Leonardo Beltrán

Phenotype-genotype analysis of CYP2C19 in Colombian mestizo individuals

BackgroundOmeprazole is metabolized by the hepatic cytochrome P450 (CYP) 2C19 enzyme to 5-hydroxyomeprazole. CYP2C19 exhibits genetic polymorphisms responsible for the presence of poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers (EMs). The defective mutations of the enzyme and their frequencies change between different ethnic groups; however, the polymorphism of the CYP2C19 gene has not been studied in Colombian mestizos. The aim of this study was to evaluate the genotype and phenotype status of CYP2C19 in Colombian mestizos, in order to contribute to the use of appropriate strategies of drug therapy for this population.Methods189 subjects were genotyped using the multiplex SNaPshot technique and a subgroup of 44 individuals received 20 mg of omeprazole followed by blood collection at 3 hours to determine the omeprazole hydroxylation index by HPLC.Results83.6%, 15.3% and 1.1% of the subjects were genotyped as EMs, IMs and PMs, respectively. The frequencies of the CYP2C29*1 and CYP2C19*2 alleles were 91.3% and 8.7% respectively whereas the *3, *4, *5, *6 and *8 alleles were not found. No discrepancies were found between the genotype and phenotype of CYP2C19.ConclusionThe frequency of poor metabolizers (1.1%) in the Colombian mestizos included in this study is similar to that in Bolivian mestizos (1%) but lower than in Mexican-Americans (3.2%), West Mexicans (6%), Caucasians (5%) and African Americans (5.4%). The results of this study will be useful for drug dosage recommendations in Colombian mestizos.

β2‐adrenoreceptor polymorphisms in asthmatic and non‐asthmatic schoolchildren from colombia and their relationship to treatment response

Asthma is a chronic and recurrent disease. Its high prevalence around the world is the result of a complex interaction between genetic and environmental factors. The genetic aspects of susceptibility, severity, and response to treatment in asthma are of great scientific interest. The purpose of the study was to establish the relationship between the Gln27Glu and Arg16Gly alleles of the β2‐adrenergic receptor (ADRB2) gene with respect to the susceptibility to and severity of asthma, as well as the response to treatment in mestizo schoolchildren. 109 schoolchildren with asthma diagnosis and 137 asymptomatic controls were genotyped for the Arg16Gly and Gln27Glu alleles of the ADRB2 gene by minisequencing. Allele, genotype, and haplotype frequencies of the ADRB2 gene between asthmatic and non‐asthmatic as well as demographic, clinical, and spirometric variables among asthmatic patients according to their genotype were compared. ADRB2 gene expression was determined by real‐time quantitative PCR. No statistical differences were found in allele, genotype, and haplotype frequencies of the ADRB2 gene between cases and controls. We did not find differences between asthmatic patients classified according to their ADRB2 genotypes and haplotypes when evaluating demographic, clinical, and spirometric variables. The ADRB2 genotype and haplotype are not associated with spirometric responses or ADRB2 gene expression after administration of a β‐2 agonist plus a glucocorticoid. These results suggest that in the group of mestizo schoolchildren studied, the Arg16Gly and Gln27Glu polymorphisms are not markers of susceptibility or severity of asthma and do not affect ADRB2 gene expression during the rescue therapy. Pediatr Pulmonol. 2012. 47:848–855.

Evaluación del programa de mantenimiento con metadona del Hospital Mental de Risaralda

BACKGROUND Psychosocial care and methadone maintenance treatment (MMT) are the preferred strategies for the management of heroin addicts, but the results are still unsatisfactory, justifying the search and intervention of the factors influencing the response to treatment. METHODOLOGY In order to determine the contribution of demographic, clinical and genetic variables on serum concentrations and response to methadone, we investigated patients on MMT, who were receiving methadone in supervised and unchanged doses at least during the previous two weeks. The age, gender, body mass index (BMI), duration of heroin abuse, addiction to other drugs, criminal background, current daily methadone doses, time spent in the TMM, comorbidity and concomitant medication were recorded. Blood samples were taken for the determination of serum levels of racemic methadone and its R and S-enantiomers, and for typing of candidate alleles of POR, CYP2B6, ABCB1, GRIN1, OPRM1, SLC6A3, DßH and ARRB2 genes, all associated with the metabolism, tissue distribution and mechanism of action of methadone. Methadone quantification was by HPLC-DAD, and the detection of genetic markers by Real Time PCR and VNTR methods. RESULTS A total of 80 subject volunteers were enrolled, with a mean age of 23.5 (5) years (86% male), all of them were addicts of multiple drugs, 60% with a criminal background, 5.1 (2.9) years taking heroin, and 5.3 (4) months on MMT, and taking a supervised dose of 41 (12) mg/day methadone. The (R), (S) and (R, S) methadone enantiomer trough plasma levels were, 84 (40), 84 (42), and 168 (77) ng/mL, respectively. All genotypes were in Hardy-Weinberg equilibrium. The two urine tests were negative for heroin in 61.3% (49/80) of the volunteers, the decline in cocaine/crack use was 83%, 30% of marijuana, and other psychoactives (inhalants, benzodiazepines, amphetamines) decreased to zero, while the consumption of snuff remained at 93.5% (75/80). Blood concentrations of racemic methadone and its enantiomers were significantly associated with the dose/day of the medication, but none of the other demographic, clinical or genetic variables impacted on serum levels of methadone. As for the results of the MMT, non-users and occasional users of heroin, as well as those who stopped taking other psychoactive drugs, and the ones who did not, were similar as regards the demographic, genetic and clinical variables. This included the blood metahdone concentrations, except for individuals who did not reduce their consumption of other psychoactives other than heroin, who had significantly (P=.03) higher blood levels of S-methadone, compared with those who did stop taking them. CONCLUSIONS There was a significant reduction in the consumption of heroin and other psychoactives, and social rehabilitation of patients. However, the extensive overlap between effective and ineffective doses of methadone suggests the presence of personal and social variables that transcend the simple pharmacological management. These probably need to be addressed more successfully from the psychosocial features, particularly as regards to identifying and overcoming relapse-trigger experiences, as well as certain features of the patient, such as their psychological distress level or their psychiatric disorders.

Detección del virus del papiloma humano (VPH) en pacientes con citología anormal: un estudio preliminar

El cancer de cuello uterino es una de las formas mas comunes de cancer en mujeres a nivel mundial y su desarrollo es un proceso multifactorial; sin embargo, los virus del papiloma humano (VPH) de alto riesgo se han asociado con su etiologia. El objetivo del presente trabajo fue determinar la prevalencia de la infeccion con VPH en pacientes con citologia anormal que consultaron para colposcopia en la unidad intermedia de Kennedy de la ciudad de Pereira. Se estudiaron 129 pacientes en el periodo Mayo-Noviembre de 2005 a las cuales se les tomo citologia con citocepillo y se realizo extraccion de ADN con el material obtenido. Se obtuvo ADN en las muestras de 123 pacientes, procediendose entonces a realizar la deteccion del ADN viral mediante PCR con los primers GP5+/GP6+ y con la PCR anidada que emplea los primers MY09/11 y GP5+/GP6+. Bajo nuestras condiciones experimentales, la PCR anidada detecto un mayor numero de infecciones por VPH que la PCR convencional. La prevalencia de la infeccion segun diagnostico citologico fue de 59.2% para ASCUS, 66.1% para LBG y 87.5% para LAG. La prevalencia global de la infeccion fue de 63.4%, con predominio significante en mujeres con edades entre 15 y 34 anos (p menor 0.05). En conclusion, los resultados muestran una prevalencia de infeccion por VPH similar a la observada en otros paises en pacientes con citologia anormal. Este es el primer estudio de su tipo realizado en Pereira y el eje cafetero.

Artículo originalEvaluación del programa de mantenimiento con metadona del Hospital Mental de RisaraldaEvaluation of the Methadone Maintenance Program of the Risaralda Mental Hospital

BACKGROUND Psychosocial care and methadone maintenance treatment (MMT) are the preferred strategies for the management of heroin addicts, but the results are still unsatisfactory, justifying the search and intervention of the factors influencing the response to treatment. METHODOLOGY In order to determine the contribution of demographic, clinical and genetic variables on serum concentrations and response to methadone, we investigated patients on MMT, who were receiving methadone in supervised and unchanged doses at least during the previous two weeks. The age, gender, body mass index (BMI), duration of heroin abuse, addiction to other drugs, criminal background, current daily methadone doses, time spent in the TMM, comorbidity and concomitant medication were recorded. Blood samples were taken for the determination of serum levels of racemic methadone and its R and S-enantiomers, and for typing of candidate alleles of POR, CYP2B6, ABCB1, GRIN1, OPRM1, SLC6A3, DßH and ARRB2 genes, all associated with the metabolism, tissue distribution and mechanism of action of methadone. Methadone quantification was by HPLC-DAD, and the detection of genetic markers by Real Time PCR and VNTR methods. RESULTS A total of 80 subject volunteers were enrolled, with a mean age of 23.5 (5) years (86% male), all of them were addicts of multiple drugs, 60% with a criminal background, 5.1 (2.9) years taking heroin, and 5.3 (4) months on MMT, and taking a supervised dose of 41 (12) mg/day methadone. The (R), (S) and (R, S) methadone enantiomer trough plasma levels were, 84 (40), 84 (42), and 168 (77) ng/mL, respectively. All genotypes were in Hardy-Weinberg equilibrium. The two urine tests were negative for heroin in 61.3% (49/80) of the volunteers, the decline in cocaine/crack use was 83%, 30% of marijuana, and other psychoactives (inhalants, benzodiazepines, amphetamines) decreased to zero, while the consumption of snuff remained at 93.5% (75/80). Blood concentrations of racemic methadone and its enantiomers were significantly associated with the dose/day of the medication, but none of the other demographic, clinical or genetic variables impacted on serum levels of methadone. As for the results of the MMT, non-users and occasional users of heroin, as well as those who stopped taking other psychoactive drugs, and the ones who did not, were similar as regards the demographic, genetic and clinical variables. This included the blood metahdone concentrations, except for individuals who did not reduce their consumption of other psychoactives other than heroin, who had significantly (P=.03) higher blood levels of S-methadone, compared with those who did stop taking them. CONCLUSIONS There was a significant reduction in the consumption of heroin and other psychoactives, and social rehabilitation of patients. However, the extensive overlap between effective and ineffective doses of methadone suggests the presence of personal and social variables that transcend the simple pharmacological management. These probably need to be addressed more successfully from the psychosocial features, particularly as regards to identifying and overcoming relapse-trigger experiences, as well as certain features of the patient, such as their psychological distress level or their psychiatric disorders.

Caracterización genotípica y fenotípica CYP2C19 de población mestiza colombiana

La isoenzima CYP2C19, que metaboliza algunos farmacos de uso frecuente, es codificada por un gen polimorfico, algunos de cuyos alelos producen enzima con actividad catalitica defectuosa o nula. Este fenomeno es el responsable de la existencia de individuos que metabolizan los farmacos sustratos de la enzima a velocidad baja (PM, poor metabolizer), intermedia (IM, intermediate metabolizer) o alta (EM, extensive metabolizer). Las mutaciones defectuosas de la enzima y las frecuencias con que ellas se presentan varian entre los diferentes grupos etnicos; sin embargo, el polimorfismo del gen CYP2C19 no ha sido estudiado en mestizo suramericano, de modo que desconocemos no solo las mutaciones prevalentes del gen, sino los porcentajes de personas de este grupo etnico cuyo metabolismo es lento, intermedio o rapido y, por lo mismo, carecemos de informacion acerca de las dosis de los farmacos sustratos de la CYP2C19 que mas se ajustan al perfil farmacogenetico del mestizo suramericano. En este estudio se determinaron las frecuencias del alelo nativo CYP2C19*1 y de las mutaciones *2, *3, *4, *5, *6 y *8 en una muestra de 189 adultos colombianos de rasgos fenotipicos mestizos, de ambos sexos, no consanguineos y clinicamente sanos y se confirmaron los fenotipos EM, IM y PM para la enzima CYP2C19 en un subgrupo de 44 personasseleccionadas entre las previamente genotipificadas. Para el estudio de los diferentes alelos se empleo la tecnica de mini-secuenciacion con ABI Prism SNaPshot Multiplex System; la fenotipificacion se realizo por HPLC, usando omeprazol como farmaco de prueba de actividad de la enzima in vivo. Se encontro 83,6% de portadores de los dos alelos nativos (fenotipo EM, metabolizador rapido), 15,3% de heterocigotos para un alelo no funcional (fenotipo IM, metabolizador intermedio) y dos personas (1,1%) portadoras de los dos alelos no funcionales (fenotipo PM, metabolizador pobre). El equilibrio de Hardy-Weinberg se confirmo para la distribucion genotipica CYP2C19. La variante *2 fue la unica identificada entre las mutadas, pues los alelos *3, *4, *5, *6 y *8 no se hallaron en el grupo de estudio. No se encontraron discrepancias entre el genotipo y el fenotipo CYP2C19. La frecuencia de individuos PM entre los mestizos colombianos incluidos en este estudio es similar a la reportada entre mestizos olivianos (1%), pero menor que la reportada entre mexicano-americanos (3,2%), caucasicos (5%) y afroamericanos (5,4%), lo cual permite suponer que tambien existan ligeras diferencias en las respuestas farmacogeneticas relacionadas con el metabolismo de los medicamentos que son sustratos de la enzima CYP2C19.