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. 2010 Mar;12(2):177-83.
doi: 10.2353/jmoldx.2010.090106. Epub 2010 Jan 21.

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

Natsumi Irahara  1 , Katsuhiko Nosho, Yoshifumi Baba, Kaori Shima, Neal I Lindeman, Aditi Hazra, Eva S Schernhammer, David J Hunter, Charles S Fuchs, Shuji Ogino
Affiliations

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

Natsumi Irahara et al. J Mol Diagn. 2010 Mar.

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.

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Figures

Figure 1
Figure 1
Pyrosequencing to measure LINE-1 methylation. A: Microdissected colon cancer (Case #1). B: Microdissected colon cancer (Case #2). The “%” numbers (in dark shade) are proportions of C and T at each CpG site after bisulfite conversion. Thus, the methylation level of each CpG site is estimated by the proportion of C (%). An overall LINE-1 methylation level is calculated as the average of the proportions of C at the four CpG sites. The first, third and fourth CpG sites follow stretches of Ts, resulting in higher T peaks (in light shade) than the second CpG site, and the proportion of C has been adjusted accordingly. The arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion.
Figure 2
Figure 2
Strategy to assess precision of bisulfite conversion and PCR-pyrosequencing on each specimen (an example of macrodissected colon cancer Case #1). Bisulfite conversion was performed on seven different aliquots (B1 through B7) from each specimen. PCR-pyrosequencing was performed on the 7 bisulfite-treated specimens (B1–B7), and was repeated seven times on two specimens (B1 and B2) on seven different days (P1 through P7).
Figure 3
Figure 3
Results on repeated measurements of LINE-1 methylation levels. A: Bisulfite-to-bisulfite variation of LINE-1 methylation values. Each dot and vertical bar represent mean and SD [SD or SD(B) in Table 1], respectively, of LINE-1 methylation values among different bisulfite-treated DNA specimens from each case (DNA source). The x-axis indicates DNA sources, ie, 10 macrodissected colon cancers, 5 matched colon cancers collected by LCM, 5 matched normal colons, and 5 unrelated blood DNA specimens, with each number representing Case ID. B: Run-to-run (between-run) variation of LINE-1 methylation values. Each dot and vertical bar represent mean and SD [SD or SD(P) in Table 1], respectively, of LINE-1 methylation values among different runs on the same bisulfite-treated DNA specimen. The x-axis indicates DNA sources, ie, 10 macrodissected colon cancers, 5 matched colon cancers collected by LCM, 5 matched normal colons, and 5 unrelated blood DNA specimens, with each number representing Case ID. Results from B1 (bisulfite-treated specimen #1; see Figure 2) and B2 are shown side-by-side for each DNA source. C: Mean LINE-1 methylation value and SE of mean in the 5 macrodissected colon cancers and matched LCM specimens. Note that each SE of mean is small due to seven repeated measurements. When a LCM specimen showed low-level LINE-1 methylation (eg, Cases 6 and 7), a matched macrodissected specimen also showed low-level LINE-1 methylation.
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