Genome-wide analysis of alternative promoters of human genes using a custom promoter tiling array

<p>Abstract</p> <p>Background</p> <p>Independent lines of evidence suggested that a large fraction of human genes possess multiple promoters driving gene expression from distinct transcription start sites. Understanding which promoter is employed in which cellular context is required to unravel gene regulatory networks within the cell.</p> <p>Results</p> <p>We have developed a custom microarray platform that tiles roughly 35,000 alternative putative promoters from nearly 7,000 genes in the human genome. To demonstrate the utility of this array platform, we have analyzed the patterns of promoter usage in 17β-estradiol (E2)-treated and untreated MCF7 cells and show widespread usage of alternative promoters. Most intriguingly, we show that the downstream promoter in E2-sensitive multiple promoter genes tends to be very close to the 3'-terminus of the gene, suggesting exotic mechanisms of expression regulation in these genes.</p> <p>Conclusion</p> <p>The usage of alternative promoters greatly multiplies the transcriptional complexity available within the human genome. The fact that many of these promoters are incapable of driving the synthesis of a meaningful protein-encoding transcript further complicates the story.</p

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management

Netboost: boosting-supported network analysis improves high-dimensional omics prediction in acute myeloid leukemia and Huntington’s disease

State-of-the art selection methods fail to identify weak but cumulative effects of features found in many high-dimensional omics datasets. Nevertheless, these features play an important role in certain diseases. We present Netboost, a three-step dimension reduction technique. First, a boosting-based filter is combined with the topological overlap measure to identify the essential edges of the network. Second, sparse hierarchical clustering is applied on the selected edges to identify modules and finally module information is aggregated by the first principal components. We demonstrate the application of the newly developed Netboost in combination with CoxBoost for survival prediction of DNA methylation and gene expression data from 180 acute myeloid leukemia (AML) patients and show, based on cross-validated prediction error curve estimates, its prediction superiority over variable selection on the full dataset as well as over an alternative clustering approach. The identified signature related to chromatin modifying enzymes was replicated in an independent dataset, the phase II AMLSG 12-09 study. In a second application we combine Netboost with Random Forest classification and improve the disease classification error in RNA-sequencing data of Huntington's disease mice. Netboost is a freely available Bioconductor R package for dimension reduction and hypothesis generation in high-dimensional omics applications

High-pitch dual-source CT angiography of the aortic valve-aortic root complex without ECG-synchronization

Purpose: To compare image quality and radiation dose of high-pitch computed tomography angiography(CTA) of the aortic valve-aortic root complex with and without prospective ECG-gating compared to a retrospectively ECG-gated standard-pitch acquisition. Materials and Methods: 120 patients(mean age 68 ± 13years) were examined using a 128-slice dual-source CT system using prospectively ECG-gated high-pitch(group A; n = 40), non-ECG-gated high-pitch(group B; n = 40) or retrospectively ECG-gated standard-pitch(C; n = 40) acquisition techniques. Image quality of the aortic root, valve and ascending aorta including the coronary ostia was assessed by two independent readers. Image noise was measured, radiation dose estimates were calculated. Results: Interobserver agreement was good(κ = 0.64-0.78). Image quality was diagnostic in 38/40 patients(group A), 37/40(B) and 38/40(C) with no significant difference in number of patients with diagnostic image quality among all groups (p = 0.56). Significantly more patients showed excellent image quality in group A compared to groups B and C(each, p < 0.01). Average image noise was significantly different between all groups(p < 0.05). Mean radiation dose estimates in groups A and B(each; 2.4 ± 0.3mSv) were significantly lower compared to group C(17.5 ± 4.4mSv; p < 0.01). Conclusion: High-pitch dual-source CTA provides diagnostic image quality of the aortic valve-aortic root complex even without ECG-gating at 86% less radiation dose when compared to a standard-pitch ECG-gated acquisitio

Principles for the post-GWAS functional characterisation of risk loci

Several challenges lie ahead in assigning functionality to susceptibility SNPs. For example, most effect sizes are small relative to effects seen in monogenic diseases, with per allele odds ratios usually ranging from 1.15 to 1.3. It is unclear whether current molecular biology methods have enough resolution to differentiate such small effects. Our objective here is therefore to provide a set of recommendations to optimize the allocation of effort and resources in order maximize the chances of elucidating the functional contribution of specific loci to the disease phenotype. It has been estimated that 88% of currently identified disease-associated SNP are intronic or intergenic. Thus, in this paper we will focus our attention on the analysis of non-coding variants and outline a hierarchical approach for post-GWAS functional studies

Diverse histone modifications on histone 3 lysine 9 and their relation to DNA methylation in specifying gene silencing

<p>Abstract</p> <p>Background</p> <p>Previous studies of individual genes have shown that in a self-enforcing way, dimethylation at histone 3 lysine 9 (dimethyl-H3K9) and DNA methylation cooperate to maintain a repressive mode of inactive genes. Less clear is whether this cooperation is generalized in mammalian genomes, such as mouse genome. Here we use epigenomic tools to simultaneously interrogate chromatin modifications and DNA methylation in a mouse leukemia cell line, L1210.</p> <p>Results</p> <p>Histone modifications on H3K9 and DNA methylation in L1210 were profiled by both global CpG island array and custom mouse promoter array analysis. We used chromatin immunoprecipitation microarray (ChIP-chip) to examine acetyl-H3K9 and dimethyl-H3K9. We found that the relative level of acetyl-H3K9 at different chromatin positions has a wider range of distribution than that of dimethyl-H3K9. We then used differential methylation hybridization (DMH) and the restriction landmark genome scanning (RLGS) to analyze the DNA methylation status of the same targets investigated by ChIP-chip. The results of epigenomic profiling, which have been independently confirmed for individual loci, show an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing. In contrast to the previous notion, dimethyl-H3K9 seems to be less distinct in specifying silencing for the genes tested.</p> <p>Conclusion</p> <p>This study demonstrates in L1210 leukemia cells a diverse relationship between histone modifications and DNA methylation in the maintenance of gene silencing. Acetyl-H3K9 shows an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing as expected. However, dimethyl-H3K9 seems to be less distinct in relation to promoter methylation. Meanwhile, a combination of epigenomic tools is of help in understanding the heterogeneity of epigenetic regulation, which may further our vision accumulated from single-gene studies.</p

Off-pump surgery for the poor ventricle?

Severely decreased ejection-fraction is an established risk-factor for worse outcome after cardiac surgery. We compare outcomes of off-pump coronary artery bypass grafting (OPCAB) and on-pump CABG (ONCABG) in patients with severely compromised EF. From 2004 to 2009, 478 patients with a decreased EF ≤35% underwent myocardial-revascularization. Patients received either OPCAB (n=256) or ONCABG (n=222). Propensity score (PS), including 50 preoperative risk-factors, was used to balance characteristics between groups. PS adjusted logistic regression analysis was performed to assess mortality and major adverse cardiac and cerebrovascular events (MACCE). A composite endpoint for major non-cardiac complications such as respiratory failure, renal failure, rethoracotomy was applied. Complete revascularization (CR) was assumed when the number of distal anastomoses was larger than that of diseased vessels. There was no difference for mortality (2.3 vs. 4.1%; PS-adjusted odds ratio (PS-OR)=1.05; p=0.93) and MACCE (13.7 vs. 17.6%; PS-OR=1.22; p=0.50) including myocardial-infarction (1.4 vs. 4.9%; PS-OR=0.39; p=0.26), low cardiac output (2.3 vs. 4.7%; PS-OR=0.75; p=0.72) and stroke (2.3 vs. 2.7%; PS-OR=0.69; p=0.66). OPCAB patients presented with a trend to less frequent occurrence of the non-cardiac composite (12.1 vs. 22.1%; PS-OR=0.54; p=0.059) including renal dysfunction (PAOR=0.77; 95% CI 0.31-1.9; p=0.57), bleeding (PAOR=0.42; 95% CI 0.14-1.20; p=0.10) and respiratory failure (PAOR=0.39; 95% CI 0.05-3.29; p=0.39). The rate of complete revascularization was similar (92.2 vs. 92.8%; PS-OR=0.75; p=0.50). OPCAB in patients with severely decreased EF is safe and feasible. It may even benefit these patients in regard to non-cardiac complications and does not come at cost of less complete revascularizatio

Mining Methylation for Early Detection of Common Cancers

A single method that detects multiple common cancer types at an early stage would have the biggest payoff for cancer control, say Brena and colleagues