Distribution of N-acetyltransferase Type 1 (NAT1) genotypes and alleles in a Turkish population

HUMAN AND MEDICAL GENETICS

SHORT COMMUNICATION

Distribution of N-acetyltransferase Type 1 (NAT1) genotypes and alleles in a Turkish population

Serdal Arslan; Naci Degerli; Fevzi Bardakci

Molecular Biology Research Group, Department of Biology, Faculty of Science and Literature, Cumhuriyet University, Turkey

^{Correspondence}

ABSTRACT

NAT1 is an intronless gene on chromosome 8p21.3 encoding a 290-amino-acid-long protein showing acetyltransferase activity. Some 26 alleles of NAT1 gene have been identified in human populations. In the present study we determined the distributions of NAT1 genotypes and alleles in a sample of 201 individuals from the Turkish population in Central Anatolia. The most frequent genotypes were NAT1*4/NAT1*4 (51.74%), NAT1*10/NAT1*4 (22.39%), NAT1*11/NAT1*4 (7.46), NAT1*10/NAT1*10 (3.98%). Frequencies of NAT1*3, *4 (wild-type), *10 and *11 alleles were 3.73%, 69.6%, 17.66% and 7.2%, respectively. The frequency of NAT1*11 was the highest amongst the populations studied so far, the other allele frequencies being close to those described in Caucasian populations.

Key words:NAT1 gene, genetic polymorphism, molecular epidemiology, Turkish population.

NAT1 and NAT2 are isoenzymes that catalyze the N-acetylation of aromatic amine and hydrazine drugs. While substrates of NAT2 enzyme are isoniazid, sulfamethazine, 2-aminofluorene and 4-aminobiphenyl, NAT1 enzyme has para-aminosalicylic acid (PAS), para-aminobenzoic acid (PABA) and sulfanilamide as substrates (Deguchi et al., 1990; Grant et al., 1992).

The polymorphism of NAT1 gene was first described about a decade ago (Vatsis and Weber, 1993), and 26 alleles have been identified in human populations (Hein et al., 2000), NAT1*3, NAT1*4, NAT1*5, NAT1*10 and NAT1*11 being the most common alleles reported. A single mutation or a combination of multiple nucleotide substitutions and insertions/deletions are responsible for the allelic variants of NAT1. While some variants (NAT1*11, *20 and *23) do not lead to differences in activity (Hughes et al., 1998; Lin et al., 1998), others result in increase (e.g. NAT1*21, NAT1*24 and NAT1*25) (Lin et al., 1998), decrease or absence of activity (NAT1*14, *15, *17, *19 and *22) (Hughes et al., 1998; Lin et al., 1998; Butcher et al., 1998) with respect to the enzyme encoded by the wild-type allele (NAT1*4). In a German population, Bruhn et al. (1999) found that enzyme activity in carriers of NAT1*3, *4 and *10 did not differ. On the other hand, NAT1*11 and *14 appeared to be low-activity alleles while NAT1*15 was a null allele. In contrast, recent data suggest that the enzyme encoded by NAT1*11 allele exhibits higher NAT1 activity relative to the product of the wild-type NAT1*4 allele (reviewed in Zheng et al. 1999; de Leon et al. 2000; Loktionov et al. 2002).

Much emphasis has been given to NAT1*10 allele, because a change in the consensus polyadenilation signal was suggested to be correlated with higher enzyme activity in colon, bladder and liver tissues (Bell, et al., 1995a; Zenser et al., 1996). Several studies have also demonstrated an association between NAT1*10 allele and colon, gastric, urinary bladder, laryngeal and head tumors as well as the development of environmental borne diseases (Bell et al., 1995a,b; Taylor et al., 1995; Katoh et al., 2000).

In this study we determined the distribution of NAT1 genotypes and alleles in a Turkish population sample using restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) assays.

Subjects: Venous blood was taken from 201 randomly selected non-cancer volunteers (116 females and 85 males) attending the Cumhuriyet University Hospital, Sivas (Central Anatolia, Turkey) as outpatients, between January and February 2002. The mean ages of male and female individuals were 44.22 ± 14.80 (16-76) and 42.03 ± 13.96 (17-86) years, respectively. The study was carried out after approval of the hospital ethical committee.

PCR-SSCP analysis of the NAT1 gene: A 1216 bp long fragment of the NAT1 gene, consisting of the 870 bp intronless coding region, the 278 bp 3'UTR and the 68 bp 5'UTR, was amplified by PCR of genomic DNA obtained from peripheral blood leukocytes. PCR was accomplished in a total of 25 mL volume containing 0.2 mM each primer (sense 5'TAAAAGTAAAATGATTTTGCTTTCG3' and anti-sense 5'GCTTTCTAGCATAAATCACCAA3'), 75 mM Tris-HCI (pH 8.8 at 25 °C), 20 mM (NH₄)₂SO₄, 0.01% Tween 20 and 1.5 mM MgCl₂, 0.2 mM each dNTP (MBI Fermentas), 0.6 units Taq DNA polymerase (MBI Fermentas) and 250 ng genomic DNA. The mixture was subjected to 2 min initial denaturation at 94 °C followed by 32 cycles of 1 min at 94 °C, 1 min at 61 °C and 1.5 min at 72 °C with a 5 min-extension step at 72 °C, in a thermal cycler (Techne, UK). The SSCP protocol described by Lo-Guidice et al. (2000) was followed, with some modifications. PCR products were digested by TfiI (New England Biolabs, Beverly, MA, USA). Electrophoresis was performed on an 8% non-denaturing polyacrylamide gel, and the patterns analyzed after silver staining.

PCR-RFLP Analysis of the NAT1 gene: A RFLP-assay was used to confirm the presence of NAT1*11 alleles, allowing to distinguish a T (NAT1*4) from a G (NAT1*11) at the nucleotide 640, as described by Lo-Guidice et al. (2000). The PCR products were digested with AlwNI (Fermentas). This enzyme cleaves NAT1*11 into three fragments (241, 451 and 496 bp), but other alleles into two fragments (451 and 746 bp). Therefore, restriction digestion produces three fragments (241, 451 and 496 bp) for NAT1*11/NAT1*11 genotype, and four fragments (241, 451, 496 bp and 746 bp) for NAT1*11/any other allele genotypes.

The NAT1 genotype distribution of the 201 individuals from the Turkish population is shown in Table 1. The ten most frequent described genotypes, as well as eight rare genotypes involving alleles not identifiable by SSCP analysis (NAT1*4/others) were found. NAT1*4/NAT1*4 (53.89%), NAT1*4/NAT1*10 (22.39%) and NAT1*4/ NAT1*11 (7.46%) were the most common ones. The frequencies of the different alleles are shown in Table 2. The wild-type allele NAT1*4 had a frequency of 69.65%. The frequencies of NAT1*10, *11, *3 and of the non-identifiable alleles were 17.41%, 7.21%, 3.73% and 2%, respectively.

The frequencies of NAT1*3, NAT1*4 and NAT1*10 alleles observed were close to those found in German (Henning et al., 1999), Canadian (Hughes et al., 1998), and French (Lo-Guidice et al., 2000) populations. A striking difference in the Turkish population was the highest NAT1*11 frequency (7.2%). Although this allele was not found in the French population (Lo-Guidice et al., 2000), it has been reported with a lower frequency in German (2.7% by Henning et al., 1999 and 3.34% by Bruhn et al. 1999) and in Canadian (2.1% by Hughes et al., 1998) populations. These populations differ from the Japanese who present the NAT1*10 allele as the most frequent, and among whom NAT1*3 and *11 alleles were never found (Yang et al., 2000). In Indian, Malay and Chinese populations, Zhao et al. (1998) reported a much higher NAT1*10 and NAT1*3 allele frequencies than those found in Caucasians.

A small departure (significant at the 5% level) from Hardy-Weinberg genotype proportions was observed (chi-squared = 12.31; d.f. = 4; p = 0.015). Only the deficiency of NAT1*3/NAT1*4 heterozygotes seems to contribute significantly to the obtained chi-squared figure. Both the sample size and its heterogeneous ethnic composition are the best explanation for this finding.

Yang et al. (2000) reported a higher activity of the enzyme encoded by NAT1*10 allele in a Japanese population, with NAT1*4/NAT1*10 female heterozygotes having higher enzyme activity than NAT1*4/NAT1*4 females. Wikman et al., (2001) considered individuals with NAT1*10 allele as rapid acetylators unless when combined with a slow allele. In contrast, Bruhn et al. (1999) did not detect increased enzyme activities in association with NAT1*4/*4, NAT1*4/*10 and NAT1*10/*10 genotypes in a German population. Jourenkova-Mironova et al. (1999) have also found low frequencies of NAT1 homozygous rapid acetylator genotypes (NAT1*10/*11 and NAT1*10/ *10). Associations between the NAT1*10 allele and a high enzyme activity with oral (Katoh et al., 1998), colon (Bell et al., 1995b), urinary bladder (Taylor et al., 1995), head and neck (Olshan et al., 2000) and gastric (Katoh et al., 2000) cancers have been described. In addition, linkage disequilibrium between NAT1*10 and NAT2 alleles has been reported in a German population, with half of the NAT1*10 alleles being linked to mutant NAT2 alleles (Henning et al., 1999). NAT1*11 allele, with the highest frequency reported thus far in the Turkish population, has been considered as a putative rapid allele in Caucasians and Black South Africans (Zheng et al. 1999; Loktionov et al. 2002). A possibility deserving investigation is of an association between NAT1*11 allele and certain cancers as shown for NAT1*10, another rapid acetylator.

Acknowledgments

This work was supported by grants from the Scientific Research Fund of Cumhuriyet University, Sivas to Fevzi Bardakci (Grant n. F-122).

Editor Associado: Francisco Mauro Salzano

Received: July 4, 2003;

Accepted: December 16, 2003.

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Publication Dates

• Publication in this collection
  20 July 2004
• Date of issue
  2004

History

• Accepted
  16 Dec 2003
• Received
  04 July 2003