Single-track sequencing for genotyping of multiple SNPs in the N-acetyltransferase 1 (NAT1) gene
© Soucek et al; licensee BioMed Central Ltd. 2004
Received: 05 March 2004
Accepted: 25 November 2004
Published: 25 November 2004
Fast, cheap and reliable methods are needed to identify large populations, which may be at risk in relation to environmental exposure. Polymorphisms in NAT1 (N-acetyl transferase) may be suitable markers to identify individuals at risk.
A strategy allowing to address simultaneously 24 various genetic variants in the NAT1 gene using the single sequencing reaction method on the same PCR product is described. A modified automated DNA sequencing using only one of the sequence terminators was used to genotype PCR products in single-track sequencing reactions of NAT1 and was shown to be universal for both DNA sequencing using labeled primers and labeled nucleotides. By this method we detected known SNPs at site T640G, which confers the NAT1*11 allele with frequency of 0.036, further T1088A and C1095A with frequency of 0.172 and 0.188, respectively and a deletion of TAATAATAA in the poly A signal area with a frequency 0.031. All observed frequencies were in Hardy Weinberg equilibrium and comparable to those in Caucasian population. The single-track signatures of the variant genotypes were verified on samples previously genotyped by RLFP.
The method could be of great help to scientists in the field of molecular epidemiology of screening of large populations for known informative biomarkers of susceptibility, such as NAT1.
We have previously described the single sequencing reaction (SSR) protocol for assessment of a known polymorphism in the 3'utr region of CYP19 (aromatase) [1, 2]. Here we have extended and optimized the use of the method for multiple polymorphisms in the NAT1 gene. The increasing number of detected mutations in the NAT1, makes genotyping using conventional restriction fragment length polymorphism (RFLP) or allele-specific amplification complicated. We developed a rapid and universal strategy based on single-track DNA sequencing analysis of a unique PCR product encompassing the entire NAT1 coding region (contains no introns) along with the flanking 5' and 3' untranslated regions. It allows a rapid and economic characterization of NAT1 alleles. Our method brings reproducible results on both Alf Express™ (Pharmacia) and ABI310 PRISM sequencing instruments and may be adopted for majority of epidemiological studies with relevance of NAT1 in environmentally related diseases.
N-acetyltransferases (NAT, EC 18.104.22.168) are implicated in the biotransformation of primary arylamines (e.g. 2-naphthylamine and aminobiphenyls), heterocyclic amines, hydrazines, and their N-hydroxylated metabolites present in tobacco smoke and food [3–7]. An increased activity for p-aminobenzoic acid acetylation (marker for NAT1 activity) was observed in the breast malignant tissues compared to benign and control tissues [8, 9]. Human NATs may have adapted a common catalytic mechanism from cysteine proteases for acetyl-transfer reactions [10, 11]. The NAT1 gene is highly polymorphic (for listing of known variant alleles http://www.louisville.edu/medschool/pharmacology/NAT.html). Expression of NAT1*16 but not NAT1*10 and NAT1*11 caused a 2-fold decrease in the amount and catalytic activity of NAT1 in COS-1 cell cytosol [12–14]. All available data suggest that slow NAT1 phenotype results from NAT1 allelic variants that encode reduced expression of NAT1 and/or less-stable NAT1 protein .
Epidemiological studies suggest that the NAT1 and NAT2 acetylation polymorphisms modify the risk of developing urinary bladder, colorectal, breast, head and neck, lung, and possibly prostate cancers [16–18]. Interactions between NAT2*4 and NAT1*10 were suggested by the increased frequency of the NAT2*4/NAT1*10 haplotype [19–21]. The individual risks associated with NAT1 acetylator phenotypes/genotypes are usually small, but they increase when considered in conjunction with other susceptibility genes and/or aromatic and heterocyclic amine carcinogen exposures. Because of the relatively high frequency of the variant NAT1 genotypes in the population, the attributable cancer risk may be high. Large-scale molecular epidemiological studies that investigate the role of NAT1 genotypes and/or phenotypes together with other genetic susceptibility gene polymorphisms and biomarkers of carcinogen exposure are necessary to expand our current understanding of the role of NAT1 acetylation polymorphisms in cancer risk .
Overview of NAT1 alleles and selection for single-track sequencing
350,351 GG → CC
402 T → C
445 G → A
459 G → A
497–499 GGG → CCC
559 C → T
560 G → A
613 A → G
640 T → G
752 A → T
777 T → C
781 G → A
787 A → G
poly A region:
884 A → G
1088 T → A
1091 ins AAA
1095 C → A
NAT1*3, *10, *11,
A-track sequencing of the 3'-untranslated region of NAT1 revealed a deletion of TAATAATAA (Figure 3D,3E on Alf Express™ and Figure 4E on ABI310 PRISM). It was found in 7 individuals accounting for the frequency 0.031, which is comparable to the frequency 0.033 observed in the study on 314 control individuals by Bruhn et al. . This change is also unique for the NAT1*11 alleles and co-segregates with T640G polymorphism. Very good co-segregation of T640G and 1067-1090delTAATAATAA polymorphisms in 7 informative samples was observed (4 heterozygotes and 3 homozygotes). Despite the different chemistry, Figures 1, 2, 3, 4 demonstrate perfect concordance between the results obtained by Alf Express™ and ABI310 PRISM analysis, verified on samples with previously characterized genotypes by RFLP.
A modified automated DNA sequencing with a fluorescent label was used to genotype PCR products spanning through the whole NAT1 gene by single-track sequencing reactions in 192 control individuals. Previously, we have shown that single-track sequencing reactions performed on PCR products, with subsequent analysis on an Alf Express™ DNA Sequencer, can be as informative, sensitive, and accurate as complete sequencing reactions but more economical option for the genotyping of known polymorphisms in the human aromatase gene . Compared to analysis by full gel-based sequencing, four times more samples can be analyzed per gel in considerably shorter time. The method could be particularly efficient in cases of high density of polymorphic sites residing in a common area as in the case of NAT1.
This paper shows that single-track sequencing reactions can be used as a tool for screening for all types of known polymorphisms in field laboratories with limited or overloaded sequence capacity. Compared to standard sequencing, this single track sequencing may have better signal to noise ratio. This approach is time and cost effective and can be accommodated for high-throughput analyses in epidemiology studies.
Chemicals for PCR and sequencing were purchased from ABI (Applied Biosystems, Foster City, CA, USA) and Amersham Biosciences (Uppsala, Sweden).
The study included 192 DNA samples from Norwegian healthy individuals obtained through Norwegian Population Registry as a population-based series of residents in the Oslo area. Test samples from healthy Norwegian controls with known genotypes (previously assessed by RFLP) were available.
PCR amplification of NAT1
A single PCR amplification of the entire coding region and the 3' flanking area of NAT1 (923 bp) using forward primer: 5'-tactgggctctgaccactat-3' and reverse primer: 5'-tgctttctagcataaatcacc-3' was performed. PCR mix contained 16.9 μl dH2O, 10 × PCR buffer (2.5 μl), 1.25 mM MgCl2 (4 μl), 2.0 mM dNTP (1.0 μl mixture of each), 10 μM oligonucleotide primer, 0.2 μl Taq DNA polymerase (Perkin Elmer, 5 U/μl) and 1.0 μl of genomic DNA (100 ng/μl) in a final volume of 25 μl. Thermal cycling (GeneAmp 2400, PE, Foster city, CA) included initial denaturation 2 min at 94°C, 10 cycles of 30 sec at 94°C, 30 sec at decreasing annealing temperature 58 to 48°C, 1 min at 72°C, and 25 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. Quality of PCR products was checked on 6% acrylamide gels.
The SNPs studied by this assay are summarized in Table 1. Single A-, G- and T- track sequencing using 1 given terminator at a time was performed using both Alf Express™ system (Pharmacia Biotech, Uppsala, Sweden) and ABI310 PRISM capillary sequencer. Alf Express™ Cy5-labeled primers NAT1A-F-CY5: 5'-gggagggtatgtttacagca-3' and NAT1A-POLYA-CY5: 5'-gcataaatcaccaatttcca-3' were used for genotyping the coding region and 3'untranslated area of NAT1, respectively. The single sequencing reactions (7 μl) contained: 1.25 μl of dH2O, 0.5 μl of 10-times concentrated FS polymerase buffer (125 mM Tris-HCl, pH 9.5, 50 mM (NH4)2SO4, 150 mM MgCl2), 2 μl of ddNTP termination mix (containing 10 mM dNTPs and either ddATP or ddTTP (10 μM) for A-track or T-track sequencing respectively, 1 μl of 2 μM primer, 0.25 μl of FS polymerase (5 U/μl), and 2 μl of NAT1 PCR product. The following conditions were used for thermal cycling: initial denaturation for 5 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 50°C, and 1 min at 68°C. The single-tracks were evaluated using conventional software AlfWin, Fragment Analyzer, v. 1.02 supplied with the sequencer (Alf Express™). For the single-track sequencing by ABI310 PRISM we used the same sequencing primers as for Alf Express™ system however labeled with the fluorophor 6-FAM. Sequencing reactions (10 μl) contained: 5.0 μl dH2O, 1.0 μl of 10-times concentrated Thermo Sequenase buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl2), 2.0 μl of ddNTP termination mix (containing dNTPs and either ddATP or ddGTP for A-track or G-track sequencing respectively – for composition see Table 3), 0.5 μl of 1 μM primer, 0.5 μl of Thermo Sequenase DNA polymerase (with pyrophosphatase) 3.2 U/μl and 1.0 μl NAT1 PCR product (described above). Thermal cycling conditions were as follows: initial denaturation 20 sec at 95°C, 25 cycles of 20 sec at 95°C, 20 sec at 55°C and 1 min at 72°C, and then 10 cycles of 20 sec at 95°C, 1 min at 72°C. Results were evaluated using GeneScan Analysis software, v. 3.7.
PS performed the SSR analysis on an Alf Express™ and prepared the first draft of the paper, CFS and MS optimized the SSR analysis for an ABI system in the lab of EHK, TK designed primers and contributed with reagents, materials, and advice, VNK brought the idea, organized the study and was responsible for the revisions of the paper.
- ABPs – aminobiphenyls:
NAT1 – N-acetyltransferase 1, SSR (Single Sequencing Reaction)
The work at this project was partly supported by grants of Grant Agency of the Czech Republic, no.: 310/01/1537 and Internal Grant Agency of the Czech Ministry of Health, no.: 6747-3. The analysis is supported by grants E01085/001 and 122772/310 by the Norwegian Cancer Society, and grant D99061/004 of the Research Council of Norway. We thank David Ryberg, National Institute of Occupational Health, Oslo, for supplying samples for testing genotypes.
- Kristensen T, Nedelcheva Kristensen V, Børresen-Dale A-L: High throughput screening for known mutations by automated analysis of single sequencing reactions (SSR). BioTechniques. 1998, 24: 832-835.Google Scholar
- Kristensen T, Nedelcheva Kristensen V, Børresen-Dale A-L: High-throughput screening for known mutations by automated analysis of single sequencing reactions. In "Polymorphism Detection and Analysis". 2000, Eaton PublishingGoogle Scholar
- Kadlubar FF, Butler MA, Kaderlik KR, Chou HC, Lang NP: Polymorphisms for aromatic amine metabolism in humans: relevance for human carcinogenesis. Environ Health Perspect. 1992, 98: 69-74.View ArticleGoogle Scholar
- Hein DW, Doll MA, Rustan TD, Gray K, Feng Y, Ferguson RJ, Grant DM: Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis. 1993, 14: 1633-1638.View ArticleGoogle Scholar
- Probst-Hensch NM, Bell DA, Watson MA, Skipper PL, Tannenbaum SR, Chan KK: N-acetyltransferase 2 phenotype but not NAT1*10 genotype affects aminobiphenyl-hemoglobin adduct levels. Cancer Epidemiol. Cancer Epidemiol Biomarkers Prev. 2000, 9: 619-623.Google Scholar
- Godschalk RW, Dallinga JW, Wikman H, Risch A, Kleinjans JC, Bartsch H, Van Schooten FJ: Modulation of DNA and protein adducts in smokers by genetic polymorphisms in GSTM1, GSTT1, NAT1 and NAT2. Pharmacogenetics. 2001, 11: 389-398. 10.1097/00008571-200107000-00003.View ArticleGoogle Scholar
- Kawakubo Y, Merk HF, Masaoudi TA, Sieben S, Blomeke B: N-Acetylation of paraphenylenediamine in human skin and keratinocytes. J Pharmacol Exp Ther. 2000, 292: 150-155.Google Scholar
- Geylan YS, Dizbay S, Guray T: Arylamine N-acetyltransferase activities in human breast cancer tissues. Neoplasma. 2001, 48: 108-111.Google Scholar
- Rodrigues-Lima F, Delomenie C, Goodfellow GH, Grant DM, Dupret JM: Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop. Biochem J. 2001, 356: 327-334. 10.1042/0264-6021:3560327.View ArticleGoogle Scholar
- Upton A, Smelt V, Mushtaq A, Aplin R, Johnson N, Mardon H, Sim E: Placental arylamine N-acetyltransferase type 1: potential contributory source of urinary folate catabolite p-acetamidobenzoylglutamate during pregnancy. Biochim Biophys Acta. 2000, 1524: 143-148.View ArticleGoogle Scholar
- Williams JA, Stone EM, Fakis G, Johnson N, Cordell JA, Meinl W, Glatt H, Sim E, Phillips DH: N-Acetyltransferases, sulfotransferases and heterocyclic amine activation in the breast. Pharmacogenetics. 2001, 11: 373-388. 10.1097/00008571-200107000-00002.View ArticleGoogle Scholar
- Butcher NJ, Ilett KF, Minchin RF: Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid. Biochem Pharmacol. 2000, 60: 1829-1836. 10.1016/S0006-2952(00)00501-3.View ArticleGoogle Scholar
- Yamanaka S, Zhang XY, Maeda M, Miura K, Wang S, Farese RV, Iwao H, Innerarity TL: Essential role of NAT1/p97/DAP5 in embryonic differentiation and the retinoic acid pathway. EMBO J. 2000, 19: 5533-5541. 10.1093/emboj/19.20.5533.View ArticleGoogle Scholar
- de Leon JH, Vatsis KP, Weber WW: Characterization of naturally occurring and recombinant human N-acetyltransferase variants encoded by NAT1*. Mol Pharmacol. 2000, 58: 288-299.Google Scholar
- Fretland AJ, Doll MA, Leff MA, Hein DW: Functional characterization of nucleotide polymorphisms in the coding region of N-acetyltransferase1. Pharmacogenetics. 2001, 11: 511-520. 10.1097/00008571-200108000-00006.View ArticleGoogle Scholar
- Wikman H, Thiel S, Jager B, Schmezer P, Spiegelhalder B, Edler L, Dienemann H, Kayser K, Schulz V, Drings P, Bartsch H, Risch A: Relevance of N-acetyltransferase 1 and 2 (NAT1, NAT2) genetic polymorphisms in non-small cell lung cancer susceptibility. Pharmacogenetics. 2001, 11: 157-168. 10.1097/00008571-200103000-00006.View ArticleGoogle Scholar
- Katoh T, Boissy R, Nagata N, Kitagawa K, Kuroda Y, Itoh H, Kawamoto T, Bell DA: Inherited polymorphism in the N-acetyltransferase 1 (NAT1) and 2 (NAT2) genes and susceptibility to gastric and colorectal adenocarcinoma. Int J Cancer. 2000, 85: 46-49. 10.1002/(SICI)1097-0215(20000101)85:1<46::AID-IJC8>3.0.CO;2-0.View ArticleGoogle Scholar
- Ishibe N, Sinha R, Hein DW, Kulldorff M, Strickland P, Fretland AJ, Chow WH, Kadlubar FF, Lang NP, Rothman N: Genetic polymorphisms in heterocyclic amine metabolism and risk of colorectal adenomas. Pharmacogenetics. 2002, 12: 145-150. 10.1097/00008571-200203000-00008.View ArticleGoogle Scholar
- Cascorbi I, Roots I, Brockmoller J: Association of NAT1 and NAT2 polymorphisms to urinary bladder cancer: significantly reduced risk in subjects with NAT1*10. Cancer Res. 2001, 61: 5051-5056.Google Scholar
- Westphal GA, Reich K, Schulz TG, Neumann C, Hallier E, Schnuch A: N-acetyltransferase 1 and 2 polymorphisms in para-substituted arylamine- induced contact allergy. Br J Dermatol. 2000, 142: 1121-1127. 10.1046/j.1365-2133.2000.03536.x.View ArticleGoogle Scholar
- Krajinovic M, Richer C, Sinnett H, Labuda D, Sinnett D: Genetic polymorphisms of N-acetyltransferases 1 and 2 and gene-gene interaction in the susceptibility to childhood acute lymphoblastic leukemia. Cancer Epidemiol Biomarkers Prev. 2000, 9: 557-562.Google Scholar
- Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, Devanaboyina US, Nangju NA, Feng Y: Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev. 2000, 9: 29-42.Google Scholar
- Henning S, Cascorbi I, Munchow B, Jahnke V, Roots I: Association of arylamine N-acetyltransferases NAT1 and NAT2 genotypes to laryngeal cancer risk. Pharmacogenetics. 1999, 9: 103-111.View ArticleGoogle Scholar
- Bruhn C, Brockmoller J, Cascorbi I, Roots I, Borchert HH: Correlation between genotype and phenotype of the human arylamine N-acetyltransferase type 1 (NAT1). Biochem Pharmacol. 1999, 58: 1759-1764. 10.1016/S0006-2952(99)00269-5.View ArticleGoogle Scholar
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