The drug metabolizer phenotype of the major P450 enzyme,
CYP2D6, can be predicted by genetic analysis. The
combination of full, reduced, or no function alleles, in
addition to gene copy number, is used to determine ultrarapid,
extensive, intermediate, or poor metabolizer status.
Data from quantitative PCR (qPCR) experiments using
TaqMan™ SNP Genotyping assays and TaqMan Copy
Number assays can be translated to star allele diplotypes
associated with metabolizer phenotypes. While qPCR can
decipher most of the information necessary, phenotypes
cannot be unequivocally assigned for samples that contain a
CYP2D6 duplication or multiduplications and are
heterozygous for alleles that are known to be duplicated and
that have different functional levels.
For these samples, allele-specific copy number analysis by
digital PCR (dPCR) can be done in order to identify the
duplicated allele. Digital PCR enables single sample
analysis with high precision and sensitivity by partitioning
target molecules into 20,000 individual reactions, thus
elucidating the ratio of the heterozygous alleles. For the
allele-specific copy number variation (ASCNV) application,
TaqMan SNP assays to CYP2D6 variants that are
associated with specific duplicated alleles were run in dPCR
on samples of known SNP genotype and CNV status. Initial
validation was conducted on Coriell cell line gDNA samples.
Here we describe work done on buccal swab samples
collected from two different sites. Representative samples
carrying CYP2D6 duplications that were heterozygous were
selected to run with ASCNV dPCR.