Flow cytometric assay for genotyping cytochrome p450 2C9 and 2C19: comparison with a microelectronic DNA array.
BACKGROUND
Cytochrome p450 (CYP) 2C9 and 2C19 metabolize a wide range of therapeutically important drugs. Genetic polymorphisms in the CYP2C9 and CYP2C19 genes result in variations in drug response. To correlate the dose required for therapeutic drug efficacy with genotype, accurate and reliable methods for detecting single nucleotide polymorphisms (SNPs) of CYP2C9 and CYP2C19 are required.


METHODS
We evaluated two technologies for genotyping CYP2C9 (*2 and *3 alleles) and CYP2C19 (*2 and *3 alleles). We developed a multiplexed flow cytometric assay based on the Luminex xMAP system and oligonucleotide-tagged Universal Array microspheres. The Luminex assay was compared with the eSensor DNA detection system, provided by Motorola Life Sciences. Genotypes determined by the two methods were confirmed by sequence analysis.


RESULTS
Of the 101 whole-genome amplified DNA samples genotyped by the Luminex method, 15 (14.8%) were heterozygous and 1 was homozygous for the CYP2C9*2 polymorphism. For the CYP2C9*3 polymorphism, 13 (12.9%) were heterozygous and 1 was homozygous. Two samples had the CYP2C9*2/*3 genotype. For CYP2C19*2, 17 (16.8%) of the samples were heterozygous and one was homozygous. The CYP2C19*3 polymorphism was not found. Genotypes determined by the Luminex assay were in complete concordance with the eSensor SNP assay results. A dilution study showed that 1.5 ng of nucleic acid was adequate for PCR and subsequent detection of SNPs by the Luminex assay. The within run and between run coefficients of variance (CVs) for allelic ratios determined by the Luminex procedure were found to be <or=4.1% and <or=9.1%, respectively, for the alleles present.


CONCLUSIONS
Both the in-house Luminex method and the eSensor trade mark DNA detection system reproducibly and unambiguously genotyped SNPs of CYP2C9 and CYP2C19 in the samples tested.
Pickering
Jerry W
JW
Associated Regional and University Pathologists, Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA. pickerjw@aruplab.com
McMillin
Gwendolyn A
GA
Gedge
Friederike
F
Hill
Harry R
HR
Lyon
Elaine
E
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D017931:DNA Primers; D004563:Electrochemistry; D005434:Flow Cytometry / Q000379:methods*; D005838:Genotype; D006801:Humans; D008863:Microspheres; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19