The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that ...probes come in two different designs, characterized by widely different DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs.
Here we propose a novel model-based intra-array normalization strategy for 450 k data, called BMIQ (Beta MIxture Quantile dilation), to adjust the beta-values of type2 design probes into a statistical distribution characteristic of type1 probes. The strategy involves application of a three-state beta-mixture model to assign probes to methylation states, subsequent transformation of probabilities into quantiles and finally a methylation-dependent dilation transformation to preserve the monotonicity and continuity of the data. We validate our method on cell-line data, fresh frozen and paraffin-embedded tumour tissue samples and demonstrate that BMIQ compares favourably with two competing methods. Specifically, we show that BMIQ improves the robustness of the normalization procedure, reduces the technical variation and bias of type2 probe values and successfully eliminates the type1 enrichment bias caused by the lower dynamic range of type2 probes. BMIQ will be useful as a preprocessing step for any study using the Illumina Infinium 450 k platform.
BMIQ is freely available from http://code.google.com/p/bmiq/.
a.teschendorff@ucl.ac.uk
Supplementary data are available at Bioinformatics online.
The high-throughput analysis of microRNAs (miRNAs) circulating within the blood of healthy and diseased individuals is an active area of biomarker research. Whereas quantitative real-time reverse ...transcription polymerase chain reaction (qPCR)-based methods are widely used, it is yet unresolved how the data should be normalized. Here, we show that a combination of different algorithms results in the identification of candidate reference miRNAs that can be exploited as normalizers, in both discovery and validation phases. Using the methodology considered here, we identify normalizers that are able to reduce nonbiological variation in the data and we present several case studies, to illustrate the relevance in the context of physiological or pathological scenarios. In conclusion, the discovery of stable reference miRNAs from high-throughput studies allows appropriate normalization of focused qPCR assays.
Regular endurance exercise training induces beneficial functional and health effects in human skeletal muscle. The putative contribution to the training response of the epigenome as a mediator ...between genes and environment has not been clarified. Here we investigated the contribution of DNA methylation and associated transcriptomic changes in a well-controlled human intervention study. Training effects were mirrored by significant alterations in DNA methylation and gene expression in regions with a homogeneous muscle energetics and remodeling ontology. Moreover, a signature of DNA methylation and gene expression separated the samples based on training and gender. Differential DNA methylation was predominantly observed in enhancers, gene bodies and intergenic regions and less in CpG islands or promoters. We identified transcriptional regulator binding motifs of MRF, MEF2 and ETS proteins in the proximity of the changing sites. A transcriptional network analysis revealed modules harboring distinct ontologies and, interestingly, the overall direction of the changes of methylation within each module was inversely correlated to expression changes. In conclusion, we show that highly consistent and associated modifications in methylation and expression, concordant with observed health-enhancing phenotypic adaptations, are induced by a physiological stimulus.
Cigarette smoking is an established environmental risk factor for Multiple Sclerosis (MS), a chronic inflammatory and neurodegenerative disease, although a mechanistic basis remains largely unknown. ...We aimed at investigating how smoking affects blood DNA methylation in MS patients, by assaying genome-wide DNA methylation and comparing smokers, former smokers and never smokers in two Swedish cohorts, differing for known MS risk factors. Smoking affects DNA methylation genome-wide significantly, an exposure-response relationship exists and the time since smoking cessation affects methylation levels. The results also show that the changes were larger in the cohort bearing the major genetic risk factors for MS (female sex and HLA risk haplotypes). Furthermore, CpG sites mapping to genes with known genetic or functional role in the disease are differentially methylated by smoking. Modeling of the methylation levels for a CpG site in the AHRR gene indicates that MS modifies the effect of smoking on methylation changes, by significantly interacting with the effect of smoking load. Alongside, we report that the gene expression of AHRR increased in MS patients after smoking. Our results suggest that epigenetic modifications may reveal the link between a modifiable risk factor and the pathogenetic mechanisms.
The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, ...the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.
Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of ...lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.
We introduce and develop a method that demonstrates that the algorithmic information content of a system can be used as a steering handle in the dynamical phase space, thus affording an avenue for ...controlling and reprogramming systems. The method consists of applying a series of controlled interventions to a networked system while estimating how the algorithmic information content is affected. We demonstrate the method by reconstructing the phase space and their generative rules of some discrete dynamical systems (cellular automata) serving as controlled case studies. Next, the model-based interventional or causal calculus is evaluated and validated using (1) a huge large set of small graphs, (2) a number of larger networks with different topologies, and finally (3) biological networks derived from a widely studied and validated genetic network (E. coli) as well as on a significant number of differentiating (Th17) and differentiated human cells from a curated biological network data.
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•Use of algorithmic randomness to steer systems in dynamical space to control and reprogram them•Applying series of controlled interventions we reprogram systems, programs, and networks•The method reconstructs the phase space and generative rules of discrete systems•We validate on a number of networks with different topologies, and on biological networks
Gene Network; Systems Biology; Complex Systems; Computer Science; Algorithms
Chronic obstructive pulmonary disease (COPD) patients often show skeletal muscle dysfunction that has a prominent negative impact on prognosis. The study aims to further explore underlying mechanisms ...of skeletal muscle dysfunction as a characteristic systemic effect of COPD, potentially modifiable with preventive interventions (i.e. muscle training). The research analyzes network module associated pathways and evaluates the findings using independent measurements.
We characterized the transcriptionally active network modules of interacting proteins in the vastus lateralis of COPD patients (n = 15, FEV
46 ± 12% pred, age 68 ± 7 years) and healthy sedentary controls (n = 12, age 65 ± 9 years), at rest and after an 8-week endurance training program. Network modules were functionally evaluated using experimental data derived from the same study groups.
At baseline, we identified four COPD specific network modules indicating abnormalities in creatinine metabolism, calcium homeostasis, oxidative stress and inflammatory responses, showing statistically significant associations with exercise capacity (VO
peak, Watts peak, BODE index and blood lactate levels) (P < 0.05 each), but not with lung function (FEV
). Training-induced network modules displayed marked differences between COPD and controls. Healthy subjects specific training adaptations were significantly associated with cell bioenergetics (P < 0.05) which, in turn, showed strong relationships with training-induced plasma metabolomic changes; whereas, effects of training in COPD were constrained to muscle remodeling.
In summary, altered muscle bioenergetics appears as the most striking finding, potentially driving other abnormal skeletal muscle responses. Trial registration The study was based on a retrospectively registered trial (May 2017), ClinicalTrials.gov identifier: NCT03169270.
Regulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical ...manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.
To gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.
The data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.
Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced ...calcium store depletion in conventional CD4
CD25
T cells (Tcons) independently of IP
levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP
receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.
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