Aim: Among the genes of drug-metabolizing enzymes, CYP2D6 is notoriously difficult to characterize owing to the complexity of gene deletions, duplications, multiplications and the presence of hybrid genes composed of CYP2D6 and CYP2D7. For SULT1A1 up to five gene copies have been reported, while UGT2B17 is known for gene deletions only. Different platforms exist for copy number variation (CNV) detection; however, there are no gold standards. Robust methods are required that address specific challenges to accurately determine gene CNVs in complex gene loci.
Materials & methods: Quantitative multiplex PCR amplification (MPA) was performed on a diverse set of genomic DNA samples. Resulting PCR fragments were separated on an ABI 3730 instrument and analyzed with GeneMapper. CYP2D6 was targeted at four different gene regions and either normalized against CYP2D8 or UGT2B15 and SULT1A2. Inconsistent observations and CNVs contrasting genotype data were further characterized by long-range PCR and/or DNA sequence analysis. UGT2B17 and SULT1A1 were normalized against UGT2B15 and SULT1A2, respectively.
Results: MPA detected 0-5, 1-5 and 0-2 copies for CYP2D6, SULT1A1 and UGT2B17, respectively. The interrogation of four CYP2D6 regions resulted in robust copy number assignments that were in agreement with genotype, sequencing and extra long PCR-based data. Gene deletions, duplication, and multiplications among known and novel hybrid genes were reliably identified. Novel findings regarding allelic variation include nonfunctional CYP2D6/2D7 hybrids such as CYP2D6*4N and *68, which were consistently identified on a subset of CYP2D6*4 alleles. In addition, a novel variant, designated CYP2D6*83, was discovered. For SULT1A1, we report the first six-copy case and for UGT2B15 and UGT2B17 we have evidence for rare deletion and duplication events, respectively.
Conclusion: This MPA-based copy number platform not only allowed us to determine CNVs, but also served as a tool for allele discovery and characterization in a diverse panel of samples in a fast and reliable manner.