Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells.

Abstract

Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.

• Authors: Natsumi Irahara, Katsuhiko Nosho, Yoshifumi Baba, Kaori Shima, Neal I Lindeman, Aditi Hazra, Eva S Schernhammer, David J Hunter, Charles S Fuchs, Shuji Ogino
• Journal: The Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 12 Issue 2 Pg. 177-83 (Mar 2010) ISSN: 1943-7811 [Electronic] United States
• PMID: 20093385 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Sulfites
• sodium bisulfite

Topics

• Blood Cells (physiology)
• Colon (anatomy & histology, pathology, physiology)
• Colonic Neoplasms (genetics)
• DNA Methylation
• Genomic Instability
• Humans
• Intestinal Mucosa (physiology)
• Long Interspersed Nucleotide Elements
• Risk Factors
• Sensitivity and Specificity
• Sequence Analysis, DNA (methods)
• Sulfites (chemistry)