Toenails: An easily accessible and long-term stable source of DNA for genetic analyses in large-scale epidemiological studies.

A192

Molecular tools are increasingly applied in epidemiological studies to define the relationship between environmental exposures and disease. Analyses of genetic factors, such as polymorphisms, require DNA. However, currently used specimens, such as lymphocytes and buccal cells, have disadvantages with regard to collection, transport, storage, and processing of samples. A relatively unknown source of DNA that can overcome these problems is nail material. In several large-scale epidemiological studies, toenail material has been collected for analysis of trace elements as biomarker for intake of these compounds. In the present study, we investigated whether toenail material that was collected 20 years ago in the Netherlands Cohort study on Diet and Cancer -a large-scale epidemiological study (n=120,852) - could be used as source of DNA for analyses of multiple genetic polymorphisms. We optimized a protocol for DNA isolation from toenails. We tested the suitability of the isolated toenail DNA in a multiplexed genotyping assay using primer extension and automated capillary gel electrophoresis in subjects from a subgroup of the cohort in which also buccal DNA was available. Part of the toenail samples had been irradiated with neutrons for analyses of selenium content (not irradiated: n=33; irradiated: n=24). For comparison reasons, also freshly harvested toenail material from healthy non-NLCS volunteers was investigated (n=11). On average, 2 μg DNA could be extracted from 10 mg toenail material. There was no significant difference in quantity of extracted DNA between irradiated and not irradiated old material, nor with freshly harvested material. A complete genotype profile of 10 polymorphisms was successfully generated for 95% of the DNA samples (amplicon sizes ranging from 92 to 148 bp). Amplification of a 596 bp fragment was successful in 10 out of 10 DNA samples from not irradiated toenails and in 4 out of 10 irradiated samples. The results show that DNA can be successfully isolated from 20-year-old not irradiated and irradiated toenail material, and that this DNA can be used for state of the art high throughput genetic analyses of polymorphisms. For large-scale epidemiological studies, these results provide new opportunities for genetic analyses in cohorts for which until now no source of DNA was available. Using toenails as source of DNA can also considerably reduce the implementation costs of future molecular epidemiological studies.