Abstract 72: DNA isolated from toenails: A new and valid source for high-throughput genotyping in large-scale epidemiological studies.
Authors: Schouten Leo J. , Hogervorst Janneke GF , Simons Colinda C. , Jonkers Leonie , Goessens Joy , Godschalk Roger , Jackmaert Sigrun , Verhage Bas , Vermeesch Joris , Van Schooten Frederik-Jan , Weijenberg Matty P. , Van den Brandt Piet A. 
Published: August 2013

Abstract
Introduction: We have previously shown that DNA can be successfully isolated from 20-year-old toenail clippings, and that this DNA can be used for analyses of genetic polymorphisms. In this study we investigated whether DNA isolated from toenails can be successfully genotyped using different commercially available platforms.
Background cohort: The Netherlands Cohort Study (NLCS) is a prospective cohort study, which started in 1986 and includes 120,852 subjects aged 55-69 years at baseline. The cohort members completed a self-administered food-frequency questionnaire on dietary habits and other risk factors for cancer and approximately 90,000 participants provided toenail clippings. Follow-up is accomplished by record linkage to the Netherlands Cancer Registry, the nationwide Pathology Registry (PALGA) and the national causes of death registry (Statistics Netherlands).
Genotyping methods: We optimized a protocol for DNA isolation from toenails, and could, on average, extract 3.3 μg DNA per 15 mg toenail clippings. Although amplification of CYP1A2 (596 bp) was successful in 82% of the samples (n=550), the isolated DNA is fragmented (mostly &lt;200 bp).
We first investigated whether genotyping using the Sequenomâ Massarray platform is feasible. Sequenomâ Massarray has been especially designed for genotyping of a limited number (up to 40 in one multiplex array) of single-nucleotide polymorphisms (SNPs) in a large number of samples.
In a pilot, 30 SNPs in insulin-like growth factor (IGF) pathway genes, hypothesized to modify the association between body size, physical activity, and energy restriction during youth and colorectal cancer risk, were selected. This set of SNPs was designed to be genotyped in one multiplex. 90 DNA samples from toenails of prostate cancer cases from the NLCS were used. Samples were analyzed in quadruplicate.
Next, we investigated whether genotyping is also successful in arrays containing &gt;100,000 markers at once. For DNA isolated from Fixed Formaline Paraffin Emdedded (FFPE) tissues Illumina® has developed the Infinium® HD FFPE Quality Control Kit, to test whether the quality of the DNA is good enough, and the Infinium® HD FFPE Restore Kit to repair the fragmented DNA. The DNA that has been restored can be genotyped using selected Illumina® Infinium chips. We investigated in a pilot, whether this method developed for DNA from FFPE tissues, is also usable for DNA isolated from toenail clippings. We tested the quality of 39 DNA samples, isolated from prostate cancer cases. The samples that passed the quality control, were restored and tested on the Illumina® HumanCytoSNP_FFPE-12 chip (containing almost 300,000 SNPs).
Results: In the pilot genotyping 30 SNPs of 90 prostate cancer cases in quadruplicate using Sequenom® Massarray, the average sample call rate was 99.0%. After excluding one sample that had been contaminated during pipetting, the reproducibility (across four replications) was 99% for four SNPs and 100% for the remaining analyzed SNPs. 27 out of 30 successfully genotyped SNPs adhered to Hardy Weinberg Equilibrium (χ2-test, p&gt;0.05).
In the pilot using the Infinium® HD FFPE Quality Control Kit, 32 out of 37 samples (86%) passed the test twice. After restoration, using the Infinium® HD FFPE DNA restore kit, we genotyped these samples using the Infinium® chip. Of the first batch of twelve toenail DNAsamples, two samples had call rates &lt;90% (83 and 87% respectively). The remaining samples had an average call rate of 96.4%.
Conclusion: DNA isolated from toenails can be used for genotyping in commercially available platforms like Sequenom® Massarray and Illumina® Infinium.
Citation Format: Leo J. Schouten, Janneke GF Hogervorst, Colinda C. Simons, Leonie Jonkers, Joy Goessens, Roger Godschalk, Sigrun Jackmaert, Bas Verhage, Joris Vermeesch, Frederik-Jan Van Schooten, Matty P. Weijenberg, Piet A. Van den Brandt. DNA isolated from toenails: A new and valid source for high-throughput genotyping in large-scale epidemiological studies. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 72.