Detection of CYP2D6, SULT1A1 and UGT2B17 Copy Number Variation (CNV) Using Multiplex PCR Amplification

Background CNVs in genes such as CYP2D6, SULT1A1 and UGT2B17 contribute to variability in drug metabolism. We have developed a robust platform utilizing multiplex PCR amplification (MPA) to determine gene copy number, detect gene rearrangements that are prominent in the CYP2D6 locus and aid in the discovery and characterization of novel allelic variants. Methods The sample cohort (n=347) was enriched for CYP2D6 CNVs. Twelve amplicons were generated from CYP2D6, CYP2D7, CYP2D8, UGT2B15, SULT1A1, SULT1A2, UGT2B15 and UGT2B17 in 3 separate reactions under linear PCR conditions. The MPA products were resolved on an ABI 3730xl instrument and analyzed with GeneMapper V4.0. Copy number was determined using peak height ratios. Results UGT2B17 exhibited 3 clusters of 0, 1 and 2 copies while 5 clusters of 1 to 6 copies were observed for SULT1A1. The highest copy number for CYP2D6 was 5. The most abundant CNV was gene duplication. Subjects with hybrid genes were accurately identified. We revealed CYP2D6 * 4 alleles that carried additional CYP2D6/2D7 hybrids and identified them as CYP2D6 * 4N , * 68, * 68‐like and *novel. Sequence analysis also confirmed 2 tentative non‐functional CYP2D6 * 1 alleles with an exon 9 conversion. Additional sequence variations in CYP2D7 and CYP2D8 were found in subjects with peak abnormalities; 13 subjects had inconsistent results which may be explained by DNA quality/purity issues or the presence of yet unidentified rearrangements. Conclusions We have established a robust assay using a platform that is amenable to high‐throughput testing, and allows multiplexing to simultaneously determine the CNVs for CYP2D6, SULT1A1 and UGT2B15 . In conjunction with genotype analysis, this assay is highly valuable for routine and research‐based genetic analyses of these genes.