Evaluation of MethyLight reactions toward the methylated and unmethylated versions of Alu, LINE-1, Satα and Sat2 sequences on a panel of DNA samples

<p><b>Copyright information:</b></p><p>Taken from "Analysis of repetitive element DNA methylation by MethyLight"</p><p>Nucleic Acids Research 2005;33(21):6823-6836.</p><p>Published online 2 Dec 2005</p><p>PMCID:PMC1301596.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Levels of methylation (in black) are expressed as PMR using DNA treated with M.I as a methylated reference. Levels of unmethylated DNA (in white) are expressed as PUR in which a WGA-DNA sample was used as an unmethylated reference. Each value represents the mean of 3–6 methylation measurements, except for the ALU-M3 reaction, which is the average of two measurements. Error bars indicate the standard error of the mean and have been omitted for the ALU-M3 reaction, as indicated by an asterisk, since we have only two PMR measurements for this reaction. We detected PMR or PUR values o

Scatterplot representation of marker discovery process and ROC curves.

<p>A (top figure: HM27, bottom figure: HM450), scatterplots of the highest PBL β-value (β-PBL_H) of 10 (HM27) and 2 (HM450) healthy control samples (X-axis) against the associated 10th percentile of CRC tumor β-values (β-CRC₁₀) on the Y-axis. The blue dots represent the eliminated probes (HM27: n = 23,049; HM450: n = 367,833) and the red dots (HM27: n = 695; HM450: n = 30,207) represent the retained probes with a β-CRC₁₀>β-PBL_H or a β-PBL_H<0.2. B, scatterplots of the mean normal colon tissue β-value (β-NC_M) for the retained probes from Panel A (X-axis) against the associated β-CRC₁₀ (Y-axis). The red dots (HM27: n = 512; HM450: n = 28,428) represent the eliminated probes, the green dots represent the retained probes (HM27: n = 183; HM450: n = 1779) with a β-CRC₁₀>β-NC_M or a β-NC_M<0.2. C, scatterplots of the retained probes from Panel B (green) displayed by the difference between β-CRC₁₀ and β-PBL_H (X-axis) against the associated β-CRC₁₀ (Y-axis). The dots within the yellow square are the probes selected for additional filtering against other types of cancer. The white arrows point out the probes of the two candidate markers. D, ROC curves for the probes used in the multiplex reaction based on methylation β-values of 335 independent colorectal cancer samples and 23 independent matched normal colorectal tissue samples (the DNA methylation data of these samples were not used in the marker discovery pipeline). The dark grey color is the area under the curve.</p

Overview of samples and data sets used for biomarker discovery.

*<p>normal samples were obtained from surgical specimens of CRC patients, at least 10 cm from the tumor margins.</p>**<p>these samples were among the samples run on the HM27 platform.</p

Clinical characteristics of CRC patients and controls used for plasma and serum analysis.

<p>Clinical characteristics of CRC patients and controls used for plasma and serum analysis.</p

DNA methylation β-values of THBD and C9orf50 in various types of samples.

<p>Jitter plots representing Infinium-based DNA methylation β-values of <i>THBD</i> (left panel) and <i>C9orf50</i> (right panel) in 335 independent CRC tumors, matched normal colon tissues, a variety of other cancer types and PBL from healthy individuals. The specific number of samples for each tissue type is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050266#pone-0050266-t001" target="_blank">Table 1</a>.</p

Schematic representation of colorectal cancer marker discovery and verification pipeline.

<p>We used DNA methylation data from the Infinium HumanMethylation27 Beadchip (HM27) and HumanMethylation450 Beadchip (HM450) Infinium platforms to screen 27,578 (HM27) and 482,421 (HM450) CpG loci for their methylation status in CRC samples, PBL samples from healthy subjects, paired normal colorectal tissue samples (NC) and 15 other types of cancer (OC). We used a stepwise approach eliminating probes that failed in any of the samples, probes that contained SNPs or repeat sequences, probes with a highest PBL β-value (β-PBL_H) or a mean normal colon tissue β-value (β-NC_M) higher than the associated 10th percentile of CRC tumor β-values (β-CRC₁₀) or higher than 0.2 in any of the PBL or NC samples (Infinium panel). The remaining probes were ranked based on the difference between β-CRC₁₀ and β-PBL_H and the top 25 were selected from both datasets (HM27 and HM450) for filtering against OC samples. Probes with a mean OC β-value higher than the associated mean CRC β-value (β-CRC_M) were eliminated. A total of 15 MethyLight reactions (markers) were designed for 10 probes and tested in a sequence of verification steps (MethyLight panel). Markers were eliminated if their performance was suboptimal in controls such as <i>in vitro</i> methylated <i>Sss</i>1 DNA, PBL and plasma samples from healthy controls and CRC tumor tissues. Markers were also eliminated if they failed to detect CRC methylated DNA in pooled plasma and serum from CRC patients. Two markers met all the selection criteria and were advanced in the pipeline for further verification on individual patient samples. (*Probes that failed in any of the samples, as well as those that included SNPs and repeat sequences; **Other cancer types used in this study are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050266#pone-0050266-t001" target="_blank">Table 1</a>, ***M.<i>Sss</i>I treated DNA).</p

Region of chromosome 3 examined with genes and 3 SNPs.

<p>A total of 99 polymorphisms were examined in the Ontario samples across a 500kb region of chromosome 3 surrounding the <i>MLH1</i> gene. Genes in this region are outlined (top panel) along with their transcriptional directionality (bottom panel). The three polymorphisms of interest are indicated. Modified from Ensembl (<a href="http://www.ensembl.org" target="_blank">www.ensembl.org</a>).</p

Logistic regression model results for MSI status with various predictor combinations in the combined data.

<p>Age at diagnosis, sex, and location are covariates common to all the models described above. IHC refers to the MLH1 immunohistochemical staining variable, CH3 refers to the <i>MLH1</i> promoter methylation variable, AIC  =  Akaike's information criterion. Logistic regression models for each SNP per study population and for the combined data are shown in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013314#pone.0013314.s003" target="_blank">File S3</a></b>.</p><p>The role of three SNPs of interest, rs1800734, rs749072, and rs13098279, is explored.</p

Proposed model for genetic susceptibility to DNA methylation in sporadic MSI-H CRCs.

<p>Specific SNPs predispose the region, including the <i>MLH1</i> gene promoter, to methylation, which results in promoter silencing and loss of <i>MLH1</i> gene expression that is measured by immunohistochemical staining. Loss of the <i>MLH1</i> gene expression leads to genome-wide microsatellite instability and MSI-H colorectal cancer.</p

Single marker analysis in the combined data for 3 SNPs for CRC cases versus controls, <i>MLH1</i> promoter methylation, MLH1 IHC staining and MSI tumor status.

<p>Analyses of CRC cases versus controls are adjusted for age, sex, and site.</p><p>OR  =  odds ratio, CI  =  confidence interval.</p><p>Single marker results for the above SNPs for each study population are shown in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013314#pone.0013314.s002" target="_blank">File S2</a></b>.</p