engleski

Genotyping of poor metabolizer of CYP2D6 by multiplex allele specific PCR

The debrisoquine/spartaine polymorphism is a clinically relevant genetic variability of drug oxidation capacity with two distinct phenotypes, extensive and poor metabolizer (EMs and PMs). The PM phenotype occurs with a frequency of about 7% in Caucasian populations and is the result of autosomal recessive inheritance of nonfunctional (null) alleles of CYP 2D6. A carrier of two homozygote or two heterozygote mutant alleles is considered a poor metabolizer (PM) phenotype, while a carrier of only one demaged allele is considered an intermediate metabolizer (IM) phenotype. Six mutant alleles, *3, *4, *5, *6, *7 and *8, may account for over 99% of the PM alleles in a given population. The aim of the study was to assess the prevalence of null alleles in a group of healthy individuals (n=145) and a group of schizophrenic patients (n=51) by the method of multiplex allele specific PCR. By this method, CYP2D6 was specifically amplified as a 4666 bp fragment by long PCR using wild type or mutation-specific primers. Two separate PCR reactions were run for each DNA sample using the CYP2D6-specific 4.7 kb preamplification product as template. The PCR products were analyzed by electrophoresis in a 3% NuSieve GTG (FMC, USA): ultraPure (BRL) 3:1 agarose gel (2, 3). The percentage of mutant alleles in the control group (2D6*3 - 1.4% ; *4 - 11.0% ; and *6 – 1.0%) differed significantly from that recorded in the patient group (2D6*3 - 0% ; *4 - 21.6% ; and *6 - 2.9 %) (p=0.016). All allele frequencies were in Hardy Weinberg equilibrium (p=0.893). Evaluating the patient group as a whole, there was a significant difference in genotype distribution (p=0.003). So, 31.4% and 5.9% of 2D6*4 alleles were found to be heterozygote and homozygote genotype, respectively in the patient group versus 18.6% and 1.4% of 2D6*4 heterozygote and homozygote genotype in controls. 2D6*6 allele was found in 5.9% of patients and 1.4% of controls, only as a heterozygous genotype. There were 0.7% of heterozygote 2D6 *4/*5 genotype only in the control group. The predicted phenotype distribution showed a significant difference of IEM between the control and patient group (p=0.044), while the incidence of PM, although higher in the patient group, showed no significant difference. The odds ratio for IEM and PM was 2.02 (CI95%: 1.018 – 3.988) and 2.96 (CI96%: 0.617 – 14.183), respectively, indicating statistical relevance.

CYP2D6; phenotyping; multiplex AS PCR

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