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 the constitution of the most common single-nucleotide polymorphisms (1846G>A, 2988G>A, 100C>T, 2549delA, 2615_2617delAAG, and 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 closed-tube system and is highly specific and sensitive. We proved that this technique is highly reliable for use in routine diagnostics.

Department of Applied Neuroscience Faculty of Medicine Central European Institute of Technology Brno Czech Republic