Alternative Reliable Method for Cytochrome P450 2D6 Poor Metabolizers Genotyping
|Original title:||Alternative Reliable Method for Cytochrome P450 2D6 Poor Metabolizers Genotyping|
|Authors:||Eva Pindurová, Alexandra Žourková, Jana Zrůstová, Jan Juřica, Antonín Pavelka|
High-resolution melting curve analysis (HRM) of polymerase chain reaction (PCR) amplicons has been described as a fast, cheap and reliable closed-tube method of genotyping with no need for labeled primers or labeled probes. We adapted this melting analysis assay for the detection of the most common nonfunctional alleles of cytochrome P-450 (CYP) 2D6 in the Caucasian population that affect the metabolism of many commonly used drugs. We used this method to genotype 91 patients under paroxetine therapy. The presence and constitution of the most common single-nucleotide polymorphisms (1846G>A, 2988G>A, 100C>T, 2549delA, 2615_2617delAAG, 1707delT) in poor and intermediate metabolizers from the Caucasian population were detected in short amplicons 148 bp). After fluorescence normalization, the wild-type, homozygous and heterozygous samples were easily distinguishable from each other by their specific melting curve shape. A total of 92.6% of the 1846G>A heterozygotes, 96 % of the 100C>T heterozygotes and 100 % of the 2988G>A, 2549delA, 2615_2617delAAG and 1707delT heterozygotes have been correctly distinguished from the wild types. One hundred percent of all the homozygotes in this group of patients have been detected without any error. HRM of short amplicons is a simple tool for effective, rapid and reliable CYP2D6 genotyping that does not require real-time PCR, labeled probes, processing or any separations after PCR. The reaction is performed in a closedtube system and is highly specific and sensitive. We proved that this technique is highly reliable for use in routine diagnostics.