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
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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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