Real-time PCR analysis of the N-acetyltransferase NAT1 allele *3, *4, *10, *11, *14 and *17 polymorphism in squamous cell cancer of head and neck

Stefan Fronhoffs1,*, Thomas Brüning2,*, Elena Ortiz-Pallardo1, Peter Bröde3, Brigitte Koch1, Volker Harth2, Agapios Sachinidis1, Hermann Maximillian Bolt2, Claus Herberhold4, Hans Vetter1 and Yon Ko1,5

1 Medizinische Universitäts-Poliklinik, Wilhelmstraße 35-37, D-53111 Bonn, Germany,
2 Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin an der Ruhr Universität Bochum, Bürkle-de-la-Camp Platz 1, D-44789 Bochum, Germany,
3 Institut für Arbeitsphysiologie, Universität Dortmund, Ardeystr. 67, D-44139 Dortmund, Germany and
4 Klinik und Poliklinik für Hals-, Nasen- und Ohrenkranke, Universität Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although tobacco smoke has been established as a main risk factor in the development of head and neck squamous cell cancer (HNSCC), genetic polymorphisms of xenobiotic metabolizing enzymes are supposed to modulate an individual's susceptibility to smoking-related HNSCC. N-acetyltransferase (NAT) 1 gene is known to be polymorphic and its protein product is implicated in the activation and detoxification of carcinogens, such as aromatic amines, present in tobacco smoke. We developed a rapid and reproducible LightCyclerTM-assisted real-time polymerase chain reaction (PCR) for NAT1 genotyping, which allowed the parallel differentiation of NAT1*3, *4, *10 and *11 alleles and separately of NAT1*14 and *17 alleles within 60 min without the need for further post-PCR processing. In order to investigate the role of the NAT1 gene polymorphism as a risk-modifying factor in HNSCC, we tested for the presence of NAT1*3, *4, *10, *11, *14 and *17 alleles in a case-control study of 291 HNSCC patients and 300 healthy controls of Caucasian origin. Our findings suggest that in Caucasians, the risk of HNSCC is not associated with NAT1 polymorphism. The overall distribution of NAT1 allele frequencies was not significantly different among cases and controls. The presence of the fast acetylator NAT1*10 and NAT1*11 alleles did not significantly increase the risk of HNSCC and no modifying effect of NAT1*10 was observed among smokers. This new approach in NAT1 genotyping substantially increases throughput of sample analysis and, therefore, enhances opportunities to study NAT1 as a risk factor in different cancers in large-scale studies.

Abbreviations: CI, confidence interval; Fl, fluorescein; LCR, LightCyclerTM-Red fluorophore; NAT1, N-acetyltransferase 1 gene; OR, odds ratio; PCR, polymerase chain reaction; HNSCC, head and neck squamous cell cancer; Tm, melting temperature.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic polymorphisms of xenobiotic metabolizing enzymes appear to modulate individual susceptibility to smoking-related cancer of the oral cavity (15). Beside alcohol consumption, tobacco smoke is considered to be a main exogenous risk factor in this kind of carcinoma (69). A number of aromatic amines and heterocyclic amines are major constituents of tobacco smoke (1012). Human metabolism of these carcinogenic compounds is complex and involves acetylation as an important pathway in order to mutate DNA and initiate carcinogenesis. In humans, two N-acetyltransferases (NAT1 and NAT2) have been identified, which catalyze detoxification and activation of various amines by N-acetylation and O-acetylation, respectively (13,14). Both NAT1 and NAT2 genes are known to be polymorphic in humans, corresponding to slow and rapid acetylator phenotypes (15). These varying enzyme activities are supposed to influence the individual metabolism of carcinogenic aromatic amines, thereby modifying the susceptibility to certain cancers.

To date, at least 15 NAT1 alleles have been identified in humans with the NAT1*4 allele denoted as the wild-type (1522). The NAT1*3 allele is probably functionally comparable with the NAT1*4 allele, as the mutation does not cause an amino acid change (14). The recently described NAT1*14 and *17 alleles encode for proteins with reduced acetylation capacity (1820), whereas the NAT1*11 allele is now agreed to be associated with higher NAT1 activity (14,17,23). The prominent NAT1*10 allele contains two single-base mutations in the polyadenylation signal sequence, which probably results in elevated enzyme activity (24). NAT1*10 was found to be associated with an increased risk of colorectal, bladder and well-differentiated gastric carcinoma (2527). In patients with smoking-induced lung cancer and oral squamous cell cancer, only a few molecular epidemiological studies have investigated the importance of NAT1 polymorphism as a potential susceptibility factor and found inconclusive data. Bouchardy et al. found the rapid acetylator NAT1*10 and *11 to be associated with a decreased risk of lung cancer (28). In contrast, Abdel-Rahman et al. demonstrated a 3.7-fold risk of smoking-related lung cancer in individuals who inherited the NAT1*10 allele (29). Jourenkova-Mironova et al. (30) found no effect of NAT1 polymorphism on the risk of oral/pharyngeal and laryngeal cancer in Caucasians. In addition, Henning et al. (31) failed to demonstrate an association of NAT1*10 or any other NAT1 allele in Caucasian patients with laryngeal cancer. These studies disagree with the findings of Katoh et al. who reported a significantly increased risk of oral squamous cell cancer associated with the NAT1*10 allele in people of Japanese descent (32).

To explore the association between NAT1 genetic polymorphism and squamous cell cancer of the head and neck (HNSCC) in Caucasians, we developed a LightCyclerTM-assisted real-time PCR (3335), which allows the rapid and reproducible differentiation of NAT1*3, *4, *10, *11, *14 and *17 alleles. Using this novel approach, we tested for the presence of these alleles in 291 Caucasian patients with HNSCC and 300 control individuals.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Two hundred and ninety-one Caucasian patients with histologically confirmed and newly diagnosed squamous cell carcinoma of the oral cavity, pharynx or larynx were recruited from the Department of Otorhinolaryngology, Head and Neck Surgery at the University of Bonn from 1996 to 1999. One hundred and forty-three patients presented with oral and pharyngeal cancer and 148 patients presented with laryngeal cancer. The control group consisted of 300 Caucasian patients visiting the outpatient clinics at the Medical Policlinic in Bonn during the same time. Controls were defined as individuals without any prior diagnosis of cancer or present respiratory ailments, comprising healthy individuals seen for general health check-ups and patients with various diseases. The main diagnoses in the control group included cardiovascular diseases (such as coronary heart disease, heart failure and hypertension) diabetes mellitus, rheumatological, gastrointestinal and infectious diseases. Information concerning smoking habits (age at the beginning, daily tobacco consumption, type of tobacco and changes in smoking habits), drinking habits, family history of cancer, possible occupational exposure and medication was gathered from both patients and control individuals. Smokers were defined as having smoked one or more cigarettes per day for 6 months or more during their lifetime. The study was approved by the local medical ethics review committee.

DNA extraction
Blood samples were collected in the general ambulance of the Medical Policlinic in Bonn. Genomic DNA was isolated from whole blood using the QiAmp DNA Blood Maxi Kit (Qiagen, Hilden, Germany).

NAT1 genotype analysis by LightCyclerTM-assisted real-time PCR
PCR was performed on a LightCyclerTM (Roche, Mannheim, Germany) using hybridization probes in combination with the LightCyclerTM DNA Master Hybridization Probes Kit (Roche, Mannheim, Germany). Generally, hybridization probes consist of two different, short oligonucleotides that hybridize to two adjacent internal sequences of the amplified PCR fragment during the annealing phase of the PCR cycles. One probe is labeled at the 5'-end with a LightCyclerTM-Red fluorophore and modified at the 3'-end by phosphorylation. The other probe is labeled with fluorescein. Only after hybridization are the two probes in close proximity, resulting in fluorescence resonance energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is stimulated by the light source of the LightCyclerTM instrument, and part of this energy is transferred to LightCyclerTM-Red, the acceptor fluorophore. The emitted fluorescence of the LightCyclerTM-Red fluorophore is measured.

In order to distinguish polymorphic alleles, a melting curve analysis has to be performed after PCR. The melting temperature (Tm) is determined by PCR fragment length, G and C content and degree of homology. PCR fragments containing mismatches will, therefore, melt off at lower temperatures than fully homologous sequences, allowing the identification of the specific melting temperature associated with each genotype.

During NAT1 gene analysis, six alleles were detected: (i) NAT1*4 [denoted as the wild-type allele that contains a T at nucleotide 1088 and a C at nucleotide 1095 (25)]; (ii) NAT1*3 [C1095A substitution (25)]; (iii) NAT1*10 [T1088A and C1095A substitutions (25)]; (iv) NAT1*11 [it has a deletion of 9 nucleotides in a trinucleotide repeat between nucleotides 1065 and 1090 and C–344T, A–40T, G445A, G459A, T640G, C1095A substitutions (17,25)]; (v) NAT1*14 [G560A, T1088A and C1095A substitutions (19)]; and (vi) NAT1*17 [C190T substitution (20)].

To distinguish between the four alleles with sequence variants in the 3' region of NAT1 near the putative polyadenylation signal [NAT1*3, *4, *10 and *11 (25)], one hybridization probe labeled with fluorescein (5'-TAGCATAAATCACCAATTT CCAAGATAACCACA-Fl-3') and two hybridization probes labeled with LightCyclerTM-Red 640 (LCR 640) and 705 (LCR 705) fluorophore were used (Figure 1Go). The LCR 640 probe (5'-LCR-CCATCTTTAAAAGACATTTATTATTATTATTATTATT-3') hybridizes to the NAT1*4 allele and the LCR 705 probe (5'-LCR-CCATCTTTAAAATACATTTTTTATT ATTATTATTATT-3') hybridizes to the NAT1*10 allele. PCR was performed using the primer pairs and the hybridization probe pairs labeled with fluorescein/LCR 640 or -/LCR705 given in Table IGo. The PCR primers and the hybridization probes were commercially synthesized by TIB MOLBIOL (Berlin, Germany). The PCR conditions were 4 mM MgCl2, 3 pmol of each hybridization probe, 20 pmol of each PCR primer, 2 µl of LightCyclerTM DNA Master Hybridization Mix and from 100 pg to 10 ng genomic DNA in a final volume of 20 µl. After 2 min of denaturation at 95°C, 45 PCR cycles were performed with 3 s denaturation at 95°C, 20 s annealing at 50°C and 25 s extension at 72°C. Melting curves were achieved following a denaturation period of 3 s at 95°C at a start temperature of 45°C and an end temperature of 80°C with a temperature increase of 0.4°C/s.



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Fig. 1. NAT1 gene sequence in the 3'-untranslated region near the polyadenylation signal according to Vatsis et al. (16), GeneBankTM accession no. S78829. For NAT1 gene analysis four alleles were detected: NAT1*4 (denoted as the wild-type allele that contains a T at nucleotide 1088 and a C at nucleotide 1095), NAT1*3 (C1095A substitution), (c) NAT1*10 (T1088A and C1095A substitutions), (d) NAT1*11 (it has a deletion of 9 nucleotides in a trinucleotide repeat between nucleotides 1065 and 1090 and C–344T, A–40T, G445A, G459A, T640G, C1095A substitutions). Mutation sites are bold and underlined. It is not possible to determine exactly which 9 bp are deleted in the NAT1*11 allele. PCR primer locations are marked by lines (sense primers are above the sequence, antisense primers are below the sequence). Locations of hybridization probes are indicated.

 

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Table I. PCR primers and hybridization probes used for LightCyclerTM-assisted PCR analysis of NAT1 gene polymorphism
 
Using the hybridization probe pair labeled with fluorescein/LCR 640, four Tm were identified at 52.2, 54, 55.2 and 58.7°C which were associated with the NAT1*10, *11, *3 and *4 alleles, respectively (Figure 2Go), as verified by direct sequencing of the PCR products according to the dye terminator method (Qiagen, Hilden, Germany) as described below. With the hybridization probe pair labeled with fluorescein/LC Red 705, four Tm were identified at 50.5, 53.5, 55.0 and 58.9°C, associated with the NAT1*4, *11, *3 and *10 alleles, respectively (Figure 2Go), as again verified by direct sequencing of the PCR products.



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Fig. 2. Differentiation of NAT1*3(*3), *4(*4), *10(*10) and *11(*11) alleles by performing melting curve analysis. LightCyclerTM-assisted real-time PCR of samples was performed using the primer pairs and the hybridization probe pair labeled with fluorescein/LightCycler-Red 640 (LCR 640) or -/LightCycler-Red 705 (LCR705) as described in Table IGo. Melting curve analysis was performed by plotting the first negative derivative of the fluorescence (F) with respect to temperature (T) against temperature [– (dF/dT) versus T]. Using the hybridization probe pair labeled with fluorescein/LCR 640 (upper part), four melting temperatures (Tm) were identified at 52.2, 54, 55.2 and 58.7°C associated with the NAT1*10, *11, *3 and *4 alleles, respectively. With the hybridization probe pair labeled with fluorescein/LC Red 705 (lower part), four Tm were identified at 50.5, 53.5, 55.0 and 58.9°C, associated with the NAT1*4, *11, *3 and *10 alleles, respectively. Association between NAT1 alleles and Tm were determined by direct sequencing of the PCR products. Wt, wild-type; Del 9, deletion of 9 nucleotides.

 
This method does not distinguish between the NAT1*4 and *17 and between the NAT1*10 and *14 alleles. To detect the NAT1*14 and *17 alleles, PCR primer and hybridization probe pairs labeled with fluorescein and LCR 640 and 705 fluorophore were constructed as summarized in Table IGo. The hybridization probe pair labeled with fluorescein (5'-GAGCCCAGTACAGAAGATGATTGACCT-Fl-3') and LCR 640 (5'-LCR-640-ACCATCCACCCCGATTTCTT-3') hybridizes to the NAT1 wild-type sequence which harbors the nucleotide 190 and the hybridization probe pair labeled with fluorescein (5'-AAGAGTAAAGGAGTAGATTTTTCGGTA-Fl-3') and LCR 705 (5'-LCR-705-TTGCTGTCTTCTAGGAGATCAGAATGAAG-3') hybridizes to the NAT1 wild-type sequence which harbors the nucleotide 560 (Figure 3Go). PCR fragments containing the C190A mutation or the G560A mutation, which characterize the NAT1*17 and the *14 alleles, therefore melt off at lower temperatures than the homologous sequences. PCR was performed as described below using the primer pair given in Table IGo and both hybridization probe pairs for the parallel detection of NAT1*14 and *17.



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Fig. 3. NAT1 gene sequence according to Butcher et al. (10), GeneBankTM accession no. AF008204. For NAT1 gene analysis two alleles were detected: NAT1*14 [G560A mutation, T1088A mutation and C1095A mutation at nucleotide 1095 (56)] and NAT1*17 (C190T mutation (10). Mutation sites are bold and underlined. PCR primer locations are marked by lines (sense primers are above the sequence, antisense primers are below the sequence). Locations of hybridization probes are indicated.

 
The PCR primers and the hybridization probes were commercially synthesized by TIB MOLBIOL (Berlin, Germany) and are summarized in Table IGo. The PCR conditions were 4 mM MgCl2, 3 pmol of each hybridization probe, 20 pmol of each PCR primer, 2 µl of LightCyclerTM DNA Master Hybridization Mix and from 100 pg to 10 ng genomic DNA in a final volume of 20 µl. After 2 min of denaturation at 95°C, 45 PCR cycles were performed with 5 s denaturation at 95°C, 20 s annealing at 55°C and 25 s extension at 72°C. Melting curves were achieved following a denaturation period of 5 s at 95°C at a start temperature of 45°C and an end temperature of 80°C with a temperature increase of 0.4°C/s.

PCR and melting procedure was detected online with the LightCyclerTM instrument. Melting curve analysis for each sample was performed by plotting the first negative derivative of the fluorescence (F) with respect to temperature (T) against temperature [ – (dF/dT) versus T].

DNA sequence analysis
PCR products were subjected to a single-strand sequencing reaction following real-time PCR. Sequencing was carried out according to the dye terminator method, using the oligonucleotide 5'-CAATAAACCAACATTAAAAGCTT-3' (Qiagen, Hilden, Germany) on an automated DNA sequencer (ABI 377, Perkin Elmer, Weiterstadt, Germany).

Classification of genotypes
NAT1*10 and *11 are referred to as rapid acetylator in comparison with NAT1*3 and *4, which are referred to as normal acetylator and NAT1*14 and *17, which are referred to as slow acetylator. All individuals with NAT1*10 or *11 alleles were classified as either homozygous or heterozygous rapid acetylator, except for individuals with heterozygous NAT1*10/NAT1*14 genotypes, which were considered to be normal acetylator. Individuals with only NAT1*4 and *3 alleles were classified as normal acetylator, whereas individuals with NAT1*4/NAT1*14 or *17 genotypes were considered to be heterozygous slow acetylator. Homozygous slow acetylator could not be detected. For statistical analysis, the group of homozygous and heterozygous rapid acetylator genotypes was compared with a second group combining normal and slow acetylator genotypes.

Statistical analysis
The frequency distributions of NAT1 alleles and NAT1 genotypes among HNSCC patients and controls were examined by calculating the Cochran–Mantel–Haenszel {chi}2-statistic. Logistic regression analyses were applied to compute odds ratios for the classified NAT1 genotypes adjusting for gender and age. Stratifying by the histological types of HNSCC and smoking habits, four logistic regression models were calculated for oral/pharyngeal and laryngeal cancer, and smokers and non-smokers, respectively. All computations were performed using version 6.12 of SAS® (36).


    Results
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 Materials and methods
 Results
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 References
 
The clinical characteristics of oral/pharyngeal cancer patients (n = 143), laryngeal cancer patients (n = 148) and controls (n = 300) are summarized in Table IIGo. Because HNSCC patients and controls had different frequency distributions for age, gender and smoking status, frequency distributions of NAT1 alleles and NAT1 genotypes were adjusted for these factors.


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Table II. Distribution of age, gender and smoking habits by HNSCC patients and hospital controls
 
Table IIIGo displays NAT1 allele frequencies in Caucasian oral/pharyngeal cancer patients (n = 286), laryngeal cancer patients (n = 296) and controls (n = 600). Among control patients NAT1*3, *4, *10, *11 and the recently described NAT1*14 and *17 alleles (19,20) were found to occur at a frequency of 1.50, 77.17, 15.67, 1.83, 2.33 and 1.50%, respectively. There was no statistically significant difference in the overall distribution of allele frequencies between cases and controls (P = 0.692). Among laryngeal cancer patients the NAT1*4 and NAT1*17 alleles were less frequent, whereas NAT1*3 and NAT1*10 alleles were more frequent. However, neither of these differences reached significance.


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Table III. Distribution of NAT1 alleles by HNSCC patients and hospital controls
 
The overall distribution of NAT1 genotype frequencies (data not shown) with regard to the acetylator status was not significantly different among HNSCC patients and controls (P = 0.721). There was a trend towards a higher frequency of homozygous (3.09%) and heterozygous rapid acetylator (29.9%) and a lower frequency of heterozygous slow acetylator genotypes (3.44%) among HNSCC patients compared with controls (2.33, 29.0, 6.33%, respectively), but this trend did not reach significance. Following stratification for smoking habits (data not shown), there was neither a significant difference in the overall distribution frequencies of NAT1 genotypes between smoking HNSCC and controls (P = 0.651), nor between non-smoking HNSCC and controls (P = 0.530).

As a result of the small number of homozygous rapid acetylator and heterozygous slow acetylator genotypes, the group of homozygous and heterozygous rapid acetylator genotypes was compared with a second group combining normal and slow acetylator genotypes in order to investigate the interaction between smoking and NAT1 gene polymorphism on the risk of oral/pharyngeal and laryngeal cancer. Considering these two groups of NAT1 genotypes, the associated odds ratios of oral/pharyngeal and laryngeal cancer, adjusted for gender and age and stratified for smoking habits, are presented in Table IVGo. Among the non-smoking population, no increase in the risk of oral/pharyngeal cancer (OR = 0.98; 95% CI, 0.42–2.23) was observed with the combined homozygous and heterozygous rapid acetylator genotypes compared with normal and slow acetylator genotypes. A slight increase in the risk of laryngeal cancer was observed (OR = 1.65; 95% CI, 0.78–3.50), but this trend did not reach statistical significance. Among smokers, we failed to find an increase in the risk of oral/pharyngeal and laryngeal cancer (OR = 0.83; 95% CI, 0.44–1.54 and OR = 1.16; 95% CI, 0.65–2.06, respectively) associated with the NAT1 gene polymorphism.


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Table IV. Distribution of NAT1 genotypes by HNSCC patients and hospital controls for smokers and non-smokers
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We describe a simple, rapid and reproducible real-time PCR method for NAT1 genotyping which allows the parallel discrimination of NAT1*3, *4, *10 and *11 alleles and separately of NAT1*14 and *17 alleles within 60 min. Applying the new method, we tested in a case-control study for the presence of these alleles in 291 patients with HNSCC and 300 control individuals of Caucasian origin. Our findings suggest that in Caucasians, the risk of HNSCC is not associated with NAT1 polymorphism. The presence of the fast acetylator NAT1*10 and *11 alleles did not significantly increase the risk of oral/pharyngeal and laryngeal cancer and no modifying effect of the fast acetylator genotypes was observed among smokers.

Genetic polymorphisms of xenobiotic metabolizing enzyme, which may determine an individual's susceptibility to certain cancers, are of growing interest in molecular cancer research. Therefore, rapid and simple genotyping methods are required to perform larger epidemiological studies. We have developed a simple method for NAT1 genotyping that is based on a LightCyclerTM-assisted real-time PCR (3335). This approach enables the complete and simultaneous analysis of 32 samples for the presence of NAT1*3, *4, *10 and *11 alleles within 60 min without further post-PCR processing. LightCyclerTM-assisted real-time PCR takes advantage of sequence-specific fluorescence hybridization probes, which allow the online monitoring of rapid-cycle DNA amplification. Discrimination of the different alleles is then achieved by performing melting curve analysis. To differentiate between the NAT1*3, *4, *10 and *11 alleles, amplification was performed using one primer pair and two hybridization probes, one labeled with fluorescein and one with LCR 640 fluorophore, which hybridizes to the NAT1*4 allele. While performing melting curve analysis, four different melting temperatures were obtained which characterized the four alleles as determined by sample sequencing. To control our results a hybridization probe labeled with LCR 705 fluorophore was constructed which hybridizes to the NAT1*10 allele resulting in a different melting temperature profile for the four alleles. Using both hybridization probes, identical genotypes were obtained in all samples, suggesting that the method enables a distinct discrimination of the four alleles. Very recently, Wikman et al. described a similar LightCyclerTM-assisted real-time PCR for NAT1 genotyping which allows the parallel analyses of NAT1*3, *4 and *10 alleles (37). In contrast to our approach, this method does not distinguish between the NAT1*3 and *11 alleles and requires the additional performance of conventional PCR-restriction fragment length polymorphism for the detection of the NAT1*11 allele.

With the method described here, the frequencies of NAT1*3, *4, *10 and *11 alleles distribution in our study control population were similar to the previous observations in larger studies with Caucasian control populations (25,26,38). In particular, we observed a NAT1*10 allele frequency of 15.7% which is nearly identical to that of 16% reported by Bell et al. (25), whereas other groups found a higher value ranging from 20 to 26% (28,39,40).

In addition, we analyzed all samples for the presence of the recently described slow acetylator NAT1*14 and *17 alleles (19,20), which is of interest as the restriction to genotyping only the four NAT1 alleles with sequence variants near the putative polyadenylation signal may lead to a misclassification of NAT*14 for NAT1*10 and of NAT1*17 for NAT1*4. Using the same primer pair, but different hybridization probe pairs, both alleles could be detected in a parallel procedure. Presently very little data are available reporting a frequency of 2.0–2.3% for NAT1*14 (28,38,41) and of 1.1% for NAT1*17 (38), which is consistent with our data.

NAT1 and NAT2 are supposed to detoxify carcinogens, such as aromatic amines in tobacco smoke by N-acetylation and to activate them by O-acetylation (13,4244). In contrast to NAT2, which is predominately expressed in the liver, NAT1 expression shows a wider tissue distribution including larynx tissue (4547). Evidence for a role of NAT1 gene polymorphism in tumorigenesis is based on previous studies, which found the NAT1*10 allele to be associated with increased enzyme activity in colon and bladder tissue (24,48) and to increase the risk of colorectal cancer, smoking-related bladder cancer, well-differentiated and advanced stage gastric adenocarcinoma and possibly prostate cancer (2527,49,50). Although the exact mechanisms remain to be determined, these findings suggested that inheritance of NAT1*10 leads to increased activation of carcinogens such as aromatic amines in cigarette smoke. However, the findings concerning the relationship between NAT1 genotypes, especially NAT1*10, and phenotypes and their association with cancer risk remains inconclusive. Recent observations could not confirm a link between inheritance of NAT1*10 and increased NAT1 activity levels in tissues (41) or with increased risk for colorectal cancer (27) and colorectal adenoma (39). In patients with smoking-induced lung cancer, which is comparable with HNSCC with respect to the direct exposure to tobacco smoke, Bouchardy et al. found the rapid acetylators NAT1*10 and *11 to be associated with a decreased risk of lung cancer (28). In contrast, Abdel-Rahman et al. demonstrated an increased risk of smoking-related lung cancer in individuals who inherited the NAT1*10 allele (29).

Our study could not demonstrate any association between the risk of HNSCC and NAT1*10 or any other NAT1 allele, which is consistent with the observations of Jourenkova-Mironova et al. in Caucasian patients with oral/pharyngeal (n = 121) and laryngeal (n = 129) cancer and 172 controls (30), as well as with the findings of Henning et al. in 255 Caucasian patients with laryngeal cancer and 510 controls (31). In contrast, Katoh et al. observed a significantly increased risk for oral squamous cell cancer associated with the NAT1*10 allele in 62 cases compared with 122 controls of Japanese origin (32). The discrepancy may be a result of the small study size. In addition, ethnic differences in NAT1 allele distribution may play a role, as Katoh et al. found a higher NAT1*10 allele frequency of 42% in the Japanese control population, which was consistent with recent observations (51). Moreover, Katoh et al. could not detect any slow acetylator alleles (NAT1*14, *15, *17) in the Japanese population (32).

Tobacco smoking has been established as a major risk factor for HNSCC (69). Following stratification for smoking habits, we failed to demonstrate significant differences in the risk of oral/pharyngeal and laryngeal cancer among smokers compared with the non-smoking population, indicating that the NAT1 gene polymorphism does not modify the risk of smoking-induced HNSCC. This conclusion is consistent with the observation of Jourenkova-Mironova et al. who found no evidence of an interaction between NAT1 genotypes and smoking on the risk of oral/pharyngeal and laryngeal cancer (30).

In summary, we present a rapid and precise LightCyclerTM-assisted real-time PCR method for genotyping the NAT1 polymorphisms, which enables the parallel analysis of NAT1*3, *4, *10 and *11 alleles, and separately of NAT*14 and *17, each in a single step procedure within 60 min. This major advantage allows for a substantial increase in the throughput of sample analysis and replaces the laborious genotyping methods that are presently a main limitation in the performance of large epidemiological studies. Applying the new method, we found no significant association between the risk of HNSCC and any of the NAT1 alleles in a Caucasian population.


    Notes
 
5 To whom correspondence should be addressed Email: yonko{at}uni-bonn.de Back

* Contributed equally. Back


    Acknowledgments
 
We thank Dr Olfert Land (TIB Molbiol, Tempelhofer Weg 11-12, D-10829 Berlin, Germany, Fax: +49 30 78799499) for the construction of the LightCyclerTM hybridization probes.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 11, 2000; revised May 24, 2001; accepted May 25, 2001.