Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis

Hsiao Wei Liao, I. Lin Tsai, Guan Yuan Chen, Chun Ting Kuo, Ming Feng Wei, Tzung Jeng Hwang, Wei J. Chen, Li Jiuan Shen, Ching Hua Kuo

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes.

Original languageEnglish
Pages (from-to)67-75
Number of pages9
JournalAnalytica Chimica Acta
Volume763
DOIs
Publication statusPublished - Feb 6 2013
Externally publishedYes

Fingerprint

Capillary electrophoresis
Cytochrome P-450 CYP2D6
Multiplex Polymerase Chain Reaction
Polymerase chain reaction
Capillary Electrophoresis
Polymorphism
polymerase chain reaction
cytochrome
Single Nucleotide Polymorphism
electrokinesis
polymorphism
Nucleotides
Genes
gene
allele
Alleles
Polymerase Chain Reaction
drug
genotype
detection

Keywords

  • Capillary electrophoresis
  • Copy number variation
  • Cytochrome P450 2D6
  • Multiplex PCR
  • Single nucleotide polymorphism

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Spectroscopy
  • Environmental Chemistry

Cite this

Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis. / Liao, Hsiao Wei; Tsai, I. Lin; Chen, Guan Yuan; Kuo, Chun Ting; Wei, Ming Feng; Hwang, Tzung Jeng; Chen, Wei J.; Shen, Li Jiuan; Kuo, Ching Hua.

In: Analytica Chimica Acta, Vol. 763, 06.02.2013, p. 67-75.

Research output: Contribution to journalArticle

Liao, Hsiao Wei ; Tsai, I. Lin ; Chen, Guan Yuan ; Kuo, Chun Ting ; Wei, Ming Feng ; Hwang, Tzung Jeng ; Chen, Wei J. ; Shen, Li Jiuan ; Kuo, Ching Hua. / Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis. In: Analytica Chimica Acta. 2013 ; Vol. 763. pp. 67-75.
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AU - Tsai, I. Lin

AU - Chen, Guan Yuan

AU - Kuo, Chun Ting

AU - Wei, Ming Feng

AU - Hwang, Tzung Jeng

AU - Chen, Wei J.

AU - Shen, Li Jiuan

AU - Kuo, Ching Hua

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AB - CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes.

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KW - Single nucleotide polymorphism

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