CYP2D6 ALLELE DISTRIBUTION IN MACEDONIANS, ALBANIANS AND ROMANIES IN THE REPUBLIC OF MACEDONIA
Kuzmanovska M, Dimishkovska M, Maleva Kostovska I, Noveski P, Sukarova Stefanovska E, Plaseska-Karanfilska D*
*Corresponding Author: Dijana Plaseska-Karanfilska, M.D., Ph.D., Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov,” Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, Republic of Macedonia. Tel: +389-2-3235-410. Fax: +389-2-3115-434. E-mail: dijana@manu.edu.mk
page: 49

MATERIALS AND METHODS

DNA Samples. DNA material for genotyping from Macedonians (n = 100), Albanians (n = 100) and Romanies (n = 100), was obtained from the DNA bank of the Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov” at the Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia. We decided to analyze 100 samples from each ethnicity, in order to be able to compare and statistically process the obtained results. The number of samples from each ethnic group does not reflect the actual representation of ethnicities in the Republic of Macedonia. The Ethics Committee of the Macedonian Academy of Science and Arts approved this study. The samples were anonymized after collection. CYP2D6 Genotyping. The genotyping was performed following a recently described protocol by Sistonen et al. [21], based on a combination of long range polymerase chain reaction (PCR), to detect whole-gene deletion/ duplication and multiplex extension of unlabeled oligo-nucleotide primers with fluorescently labeled dideoxynucleotide triphosphates (ABI PRISM® SNaPshot Multiplex Kit; Life Technologies, Carlsbad, CA, USA) to characterize 11 relevant polymorphic positions in the coding region of CYP2D6. This made it possible to identify CYP 2D6 variants which are highly represented in different human populations (i.e.,*2, *4, *10, *17, *29, *39, *41), rare variants known to be responsible for low or null metabolic activity (i.e., *3, *6 and *9), and whole gene deletion (*5) and duplications (*1xN, *2xN). This permitted us to identify the mutations that are generally thought to comprise more than 90.0% of known variants in Europeans [22], and mutation 1023 (C>T) which is rare in Europe, but is common in African populations. The haplotypes which did not show any of these mutations were classified as *1. Three parallel long range-PCRs were run for each sample using Expand Long Template PCR System (Roche Diagnostics, Basel, Switzerland). We obtained a 5.1 kb fragment containing all nine CYP2D6 exons using CYP 2D6-F [21] and CYP2D6-R primers [21]. This product was used as a template in order to be able to type 11 positions in one reaction, based on single-base primer extension with fluorescentlylabelled ddNTPs (ABI PRISM® SNaPshot Multiplex Kit; Life Technologies). The 25 μL reaction mixture contained 2U enzyme mix, Expand Long Template Buffer 1, 2 × concentrated, with 17.5 mM MgCl2, 0.2 mM each dideoxynucleotide triphosphate, 0.4 μM of each primer, and 50 ng of genomic DNA. The PCR reaction was conducted as follows: denaturation at 94°C for 10 min., 10 cycles at 94°C for 30 seconds and 68°C for 30 seconds, 25 cycles at 94°C for 30 seconds and 68°C for 10 min. and 15 seconds, plus 15 seconds per cycle, and a final extension at 68°C for 30 min. The long range-PCR products were analyzed on 0.8% agarose gels, and the 5.1 kb fragments were purified by use of 1 μL of ExoSAP-IT (USB, Affymetrix Inc., San Diego, CA, USA) overnight at 37°C (2 μL PCR product + 1 μL Exo SAP IT). The reaction ended with inactivation of the enzyme at 86°C for 20 min. The purified 5.1 kb product was used as template in the SNaPshot (Life Technologies) reaction. In the following single-base extension reaction, the detection primers annealed adjacent to the single nucleotide polymorphism (SNP) position and was extended with fluorescently- labeled dideoxynucleotide triphosphates. The SNaPshot (Life Technologies) reaction contained 3.0 μL of purified PCR product, 1 μL of pooled detection primers [21], 1 μL water and 1 μL of SNaPshot (Life Technologies) ready reaction mixture in a final volume of 7 μL. The cycling profile was 25 cycles at 96°C for 10 seconds, 55°C for 10 seconds and 60°C for 30 seconds. After the reaction, 5-phosphoryl groups of unincorporated dideoxynucleotide triphosphates were removed by addition of SAP (shrimp alkaline phosphatase) (USB, Affymetrix Inc.) for 1 hour at 37°C, followed by enzyme inactivation at 86°C for 20 min. Capillary electrophoresis of samples was performed on the ABI PRISM® 3130 genetic analyzer with LIZ120 (Life Technologies) as a size standard. The obtained results were analyzed with GeneScan 4.0 software (Life Technologies). Two additional long range-PCR reactions were used to analyze the major rearrangements, i.e., duplication or deletion of the entire CYP2D6 gene [21]. Both deletion and duplication PCR reactions were performed in a reaction volume of 12 μL containing 1U enzyme mix from the Expand Long Template PCR System (Roche Diagnostics), Long Template Buffer 1, 1 × concentrated, with 17.5 mM MgCl2, and 0.2 mM each dideoxynucleotide triphosphate. The primer concentrations were as follows: for the duplication-specific reaction, 0.3 μM CYP-207-F, 0.2 μM CYP-32-R, and 0.1 μM CYP-13-F; and for the deletion-specific reaction, 0.3 μM CYP-13-F, 0.2 μM CYP-24-R, and 0.1 μM CYP-207-F. The cycling profile was as described above. The primers CYP-13-F and CYP-24-R generated a deletion-specific fragment of 3.5 kb, while primers CYP-24-R and CYP-207-F yielded a control fragment of 3.0 kb. With the deletion-specific PCR we analyzed 94 homozygous samples. We analyzed only these samples because they had only one peak for each of the 11 analyzed polymorphic sites and we could not determine the difference between a homozygous set of polymorphic sites or a deleted allele (Figure 1). In comparison, the heterozygous patients had two peaks on at least one polymorphic site, indicating the presence of two alleles. We also analyzed 60 heterozygous samples in order to confirm our hypothesis. We looked for CYP2D6 gene duplications using the CYP-207-F and CYP-32-R primer pair. With these primers, we amplified a duplication-specific fragment of 3.2 kb. Simultaneously, a control fragment of 3.8 kb was amplified with the CYP-13-F forward primer. Apart from the duplication-specific PCR, we also confirmed the detected duplications with a comparison of the ratios of the two peaks that appeared at the same position for each separate polymorphic site. By comparing two electropherograms obtained from the SNaPshot (Life Technologies) analysis, we noticed that the duplicated allele could readily be identified as the one of interest, as it displayed higher signals in the polymorphic heterozygous sites (Figure 2A and 2B) [21]. The χ2 test and Fisher’s exact test were used to compare frequencies between populations. GraphPad InStat software (version 3.1; http://www.graphpad. com) was used for statistical analysis. A p value of 0.05 was considered significant.



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