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ABSTRACT. Inter-individual variability in drug metabolism may result in adverse drug responses. Pharmacogenetic studies have shown that polymorphisms in drug metabolizing enzymes may contribute to this variability. Among these enzymes, CYP3A4 is responsible for metabolizing over 50% of the clinically used drugs. The Brazilian population is composed of people with Native American, European, and African ancestries, and is therefore considered as one of the most intermixed populations in the world. A thorough knowledge of the genetic frequencies of CYP3A4 allelic variants is useful for the establishment of better pharmacological therapies; therefore, the aim of this study was to describe the polymorphic frequencies for CYP3A4 -392A>G (rs2740574) in a sample population from Maranhão, Brazil. Our results showed that 75.1, 21.9, and 3.0% of the individuals expressed the -392AA, -392AG, and -392GG genotypes, respectively. The -392A and -392G alleles were observed in 86.1 and 13.9% of the population, respectively. Our results reiterate the need for a better understanding of the variations in the genotype and allele frequencies of CYP3A4 -392A>G polymorphisms in various Brazilian regions, in order to elucidate the variability in drug response.

Os objetivos deste trabalho foram adequar a técnica de PCR para o polimorfismo CYP3A4*1B, reduzir as amplificações inespecíficas observadas e verificar a frequência alélica do polimorfismo na população de São Luís/MA. A reação de PCR foi padronizada para conter: aproximadamente 50 ng de DNA; 10 μL de DNA polimerase mix (1 Unidade de enzima DNA polimerase a ser testada; 10mM de Tris-HCl, pH 8,3; 50mM de KCl; 2,5mM de MgCl2; 200 μM de cada desoxinucleotídeo trifosfatado - dNTP's); 10 pmol de cada iniciador e água livre de DNAse e RNAse (Prodimol) para completar o volume reacional. Optou-se por fazer a reação de PCR para um volume final de 25μL. O primeiro teste foi realizado com Platinum Taq DNA Polimerase (Invitrogen Life Technologies). Depois utilizou-se as DNA polimerases Hot Star Taq DNA Polimerase (Qiagen) e Ampli Taq Gold (Invitrogen Life Technologies) e DNA Polimerase Master Mix Red (Amplicon - Neobio). Os resultados demonstraram que a PCR para o alelo CYP3A4*1B gera bandas inespecíficas que foram minimizadas com a utilização Hot Star Taq DNA Polimerase (Qiagen). Em relação ao polimorfismo a quantidade de portadores homozigotos foi de 0,33%, a porcentagem de heterozigotos foi de 21,67% e 75% dos indivíduos foram não portadores de polimorfismo em 60 pacientes estudados. Em conclusão, este trabalho descreve a optimização e validade da análise PCR-RFLP rápida e simples, para o alelo CYP3A4*1B. Além disso, observou-se uma frequência significativa de polimorfismo para o alelo CYP3A*1B na população estudada.Palavras-chave: Polimorfismo. CYP3A4. Citocromo P450. PCR.OPTIMIZATION OF PCR TECHNIQUE FOR DETECTION OF CYP3A4 * 1B POLYMORPHISM AND ALLELE FREQUENCY AMONG 60 RESIDENTS OF SÃO LUIS / MAAbstract: The objective of this study was to adapt the PCR for CYP3A4*1B polymorphism, reduce nonspecific amplifications observed and verify the allele frequency of the polymorphism in the population of São Luis / MA. The PCR reaction was standardized to contain: approximately 50 ng of DNA, 10 uL of DNA polymerase mix (1 unit DNA polymerase being tested; 10mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 mM of each deoxynucleotide trifosfatado - dNTP's), 10 pmol of each primer and water-free DNAse and RNAse (Prodimol) to complete the reaction volume. We chose to do the PCR reaction to a final volume of 25μL. The first test was performed with Platinum Taq DNA Polymerase (Invitrogen Life Technologies). Then we used the DNA polymerase Hot Star Taq DNA Polymerase (Qiagen) and Ampli Taq Gold (Invitrogen Life Technologies) and DNA Polymerase Mix Master Red (Amplicon - Neobio). The results demonstrated that PCR allele CYP3A4 * 1B generates nonspecific bands that were minimized using Hot Star Taq DNA Polymerase (Qiagen). In the polymorphism the amount of homozygous was 0.33%, the percentage of heterozygotes was 21.67% and 75% of subjects were not carriers of polymorphism in 60 patients. In conclusion, this study described the optimization and validity of PCR-RFLP analysis fast and simple, for the CYP3A4 * 1B allele. Furthermore, there was a significant frequency of polymorphism allele * 1B CYP3A in the population.Keywords: polymorphism, CYP3A4, cytochrome P450, PCR.OPTIMIZACIÓN DE LA PCR PARA LA DETECCIÓN DEL POLIMORFISMO CYP3A4 * 1B Y FRECUENCIA DE LOS ALELOS ENTRE 60 RESIDENTES DE SÃO LUIS / MAResumen: El objetivo de este estudio fue adaptar la PCR para el polimorfismo CYP3A4*1B, reducir amplificaciones inespecíficas observadas y verificar la frecuencia de los alelos del polimorfismo en la población de San Luis / MA. La reacción de PCR fue estandarizado que contiene: aproximadamente 50 ng de DNA, 10 uL de la mezcla de ADN polimerasa (1 unidad de polimerasa de ADN se está probando; 10 mM Tris-HCl, pH 8,3, 50 mM KCl, 2,5 mM MgCl2, 200 mM de cada desoxinucleótido trifosfatado - dNTP), 10pmol de cada cebador y agua libre de DNAsa y RNAsa (Prodimol) para completar el volumen de reacción. Elegimos a hacer la reacción de PCR a un volumen final de 25 ul. La primera prueba se realizó con Hot Star Taq DNA Polymerase (Qiagen). Luego se utilizó la ADN polimerasa Hot Star Taq DNA Polymerase (Qiagen) y Ampli Taq Gold (Invitrogen Life Technologies) y DNA Polymerase Mix Master Red (Amplicon - Neobio). Los verifiresultados demostraron que el alelo CYP3A4*1B PCR genera bandas no específicas que fueron minimizadas utilizando Hot Star Taq DNA Polymerase (Qiagen).. En el polimorfismo de la cantidad de homocigotos fue de 0,33%, el porcentaje de heterocigotos fue 21,67% y el 75% de los sujetos no eran portadores del polimorfismo en 60 pacientes. En conclusión, este estudio se describe la optimización y la validez de la PCR-RFLP análisis rápido y sencillo, para el alelo CYP3A4 * 1B. Además, hubo una frecuencia significativa de CYP3A polimorfismo alelo * 1B en la población.Palabras clave: Polimorfismo CYP3A4. Citocromo P450. PCR.