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Flow cytometric assay for genotyping cytochrome p450 2C9 and 2C19: comparison with a microelectronic DNA array
Jerry W Pickering1, Gwendolyn A McMillin, Friederike Gedge
1Associated Regional and University Pathologists, Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA. pickerjw@aruplab.com
American Journal of Pharmacogenomics : Genomics-Related Research in Drug Development and Clinical Practice
|June 4, 2004
Summary
Accurate genotyping of Cytochrome P450 (CYP) 2C9 and 2C19 genetic variations is crucial for personalized medicine. Both Luminex and eSensor systems reliably detect these single nucleotide polymorphisms (SNPs), aiding in drug response correlation.
Area of Science:
- Pharmacogenomics
- Molecular Diagnostics
Background:
- Cytochrome P450 (CYP) enzymes, specifically CYP2C9 and CYP2C19, are vital for metabolizing numerous essential medications.
- Genetic variations (polymorphisms) in CYP2C9 and CYP2C19 genes significantly influence individual drug responses and therapeutic outcomes.
- Precise detection of single nucleotide polymorphisms (SNPs) in these genes is necessary to link drug dosage with genotype for optimal treatment efficacy.
Purpose of the Study:
- To evaluate the performance of two distinct genotyping technologies for identifying key polymorphisms in CYP2C9 and CYP2C19 genes.
- To compare a newly developed multiplexed flow cytometric assay utilizing the Luminex xMAP system against the established eSensor DNA detection system.
- To confirm the accuracy of both genotyping methods through direct DNA sequence analysis.
Main Methods:
- Development of a multiplexed flow cytometry assay using Luminex xMAP technology and Universal Array microspheres for simultaneous genotyping of CYP2C9 (*2, *3 alleles) and CYP2C19 (*2, *3 alleles).
- Comparative analysis of the Luminex assay with the eSensor DNA detection system for genotyping 101 DNA samples.
- Validation of genotyping results obtained from both methods using Sanger sequencing.
Main Results:
- The Luminex assay successfully genotyped CYP2C9 (*2, *3) and CYP2C19 (*2) polymorphisms in 101 samples, identifying various heterozygous and homozygous genotypes.
- No instances of the CYP2C19*3 polymorphism were detected in the tested sample set.
- Complete concordance was observed between the Luminex and eSensor genotyping results, with sequence analysis confirming accuracy.
- A minimum of 1.5 ng of nucleic acid was sufficient for reliable SNP detection using the Luminex assay.
- The Luminex method demonstrated high reproducibility, with within-run and between-run coefficients of variance for allelic ratios below 4.1% and 9.1%, respectively.
Conclusions:
- Both the in-house developed Luminex assay and the commercial eSensor system provide reproducible and unambiguous genotyping of critical CYP2C9 and CYP2C19 SNPs.
- These validated methods are suitable for clinical applications requiring accurate pharmacogenetic profiling to guide drug therapy.