PUBLISHED VERSION

Paulo Roberto Barbato, Marco Aurélio Peres, Doroteia Aparecida Höfelmann, Karen Glazer Peres Contextual and individual indicators associated with the presence of teeth in adults Revista de Saude Publica, 2015; 49(0):27-1-27-10 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Originally published at: http://doi.org/10.1590/S0034-8910.2015049005535


INTRODUCTION
Cytochrome P450 2D6 (CYP2D6) is of great importance for the metabolism of clinically used drugs, of which it metabolizes about 20%-25% (1), including antipsychotics, antidepressants, antiarrhythmics, β blockers, etc. (2,3). Recently we have shown that the number of CYP2D6 active genes is related to the dose-corrected plasma concentration of the widely used drugs thioridazine (4), risperidone (5), and fluoxetine (6) and to the QTc interval, and thus, potentially to the risk of cardiotoxicity in patients (4,7).
CYP2D6 is a highly polymorphic gene locus with more than 50 variant alleles that lead to a widely ranging enzymatic activity (www.imm.ki.se/CYPalleles/cyp2d6.htm). The polymorphism of the enzyme results in poor metabolizers (PM), efficient metabolizers (EM), or ultrarapid metabolizers (UM) of CYP2D6 substrate drugs. Subjects with multiple gene copies (UMs) will metabolize drugs more rapidly, and therapeutic plasma levels will not be reached at ordinary drug dosages. Individuals lacking functional CYP2D6 genes (PMs), however, will metabolize selective CYP2D6 substrates at a slower rate (8) and may thus be more prone to adverse effects due to elevated drug plasma levels. In PMs, there may also be a reduced effectiveness of drug therapy when pro-drugs requiring activation by CYP2D6 are used.
The variant CYP2D6 alleles can be classified into categories according to whether they cause abolished, decreased, normal, or increased activity. Although more than 50 different allelic variants have been identified, analyses of CYP2D6*3, CYP2D6*4, CYP2D6*4 duplication, CYP2D6*5, and CYP2D6*6 mutant alleles (abolished activity), and gene duplications of CYP2D6*1 and CYP2D6*2 (increased activity) have to be performed to allow a 99% sensitive prediction of PMs or UMs in clinical routine among White European and White American populations (9). The CYP2D6*10 allele is particularly common among Chinese and Japanese populations and is associated with decreased CYP2D6 activity (10). Furthermore, the presence of the CYP2D6*17 allele among African populations also shows decreased CYP2D6 activity (11). Since, with the mapping of the human genome, the concept of race has become untenable, and given the general increase in admixture of people around the world, there is a need for genotyping for the most frequently identified alleles in a given clinical situation, independent of the location of the ethnic group. In particular, the CYP2D6 allele frequency varies between populations and geographical areas (9), so that in admixed populations (e.g., in North, Central, and South America), it would be very useful to have a genotyping method that would allow the phenotype of a given patient or healthy volunteer to be predicted.
The general use of CYP2D6 genotyping may be of help to increase the use of drug therapy and, hence, of global health (12,13). However, most of the several new strategies and methods for CYP2D6 genotyping, such us single-strand conformation polymorphism (SSCP) (14,15), real-time PCR (16,17), microarrays for DNA analysis (18,19), and TaqMan ® real-time PCR (20), are expensive or labor-intensive. This situation may widen the "biotechnological gap" between developed and undeveloped countries.
There have been calls for the use of biotechnologies and pharmacogenetics for improved health in developing countries (12,13). One limitation, however, is the unaffordability of some of the current diagnostic methods. PCR is an affordable molecular diagnostic technology and is already in use in low income regions. The development of a strategy for CYP2D6 genotyping covering the most commonly described alleles could therefore promote the use of these technologies in the developing world. The aim of this study was to design a genotyping method based on extra long PCR (XL-PCR) and PCR restriction fragment-length polymorphism (PCR-RFLP) methods to allow rapid, straightforward, and inexpensive identification of 90%-95% of CYP2D6 PM or UM genotypes in a clinical routine, independent of the individual's ethnic group.

MATERIALS AND METHODS
For CYP2D6 genotyping, 10-mL blood samples were collected in EDTA tubes, and DNA was extracted using the QIAamp ® DNA blood kit (Qiagen, Hilden, Germany).

Determination of CYP2D6 Gene Duplication
To determine whether individuals were carrying duplicated CYP2D6 genes, XL-PCR was used to amplify a fragment spanning the potential crossover sites. The forward primer (2D6dupl-F) is specific for CYP2D6 3′ flanking sequences, and the reverse primer (2D6dupl-R) is specific for a CYP2D7 sequence (21). In addition, in the same PCR, the entire CYP2D6 gene was amplified by using forward (DPKup) and reverse (DPKlow) primers (22) (see Table 1). Amplification reactions of 25 μL were performed on a Mastercycler ® 384 (Eppendorf AG, Hamburg, Germany) in 0.2-mL thin-walled tubes by using 0.375 μL of an enzyme blend of Taq and Pwo DNA polymerases (Expand™ Long Template PCR System; Roche Diagnostics GmbH, Mannheim, Germany). Amplification of 50-100 ng/μL human genomic DNA was done using 2.5 μL PCR buffer 3 (2.75 mM MgCl 2 ; Expand Long Template PCR System), 0.5 mM each dNTP (Deoxynucleoside Triphosphate Set PCR Grade; Roche Diagnostics GmbH), and 0.4 μM each primer. The cycling conditions were as follows: 2 min denaturation at 94ºC, then 10 cycles of 95°C for 20 s, 68°C for 4 min, then 20 cycles of 95ºC for 20 s, 68ºC for 4 min (increasing 5 s/cycle), and then a final extension step of 7 min at 68°C. The PCR product was then analyzed directly by 0.8% agarose gel electrophoresis, and the DNA was visualized with ethidium bromide. This XL-PCR generated CYP2D6 fragments of 5.1 kb, which subsequently served for PCR-RFLP diagnostics, and 3.5 kb if the multiplication allele was present ( Table 2).

Detection of the CYP2D6*5 Allele
To determine whether individuals were carrying CYP2D6*5 allele genes, XL-PCR was performed. The 5′2D6*5 and 3′2D6*5 primers (23) are specific to identify the presence of the CYP2D6*5 allele (3.5 kb). In addition, in the same PCR, the entire CYP2D6 gene (5.1 kb) was amplified as an internal control of the reaction by using forward DPKup and reverse DPKlow primers (22) (Table1). The amplification reactions, cycling conditions, and DNA analysis were the same as used in the determination of CYP2D6 gene duplication.
Subsequently, 8 μL PCR were digested with 5-10 U restriction enzyme (New England Biolabs, Beverly, MA, USA) (see Table 2), followed by an overnight incubation to attain complete digestion. The PCR product was then analyzed directly by 3% agarose gel electrophoresis. The DNA was visualized with ethidium bromide. Table 2 summarizes all the reamplification reactions performed.

RESULTS AND DISCUSSION
The methodological procedure for the detection of CYP2D6 mutated alleles and multiplications are summarized in Figure 1. Genomic DNA is amplified in an initial XL-PCR ( Figure 1, step 1) for the determination of CYP2D6 multiplication and the 2D6*5 allele, with each reaction containing two sets of tetraprimers (Table 2). One generated a CYP2D6 fragment of 5.1 kb, which subsequently served for further PCR-RFLP diagnostics (Figure 1, steps 2-4). The other pair generated 3.5-kb fragments if the 2D6*5 deletion or the 2D6*1, *2, or *4 gene duplication was present. Then, the assays for positions 188 and 1934 were performed to determine carriers of 2D6*4 and 2D6*10 alleles (Figure 1, steps 2 and 3). Subsequently, PCR-RFLP analyses were performed to identify the 2D6*3, 2D6*6, and 2D6*17 variant alleles (Figure 1, step 4).
All DNA possessing a 2D6*1, *2, and/or *4 multiplicated allele were amplified (except DNAs homozygous for the 2D6*4 allele, because the multiplication is on this allele) producing a 10-kb fragment (Figure 1, step 5), which was amplified for further reamplifications (Figure 1, steps 6 and 7) and to identify which allele carried a duplication. PCR techniques are not capable of differentiating between a gene duplication or multiplication, and for these individuals, a Southern blot analysis needs to be performed to determine the number of CYP2D6 genes present.
While there are several strategies and methods for CYP2D6 genotyping, such us SSCP (14,15), real-time PCR (16,17), microarrays for DNA analysis (18,19), and TaqMan real-time PCR (20), they are expensive and hence unaffordable in many locations. The XL-PCR and PCR-RFLP procedures for CYP2D6 genotyping described in the present study allow the rapid, straightforward, and inexpensive identification of 90%-95% of clinically relevant CYP2D6 genotypes based on an already existing biotechnological method and are suitable for worldwide use.

Conclusions
CYP2D6 genotyping is a useful tool in clinical medicine. However one of the problems facing its routine use is its cost in terms of labor, equipment, and reagents, especially in admixed populations where a potentially large number of alleles are present. The application of the present PCR-based method to routine clinical analysis will enable PM and UM phenotypes to be predicted and identified at a reasonable cost in a large number of individuals covering all ethnic groups. Thus, it might contribute to narrowing the "biotechnological gap" between the industrial and the developing worlds.