• Involvement of MRE11A and XPA gene polymorphisms in the modulation of DNA double-strand break repair activity: a genotype-phenotype correlation study.

      Ricceri, Fulvio; Porcedda, Paola; Allione, Alessandra; Turinetto, Valentina; Polidoro, Silvia; Guarrera, Simonetta; Rosa, Fabio; Voglino, Floriana; Pezzotti, Annamaria; Minieri, Valentina; et al. (2011-10-10)
      DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation-induced DNA damage, and failure to repair them results in cell death. In order to see if any associations exist between DNA repair gene polymorphisms and phenotypic profiles of DSB repair (DSBR) we performed a genotype-phenotype correlation study in 118 young healthy subjects (mean age 25.8±6.7years). Subjects were genotyped for 768 single nucleotide polymorphisms (SNPs) with a custom Illumina Golden Gate Assay, and an H2AX histone phosphorylation assay was done to test DSBR capacity. We found that H2AX phosphorylation at 1h was significantly lower in subjects heterozygous (no variant homozygotes were observed) for the XPA gene SNP rs3176683 (p-value=0.005), while dephosphorylation was significantly higher in subjects carrying the variant allele in three MRE11A gene SNPs: rs1014666, rs476137 and rs2508784 (p-value=0.003, 0.003 and 0.008, respectively). An additive effect of low-activity DNA repair alleles was associated with altered DSBR activity, as demonstrated by both H2AX phosphorylation at 1 h (p-trend <0.0001) and γH2AX dephosphorylation at 3h (p-trend <0.0001). Our study revealed that in addition to SNPs of genes that are well-established players in DSBR, non-DSBR genes, such as the XPA gene that is mainly involved in the nucleotide excision repair pathway, can also influence DSBR in healthy subjects. This suggests that successful DSBR may require both DSBR and non-DSBR mechanisms.
    • Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial.

      Forchhammer, Lykke; Johansson, Clara; Loft, Steffen; Moller, Lennart; Godschalk, Roger W. L.; Langie, Sabine A. S.; Jones, George D. D.; Kwok, Rachel W. L.; Collins, Andrew R.; Azqueta, Amaya; et al. (2010-03)
      The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.