Download Simultaneous Determination of 7 N-Acetyltransferase-2

Clinical Chemistry 52:6
1033–1039 (2006)
Molecular Diagnostics
and Genetics
Simultaneous Determination of
7 N-Acetyltransferase-2 Single-Nucleotide
Variations by Allele-Specific Primer
Extension Assay
Yusheng Zhu,1 David W. Hein,2 Mark A. Doll,2 Kristen K. Reynolds,1 Ntei Abudu,1
Roland Valdes, Jr.,1 and Mark W. Linder1*
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 ⴝ 80),
0.4372 (0.0270) (n ⴝ 148), and 0.9331 (0.0127) (n ⴝ 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.
Departments of 1 Pathology and Laboratory Medicine and 2 Pharmacology
and Toxicology, University of Louisville School of Medicine, Louisville, KY.
* Address 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 [email protected].
Received November 3, 2005; accepted February 23, 2006.
Previously published online at DOI: 10.1373/clinchem.2005.063198
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.
© 2006 American Association for Clinical Chemistry
N-Acetyltransferase 2 (NAT2)3 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 gene4 (1, 2 ). Sequence variations in the NAT2
gene have been associated with increased risks for a variety
of cancers and drug-induced toxicities (3–7 ). 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,
3
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.
4
Human genes: NAT2, N-acetyltransferase-2.
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Zhu et al.: Allele-Specific Primer Extension Assay for NAT2
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 –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 (12–15 ). 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 singlenucleotide 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, 25–30 ), and the TaqMan realtime 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 compli-
cated 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
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/␮L, 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.
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-␮L PCR reaction was performed on a Mastercycler® Gradient thermal cycler (Eppendorf). Each 20-␮L
reaction contained 1⫻ PCR reaction buffer, 1.5 mM
MgCl2, 0.2 mM each of the deoxynucleotide triphosphates, 0.25 ␮M 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 ␮L of SAP
(1 U/␮L; USB) and 0.5 ␮L of EXO I (10 U/␮L; USB) to
each 20 ␮L 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 ␮L of SAP- and EXO I-treated
PCR product as the template for ASPE in a total of 20 ␮L
of reaction mixture containing 1⫻ PCR reaction buffer;
1.25 mM MgCl2; 5 ␮M each of dGTP, dATP, dTTP, and
biotin-14-dCTP (Invitrogen); 0.025 ␮M primer mixture
containing 14 specific tag-labeled allele-specific primers
for 7 common and 7 variant alleles (synthesized by
Clinical Chemistry 52, No. 6, 2006
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Fig. 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.
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 1.1⫻ wash
buffer (TmBioscience), and 45 ␮L of the bead mixture was
mixed with 5 ␮L 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 1⫻ wash buffer
(TmBioscience). To the washed beads we added 150 ␮L of
1⫻ wash buffer containing 2 ng/␮L streptavidin–R-phy-
coerythrin (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 allelespecific primers with specific 5⬘-tag sequences are listed in
Table 1.
statistical analysis
We used Graphpad PRISM (Ver. 3.0; Graphpad Inc.) to
perform the data analyses to compare mean homozygous
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Zhu et al.: Allele-Specific Primer Extension Assay for NAT2
Table 1. Allele-specific primers and corresponding singlenucleotide variations.
Primer sequencesa
Tm,b °C
Singlenucleotide
variation
5⬘-CTATTTTTGATCACATTGTAAGAAGAAACCG(A)-3⬘
5⬘-ACCACAATGTTAGGAGGGTATTTTTAC(T)-3⬘
5⬘-CTTCTCCTGCAGGTGACCAT(C)-3⬘
5⬘-CAGAAGAGAGAGGAATCTGGTACC(T)-3⬘
5⬘-AAAAAATATACTTATTTACGCTTGAACCTCG(A)-3⬘
5⬘-AGAGGTTGAAGAAGTGCTGAA(G)-3⬘
5⬘-GCCCAAACCTGGTGATGG(A)-3⬘
55.2
55.2
56.9
56.0
54.7
55.1
56.6
191
282
341
481
590
803
857
a
The last nucleotides of the primers correspond to the common alleles, and
the nucleotides in parentheses indicate the variant alleles.
b
Tm, melting temperature.
common, heterozygous variant, and homozygous variant
allelic ratios.
Results
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 singlenucleotide 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.
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.
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.
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
Table 2. Comparison of allelic ratios of NAT2 generated by 3 DNA polymerases.a
Homozygous common (n ⫽ 43)
Heterozygous variant (n ⫽ 16)
Homozygous variant (n ⫽ 11)
Tspb
TaKaRa
Taq
0.036 (0.020)
0.459 (0.050)
0.942 (0.014)
0.058 (0.020)c
0.443 (0.035)
0.867 (0.045)c
0.035 (0.017)
0.456 (0.045)
0.940 (0.018)
a
Differences between categories were tested by ANOVA followed by post hoc test with Bonferroni correction. Results are given as the mean (SD). If not otherwise
stated, the results are not significantly different (P ⬎0.05).
b
Tsp, Platinum GenoTYPE Tsp DNA polymerase; TaKaRa, TaKaRa Taq HS DNA polymerase; Taq, Titanium Taq DNA polymerase.
c
P ⬍0.001 compared with the allelic ratios generated by Tsp and Taq.
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Clinical Chemistry 52, No. 6, 2006
Fig. 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 (䡺) and common (f) 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).
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%).
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.
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
Table 3. Precision of the allelic ratios of the 7 singlenucleotide variations of NAT2.
Genotypea
H
Singlenucleotide
variation
n
Mean (SD), %
n
Mean (SD), %
191
282
341
481
590
803
857
6
27
7
7
14
13
6
95.33 (0.82)
92.81 (0.62)
97.14 (0.38)
93.43 (0.53)
93.71 (0.73)
91.23 (1.17)
89.50 (4.64)
12
26
25
24
21
33
7
42.08 (1.56)
37.50 (1.61)
48.32 (1.14)
46.17 (1.71)
42.71 (2.70)
58.15 (4.63)
31.14 (5.58)
a
M
W
n
Mean (SD), %
61 3.20 (0.60)
26 1.54 (0.51)
47 2.00 (0.21)
48 4.33 (0.76)
44 3.91 (1.27)
33 10.52 (3.75)
66 2.09 (0.79)
M, homozygous variant; H, heterozygous variant; W, homozygous common.
Discussion
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 3). 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
1038
Zhu et al.: Allele-Specific Primer Extension Assay for NAT2
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 (13–15, 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 – 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 ).
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.).
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