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Complete sequencing of a genetic polymorphism in NAT2 in the Korean population.

N-Acetyltransferase 2 (NAT2) metabolizes arylamines and hydrazines. The substrates of NAT2 include many therapeutic drugs, such as isoniazid (INH), as well as chemicals and carcinogens (1-3). For that reason, N-acetylation activity is associated with drug effects or toxicities and susceptibility to various cancers. The ability of NAT2 to N-acetylate arylamines is subject to a genetic polymorphism in the NAT2 gene. The acetylation rate and NAT2 genotype distribution are quite different among various populations. The genetic polymorphism in NAT2 has not been studied extensively in the Korean population. Previous reports were based only on phenotyping or restriction fragment length polymorphism analysis, leading to possible misclassification of genotypes. We therefore decided to investigate NAT2 allelic variability and genotype distributions in the Korean population by complete sequencing. We also evaluated the relationship between genotype and phenotype to understand N-acetylation pharmacogenetics.

One thousand Korean individuals who visited the health promotion center at Samsung Medical Center were anonymously studied. An additional 23 healthy volunteers and 18 patients with pulmonary tuberculosis participated in this study. DNA was extracted from peripheral blood leukocytes. We amplified a 1211-bp fragment that included an 870-bp protein-coding region of the NAT2 gene and performed full sequencing analysis (4) on an ABI Prism 377 DNA Sequencer (Perkin-Elmer). We then checked nucleotide substitutions by combined use of allele-specific-PCR (AS-PCR) and restriction enzyme digestion. Specific primers (5) for the wild-type and mutant alleles were used in separate PCRs to detect C282T, T341C, and G590A substitutions. The nucleotides at positions 190, 481, 590, 803, and 857 were explored by digesting the 1211-bp PCR fragment or AS-PCR product carrying the wild-type or mutant allele with NciI, KpnI, TagI, DdeI, and BamHI, respectively. For phenotyping, the healthy volunteers and patients gave informed consent and were given 300 mg of INH orally after an overnight fast. Participants were 18-65 years of age; had no underlying liver, kidney, or gastrointestinal diseases; did not have any drug history within the previous 2 weeks; and were not chronic alcoholics. Blood samples were obtained at 6 h, and urine samples were collected up to 6 h after INH administration. We measured urine and plasma concentrations of INH and acetylisoniazid (AcINH) by HPLC (6), using a Hewlett Packard 1090 HPLC with an ultraviolet detector. The plasma concentration ratio of AcINH to INH (pAcINH/INH) and the urinary molar ratio of INH to AcINH (uINH/AcINH) were calculated. We applied the [chi square] test and the Fisher exact test for categorical variables and the Mann-Whitney test or Kruskal-Wallis test for numerical variables.

On the basis of genotype (Table 1), 428 (42.8%) individuals were rapid, 469 (46.9%) were intermediate, and 96 (9.6%) were slow acetylators. The major genotypes were NAT2*4/*4, *4/*6A, and *4/*7B. Genotype distribution was consistent with Hardy-Weinberg equilibrium. The frequencies of rapid and slow alleles were 66.6% and 33.1%, respectively. The major alleles that led to a reduction in NAT2 activity were *6A and *7B, which contained the G590A and G857A substitutions, respectively. The frequencies of T341C and G857A explain the ethnic difference in acetylator phenotypes between Caucasians and Asians (5, 7). NAT2*5B, which contains the T341C, C481T, and A803G substitutions, is the most prevalent allele in Caucasians (40-46%), but it occurs at a very low frequency in Japanese (0.5%) (2) and Koreans (1.5% in this study).

Our results demonstrate that the G857A substitution is common (11.5%) in the Korean population, whereas it is rare in Caucasians (1-5%) (5, 7). The prevalence of the slow acetylator has been reported to be 7.6-16.7% in Koreans (8-10). However, because of the limited number of individuals studied, it is not clear whether these reported frequencies reflect a true genotypic distribution. Moreover, in the previous studies, there was a possibility that potential genotype and deduced phenotype misclassifications occurred because the PCR-restriction fragment length polymorphism analysis method used was designed to detect just a small subset of the alleles, including the C481T, G590A, and G857A substitutions, that are commonly found in other ethnic groups. Other issues in these studies were the exclusion of the T341C substitution, which was found to have independent functional significance, and the inclusion of the C481T substitution, which does not reduce enzyme activity (11). Fortunately, the effect may be small in the Korean population because of the low prevalence of the T341C substitution. In any case, the existence of unpredictable or excluded base substitutions in genotyping analysis could cause discrepancies between genotypes and phenotypes. Additionally, a special analytical technique is required to discriminate a mutation-linkage pattern (4, 5).

Considering these points, we performed full sequencing analysis in combination with AS-PCR and restriction enzyme digestion. This would allow accurate determination of all possible genotypes and allocation of nucleotide substitution to either DNA strand. In all cases with heterozygous C282T and heterozygous G590A or G857A substitutions, we found that the genotypes were *4/*6A or *4/*7B, not *6B/*13 or *7A/*13. Cases with T341C, C481T, and A803G substitutions were always found to have *5B.

Because the phenotyping tests are known to be influenced by environmental factors, including disease status and concurrent drug therapy (1), we also studied tuberculous patients receiving quadruple therapy as well as healthy individuals and used INH as an indicator drug. Contrary to our expectations, there was no significant difference in INH metabolism between the two groups. Acetylator typing is known to be helpful in patients on a once-daily INH-containing regimen (12). Our patients responded equally well with standard daily INH treatment for tuberculosis. It is unfortunate that we could not find any potential therapeutic significance of acetylator types in patients with tuberculosis because of the small number of participants.

The three groups categorized by genotypes showed significant differences in their acetylation activity (P <0.05). However, there was an overlap between rapid and intermediate types and a wide range in the rates of acetylation in both types. Although there was a good correlation between pAcINH/INH and uINH/AcINH, pAcINH/INH provided better discrimination between rapid and intermediate types than did uINH/AcINH. The concordance rate of genotyping and phenotyping by pAcINH/INH was 90.2%. Four discordant cases had genotypes of *4/*4 with intermediate acetylation activity. Genotyping seems more relevant than phenotyping because three of four discrepant cases showed conflicting results with different phenotyping methods. We believe that the discrepancies between phenotypes and genotypes suggest possible problems in phenotyping or the presence of another factor that contributes to the variability in NAT2 activity, such as the NAT1 gene.

The correlation between pAcINH/INH and nine genotypes is shown in Fig. 1. Wide variations in acetylation activities were noted in individuals with the same genotypes. Homozygous rapid acetylators with NAT2*4/*4 exhibited mostly higher activities than did heterozygotes such as *4/*6A or *4/*7B. Individuals with two *6A alleles showed significantly decreased activities compared with intermediate acetylators with *4/*6A, suggesting a gene dosage effect.

Metabolic activity data were trimodally distributed with some overlap between rapid and intermediate acetylators. It was difficult to discern intermediate from rapid acetylators by phenotyping alone. Because rapid and intermediate acetylators account for most of the general population and the two groups have different metabolic activities in spite of overlaps, it is very important to correctly discriminate the two. Accordingly, genotype analysis is indispensable to determine acetylator status rather than, or as a supplement to phenotyping. Moreover, there are differences in NAT2 activity within each genotypec group. Several studies have demonstrated significant quantitative or qualitative differences in in vivo acetylation capacities, even among slow alleles (4,13). Our aim was to determine whether mutation linkage patterns and allelic composition, as well as the presence of nucleotide changes at the NAT2 locus, may account for the diverse acetylation activities, but we lacked sufficient numbers of alleles and individuals with each allele in this study to achieve this aim. We expect that, in the near future, quantitative estimation of NAT2 activity may be possible with knowledge of the function of each variant allele that may cause subtle differences in metabolism.

[FIGURE 1 OMITTED]

The results of this study will be helpful for future epidemiologic or clinical studies and for understanding the genetic basis of acetylation polymorphisms in the Korean population. The design of a cost-effective genotyping method to search for targeted mutations and prediction or determination of ambiguous alleles and genotypes will be facilitated. We confirmed that N-acetylation activity is significantly affected by a genetic polymorphism in NAT2. However, further studies are required to define the influence of NAT2 allelic variants and the effects of other genetic factors on the phenotypic outcome.

This work was supported by the fund from The Center for Functional Analysis of Human Genome, Korea.

References

(1.) Gross M, Kruisselbrink T, Anderson K, Lang N, McGovern P, Delongchamp R, et al. Distribution and concordance of N-acetyltransferase genotype and phenotype in an American population. Cancer Epidemiol Biomark Prev 1999;8:683-92.

(2.) Weber WW, ed. Pharmacogenetics, 1st ed. New York: Oxford University Press, 1997:189-201.

(3.) Grant DM, Hughes NC, Janezic SA, Goodfellow GH, Chen HJ, Gaedigk A, et al. Human acetyltransferase polymorphisms. Mutat Res 1997;376:61-70.

(4.) Cascorbi I, Drakoulis N, Brockmoller J, Maurer A, Sperling K, Roots I. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated Caucasian individuals: correlation with phenotypic activity. Am J Hum Genet 1995;57:581-92.

(5.) Delomenie C, Sica L, Grant DM, Krishnamoorthy R, Dupret JM. Genotyping of the polymorphic N-acetyltransferase (NAT2*) gene locus in two native African populations. Pharmacogenetics 1996;6:177-85.

(6.) Seifart HI, Gent WL, Parkin DP, van Jaarsveld PP, Donald PR. High-performance liquid chromatographic determination of isoniazid, acetylisoniazid and hydrazine in biological fluids. J Chromatogr 1995;674:269-75.

(7.) Lin HJ, Han CY, Lin BK, Hardy S. Slow acetylator mutations in the human polymorphic N-acetyltransferase gene in 786 Asians, blacks, Hispanics, and whites: application to metabolic epidemiology. Am J Hum Genet 1993;52: 827-34.

(8.) Shin JG, Shin SG, Jang IJ, Kim YS, Lee KH, Han JS, et al. Comparison of isoniazid phenotyping methods and acetylator distribution in 43 native Korean subjects. Asia Pac J Pharmacol 1992;7:1-8.

(9.) Lee SW, Jang IJ, Shin SG, Lee KH, Yim DS, Kim SW, et al. CYP1A2 activity as a risk factor for bladder cancer. J Korean Med Sci 1994;9:482-9.

(10.) Kim E, Kim WJ, Lee HL, Lee MS, Kim CW, Kim RS, et al. A case-control study on the effects of the genetic polymorphisms of N-acetyltransferase 2 and glutathione Stransferase [mu] and [theta] on the risk of bladder cancer. Korean J Prev Med 1998;31:275-84.

(11.) Leff MA, Fretland AJ, Doll MA, Hein DW. Novel human N-acetyltransferase 2 alleles that differ in mechanism for slow acetylator phenotype. J Biol Chem 1999;274:34519-22.

(12.) Ellard GA. The potential clinical significance of the isoniazid acetylator phenotype in the treatment of pulmonary tuberculosis. Tubercle 1984;65: 211-27.

(13.) Meisel P, Schroeder C, Wulff K, Siegmund W. Relationship between human genotype and phenotype of N-acetyltransferase (NAT2) as estimated by discriminant analysis and multiple linear regression: 1. Genotype and N-acetylation in vivo. Pharmacogenetics 1997;7:241-6.

Soo-Youn Lee, [1] Kyung-A Lee, [1] Chang-Seok Ki, [1] O. Jung Kwon, [2] Ho Joong Kim, [2] Man Pyo Chung, [2] Gee Young Suh, [2] and Jong-Won Kim [1] * ([1] Department of Clinical Pathology and Division of Pulmonary and Critical Medicine, [2] Department of Medicine, Sungkyunkwan University School of Medicine and Samsung Medical Center, Seoul 135-710, Korea; * address correspondence to this author at: No. 50, Ilwon-dong, Kangnam-ku, Department of Clinical Pathology, Samsung Medical Center, Seou1135-710, Korea; fax 82-2-3410-2719, e-mail jwonk@smc.samsung.co.kr)
Table 1. Frequencies of NAT2 genotypes in 1000 Korean individuals.

 Genotype Deduced
 phenotype

*4/*4 Rapid
*4/*12A Rapid
*4/*12C Rapid
Rapid, total
*4/*5B Intermediate
*4/*5C Intermediate
*4/*6A Intermediate
*4/*6B Intermediate
*4/*7A Intermediate
*4/*7B Intermediate
*6A/*12A Intermediate
*6A/*12C Intermediate
*6A/*13 Intermediate
*6C/*12C Intermediate
*7B/*13 Intermediate
Intermediate, total

*5B/*6A Slow
*5B/*7B Slow
*6A/*6A Slow
*6A/*7B Slow
*7A/*7A Slow
*7B/*7B Slow
Slow, total
*4/*19 Rapid or
 intermediate
*6A/*19 Intermediate
 or slow
Undetermined, total

Total

 Genotype Observed

 n % 95% Expected,
 confidence %
 interval, %

*4/*4 419 41.9 38.8-45.0 43.2
*4/*12A 2 0.2 0.0-0.5 0.4
*4/*12C 7 0.7 0.2-1.2 0.7
Rapid, total 428 42.8 39.7-45.9 44.4
*4/*5B 23 2.3 1.4-3.2 2
*4/*5C 2 0.2 0.0-0.5 0.1
*4/*6A 264 26.4 23.7-29.1 25.5
*4/*6B 11 1.1 0.5-1.7 0.8
*4/*7A 7 0.7 0.2-1.2 0.7
*4/*7B 154 15.4 13.2-17.6 14.5
*6A/*12A 3 0.3 0.0-0.6 0.1
*6A/*12C 1 0.1 0.0-0.3 0.2
*6A/*13 1 0.1 0.0-0.3 <0.1
*6C/*12C 2 0.2 0.0-0.5 <0.1
*7B/*13 1 0.1 0.0-0.3 <0.1
Intermediate, 469 46.9 43.8-50.0 44.1
 total

*5B/*6A 4 0.4 0.0-0.8 0.6
*5B/*7B 3 0.3 0.0-0.6 0.3
*6A/*6A 39 3.9 2.7-5.1 3.8
*6A/*7B 36 3.6 2.4-4.8 4.3
*7A/*7A 1 0.1 0.0-0.3 <0.1
*7B/*7B 13 1.3 0.6-2.0 1.2
Slow, total 96 9.6 7.8-11.4 11
*4/*19 6 0.6 0.1-1.1 0.5

*6A/*19 1 0.1 0.0-0.3 0.2

Undetermined, 7 1 0.2-1.2 <0.1
 total

Total 1000 100
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Title Annotation:Technical Briefs
Author:Lee, Soo-Youn; Lee, Kyung-A; Ki, Chang-Seok; Kwon, O. Jung; Kim, Ho Joong; Chung, Man Pyo; Suh, Gee
Publication:Clinical Chemistry
Date:May 1, 2002
Words:2294
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