The N-acetylation of arylamines and acetoxy esters, a process carried out by the enzyme N-acetyltransferase, is considered to be a major detoxification step of the arylamine carcinogens in humans. N-acetyltransferase is functionally expressed by two polymorphic isoenzymes: N-acetyltansferase 1 and N-acetyltransferase 2.
N-acetyltransferase 2 is an enzyme that functions to both activate and deactivate arylamine and hydrazine drugs and carcinogens. Polymorphisms in this gene are responsible for the N-acetylation polymorphism in which human populations segregate into rapid, intermediate and slow acetylator phenotypes. Genetic polymorphism of N-acetyltransferase 2 is related with the susceptibility towards various malignant tumors, such as bladder cancer, lung cancer, and colorectal cancer.
The intronless N-acetyltransferase 2 gene maps on chromosome 8, at 8p22, covering 9.97 kb on the direct strand. Its protein has 290 amino acids (33.5 kDa), contains one N-acetyltransferase domain, and has a cytoplasmic sub-cellular location. Three major slow acetylator NAT2 alleles have been identified, and the number of minor, rare NAT2 alleles is to date greater than 30. NAT2*4 has traditionally been designated the "wild type" human N-acetyltransferase 2 allele. It is the most common occurring allele in some, but not all ethnic groups.
Hashem et al. (1969) reported that Egyptians have the highest incidence of the slow-acetylator phenotype (92%). In 2003, Hamdy et al., studied the frequencies of important allelic variants in the N-acetyltransferase 2 gene in a sample of 200 unrelated Egyptian subjects and compared them with the frequencies in other ethnic populations. N-acetyltransferase 2 gene variants (*5,*6 and *7) were detected using an allele-specific real-time PCR assay. Genotyping of the N-acetyltransferase 2 gene revealed frequencies of 0.215, 0.497, 0.260 and 0.028 for *4 (wild-type), *5 (341C), *6 (590A) and *7 (857A), respectively. Hamdy et al. (2003) found that Egyptians resemble other Caucasians with regard to allelic frequencies of the tested variants of the N-acetyltransferase 2 gene. However, the predominance of the slow acetylator genotype in their study (60.50%) could not confirm the frequency previously reported by Hashem et al. (1969), indicating the possibility of the presence of other mutations not detectable as T341C, G590A and G857A.
Najim et al., (2005) carried out a study to determine acetylator status in 35 Iraqis with allergic contact dermatitis (ACD) patients and 67 healthy controls. Among the ACD patients, there were 13 males and 22 females, aged between 17 and 43 years. Of the 35 patients, 21 were slow acetylators with a frequency of 60%, while the frequency of rapid acetylators was 40.0%. Forty-eight of 67 healthy controls were slow acetylators (72%) and the frequency of rapid acetylators was 28.4%. There was a significant difference between the rapid acetylator status in ACD patient and healthy controls. There was no significant association between the acetylator statuses, personal history of allergy, sites of dermatitis or patch-test positivity in ACD patients.
Simsek et al. (2000) performed a new method for detection of T341C polymorphism in the N-acetyltransferase (NAT2) gene of 110 unrelated Omani subjects. This technique involved two complimentary PCR-restriction fragment-length polymorphism (RFLP) methods, in which digestion of the wild alleles with Nco I was used as the first method, and digestion of the mutant alleles with Dde I was carried as the second complimentary method. A semi-nested PCR approach was followed in both methods with PCR amplification of a 998 bp fragment (with forward and reverse primers, Nat-Hu 14 and Nat-Hu 16, respectively) which acted as templates for amplification of a 360 bp fragment in the second round of PCR. A mutagenesis primer with a mismatched base was used with the Nat-Hul 14 primer in both PCR amplifications of 360 bp in the Nco I and the Dde I methods so as to generate a Nco I and a Dde I site, respectively, in the amplified DNA. The PCR products with the 360 bp were then digested with Nco I or Dde I enzymes (for the wild type and mutant alleles, respectively). DNA sequencing was performed on both DNA chains after amplification and purification of the 998 bp DNA fragment. Both reactions were expected to give inverse patterns for homozygotes and identical patterns for heterozygotes. To determine the accuracy of these tests, the results were compared with those obtained from sequencing of the DNA of 20 samples for the T341C polymorphism. These results were the first report of the allele frequency of T341C mutation in NAT2 gene for an Arab population. In all samples, a common 136 bp fragment was generated (due to a naturally occurring non-polymorphic Nco I site in the 360 bp DNA) which acted as an internal control. In the homozygous mutant alleles, a 221 bp fragment was generated, while in the wild type samples, two fragments were generated (198 bp and 23 bp), due to further digestion of the 221 bp fragment, and in the heterozygous DNA samples, both these diagnostic fragments were generated, in addition to the common fragment. Both methods, Nco I and Dde I, yielded similar genotypes for the 110 subjects studied; 38 wild types, 47 heterozygous and 25 homozygous mutants. The allele frequency was found to be 0.44, similar to that reported for Europeans and African-Americans. Sequencing for detection of the T341C polymorphism revealed similar genotypes for those homozygous for either the wild-type or the mutant type, but almost half of the heterozygous genotypes (four out of nine) were incorrectly identified as homozygous wild-type genotypes, which included the artificial heterozygous which was prepared by mixing equal amounts of amplified DNA with homozygous wild-type and homozygous mutant genotypes. This artificial heterozygous was identified correctly by both RFLP methods. Simsek et al. (2000) described these complimentary RFLP methods in their genotype analysis as having a sensitivity and specificity of 100%, and they recommended their use in order to obtain accurate genotyping results in detecting single nucleotide polymorphisms of any gene. Simsek et al. (2000) also recommended that detection of heterozygotes should be closely observed when using automated DNA sequencing.
Tanira et al. (2003) reported for the first time results of NAT2 genotyping of healthy Omani individuals. This was also the first time that all seven known polymorphisms of NAT2 were tested in any Arab population. A total of 127 apparently healthy, unrelated Omani subjects were used for the study. Genomic DNA was amplified by PCR specific for the NAT2 gene, and the amplified product was further subjected to RFLP analysis using Msp I, Fok I, Kpn I, Taq I, Dde I, Bam HI, or Nco I, as well as sequencing analysis. The most frequent polymorphisms detected were C341T and A803G (0.44 each), while the least frequent polymorphism was G857A (0.04). This pattern was similar to reports from Arab populations in neighboring countries. Interestingly, the African G191A was not be detected in any of the alleles, possibly due to the sample being from an internal Omani region, not subjected to much of ethnic mixing. The most common alleles in this population were found to be *5B, *6A, and *4. Tanira et al. (2003) also discovered a novel allele variant, containing C282T, T341C, and G590A. This new variant was named NAT2*5J, and was accepted by the NAT2 Nomenclature Committee. Tanira et al. (2003) predicted the phenotypes of the subjects based on their genotypes, and classified them as slow (0.57), intermediate (0.38) or rapid (0.05) acetylators.
Islam (1982) investigated the polymorphic acetylation of sulphamethazine among 109 Saudi males (aged between 20-24 years) of rural Bedouin origin and 126 Saudi females (aged between 16-65 years) from urban cosmopolitan areas in Jeddah. The mean incidence of slow acetylators was 67.9, 59.5, and 63.4% in male rural, female urban and total Saudi Arabs, respectively. The frequencies of the As allele, determining slow acetylation, in male rural, female urban and total Saudi Arabs were 0.824± 0.027, 0.771 ± 0.028 and 0.796 ± 0.020, respectively.
El-Yazigi et al. (1989) examined acetylator phenotypes of 296 Saudi subjects of Arabic origin by measuring the molar concentration ratio of two caffeine metabolites, 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1MX). The subjects were originally from different regions of Saudi Arabia but, at the time of the study, lived primarily in the capital city of Riyadh. The day-to-day reproducibility of the molar concentration ratio of AFMU/1MX was established in 14 randomly selected subjects. These metabolites were stable in urine at 4 degrees and -20 degrees, but AFMU was unstable at room temperature (23 degrees). The frequency distribution data indicate that 72.3% (ranging from 65-90%) of the subjects are of slow acetylator phenotype.
Attitallah et al. (2000) compared the acetylation status in the three main racial/ethnic groups living in Tunisia: Arabs, Berbers and Numides. The frequency of slow acetylators appears identical in these three groups and is different from that observed in Caucasians. However, the N-acetyltransferase type 2 activity as a whole is lower in Tunisians than in Caucasians. Attitallah et al. (2000) attributed these differences to the various population mixings which occurred in the past, given the geographical position of Tunisia.
In year 1996, Bastaki et al. studied the distribution of polymorphic N-acetyltransferase (NAT2) phenotypes in 118 unrelated, apparently healthy, Emirati schoolboys in the eastern region of Abu Dhabi. Phenotypes were assigned by determination of the 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine (AFMU/1X) urinary molar excretion ratio after oral ingestion of caffeine. The results of this study indicated a prevalence of the slow acetylator phenotype among United Arab Emirate nationals of 61%. The fast acetylator phenotype included many individuals with intermediate acetylating capacity that could be cautiously identified as heterozygotes.
Woolhouse et al. (1997) determined the NAT2 genotypes of a sample of 106 apparently healthy, unrelated Emiratis, by PCR-RFLP. Phenotypically, 63% of these subjects were slow acetylators, while genotypically too, nine of the 13 genotypes found were associated with slow acetylation. The most common of the genotypes were NAT2*5/*5 (0.25), NAT2*5/*6 (0.25), NAT2*4/*5 (0.22). Very high concordance was noticed between genotype and phenotype among both the slow (100%) and rapid (95%) acetylators. Seven 'slow' alleles were identified among the subjects, the most common mutations being C481T (0.53) and G590A (0.21). The other mutations identified were G857A, T341C, and A803G. The frequency of the wild NAT2*4 was found to be significantly lower when compared to other distant populations. In two of the 'slow' subjects, the allele was difficult to identify. One of these was later found to be a novel allele, having four mutations- T341C, C481T, A803G, and a novel mutation, C759T. Woolhouse et al. (1997) suggested that this new allele be designated NAT2*5D.
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