Simultaneous determination of 7 N-acetyltransferase-2 single-nucleotide variations by allele-specific primer extension assay

Yusheng Zhu, David W. Hein, Mark A. Doll, Kristen K. Reynolds, Ntei Abudu, Roland Valdes, Mark W. Linder

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)1033-1039
Number of pages7
JournalClinical Chemistry
Volume52
Issue number6
DOIs
StatePublished - Jun 1 2006

Fingerprint

Acetyltransferases
Assays
Nucleotides
Alleles
Genotype
Phycoerythrin
Genetic Loci
Streptavidin
Microspheres
Toxicity
Confidence Intervals
Polymerase Chain Reaction
Substrates

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Zhu, Yusheng ; Hein, David W. ; Doll, Mark A. ; Reynolds, Kristen K. ; Abudu, Ntei ; Valdes, Roland ; Linder, Mark W. / Simultaneous determination of 7 N-acetyltransferase-2 single-nucleotide variations by allele-specific primer extension assay. In: Clinical Chemistry. 2006 ; Vol. 52, No. 6. pp. 1033-1039.
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abstract = "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.",
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Simultaneous determination of 7 N-acetyltransferase-2 single-nucleotide variations by allele-specific primer extension assay. / Zhu, Yusheng; Hein, David W.; Doll, Mark A.; Reynolds, Kristen K.; Abudu, Ntei; Valdes, Roland; Linder, Mark W.

In: Clinical Chemistry, Vol. 52, No. 6, 01.06.2006, p. 1033-1039.

Research output: Contribution to journalArticle

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AU - Zhu, Yusheng

AU - Hein, David W.

AU - Doll, Mark A.

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AU - Abudu, Ntei

AU - Valdes, Roland

AU - Linder, Mark W.

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N2 - 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.

AB - 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.

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