TaqMan Real Time–Polymerase Chain Reaction Methods for Determination of Nucleotide Polymorphisms in Human N‐Acetyltransferase‐1 (NAT1) and ‐2 (NAT2)

David W. Hein1, Mark A. Doll1

1 University of Louisville School of Medicine, Louisville, Kentucky
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.15
DOI:  10.1002/0471140856.tx0415s22
Online Posting Date:  January, 2005
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Abstract

N‐acetyltransferase 1 (NAT1) and N‐acetyltransferase 2 (NAT2) exhibit allelic variation and genetic polymorphism associated with increased susceptibility towards drug toxicity and environmental disease. TaqMan allelic discrimination methods are described to rapidly determine NAT1 and NAT2 genotypes. The SNPs selected for NAT1 genotype determinations are: C97T (R33Stop), C190T (R64W), G445A (V149I), C559T (R187Stop), G560A (R187Q), A752T (D251V), T1088A (3′UTR), and C1095A (3′UTR). The SNPs selected for NAT2 genotyping determinations are: G191A (R64Q), C282T (silent), T341C (I114T), C481T (silent), G590A (R197Q), A803G (K268R), and G857A (G286T). All NAT2 and NAT1 alleles, except very rare ones, are detected with these assays. Major advantages of the methods described in this unit are that they do not require post‐PCR processing (such as enzyme digestion) or the use of radioactivity. Since the methods amplify relatively small segments of NAT1 or NAT2, they are effective for human DNA samples derived from buccal cells or paraffin‐embedded tissues.

Keywords: NAT1; NAT2; N‐acetyltransferase; acetylator genotype; allele discrimination

     
 
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Table of Contents

  • Basic Protocol 1: Real‐Time TaqMan Analysis of NAT1 and NAT2
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Real‐Time TaqMan Analysis of NAT1 and NAT2

  Materials
  • 2× TaqMan Universal PCR Master Mix (Applied Biosystems)
  • 3 µM primers (see Tables 4.15.3 and 4.15.4; Applied Biosystems)
  • 4 µM fluorescent probes: normal, turbo, and MGB (see Tables 4.15.3 and 4.15.4; Applied Biosystems)
  • 5 to 25 ng/µl high‐quality genomic DNA samples
  • Optical 96‐well plates (Applied Biosystems)
  • Optical caps (Applied Biosystems)
  • Sequence detector (e.g., 7700 Sequence Detector, Applied Biosystems) and computer
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Figures

Videos

Literature Cited

   Butcher, N.J., Boukouvala, S., Sim, E., and Minchin, R.F. 2002. Pharmacogenetics of the arylamine N‐acetyltransferases. Pharmacogenomics J. 2:30‐42.
   Butcher, N.J., Ilett, K.F., and Minchin, R.F. 1998. Functional polymorphism of the human arylamine N‐acetyltransferase type 1 gene caused by C190T and G560A mutations. Pharmacogenetics 8:67‐72.
   Cascorbi, I. and Roots, I. 1999. Pitfalls in N‐acetyltransferase 2 genotyping. Pharmacogenetics 9:123‐127.
   Cascorbi, I., Roots, I., and Brockmoller, J. 2001. Association of NAT1 and NAT2 polymorphisms to urinary bladder cancer: Significantly reduced risk in subjects with NAT1*10. Cancer Res. 61:5051‐5056.
   Deitz, A.C., Doll, M.A., and Hein, D.W. 1997. A restriction fragment length polymorphism assay that differentiates human N‐acetyltransferase‐1 (NAT1) alleles. Anal. Biochem. 253:219‐24.
   Deitz, A.C., Rothman, N., Rebbeck, T.R., Hayes, R.B., Chow, W‐H., Zheng, W., Hein, D.W., and Garcia‐Closes, M., 2004. Impact of misclassification in genotype‐exposure interaction studies. Example of N‐acetyltransferase 2 (NAT2), smoking, and bladder cancer. Cancer Epidem. Biomark. Prev. 13:1543‐1546.
   Delomenie, C., Sica, L., Grant, D.M., Krishnamoorthy, R., and Dupret, J.M. 1996. Genotyping of the polymorphic N‐acetyltransferase (NAT2*) gene locus in two native African populations. Pharmacogenetics 6:177‐185.
   Doll, M.A., Fretland, A.J., Deitz, A.C., and Hein, D.W. 1995. Determination of human NAT2 acetylator genotype by restriction fragment‐length polymorphism and allele‐specific amplification. Anal Biochem. 231:413‐420.
   Doll, M.A. and Hein, D.W. 2001. Comprehensive human NAT2 genotype method using single nucleotide polymorphism‐specific polymerase chain reaction primers and fluorogenic probes. Anal. Biochem. 288:106‐108.
   Doll, M.A. and Hein, D.W. 2002. Rapid genotype method to distinguish frequent and/or functional polymorphisms in human N‐acetyltransferase‐1 (NAT1). Anal. Biochem. 301:328‐332.
   Fretland, A.J., Leff, M.A., Doll, M.A., and Hein, D.W. 2001a. Functional characterization of human N‐acetyltransferase 2 (NAT2) single nucleotide polymorphisms. Pharmacogenetics 11:207‐215.
   Fretland, A.J., Doll, M.A., Leff, M.A., and Hein, D.W. 2001b. Functional characterization of nucleotide polymorphisms in the coding region of human N‐acetyltransferase 1. Pharmacogenetics 11:511‐520.
   Hein, D.W. 2002. Molecular genetics and function of NAT1 and NAT2: Role in aromatic amine metabolism and carcinogenesis. Mutat. Res. 506‐507:65‐77.
   Hein, D.W., Doll, M.A., Fretland, A.J., Leff, M.A., Webb, S.J., Xiao, G.H., Devanaboyina, U.‐S., Nangju, N.A., and Feng, Y. 2000a. Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidem. Biomarker Prev. 9:29‐42.
   Hein, D.W., Doll, M.A., and Nerland, D.E., In press. Methods for N‐acetyltransferase (NAT1 and NAT2) genotype determination. In Principles of Clinical Pharmacogenomics and Introduction to Pharmaco Proteomics (S.H.Y. Wong, M.W., Linder, and R. Valdes Jr., eds.), AACC Press, Washington, D.C.
   Hein, D.W., Grant, D.M., and Sim, E. 2000b. Update on consensus arylamine N‐acetyltransferase gene nomenclature. Pharmacogenetics 10:291‐292.
   Hughes, N.C., Janezic, S.A., McQueen, K.L., Jewett, M.A.S., Castranio, T., Bell, D.A., and Grant, D.M. 1998. Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p‐aminosalicylate as an in‐vivo and in‐vitro probe. Pharmacogenetics 8:55‐66.
   Leff, M.A., Fretland, A.J., Doll, M.A., and Hein, D.W. 1999. Novel human N‐acetyltransferase 2 alleles that differ in mechanism for slow acetylator phenotype. J. Biol. Chem. 274:34519‐34522.
   Lin, H.J., Probst‐Hensch, N.M., Hughes, N.C., Sakamoto, G.T., Louie, A.D., Kau, I.H., Lin, B.K., Lee, D.B., Lin, J., Frankl, H.D., Lee, E.R., Hardy, S., Grant, D.M., and Haile, R.W. 1998. Variants of N‐acetyltransferase NAT1 and a case‐control study of colorectal adenomas. Pharmacogenetics 8:269‐281.
   Lo‐Guidice, J.‐M., Allorge, D., Chevalier, D., Debuysere, H., Faxio, F., Lafitte, J.‐J., and Broly, F. 2000. Molecular analysis of the N‐acetyltransferase 1 gene (NAT1*) using polymerase chain reaction‐restriction fragment‐single strand conformation polymorphism assay. Pharmacogenetics 10:293‐300.
   Loktionov, A., Moore, W., Spencer, S.P., Vorster, H., Nell, T., O'Neill, I.K., Bingham, S.A., and Cummings, J.H. 2002. Differences in N‐acetylation genotypes between Caucasians and Black South Africans: Implications for cancer prevention. Cancer Detection Prev. 26:15‐22.
   Svensson, C.K., and Hein, D.W., In press. Phenotypic and genotypic characterization of N‐acetylation. In Drug Metabolism and Transport: Molecular Methods and Mechanisms (L.H. Lash, ed.) The Humana Press, Totowa, N.J.
   Upton, U., Johnson, N., Sandy, J., and Sim, E. 2001. Arylamine N‐acetyltransferases‐ of mice, men and microorganisms. Trends Pharmacol. Sci. 22:140‐146.
   Vatsis, K.P., Weber, W.W., Bell, D.A., Dupret, J.‐M., Price‐Evans, D.A., Grant, D.M., Hein, D.W., Lin, H.J., Meyer, U.A., Relling, M.V., Sim, E., Suzuki, T., and Yamazoe, Y. 1995. Nomenclature for N‐acetyltransferases. Pharmacogenetics 5:1‐17.
   Vaziri, S.A.J., Hughes, N.C., Sampson, H., Darlington, G., Jewett, M.A.S., and Grant, D.M. 2001. Variation in enzymes of arylamine procarcinogen biotransformation among bladder cancer patients and control subjects. Pharmacogenetics 11:7‐20.
Key References
   Butcher et al., 2002. See above.
  Review of the role of NAT1 and NAT2 polymorphisms on drug response.
   Doll and Hein, 2001. See above.
  Original report of NAT2 genotype method described in this unit.
   Doll and Hein, 2002. See above.
  Original report of NAT2 genotype method described in this unit.
   Hein et al., 2000a. See above.
  Review of the role of NAT1 and NAT2 polymorphisms on cancer risk.
   Hein et al., In press. See above.
  Review of different NAT1 and NAT2 genotyping methods.
   McQueen, C.A. 2001. Measuring the activity of arylamine N‐acetyltransferase (NAT). In Current Protocols in Toxicology., 4.6.1‐4.6.13. John Wiley & Sons, New York.
  Describes common protocols for measuring N‐acetyltransferase catalytic activity.
Internet Resources
   http://www.louisville.edu/medschool/ pharmacology/NAT.html
  Website for NAT1 and NAT2 alleles.
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