Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

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.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

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.

DNA repair-related genes in sugarcane expressed sequence tags (ESTs)

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.