RESUMEN
Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.
Asunto(s)
Humanos , Dimensión del Dolor , Adhesión en Parafina , Pruebas Diagnósticas de Rutina , Uracil-ADN Glicosidasa , Secuenciación Completa del GenomaRESUMEN
The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50â mg/kg). Animals received melatonin during the dark period for 15 days (1â mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48â h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.
Asunto(s)
Antioxidantes/administración & dosificación , Daño del ADN/efectos de los fármacos , Melatonina/administración & dosificación , Animales , Aberraciones Cromosómicas , Ciclofosfamida , Inyecciones Intraperitoneales , Masculino , Mutágenos , Oxidación-Reducción , Ratas WistarRESUMEN
The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.
Asunto(s)
Animales , Masculino , Antioxidantes/administración & dosificación , Daño del ADN/efectos de los fármacos , Melatonina/administración & dosificación , Aberraciones Cromosómicas , Ciclofosfamida , Inyecciones Intraperitoneales , Mutágenos , Oxidación-Reducción , Ratas WistarRESUMEN
Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T-DNA insertion. The 3' end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Reparación del ADN , Genes de Plantas , Proteínas de Arabidopsis/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Metilmetanosulfonato/farmacología , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutágenos/farmacología , Tolerancia a Radiación , Rayos UltravioletaRESUMEN
A identificaçäo e caracterizaçäo de genes envolvidos com reparo de DNA säo de grande interesse, dada a sua importância na manutençäo da integridade genômica. Além disso, a alta conservaçäo dos genes de reparo de DNA faz com que possam ser utilizados como fonte de informaçäo no que diz respeito à origem e evoluçäo das espécies. Os mecanismos relacionados à remoçäo de danos pelo reparo de DNA, bem como suas conseqüências biológicas, já säo bem conhecidas em bactérias, leveduras e animais. Entretanto, no que diz respeito a organismos vegetais, ainda há muito a ser investigado. No presente trabalho, apresentamos a identificaçäo dos genes envolvidos nas principais vias de reparo de DNA em cana-de-açúcar, através de uma análise de similaridade do banco de dados do projeto brasileiro Sugarcane Expressed Sequence Tag (SUCEST) com seqüências protéicas conhecidas disponíveis em outros bancos de dados públicos (National Center of Biotechnology Information (NCBI) e Munich Information Center for Protein Sequences (MIPS) Arabidopsis thaliana). Esta busca revelou que a gama de proteínas envolvidas no reparo de DNA em cana-de-açúcar é similar a de outros eucariotos. Mesmo assim, foi possível identificar algumas características interessantes encontradas apenas em vegetais, provavelmente em funçäo do seu processo evolutivo independente. As vias de reparo do DNA aqui representadas incluem fotorreativaçäo, reparo excisäo de bases, reparo excisäo de nucleotídeos, reparo mismatch, end-joinning näo homólogo, reparo por recombinaçäo homóloga e tolerância a lesões. Este trabalho descreve as principais diferenças encontradas na maquinaria de reparo de DNA de células vegetais em relaçäo àquela de organismos nos quais encontra-se bem descrita. Tais diferenças chamam a atençäo para um potencial de mecanismos distintos em vegetais, que merecem futuras investigações.
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Humanos , Animales , Etiquetas de Secuencia Expresada , Plantas , Reparación del ADN , Bases de Datos como Asunto , Programas InformáticosRESUMEN
This presentation features linguistic and terminology management issues related to the development of the Spanish version of the Systematized Nomenclature of Medicine (SNOMED). It aims at describing the aspects of translating and the difficulties encountered in delivering a natural and consistent medical nomenclature. Bunge's three-layered model is referenced to analyze the sequence of symbolic concept representations. It further explains how a communicative translation based on a concept-to-concept approach was used to achieve the highest level of flawlessness and naturalness for the Spanish rendition of SNOMED. Translation procedures and techniques are described and exemplified. Both the computer-aided and human translation methods are portrayed. The scientific and translation team tasks are detailed, with focus on Newmark's four-level principle for the translation process, extended with a fifth further level relevant to the ontology to control the consistency of the typology of concepts. Finally the convenience for a common methodology to develop non-English versions of SNOMED is suggested.