Corresponding author: Angel T. Alvarado ( eaa.alvarado@hotmail.com ) Academic editor: Georgi Momekov
© 2021 Angel T. Alvarado, Roberto Ybañez-Julca, Ana María Muñoz, César Tejada-Bechi, Roberto Cerro, Luis Abel Quiñones, Nelson Varela, César André Alvarado, Erick Alvarado, María R. Bendezú, Jorge A. García.
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Citation:
Alvarado AT, Ybañez-Julca R, Muñoz AM, Tejada-Bechi C, Cerro R, Quiñones LA, Varela N, Alvarado CA, Alvarado E, Bendezú MR, García JA (2021) Frequency of CYP2D6*3 and *4 and metabolizer phenotypes in three mestizo Peruvian populations. Pharmacia 68(4): 891-898. https://doi.org/10.3897/pharmacia.68.e75165
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Wild type genotypes (CYP2D6) and their allelic variants have been described in a sample of a Peruvian mestizo population. The global allele frequency was 0.015 for CYP2D6*3 and 0.051 for CYP2D6*4. The percentages of genotypes described were 97% CYP2D6*1/*1 and 3.0% CYP2D6*1/*3; 90.60% for CYP2D6*1/*1, 8.55% CYP2D6*1/*4 and 0.85% CYP2D6*4/*4. The allelic frequencies of CYP2D6*3 in the Lima subpopulations were 0.022 and 0.010 for Junin; CYP2D6*4 of 0.048, 0.060, and 0.050 for residents of Lima, Junín, and Tacna, respectively. The Hardy-Weinberg equilibrium test for the studied population showed that both frequencies are in equilibrium, p <.05. The metabolizer phenotype was inferred according to the genotypes: 11.54% were classified as intermediate metabolizers (*1/*3 or *1/*4) and 0.85% as poor metabolizers (*4/*4). It is concluded that the frequencies of the CYP2D6*3 and CYP2D6*4 alleles are low for the Peruvian mestizo population compared to the Latin American and tricontinental population, due to their natural population evolution, which is manifested by their decreased metabolic activity, the same that is relevant in clinical practice.
Genetic polymorphism, Pharmacogenetics, Metabolic phenotype, Peruvian mestizo population
Genetics is one of the main factors that determine the inter-ethnic variability in the pharmacological response, and this is explained by the differences in the population frequencies of genetic polymorphisms, which have clinical implications, therefore, the doses of a drug for a certain population, are not necessarily applicable to other populations or ethnic groups (Bertilsson, 1995); this polymorphism lies in the genes that encode various enzymes and among them, the family of the cytochrome P450 system, being the isoenzyme CYP2D6 (Debrisoquine 4-hydroxylase), the most representative in liver tissue (6% of all CYP450: CYP3A4, CYP2C9, and others) (
The CYP2D locus is made up of a gene cluster, a CYP2D6 gene, and two pseudogenes (CYP2D7 and CYP2D8), with 97% and 92% similarity, respectively (
The frequencies of CYP2D6*3 and *4 are high in Caucasian European and North American populations, very low in African populations, and in some regions of Asia, such as China and Japan (
CYP2D6*5 is the product of a deletion of the complete gene, CYP2D6*6 by deletion of thymine at position 1707 (1707delT) encoding a protein with null activity (
After conducting a review in the PubMed-NCBI database on the studies of CYP2D6 in Peruvian populations, it is evident that they are still scarce, being necessary to study them, due to their European migratory origin (
Our objective was to identify the frequencies of the CYP2D6*3 (rs35742686) and CYP2D6*4 (rs3892097) variants to catalog the metabolic phenotypes of drugs in a sample of a Peruvian mestizo population, given their phenotypic importance as well as their high population and interethnic variability.
Observational, descriptive, cross-sectional study, non-probabilistic convenience sampling and prospective recruitment between January 2017 and December 2020. The Peruvian inhabitants were summoned and informed about the objectives and importance of the study, after that, only those who signed the informed consent freely and voluntarily, were enrolled in the present investigation (
To include mestizo Peruvians in the present study, reports of the migratory pattern, native genes, and non-molecular markers of miscegenation (linguistic tree and surnames) were considered (
All of this allowed us to select mestizo Peruvians over 18 years of age and of coastal and Andean origin; at the medical examination, be in good health (systolic blood pressure of 110–139 mm Hg and diastolic of 60–89 mm Hg, abdominal circumference less than 95 cm in men and 82 cm in women and not having a diagnosis of diabetes); and by an explicit declaration of each volunteer not to consume alcoholic beverages or drugs of abuse, not to have a difficulty that prevents the taking of the biological sample and to give their consent in writing. All subjects, who belonged to an ethnic group, were not in a position to give their consent, and those who did not meet the inclusion criteria were excluded from the study (
Genomic DNA (gDNA) was obtained by buccal swabbing of non-keratinized stratified flat epithelial tissue, rubbing the inner cheek mucosa five to six times with the swab to ensure an adequate amount of scaly cells. Subsequently, the swab was immersed for 60s in 300 µL of lysis buffer and the resulting mixture was refrigerated at 4°C for a time not exceeding 18 hours. The DNAg was extracted using the innuPRE DNA Master kit (Analytik Jena), following the manufacturer’s protocol, a procedure performed at the USIL Pharmacology Laboratory. Genomic DNA was quantified by spectrophotometry using Denovix equipment (model DS-11, FX, Spectrophotometer Series, USA). Samples with absorbance ratios of 260/280 nm and 260/230 nm equal to or greater than 1.8 were considered suitable for the study. The samples were stored at -20°C until analysis.
The genotypes were determined using the real-time PCR technique (RT-PCR), using Buffer TE 1X reagents, for the identification of allelic variants, TaqMan probes capable of discriminating single nucleotide polymorphisms (SNPs) identified as CYP2D6*3 (rs35742686) and CYP2D6*4 (rs3892097) (TaqMan Genotyping Master Mix, catalog number 4371355-brand Thermo Fischer Scientific Inc.). The CYP2D6*1 allele was not directly determined; All alleles that were negative for SNPs CYP2D6*3 and CYP2D6*4 were designated as CYP2D6*1, which is acceptable when the sequence variations corresponding to alleles are not determined, so it is not wrong to assume that No *X = *1.
In the standard protocol, the reaction mix was 20 ng gDNA, 5µL of 2X Genotyping Master Mix (Catalog No 4371355), 0.5 µL of 20X TaqMan SNP Genotyping AssayTM (containing the two probes and primers direct and reverse) and nuclease-free molecular biology grade water (HyPure HyCloneTM) in sufficient quantity for 10 µL of final reaction volume.
To determine CYP2D6*3, the TaqMan SNP Genotyping Assay catalog number 4362691 was used; C_32407232_50, which discriminates the g.2549delA deletion; and for CYP2D6*4 the catalog number 4362691; C_27102431_D0, which discriminates the transition g.1846G > A.
For the amplification, the Stratagene Mx3000P equipment (Agilent Technologies, Waldbronn, Germany) was used, whose program consisted of an initial cycle of denaturation at 95°C for 10 min, and the second segment consisted of 50 cycles consisting of 15s of denaturation at 92°C, 90s of alignment at 60°C and 60s of elongation at 72°C.
To determine if the distribution of the studied genotypes was in Hardy-Weinberg equilibrium (HWE), the Chi-square (X2) goodness-of-fit test was performed, considering a degree of freedom and a p-value <.05. X2 values less than 3.88 in the comparison indicated acceptance of the null hypothesis, therefore, the observed frequencies did not differ significantly from those expected (
The study was carried out in strict compliance with good clinical practices (BCP), Code of Ethics of the World Medical Association (Declaration of Helsinki), the research protocol, and informed consent being approved by the Ethics Committee of Hospital Santa Rosa, by certificate No 16-19-CMI-HSR. Each volunteer was assigned a code number to guarantee their anonymity and confidentiality (
Table
Genotype frequencies for alleles *3 and *4 of the CYP2D6 gene in a sample of a Peruvian mestizo population.
Reference ID | Genotype | Nucleotide | n | % | Allele | f | X2 |
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rs35742686 | *1/*1 | A/A | 227 | 97.0 | *1 | 0.985 | |
*1/*3 | A/del | 7 | 3.0 | *3 | 0.015 | 0.054 | |
*3/*3 | del/del | 0 | 0.0 | ||||
Total | 234 | 100.0 | |||||
rs3892097 | *1/*1 | G/G | 212 | 90.60 | *1 | 0.949 | |
*1/*4 | G/A | 20 | 8.55 | *4 | 0.051 | 3.46 | |
*4/*4 | A/A | 2 | 0.85 | ||||
Total | 234 | 100.0 |
Next, the genotype and allelic frequencies of the sub-samples corresponding to inhabitants of the coast and the Andes of Peru are exposed, which are low. For Lima residents, the frequency of CYP2D6*1/*3 and *3/*3 is 0.022, and for Junín it is 0.01, but they were not observed in Tacna residents; while the frequency of CYP2D6*1/*4 and *4/*4 was similar in the three provinces (Table
Genotypic and allelic frequencies for the variants *3 and *4 of the CYP2D6 gene in the three Peruvian mestizo populations included in the present study.
Genotype | n (%) | Allelic frequency | ||||
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Lima | Tacna | Junín | Lima | Tacna | Junin | |
CYP2D6*1/*1 A/A | 128 (95.5) | 50 (100.0) | 49(98.0) | 0.978 | 1.0 | 0.99 |
CYP2D6*1/*3 A/del | 6 (4.5) | 0 (0.0) | 1 (2.0) | 0.022 | 0.0 | 0.01 |
CYP2D6*3/*3 del/del | 0 (0.0) | 0 (0.0) | 0 (0.0) | |||
Total | 134 (100.0) | 50 (100.0) | 50(100.0) | 1.00 | 1.00 | 1.00 |
CYP2D6*1/*1 | 121 (90.3) | 45 (90.0) | 44 (88.0) | 0.952 | 0.95 | 0.94 |
CYP2D6*1/*4 | 13 (9.7) | 5 (10.0) | 4 (8.0) | 0.048 | 0.05 | 0.06 |
CYP2D6*4/*4 | 0 (0.0) | 0 (0.0) | 2 (4.0) | |||
Total | 134 (100.0) | 50 (100.0) | 50 (100.0) | 1.00 | 1.00 | 1.00 |
Table
Extrapolated metabolic phenotypes in the Peruvian mestizo population, according to the combinations of CYP2D6*3 and *4.
Extrapolated phenotypes | CYP2D6*3 | CYP2D6*4 | Activity | n | % | Genotypes |
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Extensive metabolizer (gEM) | *1/*1 | *1/*1 | 2 | 205 | 87.61 | *1/*1 |
Intermediate metabolizer (gIM) | *1/*3 | *1/*1 | 0.5–1.5 | 7 | 11.54 | *1/*3 |
*1/*1 | 1*/*4 | 20 | *1/*4 | |||
Poor metabolizer (gPM) | *1/*1 | *4/*4 | 0 | 2 | 0.85 | *4/*4 |
*3/*3 | *1/*1 | 0 | *3/*3 |
Figure
Table
Studies of CYP2D6*3 and *4 in Latin American and tricontinental populations were used in the comparison with the Peruvian population studied.
Population | CYP2D6*3%(n) | CYP2D6*4%(n) | Reference |
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Mestizo Peruvian | 3.0(234) | 9.4 (234) | Present study |
Costa Ricans | 1.4(139) | 10.4(130) |
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Colombians | 1.2(121) | 10.4(121) |
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Brazilians | 3.4(89) | 17.8(89) |
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Chileans | 1.0(253) | 12.0(253) |
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Ecuadorians | 0.4 | 10.6(118) |
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Africans | 0 | 6.0 |
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East Asians | 0 | 0.0 |
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South Asians | 0 | 11.0 |
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European | 2 | 19.0 |
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Spanish people | 1(105) | 13.8(105) |
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In this study, the CYP2D6*3 and CYP2D6*4 alleles were characterized in samples of 234 mestizo inhabitants of the coastal zone (Lima and Tacna) and the Andean Province (Junín) of Peru. Observing 3% of the CYP2D6*3 genotype and 9.4% of CYP2D6*4; with a frequency of the allele CYP2D6*3 and CYP2D6*4 of 0.015 and 0051, respectively. In previous Latin American studies on CYP2D6*3 it has been reported that this genotype is rare and varies from 0.4% to 2.0%. In Europeans, it is 2% (
Regarding the extrapolated phenotypes, it is observed that 11.54% are genotypic intermediate metabolizers (gIM) with mutated heterozygous genotypes *1/*3 and *1/*4; while 0.85% are genotypic poor metabolizers (gPM) with a homozygous mutated genotype *4/*4, with an activity of 0.5–1.5 and 0, respectively (
These results are relevant in clinical practice for this group of the mestizo population, as they are susceptible to presenting side effects and even adverse reactions, since the drug not metabolized by the CYP2D6 protein, exceeds the maximum effective plasma concentration (CME), being necessary to adjust dose based on phenotype and follow pharmacotherapy.
The limitations of the present study are in the sample size and in the method of selection for convenience. Another bias, which can lead to confusion, is not having studied the other allelic variants of CYP2D6, which are being considered in future studies by our research network. In our country, pharmacogenetic studies are still limited, although there is evidence in other Latin American countries that shows that they influence the safety and efficacy of drugs. Notwithstanding the foregoing, the results presented in this study are relevant, as they contribute to generating scientific evidence and encourage clinical studies in precision medicine, especially in patients who are treated with drugs with a narrow therapeutic margin such as antidepressants, analgesics. opioids, antihypertensives (metoprolol, propranolol), antiarrhythmics (propafenone and quinidine), neuroleptics, docetaxel, paclitaxel, tamoxifen, and tamsulosin (
We recommend conducting more research on these and other CYP2D6 alleles to complement the present study and promote them as biomarkers in precision medicine in underserved populations in Peru.
Our findings indicate that the average frequencies of the CYP2D6*3 and CYP2D6*4 alleles are low for the Peruvian mestizo population compared to the Latin American and tricontinental populations, due to their natural population evolution, which is manifested by their activity decreased metabolism, the same that is relevant in clinical practice, since CYP2D6 intervenes in the metabolism of a wide variety of prescription drugs.
The interpopulation variability reported in previous Latin American studies supports the need for large-scale pharmacogenetic studies in our country.
Latin American Society of Pharmacogenomics and Personalized Medicine and Latin American Network for the Implementation and Validation of Clinical Pharmacogenomics Guidelines (RELIVAF-CYTED), Madrid, Spain.