Accuracy of various human NAT2 SNP genotyping panels to infer rapid, intermediate and slow acetylator phenotypes

Abstract

Aim: Humans exhibit genetic polymorphism in NAT2 resulting in rapid, intermediate and slow acetylator phenotypes. Over 65 NAT2 variants possessing one or more SNPs in the 870-bp NAT2 coding region have been reported. The seven most frequent SNPs are rs1801279 (191G>A), rs1041983 (282C>T), rs1801280 (341T>C), rs1799929 (481C>T), rs1799930 (590G>A), rs1208 (803A>G) and rs1799931 (857G>A). The majority of studies investigate the NAT2 genotype assay for three SNPs: 481C>T, 590G>A and 857G>A. A tag-SNP (rs1495741) recently identified in a genome-wide association study has also been proposed as a biomarker for the NAT2 phenotype.

Materials & methods: Sulfamethazine N-acetyltransferase catalytic activities were measured in cryopreserved human hepatocytes from a convenience sample of individuals in the USA with an ethnic frequency similar to the 2010 US population census. These activities were segregated by the tag-SNP rs1495741 and each of the seven SNPs described above. We assessed the accuracy of the tag-SNP and various two-, three-, four- and seven-SNP genotyping panels for their ability to accurately infer NAT2 phenotype.

Results: The accuracy of the various NAT2 SNP genotype panels to infer NAT2 phenotype were as follows: seven-SNP: 98.4%; tag-SNP: 77.7%; two-SNP: 96.1%; three-SNP: 92.2%; and four-SNP: 98.4%.

Conclusion: A NAT2 four-SNP genotype panel of rs1801279 (191G>A), rs1801280 (341T>C), rs1799930 (590G>A) and rs1799931 (857G>A) infers NAT2 acetylator phenotype with high accuracy, and is recommended over the tag-, two-, three- and (for economy of scale) the seven-SNP genotyping panels, particularly in populations of non-European ancestry.

Figure 1. Sulfamethazine N-acetyltransferase catalytic activities (mean ± standard error of the mean) in cryopreserved human hepatocytes are plotted on the ordinate versus each individual NAT2 SNP on the abscissa

The white bar is not shown for 191G>A and 857G>A because no homozygous variant samples were available. Sulfamethazine N-acetyltransferase activities for 282C>T, 341T>C, 481T>C, 590G>A, 803G>A and rs1495741G>A all differed significantly (p < 0.0001) among homozygous common, heterozygote and homozygous variant following one-way analysis of variance. For each of these SNPs, homozygous common was significantly higher than heterozygote (p < 0.05), which was significantly higher than homozygous variant samples (p < 0.05) following one-way analysis of variance and Tukey post hoc tests. Homozygous common was not significantly different than the heterozygote for 191G>A (p = 0.6522) or 857G>A (p = 0.7185).

Figure 2. Scatter plots of sulfamethazine N-acetyltransferase catalytic activities in cryopreserved human hepatocytes are plotted on the ordinate versus each NAT2 single nucleotide pair on the abscissa

Each homozygous common sample is illustrated with an open square, each heterozygote is illustrated with an open circle, and each homozygous variant is illustrated with an open triangle. Mean activities are shown by the horizontal lines. Statistical analysis of these data is shown in the legend to Figure 1.

Figure 3. Sulfamethazine N-acetyltransferase catalytic activities (mean ± standard error of the mean) in cryopreserved human hepatocytes are plotted on the ordinate versus NAT2 genotype(s) on the abscissa

The number of samples in each NAT2 acetylator genotype group is shown and related NAT2 genotypes were combined when sample sizes were less than 4.

Figure 4. Scatter plots of SMZ N-acetyltransferase catalytic activities plotted on the abscissa versus inferred acetylator phenotype as determined by various NAT2 genotyping protocols

Each NAT2 rapid acetylator sample is illustrated with an open square, each NAT2 intermediate acetylator sample is illustrated with an open circle, and each NAT2 slow acetylator sample is illustrated with an open triangle. Mean activities are shown by the vertical lines. Since the sulfamethazine N-acetyltransferase catalytic activity distribution was not explicitly trimodal in our population of samples, the identification of misclassified inferred NAT2 phenotypes was estimated by visual inspection of the data (illustrated by the inserted boxes).

Figure 5

Misclassification (percentage) of inferred acetylator phenotype as determined with various NAT2 SNPs genotyping protocols.