CYP2D6 Haplotype Determination Using Long Range Allele-Specific Amplification: Resolution of a Complex Genotype and a Discordant Genotype Involving the CYP2D6*59 Allele.
Cytochrome P450 (CYP) 2D6, a major contributor to the metabolism and bioactivation of many clinically used drugs, is encoded by a complex, highly polymorphic gene locus. To aid in the characterization of CYP2D6 allelic variation, we developed allele-specific long-range PCR (ASXL-PCR) to amplify only the allele of interest for further characterization by PCR. This development was achieved utilizing single-nucleotide polymorphisms in the upstream region of CYP2D6 and a universal CYP2D6-specific reverse primer. This approach was assessed and optimized on samples with known genotypes. The application of ASXL-PCR clarified a case with a complex genotype (CYP2D6*2x2/*4N+*4) by amplifying the duplicated gene units separately for subsequent analysis. Furthermore, ASXL-PCR and subsequent sequence analysis also resolved genotype discord in a mother/daughter relationship by revealing the presence of the CYP2D6*59 allelic variant in both individuals. Finally, we demonstrated that the 2939G>A single-nucleotide polymorphism present on CYP2D6*59 interfered with the TaqMan genotype assay that detected 2850C>T, causing false genotype assignments. Assay interference was resolved using an alternative TaqMan genotype assay currently available as a custom-made assay. These examples demonstrate the utility of ASXL-PCR for improved CYP2D6 allele/haplotype characterization. This fast, easy-to-perform method is not limited to CYP2D6 but can be adapted to any gene locus for which polymorphic sites are known.
Division of Clinical Pharmacology, Toxicology and Therapeutic Innovation, Children's Mercy Kansas City, Kansas City, Missouri; Department of Pediatrics, University of Missouri-Kansas City, Kansas City, Missouri. Electronic address: agaedigk@cmh.edu.
Andrea
A
Gaedigk
Division of Clinical Pharmacology, Toxicology and Therapeutic Innovation, Children's Mercy Kansas City, Kansas City, Missouri.
Amanda K
AK
Riffel
Division of Clinical Pharmacology, Toxicology and Therapeutic Innovation, Children's Mercy Kansas City, Kansas City, Missouri; Department of Pediatrics, University of Missouri-Kansas City, Kansas City, Missouri.
J Steven
JS
Leeder
D000483:Alleles; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
D019389:Cytochrome P-450 CYP2D6