RESUMEN
BACKGROUND: The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS: We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS: The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS: EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.
Asunto(s)
Variaciones en el Número de Copia de ADN , Reacción en Cadena de la Polimerasa/métodos , Receptores de LDL/genética , Sondas de ADN/química , Sondas de ADN/metabolismo , Colorantes Fluorescentes/química , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa Multiplex , Análisis de Secuencia de ADNRESUMEN
INTRODUCTION: Atherosclerosis is an inflammatory chronic disease influenced by multiple factors. Different prospective studies have shown that plasmatic levels of inflammatory markers were related to atherosclerosis and cardiovascular disease. OBJECTIVE: To evaluate whether plasmatic levels of interleukin 18 (IL-18) are modulated by SNPs (single nucleotide polymorphisms) of the IL 18 gene and its possible association with insulin levels and other cardiovascular risk factors. METHODS: 746 individuals were studied for a period of two years by opportunistic selection in the metropolitan area of Valencia. Parameters of lipid and glucose metabolism were analyzed by standard methodology. IL-18 was measured by ELISA. RESULTS: Individuals with insulin resistance showed significant higher levels of IL-18. IL 18 was significantly correlated with insulin levels and other cardiovascular risk factors. The CC genotype of the rs1834481 SNP was significantly associated with lower levels of IL-18. However, the GG genotype of the rs7559479 was associated with significant higher levels of IL-18. CONCLUSION: IL-18 is associated with insulin resistance and other cardiovascular risk factors, being those levels genetically regulated.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Resistencia a la Insulina/genética , Insulina/sangre , Interleucina-18/genética , Adolescente , Adulto , Anciano , Enfermedades Cardiovasculares/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Glucosa/metabolismo , Humanos , Interleucina-18/sangre , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Factores de Riesgo , Adulto JovenRESUMEN
Antecedentes. La arteriosclerosis se considera una enfermedad inflamatoria crónica influenciada por múltiples factores de riesgo. Diferentes estudios prospectivos han mostrado que los niveles plasmáticos de moléculas inflamatorias se relacionan con la presencia de arteriosclerosis y el desarrollo de enfermedad cardiovascular. Objetivo. Evaluar si los valores plasmáticos de interleucina 18 (IL-18) están modulados por single nucleotide polymorphisms (SNP) del gen de IL-18 y su posible asociación con los niveles de insulina y otros factores de riesgo cardiovascular. Materiales y métodos. Se estudiaron 746 individuos, seleccionados de forma oportunística entre los pacientes que acudieron a diversas consultas del área metropolitana de Valencia durante 2 años. Se determinaron mediante metodología estándar parámetros del metabolismo lipídico e hidrocarbonado. La IL-18 se determinó mediante ELISA. Resultados. Los sujetos con resistencia a la insulina (RI) presentaron niveles de IL-18 significativamente superiores que los individuos sin RI. Los niveles de IL-18 se correlacionaron de forma significativa con los niveles de insulina y con otros factores de riesgo cardiovascular. El genotipo CC del SNP rs1834481 se asoció de forma significativa con menores niveles plasmáticos de IL-18. Por el contrario, el genotipo GG del SNP rs7559479 se asoció con niveles significativamente superiores de IL-18. Conclusiones. Los niveles de IL-18 se relacionan con la presencia de RI y otros factores de riesgo cardiovascular, estando modulados dichos niveles genéticamente
Introduction. Atherosclerosis is an inflammatory chronic disease influenced by multiple factors. Different prospective studies have shown that plasmatic levels of inflammatory markers were related to atherosclerosis and cardiovascular disease. Objective. To evaluate whether plasmatic levels of interleukin 18 (IL-18) are modulated by SNPs (single nucleotide polymorphisms) of the IL 18 gene and its possible association with insulin levels and other cardiovascular risk factors. Methods. 746 individuals were studied for a period of two years by opportunistic selection in the metropolitan area of Valencia. Parameters of lipid and glucose metabolism were analyzed by standard methodology. IL-18 was measured by ELISA. Results. Individuals with insulin resistance showed significant higher levels of IL-18. IL 18 was significantly correlated with insulin levels and other cardiovascular risk factors. The CC genotype of the rs1834481 SNP was significantly associated with lower levels of IL-18. However, the GG genotype of the rs7559479 was associated with significant higher levels of IL-18. Conclusion. IL-18 is associated with insulin resistance and other cardiovascular risk factors, being those levels genetically regulated