Showing papers by "Benjamin Meder published in 2016"

Distribution of miRNA expression across human tissues

Abstract: We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

MiRTargetLink--miRNAs, Genes and Interaction Networks.

Abstract: Information on miRNA targeting genes is growing rapidly. For high-throughput experiments, but also for targeted analyses of few genes or miRNAs, easy analysis with concise representation of results facilitates the work of life scientists. We developed miRTargetLink, a tool for automating respective analysis procedures that are frequently applied. Input of the web-based solution is either a single gene or single miRNA, but also sets of genes or miRNAs, can be entered. Validated and predicted targets are extracted from databases and an interaction network is presented. Users can select whether predicted targets, experimentally validated targets with strong or weak evidence, or combinations of those are considered. Central genes or miRNAs are highlighted and users can navigate through the network interactively. To discover the most relevant biochemical processes influenced by the target network, gene set analysis and miRNA set analysis are integrated. As a showcase for miRTargetLink, we analyze targets of five cardiac miRNAs. miRTargetLink is freely available without restrictions at www.ccb.uni-saarland.de/mirtargetlink.

A mutation in the glutamate-rich region of RNA-binding motif protein 20 causes dilated cardiomyopathy through missplicing of titin and impaired Frank–Starling mechanism

Abstract: Aim Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin ( TTN ). The functional and physiological consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study, we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. Methods and results We identified a family with DCM carrying a mutation (RBM20E913K/+) in a glutamate-rich region of RBM20. Western blot analysis of endogenous RBM20 protein revealed strongly reduced protein levels in the heart of an RBM20E913K/+ carrier. RNA deep-sequencing demonstrated massive inclusion of exons coding for the spring region of titin in the RBM20E913K/+ carrier. Titin isoform analysis revealed a dramatic shift from the less compliant N2B towards the highly compliant N2BA isoforms in RBM20E913K/+ heart. Moreover, an increased sarcomere resting-length was observed in single cardiomyocytes and isometric force measurements revealed an attenuated Frank–Starling mechanism (FSM), which was rescued by protein kinase A treatment. Conclusion A mutation outside the mutational hotspot of RBM20 results in haploinsufficiency of RBM20 . This leads to disturbed alternative splicing of TTN , resulting in a dramatic shift to highly compliant titin isoforms and an impaired FSM. These effects may contribute to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing.

Bias in High-Throughput Analysis of miRNAs and Implications for Biomarker Studies

Abstract: A certain degree of bias in high-throughput molecular technologies including microarrays and next-generation sequencing (NGS) is known. To quantify the actual impact of the biomarker discovery platform on miRNA profiles, we first performed a meta-analysis: raw data of 1 539 microarrays and 705 NGS blood-borne miRNomes were statistically evaluated, suggesting a substantial influence of the technology on biomarker profiles. We observed highly significant dependency of the miRNA nucleotide composition on the expression level. Higher expression in NGS was discovered for uracil-rich miRNAs (p = 7 × 10–37), while high expression in microarrays was found predominantly for guanine-rich miRNAs (p = 3 × 10–33). To verify the findings, 10 identical replicates of one individual were measured using NGS and microarrays (2 525 miRNAs from miRBase version 21). Overall, we calculated a correlation coefficient of 0.414 for both technologies. Detailed analysis however revealed that the correlation was observed only for miRN...

Prioritizing and selecting likely novel miRNAs from NGS data.

Abstract: Small non-coding RNAs play a key role in many physiological and pathological processes. Since 2004, miRNA sequences have been catalogued in miRBase, which is currently in its 21st version. We investigated sequence and structural features of miRNAs annotated in the miRBase and compared them between different versions of this reference database. We have identified that the two most recent releases (v20 and v21) are influenced by next-generation sequencing based miRNA predictions and show significant deviation from miRNAs discovered prior to the high-throughput profiling period. From the analysis of miRBase, we derived a set of key characteristics to predict new miRNAs and applied the implemented algorithm to evaluate novel blood-borne miRNA candidates. We carried out 705 individual whole miRNA sequencings of blood cells and collected a total of 9.7 billion reads. Using miRDeep2 we initially predicted 1452 potentially novel miRNAs. After excluding false positives, 518 candidates remained. These novel candidates were ranked according to their distance to the features in the early miRBase versions allowing for an easier selection of a subset of putative miRNAs for validation. Selected candidates were successfully validated by qRT-PCR and northern blotting. In addition, we implemented a web-server for ranking potential miRNA candidates, which is available at:www.ccb.uni-saarland.de/novomirank.

Validating Alzheimer's disease micro RNAs using next-generation sequencing.

Abstract: Introduction Molecular biomarkers for Alzheimer's disease (AD) can support detection and improved care for patients, but novel candidates require verification. We previously reported a 12-micro RNA (miRNA) blood-based signature using next-generation sequencing (NGS) of 54 AD cases and 22 controls. Methods We performed validation of 49 AD cases and 55 controls using NGS and also included 20 mild cognitive impairment and 90 multiple sclerosis samples to identify nonspecific markers. Thus, 103 AD cases, 77 unaffected controls, and 110 diseased controls were sequenced. Although the initial cohort came predominantly from the United States, the validation samples were collected in Germany. Results Five hundred eighty miRNAs were detected in the blood. In the initial cohort, we observed 203, in the validation cohort, 146 dysregulated miRNAs at a significance level of 0.05. With 68 miRNAs, the overlap was significant ( P = .0003). Likewise, the area under the receiver operator characteristic curve values of the miRNAs correlated (correlation of 0.93; 95% confidence interval 0.89–0.96; P −16 ). Discussion MiRNAs have the potential to support AD diagnosis and patient care.

Left ventricular long axis strain: a new prognosticator in non-ischemic dilated cardiomyopathy?

Abstract: Long axis strain (LAS) has been shown to be a fast assessable parameter representing global left ventricular (LV) longitudinal function in cardiovascular magnetic resonance (CMR). However, the prognostic value of LAS in cardiomyopathies with reduced left ventricular ejection fraction (LVEF) has not been evaluated yet. In 146 subjects with non-ischemic dilated cardiomyopathy (NIDCM, LVEF ≤45 %) LAS was assessed retrospectively from standard non-contrast SSFP cine sequences by measuring the distance between the epicardial border of the left ventricular apex and the midpoint of a line connecting the origins of the mitral valve leaflets in end-systole and end-diastole. The final values were calculated according to the strain formula. The primary endpoint of the study was defined as a combination of cardiac death, heart transplantation or aborted sudden cardiac death and occurred in 24 subjects during follow-up. Patients with LAS values > −5 % showed a significant higher rate of cardiac events independent of the presence of late gadolinium enhancement (LGE). The multivariate Cox regression analysis revealed that LVEDV/BSA (HR: 1.01, p −10 % and the presence of LGE, patients with 3 points had a significantly higher risk for cardiac events than those with 2 or less points. Assessment of long axis function with LAS offers significant incremental information for the prediction of cardiac events in NIDCM and improves risk stratification beyond established CMR parameters.

miRNAs and sports: tracking training status and potentially confounding diagnoses

Abstract: The dependency of miRNA abundance from physiological processes such as exercises remains partially understood. We set out to analyze the effect of physical exercises on miRNA profiles in blood and plasma of endurance and strength athletes in a systematic manner and correlated differentially abundant miRNAs in athletes to disease miRNAs biomarkers towards a better understanding of how physical exercise may confound disease diagnosis by miRNAs. We profiled blood and plasma of 29 athletes before and after exercise. With four samples analyzed for each individual we analyzed 116 full miRNomes. The study set-up enabled paired analyses of individuals. Affected miRNAs were investigated for known disease associations using network analysis. MiRNA patterns in blood and plasma of endurance and strength athletes vary significantly with differences in blood outreaching variations in plasma. We found only moderate differences between the miRNA levels before training and the RNA levels after training as compared to the more obvious variations found between strength athletes and endurance athletes. We observed significant variations in the abundance of miR-140-3p that is a known circulating disease markers (raw and adjusted p value of 5 × 10−12 and 4 × 10−7). Similarly, the levels of miR-140-5p and miR-650, both of which have been reported as makers for a wide range of human pathologies significantly depend on the training mode. Among the most affected disease categories we found acute myocardial infarction. MiRNAs, which are up-regulated in endurance athletes inhibit VEGFA as shown by systems biology analysis of experimentally validated target genes. We provide evidence that the mode and the extent of training are important confounding factors for a miRNA based disease diagnosis.

Essential light chain S195 phosphorylation is required for cardiac adaptation under physical stress.

Abstract: Aims Regulatory proteins of the sarcomere are pivotal for normal heart function and when affected by mutations are frequently causing cardiomyopathy. The exact function of these regulatory proteins and how mutations in these translate into distinct cardiomyopathy phenotypes remains poorly understood. Mutations in the essential myosin light chain (ELC) are linked to human cardiomyopathy characterized by a marked variability in disease phenotypes and high incidences of sudden death. Here we studied the role of the highly conserved S195 phosphorylation site of ELC using heterozygous adult zebrafish lazy susan ( lazm647 ) in regulating contractile function in normal physiology and disease. Methods and results Echocardiography revealed signs of systolic dysfunction in otherwise phenotypically unremarkable heterozygote mutants. However, after physical stress, heart function of laz heterozygous zebrafish severely deteriorated causing heart failure and sudden death. Mechanistically, we show that upon physical stress, ELCs become phosphorylated and lack of S195 dominant-negatively impairs ELC phosphorylation. In vitro motility analysis with native myosin from adult heterozygous hearts demonstrates that S195 loss, specifically following physical stress, results in altered acto-myosin sliding velocities and myosin binding cooperativity, causing reduced force generation and organ dysfunction. Conclusion Using adult heterozygous zebrafish, we show that ELC S195 phosphorylation is pivotal for adaptation of cardiac function to augmented physical stress and we provide novel mechanistic insights into the pathogenesis of ELC-linked cardiomyopathy.

miRNAs in ancient tissue specimens of the Tyrolean Iceman

Abstract: The analysis of nucleic acids in ancient samples is largely limited to DNA. Small noncoding RNAs (microRNAs) are known to be evolutionary conserved and stable. To gain knowledge on miRNAs measured from ancient samples, we profiled microRNAs in cryoconserved mummies. First, we established the approach on a World War One warrior, the "Kaiserjager", which has been preserved for almost one century. Then, we profiled seven ancient tissue specimens including skeletal muscle, stomach mucosa, stomach content and two corpus organ tissues of the 5,300-year-old copper age mummy Iceman and compared these profiles to the presence of organ-specific miRNAs in modern tissues. Our analyses suggest the presence of specific miRNAs in the different Iceman's tissues. Of 1,066 analyzed human miRNAs, 31 were discovered across all biopsies and 87 miRNAs were detected only in a single sample. To check for potential microbiological contaminations, all miRNAs detected in Iceman samples and not present in ancient samples were mapped to 14,582 bacterial and viral genomes. We detected few hits (3.9% of miRNAs compared with 3.6% of miRNAs). Interestingly, the miRNAs with higher abundance across all ancient tissues were significantly enriched for Guanine (P value of 10-13) and Cytosine (P value of 10-7). The same pattern was observed for modern tissues. Comparing miRNAs measured from ancient organs to modern tissue patterns highlighted significant similarities, e.g., for miRNAs present in the muscle. Our first comprehensive analysis of microRNAs in ancient human tissues indicates that these stable molecules can be detected in tissue specimens after 5,300 years.

Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression

Abstract: Calcium signalling plays a critical role in the pathogenesis of heart failure. Here we describe a cardiac protein named Myoscape/FAM40B/STRIP2, which directly interacts with the L-type calcium channel. Knockdown of Myoscape in cardiomyocytes decreases calcium transients associated with smaller Ca(2+) amplitudes and a lower diastolic Ca(2+) content. Likewise, L-type calcium channel currents are significantly diminished on Myoscape ablation, and downregulation of Myoscape significantly reduces contractility of cardiomyocytes. Conversely, overexpression of Myoscape increases global Ca(2+) transients and enhances L-type Ca(2+) channel currents, and is sufficient to restore decreased currents in failing cardiomyocytes. In vivo, both Myoscape-depleted morphant zebrafish and Myoscape knockout (KO) mice display impairment of cardiac function progressing to advanced heart failure. Mechanistically, Myoscape-deficient mice show reduced L-type Ca(2+)currents, cell capacity and calcium current densities as a result of diminished LTCC surface expression. Finally, Myoscape expression is reduced in hearts from patients suffering of terminal heart failure, implying a role in human disease.

Deep characterization of blood cell miRNomes by NGS.

Abstract: A systematic understanding of different factors influencing cell type specific microRNA profiles is essential for state-of-the art biomarker research. We carried out a comprehensive analysis of the biological variability and changes in cell type pattern over time for different cell types and different isolation approaches in technical replicates. All combinations of the parameters mentioned above have been measured, resulting in 108 miRNA profiles that were evaluated by next-generation-sequencing. The largest miRNA variability was due to inter-individual differences (34 %), followed by the cell types (23.4 %) and the isolation technique (17.2 %). The change over time in cell miRNA composition was moderate (<3 %) being close to the technical variations (<1 %). Largest variability (including technical and biological variance) was observed for CD8 cells while CD3 and CD4 cells showed significantly lower variations. ANOVA highlighted that 51.5 % of all miRNAs were significantly influenced by the purification technique. While CD4 cells were least affected, especially miRNA profiles of CD8 cells were fluctuating depending on the cell purification approach. To provide researchers access to the profiles and to allow further analyses of the tested conditions we implemented a dynamic web resource.

Pathway-based variant enrichment analysis on the example of dilated cardiomyopathy.

Abstract: Genome-wide association (GWA) studies have significantly contributed to the understanding of human genetic variation and its impact on clinical traits. Frequently only a limited number of highly significant associations were considered as biologically relevant. Increasingly, network analysis of affected genes is used to explore the potential role of the genetic background on disease mechanisms. Instead of first determining affected genes or calculating scores for genes and performing pathway analysis on the gene level, we integrated both steps and directly calculated enrichment on the genetic variant level. The respective approach has been tested on dilated cardiomyopathy (DCM) GWA data as showcase. To compute significance values, 5000 permutation tests were carried out and p values were adjusted for multiple testing. For 282 KEGG pathways, we computed variant enrichment scores and significance values. Of these, 65 were significant. Surprisingly, we discovered the "nucleotide excision repair" and "tuberculosis" pathways to be most significantly associated with DCM (p = 10(-9)). The latter pathway is driven by genes of the HLA-D antigen group, a finding that closely resembles previous discoveries made by expression quantitative trait locus analysis in the context of DCM-GWA. Next, we implemented a sub-network-based analysis, which searches for affected parts of KEGG, however, independent on the pre-defined pathways. Here, proteins of the contractile apparatus of cardiac cells as well as the FAS sub-network were found to be affected by common polymorphisms in DCM. In this work, we performed enrichment analysis directly on variants, leveraging the potential to discover biological information in thousands of published GWA studies. The applied approach is cutoff free and considers a ranked list of genetic variants as input.

microRNA assays for acute coronary syndromes.

Abstract: microRNAs are promising biomarkers for diverse cardiovascular diseases. While quantification of the small non-coding RNAs is routinely performed in the research laboratory, clinical-grade assessment of microRNAs in central laboratory environments or point-of-care testing is still in its infancy. In this review, we provide an overview on microRNAs as biomarkers for acute coronary syndromes and highlight promising technical approaches for microRNA-based assays systems.

A Self-Taught Artificial Agent for Multi-Physics Computational Model Personalization

Abstract: Personalization is the process of fitting a model to patient data, a critical step towards application of multi-physics computational models in clinical practice. Designing robust personalization algorithms is often a tedious, time-consuming, model- and data-specific process. We propose to use artificial intelligence concepts to learn this task, inspired by how human experts manually perform it. The problem is reformulated in terms of reinforcement learning. In an off-line phase, Vito, our self-taught artificial agent, learns a representative decision process model through exploration of the computational model: it learns how the model behaves under change of parameters. The agent then automatically learns an optimal strategy for on-line personalization. The algorithm is model-independent; applying it to a new model requires only adjusting few hyper-parameters of the agent and defining the observations to match. The full knowledge of the model itself is not required. Vito was tested in a synthetic scenario, showing that it could learn how to optimize cost functions generically. Then Vito was applied to the inverse problem of cardiac electrophysiology and the personalization of a whole-body circulation model. The obtained results suggested that Vito could achieve equivalent, if not better goodness of fit than standard methods, while being more robust (up to 11% higher success rates) and with faster (up to seven times) convergence rate. Our artificial intelligence approach could thus make personalization algorithms generalizable and self-adaptable to any patient and any model.

Erratum to: miRNAs and sports: tracking training status and potentially confounding diagnoses.

Abstract: Erratum to: J Transl Med (2016) 14:219 DOI 10.1186/s12967-016-0974-x Unfortunately, the original version of this article [1] contained an error. The name of the author [Farbod Sedaghat] was incorrect, the author's surname should be Sedaghat-Hamedani. The author's name is Farbod Sedaghat-Hamedani.