Simultaneous determination of 7 N-acetyltransferase-2 single-nucleotide variations by allele-specific primer extension assay.
BACKGROUND
Genotyping of N-acetyltransferase-2 (NAT2) is useful in predicting the risk for toxicity of NAT2 substrates. Current methods cannot detect the 7 most important single-nucleotide variations in NAT2 simultaneously in 1 tube.


METHODS
We developed an assay that uses allele-specific primer extension (ASPE) and microsphere hybridization for the simultaneous detection of 7 single-nucleotide variations in NAT2. Using 12 samples previously genotyped by a TaqMan-based assay for method development and as positive controls, we amplified the genetic locus of NAT2 comprising the single-nucleotide variations of interest by PCR and then performed ASPE with allele-specific primers and biotinylated dCTP followed by bead hybridization and streptavidin-R-phycoerythrin binding. Genotypes were determined according to the allele-specific fluorescent signal ratios.


RESULTS
The mean (SD) allelic ratios for homozygous common, heterozygous variant, and homozygous variant NAT2 genotypes were 0.0394 (0.0113) (n = 80), 0.4372 (0.0270) (n = 148), and 0.9331 (0.0127) (n = 325). The assay had 100% (95% confidence interval, 99%-100%) within-run reproducibility for 12 samples repeated 6 times and 100% (98%-100%) between-run reproducibility for a 5-sample subset run on 6 different days. NAT2 genotypes of 30 blinded samples determined by this assay were 100% (98%-100%) concordant with results obtained using the TaqMan method.


CONCLUSIONS
The developed assay can simultaneously determine single-nucleotide variations in NAT2. The assay demonstrates no overlap in allele-specific signal ratios between homozygous common, heterozygous, and homozygous variant and shows agreement with a reference method and reproducibility of genotype identification.
Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA.
Yusheng
Y
Zhu
David W
DW
Hein
Mark A
MA
Doll
Kristen K
KK
Reynolds
Ntei
N
Abudu
Roland
R
Valdes
Mark W
MW
Linder
D000483:Alleles; D001191:Arylamine N-Acetyltransferase / Q000235:genetics*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results
D017931:DNA Primers; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human; D004259:DNA-Directed DNA Polymerase