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SNP genotyping using TaqMan technology: the CYP2D6*17 assay conundrum
Andrea Gaedigk1, Natalie Freeman2, Toinette Hartshorne3
11] Division of Clinical Pharmacology, Toxicology &Therapeutic Innovation, Children's Mercy Kansas City, Kansas City, MO [2] Department of Pediatrics, University of Missouri-Kansas City, Kansas City, MO.
Scientific Reports
|March 20, 2015
Summary
Genotyping the CYP2D6 gene is complex. An unexpected assay result highlighted the need for rigorous validation of genetic testing methods, especially for highly variable genes like CYP2D6.
Area of Science:
- Pharmacogenomics
- Molecular Diagnostics
Background:
- Cytochrome P450 2D6 (CYP2D6) is crucial for drug metabolism and personalized medicine.
- CYP2D6 genotyping is complex due to its intricate gene locus.
- TaqMan technology is a common, reliable, and cost-effective method for genotype analysis.
Purpose of the Study:
- To investigate unexpected diplotype calls in CYP2D6 genotyping using TaqMan technology.
- To identify the cause of assay discrepancies in specific CYP2D6 alleles.
Main Methods:
- Utilized TaqMan technology for CYP2D6 genotype analysis.
- Performed gene resequencing on affected samples.
- Developed an alternate PCR assay to overcome the observed phenomenon.
Main Results:
- An assay targeting 1023C>T (rs28371706) produced unexpected diplotype calls suggesting a CYP2D6*4 subvariant.
- Gene resequencing revealed a trio of SNPs on the CYP2D6*4 allele in affected samples, located between primer and probe binding sites.
- An alternate assay excluding the SNP trio resolved the issue.
Conclusions:
- Assay discrepancies in CYP2D6 genotyping can arise from complex genetic variations.
- Rigorous assay validation across diverse genotypes is essential for accurate pharmacogenetic testing.
- Vigilance in monitoring assay performance is critical for highly polymorphic genes like CYP2D6.
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