Detecting aberrant DNA methylation in Illumina DNA methylation arrays: a toolbox and recommendations for its use

In this study, our goal was to determine probe-specific thresholds for identifying aberrant, or outlying, DNA methylation and to provide guidance on the relative merits of using continuous or outlier methylation data. To construct a reference database, we downloaded Illumina Human 450K array data for more than 2,000 normal samples, characterized the distribution of DNA methylation and derived probe-specific thresholds for identifying aberrations. We made the decision to restrict our reference database to solid normal tissue and morphologically normal tissue found adjacent to solid tumours, excluding blood which has very distinctive patterns of DNA methylation. Next, we explored the utility of our outlier thresholds in several analyses that are commonly performed on DNA methylation data. Outliers are as effective as the full continuous dataset for simple tasks, like distinguishing tumour tissue from normal, but becomes less useful as the complexity of the problem increases. We developed an R package called OutlierMeth containing our thresholds, as well as functions for applying them to data.</p

Increasing the Capture Rate of Circulating Tumor DNA in Unaltered Plasma Using Passive Microfluidic Mixer Flow Cells

A limiting factor in using blood-based liquid biopsies for cancer detection is the volume of extracted blood required to capture a measurable number of circulating tumor DNA (ctDNA). To overcome this limitation, we developed a technology named the dCas9 capture system to capture ctDNA from unaltered flowing plasma, removing the need to extract the plasma from the body. This technology has provided the first opportunity to investigate whether microfluidic flow cell design can affect the capture of ctDNA in unaltered plasma. With inspiration from microfluidic mixer flow cells designed to capture circulating tumor cells and exosomes, we constructed four microfluidic mixer flow cells. Next, we investigated the effects of these flow cell designs and the flow rate on the rate of captured spiked-in BRAF T1799A (BRAFMut) ctDNA in unaltered flowing plasma using surface-immobilized dCas9. Once the optimal mass transfer rate of ctDNA, identified by the optimal ctDNA capture rate, was determined, we investigated whether the design of the microfluidic device, flow rate, flow time, and the number of spiked-in mutant DNA copies affected the rate of capture by the dCas9 capture system. We found that size modifications to the flow channel had no effect on the flow rate required to achieve the optimal capture rate of ctDNA. However, decreasing the size of the capture chamber decreased the flow rate required to achieve the optimal capture rate. Finally, we showed that, at the optimal capture rate, different microfluidic designs using different flow rates could capture DNA copies at a similar rate over time. In this study, the optimal capture rate of ctDNA in unaltered plasma was identified by adjusting the flow rate in each of the passive microfluidic mixer flow cells. However, further validation and optimization of the dCas9 capture system are required before it is ready to be used clinically

Comparison of 20% sulfur hexafluoride with air for intraocular tamponade in Descemet membrane endothelial keratoplasty (DMEK)

ABSTRACT Purpose: To compare the effect of 20% sulfur hexafluoride (SF6) with that of air on graft detachment rates for intraocular tamponade in Descemet membrane endothelial keratoplasty (DMEK). Methods: Forty-two eyes of patients who underwent DMEK by a single surgeon (A.S.J.) at Wilmer Eye Institute between January 2012 and 2014 were identified; 21 received air for intraocular tamponade and the next consecutive 21 received SF6. The main outcome measure was the graft detachment rate; univariate and multivariate analyses were performed. Results: The graft detachment rate was 67% in the air group and 19% in the SF6 group (p</div

Methylation of the Claudin 1 Promoter Is Associated with Loss of Expression in Estrogen Receptor Positive Breast Cancer

<div><p>Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 <i>via</i> DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA). Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+) breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.</p> </div

Methylation of claudin 1 is associated with loss of expression in human breast cancer.

<p>A: cluster analysis and heat map of claudin 1 methylation (top), histogram of claudin 1 mRNA expression (middle), and ER/HER2 status (bottom) for 217 samples of invasive breast carcinoma and 40 matched normal tissue samples from TCGA; the turquoise dashed boxes mark three clusters of samples with methylation of the claudin 1 CpG island; the orange dashed boxes mark two clusters of ER-, basal-like samples. B: box plot of methylation of the claudin 1 CpG island in the same TCGA samples; T, tumors; N, normal. C: dot plots of claudin 1 gene expression and methylation for the three CpG island sites that have significant Spearman’s correlation coefficients (the position of each probe is noted in the upper right corner); the horizontal line marks the mean methylation value of normal samples plus three standard deviations, which is taken as threshold for methylation; the curve represents the probability of methylation as a function of expression level estimated by fitting a logistic regression model using the logit link function. DNA methylation is shown as beta values, which are the ratio of methylated DNA to total DNA: a value of 1 indicates 100% methylation.</p

Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in <i>Pglyrp</i>-2, -3 and -4

<div><p>Purpose</p><p>Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for <i>Pglyrp</i>-1 in <i>Pseudomonas aeruginosa</i> keratitis. Here we investigated functions of three other related genes <i>Pglyrp</i>-2, -3 and -4 in a mouse model of <i>P</i>. <i>aeruginosa</i> keratitis.</p><p>Methods</p><p>Wild type (WT) and each of the <i>Pglyrp</i>-null genotypes were challenged with <i>P</i>. <i>aeruginosa</i> keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of <i>Pglyrp-1-4</i>, mouse beta defensins (<i>mBD)-2</i>,<i>-3</i>, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the <i>Pglyrp</i>-null mouse types.</p><p>Results</p><p>The <i>Pglyrp-2</i>^<i>-/-</i> mice showed reduced disease and lower induction of pro-inflammatory TNF-α (<i>p</i> = 0.02) than WT or the other <i>Pglyrp</i> null mice. Viable bacterial yield was significantly lower in the <i>Pglyrp</i>-2^-/- (<i>p</i> = 0.0007) and the <i>Pglyrp</i>-4^-/- (<i>p</i> = 0.098) mice. With regards to expression of these antimicrobial genes, <i>Pglyrp</i>-2 expression was induced after infection in WT mice. <i>Pglyrp</i>-3 expression was low before and after infection in WT mice, while <i>Pglyrp</i>-4 expression was slightly elevated after infection in WT, <i>Pglyrp</i>-2 and -3 null mice. <i>Pglyrp</i>-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected <i>Pglyrp-2</i>^-/- males without statistical significance.</p><p>Conclusions</p><p>Efficient resolution of keratitis in the <i>Pglyrp-2</i>^<i>-/-</i> mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of <i>Pglyrp</i>-2 must be regulated to benefit the host.</p></div

Primers used in qRTPCR experiments.

<p>Primers used in qRTPCR experiments.</p

<i>mBD2</i>, <i>mBD3</i> and <i>Cnlp</i> (CRAMP) gene expressions.

<p>The results show expression fold change (2^-ΔΔCt) ± SEM of defensins and <i>CRAMP</i> mRNA relative to <i>Hprt</i> in total RNA from <i>Pglyrp-</i>2 ^-/-, <i>Pglyrp-</i>3 ^-/-, <i>Pglyrp-</i>4 ^-/- whole eyes (6 animals per genotype). Expression of <i>mBD2</i>, <i>mBD3</i> and most notably CRAMP was increased in infected <i>Pglyrp</i>-2^-/- eyes compared to WT without reaching statistical significance.</p

Map of the claudin 1 CpG island and associated methylation probes.

<p>Claudin 1 (<i>CLDN1</i>) mRNA is depicted on top with arrows to indicate the orientation of the gene; the thicker sections represent the coding sequence, the medium sections represent the untranslated exon regions, and the thinner sections represent the introns. The genomic region encompassing claudin 1 is represented in two different scales; the numbers indicate the distance from the TSS in nucleotides. The vertical arrows mark the position of the probes included in the Infinium HumanMethylation450 microarray. The probe excluded from the analysis because of a SNP is indicated as a dashed arrow. The grey box indicates the location of the CpG island. Individual CpG sites are marked as thin vertical marks in the magnified map.</p

Histology of eyes 48 h.p.i.

<p>H and E staining of parasagittal sections through the eye showing the lens (Le), anterior chamber (Ac) and the cornea (Co) (A-E). Cellular inflammatory infiltrates (arrow head) were quantified by counting the nuclei using Image J from 3–5 animals per genotype (F) (additional details provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137129#pone.0137129.s002" target="_blank">S2 Fig</a>).</p