Genotyping of the cytochrome P450 2D6 4469 C>T polymorphism using SimpleProbes™

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Abstract
Genotyping of human cytochrome P450s is a pharmacogenetic approach to diagnosing inherited deficiencies in drug metabolizing enzymes that influence therapeutic responses. The P450 CYP2D6 (debrisoquine hydroxylase) metabolizes numerous antidepressants and neuroleptic agents and there is evidence of a relationship between gene polymorphism and variant therapeutic response. Polymorphism in CYP2D6 causes poor, intermediate, efficient or ultrarapid metabolization of substrate drugs affecting pharmacokinetic parameters and requiring dose adjustments. Predictive genotyping for broader clinical application is reliant on fast, technically simple analyses. A new genotyping method was explored. It identifies the single nucleotide polymorphism (SNP) 4469 C>T (NCBI access no. M33388) with one fluorescent hybridization probe (SimpleProbes; SP) using the LightCycler (LC). This SNP is found in 21 alleles, comprising 30% in Caucasian populations and encoding enzymes with poor, intermediate or efficient activity. The remaining 65 known alleles either harbour a C in position 4469 or are deletion mutants. Comparative detection of C>T polymorphism was done using a well-established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and PCR followed by melting-point (T(m)) analysis with an SP covering the SNP position in 144 samples encompassing alleles *2 and *41 with a T, alleles *1,*3, *4, *6, *9, *10, *15 with a C and the deletion mutant allele *5. C>T polymorphism was detected with complete concordance. T(m) of SP/target heteroduplex complexes for C was: T(m) 67, 89 degrees C to 68, 62 degrees C and for T: T(m) 60, 70 degrees C to 61, 51 degrees C. By one-step SP methodology it proved possible within 2 h to identify an SNP in genotypes comprising >90% in Caucasian populations.

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