Comprehensive analysis of the REST transcription factor regulatory networks in IDH mutant and IDH wild-type glioma cell lines and tumors.

The RE1-silencing transcription factor (REST) acts either as a repressor or activator of transcription depending on the genomic and cellular context. REST is a key player in brain cell differentiation by inducing chromatin modifications, including DNA methylation, in a proximity of its binding sites. Its dysfunction may contribute to oncogenesis. Mutations in IDH1/2 significantly change the epigenome contributing to blockade of cell differentiation and glioma development. We aimed at defining how REST modulates gene activation and repression in the context of the IDH mutation-related phenotype in gliomas. We studied the effects of REST knockdown, genome wide occurrence of REST binding sites, and DNA methylation of REST motifs in IDH wild type and IDH mutant gliomas. We found that REST target genes, REST binding patterns, and TF motif occurrence proximal to REST binding sites differed in IDH wild-type and mutant gliomas. Among differentially expressed REST targets were genes involved in glial cell differentiation and extracellular matrix organization, some of which were differentially methylated at promoters or gene bodies. REST knockdown differently impacted invasion of the parental or IDH1 mutant glioma cells. The canonical REST-repressed gene targets showed significant correlation with the GBM NPC-like cellular state. Interestingly, results of REST or KAISO silencing suggested the interplay between these TFs in regulation of REST-activated and repressed targets. The identified gene regulatory networks and putative REST cooperativity with other TFs, such as KAISO, show distinct REST target regulatory networks in IDH-WT and IDH-MUT gliomas, without concomitant DNA methylation changes. We conclude that REST could be an important therapeutic target in gliomas.

SorLA restricts TNFα release from microglia to shape a glioma-supportive brain microenvironment.

SorLA, encoded by the gene SORL1, is an intracellular sorting receptor of the VPS10P domain receptor gene family. Although SorLA is best recognized for its ability to shuttle target proteins between intracellular compartments in neurons, recent data suggest that also its microglial expression can be of high relevance for the pathogenesis of brain diseases, including glioblastoma (GBM). Here, we interrogated the impact of SorLA on the functional properties of glioma-associated microglia and macrophages (GAMs). In the GBM microenvironment, GAMs are re-programmed and lose the ability to elicit anti-tumor responses. Instead, they acquire a glioma-supporting phenotype, which is a key mechanism promoting glioma progression. Our re-analysis of published scRNA-seq data from GBM patients revealed that functional phenotypes of GAMs are linked to the level of SORL1 expression, which was further confirmed using in vitro models. Moreover, we demonstrate that SorLA restrains secretion of TNFα from microglia to restrict the inflammatory potential of these cells. Finally, we show that loss of SorLA exacerbates the pro-inflammatory response of microglia in the murine model of glioma and suppresses tumor growth.

Exome sequencing to explore the possibility of predicting genetic susceptibility to the joint occurrence of polycystic ovary syndrome and Hashimoto's thyroiditis.

A large body of evidence indicates that women with polycystic ovary syndrome (PCOS) have a higher risk of developing Hashimoto's thyroiditis (HT) than healthy individuals. Given the strong genetic impact on both diseases, common predisposing genetic factors are possibly involved but are not fully understood. Here, we performed whole-exome sequencing (WES) for 250 women with sporadic PCOS, HT, combined PCOS and HT (PCOS+HT), and healthy controls to explore the genetic background of the joint occurrence of PCOS and HT. Based on relevant comparative analyses, multivariate logistic regression prediction modeling, and the most informative feature selection using the Monte Carlo feature selection and interdependency discovery algorithm, 77 variants were selected for further validation by TaqMan genotyping in a group of 533 patients. In the allele frequency test, variants in RAB6A, GBP3, and FNDC7 genes were found to significantly (padjusted < 0.05) differentiated the PCOS+HT and PCOS groups, variant in HIF3A differentiated the PCOS+HT and HT groups, whereas variants in CDK20 and CCDC71 differentiated the PCOS+HT and both single disorder groups. TaqMan genotyping data were used to create final prediction models, which differentiated between PCOS+HT and PCOS or HT with a prediction accuracy of AUC = 0.78. Using a 70% cutoff of the prediction score improved the model parameters, increasing the AUC value to 0.87. In summary, we demonstrated the polygenic burden of both PCOS and HT, and many common and intersecting signaling pathways and biological processes whose disorders mutually predispose patients to the development of both diseases.

Supporting the Diagnosis of Fabry Disease Using a Natural Language Processing-Based Approach.

In clinical practice, the consideration of non-specific symptoms of rare diseases in order to make a correct and timely diagnosis is often challenging. To support physicians, we developed a decision-support scoring system on the basis of retrospective research. Based on the literature and expert knowledge, we identified clinical features typical for Fabry disease (FD). Natural language processing (NLP) was used to evaluate patients' electronic health records (EHRs) to obtain detailed information about FD-specific patient characteristics. The NLP-determined elements, laboratory test results, and ICD-10 codes were transformed and grouped into pre-defined FD-specific clinical features that were scored in the context of their significance in the FD signs. The sum of clinical feature scores constituted the FD risk score. Then, medical records of patients with the highest FD risk score were reviewed by physicians who decided whether to refer a patient for additional tests or not. One patient who obtained a high-FD risk score was referred for DBS assay and confirmed to have FD. The presented NLP-based, decision-support scoring system achieved AUC of 0.998, which demonstrates that the applied approach enables for accurate identification of FD-suspected patients, with a high discrimination power.

Regulatory networks driving expression of genes critical for glioblastoma are controlled by the transcription factor c-Jun and the pre-existing epigenetic modifications.

BACKGROUND: Glioblastoma (GBM, WHO grade IV) is an aggressive, primary brain tumor. Despite extensive tumor resection followed by radio- and chemotherapy, life expectancy of GBM patients did not improve over decades. Several studies reported transcription deregulation in GBMs, but regulatory mechanisms driving overexpression of GBM-specific genes remain largely unknown. Transcription in open chromatin regions is directed by transcription factors (TFs) that bind to specific motifs, recruit co-activators/repressors and the transcriptional machinery. Identification of GBM-related TFs-gene regulatory networks may reveal new and targetable mechanisms of gliomagenesis. RESULTS: We predicted TFs-regulated networks in GBMs in silico and intersected them with putative TF binding sites identified in the accessible chromatin in human glioma cells and GBM patient samples. The Cancer Genome Atlas and Glioma Atlas datasets (DNA methylation, H3K27 acetylation, transcriptomic profiles) were explored to elucidate TFs-gene regulatory networks and effects of the epigenetic background. In contrast to the majority of tumors, c-Jun expression was higher in GBMs than in normal brain and c-Jun binding sites were found in multiple genes overexpressed in GBMs, including VIM, FOSL2 or UPP1. Binding of c-Jun to the VIM gene promoter was stronger in GBM-derived cells than in cells derived from benign glioma as evidenced by gel shift and supershift assays. Regulatory regions of the majority of c-Jun targets have distinct DNA methylation patterns in GBMs as compared to benign gliomas, suggesting the contribution of DNA methylation to the c-Jun-dependent gene expression. CONCLUSIONS: GBM-specific TFs-gene networks identified in GBMs differ from regulatory pathways attributed to benign brain tumors and imply a decisive role of c-Jun in controlling genes that drive glioma growth and invasion as well as a modulatory role of DNA methylation.

The role of epigenetic modifications, long-range contacts, enhancers and topologically associating domains in the regulation of glioma grade-specific genes.

Genome-wide studies have uncovered specific genetic alterations, transcriptomic patterns and epigenetic profiles associated with different glioma types. We have recently created a unique atlas encompassing genome-wide profiles of open chromatin, histone H3K27ac and H3Kme3 modifications, DNA methylation and transcriptomes of 33 glioma samples of different grades. Here, we intersected genome-wide atlas data with topologically associating domains (TADs) and demonstrated that the chromatin organization and epigenetic landscape of enhancers have a strong impact on genes differentially expressed in WHO low grade versus high grade gliomas. We identified TADs enriched in glioma grade-specific genes and/or epigenetic marks. We found the set of transcription factors, including REST, E2F1 and NFKB1, that are most likely to regulate gene expression in multiple TADs, containing specific glioma-related genes. Moreover, many genes associated with the cell-matrix adhesion Gene Ontology group, in particular 14 PROTOCADHERINs, were found to be regulated by long-range contacts with enhancers. Presented results demonstrate the existence of epigenetic differences associated with chromatin organization driving differential gene expression in gliomas of different malignancy.

Mapping chromatin accessibility and active regulatory elements reveals pathological mechanisms in human gliomas.

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.

Unveiling new interdependencies between significant DNA methylation sites, gene expression profiles and glioma patients survival.

In order to find clinically useful prognostic markers for glioma patients' survival, we employed Monte Carlo Feature Selection and Interdependencies Discovery (MCFS-ID) algorithm on DNA methylation (HumanMethylation450 platform) and RNA-seq datasets from The Cancer Genome Atlas (TCGA) for 88 patients observed until death. The input features were ranked according to their importance in predicting patients' longer (400+ days) or shorter (≤400 days) survival without prior classification of the patients. Interestingly, out of the 65 most important features found, 63 are methylation sites, and only two mRNAs. Moreover, 61 out of the 63 methylation sites are among those detected by the 450 k array technology, while being absent in the HumanMethylation27. The most important methylation feature (cg15072976) overlaps with the RE1 Silencing Transcription Factor (REST) binding site, and was confirmed to intersect with the REST binding motif in human U87 glioma cells. Six additional methylation sites from the top 63 overlap with REST sites. We found that the methylation status of the cg15072976 site affects transcription factor binding in U87 cells in gel shift assay. The cg15072976 methylation status discriminates ≤400 and 400+ patients in an independent dataset from TCGA and shows positive association with survival time as evidenced by Kaplan-Meier plots.

Perioperative analgesia after intrathecal fentanyl and morphine or morphine alone for cesarean section: A randomized controlled study.

OBJECTIVES: Intrathecal morphine is used in the postoperative management of pain after caesarean section (CS), but might not be optimal for intraoperative analgesia. We hypothesized that intrathecal fentanyl could supplement intraoperative analgesia when added to a local anesthetic and morphine without affecting management of postoperative pain. METHODS: This prospective, randomized, double-blind, parallel-group study included 60 parturients scheduled for elective CS. Spinal anesthesia consisted of bupivacaine with either morphine 100 µg (M group), or fentanyl 25 µg and morphine 100 µg (FM group). The frequency of intraoperative pain and pethidine consumption in the 24 hours postoperatively was recorded. RESULTS: Fewer patients in the FM group required additional intraoperative analgesia (P < .01, relative risk 0.06, 95% confidence interval [CI] 0.004-1.04). The FM group was noninferior to the M group for 24-hour opioid consumption (95% CI -10.0 mg to 45.7 mg, which was below the prespecified boundary of 50 mg). Pethidine consumption in postoperative hours 1 to 12 was significantly higher in the FM group (P = .02). Postoperative nausea and vomiting (PONV) were more common in the FM group (P = .01). Visual analog scale scores, effective analgesia, Apgar scores, and rates of pruritus and respiratory depression were similar between the groups. CONCLUSIONS: Intrathecal combination of fentanyl and morphine may provide better perioperative analgesia than morphine alone in CS and could be useful when the time from anesthesia to skin incision is short. However, an increase in PONV and possible acute spinal opioid tolerance after addition of intrathecal fentanyl warrants further investigation using lower doses of fentanyl.

Maps of context-dependent putative regulatory regions and genomic signal interactions.

Gene transcription is regulated mainly by transcription factors (TFs). ENCODE and Roadmap Epigenomics provide global binding profiles of TFs, which can be used to identify regulatory regions. To this end we implemented a method to systematically construct cell-type and species-specific maps of regulatory regions and TF-TF interactions. We illustrated the approach by developing maps for five human cell-lines and two other species. We detected ∼144k putative regulatory regions among the human cell-lines, with the majority of them being ∼300 bp. We found ∼20k putative regulatory elements in the ENCODE heterochromatic domains suggesting a large regulatory potential in the regions presumed transcriptionally silent. Among the most significant TF interactions identified in the heterochromatic regions were CTCF and the cohesin complex, which is in agreement with previous reports. Finally, we investigated the enrichment of the obtained putative regulatory regions in the 3D chromatin domains. More than 90% of the regions were discovered in the 3D contacting domains. We found a significant enrichment of GWAS SNPs in the putative regulatory regions. These significant enrichments provide evidence that the regulatory regions play a crucial role in the genomic structural stability. Additionally, we generated maps of putative regulatory regions for prostate and colorectal cancer human cell-lines.

Analgesic efficacy of intrathecal fentanyl during the period of highest analgesic demand after cesarean section: A randomized controlled study.

Cesarean section (CS) is one of the most common surgical procedures in female patients. We aimed to evaluate the postoperative analgesic efficacy of intrathecal fentanyl during the period of greatest postoperative analgesic demand after CS. This period was defined by detailed analysis of patient-controlled analgesia (PCA) usage.This double-blind, placebo-controlled, parallel-group randomized trial included 60 parturients who were scheduled for elective CS. Participants received spinal anesthesia with bupivacaine supplemented with normal saline (control group) or with fentanyl 25 µg (fentanyl group). To evaluate primary endpoints, we measured total pethidine consumption over the period of greatest PCA pethidine requirement. For verification of secondary endpoints, we recorded intravenous PCA requirement in other time windows, duration of effective analgesia, pain scores assessed by visual analog scale, opioid side effects, hemodynamic changes, neonatal Apgar scores, and intraoperative pain.Detailed analysis of hour-by-hour PCA opioid requirements showed that the greatest demand for analgesics among patients in the control group occurred during the first 12 hours after surgery. Patients in the fentanyl group had significantly reduced opioid consumption compared with the controls during this period and had a prolonged duration of effective analgesia. The groups were similar in visual analog scale, incidence of analgesia-related side effects (nausea/vomiting, pruritus, oversedation, and respiratory depression), and neonatal Apgar scores. Mild respiratory depression occurred in 1 patient in each group. Fewer patients experienced intraoperative pain in the fentanyl group (3% vs 23%; relative risk 6.8, 95% confidence interval 0.9-51.6).The requirement for postoperative analgesics is greatest during the first 12 hours after induction of anesthesia in patients undergoing CS. The addition of intrathecal fentanyl to spinal anesthesia is effective for intraoperative analgesia and decreases opioid consumption during the period of the highest analgesic demand after CS, without an increase in maternal or neonatal side effects. We recommend using intrathecal fentanyl for CS in medical centers not using morphine or other opioids intrathecally at present.

A Significant Regulatory Mutation Burden at a High-Affinity Position of the CTCF Motif in Gastrointestinal Cancers.

Somatic mutations drive cancer and there are established ways to study those in coding sequences. It has been shown that some regulatory mutations are over-represented in cancer. We develop a new strategy to find putative regulatory mutations based on experimentally established motifs for transcription factors (TFs). In total, we find 1,552 candidate regulatory mutations predicted to significantly reduce binding affinity of many TFs in hepatocellular carcinoma and affecting binding of CTCF also in esophagus, gastric, and pancreatic cancers. Near mutated motifs, there is a significant enrichment of (1) genes mutated in cancer, (2) tumor-suppressor genes, (3) genes in KEGG cancer pathways, and (4) sets of genes previously associated to cancer. Experimental and functional validations support the findings. The strategy can be applied to identify regulatory mutations in any cell type with established TF motifs and will aid identifications of genes contributing to cancer.

Genetic variability of the grey wolf Canis lupus in the Caucasus in comparison with Europe and the Middle East: distinct or intermediary population?

Despite continuous historical distribution of the grey wolf (Canis lupus) throughout Eurasia, the species displays considerable morphological differentiation that resulted in delimitation of a number of subspecies. However, these morphological discontinuities are not always consistent with patterns of genetic differentiation. Here we assess genetic distinctiveness of grey wolves from the Caucasus (a region at the border between Europe and West Asia) that have been classified as a distinct subspecies C. l. cubanensis. We analysed their genetic variability based on mtDNA control region, microsatellite loci and genome-wide SNP genotypes (obtained for a subset of the samples), and found similar or higher levels of genetic diversity at all these types of loci as compared with other Eurasian populations. Although we found no evidence for a recent genetic bottleneck, genome-wide linkage disequilibrium patterns suggest a long-term demographic decline in the Caucasian population--a trend consistent with other Eurasian populations. Caucasian wolves share mtDNA haplotypes with both Eastern European and West Asian wolves, suggesting past or ongoing gene flow. Microsatellite data also suggest gene flow between the Caucasus and Eastern Europe. We found evidence for moderate admixture between the Caucasian wolves and domestic dogs, at a level comparable with other Eurasian populations. Taken together, our results show that Caucasian wolves are not genetically isolated from other Eurasian populations, share with them the same demographic trends, and are affected by similar conservation problems.

Cytochrome b gene (cytb) sequence diversity in a Microtus oeconomus population from Bialowieza Primeval Forest.

Based on published information about the glacial, postglacial, and recent distribution of the root vole, Microtus oeconomus, we hypothesized that a population inhabiting the pristine wetland in eastern Poland (Bialowieza Primeval Forest) might comprise a high diversity of haplotypes. The support for this hypothesis was provided by an analysis of partial cytb gene sequences from 149 voles sampled within a two-hectare plot during a nine-year study. In this population, we identified eight haplotypes (PLB1-PLB8), four of which were new to the root vole. These haplotypes were characterized by low nucleotide diversity (π = 0.0054, SE = 0.0019), the absence of transversional differences between sequences, and no changes in the encoded amino acid sequence: features suggesting a lack of immigration from the distant populations. The haplotype number and their frequency distribution in males and females did not differ significantly. An assessment of the persistence of matrilines in the local population throughout the study period revealed that the haplotype composition was relatively stable for only about 3 years. A more complete haplotype network for root voles in Europe was constructed by combining the newly identified haplotypes with the 45 previously described haplotypes. Two of the haplotypes detected in this study occupy key positions in this network: PLB5, as the closest link to the North European group, and PLB8, as an ancestor to many other Central European haplotypes.

Temporally stable genetic variability and dynamic kinship structure in a fluctuating population of the root vole Microtus oeconomus.

Genetic variability, kin structure and demography of a population are mutually dependent. Population genetic theory predicts that under demographically stable conditions, neutral genetic variability reaches equilibrium between gene flow and drift. However, density fluctuations and non-random mating, resulting e.g. from kin clustering, may lead to changes in genetic composition over time. Theoretical models also predict that changes in kin structure may affect aggression level and recruitment, leading to density fluctuations. These predictions have been rarely tested in natural populations. The aim of this study was to analyse changes in genetic variability and kin structure in a local population of the root vole (Microtus oeconomus) that underwent a fourfold change in mean density over a 6-year period. Intensive live-trapping resulted in sampling 88% of individuals present in the study area, as estimated from mark-recapture data. Based on 642 individual genotypes at 20 microsatellite loci, we compared genetic variability and kin structure of this population between consecutive years. We found that immigration was negatively correlated with density, while the number of kin groups was positively correlated with density. This is consistent with theoretical predictions that changes in kin structure play an important role in population fluctuations. Despite the changes in density and kin structure, there was no genetic differentiation between years. Population-level genetic diversity measures did not significantly vary in time and remained relatively high (H(E) range: 0.72-0.78). These results show that a population that undergoes significant demographic and social changes may maintain high genetic variability and stable genetic composition.