Relevance of the ancestry for the variability of the Drug-Metabolizing Enzymes CYP2C9, CYP2C19 and CYP2D6 polymorphisms in a multiethnic Costa Rican population / Relevancia de la ancestría para la variabilidad de polimorfismos de las enzimas metabolizadoras de fármacos CYP2C9, CYP2C19 y CYP2D6 en una población multiétnica de Costa Rica

Abstract:CYP2C9, CYP2C19 and CYP2D6 metabolize around 40 % of drugs and their genes vary across populations. The Costa Rican population has a trihybrid ancestry and its key geographic location turns it into a suitable scenario to evaluate interethnic differences across populations. This study aims to describe the diversity of CYP2C9, CYP2C19 and CYP2D6 polymorphisms in Costa Rican populations in the context of their ancestry. A total of 448 healthy individuals were included in the study: Bribri (n= 47), Cabécar (n= 27), Maleku (n= 16), Guaymí (n= 30), Huetar (n= 48), Chorotega (n= 41), Admixed/Mestizos from the Central Valley/Guanacaste (n= 189), and Afro-Caribbeans (n= 50) from Limón. CYP2C9 (alleles *2, *3, *6) and CYP2C19 (*2, *3, *4, *5, *17) genotypes were determined by Real-Time PCR. African, European and Native American ancestry were inferred using 87 ancestry informative markers. The frequency of the decreased activity allele CYP2C9*2 is lower in the self-reported Amerindian groups compared to the admixed population, and the highest frequencies of CYP2C19*2 (null activity) and the CYP2C19*17 (increased activity) were found in the self-reported AfroCaribbean population. Moreover, a frequency of 0.7 % CYP2C9 gPMs in the Admixed population and a variable frequency of CYP2C19 gUMs (0.0-32.6 %, more prevalent in Afro-Caribbeans) in Costa Rican populations, was found. Finally, the following alleles were positively correlated with genomic African ancestry and negatively correlated with genomic Native American ancestry: CYP2D6*5 (null activity), CYP2D6*17 (decreased activity), CYP2D6*29 (decreased activity) and CYP2C19*17 (increased activity). No correlation for CYP2C9 polymorphisms and genomic ancestry was found. Further studies assessing the CYP2C9 and CYP2C19 sequence in these populations, preferentially by sequencing these genes, are warranted. Rev. Biol. Trop. 64 (3): 1067-1076. Epub 2016 September 01.

ResumenCYP2C9, CYP2C19 y CYP2D6 metabolizan aproximadamente el 40 % de los fármacos y los genes que las codifican varían en las distintas poblaciones humanas. La población costarricense posee ancestría trihíbrida y su posición geográfica estratégica la convierten en un escenario idóneo para evaluar la variabilidad interétnica en sus poblaciones multiétnicas. El presente estudio tiene como objetivo describir la diversidad de los polimorfismos CYP2C9, CYP2C19 y CYP2D6 en las poblaciones costarricenses en el contexto de su ancestría. Un total de 448 individuos sanos fueron incluidos: Bribri (n= 47), Cabécar (n= 27), Maleku (n= 16), Guaymí (n= 30), Huetar (n= 48), Chorotega (n= 41), mestizos del Valle Central y Guanacaste (n= 189) y afrocaribeños de Limón (n= 50). Los genotipos CYP2C9 (alelos *2, *3, *6) y CYP2C19 (*2, *3, *4, *5 y *17) fueron determinados mediante PCR tiempo real. Las ancestrías africana, europea y nativa americana fueron inferidas usando 87 marcadores informativos de ancestría. La frecuencia del alelo de actividad disminuida CYP2C9*2 fue menor en los grupos autodefinidos de amerindios que en la población mestiza y las frecuencias más altas de CYP2C19*2 (actividad nula) y CYP2C19*17 (actividad incrementada) se encontraron en la población autodefinida afrocaribeña. Asimismo, se encontró una frecuencia de gPMs CYP2C9 de 0.7 % en la población mestiza y una frecuencia variable de gUMs CYP2C19 (0.0 a 32.6 %, más prevalente en afrocaribeños) en las poblaciones costarricenses. Por último, los siguientes alelos fueron positivamente correlacionados con la ancestría africana y negativamente con la ancestría nativa americana: CYP2D6*5 (actividad nula), CYP2D6*17, CYP2D6*29 (ambos de actividad disminuida) y CYP2C19*17 (actividad incrementada). No se encontró correlación entre los polimorfismos CYP2C9 y la ancestría. Se requieren estudios posteriores que evalúen la secuencia de CYP2C9 y CYP2C19 en estas poblaciones, preferiblemente mediante la secuenciación de estos genes.

Relevance of the ancestry for the variability of the Drug-Metabolizing Enzymes CYP2C9, CYP2C19 and CYP2D6 polymorphisms in a multiethnic Costa Rican population.

CYP2C9, CYP2C19 and CYP2D6 metabolize around 40% of drugs and their genes vary across populations. The Costa Rican population has a trihybrid ancestry and its key geographic location turns it into a suitable scenario to evaluate interethnic differences across populations. This study aims to describe the diversity of CYP2C9, CYP2C19 and CYP2D6 polymorphisms in Costa Rican populations in the context of their ancestry. A total of 448 healthy individuals were included in the study: Bribri (n= 47), Cabécar (n= 27), Maleku (n= 16), Guaymí (n= 30), Huetar (n= 48), Chorotega (n= 41), Admixed/Mestizos from the Central Valley/Guanacaste (n= 189), and Afro-Caribbeans (n= 50) from Limón. CYP2C9 (alleles *2, *3, *6) and CYP2C19 (*2, *3, *4, *5, *17) genotypes were determined by Real-Time PCR. African, European and Native American ancestry were inferred using 87 ancestry informative markers. The frequency of the decreased activity allele CYP2C9*2 is lower in the self-reported Amerindian groups compared to the admixed population, and the highest frequencies of CYP2C19*2 (null activity) and the CYP2C19*17 (increased activity) were found in the self-reported Afro-Caribbean population. Moreover, a frequency of 0.7 % CYP2C9 gPMs in the Admixed population and a variable frequency of CYP2C19 gUMs (0.0-32.6 %, more prevalent in Afro-Caribbeans) in Costa Rican populations, was found. Finally, the following alleles were positively correlated with genomic African ancestry and negatively correlated with genomic Native American ancestry: CYP2D6*5 (null activity), CYP2D6*17 (decreased activity), CYP2D6*29 (decreased activity) and CYP2C19*17 (increased activity). No correlation for CYP2C9 polymorphisms and genomic ancestry was found. Further studies assessing the CYP2C9 and CYP2C19 sequence in these populations, preferentially by sequencing these genes, are warranted.

Ethnic background and CYP2D6 genetic polymorphisms in Costa Ricans / Antecedentes étnicos y polimorfismo genético del CYP2D6 en los costarricenses

CYP2D6 differences have already been demonstrated within Latin American populations by the CEIBA.FP Consortium of the Ibero-American Network of Pharmacogenetics (RIBEF, as per the acronym in Spanish). However, within the population of Costa Rica, no research has been conducted until now, even though this population has a trihybrid component ancestry that represents an interesting condition. Thus, the present study was aimed to determine the frequency of Ultra-rapid Metabolizers (UMs) and Poor Metabolizers (PMs) in a Costa Rican population, as well as to determine whether there are differences in the CYP2D6-predicted phenotype frequencies among three Costa Rican groups with different ethnic backgrounds. Additionally, these frequencies of PMs and UMs obtained were compared with Ibero-American populations published data. Finally, we also aimed to describe allele frequencies among different Costa Rican ethnic groups. This research has been undertaken within the framework of the RIBEF CEIBA Consortium studies on Latin American populations. A total of 385 individuals were included in the study: 139 mestizos, 197 Amerindians, and 49 Afro-Caribbeans. CYP2D6 genotypes were determined by XL-PCR and Real-Time PCR. The CYP2D6 variant alleles *2, *3, *4, *5, *6, *10, *17, *29, *35 and *41 were also determined. For the entire Costa Rican population, the frequency of PMs and UMs was 6% and 6.5%, respectively. The percentage of UMs in the mestizo population was higher than in the Amerindian population. CYP2D6 UMs vary from 3.6% to 10.1% and PMs from 1.4% to 10.2% among three Costa Rican groups. The highest frequencies of UMs (10.1%) and PMs (10.2%) were found in the mestizo and Amerindian populations, respectively. In conclusion, the frequencies of UMs and PMs for CYP2D6 varied widely across the mestizo, Amerindian and Afro-Caribbean Costa Rican populations. Future research in this population should be oriented to identify new CYP2D6 variants through sequencing methods, as well as to determine CYP2D6 phenotype, in order to establish the phenotype-genotype relation. Finally, further studies involving genetic markers of ancestry are needed in the Costa Rican population.

El Consorcio de la Red Iberoamericana de Farmacogenética CEIBA.FP ha demostrado que existen diferencias en cuanto a CYP2D6 en las poblaciones latinoamericanas. Sin embargo, hasta ahora, se sabe poco de este gen de importancia farmacogenética en la población de Costa Rica, la cual tiene una ancestría trihíbrida. El presente estudio tiene como objetivos: determinar la frecuencia de los fenotipos extrapolados de CYP2D6 en una población costarricense y determinar si existen diferencias en cuanto a las frecuencias de metabolizadores lentos (PMs) y ultra-rápidos (UMs) entre tres grupos con distinto origen étnico. Adicionalmente, las frecuencias de PMs y UMs obtenidas en este estudio fueron comparadas con datos de poblaciones iberoamericanas. Por último, se pretende describir las frecuencias alélicas en los distintos grupos. En el estudio se incluyeron 385 muestras de individuos: 139 mestizos, 197 amerindios y 49 afro-caribeños. Los genotipos CYP2D6 fueron determinados por XL-PCR y PCR tiempo real. Se determinaron las variantes alélicas *2, *3, *4, *5, *6, *10, *17, *29, *35 y *41. Para la población total estudiada las frecuencia de PMs y UMs fueron respectivamente 6% y 6.5%. El porcentaje de individuos UMs fue mayor en la población mestiza que en la amerindia. La frecuencia de UMs varió de 3.6 a 10.1% y la de PMs de 1.4 a 10.1% en los grupos costarricenses. Las frecuencias más altas de UMs (10.1%) y de PMs (10.2%) se encontraron respectivamente en las poblaciones mestiza y amerindia. En conclusión, las frecuencias de UMs y PMs de CYP2D6 varían ampliamente en las poblaciones mestiza, amerindia y afro-caribeña de Costa Rica. Investigaciones futuras en la población de Costa Rica deberían orientarse a identificar nuevas variantes del CYP2D6 mediante métodos de secuenciación, así como a determi- nar el fenotipo de CYP2D6 con el objetivo de establecer la relación fenotipo-genotipo. Finalmente, es necesario realizar estudios adicionales que involucren marcadores genéticos de ancestría en la población costarricense.

RELAGH - The challenge of having a scientific network in Latin America: An account from the presidents.

Latin America and the Caribbean region make up one of the largest areas of the world, and this region is characterized by a complex mixture of ethnic groups sharing Iberian languages. The area is comprised of nations and regions with different levels of social development. This region has experienced historical advances in the last decades to increase the minimal standards of quality of life; however, several factors, such as concentrated populations in large urban centers and isolated and poor communities, still have an important impact on medical services, particularly genetics services. Latin American researchers have greatly contributed to the development of human genetics and historic inter-ethnic diversity, and the multiplicity of geographic areas are unique for the study of gene-environment interactions. As a result of regional developments in the fields of human and medical genetics, the Latin American Network of Human Genetics (Red Latinoamericana de Genética Humana - RELAGH) was created in 2001 to foster the networking of national associations and societies dedicated to these scientific disciplines. RELAGH has developed important educational activities, such as the Latin American School of Human and Medical Genetics (ELAG), and has held three biannual meetings to encourage international research cooperation among the member countries and international organizations. Since its foundation, RELAGH has been admitted as a full regional member to the International Federation of Human Genetics Societies. This article describes the historical aspects, activities, developments, and challenges that are still faced by the Network.

Ethnic background and CYP2D6 genetic polymorphisms in Costa Ricans.

CYP2D6 differences have already been demonstrated within Latin American populations by the CEIBA.FP Consortium of the Ibero-American Network of Pharmacogenetics (RIBEF, as per the acronym in Spanish). However, within the population of Costa Rica, no research has been conducted until now, even though this population has a trihybrid component ancestry that represents an interesting condition. Thus, the present study was aimed to determine the frequency of Ultra-rapid Metabolizers (UMs) and Poor Metabolizers (PMs) in a Costa Rican population, as well as to determine whether there are differences in the CYP2D6-predicted phenotype frequencies among three Costa Rican groups with different ethnic backgrounds. Additionally, these frequencies of PMs and UMs obtained were compared with Ibero-American populations published data. Finally, we also aimed to describe allele frequencies among different Costa Rican ethnic groups. This research has been undertaken within the framework of the RIBEF CEIBA Consortium studies on Latin American populations. A total of 385 individuals were included in the study: 139 mestizos, 197 Amerindians, and 49 Afro-Caribbeans. CYP2D6 genotypes were determined by XL-PCR and Real-Time PCR. The CYP2D6 variant alleles *2, *3, *4, *5, *6, *10, "17, *29, *35 and *41 were also determined. For the entire Costa Rican population, the frequency of PMs and UMs was 6% and 6.5%, respectively. The percentage of UMs in the mestizo population was higher than in the Amerindian population. CYP2D6 UMs vary from 3.6% to 10.1% and PMs from 1.4% to 10.2% among three Costa Rican groups. The highest frequencies of UMs (10.1%) and PMs (10.2%) were found in the mestizo and Amerindian populations, respectively. In conclusion, the frequencies of UMs and PMs for CYP2D6 varied widely across the mestizo, Amerindian and Afro-Caribbean Costa Rican populations. Future research in this population should be oriented to identify new CYP2D6 variants through sequencing methods, as well as to determine CYP2D6 phenotype, in order to establish the phenotype-genotype relation. Finally, further studies involving genetic markers of ancestry are needed in the Costa Rican population.

Detección molecular del gen BCR-ABL por RT-PCR en niños costarricenses con leucemia

Molecular detection of the BCR-ABL gen by RT-PCR In Costa Rican children with leukemia. Many leukemias could have chromosomic translocations and according to the transcripts formed by the genes involved, the patients present an specific phenotype of the leukemia. We show the first results of the investigation of the gen BCR-ABL using RT-PCR, in order to look for the t(9;22)(q34;q11) in pediatric leukemic children. We studied in total 55 patients, 6 (10.9%) of them were positive for that translocation. Two (3.63%) of the positive children had ALL and the other 4 (7.27%) presented CML, the genotyping analysis of the transcript was studied in these children. With the introduction of this methodology as part of the routine studies, the leukemic children could get in the future an specific diagnosis, that will be important to classify them in prognostic categories and to improve the detection of minimal residual disease. Rev. Biol. Trop. 56 (4): 1613-1618. Epub 2008 December 12.

Muchas leucemias pueden presentar traslocaciones cromosómicas, las cuales, de acuerdo a los transcriptos formados por los genes involucrados, originará un fenotipo leucémica variable. En este trabajo se muestran los primeros resultados de pacientes pediátricos con leucemia, a los cuales se les hizo el estudio molecular por RT-PCR y el genotipaje para el gen BCR-ABL producto de la t(9;22)(q34;q11). De las 55 muestras estudiadas, 6 (10.9%) fueron positivas para el transcripto mencionado. De las 6 positivas, 2(3.63%) de esos pacientes tenían LLA y 4 (7.27%) eran LMC. La introducción de esta metodología en el manejo rutinario de los niños con leucemia, servirá para establecer un diagnóstico más preciso, un pronóstico más certero y un seguimiento adecuado de la enfermedad mínima residual.

[Molecular detection of the BCR-ABL gen by RT-PCR in Costa Rican children with leukemia]. / Detección molecular del gen BCR-ABL por RT-PCR en niños costarricenses con leucemia.

Many leukemias could have chromosomic translocations and according to the transcripts formed by the genes involved, the patients present an specific phenotype of the leukemia. We show the first results of the investigation of the gen BCR-ABL using RT-PCR, in order to look for the t(9;22)(q34;q11) in pediatric leukemic children. We studied in total 55 patients, 6 (10.9%) of them were positive for that translocation. Two (3.63%) of the positive children had ALL and the other 4 (7.27%) presented CML, the genotyping analysis of the transcript was studied in these children. With the introduction of this methodology as part of the routine studies, the leukemic children could get in the future an specific diagnosis, that will be important to classify them in prognostic categories and to improve the detection of minimal residual disease.

The FXIIIVal34Leu, common and risk factors of venous thrombosis in early middle-age Costa Rican patients.

In this study, eight common polymorphisms associated with venous thrombosis (VT) and thrombophilia factors were analyzed in a Costa Rican case-control study. With the use of polymerase chain reaction (PCR) methods the polymorphisms were detected in 120 patients and 133 controls (mean age <40 years old). It was concluded that a high level of fibrinogen, antiphospholipid antibodies, family history, and the genotype 34LeuLeu of FXIII OR 0.42 (0.20-0.89) showed a significant effect on the risk of VT. Associations between the risk of VT and genetic polymorphisms have been established. Some of these polymorphisms are highly prevalent in Caucasians, but there is a significant geographic variation in their prevalence among different populations. The results of this study support the protective effect of FXIII Val34Leu polymorphism in VT. These findings are consistent with previous reports that included other populations.

La implementación forense de la tecnología del ADN en Costa Rica: un análisis retrospectivo: [revisión] / Forensic implementation of DNA technology in Costa Rica: a retrospective analysis: [review]

La reciente utilización de la tecnología del ADN para la identificación individual a traído consigo una revolución en las ciencias forenses, que ha alcanzado también a la America Latina. El análisis histórico muestra que en Costa Rica sehan logrado importantes avances y en la actualidad se encuentra consolidado el trabajo con los STRs, y se están en proceso de implementación los marcadores de ADNmt y del cromosoma Y. Sin embargo, la incorporación delas innovaciones de la genética forense se ha venido realizando, cíclicamente, de 5 a 10 años tarde respecto a lospaises desarrollados en este campo. Se espera un cambio de actitud en el futuro, al estar disponibles nuevas generacionesde marcadores de ADN, que permitan explotar a corto plazo todo el potencial de esta útil herramienta al servicio de la justicia.

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica.

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.

Prevalence of eight molecular markers associated with thrombotic diseases in six Amerindian tribes and two African groups of Costa Rica.

Individuals belonging to six different Amerindian tribes and two African groups of Costa Rica were genotyped for factor V Leiden (FV), factor V haplotype HR2 (FV HR2), Factor II 20210G>A (FII), the methylenetetrahydrofolate reductase (MTHFR), factor VII polymorphisms (FVII IVS7, FVII R353Q), factor XIII (FXIII V34L), and the insertion/deletion (I/D) polymorphism of the gene of angiotensin converting enzyme (ACE). Clear differences in the prevalence were found and are first reported. The prevalence of some of the established genetic risk factors was low in Amerindians of Costa Rica (ACE) or even absent (FVL, FII), and others (MTHFR, FVHR2) had an extremely high prevalence. People of African origin carried very rare FVL or FII polymorphisms, but the DD genotype of ACE is the highest reported. Concerning the protective factors, the QQ genotype of FVII R353Q was absent in Amerindians, but the protective 7/7 genotype of FVII IVS7 frequently found. Novel alleles of FVII IVS7 (4, 8, and 9 monomers) were found. Intertribal heterogeneity was observed that may reflect the evolutionary history of these tribal groups and their admixture with other populations.

Nicaraguan population data on LDLR, GYPA, D7S8, HBGG, GC and HLA-DQA1 loci

Nicaraguans have become the most numerous and fastest increasing minority in Costa Rica: at present they represent around 6 of the total population of the country. We have analyzed the allele and genotype frequencies of six PCR-based genetic markers (LDLR, GYPA, HBGG, D7S8, GC, and HLA-DQA1) in 100 unrelated Nicaraguans living in Costa Rica. All loci studied were in Hardy-Weinberg equilibrium. Some statistical parameters of forensic interest were also calculated (h, PD and CE). Allele frequencies of the markers HLA-DQA1 and GYPA were found to be significantly different between the populations of Nicaragua and Costa Rica. Nevertheless, genetic distances showed that Nicaragua is close to other Hispanic-admixed populations like those from Argentina, Chile, Colombia, Costa Rica, and USA Hispanics. The loci set was assessed to be useful for paternity testing and individual identification in the Nicaraguan population residing in Costa Rica.

Revisión sobre la extracción de ADN a partir de huesos humanos / Review on extraction to ADN from human bones

Los huesos antiguos son una buena fuente de ADN, útil en muchos casos para el análisis forense, especialmente cuando el tejido blando está severamente descompuesto o dañado. Existen diferentes métodos para la extracción de ADN a partir de restos óseos, con diferentes eficacias, y que plantean varios problemas técnicos. Se presenta una revisión bibliográfica sobre este tema y se incluyen algunas recomendaciones prácticas. Palabras clave: Tipeo de ADN, estracción de ADN, huesos humanos, Ciencias Forenses

Identificación de restos óseos humanos mediante análisis de ADN / Identification of human bones remains using ADN analysis

En los últimos años la tecnología del ADN se ha convertido en una poderosa herramienta para la identificación individual, incluso cuando sólamente se cuenta con restos óseos. Se ilustra un caso en el cual se realizó la prueba de identificación comparando el ADN de los supuestos padres contra el ADN extraído del hueso de un menor de sexo femenino, utilizando los marcadores : HLA-DQA1, LDLR, GYPA, HBGG, D7S8, Gc, y D1S80. Los perfiles genéticos obtenidos permitieron concluir una identificación positiva de los restos como pertenecientes a la niña desaparecida. Esta metodología queda disponible para futuros casos forenses que así lo ameriten