Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10 000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation

Increased 5-hydroxymethylcytosine and decreased 5-methylcytosine are indicators of global epigenetic dysregulation in diffuse intrinsic pontine glioma

Introduction Diffuse intrinsic pontine glioma (DIPG) is a malignant pediatric brain tumor associated with dismal outcome. Recent high-throughput molecular studies have shown a high frequency of mutations in histone-encoding genes (H3F3A and HIST1B) and distinctive epigenetic alterations in these tumors. Epigenetic alterations described in DIPG include global DNA hypomethylation. In addition to the generally repressive methylcytosine DNA alteration, 5-hydroxymethylation of cytosine (5hmC) is recognized as an epigenetic mark associated with active chromatin. We hypothesized that in addition to alterations in DNA methylation, that there would be changes in 5hmC. To test this hypothesis, we performed immunohistochemical studies to compare epigenetic alterations in DIPG to extrapontine adult and pediatric glioblastoma (GBM) and normal brain. A total of 124 tumors were scored for histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3) and 104 for 5hmC and 5-methylcytosine (5mC). An H-score was derived by multiplying intensity (0–2) by percentage of positive tumor nuclei (0-100%). Results We identified decreased H3K27me3 in the DIPG cohort compared to pediatric GBM (p \u3c 0.01), adult GBM (p \u3c 0.0001) and normal brain (p \u3c 0.0001). H3K9me3 was not significantly different between tumor types. Global DNA methylation as measured by 5mC levels were significantly lower in DIPG compared to pediatric GBM (p \u3c 0.001), adult GBM (p \u3c 0.01), and normal brain (p \u3c 0.01). Conversely, 5hmC levels were significantly higher in DIPG compared to pediatric GBM (p \u3c 0.0001) and adult GBM (p \u3c 0.0001). Additionally, in an independent set of DIPG tumor samples, TET1 andTET3 mRNAs were found to be overexpressed relative to matched normal brain. Conclusions Our findings extend the immunohistochemical study of epigenetic alterations in archival tissue to DIPG specimens. Low H3K27me3, decreased 5mC and increased 5hmC are characteristic of DIPG in comparison with extrapontine GBM. In DIPG, the relative imbalance of 5mC compared to 5hmC may represent an opportunity for therapeutic intervention

Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.Published versio

High sensitivity measurements of the CMB power spectrum with the extended Very Small Array

We present deep Ka-band ($\nu \approx 33$ GHz) observations of the CMB made with the extended Very Small Array (VSA). This configuration produces a naturally weighted synthesized FWHM beamwidth of $\sim 11$ arcmin which covers an $\ell$-range of 300 to 1500. On these scales, foreground extragalactic sources can be a major source of contamination to the CMB anisotropy. This problem has been alleviated by identifying sources at 15 GHz with the Ryle Telescope and then monitoring these sources at 33 GHz using a single baseline interferometer co-located with the VSA. Sources with flux densities \gtsim 20 mJy at 33 GHz are subtracted from the data. In addition, we calculate a statistical correction for the small residual contribution from weaker sources that are below the detection limit of the survey. The CMB power spectrum corrected for Galactic foregrounds and extragalactic point sources is presented. A total $\ell$-range of 150-1500 is achieved by combining the complete extended array data with earlier VSA data in a compact configuration. Our resolution of $\Delta \ell \approx 60$ allows the first 3 acoustic peaks to be clearly delineated. The is achieved by using mosaiced observations in 7 regions covering a total area of 82 sq. degrees. There is good agreement with WMAP data up to $\ell=700$ where WMAP data run out of resolution. For higher $\ell$-values out to $\ell = 1500$, the agreement in power spectrum amplitudes with other experiments is also very good despite differences in frequency and observing technique.Comment: 16 pages. Accepted in MNRAS (minor revisions

An Inflammatory Cascade Leading to Hyperresistinemia in Humans

BACKGROUND: Obesity, the most common cause of insulin resistance, is increasingly recognized as a low-grade inflammatory state. Adipocyte-derived resistin is a circulating protein implicated in insulin resistance in rodents, but the role of human resistin is uncertain because it is produced largely by macrophages. METHODS AND FINDINGS: The effect of endotoxin and cytokines on resistin gene and protein expression was studied in human primary blood monocytes differentiated into macrophages and in healthy human participants. Inflammatory endotoxin induced resistin in primary human macrophages via a cascade involving the secretion of inflammatory cytokines that circulate at increased levels in individuals with obesity. Induction of resistin was attenuated by drugs with dual insulin-sensitizing and anti-inflammatory properties that converge on NF-κB. In human study participants, experimental endotoxemia, which produces an insulin-resistant state, causes a dramatic rise in circulating resistin levels. Moreover, in patients with type 2 diabetes, serum resistin levels are correlated with levels of soluble tumor necrosis factor α receptor, an inflammatory marker linked to obesity, insulin resistance, and atherosclerosis. CONCLUSIONS: Inflammation is a hyperresistinemic state in humans, and cytokine induction of resistin may contribute to insulin resistance in endotoxemia, obesity, and other inflammatory states

Role of TMPRSS2-ERG Gene Fusion in Negative Regulation of PSMA Expression

Prostate specific membrane antigen (PSMA) is overexpressed in prostatic adenocarcinoma (CaP), and its expression is negatively regulated by androgen stimulation. However, it is still unclear which factors are involved in this downregulation. TMPRSS2-ERG fusion is the most common known gene rearrangement in prostate carcinoma. Androgen stimulation can increase expression of the TMPRSS2-ERG fusion in fusion positive prostate cancer cells. The purpose of this investigation is to determine whether PSMA expression can be regulated by the TMPRSS2-ERG gene fusion. We employed two PSMA positive cell lines: VCaP cells, which harbor TMPRSS2-ERG fusion, and LNCaP cells, which lack the fusion. After 24 hours of androgen treatment, TMPRSS2-ERG mRNA level was increased in VCaP cells. PSMA mRNA level was dramatically decreased in VCaP cells, while it only has moderate change in LNCaP cells. Treatment with the androgen antagonist flutamide partially restored PSMA expression in androgen-treated VCaP cells. Knocking down ERG by siRNA in VCaP cells enhances PSMA expression both in the presence and absence of synthetic androgen R1881. Overexpressing TMPRSS2-ERG fusions in LNCaP cells downregulated PSMA both in the presence or absence of R1881, while overexpressing wild type ERG did not. Using PSMA-based luciferase reporter assays, we found TMPRSS2-ERG fusion can inhibit PSMA activity at the transcriptional level. Our data indicated that downregulation of PSMA in androgen-treated VCaP cells appears partially mediated by TMPRSS2-ERG gene fusion

Dysregulated Nephrin in Diabetic Nephropathy of Type 2 Diabetes: A Cross Sectional Study

Podocyte specific proteins are dysregulated in diabetic nephropathy, though the extent of their expression loss is not identical and may be subject to different regulatory factors. Quantifying the degree of loss may help identify the most useful protein to use as an early biomarker of diabetic nephropathy.Protein expression of synaptopodin, podocin and nephrin were quantified in 15 Type 2 diabetic renal biopsies and 12 control patients. We found statistically significant downregulation of synaptopodin (P<0.0001), podocin (P = 0.0002), and nephrin (P<0.0001) in kidney biopsies of diabetic nephropathy as compared with controls. Urinary nephrin levels (nephrinuria) were then measured in 66 patients with Type 2 diabetes and 10 healthy controls by an enzyme-linked immunosorbent assay (Exocell, Philadelphia, PA). When divided into groups according to normo-, micro-, and macroalbuminuria, nephrinuria was found to be present in 100% of diabetic patients with micro- and macroalbuminuria, as well as 54% of patients with normoalbuminuria. Nephrinuria also correlated significantly with albuminuria (rho = 0.89, p<0.001), systolic blood pressure (rho = 0.32, p = 0.007), and correlated negatively with serum albumin (rho = -0.48, p<0.0001) and eGFR (rho = -0.33, p = 0.005).These data suggest that key podocyte-specific protein expressions are significantly and differentially downregulated in diabetic nephropathy. The finding that nephrinuria is observed in a majority of these normoalbuminuric patients demonstrates that it may precede microalbuminuria. If further research confirms nephrinuria to be a biomarker of pre-clinical diabetic nephropathy, it would shed light on podocyte metabolism in disease, and raise the possibility of new and earlier therapeutic targets

Explaining gender differences in non-fatal suicidal behaviour among adolescents: a population-based study

<p>Abstract</p> <p>Background</p> <p>While suicide is the second leading cause of death among young people in most industrial countries, non-fatal suicidal behaviour is also a very important public health concern among adolescents. The aim of this study was to investigate gender differences in prevalence and emotional and behavioural correlates of suicidal behaviour in a representative school-based sample of adolescents.</p> <p>Methods</p> <p>A cross-sectional design was used to assess suicidal behaviour and various areas of emotional and behavioural problems by using a self-report booklet including the Youth Self-Report. One hundred sixteen schools in a region of Southern Germany agreed to participate. A representative sample of 5,512 ninth-grade students was studied. Mean age was 14.8 years (SD 0.73); 49.8% were female.</p> <p>Results</p> <p>Serious suicidal thoughts were reported by 19.8% of the female students and 10.8% of the females had ever attempted suicide. In the male group, 9.3% had a history of suicidal thoughts and 4.9% had previously attempted suicide. Internalizing emotional and behavioural problems were shown to be higher in the female group (difference of the group means 4.41) while externalizing emotional and behavioural problems slightly predominated in male students (difference of the group means -0.65). However, the total rate of emotional and behavioural problems was significantly higher in the adolescent female group (difference of the group means 4.98). Using logistic regression models with suicidal thoughts or attempted suicide as dependent variables, the pseudo-R² of gender alone was only 2.7% or 2.3%, while it was 30% or 23.2% for emotional and behavioural problems measured by the YSR syndrome scales. By adding gender to the emotional and behavioural problems only an additional 0.3% of information could be explained.</p> <p>Conclusions</p> <p>The findings suggest that gender differences in non-fatal suicidal behaviour among adolescents can to a large extent be explained by the gender differences in emotional and behavioural problems during this age.</p