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Background
Silent chromosomal translocations frequently occur in newborn children. However, the origin of these translocations remains poorly understood. Caffeine, a natural inhibitor of topoisomerase II, induces DNA double strand breaks and may be involved in the production of translocations.
Moreover, caffeine enhances error-prone repair by inhibiting ATM kinase, essential for maintaining DNA ends in repair complexes. The dose of caffeine reaching the fetus depends predominantly upon maternal intake and toxicokinetics.
The objective of the project was to improve the limited understanding of the factors affecting in utero chromosome translocation. The project aimed to demonstrate proof of principle that RNA can be extracted from Guthrie blood spots, and screened for translocations.
Methods
From a well-defined mother/baby cohort of 1,340 pregnancies, we identified 1,300 individuals and recovered 1,287 Guthrie cards (Department of Clinical Chemistry, Sheffield Children’s Hospital). These were punched and shipped to the University of Maastricht where, from a defined sub-set, RNA was isolated and amplified in preparation for mutation screening. RNA was isolated from Guthrie cards with the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. Mutation screening was performed using the Hemavision Screen kit (DNA Technology, Rosskov, Denmark).
In addition to the planned work we have evaluated the effect of sampling procedure and storage conditions of whole blood, upon levels of oxidatively damaged DNA. We have also developed methodology which allows for the comet assay (including the enzyme-modified variant) analysis of small volumes of whole blood.
Results
The amplification step resulted in a low RNA yield, which was insufficient for the mutation assay. As a positive control for the mutation assay, we performed the assay on two cord blood samples for which we knew the RNA quality was good. For one sample, the kit’s control RNA band was slightly visible but was absent for the other sample. This indicated to us that the kit was not working poorly.
From our additional work, we successfully demonstrated that small volumes of blood can be simply integrated into the comet assay (including the enzyme-modified variant) for rapid analysis of DNA damage. This significantly increased the speed and throughput of the assay by removing the need for isolation of peripheral blood mononuclear cells prior to analysis.
The assay revealed that, surprisingly, whole blood appears to act as an effective cryopreservative, when small volumes of samples are stored at -80°C. We also demonstrated that small volumes of blood, such as those obtained via a Lancet/pin-prick (~250 µL) show long-term stability at -80°C, and are amenable to our modification of the comet assay.
Conclusions
We continued to check the RNA quality from blood spots, concluding that, contrary to earlier reports in the literature, RNA quality is too low for further downstream applications. We also continued to isolate DNA from the Guthrie cards, as originally proposed, and will then perform limit mutation analyses as originally proposed in the application.
We also reported conditions that allows the storage of whole blood at -80°C, without cryopreservative, for at least one month without artefactual formation of DNA damage, as measured by the comet assay.