RAPID COMMUNICATIONSDifferential quantification of CYP2D6 gene copy number by four different quantitative real-time PCR assaysRamamoorthy, Anuradhaa b; Flockhart, David A.a b; Hosono, Naoyac; Kubo, Michiakic; Nakamura, Yusukec d; Skaar, Todd C.aAuthor Information aDepartment of Medicine, Division of Clinical Pharmacology bDepartment of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, USA cLaboratory for Genotyping Development, Center for Genomic Medicine, RIKEN, Yokohama dLaboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, University of Tokyo, Tokyo, Japan Correspondence to Todd C. Skaar, PhD, Department of Medicine, Division of Clinical Pharmacology, Indiana University School of Medicine, 1001 W. 10th Street, WD Myers Building W7123, Indianapolis, IN 46202, USA Tel: +1 317 630 2695; fax: +1 317 630 8185; e-mail: [email protected] Received 19 January 2010 Accepted 20 March 2010 Pharmacogenetics and Genomics: July 2010 - Volume 20 - Issue 7 - p 451-454 doi: 10.1097/FPC.0b013e32833a1083 Buy Metrics Abstract Copy number variations (CNVs) in the CYP2D6 gene contribute to interindividual variation in drug metabolism. As the most common duplicated allele in Asian populations is the nonfunctional CYP2D6*36 allele, the goal of this study was to identify CNV assays that can differentiate between multiple copies of the CYP2D6*36 allele and multiple copies of other CYP2D6 alleles. We determined CYP2D6 gene copy numbers in 32 individuals with known CYP2D6 CNVs from the Coriell Japanese–Chinese panel using four quantitative real-time PCR assays. These assays target different regions of the CYP2D6 gene: 5′-flanking region, intron 2, intron 6, and exon 9 (Ex9). The specific target site of the Ex9 assay was verified by sequencing the PCR amplicon. Three of the CYP2D6 CNV assays (5′-flanking region, intron 2, and intron 6) estimated CYP2D6 copy numbers that were concordant for all 32 individuals. However, the Ex9 assay was concordant in only 10 of 32 samples. The 10 concordant samples did not contain any CYP2D6*36 alleles and the 22 discordant samples contained at least one CYP2D6*36 allele. In addition, the Ex9 assay accurately quantified all of the non-CYP2D6*36 alleles in all samples. Ex9 amplicon sequencing indicated that it targets a region of CYP2D6 exon 9 that undergoes partial gene-conversion in the CYP2D6*36 allele. In conclusion, CYP2D6 Ex9 CNV assay can be used to determine the copy number of non-CYP2D6*36 alleles. Selective amplification of non-CYP2D6*36 sequence by the Ex9 assay should be useful in determining the number of functional copies of CYP2D6 in Asian populations. © 2010 Lippincott Williams & Wilkins, Inc.