Real time PCR detection of common CYP2D6 genetic variants and its application in a Karen population study

Malar J. 2018 Nov 15;17(1):427. doi: 10.1186/s12936-018-2579-8.

Abstract

Background: Plasmodium vivax malaria is characterized by relapses arising from the hypnozoite stages in the liver. The only currently registered drug for radical treatment to prevent relapse is primaquine. Primaquine, a prodrug, requires metabolism through the liver cytochrome CYP2D6 isoenzyme to its active metabolite. Mutations in the CYP2D6 gene may thus affect primaquine efficacy. A SNPs genotyping technique was developed to characterize the CYP2D6 genetic variants and tested this in the patients with Plasmodium vivax infection collected in a Karen population on the Thailand-Myanmar border, where P. vivax malaria is endemic.

Methods: Direct sequencing of PCR-reamplified products (DSP) was used to uncover exonic CYP2D6 sequence variations. Subsequently, an allele-specific oligonucleotide probe real-time SNPs genotyping (ASO) assay was developed for rapid detection of the four clinically relevant CYP2D6 variants occurring in this population. These two in-house developed assays were used to genotype CYP2D6 mutations in blood samples obtained from 70 Karen adults.

Results: Results showed a high degree of concordance between the DSP and ASO methods. Six CYP2D6 point mutations were identified within the Karen population: C100T, C1039T, G1661C, G1846A, C2850T and G4180C, at frequencies of 0.43, 0.43, 0.76, 0.02, 0.32 and 0.76, respectively. The CYP2D6*2, *4, *5, *10 and *36 allelic frequencies were 0.33, 0.02, 0.03, 0.40 and 0.01, respectively. Alleles conferring an intermediate CYP2D6 metabolizer phenotype comprised 46% of the total number of alleles.

Conclusion: The newly developed ASO assay is a reliable and rapid tool for large-scale CYP2D6 genotyping. The high frequency of the CYP2D6*10 allele in the Karen population warrants further assessment of its association with the radical curative efficacy of primaquine.

Keywords: CYP2D6; Karen; Malaria; Primaquine.

MeSH terms

  • Adolescent
  • Adult
  • Child
  • Cytochrome P-450 CYP2D6 / genetics*
  • Female
  • Genetic Variation*
  • Genotyping Techniques / methods
  • Humans
  • Malaria, Vivax / parasitology
  • Male
  • Mutation*
  • Myanmar / ethnology
  • Plasmodium vivax / isolation & purification*
  • Polymorphism, Single Nucleotide*
  • Real-Time Polymerase Chain Reaction / methods*
  • Thailand
  • Young Adult

Substances

  • Cytochrome P-450 CYP2D6