| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters to the Editor |
Departments of1
Human Genetics and
2 Plastic and Reconstructive Surgery, and
3 Division of Functional Genomics, Center for Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
4 Kyushu Medical Science, Nagasaki, Japan
5 Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan
6 Solution Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), Tokyo, Japan
aAddress correspondence to this author at:, Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki 852-8523, Japan, Fax +81-95-849-7121, E-mail kyoshi{at}nagasaki-u.ac.jp
To the Editor:
Recently, high-throughput single-nucleotide polymorphism (SNP) genotyping arrays have been used for genome-wide association study. Blood samples are commonly used for such analyses because they provide genomic DNA of high molecular weight and in high quantities. Blood sampling is often difficult, however, when patients are located a great distance from the laboratory and the sample requires careful handling for transportation. Instead, buccal swabs or urinary epithelial cells have been used as noninvasive sources of DNA(1). Fingernail clippings are also obtained noninvasively and more easily. Such samples need no special attention for transportation and can be preserved dry at room temperature for long periods; however, because fingernail clippings yield fragmented DNA that may be contaminated with certain PCR inhibitors(2), this sample type has seemed unsuitable for analyses requiring high-quality genomic DNA. In this study, we compared the precision of Affymetrix GeneChipTM array-based SNP genotyping with fingernail DNA vs blood DNA and demonstrated the usefulness of fingernail DNA in genotyping and genome copy-number analysis.
The study protocol was approved by the Ethics Committee on Human Genome and Gene Analysis, Nagasaki University, and written informed consent was obtained from every volunteer. Ten milliliters of peripheral blood and 10 fingernail clippings were obtained from 5 healthy volunteers. Blood DNA was extracted with the standard phenol-chloroform method. Clipped finger-nails were frozen in liquid nitrogen and crushed into fine powder with Multibeads ShockerTM (Yasui Kikai). The nail powder was dissolved in a urea-containing lysis solution (2 mol/L urea, 5 g/L SDS, 10 mmol/L Tris-HCl, pH 7.5, 50 mmol/L EDTA) containing 1 g/L proteinase K and 40 mmol/L dithiothreitol and was incubated overnight at 55 °C. Nail DNA was extracted by the phenol-chloroform method and suspended in 30 µL 1x Tris-EDTA buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0). Genome-wide SNP genotyping was performed with the GeneChipTM Human Mapping 250K Nsp Array according to the manufacturer’s assay manual (Affymetrix). SNP data were analyzed with GTYPE software (Affymetrix). This analysis is based on the dynamic modeling algorithm that is included in GTYPE. Genome copy-number analysis was performed with CNAG software (http://www.genome.umin.jp/)(3)(4).
The human nail plate includes soft and hard types of keratin. Hard keratin constitutes >80% of nail keratin(5) and contains large numbers of cysteine moieties linked by multiple disulfide bonds that make the nail insoluble in lysis buffer. We froze fingernail clippings in liquid nitrogen, mechanically crushed them into as fine a powder as possible, and then treated them with 2 mol/L urea. With this procedure, we were able to lyse the nail proteins almost completely without denaturing the DNA. Consequently, 10 mg (2 pieces) of fingernail clippings yielded approximately 1 µg DNA, an amount ample for the GeneChip system.
We assumed in this study that all SNP calls for the blood samples were correct, and these calls were used as baseline values for comparison with the calls for nail DNA. The mean total call rate for the blood samples was 96.26%, and that of the nail samples was 94.76%. The mean concordance rates for homozygous and heterozygous SNPs were 99.81% and 98.78%, respectively (Table 1
). The concordance rates for heterozygous SNPs in nail samples decreased as the total call rates decreased. Therefore, the discrepancy in the call rates between the 2 different sources of DNA is most likely due to some incorrect calls for heterozygous SNPs in the nail DNA. In other words, many cases of lower call rates in nail samples reflect erroneous calls as homozygous SNPs instead of as heterozygous SNPs; however, the call rates and concordance values for nail DNA were sufficient for genome-wide association studies. Moreover, we performed SNP genotyping with DNA from old fingernail clippings that had been preserved for >5 years at room temperature. The total call rate for old nail DNA was 95% or higher, showing that results for nail clippings preserved for a long time were equivalent to those obtained with fresh clippings.
|
We also used copy-number variation analysis to compare signal homogeneity for blood and nail DNA on the GeneChip. In this study, volunteer no. 5 had a duplication polymorphism in chromosome 8 that was clearly demonstrated in both sources of DNA (data not shown). The results of the present copy-number variation analysis showed that the results obtained with nail samples were equivalent to those for blood DNA with respect to the accuracy of detecting such variation.
In conclusion, our study comparing blood and fingernail DNA with respect to SNP-genotyping accuracy on the 250K Affymetrix GeneChip systems revealed that nail DNA was as useful as blood DNA for both genome-wide association studies and genome copy-number analysis. Our results reinforce the merits of using nail samples, because nails can be stored at room temperature for a long period and need not be processed immediately for DNA extraction.
Acknowledgments
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Norio Niikawa, Grants-in-Aid for Scientific Research (Nos. 17019055 and 19390095, respectively) from the Ministry of Education, Sports, Culture, Science and Technology of Japan, and was supported by Solution Oriented Research for Science and Technology (SORST) from Japan Science and Technology Agency (JST); Koh-ichiro Yoshiura, Grants-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Acknowledgments: We thank Ms. Yasuko Noguchi, Ayano Goto, and Miho Ooga for their technical assistance.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
