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Clopidogrel is widely used worldwide as an antiplatelet therapy in patients with acute coronary disease. Genetic factors influence interindividual variability in response. Some studies have explored the polygenic contributions in the drug response, generating pharmacogenomic risk scores (PgxPRS). Importantly, these factors are less explored in underrepresented populations, such as Latin-American countries. Identifying patients at risk of high-on-treatment platelet reactivity (HTPR) is highly valuable in translational medicine. In this study we used a custom next-generation sequencing (NGS) panel composed of 91 single nucleotide polymorphisms (SNPs) and 28 genes related to clopidogrel metabolism, to analyze 70 patients with platelet reactivity values, assessed through closure time (CT). Our results demonstrated the association of SNPs with HTPR and non-HTPR, revealing the strongest associations with rs2286823 (OR: 5,0; 95% CI: 1,02-24,48; p: 0,03), rs2032582 (OR: 4,41; 95% CI: 1,20-16,12; p: 0,019), and rs1045642 (OR: 3,38; 95% CI: 0,96-11,9; p: 0,05). Bivariate regression analysis demonstrated the significant association of several SNPs with the CT value, a "surrogate" biomarker of clopidogrel response. Exploratory results from the LASSO regression model showed a high discriminatory capacity between HTPR and non-HTPR patients (AUC: 0,955), and the generated PgxPRS demonstrated a significant negative association between the risk score, CT value, and the condition of HTPR and non-HTPR. To our knowledge, our study addresses for the first time the analysis of the polygenic contribution in platelet reactivity using NGS and establishes PgxPRS derived from the LASSO model. Our results demonstrate the polygenic implication of clopidogrel response and offer insights applicable to the translational medicine of antiplatelet therapy in an understudied population.
Assuntos
Plaquetas , Clopidogrel , Sequenciamento de Nucleotídeos em Larga Escala , Inibidores da Agregação Plaquetária , Polimorfismo de Nucleotídeo Único , Humanos , Clopidogrel/uso terapêutico , Clopidogrel/farmacologia , Masculino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Feminino , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores da Agregação Plaquetária/farmacologia , Pessoa de Meia-Idade , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Idoso , Herança Multifatorial/genética , Ticlopidina/análogos & derivados , Ticlopidina/uso terapêutico , Ticlopidina/farmacologiaRESUMO
Background: Colorectal cancer (CRC) is a prevalent cancer, ranking as the third most common. Recent advances in our understanding of the molecular causes of this disease have highlighted the crucial role of tumor immune evasion in its initiation and progression. CTLA4, a receptor that acts as a negative regulator of T cell responses, plays a pivotal role in this process, and genetic variations in CTLA4 have been linked to CRC susceptibility, prognosis, and response to therapy. Methods: We conducted a case-control study involving 98 CRC patients and 424 controls. We genotyped the CTLA4 c.-319C > T variant (rs5742909) and performed an association analysis by comparing allele frequencies between the patients and controls. To assess the potential functional impact of this variant, we first performed an In Silico analysis of transcription factor binding sites using Genomatix. Finally, to validate our findings, we conducted a luciferase reporter gene assay using different cell lines and an electrophoretic mobility shift assay (EMSA). Results: The case-control association analysis revealed a significant association between CTLA4 c.-319C > T and CRC susceptibility (p = 0.023; OR 1.89; 95% CI = 1.11-3.23). Genomatix analysis identified LEF1 and TCF7 transcription factors as specific binders to CTLA4 c.-319C. The reporter gene assay demonstrated notable differences in luciferase activity between the c.-319 C and T alleles in COS-7, HCT116, and Jurkat cell lines. EMSA analysis showed differences in TCF7 interaction with the CTLA4 C and T alleles. Conclusion: CTLA4 c.-319C > T is associated with CRC susceptibility. Based on our functional validation results, we proposed that CTLA4 c.-319C > T alters gene expression at the transcriptional level, triggering a stronger negative regulation of T-cells and immune tumoral evasion.
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Clopidogrel, an oral platelet P2Y12 receptor blocker, is used in the treatment of acute coronary syndrome. Interindividual variability in treatment response and the occurrence of adverse effects has been attributed to genetic variants in CYP2C19. The analysis of relevant pharmacogenes in ethnically heterogeneous and poorly studied populations contributes to the implementation of personalized medicine. We analyzed the coding and regulatory regions of CYP2C19 in 166 patients with acute coronary syndrome (ACS) treated with clopidogrel. The allele frequencies of CYP2C19 alleles *1, *2, *4, *17, *27 and *33 alleles were 86.1%, 7.2%, 0.3%, 10.2%, 0.3% and 0.3%, respectively. A new potentially pathogenic mutation (p.L15H) and five intronic variants with potential splicing effects were detected. In 14.4% of the patients, a new haplotype in strong linkage disequilibrium was identified. The clinical outcome indicated that 13.5% of the patients presented adverse drugs reactions with a predominance of bleeding while 25% of these patients were carriers of at least one polymorphic allele. We propose that new regulatory single-nucleotide variants (SNVs) might potentially influence the response to clopidogrel in Colombian individuals.
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Risk factors for venous thromboembolism in cancer vary between tumours. Leucocytosis, thrombocytosis, tumour histology and vascular compression may drive thrombosis in ovarian cancer. Thrombosis developed in 13.4% of our patients. Higher median leucocyte, neutrophil and monocyte counts were related to thrombosis. Thrombocytosis >350 × 109 /L was frequent (63.8%), but not predictive of thrombosis. Identification of prothrombotic biomarkers may help personalise preventive treatments.
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Neoplasias Ovarianas , Trombocitose , Trombose , Tromboembolia Venosa , Carcinoma Epitelial do Ovário , Feminino , Humanos , Neoplasias Ovarianas/complicações , Trombocitose/diagnóstico , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/epidemiologia , Tromboembolia Venosa/etiologiaRESUMO
PURPOSE: Warfarin is an oral anticoagulant associated with adverse reaction to drugs due to wide inter- and intra-individual dosage variability. Warfarin dosage has been related to non-genetic and genetic factors. CYP2C9 and VKORC1 gene polymorphisms affect warfarin metabolism and dosage. Due to the central role of populations' ethnical and genetic origin on warfarin dosage variability, novel algorithms for Latin American subgroups are necessary to establish safe anticoagulation therapy. PATIENTS AND METHODS: We genotyped CYP2C9*2 (c.430C > T), CYP2C9*3 (c.1075A > C), CYP4F2 (c.1297G > A), and VKORC1 (-1639 G > A) polymorphisms in 152 Colombian patients who received warfarin. We evaluated the impact on the variability of patients' warfarin dose requirements. Multiple linear regression analysis, using genetic and non-genetic variables, was used for creating an algorithm for optimal warfarin maintenance dose. RESULTS: Median weekly prescribed warfarin dosage was significantly lower in patients having the VKORC1-1639 AA genotype and poor CYP2C9*2/*2,*2/*3 metabolizers than their wild-type counterparts. We found a 2.3-fold increase in mean dose for normal sensitivity patients (wild-type VKORC1/CYP2C9 genotypes) compared to the other groups (moderate and high sensitivity); 31.5% of the patients in our study group had warfarin sensitivity-related genotypes. The estimated regression equation accounted for 44.4% of overall variability in regard to warfarin maintenance dose. The algorithm was validated, giving 45.9% correlation (R 2=0.459). CONCLUSION: Our results describe and validate the first algorithm for predicting warfarin maintenance in a Colombian mestizo population and have contributed toward the understanding of pharmacogenetics in a Latin American population subgroup.
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Resumen:
Introducción: la respuesta médica a la solicitud de la enfermera, es fundamental para la prestación de los servicios de salud.
Objetivo: determinar la percepción de la enfermera ante la respuesta médica a su llamada en la atención pediátrica y su opinión sobre estrategias de mejora.
Metodología: estudio transversal descriptivo, se utilizó un cuestionario previamente validado en apariencia y contenido con diez circunstancias de solicitud de respuesta médica; autoaplicado a personal adscrito al área de hospitalización.
Resultados: participaron 81 enfermeras. Se percibió una respuesta médica rápida (<10 minutos) para circunstancias de urgencias como: presencia de convulsiones en 65.4% de las veces, desaturación periférica de oxigeno 55.6%, reacción trans o postransfusional 46.9%, sangrado activo 44.4% y dificultad respiratoria en 37%. Para situaciones menos urgentes (10 min a 1 hr) en caso de: detección de error o duda en dosis 45.7%; queja o reclamación intensa del familiar por un procedimiento 35.8%; necesidad de un cambio o inicio de dieta 35.8%. Una respuesta más tardía (1 a 4 hrs) para: problemas con una venoclisis 39.5% y programación de procedimiento 43.2%. La estrategia de mejora más apoyada 56.8% fue la presencia obligatoria constante de un médico en sala.
Conclusiones: en situaciones urgentes la enfermera percibe una respuesta médica rápida en más de la mitad de las veces. Para situaciones menos urgentes la respuesta suele ser entre una y cuatro horas, por lo que es necesario implementar estrategias de mejora de la comunicación médico-enfermera.
Abstract:
Introduction: medical response to the nurse request is significant to provide health services. Objective: to determine the perception of nursing staff at the medical response to their call in paediatric care and their opinion on strategies for improvement.
Methodology: cross-sectional, descriptive study, a previously validated questionnaire in appearance and content of ten circumstances requiring medical response was used, auto applied to personnel assigned to the ward.
Results: 81 nurses participated. A quick medical response (<10 minutes) was perceived for emergency circumstances such as: presence of seizures in 65.4% of the time, peripheral oxygen desaturation 55.6%, trans or post-transfusion reaction 46.9%, active bleeding 44.4% and respiratory distress in 37%. For less urgent situations (10 minutes to 1 hour): doubt or error in medication 45.7%; intense parental complaint for a procedure 35.8%; need of changing or starting a diet 35.8%. A later response (1-4 hours) to: problems with an IV 39.5% and 43.2% procedure schedules. The most supported improvement strategy (56.8%) was the constant and mandatory presence of a doctor in the ward.
Conclusions: in urgent situations nursing staff perceive a quick medical response in more than half of the times. For less urgent situations the response is usually between one and four hours, so it is necessary to implement strategies to improve nurse-physician communication.
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Atitude do Pessoal de Saúde , Comportamento Cooperativo , Comunicação , Recursos Humanos de Enfermagem , Relações Interprofissionais , Relações Médico-Enfermeiro , México , HumanosRESUMO
Introducción. La distrofia muscular de Duchenne (DMD), y su forma alélica más leve, la distrofia muscular de Becker (DMB), es una entidad de herencia recesiva ligada al X, que se presenta con debilidad muscular, pérdida progresiva de las habilidades motoras y muerte precoz. Es causada principalmente por deleciones en el gen de la distrofina, el cual contiene 79 exones. Objetivo. Realizar un análisis ampliado para evaluar la presencia de deleciones en 15 exones del gen de la distrofina situados dentro y fuera del hot spot mutacional en 58 pacientes afectados con DMD/DMB sin mutación previamente identificada. Metodología. Amplificación, mediante PCR múltiplex, de 4 exones situados dentro y 11 fuera del hot spot mutacional descrito para el gen de la distrofina en 58 pacientes afectados con DMD y determinar la frecuencia de deleciones en la población analizada. Resultados. Se encontró deleción del exón 16 en uno de los pacientes estudiados, hecho que indica una frecuencia de 1,7%. No se observó ninguna deleción de los exones situados fuera del hot spot mutacional. Conclusiones. La frecuencia de deleciones en los 15 exones del gen de la distrofina analizados es baja; sólo se presentó en el exón 16, el cual se encuentra localizado en el hot spot mutacional proximal del gen. Es importante analizar este exón en los afectados, en la medida en que aumenta la tasa de detección de deleciones en un 1,7%. Se debe analizar otro tipo de mutaciones como puntuales y duplicaciones en los afectados.
Introduction. Duchenne and Becker Muscular Dystrophies (DMD/DMB) are X-linked recessive diseases characterized by progressive muscle weakness and wasting, loss of motor skills and death after the second decade of life. Deletions are the most prevalent mutations that affect the dystrophin gene, which spans 79 exons. Objective: Identify deletions on the dystrophin gene in 58 patients affected with DMD. Methods: Through multiplex PCR identify deletions on the dystrophin gene in 58 patients with DMD and observe the frequency of this mutation in our population. Results: We found deletions in 1.72% of patients (1 of 58 persons). Deletions were not the principal cause of disease in our population. It is possible that duplications and point mutations caused this illness in our patients. Conclusions: The frequency of deletions in the 15 exons analyzed from the dystrophin gene was low. The predominant types of mutation in our patients` samples were not deletions as has been observed in the literature worldwide, therefore, it is important to determine other types of mutations as are duplications and point mutations.
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Humanos , Distrofia Muscular de Duchenne , Éxons , Distrofina , Reação em Cadeia da Polimerase Multiplex , MutaçãoRESUMO
La citogenética es el estudio de los cromosomas tanto en número como en estructura, los primeros pasos en la citogenética humana se dieron a finales del siglo XIX con la publicación de Flemming en 1882 de las primeras ilustraciones del cromosoma humano a partir de observaciones al microscopio, y concluyó con Tjio y Levan en 1953 cuando se determina el número real de cromosomas humanos por célula diploide. La citogenética convencional es una herramienta de gran importancia que permite realizar el diagnóstico cromosómico de pacientes con indicación clínica de cromosomopatía, lo cual les va a permitir asesorar a las familias respecto de dicha enfermedad, su pronóstico y riesgo de recurrencia. El propósito de esta revisión es documentar a los médicos, pediatras, ginecólogos y en general al personal de la salud, de la importancia de los estudios citogenéticos en aquellos casos en que se enfrenten a un paciente con una cromosomopatía o síndrome dismórfico.
Cytogenetics is the study of chromosomes and their numerical and structural abnormalities. In the late 1800´s Flemming published his first illustrations of human chromosomes based on his observations on a microscope. In 1953 Tijo and Levan determined the number of chromosomes in a human somatic cell. From then on, conventional cytogenetics became an important tool for physicians in the diagnosis of patients with chromosomal anomalies through the use of the karyotype. The karyotype thus becomes a means to diagnose patients and provide them genetic counseling. The purpose of this review is to enlighten family doctors, pediatricians and gynecologists and other health practitioners of the importance of cytogenetics when challenged with patients with an abnormal karyotype or dysmorphic syndrome.
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La implementación de metodologías de biología molecular como la reacción en cadena de la polimerasa (PCR), ha permitido la realización de diagnósticos sensibles y específicos para múltiples enfermedades, dentro de las cuales son de gran interés las infecciosas. Hasta hoy, los métodos de identificación se basan principalmente en cultivos y serología por su sensibilidad y especificidad, pero consumen tiempo y dinero. Las muestras de orina se han constituido en una alternativa no invasiva de obtención de ADN para la realización de análisis de biología molecular. Metodología: Implementación de una estrategia para la obtención de ADN a partir de muestras de orina. Las muestras fueron tomadas de niños de guardería, para documentar la presencia o no de inhibidores de PCR a través de la amplificación de genes de Citomegalovirus humano (CMVH). Resultados: En el 27,1% de las muestras analizadas se evidenció amplificación específica para CMVH, no se encontraron diferencias significativas en la presencia del virus en los tres estratos, pero sí en la intensidad de las bandas. Conclusión: Se verificó la ausencia de inhibidores de PCR mediante la amplificación del gen de la B-globina. Se estandarizó una metodología molecular para la identificación de CMVH, la cual puede ser aplicada en diagnóstico prenatal de infección congénita.
Molecular biology methods like Polimerase Chain Reaction (PCR) has been used for diagnosis of infectious diseases. Until today, the identification methods are based mainly on cultures and serology due to their sensibility and specificity, but they are expensive and time consuming. Urine samples constitute an alternative, noninvasive, method of obtaining DNA for the accomplishment of molecular Biology analysis. Methodology: implementation of a strategy to obtain DNA from urine samples. Samples were taken from children in daycare centers, to document the presence of inhibitors, PCR amplification of genes of human Cytomegalovirus (HCMV) was done. Results: In 27.1% of the analyzed samples, specific amplification for HCMV was demonstrated. No viral significant differences were found in the three layers, although it was present in the bands. Conclusion: The inhibitor absence was verified using PCR by amplificating the gene of the B-globine. A molecular methodology for the HCMV identification was standardized, which can be applied in prenatal diagnosis of congenital infection.
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Humanos , Citomegalovirus , DNA , Diagnóstico , Coleta de Urina , Infecções , MétodosRESUMO
La distrofia muscular de Duchenne y Becker es la miopatía más común en ni ños y es causada por la ausencia de la proteína distrofina. Los afectados presentan signos de la enfermedad a edades tempranas de la vida, pierden la habilidad para caminar al comienzo de la segunda década y usualmente, fallecen alrededor de los 20 años de edad. El islamiento del gen defectuoso ha llevado a un mejor entendimiento del proceso de la enfermedad y ha permitido el diagnóstico preciso en los afectados, la posibilidad de asesoramiento genético y diagnóstico prenatal, así como la aplicación de nuevas terapéuticas basadas en el conocimiento de la patogénesis de la enfermedad. El propósito de esta revisión es presentar el progreso hecho en estas áreas, refiriéndonos particularmente a la fisiopatología y al diagnóstico molecular de la enfermedad en Colombia.
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Diagnóstico , Distrofia Muscular de Duchenne , Doenças MuscularesRESUMO
INTRODUCCIÓN: La correlación genotipo-fenotipo se estableció mediante el análisis de deleciones del gen de la distrofina en pacientes con distrofia muscular de Duchenne y Becker (DMD/DMB). OBJETIVOS: Establecer la correlación entre el genotipo molecular y el fenotipo clínico de los pacientes. MATERIALES Y MÉTODOS: Se analizaron 62 afectados mediante amplificaciones por PCR múltiplex de 18 exones ubicados en los dos puntos proclives dentro del gen. RESULTADOS: En la población analizada, 19 pacientes mostraron deleción en el gen de la distrofina con los 18 exones estudiados, esto corresponde a 31/100 de hombres afectados con deleción. CONCLUSIONES: Teniendo en cuenta la hipótesis del corrimiento del marco de lectura traduccional (CMLT) y la mutación observada en los afectados, se pudo determinar que las mutaciones out frame, resultan en pacientes con el fenotipo severo o distrofia muscular de Duchenne y las mutaciones in frame, resultan en pacientes con el fenotipo leve o distrofia muscular de Becker. Se pudo predecir un cuadro clínico de DMD o DMB en 79/100 de los casos, lo cual permite utilizar este sistema diagnóstico como una herramienta importante para ayudarle a los neurólogos en la valoración clínica de los pacientes en los cuales se encuentra deleciones