNA by PCR with 2 previously reported primers (33). A 20-µL PCR reaction was performed on a Mastercycler® Gradient thermal cycler (Eppendorf). Each 20-µL reaction contained 1x PCR reaction buffer, 1.5 mM MgCl2, 0.2 mM each of the deoxynucleotide triphosphates, 0.25 µM each primer (IDT), 1.5 U of Platinum® Taq DNA Polymerase (Invitrogen), and 25 ng of DNA. The conditions for amplification were as follows: an initial denaturing step of 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1 min, with a final elongation step of 72 °C for 5 min.

Step 2. Shrimp alkaline phosphatase (SAP) and exonuclease I (EXO I) treatment.
To remove unincorporated deoxynucleotide triphosphates and primers, we added 2 µL of SAP (1 U/µL; USB) and 0.5 µL of EXO I (10 U/µL; USB) to each 20 µL of PCR reaction mixture. After this mixture was incubated at 37 °C for 30 min, the enzymes were inactivated by heating at 99 °C for 15 min.

Step 3. ASPE.
We applied 5 µL of SAP- and EXO I-treated PCR product as the template for ASPE in a total of 20 µL of reaction mixture containing 1x PCR reaction buffer; 1.25 mM MgCl2; 5 µM each of dGTP, dATP, dTTP, and biotin-14-dCTP (Invitrogen); 0.025 µM primer mixture containing 14 specific tag-labeled allele-specific primers for 7 common and 7 variant alleles (synthesized by Sigma-genosys); and 1.5 U of Platinum GenoType Tsp DNA Polymerase (Invitrogen), or TITANIUM Taq DNA Polymerase (BD), or TaKaRa TaqTM HS DNA Polymerase (TAKARA BIO, INC.). The reaction mixture was pretreated at 96 °C for 2 min followed by 30 cycles of 30 s at 94 °C, 1 min at 55 °C, and 2 min at 74 °C.

Step 4. Luminex Flexmap microsphere hybridization and data collection.
We used 14 types of carboxylated fluorescent microspheres with covalently attached anti-tag sequences (Luminex Corporation). The bead mixture was prepared at 56 beads of each population per microliter in 1.1x wash buffer (TmBioscience), and 45 µL of the bead mixture was mixed with 5 µL of ASPE product in a PCR tube. Hybridization of ASPE products and the beads was carried out on a Mastercycler Gradient thermal cycler (Eppendorf). After DNA denaturation at 96 °C for 2 min, the mixture was incubated at 37 °C for 60 min. After hybridization, beads were transferred to a well in a filter plate (Millipore) and washed with 1x wash buffer (TmBioscience). To the washed beads we added 150 µL of 1x wash buffer containing 2 ng/µL streptavidin–R-phycoerythrin (Molecular Probes), followed by incubation at 37 °C for 15 min in the dark. Finally, beads were sorted, and the intensity of R-phycoerythrin on the beads was measured on the Luminex 100 System. Data were analyzed with Luminex IS 2.2 software.

primer sequences
Two previously reported primers were used to amplify an 866-bp fragment in the NAT2 coding region. The sequences of these 2 primers were as follows: forward primer, 5'-GGCTATAAGAACTCTAGGAAC-3'; reverse primer, 5'-AAGGGTTTATTTTGTTCCTTATTCTAAAT-3' (33). Fourteen 5'-tag–labeled allele-specific primers were used as forward primers in ASPE to determine common and variant alleles. The sequences of these 14 allele-specific primers with specific 5'-tag sequences are listed in Table 1 .


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  		Table 1. Allele-specific primers and corresponding single-nucleotide variations.

statistical analysis
We used Graphpad PRISM (Ver. 3.0; Graphpad Inc.) to perform the data analyses to compare mean homozygous common, heterozygous variant, and homozygous variant allelic ratios.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
confirmation of pcr products
We performed DNA electrophoresis on 2% agarose gels to confirm the success of PCR amplification and found that by use of forward and reverse primers, an 866-bp fragment of the NAT2 coding region containing all 7 single-nucleotide variations was amplified (data not shown). These results indicate that a NAT2 DNA fragment of the correct size was amplified consistently.

comparison of the performance of 3 dna polymerases for aspe
We used 10 DNA samples to compare the performance of 3 commercially available DNA polymerases in ASPE, including Platinum® GenoType Tsp DNA Polymerase, TITANIUM Taq DNA Polymerase, and TaKaRa Taq HS DNA Polymerase. Comparisons of the mean allelic ratios for the homozygous common, heterozygous variant, and homozygous variant generated by these 3 DNA polymerases are shown in Table 2 . Although in some cases we observed differences in allelic ratios generated by these 3 DNA polymerases, when these 3 DNA polymerases were used, the genotypes of the 7 single-nucleotide variations in all 10 DNA samples were concordant. These results indicate that all 3 DNA polymerases can be used for ASPE in NAT2 genotyping.


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  		Table 2. Comparison of allelic ratios of NAT2 generated by 3 DNA polymerases.1

aspe genotyping of 7 single-nucleotide variations of NAT2
We determined the genotypes of 7 single-nucleotide variations in NAT2 according to the ratio of the variant allele signal (M), in median fluorescence intensity (MFI), to the sum of the wild-type (W) and variant (M) allele signals: MFI [M/(W+M)]. In general, when the genotype was homozygous for the common allele, the ratio was <0.25, when the genotype was heterozygous, the ratio was 0.25–0.75, and when the genotype was homozygous for the variant allele, the ratio was >0.75. Examples of the MFIs for homozygous common and heterozygous and homozygous variants of single-nucleotide variations at nucleotides 191, 282, 341, 481, 590, 803, and 857 in the NAT2 gene are illustrated in Fig. 2 .


Figure 2
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  		Figure 2. Comparison of MFI of variant and common alleles for 7 single-nucleotide variations in the NAT2 gene.

The bar graph shows the MFI of variant ({square}) and common ({blacksquare}) alleles in homozygous variant (M), heterozygous variant (H), and homozygous common (W) genotypes for 7 single-nucleotide variations in the NAT2 gene. Each column represents the mean (SD; error bars) for determinations in DNA from 3 humans. NAT2 genotypes are clearly determined by calculating allelic ratios, using the MFI values of variant and common alleles. ANOVA followed by post hoc test with Bonferroni correction showed that the differences in allelic ratios between homozygous variant, heterozygous variant, and homozygous common genotypes are significant (P <0.001).

imprecision of the m/(w+m) ratio
We tested samples with different genotypes of NAT2 to determine the imprecision of the homozygous common, heterozygous variant, and homozygous variant M/(W+M) ratios for the 7 single-nucleotide variations in NAT2. The results are shown in Table 3 . The mean (SD) allelic ratios for homozygous common, heterozygous variant, and homozygous variant NAT2 genotypes were 0.0394 (0.0113), 0.4372 (0.0270), and 0.9331 (0.0127), respectively. For increased confidence, we suggest the following cutoff values for the allelic ratios: 0.00–0.15 for homozygous common genotypes, 0.30–0.70 for heterozygous variant genotypes, and 0.85–1.00 for homozygous variant genotypes. If the allelic ratio is 0.15–0.30 or 0.70–0.85, the sample should be retested.


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  		Table 3. Precision of the allelic ratios of the 7 single-nucleotide variations of NAT2.

reproducibility
Within-run reproducibility.
To determine within-run reproducibility, 6 replicates each of 12 samples were tested for 7 single-nucleotide variations in the same run. A total of 504 determinations were generated. Six repeats generated the same genotype (homozygous common, heterozygous variant, or homozygous variant) in all 12 samples according to the cutoff values for the allelic ratios listed above for the 3 genotypes. The within-run reproducibility was 100% [95% confidence interval (CI), 99%–100%].

Between-run reproducibility.
To determine the between-run reproducibility, we tested 5 samples in repeat runs on 6 different days. All runs generated equivalent results for all 7 single-nucleotide variations in all 5 samples, suggesting 100% between-run reproducibility (95% CI, 98%–100%).

TaqMan real-time PCR assay
To evaluate the agreement of the ASPE assay for NAT2 genotyping with an established method, we tested 30 blinded samples for all 7 single-nucleotide variations with the ASPE assay on the Luminex 100 IS System and compared the results with those obtained with the TaqMan real-time PCR on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The 2 assays generated concordant genotypes. In these 30 samples, we identified 18 slow acetylators, 11 intermediate acetylators, and 1 rapid acetylator. This comparison study shows 100% agreement (95% CI, 98%–100%) of ASPE assay NAT2 genotyping results with the results generated by the TaqMan real-time PCR assay.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ASPE assay method made it possible to readily distinguish between the 2 possible bases at each of the 7 single-nucleotide variations identified in the NAT2 coding region (191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, and 857G>A) according to the allelic ratio of the variant allele MFI to the sum of the common allele MFI and the variant allele MFI. No overlap was observed in the allelic ratios among homozygous common, heterozygous, and homozygous variant genotypes (Table 3Up ). Another advantage of this assay is the capability to detect all 7 single-nucleotide variations simultaneously in a single tube. Only 25 ng of DNA sample is required in this single-tube PCR; thus, this assay can analyze samples with very low DNA concentrations, such as buccal swabs, mouthwashes, saliva, and other forensic samples. In addition, a single-tube PCR method is less labor-intensive than one requiring 7 separate PCRs. Because results can be generated within 7 h, our method is very suitable for high-throughput applications. The assay showed 100% within-run and between-run reproducibility. Furthermore, the determination of the 7 single-nucleotide variations showed 100% concordance with TaqMan real-time PCR in 30 blinded samples, indicating the high accuracy of the assay.

Through the detection of 7 common single-nucleotide variations in NAT2, the assay provides sufficient information to deduce not only the common slow acetylation haplotypes (NAT2*5, NAT2*6, NAT2*7, and NAT2*14), but also other rapid acetylation haplotypes (NAT2*11, NAT2*12, and NAT2*13) in addition to NAT2*4. A limitation of this and most other NAT2 genotyping assays is that it does not directly phase the single-nucleotide variations and, therefore, in rare cases equivocal results are obtained that require cloning and sequencing of individual alleles. Although some investigators use allele-specific PCR and RFLP combinations after regular RFLP analysis to more accurately differentiate the genotypes (13)(14)(15)(34), these methods are labor-intensive, time-consuming, prone to experimental error, and difficult to automate; they therefore are not ideal for routine or large-scale clinical applications(35). Another bead-based approach was developed recently to phase NAT2 single-nucleotide variations(36), but this assay requires 2 separate PCRs. In addition, Sabbagh and Darlu(35) have described a computational approach for interfering haplotypes at the NAT2 locus.

Variants of the NAT2 gene are associated with adverse reactions to drugs metabolized by NAT2, such as isoniazid, sulfasalazine, and co-trimoxazole, and this ASPE assay for NAT2 genotyping provides a robust method for predicting the risk of adverse reactions to these drugs. Another important application of NAT2 genotyping is to study cancer risk related to exposure to aromatic amine and heterocyclic amine carcinogens. For example, Cartwright et al. (37) found a striking association (odds ratio = 16.7; P = 0.00005) between urinary bladder cancer and slow acetylator phenotypes in English chemical dye workers with documented exposure to aromatic amine carcinogens, a finding attributable to the deactivation of aromatic amine carcinogens by NAT2. In contrast, heterocyclic amine carcinogens are activated by NAT2. The authors of several studies(38)(39)(40)(41)(42) reported an association between the rapid NAT2 acetylator phenotype and colorectal cancer for individuals consuming well-done meat and, presumably, higher amounts of heterocyclic amine carcinogens(43). Although some reports are controversial(44), the published literature provides robust data suggesting an overall association of slow NAT2 genotype with aromatic amines in cigarette smoke, providing compelling evidence for a role of the NAT2 acetylator genotype in urinary bladder cancer(5).


   Acknowledgments
 
The Luminex 100 IS System was kindly provided on loan from TmBioscience. We would like to acknowledge the helpful discussions with Jim Gordon (TmBioscience) concerning assay design. We also thank Nancy Johnson (University of Louisville) for superb technical assistance. Support for this work was provided by US Public Health Service Grant CA34627 (to D.W.H. and M.A.D.) and by AA014235 (to M.W.L.).


   Footnotes
 
1 Nonstandard abbreviations: NAT2, N-acetyltransferase-2; RFLP, restriction fragment-length polymorphism; ASPE, allele-specific primer extension; SAP, shrimp alkaline phosphatase; EXO I, exonuclease I; MFI, median fluorescence intensity; and CI, confidence interval.

2 Human genes: NAT2, N-acetyltransferase-2.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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