Variation of the N-Acetyltransferase 2 Gene in a Romanian and a Kyrgyz Population Sylvia Rabstein, 1 Klaus Unfried, 2 Ulrich Ranft, 2 Thomas Illig, 3 Melanie Kolz, 3 Hans-Peter Rihs, 1 Chinara Mambetova, 4 Mariana Vlad, 5 Thomas Bru ¨ ning, 1 and Beate Pesch 1 1 Berufsgenossenschaftliches Forschungsinstitut fu ¨ r Arbeitsmedizin, Institute of Ruhr University of Bochum, Bochum, Germany; 2 Institut fu ¨r umweltmedizinische Forschung at Heinrich Heine University Du ¨ sseldorf, GmbH, Du ¨ sseldorf, Germany; 3 GSF-National Research Centre for Environment and Health, Institute of Epidemiology, GmbH, Neuherberg, Germany; 4 Kyrgyz Scientific Centre of Hematology, Bishkek, Kyrgyzstan; and 5 Institute of Public Health ‘‘Iuliu Moldovan’’, Cluj, Romania Abstract As part of a project on environmental disasters in minority populations, this study aimed to evaluate differences in the sequence of N-acetyltransferase 2 (NAT2 ) as a metabolic susceptibility gene in yet unexplored ethnicities. Eight single nucleotide polymorphisms (SNP) in the NAT2 coding region and a variant in the 3V flanking region were analyzed in 290 unrelated Kyrgyz and 140 unrelated Romanians by SNP-specific PCR analysis. The variants 341C, 481T, and 803G were less and 857A more prevalent in Kyrgyz (P < 0.0001). The variant at site 857 indicates Asian descent. 282C>T and 590G>A showed no significant variation by ethnicity. 364G>A and 411A>T turned out to be monomor- phic. Database comparisons of the NAT2 minor allele frequencies support that Romanians belong to Caucasians and Kyrgyz are in between Caucasians and East Asians. The distributions of predicted haplotypes differed significantly between the two ethnicities where the Kyrgyz showed a higher genetic diversity. The haplotype without mutations was more common in Kyrgyz (40.1% in Kyrgyz, 29.3% in Romanians). Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differ- ences in NAT2 genotypes with yet unknown effect on the health risks for environmental or occupational exposures in minority populations. (Cancer Epidemiol Biomarkers Prev 2006;15(1):138 – 41) Introduction The role of susceptibility genes in the development of chronic diseases is an important issue in occupational and environ- mental medicine. Arylamine N-acetyltransferase 2 (NAT2) catalyzes the addition of an acetyl group from acetyl-CoA to a terminal nitrogen on substrates (1). NAT2 is a gene with a 870-bp coding region mapped to chromosome 8p22. The GenBank accession number X14672 is commonly used as reference sequence (URL: http://www.ncbi.nlm.nih.gov). As of June 5, 2003, 36 human NAT2 alleles were reported based on variations at 15 nucleotide positions (URL: http:// www.louisville.edu/medschool/pharmacology/NAT.html). We further refer to this database as NAT database. An additional single nucleotide polymorphism (SNP) in the 3V flanking region was documented in the SNP500Cancer database of Cancer Genome Anatomy Project (ref. 2; URL: http://snp500cancer.nci.nih.gov) and in the database of Perlegen Sciences (ref. 3; URL: http://genome.perlegen.com). We further refer to these databases as SNP500 database and Perlegen database. First results of an international project on genetic susceptibility to environmental carcino- gens revealed a large variation of allele frequencies in control populations by ethnicity (4). Here, we report on the genetic variation of NAT2 in a European (Romanian) and Central-Asian (Kyrgyz) study population analyzed as a part of the European Commission-funded project ‘‘Investigation of the Risk of Cyanide in Gold Leaching on Health and Environment in Central Asia and Central Europe’’ (IRCYL; ref. 5). Materials and Methods Study Population. Unrelated subjects were selected for the analyses of NAT2 gene variants from two population surveys of IRCYL. NAT2 genotypes were obtained from whole blood samples collected with informed consent. The study was approved by the Ethics Committee of the Kyrgyz Medical Academy and by the Romanian Ministry of Health. In total, 140 Romanian and 290 Kyrgyz subjects were included. Genotyping of NAT2 Variants. Genomic DNA from frozen blood samples was prepared at the Kyrgyz Scientific Center of Haematology (Bishkek, Kyrgyzstan) and the Institute of Public Health (Cluj-Napoca, Romania) using the QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) according to the protocol of the manufacturer. DNA was then shipped to Institut fu ¨r umweltmedizinische Forschung (Du ¨ sseldorf, Germany) and tested for its applicability in PCR assays. NAT2 variants documented as polymorphic in the SNP500 database were selected for genotyping with the MassARRAY system (Sequenom, San Diego, CA; ref. 6). These variant sites comprised two synonymous polymorphisms (282C>T and 481C>T), six nonsynonymous SNPs (341T>C, 364G>A, 411A>T, 590G>A, 803A>G, and 857G>A), and a C>T polymorphism in 3V untranslated region (rs2552). Two variants (364G>A and 411A>T) turned out to be monomor- phic in the IRCYL populations. No assay could be established for the rare polymorphism 191G>A. Genotyping call rates were z96.8%. Statistical Analyses. NAT2 analyses were calculated using SAS 8.02 (Cary, NC). Deviations from Hardy-Weinberg 138 Cancer Epidemiol Biomarkers Prev 2006;15(1). January 2006 Received 4/13/05; revised 7/5/05; accepted 10/10/05. Grant support: European Commission 5th Framework Programme for the Project ‘‘Investigation of the Risk of Cyanide in Gold Leaching on Health and Environment in Central Asia and Central Europe (IRCYL)’’ (contract no. ICA2-CT-2000-10036) and Bundesministerium fu ¨ r Bildung und Forschung (German National Genome Research Net). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Sylvia Rabstein, Berufsgenossenschaftliches Forschungsinstitut fu ¨r Arbeitsmedizin, Institute of Ruhr University of Bochum, Bu ¨ rkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany. Phone: 49-234-302-4533; Fax: 49-234-302-4505. E-mail: rabstein@bgfa.de Copyright D 2006 American Association for Cancer Research. doi:10.1158/1055-9965.EPI-05-0256 Research. on October 20, 2016. © 2006 American Association for Cancer cebp.aacrjournals.org Downloaded from