Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 53: 1168-1170, 2007; 10.1373/clinchem.2006.085043
This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow 085043.Supplemental Data
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Breda, S. G.
Right arrow Articles by van den Brandt, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Breda, S. G.
Right arrow Articles by van den Brandt, P. A.
Related Collections
Right arrow General Clinical Chemistry
(Clinical Chemistry. 2007;53:1168-1170.)
© 2007 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Toenails: An Easily Accessible and Long-Term Stable Source of DNA for Genetic Analyses in Large-Scale Epidemiological Studies

Simone G. van Breda1, Janneke G. Hogervorst2, Leo J. Schouten2, Ad M. Knaapen1, Joost H. van Delft1, R. Alexandra Goldbohm3, Frederik J. van Schooten1 and Piet A. van den Brandt2,a

1 Department of Health Risk Analysis and Toxicology and
2 Department of Epidemiology NUTRIM, Maastricht University, Maastricht, The Netherlands
3 Department of Food and Chemical Risk Analysis, TNO Quality of Life, Zeist, The Netherlands

aAddress correspondence to this author at: Department of Epidemiology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Fax 31-43-3884128; e-mail PA.vandenBrandt{at}EPID.unimaas.nl.


To the Editor:

Molecular tools are increasingly applied in epidemiological studies to unravel the relationship between environmental exposures and disease (1). DNA is required for analyses of genetic factors, such as polymorphisms, but currently used specimens, such as lymphocytes and buccal cells, have disadvantages related to collection, transport, storage, and processing of samples. A relatively infrequently used source of DNA that may overcome these problems is nail material. Human toenails have been collected in several epidemiological studies, predominantly for determination of trace elements as biomarkers for the intake of these compounds (2). Until now, none of these epidemiological studies have applied human toenails as a source of DNA.

We investigated whether toenail material collected 20 years ago in the Netherlands Cohort Study on Diet and Cancer (NLCS) (3) (n = 120 852) could be used as a source of DNA for analyses of multiple genetic polymorphisms. Approximately 90 000 participants provided toenail clippings (on average, 80 mg per participant) (2). We optimized a protocol for DNA isolation from ~10 mg toenail material, based on the method of Cline et al. (4), and tested the suitability of this DNA in 2 PCR-based assays in a subgroup of the cohort (n = 57) for which buccal DNA was also available. In the 1st assay, 10 single nucleotide polymorphisms were amplified in a multiplex PCR reaction and subsequently genotyped by means of single base extension using primer extension and automated capillary gel electrophoresis as described by Knaapen et al. (5). A 2nd PCR-based test was used to investigate the maximum length of fragments that could be amplified. A portion of the toenail samples had been irradiated with neutrons for analyses of selenium content (not irradiated: n = 33; irradiated: n = 24). To assess possible effects of age of the toenails on the quality of the DNA, freshly harvested toenail material from healthy non-NLCS volunteers was investigated (n = 11) (see the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol53/issue6 for a detailed description of material and methods).

DNA was successfully isolated from all toenail samples. On average, 2 µg DNA could be extracted from 10 mg toenail material (Table 1 ). There was no significant difference in DNA quantity between the various groups of toenail samples. The large range in DNA yield was mainly due to 2 outliers. The minimum amount isolated was almost 1 µg DNA/10 mg toenail material, which is sufficient for more than 10 multiplex genotyping analyses (80 ng of DNA is sufficient per analysis).


View this table:
[in this window]
[in a new window]

  		Table 1. DNA yield and multiplex genotyping results.

A summary of quantitative results of the multiplex genotyping assays on the DNA samples is provided in Table 1Up . A genotype profile of 10 polymorphisms was successfully generated for 100%, 96%, and 94% of the DNA samples isolated from fresh, 20-year-old irradiated, and 20-year-old not irradiated toenail material, respectively (amplicon sizes 92–148 bp). The success rate using the buccal swab DNA samples was 100%. Because DNA isolation was successful for only 90% of the buccal swab material in the NLCS samples, the use of nail material as a source of DNA resulted in a higher rate of successful outcomes. For 35 individuals, we compared the outcome of the genotyping assay for both their buccal swab DNA and toenail material DNA. Surprisingly, for 1 person, the 2 sources of DNA resulted in different genetic profiles. This result could not be related to technical errors, but may be from the switching or mislabeling of a sample during the collection process 20 years ago.

The NLCS study received approval by the Medical Ethical Committees of the University Hospital Maastricht and TNO Quality of Life, Zeist, The Netherlands. Study participants gave informed consent.

Results from the 2nd PCR test showed that DNA isolated from 20-year-old (not irradiated) toenail material or from fresh toenail material could be amplified to ≤596 bp. With DNA from 20-year-old irradiated nail material, generation of the 596-bp amplicon was unsuccessful for 60% of the samples, indicating increased fragmentation of the DNA. Irradiation probably causes fragmentation of the DNA, an effect that must be taken into account when the available nail material has previously been used for trace element analyses, which require irradiation.

In conclusion, we showed that 20-year-old nonirradiated and irradiated toenails can be a source of DNA for state of the art high-throughput genetic analyses of polymorphisms. For existing large-scale epidemiological studies, our results demonstrate that toenail material can be used for genetic analyses in cohorts for which no other source of DNA is available. The use of toenails as source of DNA may be of considerable relevance in future molecular epidemiological studies, because toenail clippings can be stored for long periods at low costs while DNA quality remains constant.


Acknowledgments

Grant/funding support: This study was supported by the Network of Excellence "Environmental Cancer Risk, Nutrition and Individual Susceptibility" (ECNIS), which operates in the context of the 6th European Union Framework Program for Research and Development (FP6). A.M.K. is supported by a postdoctoral fellowship from the Netherlands Organization for Scientific Research (NWO, VENI-Grant 916.46.092).

Financial disclosures: None declared.

Acknowledgments: We thank Lucy van de Vijver and Erik Busink from TNO Quality of Life for the collection of buccal swabs; Kim Wouters from the Department of Pathology of the University Hospital Maastricht; and Prof. Harry Ostrer from the Departments of Pediatrics (Genetics) and Pathology and Medicine (Genetics) of the New York University Medical Center for DNA isolation from the buccal swabs.


References

  1. Perera FP, Weinstein IB. Molecular epidemiology: recent advances and future directions. Carcinogenesis 2000;21:517-524.[Abstract/Free Full Text]
  2. van den Brandt PA, Goldbohm RA, van ’t Veer P, Bode P, Dorant E, Hermus RJ, et al. A prospective cohort study on selenium status and the risk of lung cancer. Cancer Res 1993;53:4860-4865.[Abstract/Free Full Text]
  3. van den Brandt PA, Goldbohm RA, van ’t Veer P, Volovics A, Hermus RJ, Sturmans F. A large-scale prospective cohort study on diet and cancer in The Netherlands. J Clin Epidemiol 1990;43:285-295.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  4. Cline RE, Laurent NM, Foran DR. The fingernails of Mary Sullivan: developing reliable methods for selectively isolating endogenous and exogenous DNA from evidence. J Forensic Sci 2003;48:328-333.[ISI][Medline] [Order article via Infotrieve]
  5. Knaapen AM, Ketelslegers HB, Gottschalk RW, Janssen RG, Paulussen AD, Smeets HJ, et al. Simultaneous genotyping of nine polymorphisms in xenobiotic-metabolizing enzymes by multiplex PCR amplification and single base extension. Clin Chem 2004;50:1664-1668.[Free Full Text]




This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow 085043.Supplemental Data
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Breda, S. G.
Right arrow Articles by van den Brandt, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Breda, S. G.
Right arrow Articles by van den Brandt, P. A.
Related Collections
Right arrow General Clinical Chemistry

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS